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Simple Summary: The lysosomal lipid hydrolase acid ceramidase is upregulated in acute myeloid leukemia and promotes leukemic blast survival, underscoring its potential for therapeutic targeting. B-13 is an established ceramidase inhibitor with demonstrated efficacy in multiple solid cancers. Next-generation lysosome-localizing prodrugs of B-13 have been developed but have not been evaluated in AML. This study characterizes the in vitro anti-leukemic efficacy and cell death mechanisms of the B-13 analog and acid ceramidase inhibitor LCL-805 in acute myeloid leukemia. Acute myeloid leukemia (AML) is an aggressive hematologic malignancy requiring urgent treatment advancements. Ceramide is a cell-death-promoting signaling lipid that plays a central role in therapy-induced cell death. We previously determined that acid ceramidase (AC), a ceramide-depleting enzyme, is overexpressed in AML and promotes leukemic survival and drug resistance. The ceramidase inhibitor B-13 and next-generation lysosomal-localizing derivatives termed dimethylglycine (DMG)-B-13 prodrugs have been developed but remain untested in AML. Here, we report the in vitro anti-leukemic efficacy and mechanism of DMG-B-13 prodrug LCL-805 across AML cell lines and primary patient samples. LCL-805 inhibited AC enzymatic activity, increased total ceramides, and reduced sphingosine levels. A median EC50 value of 11.7 μM was achieved for LCL-805 in cell viability assays across 32 human AML cell lines. As a single agent tested across a panel of 71 primary AML patient samples, a median EC50 value of 15.8 μM was achieved. Exogenous ceramide supplementation with C6-ceramide nanoliposomes, which is entering phase I/II clinical trial for relapsed/refractory AML, significantly enhanced LCL-805 killing. Mechanistically, LCL-805 antagonized Akt signaling and led to iron-dependent cell death distinct from canonical ferroptosis. These findings elucidated key factors involved in LCL-805 cytotoxicity and demonstrated the potency of combining AC inhibition with exogenous ceramide. [ABSTRACT FROM AUTHOR]

Simple Summary: The sphingolipid ceramide is a key player in cytotoxic responses elicited by many anticancer drugs. Confounding this asset, however, are enzymes that promote ceramide clearance, thus limiting the propagation of ceramide-driven cancer cell death. Because several key ceramide-metabolizing enzymes have been shown to be upregulated in chemotherapy-resistant acute myeloid leukemia (AML) cells compared to chemotherapy-naïve counterparts, the current study was designed to determine the effects of blocking ceramide clearance in drug-resistant AML. For this, we employed simultaneous inhibition of ceramide hydrolysis and ceramide glycosylation and demonstrated that this dual blockade produced multi-fold elevations in intracellular ceramide levels, corrupted mitochondrial function, enhanced caspase activation, and elicited cell death in models of drug-resistant AML. We have herein identified sphingolipid metabolic junctures that can be targeted to enhance leukemia cell vulnerability in the drug-resistant setting, thus providing a novel therapeutic modality in difficult-to-treat cancers. Acute myelogenous leukemia (AML), the most prevalent acute and aggressive leukemia diagnosed in adults, often recurs as a difficult-to-treat, chemotherapy-resistant disease. Because chemotherapy resistance is a major obstacle to successful treatment, novel therapeutic intervention is needed. Upregulated ceramide clearance via accelerated hydrolysis and glycosylation has been shown to be an element in chemotherapy-resistant AML, a problem considering the crucial role ceramide plays in eliciting apoptosis. Herein we employed agents that block ceramide clearance to determine if such a "reset" would be of therapeutic benefit. SACLAC was utilized to limit ceramide hydrolysis, and D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (D-threo-PDMP) was used to block the glycosylation route. The SACLAC D-threo-PDMP inhibitor combination was synergistically cytotoxic in drug-resistant, P-glycoprotein-expressing (P-gp) AML but not in wt, P-gp-poor cells. Interestingly, P-gp antagonists that can limit ceramide glycosylation via depression of glucosylceramide transit also synergized with SACLAC, suggesting a paradoxical role for P-gp in the implementation of cell death. Mechanistically, cell death was accompanied by a complete drop in ceramide glycosylation, concomitant, striking increases in all molecular species of ceramide, diminished sphingosine 1-phosphate levels, resounding declines in mitochondrial respiratory kinetics, altered Akt, pGSK-3β, and Mcl-1 expression, and caspase activation. Although ceramide was generated in wt cells upon inhibitor exposure, mitochondrial respiration was not corrupted, suggestive of mitochondrial vulnerability in the drug-resistant phenotype, a potential therapeutic avenue. The inhibitor regimen showed efficacy in an in vivo model and in primary AML cells from patients. These results support the implementation of SL enzyme targeting to limit ceramide clearance as a therapeutic strategy in chemotherapy-resistant AML, inclusive of a novel indication for the use of P-gp antagonists. [ABSTRACT FROM AUTHOR]

