Targeting Acute Myeloid Leukemia Stem Cells Through Perturbation of Mitochondrial Calcium.

We previously reported that acute myeloid leukemia stem cells (LSCs) are uniquely reliant on oxidative phosphorylation (OXPHOS) for survival. Moreover, maintenance of OXPHOS is dependent on BCL2, creating a therapeutic opportunity to target LSCs using the BCL2 inhibitor drug venetoclax. While venetoclax-based regimens have indeed shown promising clinical activity, the emergence of drug resistance is prevalent. Thus, in the present study, we investigated how mitochondrial properties may influence mechanisms that dictate venetoclax responsiveness. Our data show that utilization of mitochondrial calcium is fundamentally different between drug responsive and non-responsive LSCs. By comparison, venetoclax-resistant LSCs demonstrate a more active metabolic (i.e., OXPHOS) status with relatively high steady-state levels of calcium. Consequently, we tested genetic and pharmacological approaches to target the mitochondrial calcium uniporter, MCU. We demonstrate that inhibition of calcium uptake sharply reduces OXPHOS and leads to eradication of venetoclax-resistant LSCs. These findings demonstrate a central role for calcium signaling in the biology of LSCs and provide a therapeutic avenue for clinical management of venetoclax resistance.

A Novel Type of Monocytic Leukemia Stem Cell Revealed by the Clinical Use of Venetoclax-Based Therapy.

The BCL2 inhibitor venetoclax has recently emerged as an important component of acute myeloid leukemia (AML) therapy. Notably, use of this agent has revealed a previously unrecognized form of pathogenesis characterized by monocytic disease progression. We demonstrate that this form of disease arises from a fundamentally different type of leukemia stem cell (LSC), which we designate as monocytic LSC (m-LSC), that is developmentally and clinically distinct from the more well-described primitive LSC (p-LSC). The m-LSC is distinguished by a unique immunophenotype (CD34-, CD4+, CD11b-, CD14-, CD36-), unique transcriptional state, reliance on purine metabolism, and selective sensitivity to cladribine. Critically, in some instances, m-LSC and p-LSC subtypes can co-reside in the same patient with AML and simultaneously contribute to overall tumor biology. Thus, our findings demonstrate that LSC heterogeneity has direct clinical significance and highlight the need to distinguish and target m-LSCs as a means to improve clinical outcomes with venetoclax-based regimens. SIGNIFICANCE: These studies identify and characterize a new type of human acute myeloid LSC that is responsible for monocytic disease progression in patients with AML treated with venetoclax-based regimens. Our studies describe the phenotype, molecular properties, and drug sensitivities of this unique LSC subclass. This article is featured in Selected Articles from This Issue, p. 1949.

Therapy-Resistant Acute Myeloid Leukemia Stem Cells Are Resensitized to Venetoclax + Azacitidine by Targeting Fatty Acid Desaturases 1 and 2.

Recent advances in targeting leukemic stem cells (LSCs) using venetoclax with azacitidine (ven + aza) has significantly improved outcomes for de novo acute myeloid leukemia (AML) patients. However, patients who relapse after traditional chemotherapy are often venetoclax-resistant and exhibit poor clinical outcomes. We previously described that fatty acid metabolism drives oxidative phosphorylation (OXPHOS) and acts as a mechanism of LSC survival in relapsed/refractory AML. Here, we report that chemotherapy-relapsed primary AML displays aberrant fatty acid and lipid metabolism, as well as increased fatty acid desaturation through the activity of fatty acid desaturases 1 and 2, and that fatty acid desaturases function as a mechanism of recycling NAD+ to drive relapsed LSC survival. When combined with ven + aza, the genetic and pharmacologic inhibition of fatty acid desaturation results in decreased primary AML viability in relapsed AML. This study includes the largest lipidomic profile of LSC-enriched primary AML patient cells to date and indicates that inhibition of fatty acid desaturation is a promising therapeutic target for relapsed AML.

Higher-dose venetoclax with measurable residual disease-guided azacitidine discontinuation in newly diagnosed acute myeloid leukemia.

