Abstract Immunotherapy, including immune checkpoint inhibitors, antagonizes many hematologic malignancies, but has transient effects on acute myeloid leukemia (AML), as innate immune cells like macrophages remain inactive. Nucleotide metabolism is critical to regulate multiple cellular functions, and cytidine triphosphate synthase 1 (CTPS1), which catalyzes de novo CTP biosynthesis, is essential for cell proliferation. We previously (Liu, 2024, ASH) showed that high CTPS1 activity promotes cancer cell growth and suppresses anti-AML immunity. Accordingly, the administration of the CTPS1 inhibitor STP-B significantly extended the survival of immunocompetent leukemic mice, an effect requiring an immune response. Analysis of TCGA cohorts, including AML, glioblastoma, and non-small cell lung cancer, consistently demonstrated a negative correlation between CTPS1 expression and macrophage M1 signature. Herein, we show that targeting CTPS1 by STP-B exerts potent anti-AML activity by 1) promoting myeloid differentiation, in particular, macrophage M1 polarization triggered by dNTP imbalance, and 2) stimulating IFN-I signaling by inhibiting CTPS1-mediated deamidation of IRF3 and histone H1. To further define these mechanisms, we first employed a syngeneic MLL-AF9 (MA9) leukemia transplant model. Leukemic mice were administered STP-B orally (100 mg/kg/i. g. /day) for 3 weeks. Then, full-spectrum flow cytometry revealed that treatment decreased leukemia burden and markedly increased CD11b⁺F4/80⁺ macrophage number and the frequency of the M1-like subset. Macrophage depletion by liposomal clodronate before STP-B treatment completely abolished survival advantages seen in STP-B-treated mice. Moreover, in the MA9 mouse model, treatment with STP-B and anti-CD47 in vivo synergized to antagonize AML, highlighting the significance of macrophage function. Transcriptomic profiling of MA9 cells (GFP+) and non-malignant myeloid cells (GFP-CD11b+) showed that STP-B treatment significantly upregulated macrophage M1SIGNATURE₁ gene set, including Il6, Il1a, Cxcl9, and Cxcl10. As confirmation, we performed an ex vivo phagocytosis assay using STP-B–pretreated bone marrow-derived macrophages (BMDMs) co-cultured for 4 hours with MA9 cells and found that STP-B–treated BMDM cells showed significantly increased phagocytic activity. To assess how STP-B impacts human hematopoiesis, we transplanted CD34⁺ cord blood cells into sublethally-irradiated NSG mice and administered STP-B 8-12 weeks post-transplant. While total human CD45⁺ cellularity was unchanged, treatment markedly increased the size of myeloid (CD33⁺CD11b⁺) and monocyte (CD14⁺CD64⁺) subsets, and increased HLA-DR⁺CD86⁺ M1-like macrophages. RT-qPCR showed increased expression of myeloid transcription factors and human M1-associated genes in STP-B-treated NSG mice. We then performed metabolomic profiling of monocytic THP-1 cells, a model of myeloid differentiation, to assess intracellular NTP and dNTP levels. STP-B treatment drastically decreased intracellular CTP levels, suggesting nucleotide imbalance. Co-treatment with a ribonucleotide reductase (RNR) inhibitor partially rescued this imbalance and reversed THP-1 cell differentiation. Importantly, STP-B-mediatedupregulation of M1-associated geneswas reduced by RNR inhibition. These results support the idea that STP-B–mediated differentiation is driven by disrupted nucleotide metabolism, as we have proposed (PMID: 35439288). Moreover, GSEA indicated that STP-B treatment upregulates IFN-I-responsive genes, likely due to its DNA damage-inducing effects. CTPS1 is one of 11 glutamine amidotransferases, which catalyze histone H1 deamidation to promote DNA repair. STP-B inhibits these activities and promotes DNA damage, based on increased nuclear S9. 6 staining and high γH2AX levels seen in STP-B-treated THP-1 cells. CTPS1 also deamidates IRF3, suppressing its transcriptional activity, an effect blocked by STP-B, enhancing expression of IFN-stimulatory genes (ISGs). To investigate these activities, we reconstituted CTPS1-knockout THP-1 cells with wild-type or glutaminase-deficient mutant CTPS1. Relative to wild-type CTPS1, expression of the mutant form exhibited ISG upregulation, suggesting that deamidation activity is critical to suppress IFN signaling. Together, these findings reveal that systemic STP-B treatment drives macrophage specification and innate immune activation, highlighting STP-B as a leukemia-ablating agent with immune-stimulatory potency.
He et al. (Mon,) studied this question.