Heterogeneity among early-stage K-Ras driven lung adenocarcinoma predicts tumour aggressiveness and identifies novel therapeutic targets

Transcription

1 Heterogeneity among early-stage K-Ras driven lung adenocarcinoma predicts tumour aggressiveness and identifies novel therapeutic targets Canceropole

2 Introduction: Ras and human cancer Ras Mutations in Human Tumors (COSMIC, 2013) Tissue/Organ K-Ras N-Ras H-Ras TOTAL: Samples tested 130,500 56,300 33,500 TOTAL: Percentage 22% 6% 3% Pancreas 58% 1% 0% Intestine/Colon 28% 2% <1% Billiary tract 26% 2% 1% Lung 23% 1% 1% Ovary 12% 1% 0% Endometrium 15% 3% <1% All Others 0-7% 0-15% 0-12%

3 Introduction: lung cancer mutations NO TARGETED THERAPY Crizotinib Erlotinib Rudin, C, et al. (2012) Sun, JM, et al. (2013)

4 Introduction: Ras activation Mitogens RTK PIP2 PTEN PIP3 GRB2 SOS1/2 GE F PI3K PDK1 RHO RAC p190 TIAM RAL-GDS Ras-GTP RAF GAP Ras-GDP cell motility AKT translation survival EXOCYST membrane dynamics, vesicle formation PLD RAL MEK ERK cell cycle progression

5 Targeting MAPK signalling RTK Mitogens K-Ras H-Ras N-Ras RAF MEK Cell cycle progression ERK

6 Targeting MAPK signalling Small Intestine Colon Survival(%) P30 mice RTK Mitogens Mek1 D/D ;Mek2 +/ Mek1 D/D ;Mek2 / OHT Diet (days) K-Ras H-Ras N-Ras Mek1 +/+ ;Mek2 +/+ Mek1 D/D ;Mek2 / A-RAF B-RAF C-RAF MEK1 MEK2 ERK1 ERK2

7 Targeting MAPK signalling RTK Mitogens K-Ras G12V A-RAF B-RAF C-RAF MEK1 MEK2? ERK1 ERK2

8 Targeting MAPK signalling RTK Mitogens K-Ras G12V A-RAF B-RAF C-RAF MEK1 MEK2? ERK1 ERK2

9 Targeting MAPK signalling RTK Mitogens K-Ras G12V A-RAF B-RAF C-RAF MEK1 MEK2? ERK1 ERK2

10 Targeting the cell cycle STOP ATG V12 * TGA IRES LacZ-neo Cre + 4OHT ATG V12 * TGA IRES LacZ-neo + 4OHT Ther. target

11 Target validation: Raf kinases K-Ras G12V Tumor Burden at 6 months of age A-Raf B-Raf c-raf C-Raf +/+ C-Raf, but not B-Raf or A-Raf, is essential for initiation of K-Ras G12V Mek1/2 C-Raf lox/lox oncogene-driven lung adenocarcinomas, yet dispensable for adult homeostasis 100 C-Raf lox/lox Survival (%) C-Raf +/+ C-Raf lox/lox C-Raf C-Raf lox C-Raf 20 Gapdh Age (weeks)

12 MAPK: Therapeutic approach Flp FSF ATG V12 * TGA RNAPol II CreERT pa 3 UTR

13 MAPK: Therapeutic approach ATG V12 * TGA RNAPol II CreERT pa 3 UTR CreERT 4-OHT CreERT 4-OHT Ther. target

14 MAPK: Therapeutic approach A Genetic Approach to Target Validation: Tumor Progression K-Ras +/FSFG12V Ad-Flpe infection NR K-Ras G12V +4OHT (active Cre) K-Ras +/FSFG12V ; Raf lox/lox PolII CreERT2 PET/CT+ tumors Systemic target ablation PR Raf lox/lox PolII CreERT2 CR

15 Fold change of SUV Max Fold change of the number of tumors uptaking FDG Fold change of tumor volume Fold change of SUV Max MAPK: Therapeutic approach A Genetic Approach to Target Validation: Tumor Progression TMX treatment (Months) 0 2 B-Raf +/+ ; C-Raf +/+ H H 2 1,5 B-Raf +/+ C-Raf +/+ B-Raf +/+ ; C-Raf lox/lox H H 1 0,5 B-Raf +/+ C-Raf lox/lox B-Raf lox/lox ; C-Raf lox/lox H H TMX treatment (months) p<0,0001 p<0, p=0.002 p= p<0,0001 p<0, B-Raf: C-Raf: 0 +/+ +/+ +/+ lox/lox lox/lox lox/lox +/+ +/+ +/+ lox/lox lox/lox lox/lox 0 +/+ +/+ +/+ lox/lox lox/lox lox/lox

