ack1 tyrosine kinases: a critical regulator of prostate · pdf filelearners objectives ......
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Learners Objectives
� How Androgen Receptor (AR) signaling is accomplished in absence of androgen
� What are the epigenetic events that takes place to achieve androgen-independent AR signaling
� What are the new therapeutic options being developed to overcome enzalutamide-resistant prostate cancer
Prostate Cancer
� Prostate cancer is second most common cancer in American men
� Almost 225,000 men are diagnosed and ~25,000 die due to prostate cancer in US every year
� Androgen receptor (AR) is a key regulator of both initiation and progression of prostate cancer to metastatic stage
Androgen Receptor Signaling
AR
TATA ARE
PSA/hK2/TMPRSS2
+DHT
Nuclear Translocation
AR Coactivator RNA Pol
AR D
AR D
Cell survival and growth
Transcription
Castration Resistant Prostate Cancer
AR + Androgen
Androgen dependent or Hormone-sensitive
Tumor Growth
18-24 M
AR + Androgen
Tumor Growth
Castration Resistant Prostate Cancer
CRPC
Second generation of anti-androgen
� Enzalutamide (Xtandi), is a new type of anti-androgen. It prevents AR entry into the nucleus, lowers PSA levels, slow the growth of tumors
� It is effective in a subset of CRPC patients (1/3rd ) and survival advantage is modest (4-6 months).
� Even the most responding patients relapsed within 2 years, often with lethal consequences
Tran et al., Science, 2009
Mechanism of Enzalutamide-resistance � AR-V7 splice variant expression has emerged to be the major cause
of Enzalutamide and Abiraterone resistance
� AR-V7 lacks the C terminal ligand-binding domain (& Enzalutamide binding) and remains constitutively active as a transcription factor.
Antonarakis et al., NEJM, 2014
CRPC: AR dependence
� Overall, the most common genetic aberration in CRPC is AR mRNA upregulation, also reflected in AR-V7 increase in Enzalutamide resistant
� These data have opened a new paradigm:
� To achieve complete remission of CRPC, ablation of AR is the key.
� However, targeted inhibition of AR transcription with small molecule inhibitors has not yet been accomplished.
ACK1 or TNK2
� Ubiquitously expressed non-receptor tyrosine kinase
� Plays a central role in integrating signal from receptor tyrosine kinases e.g. EGFR, HER2 and IR
� ACK1 binds to AR and ACK1/AR complex translocate to nucleus activating transcription of AR target genes
SAM Kinase SH3 C CL MHR UB
Novel ACK1 inhibitor, (R)-9bMS
� We identified (R)-9bMS as a potent ACK1 inhibitor
� In vitro IC50=48 nM � Mesylate salt is highly
soluble in aqueous media
� (R)-9bMS exhibit significant selectivity
� Inhibited 11 kinase out of 369 kinase (90% inhibition)
� Except ACK1 others not known to express in prostate
Lawrence. et al., J Med Chem 2015
AR
pACK1
DM
SO(R
)-9bM
S
DH
T
Enza
luta
mid
e
1.0 0.1 2.9 3.0
Actin
siRNA
Con
trol
ACK1
VCaP
1.0 0.3 2.0 0.37
DM
SO
(R)-9bM
S
MG
132
MG
132+
(R)-9bM
S
AR
Actin
AR
Actin
LAPC4
.ACK1
1.0 0.30
Con
trol
ACK1
C4-2B
1.0 0.34
AR
DM
SO
(R)-9bM
S
DH
TEn
zalu
tam
ide
1.0 0.13 0.65 1.9
Actin
C4-2B
pACK1
(R)-9b suppresses AR expression
(R)-9b inhibits AR transcription
0.00.20.40.60.81.01.21.41.6
(R)-9b
DM
SO
DH
TEn
zalu
t
(R)-9b
0.0
0.5
1.0
1.5
2.0
2.5(R
)-9b
DM
SO DH
TEn
zalu
t
(R)-9b
VCaPAR
/Act
in R
atio
0.0
0.5
1.0
1.5
2.0
* **D
MSO DH
TEn
zalu
t
(R)-9b
(R)-9b
0.0
0.4
0.8
1.2
1.6
DM
SO DH
TEn
zalu
t
(R)-9b
(R)-9b
* **
PSA
/Act
in R
atio
DM
SO DH
TEn
zalu
t
(R)-9b
(R)-9b
LNCaP
0
1
2
3
4
DM
SO DH
TEn
zalu
t
(R)-9b
(R)-9b
LAPC4
0.0
0.51.0
1.5
2.02.5
3.0
3.5
* *** **
* **
0
1
1.5
2
DM
SO(R
)-9b
C4-2B
*
0
1
1.5
2
DM
SO(R
)-9b
*2.5
2.5
*
*****
1.8VCaP LNCaP LAPC4 C4-2B
0.00.2
0.40.6
0.8
1.0
1.2D
MSO
(R)-9b
AR-V7/
Act
in R
atio
AR-V7
AR
Actin
VCaP
AR-V
DM
SO
(R)-9b
.
