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Purpose: Although tyrosine kinase inhibitors (TKI) can be effective therapies for leukemia, they fail to fully eliminate leukemic cells and achieve durable remissions for many patients with advanced BCR-ABL+ leukemias or acute myelogenous leukemia (AML). Through a large-scale synthetic lethal RNAi screen, we identified pyruvate dehydrogenase, the limiting enzyme for pyruvate entry into the mitochondrial tricarboxylic acid cycle, as critical for the survival of chronic myelogenous leukemia (CML) cells upon BCR-ABL inhibition. Here, we examined the role of mitochondrial metabolism in the survival of Ph+ leukemia and AML upon TK inhibition. Experimental Design: Ph+ cancer cell lines, AML cell lines, leukemia xenografts, cord blood, and patient samples were examined. Results: We showed that the mitochondrial ATP-synthase inhibitor oligomycin-A greatly sensitized leukemia cells to TKI in vitro. Surprisingly, oligomycin-A sensitized leukemia cells to BCR-ABL inhibition at concentrations of 100- to 1,000-fold below those required for inhibition of respiration. Oligomycin-A treatment rapidly led to mitochondrial membrane depolarization and reduced ATP levels, and promoted superoxide production and leukemia cell apoptosis when combined with TKI. Importantly, oligomycin-A enhanced elimination of BCR-ABL+ leukemia cells by TKI in a mouse model and in primary blast crisis CML samples. Moreover, oligomycin-A also greatly potentiated the elimination of FLT3-dependent AML cells when combined with an FLT3 TKI, both in vitro and in vivo. Conclusions: TKI therapy in leukemia cells creates a novel metabolic state that is highly sensitive to particular mitochondrial perturbations. Targeting mitochondrial metabolism as an adjuvant therapy could therefore improve therapeutic responses to TKI for patients with BCR-ABL+ and FLT3ITD leukemias. Clin Cancer Res; 21(6); 1360–72. ©2014 AACR.

Activating mutations in FMS-like tyrosine kinase 3 (FLT3) are common in acute myeloid leukemia (AML) and drive leukemic cell growth and survival. Although FLT3 inhibitors have shown considerable promise for the treatment of AML, they ultimately fail to achieve long-term remissions as monotherapy. To identify genetic targets that can sensitize AML cells to killing by FLT3 inhibitors, we performed a genome-wide RNA interference (RNAi)-based screen that identified ATM (ataxia telangiectasia mutated) as being synthetic lethal with FLT3 inhibitor therapy. We found that inactivating ATM or its downstream effector glucose 6-phosphate dehydrogenase (G6PD) sensitizes AML cells to FLT3 inhibitor induced apoptosis. Examination of the cellular metabolome showed that FLT3 inhibition by itself causes profound alterations in central carbon metabolism, resulting in impaired production of the antioxidant factor glutathione, which was further impaired by ATM or G6PD inactivation. Moreover, FLT3 inhibition elicited severe mitochondrial oxidative stress that is causative in apoptosis and is exacerbated by ATM/G6PD inhibition. The use of an agent that intensifies mitochondrial oxidative stress in combination with a FLT3 inhibitor augmented elimination of AML cells in vitro and in vivo, revealing a therapeutic strategy for the improved treatment of FLT3 mutated AML.

Background We investigated the utility of bioluminescence imaging (BLI) using firefly luciferase in monoclonal and polyclonal populations of leukemia cells in vitro and in vivo. Methods Monoclonal and polyclonal human lymphoid and myeloid leukemia cell lines transduced with firefly luciferase were used for BLI. Results Kinetics and dynamics of bioluminescence signal were cell line dependent. Luciferase expression decreased significantly over time in polyclonal leukemia cells in vitro. Transplantation of polyclonal luciferase-tagged cells in mice resulted in inconsistent signal intensity. After selection of monoclonal cell populations, luciferase activity was stable, equal kinetic and dynamic of bioluminescence intensity and strong correlation between cell number and light emission in vitro were observed. We obtained an equal development of leukemia burden detected by luciferase activity in NOD-scid-gamma mice after transplantation of monoclonal populations. Conclusion The use of monoclonal leukemia cells selected for stable and equal luciferase activity is recommended for experiments in vitro and xenograft mouse models. The findings are highly significant for bioluminescence imaging focused on pre-clinical drug development.

