TKCC/Garvan Cancer Biology Seminars Triple Negative Breast Cancer

TKCC/Garvan Cancer Biology Seminars Triple Negative Breast Cancer Elgene Lim Lab Head: Connie Johnson Breast Cancer Laboratory Snr Medical Oncologist: TKCC

Breast cancer is a molecularly heterogeneous disease TCGA, Nature 2012

BRCA mutation and cancer A C Basal Lum A Lum B HER2 BRCA1, Chrom 17, 220 kda nuclear protein BRCA1 BRCA2 Sorlie et al. PNAS 2003 B BRCA2, Chrom 13, 384 kda nuclear protein Lifetime risk General Population BRCA1 BRCA2 C Breast Ca 10% A 70-80% B 50-60% Biological questions 1) Is the differential breast cancer subtype distribution between WT, BRCA1, and BRCA2 mutants due to hormonal signaling? ER+ 65-70% 20% 60-65% TNBC 10-15% 80% 10-15% Ovarian Ca 1-2% 50% 30% A Similar prognosis to BRCA tumors at same stage. B Younger, high grade. C Asso with increased risk of pancreatic, prostate & male breast cancer. 2) How does modulating ER signaling reduce the risk of BRCA1 and BRCA2 breast cancer? Oophorectomy & Tamoxifen chemoprevention BC risk 50% in both BRCA1 and 2 carriers Roy et al. Nat Rev Can 2011

Medullary Breast Cancer Characteristics 1) High Grade, frequent mitosis, pleomorphic nuclei, poorly differentiated 2) Pushing borders 3) Lymphocytic infiltrate 4) Associated with BRCA1 mut 4

Germline mutations in DNA repair genes in TNBC Domagala et al, Plos One 15

Germline mutations in DNA repair genes in TNBC Domagala et al, Plos One 15

Overview Heterogeneity of TNBC Therapeutic approaches to TNBC Standard chemotherapy Platinums PAPR inhibitors Bevacizumab Novel targets in TNBC CDK inhibitors Immunotherapy 7

TNBC Molecular subtypes Publically available breast cancer expression datasets Training set 386 TNBC Validation 201 TNBC (1) AR-positive (LAR), (2) claudin-low enriched mesenchymal (M), (3) mesenchymal stem like (MSL) (4) immune response (IM) and (5) 2 cell-cycle disrupted basal subtypes: (a) BL-1 and (b) BL-2. Lehmann et al. JCI 2011 8

Differential therapeutic response according to TNBC subtypes Lehmann et al. JCI 9 2011

TNBC Molecular subtypes 10 Burstein et al. CCR 2015

TNBC Molecular subtypes 11 Burstein et al. CCR 2015

Potential Therapeutic targets in TNBC subtypes 12 Burstein et al. CCR 2015

Potential Therapeutic targets in TNBC subtypes 13 Burstein et al. CCR 2015

Overview Heterogeneity of TNBC Therapeutic approaches to TNBC Standard chemotherapy Platinums PAPR inhibitors Bevacizumab Novel targets in TNBC CDK inhibitors Immunotherapy 14

Standard Chemotherapy

TNBC and HER2+ have the highest rates of pcr to chemotherapy based regimens 60 50 40 30 20 10 0 50 30 31 18 16 7 G 1-2 HR+ G 3 No Tras Tras No Tras Tras HER2+/HR+ HER2+/HR-. 34 TNBC Meta-analysis 12 trials, n=11,955 Med FU 5.4yr All except 2 trials included Anthra/Taxane regimens pcr=ypt0/is ypn0 16 Cortazar et al, Lancet 14

pcr is associated with improved outcomes compared to no pcr HR 0.48 (0.43 0.54) HR 0.36 (0.31 0.42) In analyses by subtype, the relationship held most true for the aggressive subtypes, triple negative, HER2+ (especially ER-, HER2+) and high grade ER+ 17 Cortazar et al, Lancet 14

Platinums in TNBC Platinums in treatment naïve TNBC Presented at SABCS 2015 W Sikov, CALGB 40603 trial G Von Minckwitz, GeparSixto trial O Gluz, Adapt trial 18