Venetoclax+azacitidine is the standard of care for newly-diagnosed patients with acute myeloid leukemia (AML) for whom intensive chemotherapy is inappropriate. Efforts to optimize this regimen are necessary. We designed a clinical trial to investigate two hypotheses: i) higher doses of venetoclax are tolerable and more effective, and ii) azacitidine can be discontinued after deep remissions. Forty-two newly diagnosed AML patients were enrolled in the investigator-initiated High Dose Discontinuation Azacitidine+Venetoclax (HiDDAV) Study (clinicaltrials gov. Identifier: NCT03466294). Patients received one to three "induction" cycles of venetoclax 600 mg daily with azacitidine. Responders received MRD-positive or MRDnegative "maintenance" arms: azacitidine with 400 mg venetoclax or 400 mg venetoclax alone, respectively. The toxicity profile of HiDDAV was similar to 400 mg venetoclax. The overall response rate was 66.7%; the duration of response (DOR), event-free survival (EFS) and overall survival were 12.9, 7.8 and 9.8 months, respectively. The MRD negativity rate was 64.3% by flow cytometry and 25.0% when also measured by droplet digital polymerase chain recation. MRD-negative patients by flow cytometry had improved DOR and EFS; more stringent measures of MRD negativity were not associated with improved OS, DOR or EFS. Using MRD to guide azacitidine discontinuation did not lead to improved DOR, EFS or OS compared to patients who discontinued azacitidine without MRD guidance. Within the context of this study design, venetoclax doses >400 mg with azacitidine were well tolerated but not associated with discernible clinical improvement, and MRD may not assist in recommendations to discontinue azacitidine. Other strategies to optimize, and for some patients, de-intensify, venetoclax+azacitidine regimens are needed.

The STAT3-MYC axis promotes survival of leukemia stem cells by regulating SLC1A5 and oxidative phosphorylation.

Acute myeloid leukemia (AML) is characterized by the presence of leukemia stem cells (LSCs), and failure to fully eradicate this population contributes to disease persistence/relapse. Prior studies have characterized metabolic vulnerabilities of LSCs, which demonstrate preferential reliance on oxidative phosphorylation (OXPHOS) for energy metabolism and survival. In the present study, using both genetic and pharmacologic strategies in primary human AML specimens, we show that signal transducer and activator of transcription 3 (STAT3) mediates OXPHOS in LSCs. STAT3 regulates AML-specific expression of MYC, which in turn controls transcription of the neutral amino acid transporter gene SLC1A5. We show that genetic inhibition of MYC or SLC1A5 acts to phenocopy the impairment of OXPHOS observed with STAT3 inhibition, thereby establishing this axis as a regulatory mechanism linking STAT3 to energy metabolism. Inhibition of SLC1A5 reduces intracellular levels of glutamine, glutathione, and multiple tricarboxylic acid (TCA) cycle metabolites, leading to reduced TCA cycle activity and inhibition of OXPHOS. Based on these findings, we used a novel small molecule STAT3 inhibitor, which binds STAT3 and disrupts STAT3-DNA, to evaluate the biological role of STAT3. We show that STAT3 inhibition selectively leads to cell death in AML stem and progenitor cells derived from newly diagnosed patients and patients who have experienced relapse while sparing normal hematopoietic cells. Together, these findings establish a STAT3-mediated mechanism that controls energy metabolism and survival in primitive AML cells.

The Hepatic Microenvironment Uniquely Protects Leukemia Cells through Induction of Growth and Survival Pathways Mediated by LIPG.

Due to the disseminated nature of leukemia, malignant cells are exposed to many different tissue microenvironments, including a variety of extramedullary sites. In the present study, we demonstrate that leukemic cells residing in the liver display unique biological properties and also contribute to systemic changes that influence physiologic responses to chemotherapy. Specifically, the liver microenvironment induces metabolic adaptations via upregulating expression of endothelial lipase in leukemia cells, which not only stimulates tumor cell proliferation through polyunsaturated fatty acid-mediated pathways, but also promotes survival by stabilizing antiapoptotic proteins. Additionally, hepatic infiltration and tissue damage caused by malignant cells induces release of liver-derived enzymes capable of degrading chemotherapy drugs, an event that further protects leukemia cells from conventional therapies. Together, these studies demonstrate a unique role for liver in modulating the pathogenesis of leukemic disease and suggest that the hepatic microenvironment may protect leukemia cells from chemotherapeutic challenge. SIGNIFICANCE: The studies presented herein demonstrate that the liver provides a microenvironment in which leukemia cells acquire unique metabolic properties. The adaptations that occur in the liver confer increased resistance to chemotherapy. Therefore, we propose that therapies designed to overcome liver-specific metabolic changes will yield improved outcomes for patients with leukemia.This article is highlighted in the In This Issue feature, p. 211.

