Targeting Cytidine Triphosphate Synthase 1 Promotes Anti-AML Immunity

Clinical outcomes for patients with acute myeloid leukemia (AML) remain unfavorable. Use of immune checkpoint inhibitors (ICIs) to re-activate the immune system has proven successful against other hematological malignancies, although use of ICIs as AML treatment remains challenging, likely due to a lack of functional T cells or other innate immune cells such as macrophages. Hence, eradicating AML cells requires development of an effective leukemia-targeting drug with immune-stimulating activity to use in combination with ICIs.

CTPS1, which catalyzes the conversion of UTP to CTP, is the predominant CTPS isoenzyme in human AML. CTPS1 function is vital for cell proliferation, particularly of rapidly growing cancer cells. We assessed CTPS1 lineage specificity by comparing median essentiality scores of CTPS1 in different cancers. Analysis of Depmap datasets showed that, unlike other reportedly essential genes (PMID: 34531254), CTPS1 activity is preferentially required for AML cell viability. Follow-up of AML patients from existing datasets (TCGA, TARGET) revealed that patient specimens showing higher CTPS1 levels correlated with decreased overall survival (TCGA: P=0.026; TARGET: P<0.0001). To analyze a potential correlation between CTPS1 activity and immunity, we analyzed the BEAT AML dataset to evaluate reported cytotoxic T lymphocyte (CTL) scores and found those scores were negatively correlated with CTPS1 levels, suggesting an immunosuppressive role of CTPS1.

To evaluate CTPS1 function in AML, we utilized the CTPS1 inhibitor STP-B (PMID: 37008165), which is >1000-fold more selective for CTPS1 than CTPS2. Notably, exposing AML cells to STP-B reduced cell viability, including that of THP1 cells and AML PDX blasts [n=3]), concentration-dependently (cell lines [n=6], with absolute IC50 values of <1000nM. These effects were rescued by cytidine (100uM) treatment, suggesting compound specificity. We then assessed CTPS1 loss-of-function in MLL-AF9 (MA9) transgenic AML mice, first by transplanting c-Kit+ BM cells from sick animals into either WT immunocompetent or immunodeficient (Rag2^-/-) recipients to promote AML development. When leukemic cells engrafted (>1% GFP in PB), mice were divided into two groups and treated 3 weeks with either vehicle control or STP-B (50mg/kg, oral gavage. bid, starting on day 10). STP-B-treated Rag2^-/- mice bearing AML transplants exhibited marginal survival advantages (Median survival: control 24 days vs STP-B 27 days). However, STP-B-treated WT recipients bearing AML donor cells survived significantly longer than corresponding controls (Median survival: control 25 days vs STP-B 34 days, p=0.0224, n=6). Moreover, STP-B treatment significantly increased both frequency (control 2.1±1.1%, SPT-B 4.8±0.5%, p=0.03) and number (control11.8±9.5 x10E3 vs STP-B 46.2±6.7 x10E3, p=0.01) of CD11b+/F480+ monocytes, suggesting macrophage involvement in these outcomes. Furthermore, depletion of macrophages and T cells partially blocked survival advantages seen in leukemic animals receiving STP-B treatment. STP-B treatment also induced leukemia ablation in a HOXA9-Meis1 transduction/transplantation AML model.

GSEA revealed that ex-vivo STP-B treatment remarkably upregulated IFN-I and -g response genes in THP1 or MA9 cells. IFN responses stimulate innate immune cell (macrophage and T cell) cross-priming, rendering AML cells more susceptible to ICIs, including anti-CD47 monoclonal antibody (aCD47) treatment. Thus we asked whether combining STP-B with an aCD47 would act synergistically in an MA9 leukemia transplant model. Following leukemia development, we treated mice with isotype control (Ctrl), anti-CD47 (BE0283 [BioXCell], 10mg/kg, i.p. qd, 3 weeks, starting on day 10), STP-B (50mg/kg, oral gavage bid, 3 weeks, starting on day 10) or the STP-B/anti-CD47 combination. Notably, combination treatment remarkably inhibited leukemia progression (Median Survival: Ctrl 26 days vs Combination: 42 days, P<0.0001, n=5). Moreover, following secondary transplantation, recipient mice receiving cells from the combination-treated group exhibited significantly reduced AML engraftment.

These results suggest that targeting CTPS1 promotes AML immunity and when combined with ICIs may provide a novel therapeutic strategy against AML.