Acute myeloid leukemia (AML) is the most common adult acute leukemia and accounts for approximately 20% of childhood leukemias. Although frontline treatment of AML with cytotoxic chemotherapy is capable of achieving high remission rates, 75-80% of patients will either not respond to or will relapse after initial therapy, and most patients will die of their disease. More effective and better-tolerated therapies for AML are required. Activating mutations in fms-like tyrosine kinase 3 (FLT3) are the most frequently observed genetic defect in AML and drive leukemic cell growth and survival. FLT3 tyrosine kinase inhibitors have shown impressive anti-leukemic activity in clinical trials, however, sustained remissions using these inhibitors as monotherapy have not been achieved. In order to identify genetic targets that can sensitize AML cells to killing by FLT3 inhibitors, we performed a large-scale RNA interference-based screen. The screen identified several genes involved in metabolic regulation, including ataxia telangiectasia mutated (ATM), as being synthetic lethal with FLT3 inhibition in FLT3 mutated AML. Genetic or pharmacological inactivation of ATM or its downstream effector glucose-6-phosphate dehydrogenase (G6PD) sensitized AML cells to FLT3 inhibition through enhancing apoptosis. Whole metabolome profiling revealed that FLT3 inhibition causes severe and widespread metabolic deficiencies, including depletion of the antioxidant factor glutathione. Inactivation of either ATM or G6PD exacerbated glutathione depletion upon FLT3 inhibition. Subsequent analyses revealed that FLT3 inhibition elicits severe mitochondrial oxidative stress that is causative in apoptosis and accentuated by ATM or G6PD inhibition. The use of a drug that promotes the production of mitochondrial reactive oxygen species (ROS) in combination with a FLT3 inhibitor augmented elimination of AML cells both in vitro and in vivo. Our data support the hypothesis that FLT3 mutated AML cells are highly dependent on FLT3 activity to maintain glutathione levels and sufficient mitochondrial antioxidant capacity to sustain cell survival. Moreover, these data support the novel strategy of employing mitochondrial ROS inducing agents as adjuvant to FLT3 inhibitor therapy to more effectively treat FLT3 mutated AML. Citation Format: Mark A. Gregory, Angelo D'Alessandro, Francesca Alvarez-Calderon, Jihye Kim, Travis Nemkov, Aik Choon Tan, Kirk C. Hansen, James DeGregori. ATM/G6PD-dependent metabolic pathways promote mitochondrial redox homeostasis and resistance to FLT3 inhibition in acute myeloid leukemia. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr C78.

Bcr-Abl kinase inhibitors are the standard therapy for chronic myelogenous leukemia (CML). While effective in controlling disease in chronic phase, these inhibitors often fail to completely eliminate Bcr-Abl+ CML cells and also fail to achieve durable remissions for advanced CML and Bcr-Abl+ acute lymphoblastic leukemias (ALL). Our lab employed a large-scale synthetic lethal shRNA screen to identify genes involved in CML survival upon treatment with the Bcr-Abl kinase inhibitor imatinib (IM) [Cancer Cell. 2010;18(1):74-87]. This screen identified several genes involved in energy utilization including a component of pyruvate dehydrogenase, dihydrolipoyllysine-residue acetyltransferase. CML proliferation depends on Bcr-Abl-driven glycolysis for energy production. Upon IM treatment, we hypothesized that these cells are forced to rely on other sources of energy including the TCA cycle and the electron transport chain, and that targeting alternative energy production pathways will enhance IM-mediated elimination of CML cells. Thus, we predicted that inhibition of mitochondrial respiration by knockdown or pharmacologic inhibition would cooperate with tyrosine kinase inhibitors (TKI) to eliminate Bcr-Abl+ leukemia. Indeed, our experiments have shown that knocking down pyruvate dehydrogenase in human blast crisis Bcr-Abl+ CML cells (K562) greatly sensitized cells to IM in vitro. Conversely, supplementing the cell culture media with increasing concentrations of methyl-pyruvate protected K562 cells from IM treatment. Moreover, we found that the mitochondrial ATP-synthase inhibitor Oligomycin A (OA) also synergized with IM to kill CML cells in vitro. Interestingly, the doses of OA are far below the IC50 and do not affect the oxygen consumption rates, indicating that mitochondrial respiration is not inhibited. These OA doses alter mitochondrial transmembrane potential and increasing superoxide levels. In addition, OA enhanced elimination of Bcr-Abl+ cells by a Bcr-Abl TKI in vivo and prolonged disease-free survival in a mouse model of Bcr-Abl+ B-ALL. Notably, drug doses of OA used had no apparent adverse effects on these mice. Targeting mitochondrial metabolism as an adjuvant drug therapy may prevent the survival of residual Bcr-Abl+ CML cells upon tyrosine kinase inhibitor treatment. This project is supported by pre-doctoral fellowship number F31CA157166 from the National Cancer Institute, and Leukemia and Lymphoma Society Grant Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1127. doi:1538-7445.AM2012-1127