Nicotinamide Metabolism Mediates Resistance to Venetoclax in Relapsed Acute Myeloid Leukemia Stem Cells.

We previously demonstrated that leukemia stem cells (LSCs) in de novo acute myeloid leukemia (AML) patients are selectively reliant on amino acid metabolism and that treatment with the combination of venetoclax and azacitidine (ven/aza) inhibits amino acid metabolism, leading to cell death. In contrast, ven/aza fails to eradicate LSCs in relapsed/refractory (R/R) patients, suggesting altered metabolic properties. Detailed metabolomic analysis revealed elevated nicotinamide metabolism in relapsed LSCs, which activates both amino acid metabolism and fatty acid oxidation to drive OXPHOS, thereby providing a means for LSCs to circumvent the cytotoxic effects of ven/aza therapy. Genetic and pharmacological inhibition of nicotinamide phosphoribosyltransferase (NAMPT), the rate-limiting enzyme in nicotinamide metabolism, demonstrated selective eradication of R/R LSCs while sparing normal hematopoietic stem/progenitor cells. Altogether, these findings demonstrate that elevated nicotinamide metabolism is both the mechanistic basis for ven/aza resistance and a metabolic vulnerability of R/R LSCs.

Monocytic Subclones Confer Resistance to Venetoclax-Based Therapy in Patients with Acute Myeloid Leukemia.

Venetoclax-based therapy can induce responses in approximately 70% of older previously untreated patients with acute myeloid leukemia (AML). However, up-front resistance as well as relapse following initial response demonstrates the need for a deeper understanding of resistance mechanisms. In the present study, we report that responses to venetoclax +azacitidine in patients with AML correlate closely with developmental stage, where phenotypically primitive AML is sensitive, but monocytic AML is more resistant. Mechanistically, resistant monocytic AML has a distinct transcriptomic profile, loses expression of venetoclax target BCL2, and relies on MCL1 to mediate oxidative phosphorylation and survival. This differential sensitivity drives a selective process in patients which favors the outgrowth of monocytic subpopulations at relapse. Based on these findings, we conclude that resistance to venetoclax + azacitidine can arise due to biological properties intrinsic to monocytic differentiation. We propose that optimal AML therapies should be designed so as to independently target AML subclones that may arise at differing stages of pathogenesis. SIGNIFICANCE: Identifying characteristics of patients who respond poorly to venetoclax-based therapy and devising alternative therapeutic strategies for such patients are important topics in AML. We show that venetoclax resistance can arise due to intrinsic molecular/metabolic properties of monocytic AML cells and that such properties can potentially be targeted with alternative strategies.

The Hematopoietic Oxidase NOX2 Regulates Self-Renewal of Leukemic Stem Cells.

The NADPH-dependent oxidase NOX2 is an important effector of immune cell function, and its activity has been linked to oncogenic signaling. Here, we describe a role for NOX2 in leukemia-initiating stem cell populations (LSCs). In a murine model of leukemia, suppression of NOX2 impaired core metabolism, attenuated disease development, and depleted functionally defined LSCs. Transcriptional analysis of purified LSCs revealed that deficiency of NOX2 collapses the self-renewal program and activates inflammatory and myeloid-differentiation-associated programs. Downstream of NOX2, we identified the forkhead transcription factor FOXC1 as a mediator of the phenotype. Notably, suppression of NOX2 or FOXC1 led to marked differentiation of leukemic blasts. In xenotransplantation models of primary human myeloid leukemia, suppression of either NOX2 or FOXC1 significantly attenuated disease development. Collectively, these findings position NOX2 as a critical regulator of malignant hematopoiesis and highlight the clinical potential of inhibiting NOX2 as a means to target LSCs.

