PRC2 Inactivation Induces Resistance to Chemotherapy-Induced Apoptosis By Upregulating the TRAP1 Mitochondrial Chaperone 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. Fully exploiting this finding will require unraveling the molecular genetics underlying phenotypic variability in mitochondrial priming.

We analyzed pre-treatment T-ALL clinical specimens from a cohort of 47 patients (enriched for treatment failure, but with sufficient controls) treated on the COG AALL0434 or DFCI 05001 clinical trials using BH3 profiling analysis to assess mitochondrial apoptotic priming. We found that there was a strong association between resistance to mitochondrial apoptosis and a poor response to induction chemotherapy (P = 0.008). Furthermore, mitochondrial apoptosis resistance predicted significantly inferior event-free survival (65% vs. 91% at 5 years; P = 0.0376).

To define the molecular determinants of this mitochondrial apoptosis resistance, we performed targeted exon sequencing and array CGH copy number analysis. This revealed that loss-of-function mutations in the polycomb repressive complex 2 (PRC2) core subunits (EZH2, EED or SUZ12) were associated with mitochondrial apoptosis resistance (P = 0.007) in clinical specimens. PRC2 is a chromatin modifying complex best known for its role in transcriptional repression, which functions as a tumor suppressor in T-ALL, but whether PRC2 regulates mitochondrial apoptosis is unknown.

Using shRNA knockdown in human T-ALL cells, we found that depletion of PRC2 subunits in T-ALL cells induced mitochondrial apoptosis resistance, as assessed by BH3 profiling analysis (P < 0.001). PRC2 inactivation also induced resistance to chemotherapy-induced apoptosis (P < 0.0001), and increased T-ALL fitness following treatment with the antileukemic drug vincristine (P = 0.0001). Apoptosis resistance upon inactivation of EZH2 (a PRC2 catalytic subunit) was reversed by transduction of wild-type EZH2, but not by an EZH2 point mutant with impaired methyltransferase activity, indicating that this effect is mediated by the enzymatic activity of PRC2. In normal mouse thymocytes, heterozygous deletion of the PRC2 subunits Ezh2 or Eed was sufficient to induce apoptosis resistance in non-transformed double-negative T-cell progenitors (P < 0.010), indicating that apoptosis resistance can arise prior to oncogenic transformation.

The best-known regulators of mitochondrial apoptosis are BCL2-family genes, but RNA-seq analysis of shRNA knockdown of the PRC2 subunits in a T-ALL cell line revealed that PRC2 did not regulate expression of any of the known BCL2 family members. Instead, PRC2 loss led to upregulation of TRAP1, a mitochondrially localized chaperone of the HSP90 family. TRAP1 upregulation was necessary for induction of apoptosis resistance following PRC2 inactivation, because shRNA knockdown of TRAP1 in the human CCRF-CEM cell line completely blocked induction of apoptosis resistance following PRC2 inactivation (P < 0.0001). Moreover, pharmacologic TRAP1 inhibition synergized with the antileukemic drugs dexamethasone and doxorubicin (combination index = 0.37 and 0.42, respectively).

To define how PRC2 regulates TRAP1, we performed ChIP-seq analysis, which revealed that TRAP1 regulation by PRC2 is indirect. Combined ChIP-seq and RNA-seq analysis revealed a number of direct targets of PRC2, all of which were tested for their ability to upregulate TRAP1 and induce apoptosis resistance. This showed that the LIM domain transcription factor CRIP2 is a direct target of PRC2 that is necessary and sufficient for regulation of TRAP1, and for induction of apoptosis resistance downstream of PRC2 inactivation. To confirm the relevance of our findings, we used the EZH2 inhibitor GSK126 to inhibit enzymatic activity of PRC2, which revealed that EZH2 normally represses CRIP2 and TRAP1 expression in primary patient-derived xenografts. Finally, we found that increased TRAP1 expression correlates with treatment failure in T-ALL clinical specimens (P = 0.028).

Taken together, our findings support a model in which loss of PRC2 induces transcriptional upregulation of its direct target CRIP2, which subsequently activates expression of TRAP1, leading to resistance to chemotherapy-induced mitochondrial apoptosis.