Complexity of intra- and inter-pathway loops in colon cancer and melanoma: implications for targeted therapies

Transcription

1 Complexity of intra- and inter-pathway loops in colon cancer and melanoma: implications for targeted therapies René Bernards The Netherlands Cancer Institute Amsterdam The Netherlands

2 Molecular versus conventional diagnostics Histological analysis RNA: Gene expression analysis DNA: Mutation analysis

3 Colon cancer subtypes: identified through gene expression

4 Targeted agents: dramatic, but short-lived responses Lung cancer/gefitinib Melanoma /Vemurafenib Progression Free Survival Progression Free Survival Maemondo et al., N Engl J Med 2010 Chapman et al., N Engl J Med 2011

5 Endless two-way combinations of cancer drugs 1,000 x1, = 499,000 combinations Test each combination in 1,000 patients: 499,000,000 patients needed

6 Differential response of BRAF inhibition in BRAF mutant melanoma versus colon cancer 90% 80% 81% 70% 60% 50% 40% 30% 20% 10% 0% Melanoma 5,20% Colon cancer N Engl J Med : Kopetz et al., ASCO 2010

7 Is feedback regulation responsible for the resistance of CRC cells to BRAF inhibitors?

8 BRAF V600E mutant CRC cell lines are also less responsive to PLX4032 than melanomas having the same mutation Short-term cell viability assay Long-term colony formation assay CRC Melanoma

9 Synthetic lethal shrna screen: Inhibition of which kinase synergizes with PLX in BRAF mutant CRC? infect with shrna kinome library WiDr (BRAFV600E) culture to allow selection control vemurafenib PCR amplify bar codes Deep sequence Quantify shrnas

10 Inhibition of EGFR makes BRAF mutant CRC cells vulnerable to BRAF inhibition Cetuximab EGFR Grb RAS BRAF Mutant MEK ERK

11 Synergistic response of BRAF V600E colon cancer to EGFR and BRAF inhibition BRAF inhibitor EGFR inhibitor 1 BRAF mutant Colon cancer cells +EGFR inhibitor 2 Cetuximab 1.25 mg/ml Gefitinib mm

12 EGFR and BRAF inhibition synergize to suppress BRAF mutant colon cancer growth Human colon cancer growth in mice No drug EGFR inhibitor BRAF inhibitor BRAF+EGFR inhibitor Prahallad et al, Nature 2012

13 Timeline BRAF colon cancer project Publication March 2012 First clinical responses March Start funding research project January 2011 First patient in trial at NKI November

14 BRAF mutant CRC: Design clinical study Courtesy: Jan Schellens, Robin van Geel

15 Initial results from clinical studies in patients Courtesy: Jan Schellens, Robin van Geel

16 Combinations yield durable responses Courtesy: Jan Schellens, Robin van Geel

17 Is the combination of MEK and EGFR inhibitors also effective in KRAS mutant cells? Selumetinib (um) Selumetinib(μM) H358 H2030 H2122 NSCLC +Gefitinib (1uM) H23 CRC: SW620(KRAS G12V ) H747 SW480 SW620 CRC Selumetinib (um) SW837 Gefitinib (1uM) NSCLC: H358 (KRAS G12C )

18 Enrichment Enrichment 100 RNAi screen for enhancers of MEK inhibitors CRC-SW ,1 0, NSCLC-H358 sherbb ERBB3 activation is mostly dependent on heterodimerization ERBB3 binding partners 10 ERBB family 1 0,1 0,01 sherbb Intensity

19 Targeting EGFR and ERBB2 sensitizes KRAS mutant cells to MEK inhibitor Selumetinib (um) Gefitinib (0.5uM) +Pertuzumab (2.5ug/ml) +Afatinib (0.5uM) +Dacometinib (0.5uM) Gefitinib: EGFR inhibitor H358 (KRAS G12C ) Pertuzumab: ERBB2-targeting monoclonal antibody Also seen in H2030, H2122,SW837, SW480 and SW620 Afatinib: EGFR and ERBB2 inhibitor Dacometinib: EGFR, ERBB2 and ERBB4 inhibitor

20 % Change Tumor Volume % Change Tumor Volume 250% 343% 306% Targeting EGFR and ERBB2 sensitizes KRAS mutant cells to MEK inhibitor in a xenograft model VEHICLE AFATINIB VEHICLE AFATINIB TRAMETINIB 50 TRAMETINIB 100 TRA + AFA 0 TRA + AFA Days since treatment start 0-50 H2122 xenografts

21 Ongoing combination trials based on our synthetic lethality screens NCT : LGX818 and Cetuximab or LGX818, BYL719, and Cetuximab in BRAF mutant metastatic CRC NCT : Dabrafenib plus Trametinib plus Panitumumab in BRAF mutant CRC NCT : Vemurafenib and Panitumumab in BRAF mutant Metastatic CRC NCT : Dacomitinib Plus PD in advanced KRAS mutant cancers First responder in clinic: 2 months after publication!

