Mechanisms of Resistance to BRAF Inhibitors and possible treatments to overcome resistance

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1 Mechanisms of Resistance to BRAF Inhibitors and possible treatments to overcome resistance Maria González Cao IOR. Institut Universitari Dexeus. Barcelona Simposium GEM Junio Sevilla

2 Oncogenes: CDK4 50%, BRAF 50%, RAS 20%, PTEN 30%, TERT 70%, KIT, CCND1, AKT; Others: erb4, NEDD9, GNAQ, GNA11 Transcription factors: MITF, MYC, ETV1 Tumor Supressor: high level expression without amplification MDM4, CDKN2A, BAP1,PTEN Others: epha3, map3k5, map3k9, prex2 (regulator of PTEN and the Pi3k) McArthur G A, and Ribas A JCO 2013;31:

3 BRAF mutations First described by Davies et al (Nature 2002) More frequent in young patients BRAFV600E: 75% under 40y old In old people more frequent mut V600K/D/R NonV600E : Dabra tested activity and Vemu in phase 3 BRAFV600K PFS 6m (V600E 7m), OR 45% (V600E 60%), OS 14.5m (V600E 13.3) 1. Ongoing phase 2 trial of vemu in non V600E mutations 10% of cases have non-brafv600 exon 15 mutation (L597, K601, D594). L597 and K601 are in the activating segment of the kinase domain close to V600, D594 results in an inactive kinase. These mutation are less sensitive to BRAFi, MEKi are better 2 NRAS mutation can concurrer with these non v600 BRAF McCarthur. SMR 2012 Dahlman. Cancer Disc 2012

4 BRIM 3 and BREAK3

5 Time to response and progression BRIM2 study Ribas et al. ASCO 2011 Time on study Time to response Progressive disease Continued response 0 2 Approx timing of CT assessments Time (months) Median duration of response = 6.7 months (95% CI: 5.6, 9.8; range ) 16

6 Mechanisms of Resistance to BRAF i PDGFRb or IGF1R Nazarian et al. Nature 2010 NRASQ61 Nazarian et al. Nature 2010 Villanueva et al. Cancer Cell 2010 BRAF inh CRAF COT PI3Ki or AKTi BRAFV600E Johannessen et al. Nature 2010 MEK MEK-dependent progression PI3K P MEKi Poulikakos et al. Nature 2011 Shi et al. Nature Com 2012 Wagle et al. JCO 2011 ERK P Survival AKT MEK-independent progression

7 Mechanisms of resistance to BRAF inhibitors Develops Developsat ataamedian medianof of months months Intrinsic Acquired Series Seriesof ofbiomarkers biomarkers identifiable identifiableat atthe the commencement commencementof of therapy therapy that thatcan can predict predictdifferential differential benefit of benefit oftreatment treatment CDK CDK44 Amplified Amplified cyclin cyclind1 D1 (CCN1) (CCN1) 15-20% 15-20% melanomas melanomas RESISTANT RESISTANT PTEN PTENloss, loss, bcl2a1 bcl2a1ampl ampl RESISTANT RESISTANT to tobrafi BRAFi No Noone onemechanism mechanismof of resistance resistancehas hasbeen been seen consistently seen consistentlyin inthe the majority majorityof ofpatients patientsat at the thetime timeof ofdisease disease progression progression HGF, HGF,CMET CMET RESISTANT RESISTANT to tobrafi BRAFi Non-ERK Non-ERK dependent dependent (ERK (ERKstill still inhibited) inhibited) ERKERKdependent dependent (ERK (ERK reactivated) reactivated) Sullivan et al. Eur J Cancer. 2013;

8 Diverse mechanisms of acquired B-RAF inhibitor resistance and common denominators Nazarian, Shi... Ribas, Lo (2010) Nature Shi... Ribas, Lo (2011) Cancer Res Poulikakos... Ribas, Lo, Rosen, Solit (2011) Nature Shi... Ribas, Lo (2012) Nature Comm Shi, Moriceau... Ribas, Lo (2012), Cancer Discovery, in press

9 BRAFi Resistant mechanisms Confirmed mechanism p61brafv % NRAS mutation 15% MEK mutation 15% Other mechanism of uncertain frequence Activation through RTKS: MET, IGF-R, PDGFRbeta BRAF amplification COT increased expression NF1 loss (mutations) Erb3 Speculative MER, AXL/GAS6 MITF/bcl2A1/PDE4D MED12 SMURF Loss PTEN and Rb1

