Loss of Cystathionine-β-Synthase Activity Results in Increased Oxidative Phosphorylation and Cytarabine Resistance in Myeloid Leukemia Associated with Down Syndrome

Acute myeloid leukemia (AML) and myelodysplasia in children with Down syndrome (DS) collectively define myeloid leukemia associated with DS (ML-DS) according to the WHO 2016 classification. DS individuals are genetically predisposed to hematologic malignancies, including AML before the age of 5. Previous clinical data showed ML-DS patients being highly sensitive to cytarabine (AraC)-based chemotherapy resulting in significantly higher overall survival rates compared to non-DS children with AML. However, relapsed/refractory ML-DS patients have dismal clinical outcomes, worse than their non-DS AML counterparts, highlighting the need for a better understanding of ML-DS AraC-resistance mechanisms and the development of effective therapies for this vulnerable group of patients.

RNA sequencing data has shown a high enrichment of oxidative phosphorylation (OXPHOS) related genes in AraC-Resistant ML-DS cell lines compared to an AraC-sensitive ML-DS cell line. We have previously demonstrated significant overexpression of a chromosome 21-localized gene, cystathionine-β-synthase (CBS), in ML-DS cells compared to non-DS AML cells. Metabolomic and enzymatic activity assays confirm a loss of CBS activity in AraC-resistant ML-DS cells compared to sensitive cells. CBS is well studied in its role in producing hydrogen sulfide (H₂S) and regulating OXPHOS. We hypothesize that the hyper AraC sensitivity in ML-DS patients is due to increased CBS activity and decreased OXPHOS; loss of CBS activity in the AraC-resistant cells is associated with increased OXPHOS and AraC resistance. Ectopic overexpression of CBS in an AraC-resistant ML-DS cell line resulted in significantly increased levels of H₂S, decreased OXPHOS, and increased sensitivity to AraC. The opposing pattern was proven when CBS was knocked down in AraC-sensitive cells rendering the cells more resistant to treatment. Similarly, inhibition of OXPHOS partially re-sensitized AraC-resistant cells to AraC treatment. However, complete reversal of the resistant phenotype using OXPHOS inhibitors cannot be achieved. The activity of AraC is dependent on activation via phosphorylation by deoxycytidine kinase (dCK) once inside the cell. We found that dCK expression is lost in the AraC-resistant ML-DS cells, requiring the use of alternative therapeutic strategies. Since OXPHOS is a known vulnerability of AraC resistance, we pursued a novel treatment strategy to target OXPHOS and induce cell death using a combined therapeutic approach.

Recent studies from our lab have found that the imipridone, ONC213, has activity against OXPHOS via inhibition of the TCA cycle enzyme alpha-ketoglutarate dehydrogenase. This compound is also known to downregulate the anti-apoptotic protein MCL-1. Overexpression of MCL-1 as well as other members of the BCL-2 protein family are associated with drug resistance and poor prognosis in leukemia, including ML-DS. We have reported that ONC213 synergizes with the selective BCl-2 inhibitor, Venetoclax (VEN) to induce cell death in drug resistant non-DS AML, representing an excellent drug combination for combating AraC-resistant ML-DS cells. In vitro experiments have proven this combination therapy to be effective in the AraC-resistant ML-DS cells as well. Flow cytometry data confirmed a synergistic effect of ONC213 and VEN in inducing apoptosis after 24 hours. Sequential treatment of ONC213 (8 hours) and VEN (1 hour) significantly decreased mitochondrial respiration without inducing cell death. Ongoing studies involve in vivo evaluation of the efficacy of ONC213 alone and in combination with VEN in an AraC-resistant ML-DS cell line derived xenograft model.

This study highlights CBS, a key regulator of OXPHOS, to be vital to cellular response to AraC-based chemotherapy. ONC213 combined with VEN has proven efficacy in relapsed/refractory ML-DS cells. By targeting the vulnerabilities of AraC-resistant ML-DS cells we can develop alternative treatment strategies to improve patient outcomes of ML-DS.

Crispino:Syndax: Research Funding; Cellarity: Consultancy; Alethiomics: Membership on an entity's Board of Directors or advisory committees.