ATM/G6PD-driven redox metabolism promotes FLT3 inhibitor resistance in acute myeloid leukemia.

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.

Domain-specific c-Myc ubiquitylation controls c-Myc transcriptional and apoptotic activity.

The oncogenic transcription factor c-Myc causes transformation and tumorigenesis, but it can also induce apoptotic cell death. Although tumor suppressors are necessary for c-Myc to induce apoptosis, the pathways and mechanisms are unclear. To further understand how c-Myc switches from an oncogenic protein to an apoptotic protein, we examined the mechanism of p53-independent c-Myc-induced apoptosis. We show that the tumor suppressor protein ARF mediates this switch by inhibiting ubiquitylation of the c-Myc transcriptional domain (TD). Whereas TD ubiquitylation is critical for c-Myc canonical transcriptional activity and transformation, inhibition of ubiquitylation leads to the induction of the noncanonical c-Myc target gene, Egr1, which is essential for efficient c-Myc-induced p53-independent apoptosis. ARF inhibits the interaction of c-Myc with the E3 ubiquitin ligase Skp2. Overexpression of Skp2, which occurs in many human tumors, inhibits the recruitment of ARF to the Egr1 promoter, leading to inhibition of c-Myc-induced apoptosis. Therapeutic strategies could be developed to activate this intrinsic apoptotic activity of c-Myc to inhibit tumorigenesis.

Distributed efficient similarity search mechanism in wireless sensor networks.

The Wireless Sensor Network similarity search problem has received considerable research attention due to sensor hardware imprecision and environmental parameter variations. Most of the state-of-the-art distributed data centric storage (DCS) schemes lack optimization for similarity queries of events. In this paper, a DCS scheme with metric based similarity searching (DCSMSS) is proposed. DCSMSS takes motivation from vector distance index, called iDistance, in order to transform the issue of similarity searching into the problem of an interval search in one dimension. In addition, a sector based distance routing algorithm is used to efficiently route messages. Extensive simulation results reveal that DCSMSS is highly efficient and significantly outperforms previous approaches in processing similarity search queries.

Inhibition of calcineurin combined with dasatinib has direct and indirect anti-leukemia effects against BCR-ABL1(+) leukemia.

Treatment of BCR-ABL1(+) leukemia has been revolutionized with the development of tyrosine kinase inhibitors. However, patients with BCR-ABL1(+) acute lymphoblastic leukemia and subsets of patients with chronic myeloid leukemia are at high risk of relapse despite kinase inhibition therapy, necessitating novel treatment strategies. We previously reported synthetic lethality in BCR-ABL1(+) leukemia cells by blocking both calcineurin/NFAT signaling and BCR-ABL1, independent of drug efflux inhibition by cyclosporine. Here, using RNA-interference we confirm that calcineurin inhibition sensitizes BCR-ABL1(+) cells to tyrosine kinase inhibition in vitro. However, when we performed pharmacokinetic and pharmacodynamic studies of dasatinib and cyclosporine in mice, we found that co-administration of cyclosporine increases peak concentrations and the area under the curve of dasatinib, which contributes to the enhanced disease control. We also report the clinical experience of two subjects in whom we observed more hematopoietic toxicity than expected while enrolled in a Phase Ib trial designed to assess the safety and tolerability of adding cyclosporine to dasatinib in humans. Thus, the anti-leukemia benefit of co-administration of cyclosporine and dasatinib is mechanistically pleiotropic, but may not be tolerable, at least as administered in this trial. These data highlight some of the challenges associated with combining targeted agents to treat leukemia.

Therapeutic resistance in acute myeloid leukemia cells is mediated by a novel ATM/mTOR pathway regulating oxidative phosphorylation.

While leukemic cells are susceptible to various therapeutic insults, residence in the bone marrow microenvironment typically confers protection from a wide range of drugs. Thus, understanding the unique molecular changes elicited by the marrow is of critical importance toward improving therapeutic outcomes. In this study, we demonstrate that aberrant activation of oxidative phosphorylation serves to induce therapeutic resistance in FLT3 mutant human AML cells challenged with FLT3 inhibitor drugs. Importantly, our findings show that AML cells are protected from apoptosis following FLT3 inhibition due to marrow-mediated activation of ATM, which in turn upregulates oxidative phosphorylation via mTOR signaling. mTOR is required for the bone marrow stroma-dependent maintenance of protein translation, with selective polysome enrichment of oxidative phosphorylation transcripts, despite FLT3 inhibition. To investigate the therapeutic significance of this finding, we tested the mTOR inhibitor everolimus in combination with the FLT3 inhibitor quizartinib in primary human AML xenograft models. While marrow resident AML cells were highly resistant to quizartinib alone, the addition of everolimus induced profound reduction in tumor burden and prevented relapse. Taken together, these data provide a novel mechanistic understanding of marrow-based therapeutic resistance and a promising strategy for improved treatment of FLT3 mutant AML patients.

p19ARF directly and differentially controls the functions of c-Myc independently of p53.

Increased expression of the oncogenic transcription factor c-Myc causes unregulated cell cycle progression. c-Myc can also cause apoptosis, but it is not known whether the activation and/or repression of c-Myc target genes mediates these diverse functions of c-Myc. Because unchecked cell cycle progression leads to hyperproliferation and tumorigenesis, it is essential for tumour suppressors, such as p53 and p19ARF (ARF), to curb cell cycle progression in response to increased c-Myc (refs 2, 3). Increased c-Myc has previously been shown to induce ARF expression, which leads to cell cycle arrest or apoptosis through the activation of p53 (ref. 4). Here we show that ARF can inhibit c-Myc by a unique and direct mechanism that is independent of p53. When c-Myc increases, ARF binds with c-Myc and dramatically blocks c-Myc's ability to activate transcription and induce hyperproliferation and transformation. In contrast, c-Myc's ability to repress transcription is unaffected by ARF and c-Myc-mediated apoptosis is enhanced. These differential effects of ARF on c-Myc function suggest that separate molecular mechanisms mediate c-Myc-induced hyperproliferation and apoptosis. This direct feedback mechanism represents a p53-independent checkpoint to prevent c-Myc-mediated tumorigenesis.

Targeting Glutamine Metabolism and Redox State for Leukemia Therapy.

PURPOSE: Acute myeloid leukemia (AML) is a hematologic malignancy characterized by the accumulation of immature myeloid precursor cells. AML is poorly responsive to conventional chemotherapy and a diagnosis of AML is usually fatal. More effective and less toxic forms of therapy are desperately needed. AML cells are known to be highly dependent on the amino acid glutamine for their survival. These studies were directed at determining the effects of glutaminase inhibition on metabolism in AML and identifying general weaknesses that can be exploited therapeutically. EXPERIMENTAL DESIGN: AML cancer cell lines, primary AML cells, and mouse models of AML and acute lymphoblastic leukemia (ALL) were utilized. RESULTS: We show that blocking glutamine metabolism through the use of a glutaminase inhibitor (CB-839) significantly impairs antioxidant glutathione production in multiple types of AML, resulting in accretion of mitochondrial reactive oxygen species (mitoROS) and apoptotic cell death. Moreover, glutaminase inhibition makes AML cells susceptible to adjuvant drugs that further perturb mitochondrial redox state, such as arsenic trioxide (ATO) and homoharringtonine (HHT). Indeed, the combination of ATO or HHT with CB-839 exacerbates mitoROS and apoptosis, and leads to more complete cell death in AML cell lines, primary AML patient samples, and in vivo using mouse models of AML. In addition, these redox-targeted combination therapies are effective in eradicating ALL cells in vitro and in vivo. CONCLUSIONS: Targeting glutamine metabolism in combination with drugs that perturb mitochondrial redox state represents an effective and potentially widely applicable therapeutic strategy for treating multiple types of leukemia.

Glutaminase inhibition improves FLT3 inhibitor therapy for acute myeloid leukemia.

Acute myeloid leukemia (AML) is a blood cancer that is poorly responsive to conventional cytotoxic chemotherapy and a diagnosis of AML is usually fatal. More effective and better-tolerated therapies for AML are desperately needed. Activating mutations in FMS-like tyrosine kinase 3 (FLT3) are one of the most frequently observed genetic defects in AML. FLT3 inhibitors have shown impressive anti-leukemic activity in clinical trials; however, sustained remissions using these inhibitors as monotherapy have not been achieved. Our previous studies have implicated impaired glutamine metabolism in response to FLT3 inhibitors as a dominant factor causing AML cell death. In this study, we have employed metabolic flux analysis to examine the effects of FLT3 inhibition on glutamine utilization in FLT3-mutated AML cells using stable isotope tracers. We found that the FLT3 inhibitor AC220 inhibited glutamine flux into the antioxidant factor glutathione profoundly due to defective glutamine import. We also found that the glutaminase inhibitor CB-839 similarly impaired glutathione production by effectively blocking flux of glutamine into glutamate. Moreover, the combination of AC220 with CB-839 synergized to deplete glutathione, induce mitochondrial reactive oxygen species, and cause loss of viability through apoptotic cell death. In vivo, glutaminase inhibition with CB-839 facilitated leukemic cell elimination by AC220 and improved survival significantly in a patient-derived xenograft AML mouse model. Therefore, targeting glutaminase in combination with FLT3 may represent an effective therapeutic strategy for improving treatment of FLT3-mutated AML.

MCL1 is phosphorylated in the PEST region and stabilized upon ERK activation in viable cells, and at additional sites with cytotoxic okadaic acid or taxol.

BCL2 family members are subject to regulation at multiple levels, providing checks on their ability to contribute to tumorigenesis. However, findings on post-translational BCL2 phosphorylation in different systems have been difficult to integrate. Another antiapoptotic family member, MCL1, exhibits a difference in electrophoretic mobility upon phosphorylation induced by an activator of PKC (12-O-tetradecanoylphorbol 13-acetate; TPA) versus agents that act on microtubules or protein phosphatases 1/2A. A multiple pathway model is now presented, which demonstrates that MCL1 can undergo distinct phosphorylation events - mediated through separate signaling processes and involving different target sites - in cells that remain viable in the presence of TPA versus cells destined to die upon exposure to taxol or okadaic acid. Specifically, TPA induces phosphorylation at a conserved extracellular signal-regulated kinase (ERK) site in the PEST region (Thr 163) and slows turnover of the normally rapidly degraded MCL1 protein; however, okadaic acid and taxol induce ERK-independent MCL1 phosphorylation at additional discrete sites. These findings add a new dimension to our understanding of the complex regulation of antiapoptotic BCL2 family members by demonstrating that, in addition to transcriptional and post-transcriptional regulation, MCL1 is subject to multiple, separate, post-translational phosphorylation events, produced in living versus dying cells at ERK-inducible versus ERK-independent sites.

Tyrosine kinase inhibition in leukemia induces an altered metabolic state sensitive to mitochondrial perturbations.

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 FLT3(ITD) leukemias.