10 NRAS mutations BRAFV600 mutation truncation / amplificatio n MEK mutations downtream of BRAF CRAF/COT overexpressi on

11 Splicing forms p61brafv600 6/19 patients: 61kd variant form of BRAF(V600E) that lacks exons 4-8, a region that encompasses the RAS-binding domain. Exhibits enhanced dimerization in cells with low levels of RAS activation, as compared to full length BRAF(V600E) Truncated form of of the mutated BRAF kinase in wich interaction with RAS is enhanced This leads to dimeritation between the truncated, activated BRAF kinase and wild type RAF kinases. Once dimerized, BRAFi induces trans-activation and subsequent reactivation of MAPK pathway Clinical data from different labs Poulikakos P. Nature 2011

12 Response and relapse with vemurafenib 10/02/08 (Pre) 11/26/08 (2+ mo) 02/20/09 (4+ mo) Pt #43, UCLA melanoma stroma Nature Dec 16; 468 (7326):

13 NRAS mutation 1/16 (Nazarian. Nature 2010) 1/11 (McCarthur. JCO 2011 abs 8502) 3/13 patients (Ribas,BRIM2. JCO 2013) NRAS Mutations in Resistant Cells

14 Escape mechanisms: NRAS mutations Results from Sequenom Oncocarta Panels ERK phosphorylation at progression BOR Archival/ baseline (n=90) Progression (n=13) Case Sex Age group 8 Male 40 M1C PR WT Q61K 9 Male M1B PR WT Q61L 10 Male M1C PR Q61K 11 Male 40 M1C SD Q61K 1/90 (1) 3/13 (23) Stage Frequency of mutation in subgroup n/n (%) Pre-therapy, NRAS, and BRAF mutations co-occurred at very low frequency At disease progression: NRASQ61 mutations were noted in 3/13 patients persistence of BRAFV600 mutations were detected in 13/14 samples Sosman et al. JCO 2012; 30 (suppl): 8503 ASCO 2012

15 Escape mechanisms: MEK1 mutations Results from Sanger Sequencing (exons 2, 3, 6) Age group Stage BOR (IRC) Archival/ baseline (n=92) Progression (n=20) MEK1 mutations pre-therapy 1 Female 2 Female 3 Female 4 Female 5 Male 6 Female M1C M1A M1C M1A M1A M1B PR PR SD SD PD PD P124L P124L P124S P124S P124L P124L Unknown Unknown Unknown Unknown Unknown Unknown MEK1 mutations at progression 7 Female 8 Male 9 Male 10 Male M1A M1A M1C M1A PD PR PR CR WT WT WT WT P124L Q56P; K57E Q56P E203K Case Gender MEK1P124 mutations co-occur at low frequency (~7%) with BRAFV600 mutations. Pre therapy MEK1 codon 124 6/87; OR 2/6 MEK1 mutation was identified at progression in 3/20 cases (codon 203 and 56): This mutation activates MEK1 and is known to be resistant to both BRAF Sosman et al. JCO 2012; 30 (suppl): 8503 ASCO 1 and MEK inhibitors 2012 Emery et al. Proc Natl Acad Sci USA,

16 RAS MUTANT. MEK162 Phase 2 Ascierto. Lancet Oncol 2013

17 Figure 1 Source: The Lancet Oncology 2013; 14: (DOI: /S (13)70024-X)

18 Figure 2 Source: The Lancet Oncology 2013; 14: (DOI: /S (13)70024-X)

19 Genotype-Selective combination therapies by High-Throughput drug screening Held M A et al. Cancer Discovery 2013;3:52-67 Limited number of single agents efficacy, incompletely growth inhibition 52% of RAS selective combinations involved simvastatin

20 Confirmation of synergy and cytotoxicity of genotype-selective drug pairs BRAFmut BRAF-R NRAS mut BRAF-R Lapa+AKTi (MK 2206) Lapa+AKTi+BRAFi NRAS Sinv+Flavopi Sinv+CDK4i Simvastatin indirectly interferes with postranslational procesing of RAS prot Flavopiridol: pan cyclin dependent kinase (CDK) inhibitor Held M A et al. Cancer Discovery 2013;3:52-67

