Abstract The tendency of mitochondria to undergo or resist BCL2-controlled apoptosis (so-called mitochondrial priming) is a powerful predictor of the outcome of cytotoxic chemotherapy for cancer. 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) and consequent downstream upregulation of the TRAP1 gene, which encodes a mitochondrial chaperone protein of the HSP90 family. In clinical samples from 47 T-ALL patients, 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, as assessed by BH3 profiling (P = 0.015). In human T-ALL cells, PRC2 depletion induced resistance to mitochondrial apoptosis induction, as assessed by caspase 3/7 activation or annexin V/PI staining, in response to multiple antileukemic drugs with distinct mechanisms of action, including dexamethasone, doxorubicin, vincristine, and asparaginase (P < 0.01). In mouse immature T-cell progenitors, haploinsufficiency for the PRC2 components Ezh2 or Eed was sufficient to induce resistance to mitochondrial apoptosis, as assessed by BH3 profiling analysis (P ≤ 0.01). PRC2 is a histone-modifying complex whose activity is strongly associated with transcriptional repression. We found that PRC2 represses transcription of TRAP1, a nuclearly encoded, mitochondrially localized chaperone of the HSP90 family. Importantly, TRAP1 overexpression was necessary to induce resistance to chemotherapy-induced apoptosis downstream of PRC2 inactivation (P < 0.001), while pharmacologic inhibition of TRAP1 synergized with antileukemic drugs in PRC2-deficient leukemic cells. 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. This abstract is also being presented as Poster 07. Citation Format: Ingrid Aries, Triona Ni Chonghaile, Salmaan Karim, Sebastian Balbach, Melissa Burns, Gayle Pouliot, Stevenson Kristen, Donna Neuberg, Meenakshi Devidas, Loh Mignon, Stephen Hunger, Stuart Winter, David Teachey, Karen Rabin, Kimberly Dunsmore, Brent Wood, Lewis Silverman, Stephen Sallan, Peter Van Vlierberghe, Stuart H. Orkin, Anthony G. Letai, Alejandro Gutierrez. Polycomb repressive complex 2 inactivation induces primary chemotherapy resistance in T-ALL by upregulating the TRAP1 mitochondrial chaperone [abstract]. In: Proceedings of the Second AACR Conference on Hematologic Malignancies: Translating Discoveries to Novel Therapies; May 6-9, 2017; Boston, MA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(24_Suppl):Abstract nr PR14.
SUMMARY Resistance to asparaginase, an antileukemic enzyme that depletes asparagine, is a common clinical problem. Using a genome-wide CRISPR/Cas9 screen, we found a synthetic lethal interaction between Wnt pathway activation and asparaginase in acute leukemias resistant to this enzyme. Wnt pathway activation induced asparaginase sensitivity in distinct treatment-resistant subtypes of acute leukemia, including T-lymphoblastic, hypodiploid B-lymphoblastic, and acute myeloid leukemias, but not in normal hematopoietic progenitors. Sensitization to asparaginase was mediated by Wnt-dependent stabilization of proteins (Wnt/STOP), which inhibits GSK3-dependent protein ubiquitination and degradation. Inhibiting the alpha isoform of GSK3 phenocopied this effect, and pharmacologic GSK3α inhibition profoundly sensitized drug-resistant leukemias to asparaginase. Our findings provide a molecular rationale for activation of Wnt/STOP signaling to improve the therapeutic index of asparaginase. SIGNIFICANCE The intensification of asparaginase-based therapy has improved outcomes for several subtypes of acute leukemia, but the development of treatment resistance has a poor prognosis. We hypothesized, from the concept of synthetic lethality, that gain-of-fitness alterations in drug-resistant cells had conferred a survival advantage that could be exploited therapeutically. We found a synthetic lethal interaction between activation of Wnt-dependent stabilization of proteins (Wnt/STOP) and asparaginase in acute leukemias resistant to this enzyme. Inhibition of the alpha isoform of GSK3 was sufficient to phenocopy this effect, and the combination of GSK3α-selective inhibitors and asparaginase had marked therapeutic activity against leukemias resistant to monotherapy with either agent. These data indicate that drug-drug synthetic lethal interactions can improve the therapeutic index of cancer therapy.
Abstract Background: Fetal haemoglobin (HbF) remains a major sickle cell disease modifier. The mechanism of HbF synthesis has been studied for several decades with the intention of increasing interventions for sickle cell disease (SCD), including drugs. However, the complex mechanism of HbF synthesis is influenced by multiple genetic factors interacting with environmental factors. In order to capture useful genetic information, especially with limited resources, one has to carefully design the study. This includes choosing the relevant participants, the correct phenotyping, the choice of samples, and the right genomic assays. This paper describes the approach undertaken as part of preparations for a reticulocyte transcriptome study intended to discover genes associated with HbF decline in newborns in Tanzania. Results: Of the 152 newborns enrolled in the larger study, 40 babies were selected for the reticulocyte transcriptome study based on their HbF levels at birth and later stage of life. Of these, 30 individuals were included under the category of high HbF levels ranging from 72.6-90% and the remaining 10 under the category of low HbF levels ranging from 5.9 - 10.3%. The reticulocyte enrichment recovery purity ranged from 85% - 97%. The total RNA concentrations obtained were >250 ng total RNA, with the average purity of 1.9 (A 260/280) respectively. The total concentration obtained was sufficient for the transcriptome and other downstream assays. Conclusion: We have documented important steps and factors to consider in identifying the relevant participants and required laboratory sample processes prior to the final stage, which involves total reticulocyte RNA sequencing.
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. 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 specimens, loss-of-function mutations of PRC2 core components (
