Abstract Immunotherapy has limited efficacy in acute myeloid leukemia (AML), partly because innate immune cells such as macrophages remain inactive. Nucleotide metabolism regulates key cellular processes, and cytidine triphosphate synthase 1 (CTPS1), the enzyme responsible for de novo CTP synthesis, is essential for cell proliferation. We previously showed (Liu, 2024 ASH) that high CTPS1 activity promotes AML growth and suppresses antitumor immunity. Accordingly, the CTPS1 inhibitor STP-B significantly prolonged the survival of immunocompetent leukemic mice in an immune-dependent manner. Across TCGA cancers, CTPS1 expression negatively correlated with M1-macrophage signatures.Here, we show that STP-B exerts anti-AML activity by (1) inducing myeloid differentiation, especially M1-like macrophage polarization through dNTP imbalance, and (2) activating IFN-I signaling by blocking CTPS1-mediated deamidation of IRF3 and histone H1. In a syngeneic MLL-AF9 (MA9) model, daily oral STP-B (100 mg/kg, 3 weeks) reduced leukemia burden and markedly increased CD11b+F4/80+ macrophages, enriching the M1-like subset. Macrophage depletion completely abolished the survival benefit. Combination with anti-CD47 produced strong synergy. Transcriptomic analysis of MA9 cells and non-malignant myeloid cells showed induction of M1-associated genes (Il6, Il1a, Cxcl9, Cxcl10). Ex vivo, STP-B-treated BMDMs displayed significantly enhanced phagocytosis of MA9 cells. To evaluate human hematopoietic effects, CD34+ cord blood-engrafted NSG mice were treated with STP-B. While total human CD45+ levels were unchanged, myeloid (CD33+CD11b+), monocyte (CD14+CD64+), and HLA-DR+CD86+ M1-like macrophage populations increased, with higher expression of myeloid transcription factors and human M1 genes. Metabolomic profiling of THP-1 cells confirmed that STP-B markedly reduced intracellular CTP, indicating nucleotide imbalance. Ribonucleotide reductase inhibition partially restored balance, reversed differentiation, and suppressed STP-B-induced M1-gene expression, supporting a nucleotide-driven differentiation mechanism. GSEA demonstrated induction of IFN-I-responsive genes. STP-B increased γH2AX and nuclear S9.6 staining, consistent with DNA damage caused by inhibition of CTPS1-mediated histone H1 deamidation. STP-B also blocked CTPS1-dependent IRF3 deamidation, enhancing ISG expression. Reconstitution of CTPS1-knockout THP-1 cells with a glutaminase-deficient CTPS1 mutant similarly increased ISGs, indicating that CTPS1 deamidation activity suppresses IFN signaling. Together, these findings show that STP-B promotes macrophage specification and innate immune activation, defining STP-B as a leukemia-ablating agent with strong immunostimulatory properties. Citation Format: Meng Liu, Lei Zhang, Xin He, Haojie Dong, Yang Li, Shuaishuai Ge, Guohua Wu, Yadav P. Umesh, Wei Chen, Pinghui Feng, Guido Marcucci, Ling Li. Pharmacological targeting of CTPS1 elicits macrophage-mediated anti-leukemia immunity abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 2572.
Liu et al. (Fri,) studied this question.