Low ferroportin expression in AML is correlated with good risk cytogenetics, improved outcomes and increased sensitivity to chemotherapy.

Iron metabolism is altered in a variety of cancers; however, little is known about the role of iron metabolism in the biology and response to therapy of acute myeloid leukemia (AML). Here we show that SLC40A1, the gene encoding the iron exporter ferroportin (FPN), is variably expressed among primary AMLs and that low levels are associated with good prognosis and improved outcomes. In particular, core binding factor (CBF) AMLs, which are associated with good outcomes with chemotherapy, consistently have low level of SLC40A1 expression. AML cell lines that expressed relatively low levels of FPN endogenously, or were engineered via gene knockdown, had an increased sensitivity to chemotherapy relative to controls expressing high levels of FPN. Primary FPNlow AML bulk cells also had increased sensitivity to Ara-C treatment, iron treatment and the combination of Ara-C and iron relative to FPNhigh cells. FPNlow leukemic stem cells (LSCs) had decreased viability following addition of iron alone and in combination with Ara-C treatment relative to FPNhigh LSCs. Together these observations suggest a model where FPN mediated iron metabolism may play a role in chemosensitivity and outcome to therapy in AML.

Venetoclax with azacitidine disrupts energy metabolism and targets leukemia stem cells in patients with acute myeloid leukemia.

Acute myeloid leukemia (AML) is the most common acute leukemia in adults. Leukemia stem cells (LSCs) drive the initiation and perpetuation of AML, are quantifiably associated with worse clinical outcomes, and often persist after conventional chemotherapy resulting in relapse1-5. In this report, we show that treatment of older patients with AML with the B cell lymphoma 2 (BCL-2) inhibitor venetoclax in combination with azacitidine results in deep and durable remissions and is superior to conventional treatments. We hypothesized that these promising clinical results were due to targeting LSCs. Analysis of LSCs from patients undergoing treatment with venetoclax + azacitidine showed disruption of the tricarboxylic acid (TCA) cycle manifested by decreased α-ketoglutarate and increased succinate levels, suggesting inhibition of electron transport chain complex II. In vitro modeling confirmed inhibition of complex II via reduced glutathionylation of succinate dehydrogenase. These metabolic perturbations suppress oxidative phosphorylation (OXPHOS), which efficiently and selectively targets LSCs. Our findings show for the first time that a therapeutic intervention can eradicate LSCs in patients with AML by disrupting the metabolic machinery driving energy metabolism, resulting in promising clinical activity in a patient population with historically poor outcomes.

Subversion of Systemic Glucose Metabolism as a Mechanism to Support the Growth of Leukemia Cells.

From an organismal perspective, cancer cell populations can be considered analogous to parasites that compete with the host for essential systemic resources such as glucose. Here, we employed leukemia models and human leukemia samples to document a form of adaptive homeostasis, where malignant cells alter systemic physiology through impairment of both host insulin sensitivity and insulin secretion to provide tumors with increased glucose. Mechanistically, tumor cells induce high-level production of IGFBP1 from adipose tissue to mediate insulin sensitivity. Further, leukemia-induced gut dysbiosis, serotonin loss, and incretin inactivation combine to suppress insulin secretion. Importantly, attenuated disease progression and prolonged survival are achieved through disruption of the leukemia-induced adaptive homeostasis. Our studies provide a paradigm for systemic management of leukemic disease.

AMPK/FIS1-Mediated Mitophagy Is Required for Self-Renewal of Human AML Stem Cells.

Leukemia stem cells (LSCs) are thought to drive the genesis of acute myeloid leukemia (AML) as well as relapse following chemotherapy. Because of their unique biology, developing effective methods to eradicate LSCs has been a significant challenge. In the present study, we demonstrate that intrinsic overexpression of the mitochondrial dynamics regulator FIS1 mediates mitophagy activity that is essential for primitive AML cells. Depletion of FIS1 attenuates mitophagy and leads to inactivation of GSK3, myeloid differentiation, cell cycle arrest, and a profound loss of LSC self-renewal potential. Further, we report that the central metabolic stress regulator AMPK is also intrinsically activated in LSC populations and is upstream of FIS1. Inhibition of AMPK signaling recapitulates the biological effect of FIS1 loss. These data suggest a model in which LSCs co-opt AMPK/FIS1-mediated mitophagy as a means to maintain stem cell properties that may be otherwise compromised by the stresses induced by oncogenic transformation.

