P598: disruption in one-carbon metabolism through mthfd1 inhibition as novel therapeutic strategy in chronic lymphocytic leukemia

Background: Chronic Lymphocytic Leukemia (CLL), the most common type of leukemia in adults, is characterized by the clonal expansion of CD5+ CD19+ B cells. Despite great advance in the standard of care in the last decades, there are still unmet medical needs (long-life treatment, resistance…). Altered cellular metabolism has emerged as a hallmark of cancer by sustaining the uncontrolled growth of cancer cells. The one-carbon (1C) pathway is a major driver for tumor proliferation, providing building blocks for biosynthesis of nucleotides through pyrimidines and purines synthesis (A). Understanding the metabolic reprogramming occurring in cancer, notably in CLL, may provide insights to support the development of novel therapies. Aims: We aim to pre-clinically test a novel nanomolar MTHFD1/2 inhibitor (MTHFD1/2i) for CLL treatment. Recently, our collaborators and us described that this compound mainly inhibits the dehydrogenase/cyclohydrolase (DC) activity of MTHFD1, leading to a complete block in thymidylate synthesis in SW620 colon cancer cells (Green et al, Nature Metabolism, in press). Methods: Here we evaluate the cytotoxic activity of MTHFD1/2i on a panel of murine and human CLL cells, and also on others B cell malignancies. The molecular mechanisms sustaining the cytotoxic activity were evaluated by performing metabolic tracing, rescue experiments and CRISPR/Cas9 KO. Finally, the impact of MTHFD1/2i on CLL development in vivo was assessed in a xenograft mouse model. Results: Higher expression of MTHFD2 was observed in patients and murine CLL cells compared to normal B cells, suggesting 1C metabolism over-activation. Moreover, we showed a strong expression of both MTHFD1 and 2 enzymes in all the CLL, MCL, DLBCL and MM cancer cell lines tested. In vitro treatment with MTHFD1/2i efficiently reduced cell viability of CLL cell lines, as for MCL and DLBCL cell lines, at low nanomolar dose while no effect was observed on MM cell lines (B). We identified specific mechanism of resistance in non-responding MM cells. In CLL cells, the cytotoxic effect of MTHFD1/2i is associated with a blockade of cell proliferation. Using 13C-serine isotope tracing, we showed that MTHFD1/2i did not prevent formate release but significantly reduced ATP production from serine. By performing rescue experiments, we confirmed that the cytotoxic effect of MTHFD1/2i on CLL cells is mediated through the inhibition of the DC domain of MTHFDH1, resulting in a defect in thymidylate synthesis (C). Some 1C metabolites, notably thymidine and folate, are much higher in mouse plasma compared to human. We demonstrated in vitro that thymidine and chronical exposure with folic acid compromised MTHFD1/2i cytotoxic activity. To validate the in vivo efficacy of MTHFD1/2i, we developed a murine model in which we aim (i) to reduce the use of plasmatic thymidine, and (ii) to prevent folate to fuel the 1C cycle. To do so, we performed our in vivo experiment by using custom diet and a genetically engineered CLL cell line model (CRISPR-Cas9 KO). We demonstrated that in vivo treatment of NSG mice with MTHFD1/2i after subcutaneous engraftment of CRISPR-Cas9 KO OSU-CLL cells significantly increased survival and completely eradicated established primary tumor (D and E) Summary/Conclusion: We identified a novel nanomolar MTHFD1/2i displaying a high cytotoxic activity in CLL and other B-cell malignancies by impairing cell proliferation through a defect in pyrimidine synthesis. This inhibitor also exhibits a potent anticancer activity in a pre-clinical murine model of CLL, reinforcing its therapeutic potential for CLL treatment in clinic.Keywords: B cell chronic lymphocytic leukemia, Inhibitor