Myelodysplastic syndromes are premalignant diseases characterized by cytopenias, myeloid dysplasia, immune dysregulation with association to autoimmunity, and variable risk for acute myeloid leukemia transformation. Studies of FOXP3+ regulatory T cells (Tregs) indicate that the number and/or activation state may influence cancer progression in these patients. Focusing on patients with a lower risk for leukemia transformation, 18 (34.6%) of 52 patients studied displayed an altered Treg compartment compared with age-matched controls. Delineation of unique Treg subsets revealed that an increase in the absolute number of CD4+FOXP3+CD25+CD127lowCD45RA-CD27- Tregs (effector memory Tregs [TregEM]) was significantly associated with anemia (p = 0.046), reduced hemoglobin (p = 0.038), and blast counts ≥ 5% (p = 0.006). In healthy donors, this TregEM population constitutes only 2% of all Tregs (one to six Tregs per microliter) in peripheral blood but, when isolated, exhibit greater suppressive activity in vitro. With a median follow-up of 3.1 y (range 2.7-4.9 y) from sample acquisition, increased numbers of TregEM cells proved to have independent prognostic importance in survival estimates, suggesting that enumeration of this Treg subset may be a more reliable indicator of immunological escape than FOXP3+ T cells as a whole. Based on multivariate analyses, TregEM impacted survival independently from myeloblast characteristics, cytopenias, karyotype, and comorbidities. Based on these findings, TregEM cell expansion may be synonymous with human Treg activation and indicate microenvironmental changes conducive to transformation in myelodysplastic syndromes. [ABSTRACT FROM AUTHOR]

Simple Summary: Acute myeloid leukemia (AML) is a rapidly progressing cancer of the blood and bone marrow with high relapse rates. Standard AML treatment has evolved to yield more frequent remission for patients, with little effect on the disease's low five-year survival rate. Patients exhibit a wide variation of molecular alterations, driving efforts to profile patients based on these genetic mutations. Previously, our group developed a novel Rho-associated protein kinase (ROCK) inhibitor, DJ4, and biochemical analysis demonstrated its potency in human cancer cell lines. This work targets the overactive ROCKs, which will help patients that experience abnormalities with ROCK-related processes that have been correlated to various cancers. We provide evidence to support the therapeutic efficacy of DJ4 and indicate its promise to improve AML therapy. Our results indicate that inhibiting ROCK makes AML cells susceptible to cell death and, in leukemia mouse models, reduces disease progression and enhances survival. The poor prognosis of acute myeloid leukemia (AML) and the highly heterogenous nature of the disease motivates targeted gene therapeutic investigations. Rho-associated protein kinases (ROCKs) are crucial for various actin cytoskeletal changes, which have established malignant consequences in various cancers, yet are still not being successfully utilized clinically towards cancer treatment. This work establishes the therapeutic activity of ROCK inhibitor (5Z)-2–5-(1H-pyrrolo[2,3-b]pyridine-3-ylmethylene)-1,3-thiazol-4(5H)-one (DJ4) in both in vitro and in vivo preclinical models of AML to highlight the potential of this class of inhibitors. Herein, DJ4 induced cytotoxic and proapoptotic effects in a dose-dependent manner in human AML cell lines (IC50: 0.05–1.68 μM) and primary patient cells (IC50: 0.264–13.43 μM); however, normal hematopoietic cells were largely spared. ROCK inhibition by DJ4 disrupts the phosphorylation of downstream targets, myosin light chain (MLC2) and myosin-binding subunit of MLC phosphatase (MYPT), yielding a potent yet selective treatment response at micromolar concentrations, from 0.02 to 1 μM. Murine models injected with luciferase-expressing leukemia cell lines subcutaneously or intravenously and treated with DJ4 exhibited an increase in overall survival and reduction in disease progression relative to the vehicle-treated control mice. Overall, DJ4 is a promising candidate to utilize in future investigations to advance the current AML therapy. [ABSTRACT FROM AUTHOR]