21 Study workflow for unbiased combinatorial screening, leading to effective genotype-selective combinations discovered by Held and colleagues (5), and the emerging pathways and interaction network derived from their data. Held M A et al. Cancer Discovery 2013;3:52-67 Al-Lazikani B, and Workman P Cancer Discovery 2013;3:14-19

22 Nude mouse xenograft model of NRAS mutant: combination of a MEKi and PI3K/mTORi1,2 Posch C et al. PNAS 2013;110:

23 PD (CD4/6i)+GSK (MEKi) CR in 33% of mice Kwong. Nature 2012

24 RAS mutated treatment Huang1 Simvast+Falvop/MEKi Kwong2 MEKi+CDK4i Corcoran3 MEKi+abt-263 (bcl-xli) Posch4 MEKi+PI3Ki Greger5 MEKi+BRAFi+PI3Ki Means-Powell6 METi+Sorafenib 1.Cancer Discovery Nature Cell PNAS Mol Cancer Th ASCO 2012

25 Nf1 mutations desensitize mouse melanomas to BRAF inhibitors Maertens O et al. Cancer Discovery 2013;3: by American Association for Cancer Research

26 The putative role of NF1 loss 16/121 melanomas harbored a NF1 missense or nonsense mutation But clinically resistance has been only demonstrated in one patient that had it in the initial byopsy and had a short PFS Sensitive to pan-raf inhibitor AZ628, the ERK inhibitor VTX11e, and the combination of a MEK + mtor inhibitor (PD and rapamycin) Gibney G T, and Smalley K S Cancer Discovery 2013;3:

27 4/20 patients Only one clinical study and mouse models

28 Whole exome sequencing identified B-RAFV600E amplification In vitro testing: growth inhibition could be achieved with higher dose of BRAFi

29 Increased BRAF(V600E) expression MD Thakur et al. Nature 000, 1-5 (2013) doi: /nature11814

30 Discontinuous dosing strategy attenuates continued dependency on BRAF (V600E)-MEK-ERK signaling in resistant tumors, akin to reintroducing EGFR TKIs in EGFR mutant NSCLCs following chemotherapy (Sequist et al. Science Trans Med 2011) Das Thakur et al. Nature 2013

31 BRAF serum/plasma determination Unpublished data. IOR-CUN

32 Increased expression of COT COT encoded by MAP3K8 gene 2/3 patients BRAFi resistant Acquired and de novo Johannessen. Nature 2010

33 RTK overexpression (PDGFR, IGFR, ERBB4) RTK ligand overexpression PTEN loss AKT amplification Kwong et al. Oncogene. 2013; 1-9

34 Erb3 increase protein and RNA levels Enhanced ERB3 activity pathway is dependent on activation of ERB2 coreceptor Combination BRAFi +Lapatinib Abel E. J Clin Invest 2013

35 Model of HER2/HER3-induced primary resistance to MAPK inhibitors in BRAF-mutant thyroid cancer cells. RAF or MEK inhibitors release transcription repressor CTBP proteins from the HER3 promoter and induce HER3 gene expression Autocrine-secreted NRG1 binds to HER3, triggers HER3/HER2 heterodimerization Montero-Conde C et al. Cancer Discovery 2013;3:

36 Model of MEK inhibitor induced feedback on ERBB receptor signaling pathways. Turke A B et al. Cancer Res 2012;72: by American Association for Cancer Research

37 EGFRp increased: Dasatinib overcomes the growth and metastatic spread of vemurafenib-resistant tumors Girotti M R et al. Cancer Discovery 2013;3:

38 Combined targeting of EGFR and AKT re-establishes vulnerability Held M A et al. Cancer Discovery 2013;3:52-67

39 HGF is present in the stromal cells of melanoma and correlates with a poor response Stromal cell lines secrete HGF in co-culture with BRAFmut cell lines Innate resistance BRAFi+METi Straussman Nature 2012

40 Phospho-PDGFRβ in Acquired Resistant Cells ppdgfrβ Control 5/12 pat Nazarian. Nature 2010

41 IGF-R mediates PI3K signaling in BRAFinhibitor resistant cells 1/5 pat co-targeting MEK and IGF-1R/PI3K Villanueva. Cancer Cell 2010

