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EXPRESSION OF FIBROBLAST GROWTH FACTOR RECEPTOR 3 KN MULTIPLE MYELOMA CAUSES IL-6-INDEPENDENCE
AND ENHANCED SURWAL
Elizabeth Emma Plowright
A thesis submitted in confôrmity with the requirernents for the degree of Master of Science
hstitute of Medical Sciences University of Toronto
O by Elizabeth Emma Plowright, 1999
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Expression of fibroblast growth factor receptor 3 in Multiple Myeloma causes IL-6-independence and enhanced survival
Master of Science 1999 Elizabeth Plowright
Institute of Medical Science University of Toronto
Absîract
Multiple myeloma (MM) is a common hematologic malignancy. A t(4;14)
chromosomal translocation has been identified in 25% of multiple myeloma which results
in dysregulated expression of fibroblast growth factor receptor 3 (FGFR.3) on der(l4) and
an IgH-MMSET hybrid mRNA on der(4). Activating mutations of the translocated
FGFR3 have been identified in MM cells suggesting that FGFR3 plays an important role
in tumor development. In this thesis the potential role of FGFR3 in myeloma was
investigated in an interleukin-6-dependent murine myeloma ce11 line. Overexpression of
wild-type FGFR3 stimulated with ligand or mutant FGFR3 resulted in interleukin-6-
independence. decreased apoptosis in the absence of Uiterleukin-6 and an enhanced
proliferative response to interleukin-6. These findings provide an explanation for the
rnechanism by which f&3 contributes to both the viability and propagation of the
myeloma clone and may provide a basis for the development of therapies targeting
myeloma patients harbouring the t(4; 14) translocation.
Acknowledgments
1 would like to thank my supe~so r s , Dr. A. Keith Stewart and Dr. Ian Dubé, for
providing me with the opportunity to pursue the research contained within this thesis.
My supervisors, as well as my thesis cornmittee members, Dr. Brent Zanke and Dr.
Robert Hawley, have consistently challenged me throughout this project with intriguing
questions and have provided me with vaiuable advise and direction.
1 would also like to thank Dr, Dwayne Barber and Dr. Donald Branch, who both
provided valuable research supplies, as well as guidance through technical difficulties.
Both individuals were endlessly patient with my consistent questions and have
contributed significantly to my understanding of several techniques.
1 also need to thank the many members of the lab who have provided both
assistance with technical matters and provided moral support throughout the duration of
my studies. 1 especiaiiy offer my thanks to Darrin Cappe who made my first few months
in the 1ab easier by dways assisting me in figuring out cloning problems, Christine
Dodgson who shared her own experiences to guide me and Zhihu Li who has assisted me
technicdly. My experience within the lab was exceptiond due to al1 the interesting and
arnazing people who worked with me.
1 also need to extend my gratitude to my family and fiends who have always been
available to listen to me in times of need. My fiiends have reminded me to stop and
smell the roses. My family has constantly stood behind my decisions in life, encouraging
hard work and dedication but recognïzing the need for individuality. Their support has
been vital to me.
iii
Table of Contents
. . Abstract ...................................................................................................................... il
... ............................................................................. ............ Acknowledgrnents ..... ...... .. rii
....................................................................................................... Table of Contents iv
............................................................................................................. List of Tables vii
... ................ List of Figures .. .......................................................................................... viu
List of Abbreviations ................................................................................................. x
CHAPTER 1 : Introduction
Multiple myeloma ............................................................................................. 1
1 . 1 . 1 Growth factors in multiple myeloma .................................................. 3
1.1.2 Genetic rearrangements in multiple myeloma .................................... 4
1.1.2.1 Translocations into immunoglobulin
.............. heavy chah locus ... .................................... 6
1.1.2.2 Fibroblast growth factor receptor 3 translocations
................................ to immunoglobulin heavy chah locus 9
................ Fibroblast growth factor receptor 3 ..................................--.. 10
1.2.1 Function of fibroblast growth factor receptor 3 ................................ 16
.................................................................... 1.2.2 Thmatophonc dysplasia 1 8
Interleukin-6 ...................................................................................................... 20
Jak-STAT signal transduction pathway ............................................................. 25
............................................................... ............................... Jaks ....
STATs .............................. ... .............................................................
...................................................................................................... MAP kinases
Apoptosis ...........................................................................................................
1.6.1 B ~ 1 - x ~ ................... .. ..... .. .............................................................
1.6.2 Bcl-2 family members and multiple myeloma ....................................
.................................................................................... S tudy rationale ............. ..
.......................................................................................... Hypothesis .......... ..
Experimental objectives .................................... .... ...........................................
CHAPTER 2: Materials and Methods
Retrovirai vector construction ............................................................................ 43
Retroviral production ..................... ... ............................................................. 43
Expression of wild-type f@3 and fgfi-TD in B9 cells .........................~~~~~~~~~.. 46
..................................................................................................... Tissue culture 46
. * Ce11 viability and proliferation .......... ... ........................................................ 47
Ligand stimulated proliferation assay ................................................................ 47
Ce11 cycle and apoptosis analysis ....................................................................... 48
Western blots ................................................................................................. 49
...................... 2.9 Irnmunoprecipitation and NI viîro kinase assay ...................... ... 50
CHAPTER 3 : Results
..................................................... 3.1 Generation of FGFW expressing ce11 lines 52
3.2 Function of hurnan FGFR3 in transfected ce11 iines .......................................... 52
3 -3 Growth respoose to IL6 .................................................................................... 59
........ 3.4 Expression of human FGFR3 decreases apoptosis in the absence of IL-6 67
3 -5 FGFR3 signaling induces phosphorylation of STAT3 ...................................... 70
3.6 FGFEU survivd signal is mediated by upreguiation of bcl-xL ......................... 73
3.7 Enhanced proliferative response to IL-6 is not mediated by
ERK or SAPK ................................................................................................... 82
CHAPTER 4: Discussion and Future Investigations
4.1 Discussion .......................................................................................................... 95
. . ................ ................................................................ 4.2 Future Investlgaiions .......... 106
..................................................... References .. .......................................... 108
List of Tables
Table 1 : Percentage of ceiis in various stages of the ceii cycle after .............................. 72 hours of culture in the presence of absence of I L 6 71
Table 2: Percentage of apoptotic B9 ceiis as assessed by Tunel assay ................... after five days of culture in the presence or absence of IL-6 ... 72
vii
List of Figures
Figure 1:
Figure 2:
Figure 3:
Figure 4:
Figure 5:
Figure 6:
Figure 7:
Figure 8:
Figure 9:
. . ..................................................................... FGFR3 protein orgafllzation 12
Schematic diagram representing IL-6 signaling cascade ......................... 22
Schematic diagram of the three MAP kinase cascades ............................ 30
Vector diagram of MFINV-WT and MFW-TD .................................. 44
Expression of FGFR3 in GP+E packaging ceU h e s ............................... 53
Expression of FGFR3 is achieved at variable ievels in B9-WT and B9-TD cells ...................................................... .... 55
FGFR3 is phosphorylated in the presence of ligand (aFGF+heparin) in BPWT and constitutively phosphorylated in B9-'ID .......................... 57
.............................................. FGFR3 expressed in B9 cells is fùnctional 60
Cells expressing mutant FGFEU have an enhanced proliferative rate in the presence of IL-6 and enhamed survival in the absence of IL-6 ...................................................................................................... 62
Figure 10: B9-TD cells are viable in the absence of IL-6 ....................................... 65
Figure 1 1 : Ligand stimulated cells expressing wiid-type FGFR3 are more metabolically active in the absence of IL-6 ............................. .............. 68
Figure 12: STATS is tyrosine phosphorylated independently of IL.6.- .......... .. .... 74
Figure 13 : STATl is only tyrosine phosphorylated upon IL-6 stimulation .............. 76
Figure 1 4: STAT3 is constitutively tyrosine phosphory lated in FGFR3 expressing cells ........................................................................................ 78
Figure 15: STAT3 is phosphorylated in B9-WT and B9-TD upon stimulation ................................................ with ligand (aFGF+heparin) ................ ... 80
Figure 16: Expression of Bcl-xL protein is elevated in FGFR3 overexpressing cells ................................................................................ 83
Figure 17: Bax protein levels are not aitered due to FGFR3 expression
viii
............................................................................................... in B9 cells 86
.................. .................... Figure 18: ERK activity is induced by IL-6 stimulation .. 89
Figure 19: SAPK activity is not altered due to FGFIU expression .......................... 91
.............................. Figure 20: p38 activity is induced by ligand binding to FGFR3 93
List of Abbreviations
ACH aFGF ATP
CNTF
ERK
FADD FCS FGF FGFR FGFR.3 FISH
G-CSF GCK GDP GM-CSF Grb2 GTP
HCH hnRNPs HPKl HEU'
Achondroplasia Acidic fibroblast growth factor Adenosiue triphosphate
Basic fibroblast growth factor Bcl-2 homology 1 Bcl-2 homology 2 Bcl-3 homology 3
Ciliary neurotrophic factor
Extracellular signal-regulated kinase
Fas-associated protein with death domain Fetal calf semm Fibroblast growth factor Fibroblast growth factor receptor Fibroblast growth factor receptor 3 Fluorescence in situ hybridization
Granulocyte colony-stinulating factor Germinal center kinase Guanosine diphosphate Granulocyte-macrophage colony-stimulating factor Growth factor receptor bound protein 2 Guanosine triphosphate
Hypochondroplasia Heterogeneous nuclear ribonucleoprotein particles Hematopoietic progenitor kinase 1 Horseradish peroxidase
Ixnmunoglobulin lmmunoglobulin heavy chain immunoglobulin light chain Interleukin-6 Interleukin-6 receptor Iscove's modified Dulbecco's medium Interferon regulatory factor 4
Jak Janus kinase
LIF L S W
MAP MBP MDR mFGFR3 MGUS M X 3 MM MMSET MTT MUM1
PARP PBS PCR PLC--{ PR^ P-w
SAPK SDS-PAGE SH2 SH3 SKY SOS 1 STAT
TD TdT TGF-P
VAD
Jun N-terminal kinase
Leukemia inhibitory factor Lymphocyte-specific interferon regdatory factor
Mitogen-activated protein Myelin basic protein Multi-drug resistance Murine fibroblast growth factor receptor 3 Monoclonal gammopathy of undetermined significance Mixed lineage kinase 3 Multiple myeloma Multiple myeloma SET domain protein 3 -(4,5-dimethy lthiazo 1-2-y1)-2,s-dipheny lteo bromide Multiple myeloma oncogene 1
Nuclear magnetic resonance
Oncostatin M
Pol y ( ADP-ribose) po 1 ymerase Phosphate buffered saline Polymerase chah reaction P hospholipase C-y Retino blastoma protein Phosphotyrosine
Retinoblastoma
Stress activated protein b a s e Sodium dodecyl sulfate polyacrylamide gel electrophoresis Src homology domain 2 Src homology domain 3 Spectrd karyotyping Son of sevenless Signal transducea and activators of transcription
Thanatophoric dysplasia Terminal deoxynucleotidyl transferase Transforming growth factor+
Uitravio let
Vincristhe, doxorubicin, and dexamethasone
Chapter 1 Introduction
1.1 Multiple Myeloma
Multiple myeloma (MM) is an uniformly fatal fonn of human cancer that involves
clonai expansion of differentiated B cells. It normally affects individuals over the age of
40 and accounts for 10% of aii hematopoietic malignanciesl. The pathogenesis of the
disease is currentiy unknown and there is no cure- Clinical features of MM inchde
recurrent bacterial infections, anemia, osteolytic lesions and rend insufficiency2. Despite
some recent advances in therapy for younger patients undergoing autologous stem ce11
tran~~lantatiod, treatment outcomes have improved little over the past 20 yean4 and
median survival remaias only 3-4 years3.
Myeloma cells are long-lived but have a low proliferative potentialS with only a
rmall percentage of myeloma cells, less than 1%, dividing at any one Urne5; 6. The
malignant cells produce immunoglobulins, usually monoclonal IgG or IgA. T k bone
marrow is colonized by myeloma cells, where they cause "punched-out" lesions in the
bones. Although MM is usually disseminated throughout canceilous bone, circulating
rnalignant cells are only clinically evident in terminal phases of the disease7. In the bone
marrow environment the bone marrow stromal cells are thought to provide essential
growth factors and cytokines to MM cells.
The absence of proliferating rnalignant cells in most MM patients makes it
difficult to fully identify the phenotype and abnormaiities of the MM stem ce118. It has
been postulated that the precursor MM ce11 is a pst-gemiinal center ce11 that selectively
homes to the bone mamod; 'O. However, the smaii population of memory pre-switch B
cells expressing tumor-specific V@)J genes that c m be detected in MM likely represents
ancestral footprints of the malignant clone but may also suggest the presence of a pre-
11; 12 switch B stem ce11 - Myeloma cells have productively rearranged V@)J sequences
of both heavy and light chahs and the presence of stable somatic hypermutations of the
expressed immunoglobulin (Ig) gene products 10; 13-15 demonstrates that such B cells have
responded to antigen in the past. These cells have also undergone isotype switch
recombination of the heavy chain, usually IgM to IgG or I~A'. This exclusive isotype
bias supports the hypothesis that the critical event in MM occurs after antigen selection
and class switching in B ce11 development has taken place16.
Treatrnent of MM has progressed little over the past 20 years4 except for younger
patients undergohg autologous stem ce11 transplants3. Standard treatment for older
3; 17 patients remains a combination of melphalan and prednisone , however, in newly
diagnosed younger patients it is common to use a combination of vincristhe, doxorubicin
and dexamethasone (VAD). Even though VAD treatment does not prolong survival, it
tends to induce rapid remissions". Despite extensive study the use of combination
chemotherapy has consistently failed to improve outcome when compared to melphalan
and prednisone. The lack of advance with conventional chemotherapy has resulted in
interest in high dose melphalan therapy4. High doses of melphalan can induce complete
remission in 20-30% of patients 3; 18-20 but this treatment ofien causes severe
myelosuppression4. After high dose therapy, autologous stem ce11 transplants increase the
18; 21 rate of restoration of hematopoiesis , but contamination of the gr& by myeloma cells
is a concem. Despite evidence that high dose melphalan foilowed by autologous stem
ce11 transfer can improve suMval for selected patients3, all patients continue to relapse
after transplant. Novel therapies are therefore of utmost importance in improving
outcornes for MM patients.
1.1.1 Growth Factors in Multiple Myeloma
1nterleuk.b-6 (IL-6) is the most important cytokine for MM ce11 growth and
22-24 survival. IL-6 can support in vitro growth of fieshly explanted myeloma cells .
Addition of ad-Il-6 monoclonal antibodies to myeloma ce11 cultures almost completely
inhibits ceIl growthu. IL-6 has also been s h o w to be capable of preventing apoptosis in
25; 26 MM cells . Some myeloma cells are capable of producing I L 6 and most express the
22; 23 IL-6 receptor . Immature MM cells ( ~ ~ 4 5 ' ) express and produce IL-6 and its
receptor while mature MM cells (CD453 no longer express IL-6 and seldom express the
IL-6 receptorZ7.
Some other growth factors involved in MM include eorandocyte colony-
stimulating factor (G-CSF), granulocyte/macrophage colony-stirnulating factor (GM-
CSF) and interferon-a. G-CSF is a growth factor for freshly explanted myeloma cel~s'~.
