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  • Melnick DeptRetreat2012Poster Conversion of EGFR Mutant ......of EGFR Mutant Lung Adenocarcinomas to Small Cell Lung Cancer Upon Treatment with Tyrosine Kinase Inhibitors Department
1
• Somatic mutations in the Epidermal Growth Factor Receptor (EGFR) are seen in approximately 15% of lung adenocar- cinomas • Mutant EGFR lung cancers are sensitive to tyrosine kinase inhibitor (TKI) treatment • Resistance emerges on average 10-14 months after initiation of TKI treatment • Phenotypic switch from lung adenocar- cinoma to Small Cell Lung Cancer (SCLC) is seen in 6% of acquired resist- ance cases 1 • EGFR mutant SCLC is sensitive to chemotherapies used to treat SCLC To understand the cellular and molecular mechanisms underlying the transforma- tion of mutant EGFR lung adenocarcino- mas to SCLC upon TKI treatment. • Nkx2-1 is a lineage-specific transcription factor involved in lung morphogenesis • Nkx2-1 expression is common in lung adenocarcinomas and SCLC • Mutant EGFR expression is targeted to Nkx2-1-expressing cells in the mouse lung • Expression of mutant EGFR is directed to a wide variety of cell types in the lung • Preliminary data indicate suboptimal reporter and rtTA activity from the Rosa-LSL-rtTA-eGFP reporter strain • Induction of mutant EGFR expres- sion is much lower than observed in other mutant EGFR inducible mouse models used in our lab CONCLUSIONS REFERENCES BACKGROUND OBJECTIVE EXPERIMENTAL STRATEGY 1. Oxnard GR, Arcila ME, Chmielecki J, Ladanyi M, et al. New strategies in overcoming ac- quired resistance to epidermal growth factor receptor tyrosine kinase inhibitors in lung can- cer. Clin Cancer Res 2011; 17:5530-37. 2. Tagne JB, Gupta S, Gower AC, Shen SS, et al. Genome-wide analyses of Nkx2-1 binding to transcriptional target genes uncover novel regulatory patterns conserved in lung devel- opment and tumors. PLoS ONE 2012; 7(1): e29907. 3. Politi K, Zakowski MF, Fan PD, Schonfeld EA, et al. Lung adenocarcinomas induced in mice by mutant EGF receptors found in human lung cancers respond to a tyrosine kinase inhibitor or to down-regulation of the recep- tors. Genes & Dev 2006; 20:1946-1510. 4. Belteki G, Haigh J, Kabacs N, Haigh K, et al. Conditional and inducible transgene expres- sion in mice through combinatorial use of Cre- mediated recombination and tetracycline in- duction. Nucleic Acids Res 2005; 33(5): e51. Understanding the Molecular and Cellular Mechanisms That Underlie the Conversion of EGFR Mutant Lung Adenocarcinomas to Small Cell Lung Cancer Upon Treatment with Tyrosine Kinase Inhibitors Department of Pathology and Yale Cancer Center, Yale School of Medicine, New Haven, CT Mary Ann C. Melnick, MS and Katerina Politi, Ph.D T790M & rare second site mutations (60%) Unknown Mechanism* (30%) SCLC transformation (6%) MET amplification (4%) FUTURE PLANS • Confirm correct cre-mediated recom- bination by crossing the Nkx2-1-cre mouse to the Rosa-loxP-tdTomato- loxP-eGFP cre-reporter mouse • Use an alternate rtTA reporter strain (CAGs-LSL-rtTA3IRESKate) to induce mutant EGFR expression 0 20 40 60 80 100 120 140 160 180 200 1519 1716 1234 1949 1950 1957 1958 1961 1713 1715 1235 1953 1717 1959 TetO-EGFR L858R ;Nkx2-1-cre; Rosa-LSL-rtTA-eGFP(+/-) TetO-EGFR L858R ; Rosa-LSL-rtTA-eGFP(+/-) Nkx2-1-cre; Rosa-LSL-rtTA-eGFP(+/-) 3 months 4 months 7 months human EGFR expression (relative quantification) Mutant EGFR is expressed at variable levels in TetO-EGFR L858R ; Nkx2-1-cre;Rosa-LSL-rtTA-eGFP lung tissues Time on dox EGFR actin Mutant EGFR is detected in lung samples of TetO-EGFR L858R + animals time on dox (months) 3 4 7 3 4 7 3 7 3 genotype TetO-EGFR L858R ;Nkx2-1-cre; Rosa-LSL-rtTA-eGFP(+/-) TetO-EGFR L858R ; Rosa-LSL-rtTA-eGFP(+/-) Nkx2-1-cre; Rosa-LSL-rtTA-eGFP(+/-) TetO-EGFR L858R ; CCSP-rtTA negative control RESULTS FUNDING Translational-Targeted Area of Research Excellence TetO-mutEGFR;Nkx2-1-cre; Rosa-LSL-rtTA-eGFP Mutant EGFR expressed in Nkx2-1-expressing cells when mice are given doxycycline *mutEGFR = mutant EGFR Nkx2-1 is expressed in most epithelial cells of developing lung buds of TetO-EGFR L858R ;Nkx2-1-cre; Rosa-LSL-rtTA-eGFP(+/-) embryos (E11.5) IHC: Nkx2-1 Cre expression is found only in Nkx2-1-cre+ mouse tissues that express Nkx2-1 TetO-EGFR L858R ;Nkx2-1-cre; Rosa-LSL-rtTA-eGFP(+/-) Nkx2-1-cre; Rosa-LSL-rtTA-eGFP(+/-) TetO-EGFR L858R ; Rosa-LSL-rtTA-eGFP(+/-) L H Li SpK Bl I Br T 1235 1715 L H Li K Sp Br 1713 L H Li K Sp Br 1717 L Li K Sp Br Genotype L H Li Sp K Bl I Br T 1234 L H Li K Sp Br 1519 L H Li K Sp Br 1716 Cre actin Cre actin Cre actin Mutant EGFR is expressed in lung tissue of TetO-EGFR L858R ; Nkx2-1-cre;Rosa-LSL-rtTA-eGFP(+/-) mice 4 months on dox 7 months on dox IHC: L858R TetO-EGFR L858R ;Nkx2-1-cre; Rosa-LSL-rtTA-eGFP(+/-) TetO-EGFR L858R ; Rosa-LSL-rtTA-eGFP(+/-) GFP-reporter expression is detectable at low levels in lungs of animals upon cre-mediated recombination *includes epithelial-mesenchymal transition (present at an uncertain prevalence) Frequency of acquired resistance mechanisms to EGFR TKIs TetO-mutEGFR* 3 ;Nkx2-1-cre 2 Rosa-LSL-rtTA-eGFP 4