42 TAM Receptors (TYRO, AXL, MERTK) AXL was commonly expressed in NRAS-mutant melanomas lacking MITF expression and contributed to a migratory and invasive phenotype Sensi et al. found that melanoma cells often secrete GAS6, a ligand of TAM receptors, indicating a mechanism of TAM autocrine signaling in melanoma. These observations support the investigation of MERTK as a potential therapeutic target in melanoma. Nazarian 2010

43 MERTK C-MER proto-oncogene tyrosine kinase (MERTK) Therapeutic target in hematopoietic malignancies and several solid tumors including lung, prostate, and brain MERTK is overexpressed in approximately half of melanoma cell lines, irrespective of BRAF and NRAS status, and is an active receptor. Potent mediator of prosurvival and antiapoptotic signaling pathways MERTK is an upstream activator of both ERK1/2 and AKT. MERTK expression increases with nevus-to-melanoma disease progression Oncogenic role for MERTK in melanoma. Targeting of MERTK in melanoma cells using UNC1062 Schlegel 2013 Cancer Discov

44 BCL2A1 and MITF mediate resistance to BRAFi Normalized expression was compared in patients who achieved OR (n = 12) versus those without objective responses (n = 7). Haq R et al. PNAS 2013;110:

45 BCL2A1 is dysregulated in melanoma Haq R et al. PNAS 2013;110: by National Academy of Sciences

46 Oncogene MITF directly regulates BCL2A1 It may also contribute to resistance to BRAF inhibitors. MITF is amplified in 15 20% Bcl2A1 amplified 30% melanoma Although there are no small molecules that target MITF, targeting downstream pathways such as BCL2A1 may be of clinical utility to this group of melanomas. In contrast, MITF-negative melanomas do not express BCL2A1 and are not sensitive to BCL2A1 suppression.

47 MED 12 loss induces an EMTlike phenotype through TGFbeta Resignaling Complete supression of MED12 is not viable for cells Only MED12 loss confers an advantage under drug pressure TGFBeta R 1 inhibition: YR-290 (JNCI oct 2012) R Bernards Cell 2012

48 SMURF2 depletion sensitizes melanoma cells to MEKi SMURF (Smad ubiquitination regulatory factor 2) is a E3 ubiquitin ligase High levels of SMUR and MITF correlates with MEKi resistance Supressing SMURF sensitices to MEKi. Smith M P et al. JNCI J Natl Cancer Inst 2013;105:33-46

49 % Primary/acquir Treatment NRAS mut 15 P/A Mek+simvastatin, Meki+ CDK4i, MEKi+EGFRi, MEKi+abt263, smvastatin+flavopiridol, simvastatin+cdk4i, MEKi+PI3Ki Braf ampl 15 A BRAFi intemitente, Dosis de braf mayor, MEK+BRAF Splicing 20 A MEK+BRAFi, MEKi+PI3Ki MEK mut 15 A - NF? A/P MEKi+MTORi, CRAFi+BRAFi, panrafi (AZ628) COT sobre? A - IGFR, PDGFR? A MEKi+IGFRi, BRAFi+IGFRi EGFR,? A/P BRAFi+EGFRi, Dasatinib, AKTi+EGFRi FGFR3? A MEK+FGFRi HGF? P BRAFi+METi PTEN loss, or RB inact? P BRAFi+everolimu BRAFi+MEKi ERB2, ERB3? A/P BRAFi+Lapa MED12? Mediator A BRAFi+TGFBi (YR-290) BCL2A1-MITF overexpress? P BRAFi+bcla2i, obatoclax

50 Clinical Trials in Spain Pimasertib (MEKi) in NRAS mut ( ) Phase II randomized EMD Serono MEK162+AMG479 (MEKi+ Anti IGFR1)( ) Phase Ib Novartis Pimasertib (MSC B) + SAR (MEKi+PI3Ki)( NCT ) Phase Ib EMD Serono LEE011+ LGX818 (CDK4/6i+BRAFi) Phase Ib Novartis MEK162 vs Dacarbazine (MEKi) in NRAS mut. Phase III Novartis

51 Conclusions Resistance to BRAF inhibitors is mediated by different mechanisms. Not well known frequence Secondary NRAS mutations Upregulation of RTKs (PDGFRβ, IGF1R) BRAF truncations or amplification The mechanism of resistance may predict for sensitivity to the addition of secondary treatments. Limited clinical data MEK inhibitors PI3K/AKT/mTOR inhibitors Combinations

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