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CALGB 40603 Schema 20

Response: pcr 21

Survival outcomes 22

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GeparSixto Schema Concurrent Trastuzumab and Lapatinib in HER2+ subgroup. Concurrent Bev in TNBC subgroup. 24

TNBC Schema 25

pcr by subtype 26

pcr by BRCA status in TNBC 27

DFS by subtype 28

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ADAPT Schema PEP pcr (ypt0/is ypn0) pcr in early responders vs non responders SEP EFS and OS Toxicity 4 x EC given as adjuvant tx to those without pcr 30

Hypothesis 31

Consort Diagram Pt characteristics 32

Toxicity Grade 3 33

Response: pcr Subgroup Analysis 34

Conclusions CALGB 40603 Achievement of pcr is asso with significant improvements in EFS and OS, however study was underpowered to determine if addition of Carbo or Bevacizumab improves EFS/OS Previous studies (Beatrice, GeparQuinto, NSABP B-40) failed to demonstrate improvements in long term outcomes with addition of Bevacizumab in neoadjuvant setting. Despite high pcr rates, neither Carbo nor Bev have been shown to improve RFS or OS when given as part of a neoadj regimen 35

Conclusions GeparSixto Carbo improved EFS in TNBC (HR = 0.65, p<0.5) but not in HER2+ BC DFS benefit of Carbo was predicted by pcr Unexpected improvement in EFS in BRCA WT pts and not in BCRA mut carriers. Favorable prognosis after pcr is independent of BRCA status 36

Conclusions - Adapt Nab-Pac/Carbo is asso with less toxicity and is superior to Nab-Pac/Gem in achieving pcr (45.9% vs 28.7%) Observed efficacy is comparable to longer and more toxic anthracycline-taxane combination tx Early morphological changes predict for pcr irrespective of treatment arm No predicitve factors for Carbo efficacy have been identified so far 37

Survival outcomes across trials GeparSixto More intensive backbone Weekly Carbo -? Less time for DA repair Concurrent Tx -?Synergy 38

Do these data warrant routine use of Carbo in TNBC? Hazard ratios suggest benefit, but not enough data to be conclusive Chemo backbone and Carbo dose/schedule may be critical to optimal efficacy CALGB 40603: Taxol GeparSixto: Taxol, Peg Doxo Adapt: Peg Taxol LT effects of added toxicity not known 39

Bevacizumab in TNBC CALGB 40603: Neoadj Chemotherapy +/- Bev in TNBC BEATRICE: Adjuvant Bev in TNBC Odds ratio: 1.58 p = 0.0089 48% (41-54%) 59% (52-65%) n=218 n=215 Sikov et al, JCO 14. 9 Cameron et al, Lancet Onc 13

PARP inhibitors Sonnenblick et al, Nat RV Clin Onc 15

PARPi in clinical development Sonnenblick et al, Nat RV Clin Onc 15

PARPi in clinical development Livarghi and Garber, BMC Med 15

PARPi in clinical development Livarghi and Garber, BMC Med 15

Resistance to PARPi Livarghi and Garber, BMC Med 15

Overview Heterogeneity of TNBC Therapeutic approaches to TNBC Standard chemotherapy Platinums PARP inhibitors Bevacizumab Novel targets in TNBC CDK inhibitors Immunotherapy 46

CDK complexes are involved in cell cycle and other biological processes Palbociclib Ribociclib Abemaciclib Flavopiridol Seliciclib Dinaciclib TZH1/2 CycK CDK12 Asghar et al. Nat Rv Drug Disc 2015 47