29; 30 This cytokine has stnictural homology to IL-6 and its receptor shares homology with
gp1303'. Studies which have demonstrated that G-CSF-mediated growth in MM can be
inhibited by anti-IL-6 and anti-IL-6 receptor (IL-6R) an t ib~dies~~ . GM-CSF can
synergize with IL-6 to support myeloma ce11 proliferatio$2. Reports suggest that the
addition of GM-CSF and IL-6 to fkeshly explanted myeloma ceils enables the generation
of IL-6-dependent ceii hes4' '. interferons stimulates proliferation of fiesh myeloma
33-35 cells in 1940% of patients however, at high doses it has k e n found to inhibit
proliferatio~3. It has also been reported to stimulate the proliferatioo of MM ce11 liues by
inducing autocrine production of ~ - 6 ~ ~ .
1.1 -2 Genetic Rearrangements in Muitiple Myeloma
In MM it is dificult to determine the initiating cytogenetic event because at the
t h e of diagnosis most karyotypes have already evolved."and because karyotypes can
only be obtained in 3040% of patients. Most MM cens are found to be aneuploid 38; 39
and there are extensive chromosome abn~rmalities'~. As well, chromosomal aberrations
tend to increase as the disease progresses41' 42. Karyotypic abnormalities in MM have
40-42 been detected at a fiequency of 30-50% by conventional analysis . However,
conventional cytogenetics underestimates the fiequency and extent of abnormalities since
these techniques rely on obtaining metaphase spreads that are difficult to obtain in MM
5; 43 due to the low proliferative potential of the tumor cells . More recent cytogenetic
techniques, such as fluorescence in siru hybridization (FISH) and spectral kqotyping
(SKY), detect chromosomal aneuploidy in 80-90% of MM patients 39; 43; 44
The most common cytogenetic abnomaiities in MM are translocations into the
immunoglobulin heavy chah (IgH) locus 16; 40; 4s (see section 1.1.2.1 ), monosomy 13 41; 42;
44; 44; 46; 47 and trisomies of chromosomes 3, 5, 7, 9, 1 1, 15 and 19 41; 42; 44; 47 . These
abnormalities in MM tend to involve oncogenes or tumor suppressors. For example,
alterations of the c-myc oncogew locus concomitant with elevated expression of c-ntyc
mRNA has been detected in MM? High expression of c-myc mRNA has been detected
in approximately 90% of MM patients48.
Rus oncogene mutations have been identified in 39% of newly diagnosed patients
with MM'~. The expression of N-rus in the IL-6-dependent myeloma ce11 line, ANBL6,
resulted in significant IL-6-independent proliferation and reduction in apoptosis"9. Thus
ras mutations may enable MM ceils to be less dependent on cytokines to proiiferate and
are likely capable of escaping apoptosis generating a long-lived cell.
Mutations of the tumor suppressor p53 also fiequently occur in MM and appear
50; 5 1 to be associated with more advanced foms of MM . In one study point mutations in
p j 3 were detected in 7 of 53 patients5o. Overexpression of p53 in a myeloma ce11 line,
U266, suppresses autocrine IL-6 production and proliferation52 suggesting that loss of
pS3 in MM may lead to enhanced proliferation of MM cells.
Mutations of the turnor suppressor Rb have k e n described in up to 70% of
patients with MM 51; 53; 54 . Urashima et al. (1996) demonstrated that incubation of MM
cells with antisense Rb oligonucleotides triggered IL-6 secretion and ce11 proliferation.
Monoallelic deletions of chromosome 13, which contains the Rb gene, are one of the
most cornmon cytogenetic aberrations in Mhd 1; 42; 44 but do not seem to be associated
with altered Rb expression16. This suggests that another tumor suppressor besides Rb
may be located on chromosome 13 in MM? Thus the high fkequency of change in
known tumor suppressors and oncogenes underscores the dificulty in defining molecular
pathways relevant to myeloma growth amongst considerable genetic chaos.
1-1 -2.1 Translocations into the immunoglobulin heavy chah locus
The hallmark lesion of most 9-ceii tumors involves dysregulation of an oncogene
by a chromosomal translocation involving the IgH locus (14q32.3) or less fiequently the
5 5-5 7 immunoglobului light chah (IgL) loci(2p 1 2 ~ or 22ql1 A ) . These translocations are
the results of errors in t w ~ different gene rearrangement mechanisms, V(D)J
recombination (occurs early in B ce11 development) and isotype switch recombination
(occurs late in B ce11 deve~o~rnent)~~. Breakpoints near JH segments are thought to occur
during V@)J recombination while those near switch are due to errors occurring in
isospe switch recombination. Switch regions consist of tandem pentamenc repeats that
are 1-3 kb long. These sequences are located at the 5' end of each constant region gene
segment and mediate isotype ciass switching. In MM ce11 lines the translocations into IgH
58-60 are preferentially found to involve switch regions .
Translocations into IgH switch regions are considered illegitimate switch
recombinations because the recombination events involve only one switch region. These
translocations result in dysregulated gene expression by juxtaposition of endogenous
pmmoters to the powerful regulatory regions of the IgH locus6'. They typically result in
cis dysregulation of the oncogene located on the partner chromosome involved in the
translocation while the aHelic oncogene on the unaffected chromosome is not
dysregulated62. In MM it is hypothesized that these translocations affect the non-
productive Ig allele'6.
It has been proposed that translocations into switch regions are early events in the
58; 63 development of MM - Monoclonal gammopathy of undetermined signifïcance
(MGUS) is a plasma cetl disorder that may progress to MM. Lf MGUS progresses to MM
it does so without a change in IgH i ~ o t ~ ~ e ~ ~ , supporting the theory that the translocations
in MM are early events. Also the IgH isotype in MM remains identicai throughout
myeloma developments8 and there is a similar incidence of illegitimate translocations in
al1 three stages of M.
Studies using traditional cytogenetics detect translocations i n v o l ~ g l4q32 in 1 O-
60% of M M ~ ~ , however, these techniques are limited due to the dificulty of obtaining
metaphase spreads in MM. To increase the sensitivity of detecting these translocations
Bergsagel et al. (1996) combined traditional cytogenetics with a Southem blot technique
in order to identiQ breakpoints on IgH locus. From this analysis it became apparent that
most MM ce11 lines (1 9 of 2 1) contained IgH translocations 58; 59; 6s . By analysing patient
samples it was evident that this phenornenon was not just a ce11 line artifact 16; 63; 64; 66; 67
Richelda et al. (1997) detemiined that 21 of 88 (24%) MM tumor biopsies fiom
MM patients displayed rearranged fiagrnents that were potential switch-mediated
translocations by a Southern blot technique based on the linkage of the joining and the
constant regions. Nishida et al. (1997) determined that 3 1 of 42 (74%) patients with MM
or plasma ce11 malignancy harboured a translocation involving IgH by double colour
FISH. Independently Avet-Loiseau et al. (1 998) detected IgH translocations in 57% of
MM patients by FISH.
The translocation partners in MM are heterogeneous and non-random. nie most
63; 65 fiequent translocation partnea include cyclin DI (1 lq13) , fibroblast growth factor
58; 62 receptor 3 (FGFR3) (4p 1 6)6': 66, and c-maf(16q23) . Other genes (and their loci) that
58; 63 have been identified in these translocations include MUMI (6p2~)~', bcl-2 (18921) ,
c-mye (gq24)I6, M L - 1 (1 lq23)16, 6p215', 7p3263, bd7 (12~24)~', 20ql 169, bc16 (3@n6',
and 2 1 ~ 2 2 ' ~ . Only of a few of these chromosomal partners have been identified in more
than one sample 47; 58; 63
Cyclin DI (bcl-I/PRAD-2) is involved in 20-25% of the translocations invofving
the IgH locus in Mt; 63. The c y c h DI gene was found to be located 1 00-400 kb from
the breakpoint identified in M b l 55; 65; 70 . The t(11; 14) translocation has been found in 6 of
2 1 ce11 lines and there is a 1 : 1 correlation between the presence of the translocation and
58; 65 overexpression of cyclin Dl . As well a variant translocation into a light cha i . has
been described for this gene7'.
The 6p25 breakpoint was found to involve MuMMRF4 which is a member of
72; 73 the interferon regdatory factor family of transcription factors . IRF4 is a lymphoid
specific gene that is rapidly induced in T-ce11 receptor cross-linking that is basally
expressed in most B-cells but not Tcells". This t(6; 14) translocation was identified in
two MM ce11 lines6'. The expression of WMl/IRF4 was found to be higher in IL-6-
dependent ce11 lines than IL-6-independent cells but the highest expression was detected
in the two lines with (6; 14) translocati~n~~.
c-maf translocation appears to occur 25% of the time in M M ~ ~ . The expression
of c-maf was elevated in only the 6 (of 21) ce11 lines that harboured the c-maf
trans~ocation~~. This translocation has also been identified in prirnary patient
sarnples 58; 63; 64 . Two of the ce11 lines with t(14;16) translocation possess a second
translocation involving an IgH locus. The coincidence of two IgH translocations or an
IgH translocation and a variant translocation has previously been reported in a number of
MM ce11 lines and tumors 47; 63; 75; 76
1.1.2.2 FGFR3 translocations into immunoglobulin heavy chain locus
FGFR3 is reported to be involved in 25% of translocations occurring into the IgH
locus in MM6! This translocation may be unique to MM since it has not been previously
descnbedl6: 67 however, a translocation of FGFRI, t(8;13), in stem-ce11
myeloproliferative disorder has k e n de~cr ibed~~. Breakpoint aaalysis of ce11 lines
established that FGFR3 is localized about 100 kb fiom the most centromeric breakpoint
on chromosome It was found that FGFR3 is located on der(l4) chromosome
containing the 3' IgH enhancer but not the intronic e n h a n ~ e r ~ ~ .
FGFR3 translocations into IgH were detected in 5 of 21 ce11 lines and 3 of 11
primary tumors and expression of the protein has been detected in twelve MM ceil iines
and five -or ~ a r n ~ l e s ~ ~ . However, FGFR3 is only expressed at high levels in myeloma
cells containing a t(4:14) Richelda et al. (1997) report amplification of
FGFR3 rnRNA only in tumors harboring t(4; 14) translocation but not in bone marrow of
normal individuals. Avet-Loiseau et al. (1998) found that the translocation of 4p16
occurred in 12% of patients with MM, making it the second most cornmon rearrangement
in patient samples.
Interestingly, activating mutations of the translocated FGFR3 have also been
66; 67 detected in ce11 lines and patient samples . The activating point mutations identified
Include Y373C (in KMS 1 l), K650E (in OPM2), and K650M (in a primary t u s n ~ r ) ~ ~ .
These mutations of FGFR3 have been identified in 3 of 6 ce11 lines and 1 of 3 primary
turnors which contain t(4:14) translocation suggesting an important role in tumor
deve~o~rnent~~. It is likely that these are acquired somatic mutations because if these
mutations arise within the gennline they would probably cause thanatophoric dysplasia, a
lethal form of dwarfism (see section 1.2. I )~ ' . So far it has been demonstrated that in one
patient sample the translocation occurred prior to the mutation of the gme6'.
The translocation dysregulating FGFR3 has also been found to dsyregulate
another gene, multiple myeloma set domain protein ( u M s E Z " ) ~ ~ . The weaker intronic
enhancer of IgH is localized to chromosome 4 adjacent to M M S E ~ ' upon the
translocation of chromosomes 4 and 14 in myeloma MMSET encodes a protein with
domains homologous to trithorax group genes6'. MMSET RNA was detected in 14 of 2 1
MM cells with a higher expression level occurring in 5 of 6 lines containing t(4:14)
translocation6'. It is hypothesized that this protein also plays a role in neoplastic
transformation since the protein contains a nurnber of domains present on nuclear
proteins, such as SET domains and PHD fingers6'. Proteins containing these domains
78-80 have been implicated in chromatin remodeling . However the hct ion of this protein
is currently unknown and expenizentation is required to clearly elucidate its role.
1.2 Fibrobiast Growth Factor Receptor 3
The fibroblast growth factor receptor (FGFR) family is composed of at least four
tyrosine kinase recepton that have high sequence homology 81; 82; 82; 83 . FGFR3 was
originally cloned by two independent groups almost simultaneously. Keegan et al.
(1991) cloned FGFR3 fiom a human chronic myelogenous leukemia ce11 K562 cDNA
library while Thompson et al. (1991) identined FGFR3 while examining chromosome 4
for candidate genes for Huntington diseasew.
FGFR3 human mRNA is 4.5 kb in sizes2. There is extensive homology between
murine and hurnan structural and fimctional domains of FGFR~? There is 84%
hornology at the DNA level, and 92% homology at the amho acid levefs. The size of
introns and exons are nearly identical between human and mouse FGFR3 and the
prornoter region has 60% nucleotide similaritYsS.
Keegan et al. (1991)~~ introduced mammalian FGFR3 into COS cells and
identified three resulting protein products, of 97 kDa, 125 kDa, and 135kDa The 97 kDa
f o m is consistent with the predicted amino acid sequence; this form is unglycosylated
and represents the soluble fom of the receptor8! The 125 kDa and 13 5 kDa forms have
undergone hirther post-translational modifications by addition of N-linked
polysacchrides86; the 125 kDa f o m is likely the precursor to the 1 35 kDa fom, although,
Kami er al. (1997) suggested that these two forms acnially represent two splice variants.
FGFR.3 is composed of three extracellular immunoglobulin-like domains, a
msmembrane domain, a tyrosine kinase domain that is separated by an acidic insert
84; 86 sequence, and a carboxyl terminal tail (Figure 1). The FGFRs are part of subclass IV
of tyrosine kinase receptors due to the presence of the insert sequence in the h a s e
domain and the number of Ig-like domains?
FGFRs undergo alternative splicing of mRNA generating several different
Figure 1 : FGFR3 protein organization. The extracellular domain is characterized by amino terminal domain and three Ig-like domains, followed by trammembrane domain (TM). The kinase domain (TKl and TK2) is separated by an insert sequence. The TD II mutation (K650E) occurs within the second kinase domain as indicated.
88-9 1 isoforms that dif3er in their afïbity and specificity for ligands . The main structural
88; 91 variants include receptors with ody two Ig-like domains , receptors with Fnuicated
89: 91 kinase domainsS8, secreted forms of the receptors , and intracellular form~'~. There are
two different exons that encode the C-terminai half of the third Ig-like domain 90; 92
resulting in splice varïants, IIIb and iIIc which are variant forms of the third Ig-like
domaingo.
The expression of FGFR3 is highly regulated with a distinct ce11 type-specific
spatial and temporal pattern of expression. In mouse embryos the expression pattern of
FGFR3 mRNA alters during d e ~ e l o ~ r n e t n ~ ~ . In the human fetus FGFR3 was detected at
high levels in kidney, lung, srnall intestine, and braing4. Human and muMe adults have
high expression of FGFIU in centrai nervous system, kidney, intestine, bone and
lmg'"' 93 while there is little to no FGFR3 detected in the stomach, ureter, or
Splice variants also display different expression patterns with murine FGFR3 UIb being
expressed mainly in the skin, h g , kidney and liver while FGFR3 IIIc is expressed at the
highest levels in the brain*.
FGFR3 expression is absent in most hematopoietic progenitor/stem cellsgs.