16 Anti-pCofilin Anti-Active Caspase-3 Anti-Ki67 MAPK: Therapeutic approach A Genetic Approach to Target Validation: Tumor Progression Apoptosis Differentiation B-Raf +/+ ;C-Raf +/+ B-Raf +/+ ;C-Raf lox/lox B-Raf lox/lox ;C-Raf lox/lox Ki67 + cells (%) p< p< K-Ras G12V 10 0 C-RAF B-Raf +/+ ;C-Raf +/+ B-Raf +/+ ;C-Raf lox/lox B-Raf lox/lox ;C-Raf lox/lox 1.5 Active Casp 3 + cells (%) p< p= MEK1 MEK ERK1 ERK2 B-Raf +/+ ;C-Raf +/+ B-Raf +/+ ;C-Raf lox/lox B-Raf lox/lox ;C-Raf lox/lox 80 pcofilin + cells (%) p< Proliferation p<0.0001

17 MAPK: Genetic modelling of kinase inhibition K (ATP Binding Site) P P D (DFG): proton acceptor P Mg 2 + D (HRD) KD loxp loxp a b SA cdna pa (ex c-x) STOP c d e f x loxp KD c-raf D468A Cre Recombinase

18 MAPK: Genetic modelling of kinase inhibition Ad-Flpe infection K-Ras +/FSFG12V C-Raf LmLD468A CreERT2 K-Ras G12V PET/CT+ tumors +4OHT (active Cre) Systemic C-RafD468A induction?

19 SUV Max (Fold change) Tumor Volume (Fold change) MAPK: Genetic modelling of kinase inhibition 8 weeks Tamoxifen diet * * C-Raf: +/+ lml/lml C-Raf: +/+ lml/lml C-Raf LmLD468A/LmLD468A ;Tg.hUBC-CreERT2 +/T show no phenotype after 6 months of Tamoxifen diet

20 Conclussions (I) Overall MAPK signalling is essential for mouse homeostasis and individual mediators are functionally redundant; their function is specialized in K-Ras G12V -driven NSCLC. C-Raf is a valuable therapeutic target for K-Ras G12V -driven NSCLC treatment with potentially low systemic toxicity. Inhibition of C-Raf catallytic activity induces regression of advanced K-Ras G12V -driven NSCLC.

21 Combination therapy: choosing novel targets Hanahan & Weinberg, Cell 144 (2011)

22 Combination therapy: choosing novel targets

23 Combination therapy: choosing novel targets EARLY LESIONS ADVANCED TUMORS Study EARLY lesions to: identify the nature of K-Ras-dependent essential mediators in NSCLC discover new druggable targets for K-Ras-driven NSCLC treatment

24 PC2 PC Early stages in NSCLC progression K-Ras G12V early NSCLC lesions N T T 1 T 2 Ddr1 NFR Pre-LCM Post-LCM (BIDMC, Boston) T2 T2 T2 T2 N N T1 T1 N N T1 N N N N p<0, PC1 FDR<0.05 Log 2 signal -3 3 FDR<0.15 Log 2 signal -3 3

25 Ddr1 as a novel therapeutic target DDR1 is up-regulated in NSCLC compared to normal lung tissue, and in metastasis compared to primary tumors Imatinib DDR1 IS DRUGGABLE Desatinib DDR1 is the most abundantly phosphorylated protein in NSCLC Nilotinib Miao, L et al. (2013) Bafetinib DDR1 high expression correlates with poor survival in NSCLC Specific DDR1 inhibitors Rikova, K et al. (2007) Valencia, K et al. (2012)

26 IHC: TTF1 Ddr1 as a novel therapeutic target: genetic validation Survival (%) Absolute tumor number/lung Ddr1 +/ + Ddr1 +/ Ddr1 / x K-Ras +/LSLG12V Ddr1 / 20 p< Months K-Ras G12V ;Ddr1 +/+ K-Ras G12V ;Ddr1 / 40 * Ddr1 +/+ Ddr1 +/ Ddr1 /

27 Ddr1 as a novel therapeutic target: pharmacologic validation VEHICLE TREATED* Months 0 2 = = = * 7rh inhibitor treatment, 50mg/kg, daily oral administration

28 IHC: Ddr1 IHC: KI67 IHC: C3A Ddr1 as a novel therapeutic target: pharmacologic validation DRUG-INSENSITIVE TUMORS DRUG-SENSITIVE TUMORS

