Functional Genomics: Implications in Tissue Pathology
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Functional Genomics: Implications in Tissue Pathology

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Unresponsiveness to therapy remains a significant problem in the treatment of cancer, also with the new classes of cancer drugs. In my laboratory, we use functional genetic approaches to identify ...

Unresponsiveness to therapy remains a significant problem in the treatment of cancer, also with the new classes of cancer drugs. In my laboratory, we use functional genetic approaches to identify biomarkers that can predict responsiveness to targeted cancer therapeutics. Nevertheless, it remains poorly explained why a significant number of tumors do not respond to these therapies. We aim to elucidate the molecular pathways that contribute to unresponsiveness to targeted cancer therapeutics using a functional genetic approach.
To identify biomarkers that control tumor cell responsiveness to cancer therapeutics, we use multiple complementary approaches. First, we use genome wide loss-of-function genetic screens (with shRNA interference libraries) in cancer cells that are sensitive to the drug-of-interest to search for genes whose down-regulation confers resistance to the drug-of-interest (resistance screens). In addition, we use shRNA screens to screen for genes whose inhibition enhances the toxicity of cancer drugs (sensitizer screens). As a third approach, we use gain of function genetic screens in which we search for genes whose over-expression modulates drug responsiveness. Once we have identified candidate drug response biomarkers in relevant cell line models, we ask if the expression of these genes is correlated with clinical response to the drug-of-interest. For this, we use tumor samples of cancer patients treated with the drug in question and whose response to therapy is documented.
In a fourth and distinct approach we perform high throughput sequencing of the “kinome” (some 600 genes) of tumor samples to identify connections between cancer genotype and drug responses.
Examples of some of these approaches to identify biomarkers of response to different cancer drugs will be presented.

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  • Phase I clinical trial data with RAF inhibitors has demonstrated a significant response rate (81% response rate) in metastatic melanoma patients with B-RAFV600E-positive tumors (N Engl J Med. 2010 363:809-19). In contrast, the response rates in B-RAFV600E-positive colon tumors have been reported as 5.2% (Kopetz et al., ASCO 2010).
  • Mitogen-activated protein kinase (MAPK) cascades are key signaling pathways involved in the regulation of normal cell proliferation, survival and differentiat. Cancers arise owing to the accumulation of mutations in the critical genes in the MAPK cascades. BRAF mutation are frequently detected in melanma thyroid and colon cancer
  • Pharmacodynamic biomarker-driven trial of MK-2206, an AKT inhibitor, with selumetinib (selumetinib), a MEK inhibitor, in patients with advanced colorectal carcinoma (CRC) Tolerable doses for the combination in solid tumors (ASCO 2011, abstr 3004) are less than single agent MTDs. (ASCO 2011, abstr 3004) Although 4/8 pts demonstrated biologically significant inhibition in one marker, at the MTD for this combination no pt had ≥ 70% inhibition of both targets. If repeated dosing does not produce the desired inhibition of pERK and pAKT, we will conclude that the dose reductions for each agent necessitated by the toxicity of the agents used in combination preclude the possibility of providing adequate dual pathway inhibition.