Actin
LAPC4
AR-V
DM
SO(R
)-9b
Enza
luta
mid
e
DH
T
*
(R)-9b suppresses AR-V expression
TATA
AR Coactivator
Transcription Androgen
AR
ACK1
• ACK1 `piggybacks’ AR to nucleus • DNA is not exposed, but bound to histones to form chromatin
• Does ACK1 phosphorylate histone to activate AR transcription?
P
P
ACK1 translocates to nucleus
Histone H4 phosphorylation at Tyr88
Antibody generation Mass Spectrometry
pY88-H4
H4
pY88-H4 Ab
pY88-H4 Ab+phosphopeptide
Biotin
100 400 700 1000 1300 1600m/z0
50
100
Intensity
b1
558.8 559.5 560.2m/z
[M+3H]3+, 558.9605
MS1
y1
y2 y4
y5
b7
y6y7
KT V T A M D V V Y AL KRMS/MS
pY Immonium Ion
CRPC Sample #1
ACK1 phosphorylates H4 at Tyr88
Endogenous H4 Y88-phosphorylation
+ + + H4
pY88-H4
+ + ACK1 _ _ + (R)-9bMS
ACK1
H4
_
Inpu
t
+ + _ + _ _
LAPC4(PDGF)
pY88-H4
+ + Ligand
pACK1
H4
_ + (R)-9bMS _ _
LNCaP(IGF)
pY88-H4
H4
ACK1
Y88F
Y72F
Vec
+ ACK1
Myc
Inpu
t
Inpu
t
In vitro Kinase assay
+ + + H4
pY88-H4
+ + ACK1 _ _ + (R)-9bMS
ACK1
H4
_
Inpu
t
+ + _ + _ _
LAPC4(PDGF)
pY88-H4
+ + Ligand
pACK1
H4
_ + (R)-9bMS _ _
LNCaP(IGF)
pY88-H4
H4
ACK1
Y88F
Y72F
Vec
+ ACK1
Myc
Inpu
t
Inpu
t
+ IGF
DM
SO
DH
T
(R)-9b
MS
Enz
alut
C4-2B
pY88-H4
H4
pY-ACK1
Input
H4 Y88-phosphorylation is insensitive to Enzalutamide and DHT
pY88-H4 marks deposited upstream of AR
Exon 1 2 3 4 5 6 7 866,7
63,8
74
66,6
69,6
00
66,6
49,5
50
66,6
31,3
00
AREM1 AREM2 AREM3
ChIP:pY88ChIP:IgG
nt position
Peak regions
Primers 4 137
AREM 3 2 1
(R)-9b compromises pY88-H4 deposition
ChIP: pY88-H4 IgG
AREM1
Per
cent
ofIn
put
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6
DMSO(R)-9b
AREM2
0.0
0.5
1.0
1.5
2.0
2.5AREM3
DMSO(R)-9b
ChIP: pY88-H4 IgG ChIP: pY88-H4 IgG
* *
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
DMSO(R)-9b
*** *
Control Primer
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6
DMSO(R)-9b
ChIP: pY88-H4 IgG
WDR5-MLL2 complex is an epigenetic reader of pY88-H4 marks
� pY88-H4 peptide used as a bait to identify interacting proteins:
Ø MLL2 (KMT2D)- H3K4 lysine methyltransferase, modifies histones to activate transcription
Ø WDR5- WD repeat domain 5
WDR5 `reads’ pY88-H4 marks, MLL2 acts as a `scribe’ to regulate deposition of H3K4me3 activating marks
ChIP: H3K4me3ChIP: MLL2
Per
cent
ofIn
put
0.0
0.2
0.4
0.6
0.8
1.0
1.2
DMSO(R)-9b
0.0
0.2
0.4
0.6
