Targeting IMPDH to inhibit SAMHD1 in KMT2A-rearranged leukaemia

This study demonstrates that the combination of Smac mimetic and oncolytic virotherapy (OVT) induces cell death in acute myeloid leukemia (AML) cell lines and primary patient samples; this is the first time the efficacy of this combination approach has been investigated in a human cancer model and for the treatment of AML. Methods: The Smac mimetic LCL161 and several oncolytic viruses (OVs) were tested for in vitro cytotoxicity against a panel of diverse AML cell lines. Bystander killing of LCL161-treated AML cells was explored using conditioned media from OV-treated PBMCs. Multiplex immunoassays were used to detect potential mediators of bystander cytotoxicity produced following OV treatment. Several upregulated cytokines were tested for their ability to enhance LCL161 toxicity in AML cell lines by MTS assay. The activation of healthy donor peripheral blood mononuclear cells (PBMC) was investigated by flow cytometry and immune cell-mediated death of AML cells was measured using chromium release assays. In vitro direct and bystander toxicity was further verified using fresh blood samples from AML patients. Results: A rhabdovirus, MG1, showed the greatest cytotoxicity across a panel of AML cell lines compared to several other OVs. In resistant cell lines the addition of LCL161 treatment led to increased cell death, indicating the potential merits of a combination strategy. Primary AML samples exposed to both LCL161 and MG1 showed greater levels of cell death than either treatment alone, confirming combinational efficacy. Treating healthy donor PBMCs with virus induced the activation of innate immune effector cells, as indicated by an increase in CD69 on NK cells as well as stimulating CD14+ monocytes to produce membrane-bound TNFα-related apoptosis-inducing ligand (TRAIL). Immune cell-mediated death was increased following activation by MG1, and LCL161 sensitized AML cell lines to death by the membrane mimicking KillerTRAIL. Conditioned media from MG1-treated healthy and patient-derived PBMCs displayed an inflammatory milieu that was toxic to AML cell lines. Further analysis identified several cytokines relevant to AML therapy, including the anti-viral interferon alpha (IFNα), soluble TRAIL, and tumor necrosis factor alpha (TNFα); LCL161 was able to increase the sensitivity of AML cells to MG1-conditioned media and recombinant versions of IFNα and TNFα. Discussion: OVT has shown positive clinical efficacy in many solid tumors; however, it is underexplored in the context of hematologic malignancies. Many cases of spontaneous remission following viral infection, the correlation of cytomegalovirus reactivation and positive prognosis following hematologic stem cell transplant, along with the identification of OVs which target leukemic cells without harming healthy hematologic stem cells, suggest that AML patients may be suitable for OVT. Indeed, data from our lab suggest that PBMCs from AML patients are able to recognize and respond to OV, to induce antitumor cytotoxicity. MG1 is cytotoxic to AML cells and acts as an immunogenic agent to induce the release of soluble inflammatory mediators by PBMCs that lead to the death of bystander cells without the need for direct infection. Recent studies into the use of Smac mimetics in hematologic malignancies have demonstrated the synergistic action of combining treatment with inflammatory cytokines, suggesting that combination with an immunogenic agent may be beneficial. Indeed, LCL161 is able to sensitize both AML cell lines and patient-derived AML blasts to MG1 treatment. The release of inflammatory mediators following MG1 treatment also stimulates the activation of innate immune effector cells to induce AML cell death, indicating the ability to initiate a cell-mediated innate immune attack on AML cells using this regime. In conclusion, these data suggest that MG1 should be considered in combination with the Smac mimetic, LCL161, for the treatment of AML. Citation Format: Joanne L. Hopper, Louise ME Müller, Victoria A. Jennings, Gina B. Scott, Stewart McConnell, Richard Kelly, Fiona Errington-Mais. Oncolytic virotherapy enhances Smac mimetic treatment of acute myeloid leukemia [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 20.

LP-118, a Selective Bcl-2 Inhibitor with Tuned Bcl-Xl Activity, Causes Myeloid Differentiation and Cell Death in Acute Myeloid Leukemia (AML)

BackgroundEnhanced glycolysis plays a pivotal role in fueling the aberrant proliferation, survival and therapy resistance of acute myeloid leukemia (AML) cells. Here, we aimed to elucidate the extent of glycolysis dependence in AML by focusing on the role of lactate dehydrogenase A (LDHA), a key glycolytic enzyme converting pyruvate to lactate coupled with the recycling of NAD+.MethodsWe compared the glycolytic activity of primary AML patient samples to protein levels of metabolic enzymes involved in central carbon metabolism including glycolysis, glutaminolysis and the tricarboxylic acid cycle. To evaluate the therapeutic potential of targeting glycolysis in AML, we treated AML primary patient samples and cell lines with pharmacological inhibitors of LDHA and monitored cell viability. Glycolytic activity and mitochondrial oxygen consumption were analyzed in AML patient samples and cell lines post-LDHA inhibition. Perturbations in global metabolite levels and redox balance upon LDHA inhibition in AML cells were determined by mass spectrometry, and ROS levels were measured by flow cytometry.ResultsAmong metabolic enzymes, we found that LDHA protein levels had the strongest positive correlation with glycolysis in AML patient cells. Blocking LDHA activity resulted in a strong growth inhibition and cell death induction in AML cell lines and primary patient samples, while healthy hematopoietic stem and progenitor cells remained unaffected. Investigation of the underlying mechanisms showed that LDHA inhibition reduces glycolytic activity, lowers levels of glycolytic intermediates, decreases the cellular NAD+ pool, boosts OXPHOS activity and increases ROS levels. This increase in ROS levels was however not linked to the observed AML cell death. Instead, we found that LDHA is essential to maintain a correct NAD+/NADH ratio in AML cells. Continuous intracellular NAD+ supplementation via overexpression of water-forming NADH oxidase from Lactobacillus brevis in AML cells effectively increased viable cell counts and prevented cell death upon LDHA inhibition.ConclusionsCollectively, our results demonstrate that AML cells critically depend on LDHA to maintain an adequate NAD+/NADH balance in support of their abnormal glycolytic activity and biosynthetic demands, which cannot be compensated for by other cellular NAD+ recycling systems. These findings also highlight LDHA inhibition as a promising metabolic strategy to eradicate leukemic cells.

Enhanced glycolysis plays a pivotal role in fueling the aberrant proliferation, survival and therapy resistance of acute myeloid leukemia (AML) cells. Here, we aimed to elucidate the extent of glycolysis dependence in AML by focusing on the role of lactate dehydrogenase A (LDHA), a key glycolytic enzyme converting pyruvate to lactate coupled with the recycling of NAD+. We compared the glycolytic activity of primary AML patient samples to protein levels of metabolic enzymes involved in central carbon metabolism including glycolysis, glutaminolysis and the tricarboxylic acid cycle. To evaluate the therapeutic potential of targeting glycolysis in AML, we treated AML primary patient samples and cell lines with pharmacological inhibitors of LDHA and monitored cell viability. Glycolytic activity and mitochondrial oxygen consumption were analyzed in AML patient samples and cell lines post-LDHA inhibition. Perturbations in global metabolite levels and redox balance upon LDHA inhibition in AML cells were determined by mass spectrometry, and ROS levels were measured by flow cytometry. Among metabolic enzymes, we found that LDHA protein levels had the strongest positive correlation with glycolysis in AML patient cells. Blocking LDHA activity resulted in a strong growth inhibition and cell death induction in AML cell lines and primary patient samples, while healthy hematopoietic stem and progenitor cells remained unaffected. Investigation of the underlying mechanisms showed that LDHA inhibition reduces glycolytic activity, lowers levels of glycolytic intermediates, decreases the cellular NAD+ pool, boosts OXPHOS activity and increases ROS levels. This increase in ROS levels was however not linked to the observed AML cell death. Instead, we found that LDHA is essential to maintain a correct NAD+/NADH ratio in AML cells. Continuous intracellular NAD+ supplementation via overexpression of water-forming NADH oxidase from Lactobacillus brevis in AML cells effectively increased viable cell counts and prevented cell death upon LDHA inhibition. Collectively, our results demonstrate that AML cells critically depend on LDHA to maintain an adequate NAD+/NADH balance in support of their abnormal glycolytic activity and biosynthetic demands, which cannot be compensated for by other cellular NAD+ recycling systems. These findings also highlight LDHA inhibition as a promising metabolic strategy to eradicate leukemic cells.

The Mitochondrial Unfolded Protein Response (UPRmt) Is Upregulated in Acute Myeloid Leukemia (AML) and Inhibiting the UPRmt Protease, LONP1, Leads to Mitochondrial Protein Aggregation and Cell Death Selectively in AML

Inhibition of ATR Potentiates Acute Myeloid Leukemia Cells to the RNA Pol I Transcription Inhibitor CX-5461

The RET Receptor Tyrosine Kinase Promotes Acute Myeloid Leukemia through Protection of FLT3-ITD Mutants from Autophagic Degradation

