Book Citations: Authors, Title, HemaSphere, 2023;7(S3):pages. The individual abstract DOIs can be found at https://journals.lww.com/hemasphere/pages/default.aspx. Disclaimer: Articles published in the journal HemaSphere exclusively reflect the opinions of the authors. The authors are responsible for all content in their abstracts including accuracy of the facts, statements, citing resources, etc. 94 analysis consisting of pulsed SILAC, RNA sequencing and polysome profiling performed in CLL patient samples and cell lines treated with FL3 revealed the decreased translation of the MYC oncogene (C). Furthermore, inhibition of translation was associated with a block of proliferation (D) and a profound rewiring of MYC-driven metabolism. Interestingly, contrary to other models, in CLL, the RAS-RAF-(PHBs)-MAPK pathway is neither impaired by FL3 nor implicated in translation regulation. We rather showed that PHBs are directly associated with the translation initiation complex (E). Knock-down of PHBs resembled FL3 treatment (F), confirming the direct involvement of PHBs in translation initiation. Importantly, inhibition of translation was efficient in controlling CLL development in vivo (G). Finally, high expression of translation initiation-related genes and PHBs genes correlated with poor survival and unfavorable clinical parameters in CLL patients (H). Summary/Conclusion: We demonstrated that translation inhibition is a valuable strategy to control CLL development by blocking the translation of several oncogenic pathways including MYC. We also unraveled a new and direct role of PHBs in translation initiation, thus creating new therapeutic opportunities for CLL patients. HemaSphere | 2023;7(S3) EHA2023 Hybrid Congress Copyright Information: (Online) ISSN: 2572-9241 © 2023 the Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the European Hematology Association. This is an open access Abstract Book distributed under the Attribution-NonCommercial-NoDerivs (CC BY-NC-ND) which allows third parties to download the articles and share them with others as long as they credit the author and the Abstract Book, but they cannot change the content in any way or use them commercially. Abstract Book Citations: Authors, Title, HemaSphere, 2023;7(S3):pages. The individual abstract DOIs can be found at https://journals.lww.com/hemasphere/pages/default.aspx.Book Citations: Authors, Title, HemaSphere, 2023;7(S3):pages. The individual abstract DOIs can be found at https://journals.lww.com/hemasphere/pages/default.aspx. Disclaimer: Articles published in the journal HemaSphere exclusively reflect the opinions of the authors. The authors are responsible for all content in their abstracts including accuracy of the facts, statements, citing resources, etc. 95

Dysregulation of mRNA translation, including preferential translation of mRNA with complex 5'-UTRs such as the MYC oncogene, is recognized as an important mechanism in cancer. In this study, we show that both human and murine chronic lymphocytic leukemia (CLL) cells display a high translation rate, which can be inhibited by the synthetic flavagline FL3, a prohibitin (PHB)-binding drug. A multiomics analysis consisting of pulsed SILAC, RNA sequencing and polysome profiling performed in CLL patient samples and cell lines treated with FL3 revealed the decreased translation of the MYC oncogene and of proteins involved in cell cycle and metabolism. Furthermore, inhibition of translation was associated with a block of proliferation and a profound rewiring of MYC-driven metabolism. Interestingly, contrary to other models, the RAS-RAF-(PHBs)-MAPK pathway is neither impaired by FL3 nor implicated in translation regulation in CLL cells. Here, we rather show that PHBs are directly associated with the translation initiation complex and can be targeted by FL3. Knock-down of PHBs resembled FL3 treatment. Importantly, inhibition of translation was efficient in controlling CLL development in vivo either alone or combined with immunotherapy. Finally, high expression of translation initiation-related genes and PHBs genes correlated with poor survival and unfavorable clinical parameters in CLL patients. In conclusion, we demonstrated that translation inhibition is a valuable strategy to control CLL development by blocking the translation of several oncogenic pathways including MYC. We also unraveled a new and direct role of PHBs in translation initiation, thus creating new therapeutic opportunities for CLL patients.

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