29 Ddr1 as a novel therapeutic target: in search of a mechanism Collagen Adapted from Borza et al, Matrix Biology (2013)

30 Ddr1 as a novel therapeutic target: in search of a mechanism IHC: C3A % C3A positive cells/tumor VEHICLE CHEMO 2-weeks treatment 7rh 7rh+ CHEMO **

31 Ddr1 as a novel therapeutic target: in search of a mechanism Collagen

32 IHC: Hes1 Ddr1 as a novel therapeutic target: in search of a mechanism CHEMO + 7rh (2 weeks) 7rh (2 months) 7rh (2 weeks) VEHICLE CHEMO (2 weeks) % Hes1 positive cells/tumor TUMOR K-Ras G12V ;Ddr1 +/+ BRONCHIOLES *** ***

33 Ddr1 as a novel therapeutic target: in search of a mechanism Collagen Adapted from Borza et al, Matrix Biology (2013)

34 Ddr1 as a novel therapeutic target: in search of a mechanism CHEMO + LY rh+ LY rh+LY CHEMO VEHICLE LY IHC: C3A IHC: Hes1 Hes1 Gapdh

35 Ddr1 as a novel therapeutic target in aggressive NSCLC GENETIC DELETION OF Ddr1 Increase in overall survival Decrease in tumor number Decrease in tumor size Effect in tumor grading p53 +/+ p53 lox/lox PHARMACOLOGICAL INHIBITION OF Ddr1 Efficacy of 7rh as single agent Efficacy of 7rh in combination with chemotherapy Efficacy of 7rh in combination with LY

36 % C3A positive cells/tumor Ddr1 as a novel therapeutic target in aggressive NSCLC CHEMO + LY rh+ LY rh+LY CHEMO VEHICLE LY ** * IHC: C3A IHC: Hes Hes1 Gapdh

37 Is this applicable to human NSCLC? IHC: HES1 IHC: DDR1 Human TMA IHC: DDR1 Gene K-RAS DDR1 HES1 K-RAS * 0.025* DDR1 HES ** n = 554 patients. *Tendency towards co-occurrence (2<Odds ratio<10) ** Strong tendency towards co-occurrence (Odds ratio>10)

38 Conclussions (II) Upon K-Ras G12V expression in lung, there are at least two different types of early lesions based on gene expression profiling (T1 and T2). T1 signature is closer to normal lung epithelial cells and could help to identify new tumor suppressor genes. T2 overlaps with advanced murine and human NSCLC signature, thus suggesting that the aggressiveness of lesions could be determined early during tumor onset. By an unbiased approach we identified Ddr1 as a valuable therapeutic target for K- Ras mutated NSCLC. Genetic ablation or pharmacological inhibition of Ddr1 impairs tumor growth in p53- proficient K-Ras G12V NSCLC. Combined inhibition of Ddr1 and Notch signaling efficiently triggers an apoptotic response even in p53-deficient K-Ras G12V NSCLC.

39 Take home message 1 There are >800 drugs targeting signaling pathways; finding the right drug (or combination of) for personalized treatment is challenging. Tumor evolution, heterogeneity and resistance. Kreso & Dick, Cell Stem Cell 2014 Swanton, Cancer Res 2012

40 Take home message 1 Cancer cell line encyclopedia K-RAS Human NSCLC

41 Take home message 1 Kreso & Dick, Cell Stem Cell 2014 Swanton, Cancer Res 2012

42 Take home message 2 DDR1/NOTCH inhibitors NO TARGETED THERAPY Crizotinib Erlotinib

43 Acknowledgements Experimental Oncology Group: Mariano Barbacid Chiara Ambrogio Rafael Blasco Emilie Bousquet Sarah Francoz Patricia Nieto Collaborators: Ke Ding (GIBG, Guangzhou) Pierre Dubus (Univ. Bordeaux) Montserrat Sánchez-Céspedes (Idibell, Barcelona) Manuel Serrano (CNIO) Gonzalo Gómez (CNIO)

44 Combination therapy: validating novel targets (II) N T T 1 T 2 T 2 T 1

45 Combination therapy: validating novel targets (II) CpG TSS Tumor #1 Tumor #2 T1 Tumor #3 Tumor #4 T2 Tumor #5 Tumor #6 IHC: SERPINB5

46 Combination therapy: validating novel targets (II) % Survival Placebo 5-Aza + Ent. 5-Aza Entinostat : reversible 5d 8w K-Ras G12V 3 m Placebo 5-Aza + Ent. Age (months)