Bcr-Abl kinase inhibitors are the standard therapy for chronic myelogenous leukemia (CML). While effective in controlling disease in chronic phase, these inhibitors often fail to completely eliminate Bcr-Abl+ CML cells and also fail to achieve durable remissions for advanced CML and Bcr-Abl+ acute lymphoblastic leukemias (ALL). Our lab employed a large-scale synthetic lethal shRNA screen to identify genes involved in CML survival upon treatment with the Bcr-Abl kinase inhibitor imatinib (IM) [Cancer Cell. 2010;18(1):74-87]. This screen identified several genes involved in energy utilization including a component of pyruvate dehydrogenase, dihydrolipoyllysine-residue acetyltransferase. This target was identified by two independent shRNAs, each underrepresented over 22 fold in treated cells compared to untreated. CML proliferation depends on Bcr-Abl-driven glycolysis for energy production. Upon IM treatment, I hypothesized that these cells are forced to rely on other sources of energy including the TCA cycle and mitochondrial respiration, and that targeting alternative energy production pathways will enhance IM-mediated elimination of CML cells. Thus, I predicted that inhibition of mitochondrial respiration by knockdown or pharmacologic inhibition would cooperate with tyrosine kinase inhibitors to eliminate Bcr-Abl+ leukemias. My experiments have shown that knocking down pyruvate dehydrogenase in human blast crisis Bcr-Abl+ CML cells (K562) greatly sensitizes cells to IM in vitro. Conversely, supplementing the cell culture media with increasing concentrations of methyl-pyruvate protects K562 cells from IM treatment. Moreover, I find that the mitochondrial ATP-synthase inhibitor Oligomycin A also synergizes with IM to kill CML cells in vitro. In addition, inhibition of mitochondrial respiration with Oligomycin A enhances elimination of Bcr-Abl+ cells by a Bcr-Abl tyrosine kinase inhibitor in vivo and prolongs disease-free survival in a mouse model of Bcr-Abl+ B-ALL. The drug doses of Oligomycin A used are over 100-fold below the LD50, and have no apparent adverse effects on these mice. Targeting mitochondrial metabolism as an adjuvant drug therapy may prevent the survival of residual Bcr-Abl+ CML cells upon tyrosine kinase inhibitor treatment. *This project is supported in part by pre-doctoral fellowship number F31CA157166 from the National Cancer Institute. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3796. doi:10.1158/1538-7445.AM2011-3796

Summary Although Bcr-Abl kinase inhibitors have proven effective in the treatment of chronic myeloid leukemia (CML), they generally fail to eradicate Bcr-Abl + leukemia cells. To identify genes whose inhibition sensitizes Bcr-Abl + leukemias to killing by Bcr-Abl inhibitors, we performed an RNAi-based synthetic lethal screen with imatinib mesylate in CML cells. This screen identified numerous components of a Wnt/Ca 2+ /NFAT signaling pathway. Antagonism of this pathway led to impaired NFAT activity, decreased cytokine production, and enhanced sensitivity to Bcr-Abl inhibition. Furthermore, NFAT inhibition with cyclosporin A facilitated leukemia cell elimination by the Bcr-Abl inhibitor dasatinib and markedly improved survival in a mouse model of Bcr-Abl + acute lymphoblastic leukemia (ALL). Targeting this pathway in combination with Bcr-Abl inhibition could improve treatment of Bcr-Abl + leukemias.