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Page 1: Melnick DeptRetreat2012Poster Conversion of EGFR Mutant ......of EGFR Mutant Lung Adenocarcinomas to Small Cell Lung Cancer Upon Treatment with Tyrosine Kinase Inhibitors Department

•  Somatic mutations in the Epidermal Growth Factor Receptor (EGFR) are seen in approximately 15% of lung adenocar-cinomas

•  Mutant EGFR lung cancers are sensitive to tyrosine kinase inhibitor (TKI) treatment

•  Resistance emerges on average 10-14 months after initiation of TKI treatment

•  Phenotypic switch from lung adenocar-cinoma to Small Cell Lung Cancer (SCLC) is seen in 6% of acquired resist-ance cases1

•  EGFR mutant SCLC is sensitive to chemotherapies used to treat SCLC

To understand the cellular and molecular mechanisms underlying the transforma-tion of mutant EGFR lung adenocarcino-mas to SCLC upon TKI treatment.

•  Nkx2-1 is a lineage-specific transcription factor involved in lung morphogenesis

•  Nkx2-1 expression is common in lung adenocarcinomas and SCLC

•  Mutant EGFR expression is targeted to Nkx2-1-expressing cells in the mouse lung

•  Expression of mutant EGFR is directed to a wide variety of cell types in the lung

•  Preliminary data indicate suboptimal reporter and rtTA activity from the Rosa-LSL-rtTA-eGFP reporter strain

•  Induction of mutant EGFR expres-sion is much lower than observed in other mutant EGFR inducible mouse models used in our lab

CONCLUSIONS

REFERENCES

BACKGROUND

OBJECTIVE

EXPERIMENTAL STRATEGY

1.  Oxnard GR, Arcila ME, Chmielecki J, Ladanyi M, et al. New strategies in overcoming ac-quired resistance to epidermal growth factor receptor tyrosine kinase inhibitors in lung can-cer. Clin Cancer Res 2011; 17:5530-37.

2.  Tagne JB, Gupta S, Gower AC, Shen SS, et al. Genome-wide analyses of Nkx2-1 binding to transcriptional target genes uncover novel regulatory patterns conserved in lung devel-opment and tumors. PLoS ONE 2012; 7(1): e29907.

3.  Politi K, Zakowski MF, Fan PD, Schonfeld EA, et al. Lung adenocarcinomas induced in mice by mutant EGF receptors found in human lung cancers respond to a tyrosine kinase inhibitor or to down-regulation of the recep-tors. Genes & Dev 2006; 20:1946-1510.

