GSE112147
PRC2 Inactivation Induces Primary Chemoresistance by Repressing Mitochondrial Apoptosis in T-ALL
The tendency of mitochondria to undergo or resist BCL2-controlled apoptosis (so-called mitochondrial priming) is a powerful predictor of response to cytotoxic chemotherapy.  To fully exploit this finding, it will be important to understand the molecular genetic contexts responsible for the relative mitochondrial priming of chemotherapy-sensitive versus resistant cell populations.   Here, we report that mitochondrial apoptosis resistance in T-cell acute lymphoblastic leukemia (T-ALL) is mediated by inactivation of polycomb repressive complex 2 (PRC2).  In T-ALL clinical samples from children with T-ALL treated on recent Dana-Farber Cancer Institute or Children's Oncology Group clinical trials, we found that loss-of-function mutations in any of three core components of PRC2 (EZH2, EED or SUZ12) were associated with resistance to mitochondrial apoptosis.  In human T-ALL cells, PRC2 depletion induced resistance to apoptosis induction by multiple chemotherapeutics with distinct mechanisms of action, including dexamethasone, doxorubicin and vincristine.  PRC2 loss induced apoptosis resistance via transcriptional upregulation of the LIM domain transcription factor CRIP2, and subsequent downstream upregulation of the mitochondrial chaperone TRAP1.  Importantly, TRAP1 overexpression was necessary to induce resistance to chemotherapy-induced apoptosis downstream of PRC2 inactivation, and pharmacologic inhibition of TRAP1 synergized with dexamethasone and doxorubicin.  These findings demonstrate the importance of relative mitochondrial apoptotic priming as a prognostic factor in T-ALL, and implicate mitochondrial chaperone function as a molecular determinant of response to cancer chemotherapy, suggesting a rationale for targeted therapeutic intervention. Overall design: CCRF-CEM cells transduced with shRNAs targeting control (Luciferase) or EZH2 were cross-linked using formaldehyde, and sonication was performed to fragment chromatin to 200-700 bp size range using a Covaris E220 Focused-ultrasonicator.  Immunoprecipitation was then performed on 5 million cells with anti-H3K27me3 antibody (Active Motif Cat #39155). Protein A/G dynabeads were used to pull down antibody-bound chromatin, followed by washing and elution.  Cross-linking was reversed with proteinase K treatment, and DNA was cleaned up using SPRI beads.  ChIP DNA was quantified using the Qubit system (Invitrogen).  Sequencing libraries for ChIP-seq were prepared according to Illumina's instructions.  ChIP DNA and input were sequenced with the Illumina NextSeq 500 platfom.