S251: DISRUPTION OF SUCCINATE RECEPTOR SIGNALING PROMOTES MYELOPROLIFERATION AND ACUTE MYELOID LEUKAEMIA

Abstract

Background: Unlike healthy haematopoietic stem cells (HSC), leukaemia stem cells (LSC) have high oxidative phosphorylation and hence targeting the tricarboxylic acid (TCA) cycle seems a promising therapeutic strategy in acute myeloid leukemia (AML) treatment. Accumulation of the TCA cycle oncometabolite succinate is associated with tumourigenesis, but its potential involvement in HSC function and leukaemia is unkown. Succinate stabilizes the oxygen-sensing transcription factor hypoxia-inducible factor (HIF)-1-α and activates interleukin (IL)-1β production in macrophages. Succinate may also be exported and has cell extrinsic effects through its receptor, succinate receptor-1 (SUCNR1), which may be pro- or anti-inflammatory. Aims: To investigate the in vivo role and molecular mechanisms of succinate and its receptor in normal and malignant haematopoiesis. Methods: To study the effect of succinate receptor on haematopoiesis, we characterized the immunophenotype of Sucnr1-KO mice. To dissect the role of Sucnr1 on healthy haematopoiesis via cell-autonomous effects, we transplanted fl/Sucnr1/fl control or Mx1-Cre fl/Sucnr1/fl bone marrow (BM) cells into wild-type (WT) C57BL/6J mice, which were later injected with pIpC. To study the role of Sucnr1 through non-cell autonomous effects, we transplanted WT BM into Nes-CreERT2fl/Sucnr1/fl recipients or fl/Sucnr1/fl controls, which were induced with tamoxifen. We characterized the effects of Sucnr1 deletion at the molecular level by bulk and single-cell RNA-sequencing (scRNA-seq), and aimed to uncover the molecular mechanisms mediating the effects of Sucnr1 ablation using in vivo tageting with drugs. To study the effect of succinate on leukaemic progression, we used two different mouse models, i.e. Mx1-Cre NrasG12D and MLL-AF9 knock-in (KI). In human AML, we quantified SUCNR1 expression in peripheral blood mononuclear cells (PBMC) from AML patients versus healthy volunteers, and treated xenograft recipients of CD34+ AML cells with succinate or vehicle. Results:In vivo deletion of Sucnr1 induced myeloid bias and expansion of the haematopoietic stem and progenitor cell (HSPC) compartment. In vivo deletion of Sucnr1 from Nestin+ stromal cells promoted expansion of long-term HSC (LT-HSC) versus intact recipients. Conversely, in vivo deletion of Sucnr1 from the haematopoietic system induced expansion of multipotent progenitors (MPP) versus intact haematopoietic cells. RNA-seq in HSPC from Sucnr1-KO versus WT mice showed changes in genes associated with myeloid output, and pinpointed related pathways potentially involved. scRNA-seq confirmed expansion of LT-HSC in Sucnr1-KO versus WT mice. In vivo treatments targeted signalling pathways at least partially responsible for the haematopoietic abnormalities in Sucnr1-KO. We found higher content of succinate in the BM extracellular fluid of leukaemic Mx1-Cre NrasG12D. Succinate injection to xenograft recipients of CD34+ AML cells increased leukaemic output versus vehicle, promoted myeloid bias in Mx1-Cre NrasG12D mice and reduced survival in mice transplanted with BM of MLL-AF9 leukaemic donors. PBMC from AML patients expressed lower level of SUCNR1 versus healthy donors and BM nucleated cells from Mx1-Cre NrasG12D also expressed less Sucnr1 than their healthy counterparts. Summary/Conclusion: We show a novel role for succinate and its receptor in the haematopoietic system and pinpoint this pathway as a novel means of communication within the BM HSC microenvironment, whose disruption may be involved in myeloid malignancy. These findings may help design new therapeutic strategies for AML patients.