4.  Belteki G, Haigh J, Kabacs N, Haigh K, et al. Conditional and inducible transgene expres-sion in mice through combinatorial use of Cre-mediated recombination and tetracycline in-duction. Nucleic Acids Res 2005; 33(5): e51.

Understanding the Molecular and Cellular Mechanisms That Underlie the Conversion of EGFR Mutant Lung Adenocarcinomas to Small Cell Lung Cancer Upon Treatment

with Tyrosine Kinase Inhibitors

Department of Pathology and Yale Cancer Center, Yale School of Medicine, New Haven, CT Mary Ann C. Melnick, MS and Katerina Politi, Ph.D

T790M & rare second site mutations

(60%)

Unknown Mechanism*

(30%)

SCLC transformation (6%)

MET amplification (4%)

FUTURE PLANS

•  Confirm correct cre-mediated recom-bination by crossing the Nkx2-1-cre mouse to the Rosa-loxP-tdTomato-loxP-eGFP cre-reporter mouse

•  Use an alternate rtTA reporter strain (CAGs-LSL-rtTA3IRESKate) to induce mutant EGFR expression

0 20 40 60 80

100 120 140 160 180 200

1519 1716 1234 1949 1950 1957 1958 1961 1713 1715 1235 1953 1717 1959

TetO-EGFRL858R;Nkx2-1-cre;

Rosa-LSL-rtTA-eGFP(+/-) TetO-EGFRL858

R;

Rosa-LSL-rtTA-eGFP(+/-)

Nkx2-1-cre

;

Rosa-LSL-rtTA-eGFP(+/-)

K  

3 months 4 months 7 months

hum

an E

GFR

exp

ress

ion

(rel

ativ

e qu

antif

icat

ion)

Mutant EGFR is expressed at variable levels in TetO-EGFRL858R; Nkx2-1-cre;Rosa-LSL-rtTA-eGFP lung tissues

Time on dox

EGFR

actin

Mutant EGFR is detected in lung samples of TetO-EGFRL858R+ animals

time on dox (months)

3 4 7 3 4 7 3 7 3

genotype Tet

O-EGFRL85

8R ;Nkx2-1-

cre;

Rosa-LS

L-rtTA

-eGFP

(+/-)

TetO-EGFR

L858R ;

Rosa-LS

L-rtTA

-eGFP

(+/-)

Nkx2-1-

cre;

Rosa-LS

L-rtTA

-eGFP

(+/-)

TetO-EGFR

L858R ;

CCSP-rtTA

negative control

RESULTS

FUNDING

Translational-Targeted Area of Research Excellence

TetO-mutEGFR;Nkx2-1-cre; Rosa-LSL-rtTA-eGFP

Mutant EGFR expressed in Nkx2-1-expressing cells when mice are given doxycycline

*mutEGFR = mutant EGFR

Nkx2-1 is expressed in most epithelial cells of developing lung buds of TetO-EGFRL858R;Nkx2-1-cre; Rosa-LSL-rtTA-eGFP(+/-) embryos (E11.5)

IHC: Nkx2-1

Cre expression is found only in Nkx2-1-cre+ mouse tissues that express Nkx2-1

TetO-EGFRL858R;Nkx2-1-cre; Rosa-LSL-rtTA-eGFP(+/-)

Nkx2-1-cre; Rosa-LSL-rtTA-eGFP(+/-)

TetO-EGFRL858R; Rosa-LSL-rtTA-eGFP(+/-) L H Li SpK Bl I Br T

1235 1715

L H Li K Sp Br

1713

L H Li K Sp Br

1717

L Li K Sp Br

Genotype

L H Li Sp K Bl I Br T

1234

L H Li K Sp Br

1519

L H Li K Sp Br

1716

Cre

actin

Cre

actin

Cre

actin

Mutant EGFR is expressed in lung tissue of TetO-EGFRL858R; Nkx2-1-cre;Rosa-LSL-rtTA-eGFP(+/-) mice

4 months on dox

7 months on dox

IHC: L858R

TetO-EGFRL858R;Nkx2-1-cre; Rosa-LSL-rtTA-eGFP(+/-)

TetO-EGFRL858R; Rosa-LSL-rtTA-eGFP(+/-)

GFP-reporter expression is detectable at low levels in lungs of animals upon cre-mediated recombination

*includes epithelial-mesenchymal transition (present at an uncertain prevalence)

Frequency of acquired resistance mechanisms to EGFR TKIs

TetO-mutEGFR*3;Nkx2-1-cre2

Rosa-LSL-rtTA-eGFP4

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