Mitochondrial Apoptosis Resistance Research Articles

PP2A-based triple-strike therapy overcomes mitochondrial apoptosis resistance in brain cancer cells.

Mitochondrial glycolysis and hyperactivity of the phosphatidylinositol 3-kinase-protein kinase B (AKT) pathway are hallmarks of malignant brain tumors. However, kinase inhibitors targeting AKT (AKTi) or the glycolysis master regulator pyruvate dehydrogenase kinase (PDKi) have failed to provide clinical benefits for brain tumor patients. Here, we demonstrate that heterogeneous glioblastoma (GB) and medulloblastoma (MB) cell lines display only cytostatic responses to combined AKT and PDK targeting. Biochemically, the combined AKT and PDK inhibition resulted in the shutdown of both target pathways and priming to mitochondrial apoptosis but failed to induce apoptosis. In contrast, all tested brain tumor cell models were sensitive to a triplet therapy, in which AKT and PDK inhibition was combined with the pharmacological reactivation of protein phosphatase 2A (PP2A) by NZ-8-061 (also known as DT-061), DBK-1154, and DBK-1160. We also provide proof-of-principle evidence for in vivo efficacy in the intracranial GB and MB models by the brain-penetrant triplet therapy (AKTi + PDKi + PP2A reactivator). Mechanistically, PP2A reactivation converted the cytostatic AKTi + PDKi response to cytotoxic apoptosis, through PP2A-elicited shutdown of compensatory mitochondrial oxidative phosphorylation and by increased proton leakage. These results encourage the development of triple-strike strategies targeting mitochondrial metabolism to overcome therapy tolerance in brain tumors.

JAK3 mutations and mitochondrial apoptosis resistance in T-cell acute lymphoblastic leukemia.

Resistance to mitochondrial apoptosis predicts inferior treatment outcomes in patients with diverse tumor types, including T-cell acute lymphoblastic leukemia (T-ALL). However, the genetic basis for variability in this mitochondrial apoptotic phenotype is poorly understood, preventing its rational therapeutic targeting. Using BH3 profiling and exon sequencing analysis of childhood T-ALL clinical specimens, we found that mitochondrial apoptosis resistance was most strongly associated with activating mutations of JAK3. Mutant JAK3 directly repressed apoptosis in leukemia cells, because its inhibition with mechanistically distinct pharmacologic inhibitors resulted in reversal of mitochondrial apoptotic blockade. Inhibition of JAK3 led to loss of MEK, ERK and BCL2 phosphorylation, and BH3 profiling revealed that JAK3-mutant primary T-ALL patient samples were characterized by a dependence on BCL2. Treatment of JAK3-mutant T-ALL cells with the JAK3 inhibitor tofacitinib in combination with a spectrum of conventional chemotherapeutics revealed synergy with glucocorticoids, in vitro and in vivo. These findings thus provide key insights into the molecular genetics of mitochondrial apoptosis resistance in childhood T-ALL, and a compelling rationale for a clinical trial of JAK3 inhibitors in combination with glucocorticoids for patients with JAK3-mutant T-ALL.

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. DisclosuresAries:Pfizer: Employment. Teachey:Amgen: Consultancy; La Roche: Consultancy. Letai:AstraZeneca: Consultancy, Other: Lab research report; AbbVie: Consultancy, Other: Lab research report; Flash Therapeutics: Equity Ownership; Novartis: Consultancy, Other: Lab research report; Vivid Biosciences: Equity Ownership.

PRC2 loss induces chemoresistance by repressing apoptosis in T cell acute lymphoblastic leukemia.

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 (EZH2, EED, or SUZ12) were associated with mitochondrial apoptosis resistance. In T-ALL cells, PRC2 depletion induced resistance to apoptosis induction by multiple chemotherapeutics with distinct mechanisms of action. PRC2 loss induced apoptosis resistance via transcriptional up-regulation of the LIM domain transcription factor CRIP2 and downstream up-regulation of the mitochondrial chaperone TRAP1 These findings demonstrate the importance of mitochondrial apoptotic priming as a prognostic factor in T-ALL and implicate mitochondrial chaperone function as a molecular determinant of chemotherapy response.

The novel TRAIL-receptor agonist APG350 exerts superior therapeutic activity in pancreatic cancer cells

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has raised attention as a novel anticancer therapeutic as it induces apoptosis preferentially in tumor cells. However, first-generation TRAIL-receptor agonists (TRAs), comprising recombinant TRAIL and agonistic receptor-specific antibodies, have not demonstrated anticancer activity in clinical studies. In fact, cancer cells are often resistant to conventional TRAs. Therefore, in addition to TRAIL-sensitizing strategies, next-generation TRAs with superior apoptotic activity are warranted. APG350 is a novel, highly potent TRAIL-receptor agonist with a hexavalent binding mode allowing the clustering of six TRAIL-receptors per drug molecule. Here we report on preclinical in vitro and in vivo studies testing the activity of APG350 on pancreatic ductal adenocarcinoma (PDAC) cells. We found that APG350 potently induced apoptosis of Colo357, PancTuI and Panc89 cells in vitro. In addition, APG350 treatment activated non-canonical TRAIL signaling pathways (MAPK, p38, JNK, ERK1/ERK2 and NF-κB) and induced the secretion of IL-8. Stable overexpression of Bcl-xL inhibited APG350-induced cell death and augmented activation of non-canonical pathways. Intriguingly, pre-treatment of Bcl-xL-overexpressing cells with the BH3-mimic Navitoclax restored their sensitivity to APG350. To study the effects of APG350 on PDAC cells in vivo, we applied two different orthotopic xenotransplantation mouse models, with and without primary tumor resection, representing adjuvant and palliative treatment regimes, respectively. APG350 treatment of established tumors (palliative treatment) significantly reduced tumor burden. These effects, however, were not seen in tumors with enforced overexpression of Bcl-xL. Upon primary tumor resection and subsequent APG350 treatment (adjuvant therapy), APG350 limited recurrent tumor growth and metastases. Importantly, therapeutic efficacy of APG350 treatment was more effective compared with treatment with soluble TRAIL in both models. In conclusion, APG350 represents a promising next-generation TRA for the treatment of PDAC. Moreover, our results suggest that combining APG350 with Navitoclax might be a succesfull strategy for cancers harboring mitochondrial apoptosis resistance.

Abstract PR14: Polycomb repressive complex 2 inactivation induces primary chemotherapy resistance in T-ALL by upregulating the TRAP1 mitochondrial chaperone

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 &amp;lt; 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 &amp;lt; 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.

PRC2 Mutations Induce Resistance to Conventional Chemotherapy By Inhibiting Mitochondrial Apoptosis in T-Cell Acute Lymphoblastic Leukemia

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. DisclosuresTeachey:Novartis: Research Funding. Letai:AbbVie: Consultancy, Research Funding; Tetralogic: Consultancy, Research Funding; Astra-Zeneca: Consultancy, Research Funding.