Functional Genomics: Implications in Tissue Pathology Presentation Transcript

  • 1. René BernardsFunctional genomics:Implications in Tissue PathologyThe Netherlands Cancer InstituteAmsterdamThe Netherlands
  • 2. Genotype-driven therapy of non-small cell lung cancerCrizotinibKwak et al, N Engl J Med 2010ErlotinibGefitinibMaemondo et al., N Engl J Med 2010
  • 3. Endless two-way combinations of cancer drugs1,000 x1,0002= 500,000 combinationsTest each combinationin 1,000 patients:500,000,000 patients needed
  • 4. Three vignettes on the use of functionalgenetics to find effective combination therapies• The case of BRAF mutant colon cancer• The case of KRAS mutant lung/colon cancer• The case of RTK mutant lung cancer
  • 5. N Engl J Med. 2010 363:809-19Kopetz et al., ASCO 2010Vemurafenib (PLX4032) - A selective BRAFV600EinhibitorDifferential response of BRAF inhibition in BRAFmutant melanoma versus colon cancer
  • 6. BRAFV600Emutant CRC cell lines are also lessresponsive to PLX4032 than melanomas having thesame mutationCRCMelanomaShort-term cell viability assay Long-term colony formation assay
  • 7. Is feedback regulation responsible for the resistanceof CRC cells to BRAF inhibitors?
  • 8. infect withshRNA kinome libraryPCR amplifybar codesWiDr(BRAFV600E)Deepsequence Quantify shRNAsvemurafenibculture toallow selectioncontrolSynthetic lethal shRNA screen: Inhibition of whichkinase synergizes with PLX in BRAF mutant CRC?
  • 9. Inhibition of EGFR makes BRAF mutant CRC cellsvulnerable to BRAF inhibitionGrbBRAFEGFRRASMEKERKCetuximabMutant
  • 10. Synergistic response of BRAFV600ECRCto EGFR and BRAF inhibitionCetuximab 1.25 µg/mlGefitinib 0.125 µMVACO (BRAFV600E)Also seen in WiDr andKM20 (BRAFV600E)+Cetuximab+Gefitinib
  • 11. BRAFV600Einhibition causes feedback activationof EGFRVACO (BRAFV600E)Also seen in WiDr andKM20 (BRAFV600E)
  • 12. EGFR and BRAF inhibition synergize to induce apoptosisand suppress BRAFV600ECRC tumor growthAlso seen in WiDr (BRAFV600E)Prahallad et al, Nature 2012
  • 13. Feedback regulation of EGFR by BRAF inhibitionLigandCDC25CPI3KAKTVemurafenibCetuximab
  • 14. Mechanism-based rational drug combinations:rapid translation to the clinic8 months frompublication toclinical trial!
  • 15. EGFR levels determine response to BRAFInhibition in multiple tumor types
  • 16. ControlGefitinibPLX4032PLX4032+GefitinibTrametinibTrametinib+GefitinibEGFR expression in melanoma is sufficient toconfer resistance to BRAF and MEK inhibitors
  • 17. BRAF mutant melanomas upregulate EGFRduring the development of drug resistanceEGFR IHC in brownPatient 7 Pre vemurafenibPatient 9 Pre vemurafenibPatient 7 Post vemurafenibPatient 9 Post vemurafenibTotal:5/12 patientsbecameEGFR positive(40%)
  • 18. Three vignettes on combination therapies tofight resistance• The case of BRAF mutant colon cancer• The case of KRAS mutant lung/colon cancer• The case of RTK mutant lung cancer
  • 19. Nat Rev Cancer 3: 459–465. Nat. Rev. Clin. Oncol., 6 (2009) Cancer Research, 69 (10), 4286-4293 Clin Cancer Res 2012;18:2515-2525Cell survival and proliferationInhibitorReceptorsMEK inhibitorLow efficacy (PDX assay) forKRAS mutant tumorsTargeting KRAS mutation with MEK inhibitorPotential for clinic failure:
  • 20. +Gefitinib (1uM)Is the combination of MEK and EGFR inhibitorsalso effective in KRAS mutant cells?Gefitinib (1uM)0.25 0.5 1Selumetinib (uM)CRC: SW620(KRASG12V)NSCLC: H358 (KRASG12C)0.25 0.5 1Selumetinib (uM)0 0.125 0.25 0.5 1Selumetinib(μM)H747SW480SW620SW837CRCH358H23H2030H2122NSCLC
  • 21. RNAi screen for enhancers of MEK inhibitorsERBB3 activation is mostly dependent onheterodimerizationERBB3 binding partnersERBB familyshERBB3shERBB3
  • 22. H358 (KRASG12C)Also seen in H2030, H2122,SW837, SW480 and SW620Targeting EGFR and ERBB2 sensitizes KRAS mutantcells to MEK inhibitor+Dacometinib (0.5uM)+Afatinib (0.5uM)+Gefitinib (0.5uM)+Pertuzumab (2.5ug/ml)0.25 0.5 1Selumetinib (uM)• Gefitinib: EGFR inhibitor• Pertuzumab: ERBB2-targeting monoclonalantibody• Afatinib: EGFR and ERBB2 inhibitor• Dacometinib: EGFR, ERBB2 and ERBB4 inhibitor
  • 23. MEK inhibition induces ERBB2 and ERBB3transcription