However the gene was cloned fiom both a leukemic ce11 line" and erythroleukemia
~ e l l s ~ ~ and has been detected in cultured fibroblasts and lymphoblastoid c e l ~ s ~ ~ . A
potentiai mitogenic role for FGFs in proliferation or dinerentiation of early adult
hematopoietic progenitors in vitro has also been describedg7. FGF2 is considered to be
the most important FGF in shulat ing proliferation of hematopoietic c e ~ l s ~ ~ . Interestingly
though, recent work suggests that FGFs may not play an important role in regulating
hematopoietic ce11 development since none of the FGFRs are expressed on progenitor
cells and cuituring normal bone marrow cells with FGF2 in serum fiee conditions did not
enhance groliferation or survivalgS. Furthemore, when mutations of FGFRs (such as
those in human dwarfism) are present hematopoiesis appears to be normal 99-101
suggesting that FGFRs are not involved in normal hematopoiesis. Thus dysregulation of
FGFR, in MM would likely cause the ectopic overexpression of a receptor potentially
having a profound effect on intraceIluIar signaiing.
Thee are at least thirteen fibroblast growth factors (FGFS)"~ capable of binding
FGFRs. Each receptor has a unique specificity for FGFs 89; 92; 103 . FGFR3 binds acidic
FGF (aFGF) (FGF-1) 3-4 fold higher than controls and binds basic FGF (bFGF) (FGF-2)
with low aflïnitylM. To bind the FGFRs with high affinity heparin sulfate proteoglycans
or heparin are also required 105-107 . aFGF is capable of binding murùie FGFR3 (mFGFR3)
in the absence of heparin but the association was increased in its presence104. It is
postulated that the association between aFGF and mFGFR.3 in the absence of heparin is
Iikely not a high affinity complex since BaF3 cells expressing mFGFR3 have an absolute
requirernent for heparin in order to respond to ~FGF?
Binding of the FGF ligand and heparin CO-factor induces FGFR
d imerization 108;log , which leads to autophosphory lation (aansphosphorylation) of the
kinase domain. After the receptor is autophosphorylated it is capable of phosphorylating
110-1 12 intracelluiar pro teins involved in signal transduction pathway s .
Autophosphorylation of the receptor is a rapid event occurring within 1 minute of ligand
stimulation and reaching its maximum afier 1 5 minutes' 13.
FGFR3 has been reported to be an intrinsically weak kinase 90;104 for example,
FGFIU is unable to generate a strong mitogenic signal in FGFR-negative ce~ls"~.
However, mutations of the receptor that result in constitutive activation have been shown
to increase the kinase activity of FGFR3 115; I l 6 (see section 1 -2- 1).
FGFR3 has been found to be associated with both the ce11 membrane and the
nuclear envelope. FGFR3 transfections hto COS-7 cells resulted in nuclear Iocaiization
of a portion of the receptor mole~ules"~. In primary chondrocytes fiom f e t w s with
thrnatophoric dysplasia type 1, which develops due to severai different autosomal
dominant mutations in FGFR3, FGFR3 was found to have a perinuclear as well as a
plasma membrane 10calization~~~. Johnston et al. (1995) found that treatment with ligand
did not alter the locdization of FGFR3. However, Mahr et al. (1996) demonstrated that
upon treatment with ligand, FGFRs translocate to the nucleus. Another study described
the association of FGFRl with the perinuclear region d e r treatment with ligandti9. The
appearance of receptors in the nuclear fiaction after ligand stimulation is time and
concentration dependentL20. Normdly ligand stimulation of a receptor results in
dimerkation followed by down-regulation so that the signal is transient8'. The
previously reported localization of the receptor and ligand to the nuclear region is thought
to down-regulate the biological signal since the localization of FGFR3 in the ce11 plasma
membrane is required to transduce signds to the ce11'~'.
1.2.1 Function of Fibroblast Growth Factor Receptor 3
FGFR3 can transmit signals that either inhibit or stimulate ce11 growth depending
upon the ceil type. Its primary role is as a negative regulator of bone growth I 2 î ; 123
Knockout mice display long bone overgrowth and skeletal disorders 122; l23 . The
expression of FGFR3 in chondrocytes of normal individuals inhibits chondrocyte
proliferation 116; 122; l î 3 . However, expression of FGFR3 in an L-3-dependent lymphoid
ce11 line enabled the cells to proliferate by FGF stimulation in the absence of IL-^'?
Mien FGFR3 was expressed in a pro-B ce11 line, BaF3? it induced a mitogenic
124 response -
Activation of FGFR3 leads to activation of several intracellular proteins, however,
the compIete signaling paùiway of FGFR3 is not yet fûily elucidated. Stimulation of
FGFR3 has been shown to lead to Shc phosphorylation but an interaction between
FGFR3 and Grb2 was not obser~ed"~. However, it appears that FGFIU is capable of
activating a ShcGrbZmSos complex which would provide a link to the mitogen-activated
protein (MAP) kinase signaling pathways by activatïng ras l 13. indeed, phosphory lation of
ERKI and ERK2 have been descnbed following ligand stimulation of FGFR3 113; 118
FGFR3 also ac tivates the Janus kinase(Jak)-STAT (signal transducers and activators of
transcription) pathway 116; 118 . In prirnary c hondroc ytes FGFR3 phosphory lates
STAT1116; 118 . It has been demonstrated that FGFR3 can cause phosphorylation of
phospholipase C-y (PLC-y)1 13. Further experimentation is required to Fully detennine
which signal pathways FGFR3 is capable of activating and which pathways are
responsible for which cellular respoase to FGFs.
Mutations within FGFR3 arising in the germiine cause various bone dysplasias
such as chondrodysplasias and craniosynostosis. Craniosynostosis syndromes are
heterogeneous and mutations in FGFR1, FGFR2 and FGFR3 account for various f o m
of these disorders. Muenke syndrome is caused by a P250R mutation in FGFR3 125; 126
Another craniosynostosis, Saethre-Chotzen, is caused maialy by mutations in TWIST
transcription factorL2" 12', however, in some cases mutations also occur in FGFIU or
FGFR~'~' . Chrondrodysplasias are subdivided into three forms, achondroplasia (ACH),
hypochondroplasia (HCH) and thanatophoric dy splasia (TD). Each of these disorders
represent varying forms of dwarf?sxn with TD king the most severe. These three
diseases are caused by autosomal dominant mutations in different regions of FGFR3 and
the seventy of the phenotype appears to correlate with the activation of the receptor as a
result of the mutationH5. For example the mutation responsible for TD, the most severe
of these disorders, has been found to result in a more strongly activated receptor than
thoçe that cause ACH'".
1.2.2 Thanatophoric dysplasia
TD is the most severe disorder caused by mutations in FGFR3. This disorcier was
originally described by Maroteaux et al. (1967)"'. Individuals with TD die neonatally or
shortiy &er birth 131; 132; 132 . Affected individuais display shortening of the limbs and
rïbs, reduction in vertebrae height, and facial and skull abnormalities 131; 132; 132 . TD is
M e r subclassed clinically into type 1 (TDI) and type II (TDII) based on the shape of the
fernurs and the presence and absence of a cloverleaf skull.
TDI is caused by several distinct mutations of FGFR3, which mainly occur
around the Ig-iike domains or the transmembrane domain in the protein 96; 96; 133; 134
However, TDLI is caused solely by a mutation in the kinase domain of FGFR3 ( ~ 6 5 0 ~ ) ~ ~ .
Interestingly this same mutation has been identified in the translocations occurring to the
IgH switch region in (see section 1.1.2.2).
The K650E mutation is a lysine to giutamic acid switch at codon 650 (nucleotide
1948) within the second kinase dornain of FGFR~~' . The mutation occurs within a highly
conserved activation loop and is located two amino acids downstream of an
autophosphorylation site? It has been proposed that the change in charge within the
activation domain would have a drarnatic effect on receptor activation1 ls. This group
suggests that the mutation within this dornain may release the inhibitory conformation of
the kinase region enabling the receptor to be activated in the absence of Ligand.
FGFR.3 containing the K650E mutation results in constitutive activation of
FGFR3 15. The TDII mutant exhibits 100-fold greater autophosphorylation than wild-
type FGFR3 ' 15. B y kinase assay Su et al. (1 997) demonstrated that the TDII mutant had
a higher intrinsic kinase activity than wild-type FGFR3. It has also been demonstrated
that FGFR3 containing K650E mutation is still partially ligand-responsive since it is
possible to induce m e r stimulation of the receptor (phosphorylation) by binding of the
ligand to the receptor fùrther enhancing the effect of FGFR3 116; 124
In hurnan chondrocytes FGFR3 a TDII mutation results in growth arrest of the
ce11"~. Activated FGFR3 in chondrocytes phosphorylates STATl in the absence of
ligand1I6. This phosphorylation of STATl leads to increased expression of p21, a ce11
cycle regulator, which results in growth arrest116. Legeai-Mallet et al. (1998)
demonstrated that chondrocytes expressing the TDI mutant form of FGFR3 undergo a
higher degree of apoptosis. Thus in individuals affected by activating mutations of
FGFR3 in the gennline, signaling through the receptor results in ce11 growth arrest of
chondrocytes resulting in inhibition of long bone growth 116; 1 l8 . However, the pathways
activated by FGFR3 may be cell-type specific since it has also been s h o w that FGFIU
can cause growth effects in other cells. Expression of TDII mutant form of FGFR3 in a
pro-B ce11 line, BaF3, resdted in ligand independent g r ~ w t h ' ~ ~ .
It has been proposed that FGFR3 activated by the K650E mutation may have
different cellular effects than those of wild-type F G F R ~ ~ ~ * . Indeed, a study by Webster
and Donoghue (1997) suggests that activated FGFR3 has the potential of acting as an
oncogene. The variabie effect of FGFR3 on ce11 growth may resdt fiom constitutive
versus transient activation. Zong et al. (1996) suggest that the signais induced by
transient activation likely differ fiom those induced by prolonged activation.
1 -3 Interleukin-6
IL-6 is a multi-functional cytokine produced by a wide variety of cells 136-138
which exerts growth-inducing, growth-inhibitory, and differentiation-inducing effects
depending on the target ~ e l l " ~ . IL-6 was originally identified as a by-product of
interferon-p production by human fibroblasts 140; 141
IL-6 has been found to be involved in several cellular events. It induces the final
maturation of B cells into immunoglobulin-secreting ce~ls"~' 14? IL6 has also been
s h o w to induce the production of IL-2 by murine T c e ~ l s ' ~ ~ , to induce the proliferation of
hurnan and murine T cells 144; 145 , to induce the differentiation of cytotoxic T c e l ~ s l ~ ~ , and
6; 22; 23; 26; 139; 147; 148 as a growth and survival factor for MM
The IL-6 receptor is composed of two subunits, a ligand binding a subunit, Il-
6 ~ ~ ~ ~ , and a signal tramduchg subunit, gp130 (or the P subunit) 150; 151 . IL-6 binds the a
subunit which induces homodimerizarion of g p l 3 0 ' ~ ~ fonning a complex of two &6R,
two gp130 and two IL4 molecules. Gp130 is a common signal transducing subunit €or
several cytokines including IL-6, leukemia inhibitory factor (LIF), ciliary neurotrophic
factor (CNTF), IL- 1 1, oncostatin M (OM) and cardiotrophin-l 1s-156
IL-6-induces gp130 signaling, and activates Janus kinases (Jaks) 157-162 which
leads to phosphorylation of STATI and STAT3 158; 160; 162-165 . Also gp 130 activates the
Ras-dependent MAP kinase cascade 161; 165; 166 (Figure 2).
IL-6 is important for the growth and sunrival of MM cells 6; 22; 23; 25; 26; 139; 147; 148
Exposure of freshly explanted malignant plasma cells to I L 6 often results in enhanced
proliferationu' 16'. The withdrawal of IL-6 fkom an IL-6-dependent myeloma ce11 line,
B9, causes programmed ce11 death with significant ce11 death by 72 h ~ u r s ~ ~ . Concomitant
with the induction of apoptosis is the induction of ce11 arrest in GI indicating the
importance of IL-6 in both growth and survival of MMz6. Furthemore, IL-6 can protect
against senun starvation in MM cells but it cannot protect against the ability of senun
starvation to inhibit proliferation25. IL-6 also protects MM cells against dexamethasone-
25; 168 induced ce11 death .
Since IL-6 normally acts as a differentiation factor for normal B cells it suggests
that there may be altered IL-6 signaiing in MM 169; 170 . It appears that the mitogenic
signal of IL-6 to MM cells is transrnitted via MAP kinases 165; 171 while the survival signal
Figure 2: Schematic diagram representing IL-6 signaling cascade. Association of IL-6 with IL-6R enables the recmitrnent of gp 130. Jaks, constitutively associated with gp 130, are phosphorylated and then phosphorylate specific residues on gp 1 30 that serve as binding sites for STAT 1 and STAT3. STATl and STAT3 form homodimers and heterodimers before translocating to the nucleus. As well receptor phosphorylation activates Grb2 and results in the formation of ShcaGrb2.Sos 1 complexes. SOS 1 in this complex then induces the exchange of GDP for GTP on Ras, activating the ERK signaling cascade.
/ SOS llGrb2 Ras
Raf
MEK- 1
ERK
binds SIE binds CTGGGA
Adapted fiom Kishimoto et al. (1 994)
is transmitted through Jak-STAT 165; 172; 173 . Indirect evidence suggests that the mitogenic
49; 161 signal in MM celis is mediated through Ras . Ogata et al. (1997) demonstrated that
Jaks, gp 130, STATl ancüor STAT3 were phosphorylated in response to IL-6 in al1 MM
cells studied, however, MAP kinase activation was oniy observed in those cells that
proliferated in response to IL-6. Experiments performed on truncated receptors suggest
that STAT3 is importaat for ce11 su rv i~a l '~~ . However it has been recently suggested that
there may be an interaction between the MAP kinase and JAK-STAT pathways in MM
ce11 growth. French et al. (1998) transfected an IL-6-dependent myeloma ce11 line with
various mutant forms of gp130. CelIs transfected with gp130 containing oniy the
consensus binding sequence for MAP b a s e failed to proliferate. This suggests that the
presence of the MAP kinase binding site alone is not sufficient for proliferation and the
Jak-STAT may also be required for IL-6 induced proliferation'70.
There is debate in the literature as to whether IL-6 acts as a paracnne or autocrine
growth factor in MM. This issue is important clinically since it would effect therapy2'. A
neutralizing anti-IL-6 antibody therapy wodd be an attractive option for paracrine
stimulated tumors but would not likely be as effective against autocrine hunors2'. With
paracrine stimulation the neutralizing antibody could interact with IL-6 as it moves fiom
the ce11 where it is produced to the target cell. Autocrine stimulation would decrease the
time penod within which the neutralizing antibody could interact with IL6 thus reducing
the efficacy of this treatment. There are cases of myeloma ce11 lines that produce their
own IL-6, such as U266 and RPMI 8226 23; 174; 175 - Kawano et al. (1988) detected IL-6
mRNA in four fieshiy isolated myeloma ceil cultures. Hata et al. (1993) independently
detected IL-6 mRNA in purified myeloma cells fiom 45% of patient samples suggesting
an autocrine mechanism. However in vivo the stroma1 cells of the bone marrow appear to
22; 176 be the major source of IL-6 for myeloma cells . Klein et al. (1989) only detected
small quantities of IL4 in vimially pure populations of fieshly isolated myeloma cells.
Freshly isolated myeloma celIs require IL-6 to proliferate and without this cytokine the
cultures cannot be maintained8.
1.4 Jak-STAT signal t r d u c t i o n pathway
The Jak-STAT pathway is a comrnon signaling pathway shared by a number of
cytokines and growth factors that regulate gene expression, cellular activation,
differentiation and proliferation 177; 178 . Abnomal Jak-STAT signaling has been associated
with cellular transformation 179-182 . It has been hypothesized that prolonged activation of
the pathway may lead to a gene expression pattern that differs fiom that occurring due to
trans ient a~tivation"~. This may result in constitutive activation of genes critical for
cellular growth, potentidly causing cellular transfor~nation~~~.