Abnormal expression of the transcription factor EVI1 through chromosome 3q26 rearrangements has been implicated in the development of one of the most therapeutically challenging high-risk subtypes of acute myeloid leukemia (AML). Here we integrated genomic and metabolic screening of hematopoietic stem cells to reveal that EVI1 overexpression altered cellular metabolism. A pooled shRNA screen targeting metabolic enzymes identified the ATP-buffering, mitochondrial creatine kinase CKMT1 as a druggable dependency in EVI1-positive AML.Of 18 screened AML cell lines harboring various genetic alterations, only the four EVI1-expressing lines exhibited markedly elevated CKMT1 protein expression and activity. Treatment of this cell line panel with either CKMT1-targeting shRNAs or cyclocreatine, an analog of the CKMT1 substrate creatine and inhibitor of the creatine biosynthesis pathway, showed that elevated CKMT1 protein expression correlated with sensitivity to CKMT1 pathway inhibition. Consistent with these data, flow cytometry analysis of a panel of 68 unselected primary AML patient specimens revealed that the four leukemias with the highest levels of EVI1 expression also had elevated CKMT1 protein levels and enhanced sensitivity to cyclocreatine treatment.We next established that enforced EVI1 expression increased CKMT1 protein and mRNA levels and that three independent shRNA molecules targeting EVI1 drastically reduced CKMT1 expression in two EVI1-positive AML cell lines. A luciferase-based reporter system established that RUNX1 represses CKMT1 expression through direct binding to its promoter. ChIP-qPCR approaches were then applied to dissect the sequential events involved in EVI1-induced CKMT1 upregulation and the possible role of RUNX1 as an intermediate. In both primary AML samples and cell lines, we determined that EVI1 represses RUNX1 expression by directly binding to its promoter. This, in turn, eliminates repressive RUNX1 binding at the CKMT1 promoter and thereby promotes CKMT1 expression. Based on these data, we explored the relationship between EVI1 and RUNX1 expression with CKMT1 mRNA levels in two AML transcriptional datasets (GSE14468 and GSE10358). We divided these cohorts into four subgroups with high versus low expression of EVI1 and RUNX1. Consistent with our mechanistic analysis, primary AML samples within the EVI1high/RUNX1low subgroup were significantly more likely to express high levels of CKMT1 than AML samples in the other three subgroups.CKMT1 promotes the metabolism of arginine to creatinine. To determine the effect of CKMT1 suppression on this pathway, we measured the metabolic flux of stable-isotope labeled L-arginine 13C6 through creatine synthesis using mass spectrometry. CKMT1-directed shRNAs or cyclocreatine selectively decreased intracellular phospho-creatine and blocked production of ATP by mitochondria. Salvage of the creatine pathway by exogenous phospho-creatine restored normal mitochondrial function, prevented the loss of viability of human EVI1-positive AML cells induced by cyclocreatine or CKMT1-directed shRNAs, and maintained the serial replating activity of Evi1-transformed bone marrow cells.Primary human EVI1-positive AML is frequently associated with somatic NRAS mutations. Thus, to investigate whether EVI1 over-expression sensitizes primary AMLs to CKMT1 inhibition in vivo, we transplanted primary NrasG12D mutant AMLs with and without elevated Evi1 expression into congenic recipient mice. In this system, Ckmt1 knockdown did not significantly alter the outgrowth of control Nras mutant AML cells compared to a shControl (63% versus 71%). By contrast, NrasG12D AML cells characterized by elevated Evi1 expression were profoundly depleted by Ckmt1 suppression to 2% versus 58% in shControl recipients. Consistent with these results, pharmacologic or genetic inhibition of the CKMT1-dependent pathway blocked disease progression and prolonged the survival of mice injected with human EVI1-positive cells but not with EVI1-negative cells, without noticeable cytotoxic effect on normal murine cells.In conclusion, we have integrated "omic" approaches to identify CKMT1 as a druggable liability in EVI-positive AML. This study supports a potential therapeutic avenue for targeting the creatine kinase pathway in EVI1-positive AML, which remains one of the worst outcome subtypes of AML. DisclosuresDeAngelo:Incyte: Consultancy; Novartis: Consultancy; Celgene: Consultancy; Amgen: Consultancy; Baxter: Consultancy; Pfizer: Consultancy; Ariad: Consultancy. Stone:Pfizer: Consultancy; Agios: Consultancy; Jansen: Consultancy; Celator: Consultancy; Merck: Consultancy; Amgen: Consultancy; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees; Karyopharm: Consultancy; Novartis: Consultancy; Abbvie: Consultancy, Membership on an entity's Board of Directors or advisory committees; Xenetic Biosciences: Consultancy; Sunesis Pharmaceuticals: Consultancy; Seattle Genetics: Consultancy; Roche: Consultancy; Juno Therapeutics: Consultancy; ONO: Consultancy.

Inhibition of Impdh As an Effective Treatment for MLL-Fusion Leukemia

Pacritinib Targets IRAK1 and Shows Synergy with HDAC and BET Inhibitors in Acute Myeloid Leukemia

Methylation Status of Tumor Suppressor Genes BEX2, IGSF4, RARB and TIMP3 Is Indicative for AML with Translocations of the MLL Gene.

Anti-Apoptotic BCL-2 Family Members Confer Resistance to Calicheamicin-Based Antibody-Drug Conjugate Therapy of Acute Leukemia

Expression Of FLT3-ITD Dysregulates The DBC1-Sirt1-p53 Signaling and Promotes Therapy Resistance

Identifying Immune Resistance Mechanisms to CD33/CD3 BiTE® Antibody Construct (AMG 330) Mediated Cytotoxicity

Comparison of Three Different NOD/SCID-Related Strains in Preclinical Models of Acute Leukemia.