  • 24. EGFR+ERBB2 inhibitor synergizes with MEKinhibitor to induce apoptosis in KRAS mutant cellsSelumetinibAfatinib+-+ ++-- -Clev. PARPp-BAD (S112)p-BAD(S136)BADp-ERKERK1/2p-p90RSKp90RSKp-AKTAKTH358p-BIM(S69)BIM
  • 25. Targeting EGFR and ERBB2 sensitizes KRAS mutantcells to MEK inhibitor in a xenograft model250%343%306%H2122 xenografts
  • 26. BRAFMelanomaColon cancerBRAFi/MEKiBRAFi/MEKi+EGFRiGenotype Tissue type Effective therapyKRASKRASColon cancerLung cancerMEKi+EGFRi+HER2i
  • 27. Three vignettes on combination therapies to fightresistance• The case of BRAF mutant colon cancer• The case of KRAS mutant lung/colon cancer• The case of RTK mutant lung cancer
  • 28. Resistance to TKIs is a frequent problemin the clinicCrizotinibGefitinibErlotinibAcquired resistance to EGFR inhibitorsSequist et al., Sci Transl Med 2011 Choi et al., NEJM 2010* L1196M in EML4-ALK* Other unknown mechanismsT790M(49%)
  • 29. Cell line models matching NSCLC genotypesPC9 (EGFRDelE746A750)H3255 (EGFRL858R)ErlotinibGefitinibTKI-sensitive NSCLC cell linesH3122(EML4-ALK)
  • 30. Finding genes that can cause resistance tocrizotinib in EML4-ALK positive NSCLCculture toallow selectioncrizotinibcontrolinfect withshRNA libraryPCR amplifybar codesH3122(EML4-ALK)
  • 31. Suppression of MED12 causes crizotinib resistanceshMED12#1shMED12#2MED12Transcription Mediator complexCDK8L1224F, recurrent in Prostate cancer (5%)Uterine Leiomyomas (70%)Exon 2Mäkinen et al., Science Express 2011; Barbieri et al. Nat Genet. 2012Exon 26MED12 mutations in cancers
  • 32. PC9 (EGFRDelE746A750)MED12 knockdown causes MEK and ERK activationERKMED12p-ERKpLKOshMED12#3shMED12#5pLKOshMED12#3shMED12i#5Gefitinib: - - - + + +p-MEKMEK
  • 33. infect with TRCshRNA kinome library MED12KD(EML4-ALK)+ Crizotinibculture toallow selectioncontrolResistantRe-sensitization screen: Which kinase to inhibit torestore the crizotinib sensitivity in MED12KDcells?
  • 34. infect with TRCshRNA kinome libraryPCR amplifybar codesMED12KD(EML4-ALK)Deepsequence Quantify shRNAs+ Crizotinibculture toallow selectioncontrolDepletedby CrizotinibResistantRe-sensitization screen: Which kinase to inhibit torestore the crizotinib sensitivity in MED12KDcells?
  • 35. Suppression of TGFβR2 restoresthe crizotinib sensitivity in MED12KDcells
  • 36. IIITGFβ activation is sufficient to drive drug resistanceH3122(EML4-ALK)
  • 37. Downregulation of MED12 leads toelevated TGFβR signaling in different tumor typesPC9(EGFRDelE746A750)H3122(EML4-ALK)PC9A375(BARFV6700E)MelanomaPC9SKCO-1(KRASV12)ColonNSCLCNSCLC
  • 38. TGFβ activates the RAS-RAF-MEK-ERK pathwayZhang, Y. Cell Research 2009:19 p128
  • 39. MED12KDalso causes resistance tochemotherapies through TGFβ signaling
  • 40. Generation of a “MED12KDsignature”H3122; NSCLCPC9; NSCLCSKCO1; colon cancerA375; melanomaHuh7; HCCWild type+MED12KDDifferentially expressed genesin 3 out of 5 cell lines (>2-fold);237 genes
  • 41. MED12 loss induces an EMT-like phenotype
  • 42. A MED12KDsignature predicts resistance tochemotherapy in stage III colon cancer
  • 43. Three gene profiles specific for intrinsicsubtypes• A-type profile of 32 genes• B-type profile of 53 genes• C-type profile of 102 genesThree gene profiles specific for intrinsicsubtypes• A-type profile of 32 genes• B-type profile of 53 genes• C-type profile of 102 genesColon cancer also has “intrinsic subtypes”
  • 44. 44Colon cancer subtypes differ in biology and inresponse to chemotherapy
  • 45. TGFβR2 inhibitor restoresthe crizotinib sensitivity in MED12KDcellsAnd by other means?EMT by MED12 lossTGFβ signalingresponse to targeted drugsTGFβRinhibitorspotential treatment strategy:(also true for EGFR inhibitors)H3122(EML4-ALK)
  • 46. Precision medicineAlterations in pathwaysCancer genome analysesCross talk between pathwaysFunctional genetic analyses
  • 47. AcknowledgementsThe people involved:Chong SunSid HuangAnirudh PrahalladWipawadee GrernrumLorenza MitempergherFloris GroenendijkTheo KnijnenburgAndy SchlickerLodewijk WesselsWouter NijkampRoderick BeijersbergenCollaborators:Federica Di NicolantonioAlberto BardelliPaul RoepmanPeter ten DijkeOur funding sources: