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.

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.

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.

Targeting the IDH2 Pathway in Acute Myeloid Leukemia.

Acute myeloid leukemia (AML) is an aggressive disease with a poor prognosis. A large percentage of patients succumb to this disease in spite of aggressive treatments with chemotherapy. Recent advances with mutational analysis led to the discovery of isocitrate dehydrogenase (IDH) mutations in AML. IDH2 is an enzyme that catalyzes the oxidative decarboxylation of isocitrate to α-ketoglutarate; its mutated version leads to the accumulation of the oncometabolite (R)-2 hydroxyglutarate, which disrupts several cell processes and leads to a blockage in differentiation. Targeting IDH2 is compelling, as it is an early and stable mutation in AML. Enasidenib, a specific small-molecule inhibitor of IDH2, recently gained FDA approval for the treatment of patients with relapsed/refractory IDH2-mutated AML. In this review, we will focus on the indications and efficacy of enasidenib in the treatment of patients with IDH2-mutated AML. Clin Cancer Res; 24(20); 4931-6. ©2018 AACR.

p66Alpha-MBD2 coiled-coil interaction and recruitment of Mi-2 are critical for globin gene silencing by the MBD2-NuRD complex.

Nucleosome remodeling complexes comprise several large families of chromatin modifiers that integrate multiple epigenetic control signals to play key roles in cell type-specific transcription regulation. We previously isolated a methyl-binding domain protein 2 (MBD2)-containing nucleosome remodeling and deacetylation (NuRD) complex from primary erythroid cells and showed that MBD2 contributes to DNA methylation-dependent embryonic and fetal ß-type globin gene silencing during development in vivo. Here we present structural and biophysical details of the coiled-coil interaction between MBD2 and p66α, a critical component of the MBD2-NuRD complex. We show that enforced expression of the isolated p66α coiled-coil domain relieves MBD2-mediated globin gene silencing and that the expressed peptide interacts only with a subset of components of the MBD2-NuRD complex that does not include native p66α or Mi-2. These results demonstrate the central importance of the coiled-coil interaction and suggest that MBD2-dependent DNA methylation-driven gene silencing can be disrupted by selectively targeting this coiled-coil complex.

Trypanosoma cruzi trans-sialidase operates through a covalent sialyl-enzyme intermediate: tyrosine is the catalytic nucleophile.

Modified sialic acid substrates have been used to label Trypanosoma cruzi trans-sialidase, demonstrating that the enzyme catalyses the transfer of sialic acid through a covalent glycosyl-enzyme intermediate, a mechanism common to most retaining glycosidases. Peptic digestion of labeled protein, followed by LC-MS/MS analysis of the digest, identified Tyr342 as the catalytic nucleophile. This is the first such example of a retaining glycosidase utilizing an aryl glycoside intermediate. It is suggested that this alternative choice of nucleophile is a consequence of the chemical nature of sialic acid. A Tyr/Glu couple is invoked to relay charge from a remote glutamic acid, thereby avoiding electrostatic repulsion with the sialic acid carboxylate group.