22 Relative EGFR expression EGFR level determines response to BRAF inhibitor Melanoma Colon cancer 2500 p-egfr EGFR HSP Melanoma Colon cancer TCGA database Could melanoma develop resistance to BRAF or MEK inhibitors through acquired expression of EGFR?

23 BRAF mutant melanomas upregulate EGFR during the development of drug resistance Pre- vemurafenib Post- vemurafenib Patient #1 NKI Patient #2 NKI Total: 6/16 patients became EGFR positive Patient #3 IGR EGFR IHC in Brown

24 EGFR expression confers BRAF inhibitor resistance in melanoma EGFR HSP90 Also seen in SK-MEL-28 cells

25 % Change tumor volume (Mean ± SEM) EGFR expression impairs BRAF(V600E) melanoma Proliferation through Oncogene-Induced-Senescence A375-Ctrl. EGF (ng/ml) A375-Ctrl. and A375-EGFR xenografts Empty-Vehicle A375-Ctrl. EGFR-Vehicle A375-EGFR 200 A375-EGFR Days since treatment start EGFR RB p27 p21 A375-Ctrl. A375-EGFR HSP90 SA-β-Gal staining in Blue

26 FACS-assisted shrna genetic screen for determinants of vemurafenib resistance and EGFR expression DAPI DAPI Chromatin regulator shrna library A375 Untreated control 0.5μM Vemurafenib selection (3 weeks) - Chr Library - Vemurafenib EGFR lo w 85.4% EGFR high 0.1% + Chr Library - Vemurafenib EGFR lo w 82.9% EGFR high 0.1% EGFR high Sort EGFR hgh cells - Chr Library + Vemurafenib EGFR low EGFR high 73.5% 0.1% + Chr Library + Vemurafenib EGFR low EGFR high 43.5% 18.1% EGFR Enriched shrna EGFR Deepsequencing SOX10

27 Relative mrna level SOX10 suppression induces EGFR expression and confers vemurafenib resistance 5,0 4,0 EGFR SOX10 HSP90 A375 Vemurafenib (μm) 3,0 2,0 1,0 0,0 SOX10 EGFR Control Ctrl. shsox10-1 shsox10-2 shsox10-1 shsox10-2 A375 A375 b-gal staining in Blue

28 EGFR induction by SOX10 suppression is reversible Polyclonal shsox10 cell population Ctrl. shsox vemurafenib 7 Time (day) - vemurafenib 14 EGFR A375

29 Relative mrna level Relative mrna level Inverse relation between EGFR and SOX10 in paired biopsies of BRAF mutant melanoma Patient #5 Patient #3 pre-treatment post-trametinib pre-treatment post-dabrafenib EGFR IHC in Red EGFR IHC in Brown SOX10 Pre-treatment Post-trametinib SOX10 Pre-treatment Post-dabrafenib Pre-treatment Post-trametinib 0 Pre-treatment Post-dabrafenib

30 The drug holiday effect explained through changes in RAS-BRAF-MEK signaling RTK-1 RTK-1 RTK-2 RTK-1 RTK-2 RTK-1 RAS RAS RAS RAS Drug BRAF* Drug BRAF* BRAF* Drug BRAF* MEK MEK MEK MEK Drug response Drug resistant Senescence Drug Proliferation response Drug resistance development Drug holiday Re-treat after drug holiday

31 Cancer genome analyses Functional genetic analyses Alterations in pathways Cross talk between pathways Precision medicine

32 Acknowledgements The people involved: Chong Sun Liqin Wang Sidong Huang Anirudh Prahallad Prashanth Kumar Wipawadee Grernrum Roderick Beijersbergen Our funding sources: Collaborators: Jan Schellens (NKI) Robin van Geel (NKI) Caroline Robert (IGR) John Haanen (NKI) Jelle Wesseling (NKI) Federica Di Nicolantonio (IRCC) Alberto Bardelli (IRCC)