Rationale for Combining PARP Inhibition with Agents Targeting HR HR-Proficient Cancers Goal is to selectively disrupt HR in cancer cells and sensitize to PARP inhibition HR-Deficient Cancers (germline or somatic HR gene mutation, e.g. BRCA1/2) Acquired resistance to PARP inhibition often involves restoration of HR De novo resistsance to PARP inhibition exists (hypomorphic BRCA proteins) In PARP inhibitor-sensitive tumors, goal is to augment the extent and durability of response Agents that inhibit HR HR proficient + Platinum or PARP Inhibitor HR deficient Ashworth, Cancer Res 2008; Dhillon et al. Cancer Sci 2011; Jaspers et al. Cancer Discov 2012; Johnson et al. PNAS 2013; Bouwman et al. Clin Cancer Res 2014; Bunting et al. Mol Cell 2012 48

Genome wide synthetic lethality identifies CDK12 as a determinant of PARPi sensitivity in Ov Ca Mutations in CDK12 in TCGA Serous Ov Ca 2011 (n= 316) Low CDK12 expression correlates with PARPi sensitivity in serous Ov Ca cell lines Bajrami et al., Can Res 2014 49

Dinaciclib is a potent inhibitor of CDK9/12 44% homology in Kinase Domain of CDK9 and CDK12 Dinaciclib is the most potent known inhibitor of CDK12 (IC50 68nM) 50

Dinaciclib decreases BRCA1 and DNA repair genes in DNA damage and repair pathways Transcriptome of Dinaciclib tx vs untx MDA-MD-231 cells BRCA1 in DNA repair pathway Ingenuity Pathway Analysis Can Dinaciclib induce a BRCA1 deficient phenotype and PARPi sensitivity? 51

BRCA1 proficient TNBC cell lines 10 nm Dinaciclib Vehicle 10 Gy γ irradiation Dinaciclib sensitizes BRCA1-proficient cells to PARPi In the presence of dinaciclib, the IC 50 to veliparib was reduced between 2.5 and 12.5-fold 52

BRCA mutant TNBC cells with acquired PARPi resistance are resensitized by Dinaciclib In vitro In vivo Cisplatin & Olaparib Resistant BRCA2 mut PDX derived from metastasis 53

BRCA1 mutant TNBC cells with primary PARPi resistance are sensitized to PARPi by Dinaciclib A In vitro SUM149 HCC1937 In vivo C PARPi-resistant BRCA1 mut PDX derived from metastasis B Dinaciclib 10 Gy γ irradiation Veliparib ± dinaciclib dinaciclib (nm) D dinaciclib (nm) RAD51 activity following Olaparib suggests residual HR activity Increased H2AX with combo suggest overcoming residual HR activity 54

PARPi-sensitive BRCA1 mut TNBC cells have additive antitumour effects with combination therapy 50 yo EBC. G3 TNBC Kaplan-Meier Survival Curve Patient tumor BRCA1 -/-, p53 -/- Passage 1 PDX BRCA1 -/-, p53 -/- Tumor response 55

Minimal residual tumor after combination treatment End of Experiment vehicle veliparib dinaciclib combination

TZH1 binds selectively and irreversibly to CDK7, and inhibits RNAPII CTD phosphorylation GO Term analyses of 527 cell lines 57

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Concept of Super-enhancers Typical enhancers composed of transcription factor binding sites located at a distance from the transcriptional start site that act through chromosomal looping events to enhance transcription. Super-enhancers consist of very large clusters of enhancers densely occupied by transcription factors, co-factors and chromatin regulators (e.g. BRD4) arise via gene amplification, translocation or transcription factor overexpression facilitate high level of expression of genes involved in cell identity, growth and proliferation; often genes and encoded proteins have short half-life, so high-level transcription is critical to maintenance of their expression highly sensitive to perturbation Whyte et al. Cell 2013; 153: 307-19; Lovén et al. Cell 2013; 153: 320-34; Chapuy et al. Cancer Cell 2013; 24: 777-90. 59

CDK7 inhibition selective targets TNBC A BC cell lines B Red: TNBC cell lines Blue: ER+ cell lines Selective induction of apoptosis and suppression of RNAPII CTD phosphorylation C Primary BC cell lines D E Live (green)/dead (red) Cell viability assay Wang et al., Cell 2015 60