Targeted therapy for a subset of acute myeloid leukemias that lack expression of aldehyde dehydrogenase 1A1.

Aldehyde dehydrogenase 1A1 (ALDH1A1) activity is high in hematopoietic stem cells and functions in part to protect stem cells from reactive aldehydes and other toxic compounds. In contrast, we found that approximately 25% of all acute myeloid leukemias expressed low or undetectable levels of ALDH1A1 and that this ALDH1A1- subset of leukemias correlates with good prognosis cytogenetics. ALDH1A1- cell lines as well as primary leukemia cells were found to be sensitive to treatment with compounds that directly and indirectly generate toxic ALDH substrates including 4-hydroxynonenal and the clinically relevant compounds arsenic trioxide and 4-hydroperoxycyclophosphamide. In contrast, normal hematopoietic stem cells were relatively resistant to these compounds. Using a murine xenotransplant model to emulate a clinical treatment strategy, established ALDH1A1- leukemias were also sensitive to in vivo treatment with cyclophosphamide combined with arsenic trioxide. These results demonstrate that targeting ALDH1A1- leukemic cells with toxic ALDH1A1 substrates such as arsenic and cyclophosphamide may be a novel targeted therapeutic strategy for this subset of acute myeloid leukemias.

Rational Design of a Parthenolide-based Drug Regimen That Selectively Eradicates Acute Myelogenous Leukemia Stem Cells.

Although multidrug approaches to cancer therapy are common, few strategies are based on rigorous scientific principles. Rather, drug combinations are largely dictated by empirical or clinical parameters. In the present study we developed a strategy for rational design of a regimen that selectively targets human acute myelogenous leukemia (AML) stem cells. As a starting point, we used parthenolide, an agent shown to target critical mechanisms of redox balance in primary AML cells. Next, using proteomic, genomic, and metabolomic methods, we determined that treatment with parthenolide leads to induction of compensatory mechanisms that include up-regulated NADPH production via the pentose phosphate pathway as well as activation of the Nrf2-mediated oxidative stress response pathway. Using this knowledge we identified 2-deoxyglucose and temsirolimus as agents that can be added to a parthenolide regimen as a means to inhibit such compensatory events and thereby further enhance eradication of AML cells. We demonstrate that the parthenolide, 2-deoxyglucose, temsirolimus (termed PDT) regimen is a potent means of targeting AML stem cells but has little to no effect on normal stem cells. Taken together our findings illustrate a comprehensive approach to designing combination anticancer drug regimens.

Leukemic Stem Cells Evade Chemotherapy by Metabolic Adaptation to an Adipose Tissue Niche.

Adipose tissue (AT) has previously been identified as an extra-medullary reservoir for normal hematopoietic stem cells (HSCs) and may promote tumor development. Here, we show that a subpopulation of leukemic stem cells (LSCs) can utilize gonadal adipose tissue (GAT) as a niche to support their metabolism and evade chemotherapy. In a mouse model of blast crisis chronic myeloid leukemia (CML), adipose-resident LSCs exhibit a pro-inflammatory phenotype and induce lipolysis in GAT. GAT lipolysis fuels fatty acid oxidation in LSCs, especially within a subpopulation expressing the fatty acid transporter CD36. CD36(+) LSCs have unique metabolic properties, are strikingly enriched in AT, and are protected from chemotherapy by the GAT microenvironment. CD36 also marks a fraction of human blast crisis CML and acute myeloid leukemia (AML) cells with similar biological properties. These findings suggest striking interplay between leukemic cells and AT to create a unique microenvironment that supports the metabolic demands and survival of a distinct LSC subpopulation.

Regulation of endothelial cell inflammation and lung polymorphonuclear lymphocyte infiltration by transglutaminase 2.

