Holloway, Kimberly R.

566 Background: Endocrine therapy (ET) resistance (ETR) remains a primary challenge in ER+ breast cancer. Analyzing pretreatment tumor transcriptomes across trials with early response endpoints can reveal shared and specific ETR signatures. This study utilizes baseline RNA data from the Phase III ALTERNATE trial (Alliance A011106, NCT01953588; Anastrozole [A], Fulvestrant [F], or AF) and the ACOSOG Z1031B trial (NCT00824941) to identify predictors of early Ki67 response in postmenopausal ER+/HER2– patients. Methods: ETR was defined as week-4 on-treatment Ki67 >10%. Baseline gene expression from ALTERNATE was analyzed to identify differentially expressed (DE) genes (Wilcoxon test, P<0.05) and Hallmark pathways associated with ETR, both across and within individual treatment arms. Feature selection was performed using mixOmics. A Pan-Endocrine Therapy Signature (PETS) was developed by uniting DE genes identified across all three ALTERNATE arms and Z1031B. All statistical analyses were conducted in R (P<0.05). Results: Overall ETR rate in the ALTERNATE RNA-seq cohort (n=733) was 26%. ETR was associated with high Risk of Recurrence (ROR), Oncotype RS, and Mammaprint scores (calculated from RNA-seq data in research setting). In luminal tumors (n=649), ETR was linked to chr 3q13.33, 8q24.13, and 20q13.12 cytoband upregulation and 17q21, 18q23, 3p21.1, and 10q24.32 cytoband downregulation. ETR tumors showed T-cell, E2F target, and interferon-γ enrichment; sensitive tumors favored early estrogen response and muscle differentiation. At individual gene level, high MYBL2 , PIF1 , TROAP and with low HJURP predicted ETR across all samples (AUC>0.70). A deep learning model using all protein-coding genes achieved AUC 0.82 (training) and 0.79 (test) in predicting ETR. Cross-trial integration identified ETR-associated PETS, enriched for genomic instability. PETS performed comparably to established signatures and strongly correlated with MYBL2 signature (r=0.93). Top ETR predictors were MYBL2 , AURKB , and EME1 for Arm A and IL4I1 , TNFAIP6 , and ANLN for Arm AF. AF-resistant tumors were enriched for systemic lupus and RIG-I–like receptor signaling; sensitive tumors favored PI3K–AKT, EGFR TKI resistance, AMPK, and insulin signaling. Conclusions: Baseline transcriptomics identify shared and therapy-specific ETR markers. The 15-gene PETS defines a convergent resistance signature, performing similar to established signatures in predicting ETR and correlating with MYBL2. Enrichment of cell-cycle and immune pathways in resistant tumors may suggest patient stratification approach for alternative or combinatorial strategies to overcome early ETR in ER+ breast cancer. Acknowledgement: https://acknowledgments.alliancefound.org. Support: U10CA180821, U10CA180882, U24CA1. Clinical trial information: NCT01953588 .

Abstract Significant endocrine therapy–resistant tumor proliferation is present in ≥20% of estrogen receptor–positive (ER+) primary breast cancers and is associated with disease recurrence and death. Here, we uncover a link between intrinsic endocrine therapy resistance and dysregulation of the MutL mismatch repair (MMR) complex (MLH1/3, PMS1/2), and demonstrate a direct role for MutL complex loss in resistance to all classes of endocrine therapy. We find that MutL deficiency in ER+ breast cancer abrogates CHK2-mediated inhibition of CDK4, a prerequisite for endocrine therapy responsiveness. Consequently, CDK4/6 inhibitors (CDK4/6i) remain effective in MutL-defective ER+ breast cancer cells. These observations are supported by data from a clinical trial where a CDK4/6i was found to strongly inhibit aromatase inhibitor–resistant proliferation of MutL-defective tumors. These data suggest that diagnostic markers of MutL deficiency could be used to direct adjuvant CDK4/6i to a population of patients with breast cancer who exhibit marked resistance to the current standard of care. Significance: MutL deficiency in a subset of ER+ primary tumors explains why CDK4/6 inhibition is effective against some de novo endocrine therapy–resistant tumors. Therefore, markers of MutL dysregulation could guide CDK4/6 inhibitor use in the adjuvant setting, where the risk benefit ratio for untargeted therapeutic intervention is narrow. Cancer Discov; 7(10); 1168–83. ©2017 AACR. This article is highlighted in the In This Issue feature, p. 1047

Abstract Estrogen receptor positive (ER+) breast cancer is treated with endocrine therapy but intrinsic resistance occurs in ~1/3 of patients and acquired resistance in ~1/5 of the remainder. While many resistance mechanisms have been explored, therapeutic strategies to overcome resistance in the clinical setting have seen mixed outcomes, and appear most effective in the acquired resistance setting. Understanding mechanisms of resistance and finding therapeutic strategies to target them, therefore, remain important challenges facing breast cancer researchers. In this study we systematically examine the role of DNA damage repair defects in inducing endocrine therapy resistance, a relatively understudied question of recent interest. We use in silico analysis of clinical datasets, in vitro experiments evaluating endocrine therapy resistance in response to DDR dysregulation in multiple breast cancer celllines, and in vivo validation using cellline xenograft and patient-derived xenograft models. We also use gene expression microarrays and RPPA data from cell lines, patient-derived xenografts and primary ER+ breast tumors to uncover therapeutic options that are validated in vitro and in vivo and corroborated by clinical trial data. The results of this study uncover an intriguing link between mismatch repair (MMR) deficiency, specifically of the MutL complex (MLH1/3, PMS1/2), and poor prognosis in ER+ disease. We find a direct role for MutL loss in endocrine therapy resistance in vitro and in vivo by knocking down multiple MutL genes using CRISPR and stable shRNA approaches validated using standard rescue experiments. We identify the underlying mechanism: MutL deficiency in ER+ breast cancer abrogates Chk2-mediated feedback inhibition of CDK4/6 that appears necessary for endocrine therapy responsiveness. Consequently, pharmacological targeting of CDK4/6 in vitro and in vivo significantly inhibits growth of endocrine therapy resistant MutL-deficient ER+ breast cancer cells. These results are corroborated by data from a neoadjuvant clinical trial demonstrating that cell cycle regulation of MutL-mutant tumors tends to be estrogen-independent but sensitive to CDK4/6 inhibitors. The results of this study provide important biological and clinically relevant insights. 1) MMR deficiency is unexpectedly causal to intrinsic endocrine therapy resistance 2) This causal effect appears to be mediated by abrogation of cell cycle checkpoint activation in response to endocrine therapy 3) MMR deficiency in a subset of ER+ tumors explains why CDK4/6 inhibition is effective against some de novo endocrine therapy resistant tumors. While there are currently no biomarkers to guide the use of CDK4/6 inhibitors for ER+ breast cancer, markers of MMR dysregulation could identify patients in whom CDK4/6 inhibition should be used to prevent disease recurrence. Citation Format: Svasti Haricharan, Jacob Schmelz, Cheryl Schmidt, Purba Singh, Kimberly R. Holloway, Meenakshi Anurag, Shunqiang Li, Shyam M. Kavuri, Shixia Huang, Dean P. Edwards, Vera Suman, Kelly Hunt, John A. Olson, Jeremy Hoog, Cynthia X. Ma, Matthew N. Bainbridge, Matthew J. Ellis. Mismatch repair defects and endocrine therapy resistance in estrogen receptor positive breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 489. doi:10.1158/1538-7445.AM2017-489