Differentially expressed TNBC genes are sensitive to CDK7i and are critical for TNBC survival THZ1 treatment globally affects steady-state mrna levels in TNBC Enriched GO functional categories of TNBC genes sensitive to THZ1 tx Signalling pathways and transcription factors comprising Archilles Cluster of genes Genes differentially expressed between TNBC and ER/PR+ breast cancer lines. BT549 MB-468 ZR-75-1 T47D 40% of the genes in the Achilles cluster were associated with super-enhancers in TNBC cells Wang et al., Cell 2015 61

Summary CDK7 +/- CDK9 inhibitors may perturb superenhancer complexes that govern expression of genes controlling the oncogenic state CDK7 is a relevant target in TNBC cells CDK12 is a relevant target for disrupting DNA repair pathways such as HR and for sensitizing breast cancer cells to DNA damage or PARP inhibition Dinaciclib is a highly potent inhibitor of CDK12 and sensitizes BRCA wildtype, HR-proficient TNBC cell lines to PARP inhibition Dinaciclib reverses acquired resistance and overcomes primary resistance to PARP inhibition in BRCA-mutated cells Phase 1 trial of dinaciclib/veliparib is underway, with planned expansion cohort work in BRCA WT and mutated TNBC 62

Immunotherapy and TNBC 63

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How does this compare with other solid tumours? 67

TILS in TNBC outcomes 68

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Trial & pt characteristics 70

Multivariate Cox Analyses (adjusted) 71

Conclusions 72

TNBC Subtypes: Potential Targets Basal-like 1: Cell cycle, DNA repair & proliferation genes Basal-like 2: Growth factor signaling (EGFR, MET, Wnt, IGF1R) PARPi, ± DNA damaging agents EGFR (cetuximab, lapatinib) Self-renewal pathways (Wnt, Notch) IM: Immune cell processes (medullary breast cancer) Immune check point (PD1/PDL1, CTLA4) Vaccines: MUC1, NYO-ESO1 M: Cell motility and differentiation, EMT processes MSL: Similar to M but growth factor signaling, low levels of proliferation genes (metaplastic cancers) PI3Ki, RAS/MEK/Erk, MET, PTEN etc, etc LAR: AR and downstream genes, luminal features AR modulators (enzalutamide, bicalutamide, etc)

Suggested Readings 1. Comprehensive Molecular Portraits of Human Breast tumours. TCGA. Nature 2012. 2. Identification of human triple-negative breast cancer subtypes and preclinical models for selection of targeted therapies. JCI 2011. 3. Comprehensive Genomic Analysis Identifies Novel Subtypes and Targets of Triple-Negative Breast Cancer. Clin Cancer Res 2015 4. Dent R, Trudeau M, Pritchard KI, et al. Triplenegative breast cancer: clinical features and patterns of recurrence. Clin Cancer Res 2007 5. Aromatase inhibitors versus tamoxifen in early breast cancer: patient-level meta-analysis of the randomised trials. EBCTCG. Lancet 2015. 6. Prognostic Value of Ki67 Expression After Short- Term Presurgical Endocrine Therapy for Primary Breast Cancer. Dowsett et al. JNCI 2007. 7. Randomized Trial of Letrozole Following Tamoxifen as Extended Adjuvant Therapy in Receptor + Breast Cancer. Goss et al. JNCI 2005. 8. Long-term effects of continuing adjuvant tamoxifen to 10 years versus stopping at 5 years after diagnosis of oestrogen receptor + breast cancer: ATLAS, a randomised trial. Davies et al. Lancet 2013. 9. Adjuvant Ovarian Suppression in Premenopausal Breast Cancer. Francis et al. NEJM 2015. 10. A Multigene Assay to Predict Recurrence of Tamoxifen-Treated, Node-Negative Breast Cancer. Paik et al. NEJM 2004. 11. Combination Anastrozole and Fulvestrant in Metastatic Breast Cancer. Mehta et al. NEJM 2012. 12. Everolimus in Postmenopausal Hormone-Receptor + Advanced Breast Cancer. Baselga et al. NEJM 2012. 13. Palbociclib in Hormone-Receptor + Advanced Breast Cancer. Turner et al. NEJM 2015.