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P1308: SELECTIVE SMALL MOLECULE INHIBITION OF CTP SYNTHASE 1 (CTPS1) SUPPRESSES T CELL PROLIFERATION AND CYTOKINE RELEASE, HIGHLIGHTING A NOVEL THERAPEUTIC TARGET FOR GRAFT-VERSUS-HOST DISEASE

Background: Background: Graft-versus-host disease (GvHD) significantly increases both the morbidity/mortality and economic cost of allogeneic haematopoietic stem cell transplantation. Key to successful therapy is effective disease control whilst maintaining a competent anti-infection response. Acute GvHD (aGvHD) is driven by the rapid proliferation of, and cytokine release from, donor-derived T cells. Standard of care therapy with systemic steroids is associated with broad and deleterious immunosuppression, and fails to achieve adequate disease control in a third of patients. There is currently a lack of consensus around therapy beyond steroids [1]. CTP synthases catalyse the conversion of UTP to CTP, the rate limiting step in pyrimidine synthesis. Humans have two isoforms of this enzyme (CTPS1 and CTPS2); human genetic studies have identified an essential and non-redundant role for CTPS1 in the proliferation of B and T cells [2]. Aims: Aims: To assess the impact of selective CTPS1 inhibition on the proliferation of, and cytokine release from, human T cells. Methods: Methods: Aliquots of whole blood or isolated peripheral blood mononuclear cells (PBMC) from healthy volunteer donors underwent T cell activation with CD3/CD28 beads and incubation with or without a selective CTPS1 inhibitor for 24-72 hours. Levels of intracellular CTP, ATP and GTP nucleotides were quantitated by liquid chromatography– mass spectrometry; cell proliferation was assessed by tritiated thymidine incorporation, T cell cytokine represents a novel


P1308 SELECTIVE SMALL MOLECULE INHIBITION OF CTP SYNTHASE 1 (CTPS1) SUPPRESSES T CELL PROLIFERATION AND CYTOKINE RELEASE, HIGHLIGHTING A NOVEL THERAPEUTIC TARGET FOR GRAFT-VERSUS-HOST DISEASE
Step Pharma, Saint-Genis-Pouilly, France Background: Background: Graft-versus-host disease (GvHD) significantly increases both the morbidity/mortality and economic cost of allogeneic haematopoietic stem cell transplantation. Key to successful therapy is effective disease control whilst maintaining a competent anti-infection response. Acute GvHD (aGvHD) is driven by the rapid proliferation of, and cytokine release from, donor-derived T cells. Standard of care therapy with systemic steroids is associated with broad and deleterious immunosuppression, and fails to achieve adequate disease control in a third of patients. There is currently a lack of consensus around therapy beyond steroids [1]. CTP synthases catalyse the conversion of UTP to CTP, the rate limiting step in pyrimidine synthesis. Humans have two isoforms of this enzyme (CTPS1 and CTPS2); human genetic studies have identified an essential and non-redundant role for CTPS1 in the proliferation of B and T cells [2].
[1] Blood. 2020 May 7;135 (19) Aims: Aims: To assess the impact of selective CTPS1 inhibition on the proliferation of, and cytokine release from, human T cells.

Methods:
Methods: Aliquots of whole blood or isolated peripheral blood mononuclear cells (PBMC) from healthy volunteer donors underwent T cell activation with CD3/CD28 beads and incubation with or without a selective CTPS1 inhibitor for 24-72 hours. Levels of intracellular CTP, ATP and GTP nucleotides were quantitated by liquid chromatographymass spectrometry; cell proliferation was assessed by tritiated thymidine incorporation, Ki-67 by flow cytometry and cytokine release by analysis of plasma using the Luminex platform.

Results: Results:
STP938 is a potent and reversible low molecular weight small molecule inhibitor of CTPS1, which shows >1,300-fold selectivity for human CTPS1 over CTPS2. Exposure of stimulated PBMCs to STP938 resulted in a concentration dependent depletion of CTP but had no effect on the levels of GTP or ATP, confirming selective inhibition of CTP synthase by STP938. CTPS1 inhibition by STP938 elicited a concentration dependent inhibition of CD3/CD28-induced T cell proliferation in whole blood samples after 72 hours incubation, resulting in full suppression of proliferation at the highest concentration tested (1 µM) ( Figure A). Exposure to STP938 also reduced the proportion of T cells in cell cycle, as evidenced by a decrease in the expression of Ki-67 ( Figure B). STP938 exposure induced a concentration dependent inhibition of T cell cytokine release in whole blood samples, with higher STP938 concentrations producing near-complete suppression ( Figure C). Notably, significant inhibition of T cell cytokine release was observed after only 24 hours' exposure of stimulated T cells to STP938 ( Figure D, 1 μM STP938). Given that human T cells do not undergo cell division until at least 40 hours after CD3/CD28 stimulation is initiated [3], these findings indicate that the ability of CTPS1 inhibition to prevent cytokine release is independent of its effects on cell proliferation.

Image: Image:
Summary/Conclusion: Summary/Conclusion: CTPS1 inhibition by STP938 induces a rapid suppression of T cell cytokine release along with suppression of T cell proliferation; both of these effects are concentration dependent and near maximal at exposures of STP938 that are likely to be achievable in the clinic. CTPS1 represents a novel target with the potential to effect rapid and specific disease control in aGvHD. STP938 is currently on the path to clinical development in lymphoid malignancy.