0.8
1.0DMSO(R)-9b
AREM1 ControlAREM1 Control
ChIP: Pol II
0.0
0.2
0.4
0.6
0.8
1.0DMSO(R)-9b
AREM1 Control
** * *
0
1
2
3
4
DMSO(R)-9b
ChIP: WDR5
AREM1 Control
**
Per
cent
ofIn
put
Per
cent
ofIn
put
Per
cent
ofIn
put
(R)-9bMS overcomes Enzalutamide Resistance
LNCaP-C4-2B 0.40 uM VCaP 0.45 uM LAPC4 0.75 uM LNCaP 1.75 uM RWPE 10.0 uM
(R)-9bMS Inhibits CRPC Growth
n=9
n=9
Days post injection
Vehicle(R)-9bMS
0 10 20 30
Tum
orVo
lum
e(m
m3)
0
200
400
600
800
1000
1200
C4-2B XenograftTumorss0.0
0.2
0.4
0.6
0.8
1.0DMSO(R)-9b
n=6
n=3
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4ARDMSO(R)-9b
PSA
n=6
n=3
0.0
0.5
1.0
1.5
2.0
2.5
3.0TMPRSS2DMSO(R)-9b
n=3n=6
** ** **
ACK1 AR MLL2 WDR5
P P
AREMs 1-3 AR Transcription
ACK1 Inhibitor
Mahajan K. et al., Cancer Cell, 2017
M
Epigenetic dynamics at AR locus
9b
Conclusion
� We uncovered a new signaling nexus - ACK1 mediated H4-Y88-phosphorylation that maintains AR transcript levels in absence of androgen
� H4-Y88-phosphorylation can be suppressed by small molecule inhibitor (R)-9bMS, compromising AR and AR-V7 transcription
� ACK1 inhibitor sensitizes Enzalutamide-resistant CRPC cells, opening new treatment option for patients that are:
Ø (i) not-responsive to this second generation of antiandrogen Ø (ii) those CRPC tumors that have developed resistance
� Mahajan K, Malla P, Lawrence H, Chen Z, Sinha CK, Malik R, Shukla S, Kim J, Coppola D, Lawrence N and Mahajan NP*.
ACK1 regulates histone H4 Tyr88-phosphorylation and AR gene expression in castration resistant prostate cancer. Cancer Cell, 31:790-803, 2017.
v Mahajan K and Mahajan NP*. ACK1/TNK2 tyrosine kinase: molecular signaling and evolving role in cancer. Oncogene, 34:4162-7, 2015
v Lawrence HR, Mahajan K, Liu Y, Zhang D, Tindal N, Huseyin M,
Gevariya H, Kazi S, Ozcan S, Mahajan NP*, Lawrence NJ*. Development of novel ACK1 inhibitors using a fragment based approach. J. Med. Chem., 58:2746-63, 2015.
References
v Kiran Mahajan v Nick Lawrence v Harshani Lawrence v Domenico Coppola v Pavani Malla v Chandan Sinha, Michigan
Acknowledgements
� NIH/NCI (R01 CA208258) � Bankhead Coley Award (6BC08) � Department of Defense Breakthrough Award
(W81XWH-15-1-0312) � Celgene award (02-20000-01-01) � Prostate Cancer Foundation, Challenge Award (17CHAL06)
Current Funding