Countering Development of Resistance to Treatment in AML By Targeting Metabolic Adaptations

Background:

Acute myeloid leukemia (AML), the most common leukemia in adults, is associated with a low survival rate. Combining BCL-2 targeting, using the BH3 mimetic Venetoclax, with hypomethylating agents shows promise for AML treatment. Despite advancements in treatment, the persistence of drug resistance remains an obstacle, resulting in a discouraging 5-year survival rate of less than 30%. This underscores the critical need to deepen our understanding of the mechanisms underlying the development of therapy resistance.

To discover the molecular basis of resistance to BH3 mimetic-based treatments, we have previously performed genome-wide loss-of-function CRISPR screens in AML cells treated with various BH3 mimetics and their combination. One of the top synthetic lethal targets in all the treatment groups was the gene encoding the mitochondrial citrate carrier (CIC), SLC25A1. CIC plays a crucial role in regulating glycolysis, fatty acid synthesis, sterol biosynthesis, and mitochondrial oxidative phosphorylation. Yet, the exact role of CIC in leukemia development and acquisition of resistance is unexplored. Based on our data, we hypothesize that augmented citrate transport from mitochondria to the cytosol rewires AML metabolism. At the same time, CIC targeting can enhance leukemia sensitivity to BH3 mimetics-based treatments, offering a potential avenue for improving AML therapy.

Methods:

Analysis of single-cell transcriptomics data from patients with AML and immunoblotting analyses of patient-derived xenografts and cell line models were utilized to examine CIC expression. Protein levels were also analyzed in BH3 mimetics-resistant AML cell lines compared to their isogenic, sensitive counterparts. SLC25A1 (CIC) was genetically deleted using CRISPR/Cas9 technology. Upon successful CIC ablation, we performed competition-based survival, proliferation, and apoptosis assays. Extensive metabolomics and RNA-sequencing experiments followed by pathway enrichment analyses were carried out on CIC knockout AML cell lines. Next, we pharmacologically inhibited CIC to determine its efficacy as a monotherapy or in combination with BH3 mimetics and other FDA-approved AML therapies. For this purpose, we performed CellTiter-Glo viability assays and Annexin V staining, analyzed by flow cytometry.

Results:

CIC is upregulated in AML and further induced upon the gain of BH3 mimetics resistance. Notably, SLC25A1 expression positively correlates with lower overall survival in AML patients and the degree of intrinsic BH3 mimetics-resistance in a panel of AML cell lines. Loss of CIC impedes AML cell growth and enhances sensitivity to cell death, even in therapy-resistant blasts. CIC ablation impairs AML bioenergetics, including mitochondrial respiration, glycolysis, as well as lipid metabolism by affecting citrate subcellular pools. Lastly, pharmacologic inhibition of CIC re-sensitizes AML cells to FDA-approved therapies.

Conclusion:

Our studies demonstrate that CIC is a potential prognostic marker for AML and the emergence of resistance to BH3 mimetics treatment. CIC regulates both mitochondrial and lipid metabolism, underscoring the critical role of mitochondrial function in AML biology and therapy.

No relevant conflicts of interest to declare.