We addressed the role of transglutaminase 2 (TG2), a calcium-dependent enzyme that catalyzes cross-linking of proteins, in the mechanism of endothelial cell (EC) inflammation and lung polymorphonuclear lymphocyte (PMN) infiltration. Exposure of EC to thrombin, a procoagulant and proinflammatory mediator, resulted in activation of the transcription factor nuclear factor κB (NF-κB) and its target genes, vascular cell adhesion molecule 1, monocyte chemotactic protein 1, and interleukin 6. RNAi knockdown of TG2 inhibited these responses. Analysis of NF-κB activation pathway showed that TG2 knockdown was associated with inhibition of thrombin-induced DNA binding as well as serine phosphorylation of RelA/p65, a crucial event that controls transcriptional capacity of the DNA-bound RelA/p65. These results implicate an important role for TG2 in mediating EC inflammation by promoting DNA-binding and transcriptional activity of RelA/p65. Because thrombin is released in high amounts during sepsis, and its concentration is elevated in plasma and lavage fluids of patients with acute respiratory distress syndrome, we determined the in vivo relevance of TG2 in a mouse model of sepsis-induced lung PMN recruitment. A marked reduction in NF-κB activation, adhesion molecule expression, and lung PMN sequestration was observed in TG2 knockout mice compared with wild-type mice exposed to endotoxemia. Together, these results identify TG2 as an important mediator of EC inflammation and lung PMN sequestration associated with intravascular coagulation and sepsis.

Targeting aberrant glutathione metabolism to eradicate human acute myelogenous leukemia cells.

The development of strategies to eradicate primary human acute myelogenous leukemia (AML) cells is a major challenge to the leukemia research field. In particular, primitive leukemia cells, often termed leukemia stem cells, are typically refractory to many forms of therapy. To investigate improved strategies for targeting of human AML cells we compared the molecular mechanisms regulating oxidative state in primitive (CD34(+)) leukemic versus normal specimens. Our data indicate that CD34(+) AML cells have elevated expression of multiple glutathione pathway regulatory proteins, presumably as a mechanism to compensate for increased oxidative stress in leukemic cells. Consistent with this observation, CD34(+) AML cells have lower levels of reduced glutathione and increased levels of oxidized glutathione compared with normal CD34(+) cells. These findings led us to hypothesize that AML cells will be hypersensitive to inhibition of glutathione metabolism. To test this premise, we identified compounds such as parthenolide (PTL) or piperlongumine that induce almost complete glutathione depletion and severe cell death in CD34(+) AML cells. Importantly, these compounds only induce limited and transient glutathione depletion as well as significantly less toxicity in normal CD34(+) cells. We further determined that PTL perturbs glutathione homeostasis by a multifactorial mechanism, which includes inhibiting key glutathione metabolic enzymes (GCLC and GPX1), as well as direct depletion of glutathione. These findings demonstrate that primitive leukemia cells are uniquely sensitive to agents that target aberrant glutathione metabolism, an intrinsic property of primary human AML cells.

Critical role of non-muscle myosin light chain kinase in thrombin-induced endothelial cell inflammation and lung PMN infiltration.

The pathogenesis of acute lung injury (ALI) involves bidirectional cooperation and close interaction between inflammatory and coagulation pathways. A key molecule linking coagulation and inflammation is the procoagulant thrombin, a serine protease whose concentration is elevated in plasma and lavage fluids of patients with ALI and acute respiratory distress syndrome (ARDS). However, little is known about the mechanism by which thrombin contributes to lung inflammatory response. In this study, we developed a new mouse model that permits investigation of lung inflammation associated with intravascular coagulation. Using this mouse model and in vitro approaches, we addressed the role of non-muscle myosin light chain kinase (nmMLCK) in thrombin-induced endothelial cell (EC) inflammation and lung neutrophil (PMN) infiltration. Our in vitro experiments revealed a key role of nmMLCK in ICAM-1 expression by its ability to control nuclear translocation and transcriptional capacity of RelA/p65 in EC. When subjected to intraperitoneal thrombin challenge, wild type mice showed a marked increase in lung PMN infiltration via expression of ICAM-1. However, these responses were markedly attenuated in mice deficient in nmMLCK. These results provide mechanistic insight into lung inflammatory response associated with intravascular coagulation and identify nmMLCK as a critical target for modulation of lung inflammation.