There are oflen interactions between Jak-STAT and the MAP kinase pathways. in
some systems it appears that the MAP kinase members, ERKs, and Jak-STATs are
activated by the same stimulus and either regulate different responses or
165; 184-186 s yngerize . The interaction of ERK and Jak-STAT pathways can lead to
synergistic activation of genes by interfer~n-al~~. However the molecular basis of this
interaction between these pathways is not clearly ~nders tood '~~.
1.4.1 Jaks
Jaks are a family of at least four nonreceptor tyrosine kinases that associate with
various receptors and induce phosphorylation of STATs. The Jak family was
independently identified by low stringency hybridization'89 and by polymerase chain
reaction (PCR) designed to identfi novel tyrosine kinases by PCR 190; 191 . The family
members possess two kinase domains. The more carboxy-terminal kinase domain
contains the consensus sequences associated with tyrosine kinases and is thought to be
activelg2. The more amino-terminal kinase domain lacks several residues essential for
kinase activity and is thought not to have kinase a c t i ~ i t y ' ~ ~ . These proteins have no
detectable src homology 2 (SH.2) or src homology 3 (SH3) domains 192; 193
It has been found that Jakl, Jak2 and Tyk2 are constitutively associated with the
box 1 region of gp130 152; 157 . After receptor autophosphorylation Jaks are activated by
transphosphorylation and then phosphorylate specific tyrosine residues on gp 130 to create
docking sites for STATs 157: 173; 178 . Jaks are involved in phosphorylating the STAT
moIecules that bind to gp130. However, it has been difficult to fiilly determine the role
of Jaks in signaling pathways since there are extremely few mammdian systems that
have deficiencies in .Jakdg4.
1.4.2 STATs
STATs are transcription factors activated by Jaks and some receptor tyrosine
kinases la'; Ig5; Ig6. There are at least eight proteins. STATs were origindly identified as
transcription factors responsible for interferon-a- and interferon-y-dependent gene
expression195. STATs are characterized by a carbov-terminal SH2 motif but overall
hornology between STAT molecules is lowtg7. There is also a conserved tyrosine within
the carboxy-terminus which must be phosphorylated to activate STATs 198; 199 . In the
cytopiasm STATs exist in latent f o m (monomeric). These molecules become tyrosine
phosphorylated on a single tyrosine after ligand binding to receptor, form homo- and/or
heterodimers and then migrate to the nucleus where they regulate gene expression 197; 200
Homo- or heterodimers are fonned via SH2 domains of STATs 20 1-203 - Tyrosine
phosphatases inactivate STATs directly in the nucleus2w by interacting with the amino
terminal domain of STATS~".
STAT consensus motifs, TTCNNNGAA, are found in a variety of
206307 promoters indicating the importance of these molecules in regulating gene
transcription. The STAT rnolecule recmited by each promoter depends on the specific
nucleotides present in the consensus sequence 201; 208; 209 . The recruitrnent of STATs is
dictated by the promoter rather than the availability of STATS~''.
STATs can be phosphorylated on serine as well as tyrosine. Serine
phosphorylation may be mediated by proteins of the MAP kinase signal pathway, such as
ERK2187; 210; 21 1 . Serine phosphorylation may be necessary for maximal stimulation of
gene transcription by STATs 186; 187; 210 . However, serine phosphorylation alone does not
induce DNA binding activity of STATs but seems to create a more permissive state and
increases STAT activation when there is simultaneous stimulation by a cytokine or
'1 1; 212 g o wth factorA
Different STAT molecules appear to have distinct roles in the ce11 as
demonstrated by STAT deficient mice 213-217 . For example, STAT3 deficient mice die
during early embryogenesis suggesting an important role for this transcription factor in
d e v e ~ o ~ m e n t ~ ~ ~ . iL-6 is unable to protect STAT3 deficient T cells fiom apoptosis2'8.
However, the Levels of bcl-2 and bcl-xL do not alter in T cells deficient in STAT3
suggesting that STAT3, activated by IL-6, prevents apoptosis by a mechanism
independent of bcl-2 and bcl-x, in T cells2".
STAT moIecules may be involved in both proliferative and -val
signals116' 213' 2i5' 219-U2. STAT3 has been s h o w to be invoived in reguiating cyclios and
cdks expression in a pro-B ce11 line* and thus functions in regulating ce11 cycle
progression. STATl up-regulates expression of p21 in primary chondrocytes through
FGFR3 16. These data support a role for STATs in ce11 cycle regulation. With regard to
ce11 s w i v d , several reports suggest that STATl or STAT3 may be involved in
regulating expression of bcl-2 farnily members, specifically bcl-xL in; u3; 224 suggesting a
role for STATs in anti-apoptosis. For example, it has very recently been reported that the
172 IL-6 survival signal in MM is mediated by STAT3 via upregulation of bcl-xL .
A recent report suggests that STATl is involved in the negative regulation of
transforming growth factorop (TGF-9) signaling . TGF-P activates transcription factors
Smad 2 and Smad 3 225; 226 through TGF-i3 type-1 receptor or TGF-B type-II receptor.
' Anti-Smads', such as Smad 6 or Smad 7, can interfere with TGF-B signaling 225; 226 . For
example, Smad 7 binds the TGF-P receptor complex preventing it fiom phosphorylating
and activating other Smads 227: 228 . Jaks and STATs, induced by interferon-y, can increase
the expression of Smad 7, thus inhibithg signaling via TGF-B~'. STATl is required for
this increase in Smad 7? UUoa et al. (1999) suggest that this may be a regdatory
mechanism for processes under the control of both TGF-P and interferon-y.
STAT3 appears to be involved in many cases of cellular transformation. STAT3 is
constitutively activated in cells transformed by src"' and human lyrnpbotrophic virus
~ l X v - 1 ~ ~ . The most common STATs that have been found to be activated in himors
and transformed cells are STAT3 and STATS 183; 231-235 . Most Unportantly, Catlett-
Falcone et al. (1999) demonstrate that STAT3 is constitutively activated in bone marrow
fiom myeloma patients as compared to controls. Thus it appears that STATs, in
particular STAT3, are important in mediating cellular events that lead to malignant
transformation-
1.5 MAP kinases
The MAP kinase pathways are characterized by a three-component protein kinase
cascade. MAP kinases phosphorylate substrates on serine or threonine located adjacent
to proline residues and thus are proline-directed kinases. There are three known M M
kinase pathways, ERK, SAPWJNK (stress activated protein kinasek-jun N-terminai
kinase) and p38 pathways (Figure 3). There appears to be differences in the stimuli that
leads to MAP kinase activation and the relative activation of each pathway depends upon
the stimulus used 236; 237; 237-239
The ERK pathway is initiated by the adaptor protein, Grb2, which binds tyrosine
phosphorylated receptors or Shc through its SH2 domain. Grb2 then recruits Sosl and ras
guanine nucleotide exchange factor via its SH3 domains 240-242 . When this complex is
Figure 3: Schematic diagram of the three MAP kinase cascades. These cascades are activated by GTPases; Ras, Rac or Cdc42. The three component cascade is then activated leading to the phosphorylation of ERK, SAPK or p38.
Growth factor, cvtokine
7 Ras
raf
ERK
Elk- 1 MAPKAPK- 1
Stress stimulus, cvtokines
v Rac, Cdc42
c-jun, ATF-2
Adapted fiom Schulze-Osthoff et al. (1 997)
31
assembled, Sosl causes the exchange of GDP for GTP on Ras thereby activating Ras,
which will be later deactivated by hydrolysis of GP. Activated Ras is then able to
activate the rnernbers of the Raf family of serine/threonine kinases 244; 245 , leading to
activation of ERK.
Three separate proteins are encoded by human ras genes, N-rus, K-ras, and H-ras
which are coIlectively called Ras or p21 ri1~246-250 . Activoting mutations of ras are
fiequently found in cancer- For example, in MM K-ras and N-ras genes are activated at a
fiequency of 20-50% 25 1-254 . In MM it has been shown that N-ras expression in IL-6-
dependent ce11 line causes entianced growth potential of these cells in the absence of IL-6
and a reduction in ce11 death in the absence of IL-&'.
ERKs are present in low concentrations in the cell. There are several different
isofoms of ERK, with the best characterized being ERKl @12) and ERK2 @44) 255-257
ERKs are phosphorylated on threonine and tyrosine residues in the sequence TEY 258; 2S9
ERKs are activated by mitogens and growth factors and regulate the expression of genes
associated with growth by activating transcription factors such as ELK- I /SAP- 12". It has
been suggested that the ERK pathway may be invoived in the proliferation of MM
cells16S: 171
Similar to ERK activation SAPK and p38 pathways are activated by GTP-bhding
proteins known as rho-like GTPases which are similar to ras260. The S APWJNK cascade
was discovered during studies on the cooperation between Ras and c-junz6' and when
237; 262 kinases responsible for phosphorylating c-jun were investigated . The mammalian
SAPK family is comprised of three separate genes that encode ten separate SAPK
proteins263. These proteins are aU phosphorylated on conserved theouine and tyrosine
residues in the sequence TPY. SAPWJNK is involved in responses to stress such as
ultraviolet 0 and ionizing irradiation, cytokhes or heat and serve to activate the
transcription factors c-jun, and ATF-2 261; 264
There are multiple upstream kinases that are capable of ultimately activating
SAPK/JNK. Mixed lineage kinase 3 (MLK3) is a serinehhreonine that contains multiple
interaction domains including SH3, Cdc42 bùiding sites, and ZIP domains 265; 266 - MLK3
can activate SAPWJNK and p38267. MLK3 phosphorylates SEKl or MKIO leading to
activation of SAPWJNK or p38, r e ~ ~ e c t i v e l ~ ~ ~ ~ . A Ste2O homologue, germinal center
kinase (GCK), is also capable of activating SAPWJNK, but does not activate p38 or
ERK~? Another activator of S A P W M is hernatopoietic progenitor kinase 1 (HPKl), a
Ste2O-like kinase269. No interaction was found between MEKKI and HPKl but HPKl
phosphorylates M L K ~ ~ ~ ~ . In many cases the position of these kinases within the
signaling pathway and their interactions with other kinases within the pathway is not yet
elucidated, however, it appears that there are mdtiple proteins involved in the regulation
of the SAPWJNK pathway.
Even though SAPK pathways have been implicated mainly in the response to
stress, the outcorne of activating this pathway is cell- and context-dependent and can
result in growth, differentiation or apoptosis263. For example, in Jurkat T cells inhibiting
SAPK signaiing does not prevent Fas-mediated apoptosis 270; 271 but in neuroblastorna
cells Fas-mediated apoptosis is dependent on SAPK activationzn. Several studies
çuggest that SAPK activation plays a role in inducing apoptosis in MM'~'.
Dexamethasone and anti-Fas antibody activate SAPK in myeloma ceils but this activation
of SAPK can be inhibited by IL-^'^'. Anti-serw oligonucleotides to c-jun prevent
apoptosis due to IL-6 starvation in an Il-6-dependent murine ce11 ~ine"~.
The other stress-activated protein kinase pathway involves p38, also known as
reactivating kinase or C S B ~ ' ~ . This pathway was originally identified in murine pre-B
cells transfected with CD14 and stirnulated with L P S ~ ~ ~ . p38 is closely related to the
HOGl gene product of Saccharomyces cerevisiue, that is involved in restoring osmotic
gradients across ce11 membranes 238; 275 . To be activated p38 must be phosphorylated on
tyrosine and threonine in the conserved sequence TGY. This phosphorylation occurs in
response to a wide variety of stress stimuli such as heat shock, and oxidative stress. The
physiological consequences of p38 stimulation largely remain to be defined. However it
has been determined that the p38 pathway is required for c-fos mRNA expression in
response to W light2". As well p38 inhibition prevents IL-6 mRNA synthesis induced
by tumor necrosis factor ( T N F ) ~ ~ ~ . Apparently p38 is activated by CD40 in human
tonsillar B cells and multiple B ce11 Iines and is required, at least partially, for CD40-
induced activation of NF-KB~". Evidence suggests that activation of p38 and NF-KB are
mediated by separate pathways but may converge downstream 277; 279 . As well it has been
demonstrated that in myeloma cells treated with Fas, p38 levels do not change in
response to IL-6, unlike SAPK which is dom-regulated upon [L-6 stimulation280. This
suggests that IL-6 does not act upstream of p38280.
1.6 Apoptosis
Apoptosis, or programmed ceii deaîh, is an active process regulated by a complex
genetic network. The mechanism is evolutionarily conserved and is required for the
development and maintenance of tissue homeostasis in multicellular organisms 281; 282
Apo p tosis can be distinguished fkom necrosis b y morphologicai and bioc hemical events
including: chromatin condensationzg3, nucleosomal DNA hgmentation2", caspase
activation, loss of mitochondrial membrane potentia128', and cytochrome c
redistributiod8'.
The best defined apoptotic pathway is that of Caenorhabdiris elegam. In this
organism there are two autosomal death effector genes, ced-3 and ced-4, that are required
for the death of al1 131 cells destined to die during worm d e v e l ~ ~ r n e n t ~ ~ ~ and one
autosomal death repressor, ced-9, that can repress the death pathway 286-288 - Interestingly
these proteins have k e n noted to share homokogy with death effectors and death
repressors of the mammalian system. ced-3 is homologous to IL- 1 P converting enzyme
(ICE)*~', a member of the caspase family of death effectors. ced-9 has structural and
functional hornology to bcl-2, sharing 23% amino acid identity and 49% similarity2".
In the mammalian system the induction of apoptosis appears to be regulated by
the equilibrium between death suppressors and effectors 290; 291 . The downstream
effectors of apoptosis are the caspase family members. Ail caspases are expressed as
proenzymes containhg a MI2-terminal domain, a large subunit and a small s ~ b u n i t ~ ~ ~ .
Activation is achieved by proteolysis followed by formation of heterodirners composed of
the large and small subunits. Proapoptotic signals led to the activation of initiator
caspases that in t u . activate effector caspases 293; 294 - Initiator caspases are activated by
binding to specific CO-factors. For example, procaspase 8 associates with Fas-associated
protein with death domains (FADD) through its death domainZg5. Upon activation
effector caspases degrade cellular components required for ce11 and nuclear integrity,
including lamin A and ~ ~ R N P s ~ ' ~ , cleave ICAD/DFF45 297; 298 a nuclease inhibitor, and
inactive or deregulate proetins involved in DNA repair, mRNA splicing and DNA
replication 299; 300
Another family of proteins involved in apoptosis is the bcl-2 family that contains
both suppressors and effectors of apoptosis and appear to be active upstream of
30 1 caspases . The first member of bcl-2 family, bcl-2, was originaily identi£ied at a
chromosomal breakpoint in follicular lymphoma70' 302*307. The biologicd function of
bcl-2 was determined when it was demonstrated that bcl-2 enabled IL-3-dependent cells
to escape death upon cytokine withdrawal 308; 309 . The discovery that bcl-2 suppressed ce11
308-3 10 death established a new class of oncogenes
To date several family members have been identified that include both
suppressors and activaton of apoptosis. Anti-apoptotic members include bcl-2, bcl-xL,
Mcl-1 and A-1 while pro-apoptotic members are bax, bad, bcl-xs and bak. The bcl-2
family displays homology within two conserved domains, bcl-2 homology domain 1
(BH 1) and bcl-2 homology domain 2 (BH2) 290; 311: 312 . These domains, dong with bcl-2
homology domain 3 (BH3), regulate formation of homo- and heterodimers 312-315 . As
well there is an N-terminal region that displays 57% identity between bcl-2 and bcl-~2'~.
At least part of the activity of the bcl-2 family depends on the formation of homo-
and heterodimers. Bcl-2 and bcl-xL heterodimerize with bax, bak and
bad 291; 312; 313; 316-318 . Both bcl-2 and bcl-x, bind to bax via their BHI and BH2
domains 312; 313; 315; 317 . Bax and bcl-2 are also capable of formiog hornodi~ners~~~. The
ratio of apoptosis suppressors to apoptosis activators determines the cell's fate 290; 291; 312- 9
if approximateIy half the bax within a ce11 is complexed in heterodimers the ce11 is
protected from apoptosis291. However, it is d l 1 unclear what the active moiety is that
reguiates celi death, baxhax or the heterodimers 291: 319
Bcl-2 family members tend to be localized to the outer mitochondrial, outer
nuclear and endoplasrnic reticulum membranes 320-323 . It has been postulated that this
locaiization of bcl-2 family members may enable them to regdate ion flues. Indeed,
bcl-xL prevents changes in mitocbondrial membrane potential, reduces the amount of
cytochrorne c released and prevents mitochondrial depolarization after death
Also bcl-x, is capable of forming channels in synthetic lipid vesicles in a pH sensitive
manner and exhibits cation specificity3". Bcl-2 family members may prevent apoptosis
by regulating ion fluxes since this could control mitochondrial permeability and prevent
loss of membrane integrity 325-327
The mitochondria plays an important role in reguiating apoptosis. There are three
main ways in which the mitochondria can be involved in triggering cellular death. First,
disruption of electron transport has been recognized as an early feature of ce11 death. Fas
activation Ieads to disruption of cytochrome c h c t i o n in the electron transport
One consequence of disnipting the electron transport chah is a decrease in the production
of ATP, an event that occurs later during apoptosis3". Secondly, the mitochondria
contains proteins that can activate caspases upon their release, such as cytochrome c.
Cytochrome c forms a compiex with Apaf-1 and procaspase 9 upon its release fiom the
rni to~hondr ia~~~ causing capase 9 activation. Procaspase 3 is also contained within the
mitochondria3". Thirdly, the mitochondria can also alter the cellys reduction.oxidation
potential. The mitochondria is the major source of superoxide anion.
Apoptosis is often associated with a collapse of the inner transmembrane potential
of the rnitochoodria3". This Leads to the rupture of the mitochoadria due to expansion of
the matrix, causing the release of caspase activating factors. However, in some instance
the mitochondria remains morphologically normal while the ce11 undergoes apoptosis. In
these cases it appears that bcl-2 family members are involved in regulating cytochrome c
release via their interaction with VDAC"~. VDAC is an abundant outer rnitochondria
membrane voltage-gated pore. Recent studies demonstrate that bax and bak can interact
with VDAC and cause it to open allowing cytochrorne c r e l e a ~ e ~ ~ ~ . In contrast bcl-xL can
interact with VDAC to cause its c l o s ~ r e ~ ~ ~ . Many features of the rnitochondria's role in
apoptosis remain to be elcudiated.
1.6.1 Bd-xL
BcI-x was cloned by low stringency hybridization using a murine bcl-2 probe in
chicken lyrnphoid c e l l ~ ~ ~ ~ . The clone, bcl-x, was then used to screen a human cDNA
library which isolated two distinct t r a r ~ s c r i ~ t s ~ ~ ~ . Bcl-x produces two gene products due
to alternative splicing; bcl-x, is a 233 amino acid protein that contains BH1 and BH2
domains while bcl-xs is a 170 amino acid protein which lacks 63 amino acids that
encornpasses BHI and BH2 334; 335 - The resultant proteins ciiffer fùnctionally with bcl-xL
suppressing cell death and bcl-x, promoting ce11 death334.
The pathways involved in bcl-xL gene regulation are not fully elucidated. In
myeloma it has been suggested that IL-6 induces up-regdation of bcl-xL 148: 336
Packharn et al. (1998) suggest that activation of Jak2 is sufEcient to up-regulate bcl-xL
and is required for ce11 survival. This is supported by the observation by Oshiro et al.
(1998) that inhibition of Jak2 results in down-regdation of bcl-xL. However, STATs are
also involved in regulating gene transcription of bcl-x, as STAT3 is capable of up-
reguiating bcl-xL in; 223 . B ~ 1 - x ~ levels in the myeloma ceil line, U266, decrease when
these cells are transfected with STAT3 dominant-negativeLn. Ln cardiac myocytes it was
found that LIF could induce bcl-x expression likely via STATI U4.
1.6.2 Bcl-2 farnily members and MM
The expression of bcl-2 family members in MM has k e n studied since alterations
of anti-apoptotic mechanisms are comrnon in human cancer. There are conflicting reports
on the level of bcl-2 expression in MM. One group reported that 28% of MM patients
express b ~ l - î ~ ~ ' , another report 80% of MM patients express bc1-2~~' while yet another
group report that 43% of patients have high expression of b c 1 - 2 ~ ~ ~ . Bcl-2 expression has
not been found in murine myeloma cells and instead there is expression of bcl-x, 148; 340
B ~ 1 - x ~ has aiso been found to be expressed in malignant human plasma cells and several
human myeloma ce11 ine es'^'. Harada et al. (1998) suggest that bcl-2 may be
preferentially expressed in tumor cells in early stages of MM while bcl-xL is expressed in
more advanced stages of MM. B ~ 1 - x ~ may be up-regulated at the time of relapse in
MM34 1 . In contrast, bcl-xs is seldom expressed in MM ~ e l l s l ~ ~ ' 34@342. Bax is detected
rarely in fresh MM ceiis but is detected in myeloma ce11 lines suggesting its expression
rnay be induced by in vitro c ~ l t u r i n ~ ~ ~ ~ .
Bc1-xL is an important regulator of apoptosis in MM cells 148; 340-342 - It has been
reported that bcl-xL is upregulated by I L 6 in MM 148; 336; 341; 343 . When IL-6 is
withdrawn from B9 myeloma ce1 1s the leveis of bci-xL decline over 48 hours but return to
steady States within 8 hours of re-addition of IL-6'". Also it appears that up-regdation
of bcl-x, protects MM cells fiom chemotherapy since it has been shown that bci-xL
overexpression in MM cek makes these ceils resistant to doxombd4'. Finally, MM
sarnples expressing bcl-xL are more resistant to ce11 death induced by melphalan and
prednisone or vincristine, adriamycin and dexa~nethasone~~'.
1.7 Study Rationale
Multiple myeloma is a fatal form of human cancer that affects tenninally
differentiated B cells. The pathogenesis of this disease remains unknown and there is no
cure. Translocations into IgH switch regions occur universdy in MM, and are
heterogeneous and non-random. Translocations of FGFR3 into the lgfI locus occur at a
fiequency of 25% in MM; and thus comprise one of the most comrnon translocations
detected in MM patients and cell luies. Furthemore, activating mutations of translocated
FGFR3 have been noted at a high fkequency in MM suggesting an important role of this
receptor in tumor development and progression. The downstream effect of
overexpression of FGFEU in MM is unknown and a better understanding of the signahg
pathways of FGFR3 on myeloma cells codd contribute to the understanding of MM
development and progression, as well as potentially defining targets for the development
of rational molecular based therapies.
1.8 Hypothesis
We postulated that the overexpression of FGFR3 in myeloma cells due to
iilegitimate switch recombination would cause the phosphorylation of STATs and would
up-regulate bcl-xL. These cellular events would cause both increased proliferation and
enhanced survival potential of myeloma cefls in vivo.
1 -9 Experimental Objectives
The initial objective of this project was to examine whether the expression of
FGFR3 in IL-6-dependent myeloma cells would alter the growth characteristics of these
cells, especidy with respect to IL-6. We were interested in determing whether wild-type
and constitutively active FGFR3 would cause similar cellular effects. We also airned to
examine the potential downstream targets of FGFIU in MM ceus, particularly STAT
phosphorylation and the expression level of bcl-xL in ceils grown in the absence of IL-6.
Chapter 2
Materials and Methods
2.1 Retroviral vector construction
Two pcDNA3 plasmids containing the full tength human cDNA of wild-type or
TD II mutant form (FGFR3-TD) of FGFR3 were obtained fiom Daniel. J. Donoghue
(San Diego, d al if or nia)"? FGFR3 was released by restriction cutting with Hindm and
SpeI, which aiso removed the 3' untranslated region. The recessed 3' termini ends of the
cDNA fiagxnents were filled and the hgments ligated into a HpaVSnaBI-digested MMV
r e t r ~ v i n i s ~ ~ containing the neomycin resistance gene as the selectable marker. The
resulting vectors were termed M F W - W T (wild-type FGFR3) and M F W - T D (TD II
K6SOE mutant form of FGFR3) (Figure 4). The parental MINV vector (neor only) was
used as a negative vector control.
2.2 Retrovirus production
Twenty micrograms of the constructed retroviral vectors and the parental MlNV
(neor only) vector were electroporated into 1.5 x 106 PA3 17 amphotropic packaging
ce~ l s '~~ . M e r two days of culture the supematant was collected, passed through a 0.45
micron filter, and overlaid on subconfluent monolayers of Gf +E 86 ecotropic packaging
~ e l l s ' ~ ~ in the presence of 8 ug/rnL of polybrene (Sigma, St-Louis, MO). On day three
and four of culture this process was repeated. Transduced GP+E 86 cells were seIected in
fiesh medium containing 1.2 mg/mL of G4 18 (Gibco, Grand Island, NY) over a penod of
14 days. Vector supematant was collected fiom GP+E 86 producer cells 24 hours after
Figure 4: Vector diagram of MFINV-WT and MFINV-TD. cDNA of either wild-type FGFR3 or FGFR3-TD was digested fiom a pcDNA3 plasmid with Hindm and SpeI. This fiagment was blunt-ended into HpaVSnaBI-digested MlNV vector. The vector contains ampicillin resistance for bacterial selection and neomycin resistance for selection of stable clones.
the medium was changed to Iscove's Modifïed Dulbecco's Medium (IMDM) plus 5%
fetal calf s e m (FCS) (Gibco, Grand Island, NY).
2.3 Expression of wild-type FGFRJ and FGFR3-TD in B9 cells
B9 cetls were subcloned Tom an IL-6-dependent plasmacytoma ce11 line when a
ce11 line more sensitive to IL-6 was d e ~ i r e d ~ ~ ~ . This ceIl line is commonly used in a
biologicai assay for quantimg IL-6 activity since they have a strict requirement for I L 6
26; 347 for growth and survival . ~ 9 ~ ~ ' cells were plated at a demity of 1 x 106 cells in 60
mm dishes. Supernatant fiom the G418 selected GP+E 86 producer lines was added to
the cells dong with 2% IL-6 conditioned media fiom the SP2/mlL-6 ce11 lines4* and 8
ug/mL of polybrene (Sigma, St. Louis, MO). After 2 days, fkesh retroviral supernatant
was added. M e r an additional 72 hours of incubation, B9 cells were selected in 2% IL-6
conditioned medium and 1 . 2 m g / d G418 for 2 weeks, generating the ce11 lines B9
MMV (empty vector), B9-WT (wild-type FGFR3), and B9-TD (FGFR3-TD). After
selecting for stably transfected cells a limiting ce11 dilution was pexformed to generate
single cell clones of B9-WT and B9-TD. initial IL-6 independence assays and apoptosis
studies utilized pooled cells. Subsequent western blot analysis utilized individual high or
Iow expressing clones of B9-WT and B9-TD.
2.4 Tissue culture
Cells were cultured at 37OC in a humidified atmosphere containhg 5% CO2. B9
cells were grown in IMDM supplemented with 5% FCS, 2% IL-6 conditioned medium
and penicillin-streptomycin (Gibco, Grand Island, NY). KMS 1 1349, a human myeloma
ce11 line containhg a FGFR3 translocation, was kindly provided by Masayoshi Namba
(Okayama, Japan). KMSll was grown in RPMI Medium 1640 supplemented with 10%
FC S and peniciIlin-streptornycin. Ut66 350; 351 , a human myeloma ce11 line, was grown in
IMDM with 10% FCS and peniciliin-streptomycin. GP+E 86 and PA317 cells were
grown in Dulbecco modified Eagle medium (DMEM) supplemented with 10% FCS and
penicillin-streptomycin-
2.5 Ce11 viability and proliferation
To examine ce11 viability and proliferation B9, B9 MINV, F39-WT and B9-TD
were washed 4 times in iMDM without FCS and then plated in triplicate in a 96 well
plate at a density of 1 x 104 cellslwell in varying concentrations of iL-6 and cultured for
48-96 hours. Total cell number and percent viability were detennined every 24 hours by
trypan blue staining and enurneration with a hernocytometer. For proliferation assays 0.5
pCi of '~-th~midine (Amersham, Arlington Heights, IL) was added to each well in the
last 18 hours of the assay. Cells were harvested ont0 g l a s filter paper (Gelman Science,
Ann Arbor, MI), and 3~-thymidine counts determined by liquid scintillation.
2.6 Ligand stimulated proliferation assay
B9-WT cells were plated at a concentration of 2 x 10' cells/ml in 96 well plates
in the presence of increasing concentrations of acidic aFGF (R&D Labs, Minneapolis,
MN) and heparin (Sigma, St.Louis, MO). Cells were subsequently incubated for two or
three days and then analysed using the chromogenic dye 3-(4,s-dimethylthiaml-2-y1)-2,s-
diphenyltetrazolium bromid (MTT) (Boehringer Mannheim, Mannheim, Germany).
Absorbante at 570nm-650nm was read on an ELISA plate reader (Molecular Devices).
M e r optimal concentrations of aFGF (40 ng/mL) and heparin (30 ug/mL) for the support
of ce11 proliferation were determined, B9, B9 W, B9-WT and B9-TD were washed 4
times in IMDM lacking FCS and plated in triplkate at a concentration of 2 x 1o5 cells/ml
in 96 well plates. Each ceil line was anaiyzed uader four different conditions, 40 ng/mL
aFGF and 30 ug/mL cf heparin, 40 ng/mL aFGF and 30 ug/mL of heparin plus 2% IL-6,
2% IL-6 without ligand and 0% IL6 without ligand. Cells were incubated for 48-72
hours and then anaiyzed by MTT. MTT (3-[4,5-dimethylthiazo1-2-yl]-2,5-diphenyl) is a
tetrazolium salt that is cleaved to formazan dye by metabolically active cells. This
cleavage resuits in a colour change that c m be rneasured by spectrophotornetry.
2.7 Ce11 cycle and apoptosis analysis
Ce11 lines were washed and then plated in iMDM at a density of 0.5 x 1o6
cells/mL in 0% IL-6 or 0.1 x 106 cells/mL in the presence of IL-6. Cells were cultured for
48 or 72 hours. After harvesting the cells, 500 uL of a hypotonie fluorochrome solution
(0.1% sodium citrate, 0.1% Triton X-100, 50 u g / d of propidium iodide) was added to
each ce11 pellet. The samples were incubated overnight in the dark at 4OC. Flow
cytometry was perfomed on a FACScan (Becton Dickinson, Meylan, France) the
following day using Ce11 Quest software to acquire ce11 cycle data and ModFit LT
software for analysis. Tunel assays (Promega, Madison, Wisconsin) were perfomed on
cells grown in 100 mm plates in the presence or absence of 2% IL-6. Four million celis
were fixed with 1% parafomialdehyde in phosphate buffered saline solution (PBS) for 20
minutes on ice. M e r incubation in ethanol and washing Two times in PBS, ceiis were
incubated at 37OC for one hour with tenninal deoxynucloetidyl tramferase (TdT) enzyme
and nucleotide mix containing fluorescein-12-dUTP in order to end label the 3'OH end of
the fiagmented DNA. The reaction was ceased by addition of 20mM EDTA and cells
were washed twice in 0.1% Triton X-100 in PBS and stained with propidiurn iodide for
30 minutes. Anaiysis was performed by flow cytometry and apoptotic cells were
fluorescein- 12-dUTP positive.
