Ctr9 as a Predictive Biomarker for EZH2 Inhibitor Sensitivity

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PROJECT SUMMARY Approximately 70% of breast cancers (BC) are estrogen receptor (ER)-positive, also characterized as luminal- type BC; thus, are treated with endocrine therapy (ET). The transition from ER+ luminal cells to ER-negative ‘stem-like cells’ (SLCs) is a major mechanism of ET resistance; however, the underlying mechanism remains largely unknown. We discovered that Ctr9, a subunit of PAFc transcriptional activator complex, is a determi- nant of luminal cell identity. Using inducible and stable Ctr9 knockdown (KD) BC cell lines, a substantial decrease of ER stability and increase of H3K27me3, a repressive histone mark on chromatin, were observed upon Ctr9 depletion, indicating of a transition from luminal BC cells to SLCs. The cellular plasticity manifested by Ctr9 engages two downstream effector proteins, Jarid2 and KDM6A, that control the levels of repressive histone mark, H3K27me3. Ctr9 silencing leads to decreased Jarid2, a subunit of PRC2, and triggers a PRC2 subtype switch from less active PRC2.2 to PRC2.1 with higher H3K27me3 activity. In addition to enhancing PRC2 H3K27me3 deposition activity, Ctr9 KD results in a decrease of KDM6A, the ‘eraser’ enzyme for H3K27me3. Furthermore, Ctr9 depletion generates vulnerability that renders BC cells hypersensitive to EZH2 inhibitors (EZHi). EZH2 is highly expressed in SLCs for stem cell maintenance and expansion. Recently, tazemetostat, an EZH2i, is FDA-approved for the treatment of follicular lymphoma. We hypothesize that loss of Ctr9 leads to the transition from a Ctr9-expressing luminal lineage to an EZH2-governed SLC, thus the cells become sensitive to EZH2i. We will test our hypothesis by pursuing three aims: (1) Dissect how Ctr9 depletion results in transition from ER+ luminal cells to SLCs; (2) Determine the roles of Jarid2 and KDM6A during luminal to SLC transition and sensitivity to EZH2i; (3) Determine whether Ctr9 and H3K27me3 are predictive biomarkers for EZH2i sensitivity in vivo and study the inverse correlation of Ctr9 levels with H3K27me3 in human specimens. Because of the higher levels of expression of EZH2 in ER-negative BC as compared to ER+ BC, EZH2 inhibition has been extensively investigated in ER-negative BC. The study is of high impact because our findings that Ctr9 demarcates EZH2 activity and H3K27me3 levels in luminal cells opens an excit- ing possibility to target Ctr9low/H3K27me3high ER+ BC using EZH2i. Successful completion of this study will justify a clinical trial to use Ctr9/H3K27me3 as independent biomarker to select ER+ BC patients for treatment with EZH2i.