Harnessing the Power of Sphingolipids: Targeting Acid Ceramidase as a Ceramide-Centric Therapeutic Approach for Acute Myeloid Leukemia

Acute myeloid leukemia (AML) is an aggressive hematologic malignancy characterized by uncontrolled proliferation of myeloid blasts which results in anemia, immunodeficiencies, increased infection risk, and bone marrow failure. AML is the most common acute leukemia in adults, and approximately 20,000 individuals are diagnosed annually. Each year, an estimated 10,000 individuals die in the United States alone. Although individuals of all ages develop AML, the median age at diagnosis is 69. Survival rates vary between younger patients less than 60 years (~50% 5-year survival) versus older patients over 60 years (~20% 5-year survival). Recent advancements in the molecular underpinnings of AML have supported the approval of several targeted therapeutics to supplement intensive chemotherapy. One notable example is the approval of venetoclax, a BH3 mimetic and an inhibitor of the anti-apoptotic protein BCL-2. Venetoclax is prescribed in combination with low-dose cytarabine or hypomethylating agents and has transformed the treatment landscape for chemotherapy-ineligible AML patients. However, responses are incomplete due in part to primary or adaptive drug resistance, which underscores an unmet need for additional therapeutics and strategies to circumvent resistance.

Metabolic rewiring is an important contributing factor to AML initiation and progression. Dysregulated sphingolipid metabolism is an emergent AML hallmark. Central to sphingolipid metabolism are ceramides, a class of cytotoxic, tumor suppressor sphingolipid. Ceramide accumulation is toxic to cancer cells including AML and supports the cytotoxic mechanism of various anti-cancer drugs. In AML, overexpression of acid ceramidase (AC), a ceramide-catabolizing enzyme, is associated with poorer survival outcomes and contributes to leukemic survival and therapy resistance. Here, we evaluated AC targeting as a ceramide-centric therapeutic strategy in AML. In Chapter 2, we evaluated the efficacy and cytotoxic mechanism of the small molecule AC inhibitor, LCL-805. The AC inhibitory effects of LCL-805 were confirmed using fluorogenic AC activity assays, sphingolipid profiling, and immunoblotting assays. LCL-805 effectively killed human AML cell lines and primary patient samples. Mechanistically, LCL-805 reduced levels of phosph-Akt (Ser473), as part of its cytotoxic mechanism as Akt reactivation blunted cell death. Iron chelation also blunted LCL-805 cytotoxicity. LCL-805 improved the cytotoxic efficacy of exogenous ceramide supplementation via C6-ceramide nanoliposomes (CNL) when tested in a panel of primary AML patient samples. CNL recently entered phase I clinical trial for relapsed/refractory AML. In Chapter 3, we tested the therapeutic efficacy of inhibiting AC to enhance venetoclax cytotoxicity in venetoclax-resistant AML. Previous work demonstrated that AC overexpression increased BH3 mimetic resistance. In our current work, analysis of the BeatAML 2.0 dataset revealed a positive relationship between AC gene expression and increased venetoclax resistance. Pharmacologic inhibition of AC enhanced the cytotoxicity of venetoclax in resistant AML cell lines and primary samples. AC inhibition also enhanced the cytotoxicity of the clinically utilized venetoclax + cytarabine combination. Mechanistically, AC inhibition and venetoclax induced a cytotoxic integrated stress response leading to NOXA accumulation, mitochondrial dysfunction, and caspase-dependent cell death. In Chapter 4, we developed analogs of the ceramide analog and AC inhibitor, SACLAC, to enhance its stability and drug-like properties. The SACLAC molecule relies on a chloride-based cysteine reactive group to covalently bind the catalytic Cys143 of AC to inhibit its activity. Though SACLAC potently inhibits AC activity and kills AML cells, the molecule exhibits poor pharmacokinetic properties due to the unstable chloride group. To improve SACLAC’s drug-like properties, we characterized five SACLAC analogs harboring different cysteine reactive groups and two methoxy polyethylene glycol-linked SACLAC molecules (2 kDa and 750 Da mPEG-SACLAC). The MO-1-114 analog, which harbors a dimethylamine-linked acrylamide group to replace the chloride group, emerged as the top analog as most potently reduced AC activity and cell viability. The 750 Da mPEG-SACLAC molecule was similarly potent to free SACLAC in cell viability assays and fluorogenic AC activity assays. Preliminary pharmacokinetic testing of 750 Da mPEG-SACLAC enhanced SACLAC retention and serum concentration compared to free SACLAC. These data support additional characterization of sphingolipid dysregulation in AML and the development of novel sphingolipid-modulating drugs as standalone therapeutics or to augment existing AML drugs.