2.8 Western blots
Cells were lysed with RIPA buffer (50 mM Hepes pH 7.23, 150 mM NaCl,
50 FM ZnCI,, 50 p M NaF, 50 p M O-phosphate, 2 mM EDTA, 1% Nonidet P40, 0.1%
sodium deoxycholatc, 0.1% SDS) containing 2 m M phenylmethylsulfonyl fluoride and 2
rnM sodium orthovanadate. After a 20 minute incubation on ice, lysates were cleared by
centrifugation for 15 minutes. Lysates were combined with an equal quantity of 2 X SDS
loading b a e r (5% SDS, 50 m M Tris pH 6.8, 200 mM DTT, 0.02% glycerol, a pinch of
bromphenol blue) and denatured at 95OC for 5 minutes. Sampies were hctionated by
SDS-PAGE. Separated proteins were transferred to nitrocellulose (Costar, Acton, MA).
Membranes were probed with antibodies to the C-terminus end of FGFR3, murine
bcl-xsn, murine bcl-2, murine bax, anti-phosphotyrosine (dl Santa Cruz Biotechnology,
Santa Cruz, CA), anti-phospho-STAT1 (Upstate Biotechnology, Lake Placid, NY),
monoclonal anti-STAT3 and anti-STATl (Transduction Laboratones, Lexington, KY) or
polyclonal anti-phospho-STATZBTATS, anti-phospho-STATS, anti-phospho-STAT3
(lcindly provided by David A. Frank, Dana-Farber Cancer Institute, Boston, MA), and
anti-STATS (kindly provided by Dr. J. N. Me, Memphis, IN). Goat 4 - r abb i t IgG
horseradish peroxidase (HRP) (PharMingen, Mississauga, ON) or sheep anti-mouse IgG
HRP (Amersham, Arlington Heights, IL) were used as secondary antibodies and blots
were developed by enhanced chemiluminescence (ECL; Amersham, Arlington Heights,
IL) according to manufacturer's instructions.
2.9 Immunoprecipitation and in vitro kinase assay
Twenty-five rnicroliters/reaction of 20% (v/v) Protein-A Sepharose (Pharmacia,
Uppsala, Sweden) was incubated with 12 uL of anti-FGFIU, anti-ERIC1 (recognizes
ERKl and ERK2), anti-p38 or anti-JNK1 (SAPK) (ail fiom Santa C m Biotechnology,
Santa Cruz, CA) overnight at 4 ' ~ . M e r washing the bead-antibody complex four times
with RIPA, 1250 ug of total ce11 lysate was incubated with the bead complex for 2 hours
at 4 ' ~ . Protein content of lysates were quantified using BCA* protein assay reagent
(Pierce, Rockford, Illinois). hunoprecipitates were washed with RIPA and the
samples split into equal aliquots. One aliquot was washed four times with kinase buffer
and subjected to in vitro kinase assay in kinase bufTer (50 mM Hepes pH 7.23, 150 mM
NaCl, 0.5% Nonidet P40, 1 m M MgCl,, 1 m M MnC12, 5 pCi [ a 3 2 ~ ] ATP) including
rabbit enolase as a substrate for FGFR3, myeiin basic protein (MBP) as a substrate for
ERK or p38 or c-jun as a substrate for M l (SAPK). Samples were incubated at 3 7 ' ~
for 15 minutes, the reaction was stopped by the addition of 2 X SDS loading buffer and
sarnples boiled for 10 minutes. Samples were fiactionated by SDS-PAGE and
phosphory lated proteins visualized by autoradiography . The remaining aliquot of the
irnrnunoprecipitate was washed with RIPA, mixed with 2 X SDS loading bufTer , boiled
and then electrophoresed by SDS-PAGE and the membranes probed with anti-FGFR3,
ERKI, p38 or JNK1(SAPK).
Chapter 3
Results
3.1 Generation of FGFR3 expressing ce11 [ines
Human FGFRJ cDNAs, wild-type and TDII mutant form, were cloned into the
MSCV-based retroviral vector M I N V ~ ~ ~ (Figure 4). GP+E packaging cell Iines exporthg
these vimes were generated. Viral titer for wild-type FGFR3 packaging cells was 2 x
10' viral partic1edm.L and for FGFR3-TD packaging cells was 2 x 10' viral particles/ml.
Westem blots of packaging cell lysates confinned the expression of FGFR3 in only wild-
type and mutant lines and not in GP+E parental cells or those idected with neor only
vector (Figure 5). Subsequently IL-6-dependent B9 ce11 lines were ùifected with the
retroviruses and selected for 10 days in G4 18-containhg medium, generating the ce11
iines B9 MINV (empty vector), B9-Wï (wild-type FGFR3) and B9-TD (TDII mutant
FGFR3). M e r stably transfected cells were selected, a limiting ce11 dilution was
performed to generate single ce11 clones of B9-WT and B9-TD. Westem blots of these
clones confirmed expression of FGFR3 in B9-WT (Figure 6A) and B9-TD (Figure 6B)
and demonstrated that clones expressed varying levels of FGFR3.
3.2 Function of human FGFR3 in transfected ce11 lines
Phosphotyrosine @-tyr) western blots demonstrated that FGFR3 was
phosphorylated in the absence of ligand in B9-TD (Figure 7). Upon addition of ligand
(aFGF and heparin) phosphorylation of wild-type FGFR3 was induced in B9-WT. This
phosphorylation pattern has k e n previously described 115; 124 suggesting that the mutant
Figure 5: Expression of FGFR3 in GP+E packaging ce11 lines. FGFR3 is translated to a 125kDa and 135kDa, which represent different glycosylation States. Lane 1 and lane 2 demonstrate FGFR3 is not expressed in parental GP+E line or GP+E infected with neor vector ody, respectively. Wild-type FGFR3 is expressed in GP+E infected with MFINV-WT (lane 3) and the TD mutant form in GP+E infected with MFW-'TD(1ane 4). Lane 5 represents KMS 1 1 lysate which is a positive control for FGFR3.
Figure 6: Expression of FGFR3 is achieved at variable levels in B9-WT and B9-TD cells. (A) Western blot analysis of FGFR3 expression in B9 clones: KMS 1 1 myeloma ce11 line positive control for FGFR3 (lane l), parental B9 (lane 2) and B9-WT sub-clones (lanes 3- 15). (B) KMS 1 1 positive control for FGFIU (lane l), U266 myeloma ce11 line negative control (lane 2), parental B9 (lane 3), and B9-TD sub-clones (lanes 4-1 8).
Figure 7: FGFIU is phosphorylated in the presence of ligand (aFGF+heparin) in BPWT and constitutively phosphorylated in B9-TD. FGFR3 expressing and control ce11 lines were depleted of cytokine for 18 hours and then stimulated with or without EL- 6 for 10 minutes at 37OC. Total ce11 lysates were resolved via SDS-PAGE and the membrane was probed with phospho- tyrosine antibody (lower panel). The membrane was then reprobed with anti-FGFR3 (upper panel). Pooled cells are denoted as pool and single ce11 clones as clone.
8 9 Cr,
11
receptor was active in the absence of ligand. An in vitro kinase assay confïrmed that both
the wild-type and mutant receptors were functional since both wild-type and mutant
FGFR3 exhibited the ability to autophosphory late (Figure 8). The decreased
phosphorylation of wild-type FGFR3 in the presence of ligand is thought to occur due to
receptor downregulation. This assay also demonstrated that mutant FGFR3 exhibited a
higher level of autophosphorylation than wild-type FGFR3, as previously
described 115; 116
3.3 Growth response to interleukin-6
To deterrnine whether the overexpression of FGFR3 would ùifluence IL-6-
induced proliferation of the transfected B9 celis, proliferation and ce11 viability were
examined by a variety of techniques, including '~-th~midine, trypan blue staining, and
M ï T assays. Initiaily '~- th~midine assays were used to examine the proliferative
response of B9 cells and the transfected lines to the presence and absence of IL-6. B9, B9
MINV, B9-WT and B9-TD were plated in ûiplicate, in the absence of S e m , at equai ce11
concentrations in various concentrations of IL-6 and grown for 72 or 96 hours. In the last
18 hours of the assay 0.5 pCi of '~-th~rnidine was added to die ce11 culture medium. The
proliferation of B9, B9 MINV and B9-WT were similar over 96 hours (Figure 9).
B9-WT was expected to display the same proliferative response as B9 since these cells
were not stimulated with ligand, thus FGFR3 should not be active. Interestingly, B9-TD
displayed a higher proliferation than the other ceil lines at al1 concentrations of IL-6. In
the absence of IL-6 B9-TD proliferation was 3-fold higher than B9, B9 MiNV or B9-WT
Figure 8: FGFIU expressed in B9 is functional. An in vitro kinase assay in the presence and absence of ligand (aFGF + heparin) stimulation indicates that wild-type FGFR3 and TD-FGFR3 are functional. Also mutant FGFR3 displays a higher level of autophosphorylation than wild-îype FGFR3.
Figure 9: Cells expresshg mutant FGFR3 have an enhanced proliferative rate in the presence and absence of IL-6. Parental B9, B9 MINV, B9-WT pooled cells, and B9-Ti3 pooled cells were ùicubated in the presence of increasing concentrations of IL-6 and a 3H- thymidine assay was performed. Data points represent the average of three samples and error bars represent the standard deviation at each point.
Enhanced proliferation of BO-TD cells in the presence and absence of IL4
1 1 1 1 i I 1 1 1 1 1
O 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 2
IL4 concentration (%)
+ B9 4 B 9 MINV + 69-WT * 69-TD
suggesting the ability to become IL-6-independent.
To M e r examine the response of these ce11 lines to IL-6, viability assays using
trypan blue stauiing were perforrned over 72 and 96 hours. B9, B9 M I N V , B9-WT and
B9-TD were plated in triplicate in the presence and absence of IL-6, in the absence of
senim. Total cell number and percent viability was detennined at 72 or 96 hours by
trypan blue staining and enurneration was perfomied with a hemocytometer. AI1 four ce11
iines displayed lh i ted ce11 death in the presence of L-6 after 96 hours (Figure 10).
However in the absence of IL-6, B9, B9 MTNV, B9-WT and B9-TD were 25%, 20%,
47% and 80% viable, respectively. These data demonstrate that B9-TD cells remain
more viable in the absence of IL-6.
Also of interest is that, unlike parental B9 cells, B9-TD cells could be maintained
indefinitely in culture in the absence of IL-6, especially those clones expressing high
levels of FGFR3. Also B9-WT clones expressing high levels of FGFR3, for example
clone #3 and clone #6, also exhibited IL-6-independence in the absence of ligand.
Since the above experiments were carried out in the absence of ligand (aFGF and
heparin) and not al1 myeloma cells immediately acquire activating mutations of FGFR3,
the response of the B9-WT and B9-TD cells to ligand was examined. Initially the
response of B9-WT to Ligand in the absence of IL-6 was examined. B9-WT cells were
plated in triplicate and both the concentrations of heparin and aFGF were varïed between
O ng/mL to 100 ng/mL aFGF and O ug/mL to 30 udmL of heparin based on previous
reports in the literature 90; I 1 8; 352 . Metabolic activity was rneasured by MTT assay and it
was apparent that for B9-WT this increased until 40 n g M of aFGF and 30 ug/mL of
Figure 10: B9-TD cells are viable in the absence of IL-6. Parental B9, B9 MINV, B9-WT pooled cells and B9-TD pooled cells were incubated in the presence and absence of IL-6. Trypan blue staining and enurneration on a hemacytometer was perfonned to determine ce11 viability. Data points represent the average of three sarnples and error bars represent the standard deviation at each point.
lncreased viability of BO-TD in the absence of IL4
IL4 concentration
heparin was reached after which no increase in proliferation was observed (data not
shown).
After deteminhg the optimal concentrations of aFGF and heparin to stimulate
BPWT cells, dl three ceii h e s , B9, B9 MINV, B9-WT and B9-TD were plated in
triplicate, in the absence of s e m , and allowed to grow for 48 or 96 hours. Cells were
grown under four different conditions; 40 ng/rnL aFGF and 30 ug/mL heparin lacking iL-
6, 40 ng/mL aFGF and 30 ug/mL heparin plus 2% IL-6 conditioned medium, 2% IL-6
conditioned medium without Ligand and OYO IL-6 without ligand. The metabolic activity
of al1 the cells were similar in the presence of ligand plus 2% IL6 and in the presence of
2% IL-6 except that B9-TD displayed a higher metabolic rate in both conditions than the
other ce11 lines. In the absence of IL-6 and ligand it was confirmed that B9-ID remained
more metabolically active than the other three ce11 lines (Figure I I ) . In the presence of
ligand and absence of IL-6, B9-WT have a higher metabolic rate than that of parental B9
and B9 MINV suggesting that the activation of wild-type FGFR3 couid support ce11
proliferation in the absence of IL-6. As well the mebbdic activity of B9-TD was M e r
enhanced in the presence of ligand.
3 -4 Expression of FGFR3 decreases apoptosis in absence of IL-6
It has previously been demonstrated that the withdrawal of IL-6 Fom B9 results in
the arrest of these celis in ~1~~ so ceii cycle analysis was performed on B9, B9 MINV,
B9-WT and B9-TD cells grown in the presence and absence of IL-6 for 72 hours. This
tirnepoint was chosen based on the previously described expehents with '~-th~midine
Figure 1 1 : Ligand stimulated cells expressing wild-type FGFR.3 are more metabolically active in the absence of IL-6. An M ï T assay was perforrned on IL-6 starved FGFR3 expressing ce11 lines and controls in the presence and absence of ligand (aFGF and heparin). Data points represent the average of three samples and error bars represent the standard deviation at each point. Optical density measurements correlate to metabolic activity.
Ligand stimulation increases the metabolic activity -
of cells expressing wild-type FGFR3
2% IL-6 40 nglmL afgf + 0% 11-6 40 ng/mL afgf + heparin + 2% heparin + 0%
1 1-6 I L-6
and previous d y s i s by Sabourin and ~ a w l e ~ ~ ~ . As expected parental B9 cells enter cell
growth arrest upon removai of IL-6, however, it appeared that to some degree so did B9-
WT md B9-TD (Table 1 ). B9-TD had more cells in S phase and fewer sub-Go apoptotic
cells in the absence of IL6 and aFGF but is likely not significant.
To better characterize the amount of apoptosis occurring in these cultures in the
absence of IL-6, Tunel assays were perfonned. B9, B9 W, B9-WT and B9-TD were
cultured in the presence and absence of IL-6 for five days before analyzhg the fiequency
of apoptotic cells. This t h e penod was chosen based on the results of the 72 hour ce11
cycle analysis (see above). B9 cells were essentially al1 apoptotic after five days in the
absence of IL-6 while only 53% of B9-TD cells were undergoing apoptosis (Table 2). B9
MINV and B9-WT had between 85-88% apoptotic cells in the absence of IL-6. In the
presence of IL-6 B9-TD had the highest ce11 death of the four ceil lines analysed. It is
thought that this high amount of ce11 death reflects a high ce11 turnover. Since this assay
was carried out in the absence of ligand it was expected that B9-WT cells wouid not be
highly viable without IL-6. These data demonstrate that activated FGFR3 is capable of
preventing apoptosis of an IL-6-dependent ce11 line in the absence of IL-6.
