Abstract
Although contemporary combination chemotherapy can cure a substantial fraction of patients with T-cell acute lymphoblastic leukemia (T-ALL), front-line therapy fails in 15-20% of children and 50-70% of adults, and these patients have a poor prognosis. Strikingly, half of treatment failure events in childhood T-ALL are induction failure, suggesting pre-existing resistance to chemotherapeutics with distinct molecular targets. The molecular basis for induction failure remains poorly understood. Recent work has shown that mitochondrial apoptosis resistance is a cellular phenotype that predicts chemotherapy failure in some tumor types. However, the molecular mechanisms responsible for the striking variability in chemotherapy response among different patients with seemingly identical tumors remain largely unknown.
Using a technique known as BH3 profiling, we analyzed mitochondrial apoptosis sensitivity or resistance in pre-treatment clinical specimens from a cohort of 47 children and adolescents treated on the COG AALL0434 or DFCI 05001 clinical trials. We found that mitochondrial apoptosis resistance was strongly associated with a poor response to induction chemotherapy (P = 0.008), as well as inferior 5-year event-free survival (65% vs 88%; P = 0.036 by log-rank test). Apoptosis resistance was weakly associated with the early T-cell precursor (ETP) immunophenotype (P = 0.08), but univariate and multivariable Cox regression analysis including both revealed that apoptosis resistance predicts clinical outcome more strongly than ETP status.
To identify molecular lesions underlying mitochondrial apoptosis resistance, we applied targeted exome sequencing and array CGH to this cohort. We found that loss-of-function mutations in genes encoding core components of the polycomb repressive complex 2 (PRC2), including EZH2, EED or SUZ12, are associated with resistance to mitochondrial apoptosis (P = 0.015). PRC2 is a chromatin-modifying complex best known for its role in transcriptional repression. The PRC2 complex has been implicated as a tumor suppressor in T-ALL, but whether PRC2 plays a direct role in chemotherapy response is unknown.
To test whether PRC2 regulates mitochondrial apoptosis in human T-ALL, we performed shRNA knockdown of the PRC2 core components EZH2, EED or SUZ12 in human T-ALL cell lines. Knockdown of each of these genes significantly induced mitochondrial apoptosis resistance, as assessed by BH3-profiling. This effect was dependent on the lysine methyltransferase activity of the PRC2 complex, because the effect of EZH2 knock-down was rescued by expression of wild-type EZH2, but not a point mutant that is methyltransferase-defective (P < 0.001). PRC2 knockdown also induced significant resistance to apoptosis induction (assessed using caspase 3/7 activation or annexin V/PI staining) in response to various chemotherapeutics with distinct molecular targets, including vincristine, dexamethasone, asparaginase, methotrexate, mercaptopurine, nelarabine, cytarabine and etoposide.
To test whether PRC2 regulates mitochondrial apoptosis during normal T-cell development, we took advantage of mice heterozygous for a floxed Ezh2 or Eed allele, and induced deletion of one allele in hematopoietic cells using Mx-Cre activation by pIpC. Controls were Ezh2 and Eed wild-type mice with Mx-Cre activation. BH3 profiling analysis revealed that loss of one Ezh2 or Eed allele is sufficient to induce apoptosis resistance in non-transformed double-negative thymocytes (P = 0.003 for Ezh2 and P = 0.008 for Eed), suggesting that chemotherapy resistance can develop prior to oncogenic transformation.
To define the transcriptional consequences of PRC2 inhibition in T-ALL, we performed RNA sequencing of T-ALL cells infected with shRNAs targeting EZH2, EED or SUZ12 (2 independent hairpins for each gene), or two control shRNAs. RNA sequencing analysis revealed a number of candidate transcriptional targets linking PRC2 to the mitochondrial apoptotic machinery, which are currently being investigated using functional genetics and small molecule inhibitors.
Collectively, these data implicate polycomb repressive complex 2 function as a key determinant of chemotherapy response in childhood T-ALL. Defining the mechanism linking PRC2 to the mitochondria will provide a rational target for therapeutic intervention.
Disclosures
Teachey: Novartis: Research Funding. Letai:AbbVie: Consultancy, Research Funding; Tetralogic: Consultancy, Research Funding; Astra-Zeneca: Consultancy, Research Funding.
PRC2 loss induces chemoresistance by repressing apoptosis in T cell acute lymphoblastic leukemia