3.5 FGFR3 signalhg induces phosphorylation of STAT3
S i n e STATs have been suggested to play a role in anti-apoptosis ~ i ~ n a l i n ~ " ~ '
the tyrosine phosphorylation of STATI, STAT3 and STATS in the presence and
absence of IL-6 was examined in al1 the cell lines by western blots utilizing phospho-
specific antibodies. STAT phosphorylation was anaiyzed &er 10 minutes of stimulation
Table 1 : Percentage of cells in various stages of the ce11 cycle after 72 hours o f culture in the presence and absence of IL6
Go-G, 34.5% S 34.9% G2-M 23.8% Apoptosis 15.3%
GoœG, 67.5% S 26.3% G,-M 6.1% Apoptosis 54.3%
Go-G, 71.2% S 26.4% G2-M 15.5% Apoptosis 40.8%
Go-G1 31.1% S 42.2% G2-M 26.6% Apoptosis 20.4%
Go-G, 67.3% s 28.1% Gz-M 4.6% A~o~tos is 32.9%
Go-G, 24.9% S 40.9% G2-M 34.3% Apoptosis 24.4%
Go-G, 24.5% S 49.9% G2-M 25.4% Apoptosis 8.2%
Table2: Percentage of apoptotic B9 cells as assessed by Tunel assay after five days of culture in the presence or absence of IL6
IL-6 concentration
with IL-6 since it has k e n previously demonstrated by others that this is the point of
maximal STAT phosphorylation Ui plasma cells stimulated by JL-6'? STATS was
found to be tyrosine phosphorylated constitutively in ail the celi lines independent of IL6
(Figure 12). The level of phosphorylation was not M e r enhanced due to activation of
FGFR3. STATl was induced in the cell lines only in the presence of IL-6 (Figure 13).
However, STAT3 was found to be constitutively tyrosine phosphorylated in the absence
of IL-6 and ligand in B9-WT and B9-TD clones expressing high levels of FGFR3 (Figure
14). The level of STAT3 phosphorylation in the absence of IL-6 and ligand was higher in
B9-TD clones than B9-WT clones without IL-6. When stimulated with ligand in the
absence of IL-6 STAT3 phosphorylation is detected in B9-WT clones and in B9-TD
clones (Figure 15).
3.6 FGFR.3 survival signai is mediated by up-regulation of bcl-x,
Since it has previously been s h o w that the IL-6 survival signal in myeloma is
mediated by up-regdation of bc1-xL 148; 340-342 and that STATs may be involved in up-
regulating bcl-xL 172; 2î3; 224 the expression of bcl-x, was next examined by western blots.
B9, B9 MINV, B9-WT and B9-TD cells were starved of cytokine for 48 hours and
lysates generated at O hours, 8 hours, 24 hours and 48 houn. Lysates were quantified for
protein content and 20 ug of protein loaded for each sample so that comparisons of
protein levels could be made directly fiom the western blot. These blots demonstrated
that the baseline level of bd-xL is higher in B9-TD cells expressing activated FGFR3 than
in B9 controls (Figure 16A). As previously dem~ostrated~~', the level of hl-xL in B9
Figure 12: STATS is tyrosine phosphorylated independently of IL-6. FGFR3 expressing and control ce11 lines were depleted of cytokine for 18 hours and then stimulated with or without IL-6 for 10 minutes at 37°C. Total ce11 lysates were resolved via SDS-PAGE and the membrane was probed with a phospho- specific STATS antibody (upper panel). The membrane was reprobed with a peptide-specific antibody that recognizes total STATS Oower panel). Subscript p indicates pooled cells and single ce11 clones are designated with a number.
B9 B9 MINV B9-WT B9-WT#3 B9-TD B9-TD#14 P P
Figure 13: STATl is only tyrosine phosphorylated upon IL-6 stimulation. FGFR3 expressing and control ce11 lines were depleted of cytokine for 18 hours and then stimulated with or without IL-6 for 10 minutes at 37OC. Total cell lysates were resolved via SDS-PAGE and the membrane was probed with a phospho- specific STATl antibody (upper panel). The membrane was reprobed with a peptide-specific antibody that recognizes total STATl (lower panel). Subscript p indicates pooled cells and single ce11 clones are designated with a number.
B9 B9 MINV B9-WT, B9-WT#3 B9-TD, B9-TD#14
Figure 14: STAT3 is constitutively tyrosine phosphorylated in FGFIU expressing cells. FGFR.3 expressing and control ce11 lines were depleted of cytokine for 18 hours and then stimulated with or without IL-6 for 10 minutes at 37°C. Total ceIl lysates were resolved via SDS-PAGE and the membrane was probed with an activation-specific STAT3 antibody (upper panel). The membrane was reprobed with a peptide-specific antibody that recognizes total STAT3 (lower panel). Pooled cells are denoted as pool and single-ce11 clone expressing high levels of FGFR3 as clone.
Figure 15: STAT3 is phosphorylated in B9-WT and B9-TD upon stimulation with ligand (aFGF+heparin). Ce11 lines were depleted of ILd for 1 8 hours and then stimulated with IL6 or aFGF+heparin for 10 minutes at 37OC. Total ce11 lysates were resolved via SDS-PAGE and the membrane was probed with a phospho-specific STAT3 antibody (upper panel). The membrane was then reprobed with an antibody that recognizes total STAT3 (lower panel). Pooled cells are denoted as pool and single ce11 clones are designated with a number.
cells was found to decline over the 48 hours after cytokine withdrawai and dso in the
control ce11 line B9 M W . In contrast, the expression of this protein remains relatively
constant in B9-TD ceils d e r the removai of IL-6 fiom the culture medium- B9-WT
clones expressing high levels of FGFR3 also exhibited an increased baseline level of
bcl-xL and the protein expression remained constant afler the removal of IL-6 (Figure
16B). These same clones that exhibited high levels of bcl-xL were previously shown to
constitutively express STAT3. Of interest was the observation that the addition of ligand
to these clones actually resuited in a slight decrease in the expression of bcl-x, however,
the levef of expression still remained higher than that seen in B9 cells without IL-6 over
48 hours(Figure 16B).
The expression of bcl-xs, bax and bcl-2 was also exarnined by western blots. Bax
expression levels did not change due to the expression of FGFR3 in either the presence or
absence of IL-6 (Figure 17). Expression levels remained constant and essentially the
same in al1 ce11 lines regardless of IL-6. Expression of bcl-xs and bcl-2 were not detected
in any of the ceil lines indicathg these are likely not regulated by FGFR3 signaling (data
not shown). It has previously been noted that B9 cells do not express endogenous
bd-2 148.
3.7 Enhanced proiiferative response to IL-6 is not mediated by ERK, or SAPK
The up-regdation of bcl-xL may explain the enhanced sumival of B9-WT and B9-
TD cells in the absence of IL-6 but it does not explain the enhanced proliferation of these
cells in response to IL-6. It has previously been suggested that MAP kinases are involved
Figure 16: Expression of B ~ 1 - x ~ protein is elevated in FGFR3 overexpressing cells. Total ce11 lysates were prepared at varying time points fkom FGFR3 expressing clones and control cells grown in the absence of IL6 over 48 hours. For each sample 20 ug of total protein was loaded in each lane and than resolved via SDS-PAGE and the membrane probed with an anti-bcl-x, antibody. Membranes were then reprobed with anti-ERK as a protein loading control. (A) B ~ 1 - x ~ is overexpressed at baseline in cells expressing mutant FGFR3 and remains high f i e r IL-6 withdrawal (top panel). ERIC expression remains consistent in al1 lines(lower panel). (B) B ~ 1 - x ~ is overexpressed at baseline and remains high in cells overexpressing wild-type FGFR.3. With the addition of ligand there is a down-regulation of bcl- x,(first and third panel). ERIC expression remains consistent in the ce11 lines (second and fourth panels). Top two panels represent one single ce11 clone and the bottom two panels a different single ce11 clone.
B9 B9 MINV B9-TD#S 4
Figure 17: Bax protein levels are not altered due to FGFR3 expression in B9 cells. Ce11 lines were grown in the presence of IL-6 or in the absence of I L 6 for 48 hours. Total cellular lysates were resolved via SDS-PAGE and the membrane probed with anti- bax.
B9 B9 MINV B9-WT#3 B9-WT#6 B9-TD#7 B9-TD#14
IL-6 nnnnnn + - + - + - + - + - + -
in regulating rnitogenic responses in MM 161; 165; 171 . As weii it has previously been
demonstrated that SAPK is involved in inducing apoptosis in so its down-
regdation couid potentially lead to decreased ce11 death. The potentiai role of these
molecules in FGFR3 signaling was examined by in v i ~ o kinase assays. These assays
demonstrate that ERKI, ERK2, or SAPK activity does not change in the presence of
FGFR3 expressing cells as compared to controls. ERKl and ERK2 activation, measured
by phosphorylation of MBP, was induced in response to IL-6 in dI ce11 Iines but the
increase in activity remained consistent in ail ce11 lines (Figure 18). SAPK activation,
measured by phosphorylation of c-jun, remained constant in al1 ce11 Iines independent of
IL-6 (Figure 19). It was noted that p38 activity was induced in B9, B9-WT and B9-TD in
the presence of IL-6 and also following ligand stimulation in B9-WT and BI-TD (Figure
20) suggesting that ligand stimulation of FGFR3 might lead to increased p38 activity.
This increased activity of p38 by aFGF and heparin suggests that M e r evaluation of
p38 shouid be undertaken. There is an inhibitor of p38 that could be used in order to
determine whether this kinase is involved in proliferative signaling. M e r inhibithg the
p38 pathway the proliferation of the cells couid be examined by 3~-thymidine assays.
Figure 18: ERK activity is induced by IL6 stimulation. Cells were depleted of I L 6 for 18 hours and then stirnulated with or without IL-6 for 10 minutes at 37OC. Total ce11 lysates were irnmunoprecipitated using anti-ERK antibody and then subjected to an in vitro kinase assay with MBP as a substrate for ERK. Phosphorylation of MBP is indicative of ERK activity.
Fi-gure 19: SAPK activity is not altered due to FGFM expression. Cells were depleted of IL-6 for 18 hours and then stimulated with or without IL-6 for 10 minutes at 37°C. Total ce11 lysates were immunoprecipitated ushg anti-SAPK antibody and then subjected to an in vitro kinase assay with c-jun as a substrate for SAPK. Phosphorylation of c-jun is indicative of SAPK activity.
B9 B9 MINV BPWT
Figure 20: p38 activity is induced by ligand (aFGF+heparin) stimulation of FGFR3. Cells were depleted of IL-6 for 18 hours and then stimulated with or without IL-6 for 10 minutes at 37OC. Total ce11 lysates were immunoprecipitated using anti-p38 antibody and then subjected to an in vitro kinase assay with MBP as a substrate for p38. p38 is activity is induced in al1 cells in the presence of IL,-6 but also appears to be stimulated by ligand activation of FGFR3. MBP phosphorylation is indicative of p38 activity.
Chapter 4
Discussion and Future Investigations
4.1 Discussion
The t(4; 14) translocation occurs at a frequency of 25% in multiple myeloma cell
lines and patient samples resuiting in dysregulated expression of FGFR3 on der(l4) 61; 66
and IgH-MMSET hybrid mRNA transcripts on d t~ (4 )~ ' . Furthemore, activating
mutations of FGFR3 have been identifieci fiequently in myeloma samples. One such
activating mutation, K650E, is known to cause thanatophonc dysplasia type II when the
mutation arises within the germline133' 3" suggesting that the activating mutations of
16; 61 FGFR3 in MM aise afler the translocation of FGFR3 . To date numerous studies
have analyzed the fiequency of IgH translocations in MM but the downstrearn effects of
dysregulated gene expression of FGFR3 have not been examined. Thus the potential role
of FGFR3 in MM was investigated in this thesis.
FGFW has been shown to be involved in negatively regulating bone
growth'u; IL). Pnor investigations of downstream signaling patterns of FGFR.3, and its
various mutant forms, have been performed in relation to its role in human
chondrodysplasias (dwarfism). It has k e n demonstrated that in chondrocytes the TDII
form of FGFR3 phosphorylates STATl, which up-regulates p2 1 and results in ce11
growth arrest116. As weli an activating T'DI mutation of FGFR3 has been found to
phosphorylate STATl in chondrocytes dong -aith a decline in bcl-2 levels and increase in
bax expression resulting in increased apoptosis' ". Thus in chondrocytes it appears that
expression of mutant FGFR3 results in ce11 growth arrest and increased apoptosis. If the
sarne pathways were activated in myeloma cells it rnight suggest that a translocation of
FGFR3 in myeloma would result in increased apoptosis of the maiignant clone.
However, this is unlikely in myeloma since viable cells harbouring the t(4;14)
translocation and activating mutation of translocated FGFR3 are detected in both cell
lines and patient samples. Thus it appears that FGFR3 signalhg may differ in different
ce11 types, a hypothesis which is supported by the finding that FGFR3 expression in a
pro-B ce11 line renilts in ce11 proiiferation in response to
B9 cells were subcloned fiom an IL-6-dependent plasmacytoma ce11 line when a
ceIl more sensitive to IL-6 was desiredJ4'. This ce11 line is commonly used in a biological
assay for quantifying IL-6 activity since they have a strict requirement for IL-6 for
26; 347 growth and survival . This made it an ideal ce11 line to examine the effect of FGFR3
expression on the growth response of rnyeloma cells to IL-6. To examine the role of
FGFR3 in myeloma Il-6-dependent murine B9 cells were engineered to express either
wild-type hurnan FGFR3 or FGFR3-TD mutant (K650E).
Mthough human FGFR3 was introduced into murine cells, expression of human
FGFR3 in this murine ce11 line should still be representative of the pathways involved in
human myeloma since hurnan FGFR3 and mouse fafi3 are highly homologous. The
clustering of exons and introns are nearly identical, both human FGFR3 and mouse fgfr3
contain the same altematively spliced introns, and the binding sites in the promoters are
similar? As well there is 84% homology at the DNA level and 92% hornology at the
arnino acid leve18'.
B9 cells do not express murine FGFR3 as show by western blotting and in vitro
kinase assays in this thesis. It is possible that they do express other FGFRs but it would
seem d i k e l y since these celis also do not respond to aFGF; a common ligand for al1
FGFRs. It appears that most hematopoietic celIs may express FGFRs but FGFs are not
an important growth factor for hematopoietic cells9'. Thus there is no endogenous
FGFR3 and likely no other FGFRs that would contribute to the grow of B9 in these
experiments suggesting that the effects observed are solely due to FGFR3 expression in
these cells.
In these experiments pooled and cloned B9-WT and B9-TD cells were compared
to pooled parental cells. It is possible that cloned B9 cells could display bcl-x,
upregulation and STAT3 activation. Cloned B9 celis were not analysed in these
experiments since we were unable to maintain B9 cells in the absence of IL-6.
Expression of FGFR3, particularly the TD mutant, enabled B9 cells to grow
independently of IL6 and to exhibit an enhanced proliferative response in the presence of
IL-6. While these responses could be obtained soiely by the expression of TDII mutant
FGFR3, wild-type FGFIU either had to be stimulated by ligand or overexpressed. When
wild-type FGFR3 is stimulated by its ligand 89 cells were capable of proliferating in
response to aFGF instead of IL-6. This has also previously been demonstrated with an
IL-3-dependent ceil Iine that could grow in the presence of aFGF and heparin instead of
IL-3 when it was transfected with FGFRS'~. Overexpression of wild-type FGFR3
supported proliferation of B9 cells in the absence of IL-6 and ligand. Overexpression of
wild-type FGFR3 would increase the concentration of the receptors on the ce11 membrane
and likely contribute to çpontaneous dimerization in the absence of ligand. Li et al.
(1 997) observed activation of wild-type FGFR3 in the absence of ligand also thought to
be due to spontaneous dimerization. The activation of wild-type receptors in the absence
of ligand has also been noted to occur with overexpression of other ce11 surface
receptors 183; 354356 . Other groups suggest that overexpression might be respomible for
increasing the level of constitutive tyrosine activity to a threshold required for
~ i ~ n a l i n ~ ~ ' ~ or by causing stable non-covalent intermolecular interactions that result in
constitutive activation)56. Thus high expression of wild-type FGFR3 in myeloma cells
would support proliferation in the absence of growth factors.
Similar to the dose-dependent activation of wild-type receptor in the absence of
ligand, it has been suggested that the concentration of mutant receptors determines the
strength of the response. It has been noted for example that increasing the number of
rnutated receptors in vivo by two alleles produces a more severe dwarfism phenotype3s7.
This phenornenon also appears to occur in myelorna cells since cells expressing the
highest levels of TDlI mutant FGFR3 displayed the most pronounced ability to
proliferate and survive in the absence of IL-6, f i e r suggesting that the expression Ievel
of FGFIU is important. Thus for both wild-type and TD mutant FGFR3 there appears to
be a dose response in that cells with the highest expression levels display the most
pronounced phenotype as seen in this study in vitro.
Several reports suggest that STATl is involved in FGFR3 signaling in
c hondrocytes 116; 118 . However, FGFR3 expression in B9 did not alter STATl
phosphorylation, supporting the idea that pathways activated by FGFR3 may be cell-
specific. STATl was only phosphorylated in B9, B9-WT and B9-TD in response to IL-
6158; 160; 162 , while, S T A n was constitutively phosphorylated in cells expressing mutant
FGFR3 and cells expressing high levels of wild-type FGFR3. Since senun was present in
the medium during these experiments it is possible that the senun may account for some
of the STAT3 activation seen. However, there was a correlation between the amount of
FGFR3 expressed and the activation of STAT3. STATs have been irnplicated in ce11
growth, suppression of ce11 growth and survival 116; 172; 213; 219 224 suggesting that the
activation of STAT3 may be significant in myeloma cells.
Since cells expressing FGFR3 exhibit an enbanced survival potential in the
absence of IL-6, and since STAT3 was found to be phosphorylated in the absence of IL-6,
the expression levels of bcl-2 family members were examined. It appears that STAT l
and STAT3 are capable of upregulating bcl-xL In; zu; 224 , which mediates the survival
response of B9 cells in the presence of IL-^'^*. in cells expressing mutant FGFR3 or high
levels of wild-type FGFR3, bcl-xL was maintained in the absence of IL-6 while the levels
of bcl-xL in B9 cells declined rapidly in the absence of IL-6. Also it was noted that
STAT3 phosphorylation correlated with the up-regulation of bcl-xL.
This suggests that bcl-xL might be mediating the s d v a l of cells expressing
FGFR3 but it was necessary to examine the expression of other important bcl-2 family
members. It has previously k e n reported that B9 cells express high levels of the pro-
apoptotic gene baxt4*, thus if bax expression is altered due to FGFR3 it may potentially
counter the effects of bcl-x,. However, we observe that in B9 cells FGFR3 expression
does not alter bax expression levels in the absence of IL-6. As well bcl-xs and bcl-2
expression are not detected in these cells. This suggests bcl-xL is likely involved in
preventing apoptosis in the absence of IL6 in ceils overexpressing FGFR3.
In £39 cells the reduction in apoptosis in the absence of IL-6 rnight be mediated by
the up-regulation of bcl-x. Furthemore, it is plausible that STAT3 is mediating a
proliferative response in the absence of IL-6 since STAT signaling has previously been
implicated in growth responses 181; 213; 215; 221; 222 . IL-6 enhances the proliferation of the
cells expressing FGFR3 but the phosphorylation of STAT3 is not increased in the
presence of IL-6 suggesting that another pathway is involved in the proliferative effect, It
was initially thought that activation of MAP kinases may be involved in the enhanced
proliferative response to IL6 since it has been suggested that these pathways regulate the
rnitogenic response of myeloma cells to IL-6 165; 171 . However in B9 cells expressing
FGFR3 there was no differential activation of these pathways in the presence of IL-6
suggesting that these proteins were not mediating the proliferative response to IL-6.
Interestingly it was observed that p38 activity appeared to increase when both wild-type
and TD mutant expressing cells were stimulated with ligand. p38 signaling remains to be
fully defined but it appears to be capable of inducing proliferation or apoptosis depending
upon ce11 type and stimulus 276278. Further experimentation is required to fully understand
the potential role of p38 in ligand mediated signaling but the p38 in viîro kinase assay
results suggest that this pathway may be involved in either proliferation or prevention of
apoptosis in myeloma cells expressing FGFR3.
We cannot exclude the possibility that signaling by mutant FGFR3 may result in
slightly different cellular effects, such as more pronounced proliferation in the presence
and absence of ILA. It has previously been suggested by others that the intensity of the
signal from K650E mutant FGFR3 may have different cellular effects thaa wild-type
FGFR3 116; 121; 124 . This may be related to the length of t h e the receptor remains active
since prolonged signaling likely causes a unique cellular response 135; 183 . Signaling
through mutated FGFR3 is likely prolonged because there is no ligand regulating
activation. Normally ligand stimulation of a receptor results in dimerization and down-
87; 358 regulation of the receptor so that the signal is transient . For exampie it has been
shown that in hepatocytes gp t 30 is internalized in response to IL-6 binding which down-
regulates the signal3". As well it has k e n demonstrated that FGFR3 translocates to the
nucleus upon ligand stimuiaiion 119; 120 and this intemalization likely down-regulates the
biological signai of FGFIU'~'.
In the curent study it appears that ligand binding to FGFR.3 regulates intracellular
signaling in a manner distinguishable fiom spontaneous dimerization due to
overexpression of wild-type FGFR3. When wild-type clones were treated with ligand a
decline in bcl-xL was observed, although the level of expression stiii remained higher that
that seen in B9 parental cells. Both of these initially puzzling results rnight be best
explained by dom-regulation of the receptor in response to ligand bindingS7. As
expected the signd generated by ligand binding results in less pronounced activation of
intracellular proteins. This suggests that wild-type FGFIU would cause a less
pronounced phenotype in vivo as seen in vitro.
We observed that cells expressing mutant FGFR3 displayed a stronger
proliferative response and enhanced survival in the absence of IL-6 than cells expressing
wild-type FGFR3 stimulated with aFGF. In this regard it has been demonstrated that the
K650E mutant of FGFR3 appears to have stronger intrinsic kinase activity than wild-type
FGFR3 114; 116. , also shown by in vitro kinase assay in this report (Figure 3). This suggests
that mutant FGFR3 could potentiaily transmit signals through the ce11 more effectively
due to a stronger activation of cellular proteins. This is supported by the hding that
STAT3 was phosphorylateà at a higher level in B9-TD cells than ia B9-WT cells in the
absence of IL-6. In other studies it has been demonstrated that constitutive activation of
FGFW results in more rapid phosphorylation of ERK in chondr~c~tes'". Thus it is
predicted that in myeloma the cells expressing wild-type FGFR3 would activate the same
pathways as those expressing TDII mutation but the downstream effects are expected to
be less pronounced.
As previously mentioned, B9 cells require IL-6 for both growth and survival 26; 148;
347 . Removal of IL-6 fiom B9 cells has been shown to cause apoptosis with significant
ce11 death by 72 hours and ce11 arrest in ~ 1 ~ ~ . Analysis of overexpression of FGFR3 in
myeloma cells suggests that FGFR3 can provide both a proliferative and survival signai
to B9 cells in the absence of IL-6. A similar result was obtained when constitutively
active N-ras was introduced into an IL-6-dependent myeloma ce11 l i ~ ~ e ' ~ . The expression
of N-ras resulted in IL-6-independent proliferation as well as a reduction in the
percentage of ce11 undergohg apoptosis in the absence of IL-6. This suggests that other
receptors or intracellular signaling proteins may be capable of activating responses
similar to those induced by IL6 and thus may be able to replace the requirement for IL-6.
This appears to occur with FGFR3 since it activates STAT3 and up-regulates bcl-xL both
events previously attributed to IL-6 activity in myeloma cells 148; 165
The ability of FGFR3 to provide both a proliferative and survivai signal to
myeloma cells is significant. It is postulated that a ce11 only acquiring mutations that
provide for enhanced proliferation may die in the absence of growth factors but when
there is the additional or sixnultaneous acquisition of events that suppress apoptosis the
ce11 c m become transfom~ed~'~. As well acquisition of a mutation that enables a ce11 to
survive better in the absence of growth factors, such as up-regulation of an anti-apoptotic
gene, makes them more vuinerable to M e r oncogenic changes since they are Iess Iikely
to die after acquiring M e r genetic insdts. Thus translocation of FGFR3 would provide
myeloma cells with the ability to acquire fûrther mutations contributing to enhanced
malignant transformation potential.
It is interesthg that some genetic alterations in myeloma cells, such as FGFR3
overexpression, result in [L-6-independence. Even though it has been suggested that LL-6
26; 148 is critical for the growth and sunival of MM cells it has also been suggested that
mature myeloma cells no longer express the ILdR and are not responsive to IL-^^'.
Kawano et al. (1995) found that immature MM cells ( ~ ~ 4 5 3 proliferated in response to
IL-6 in vitro while mature MM cells (CD453 had no response to IL-6. As well malignant
cells are normally only found to be circulating in the terminal phases of the disease6; l 8
which is when myeloma cells are likeiy no longer dependent on IL-6. This suggests that
genetic alterations that cause ILdindependence may be the events that trigger the
maturation of myeloma cells or result in a more aggressive form of rnyeloma.
Overexpression of wild-type FGFR3 and TD mutant FGFR3 in myeloma would result in
loss of reliance on IL-6, and in the case of mutant FGFR3 wouid decrease the reliance on
any ligand, enabling these cells to more effectively disseminate throughout the body since
they no longer require ligand stimulation to proliferate. However, there is currently no
information about the circulating plasma cells in patients habouring a t(4: 14)
translocation.
An interesting feature of FGFR3 translocation in MM is the presence of mutations
within the translocated gene that appear to occur independently of the translocation6'.
There are other cases of lymphoid neoplasms where mutations fiequently arise
independent of translocations such as c-myc in Burkitt lymphoma359 and the bcl-6 gene in
Non-Hodgkin's iymphoma360. Both of these mutations tend to mainly aEect the
regdatory sequences of these genes unlike the mutations identified in FGFR3 that occur
within the coding sequence. Analysis of the bcl-6 and the c-mye mutations suggest that
like FGFR3 they are somatic mutations 359; 360 . For c-myc alterations in Burkitt
lyrnphoma it is thought that the alterations of the noncoding region may be the primary
cause of deregulated gene expression35g, however, for FGFIU it is postulated that the
placement next to the IgH enhancers causes the deregulated gene expressions8. Even
though a few other cases do exist it is uncornmon for the same gene to undergo multiple
alterations within a single cell. This apparent selection for activating mutations suggests
that mutations of FGFR3 likely contribute significady to the growth a d o r survival of
MM. This is consistent with the findings of this study since it is apparent that
constitutiveiy active FGFR3 induces a more pronounced growth and survival effect on
myeloma cells in the absence of Ligand than wild-type FGFR3 activated by ligand
perhaps providing the selection pressure for activating mutations of FGFR3.
In conclusion, FGFR3 overexpressing myeloma cells proliferate in the absence of
IL-6, exhibit an enhanced proliferative response to iL-6 and exhibit decreased apoptosis
upon IL-6 withdrawal. It appears that the survival of these cells in the absence of IL-6
might be mediated b y up-regdation of bcl-xL. The mechanism of enhanced pro liferation
is not yet elucidated but appears not to be mediated by ERK or SAPK.
It is specuiated that the initial translocation of FGFR3 into the immunoglobulin
heavy chah swïtch region in a germinal center B ceii likely provides the af5ected B ce11
with enhanced growth and swiva l potential with signaling potentially occurring through
the IL-6 receptor and FGFR3. Further activathg mutations of FGFR3 would enable
these cells to escape entirely Erom the need for ligand stimulation and would result in
M e r enhancement of proliferation providing a cornpetitive advantage over other B
cells. These findings provide a plausible, albeit partial, explanation for the myeloma
promoting properties of FGFR3.
4.2 Future Investigations
It rernains to be M e r elucidated which signalhg pathways are involved in the
enhanced proliferative response of FGFR3 expressing cells to IL-6. The pathways
activated by FGFR3 have not k e n extensively studied to date and moa work suggests
that FGFR3 activates STATl 116; 118 or ERKs 113; 1 18 . There are other signaling pathways
that need to aiso be examined. It wodd be interesting to M e r identify the role of p38
in ligand stirnulated ceîis expressing FGFR3 since it is possible that this stress-activated
pathway is causing cellular proliferation. The p38 kinase pathway couid be inhibited by a
specific inhibitor and then the proliferation of these cells could be measured by 3~
thymidine. If p38 is involved in the proliferation of these cells it would be expected that
when the pathway is inhibited the amount of proliferation observed wodd decline.
The involvement of Jaks was not examined in this study. It is possible that Jaks
mediate the binding of STAT3 to FGFR3. However, it has been suggested that since
tyrosine h a s e recepton have intrinsic kinase activity they may not require Jaks to
phosphorylate STATS)~'. It would be possible to analyze Jak kinase activation following
stimulation with aFGF to determine whether they might be involved in downstream
signaling pathways. If Jaks aie actived than this pathway could be inhibited by a Jak
inhibitor, tyrophostin AG490, and then STAT3 phosphorylation codd be examined in the
cells.
This study also suggests the involvement of STAT3 in up-regulating bcl-xL but
M e r experimentation couid more dennitively define the role of STAT3 with respect to
bcl-xL up-regulation. Electrophoretic gel mobility shift assays could be carried out to
determine whether phosphorylated STAT3 does translocate to the nucleus. As well it
would be possible to transfect the cells with a dominant negative STAT3 and then
examine whether bcCxL remains elevated after cytokine withdrawal.
Since this work was undertaken it was subsequently described that the
translocation of FGFR3 also results in the dysreguiation of another gene, M M S E ~ ' . The
potentid consequences of overexpression of both of these proteins in MM needs to be
examined. This could be studied by transfecthg MMSET into clones expressing wild-
type FGFR3 and TDII mutant FGFR3 that were generated in this expriment. It would
also be interesting to generate some ceiis expressing MMSET alone to examine its
potential effects in MM.
It has been suggested that the array of different chromosomal partners uivolved in
IgH translocations in MM may provide a basis for sub-dividing patients and then
targeting therapies based upon the translocation involveds8. Io the case of FGFIU
translocations therapies may involve targeting bcl-xL. It has been suggested that cells
overexpressing anti-apoptosis genes may be more cesistant to cytotoxic agents3" and by
aitering the amount of bcl-xL expressed this may make the cells more susceptible to
chemotherapy. As well it may be possible to target the activated STAT3 by transfecting
cells with dominant-negative STAT3. This would prevent the binding of STAT3 to bcl-x
promoter and thus cause the cells to be more susceptible to cellular death.
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