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.

ABSTRACT:Arginine methylation catalyzed by protein arginine methyltransferases (PRMTs) is required for cancer cell proliferation, but whether PRMTs mediate resistance to therapy remains unclear. Here, we performed loss-of-function screens in venetoclax-resistant (VEN-R) acute myeloid leukemia (AML) patient-derived xenograft cells and found that PRMT9 plays a critical role in promoting VEN resistance. Specifically, VEN-R AML samples exhibited high levels of PRMT9, and PRMT9 inhibition resensitized AML cells to VEN treatment. In preclinical resistant models, genetic ablation of PRMT9 synergized with VEN to eradicate AML cells. Consistently, pharmacologic inhibition of PRMT9 combined with VEN produced similar effects in VEN-R AML mouse models. Mechanistically, PRMT9 ablation disrupted RNA splicing by inducing exon skipping in mRNA encoding ALG13, an uridine diphosphate (UDP)-N-acetylglucosaminyltransferase subunit, thereby downregulating expression of the VEN efflux transporter encoded by the adenosine triphosphate-binding cassette subfamily C member 1 gene. PRMT9 inhibition also suppressed protein synthesis, leading to downregulation of short-lived oncoproteins such as MCL1. These findings establish a connection between PRMT9-mediated arginine methylation and poor VEN responsiveness and demonstrate that targeting PRMT9 may represent a viable strategy to overcome VEN resistance.

De novo purine synthesis is required to maintain tumor growth; however, its impact on therapy resistance remains unclear. Here, through a dynamic BH3-priming-based CRISPR screen, we found that deletion of ADSS2, which encodes the adenylosuccinate synthase 2 enzyme essential for adenosine monophosphate (AMP) synthesis, re-sensitizes drug-resistant acute myeloid leukemia cells to venetoclax and a myeloid cell leukemia-1 (MCL1) inhibitor. Single-cell sequencing analysis of patient-derived xenograft samples revealed a positive association of high ADSS2 activity in TP53-mutant cells with poor responsiveness to venetoclax. We developed an ADSS2 antagonist, which synergized with BH3 mimetics to promote apoptosis in preclinical models. Mechanistically, sensitization mediated by ADSS2 targeting correlated with downregulated AMP-activated protein kinase activity, which in resistant cells promotes mitophagy to eliminate damaged mitochondria after BH3 mimetic treatment. These data show that AMP synthesis promotes BH3 mimetic resistance and that combining ADSS2 targeting with BH3 mimetics represents a promising anti-cancer approach.

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 M1_SIGNATURE_1 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.

Abstract Primary or acquired resistance to immunotherapies, including immune checkpoint inhibitors (ICIs) and CAR-T cells, remains a major clinical challenge. This resistance is particularly prevalent in "immune-cold" tumors like acute myeloid leukemia (AML), which are characterized by low immunogenicity, an immunosuppressive microenvironment, and immune escape by leukemia stem cells (LSCs). Activating the tumor-intrinsic cGAS–STING pathway to induce Type I interferon (IFN-I) responses is a promising strategy to convert "cold" tumors to "hot". However, achieving this selectively in cancer cells while sparing normal tissues remains a critical, unsolved barrier. We sought to identify novel mechanisms controlling innate immune signaling in OXPHOS-heightened cancers like AML to develop a strategy for overcoming immunotherapy resistance.We employed an inducible CRISPR-KO screen to identify regulators of mitochondrial homeostasis and cGAS-induced innate immunity. The screen identified a subset of minor mitochondrial dehydrogenases (including DHODH, SDHs) as critical regulators of mitochondrial redox balance and mtDNA integrity. We demonstrate that genetic or pharmacologic inhibition of these enzymes reroutes metabolic flux toward the Electron Transport Chain (ETC) Complex I (C-I). This paradoxically hyperactivates C-I, amplifying mtROS, inducing mtDNA instability, and causing mtDNA leakage into the cytosol. The cytosolic mtDNA is sensed by cGAS, leading to robust STING activation and systemic IFN-I responses in vivo. Crucially, this immune-stimulatory mechanism is independent of the canonical metabolic roles of these enzymes; for instance, this response after DHODH inhibition was not rescued by uridine supplement. In immunocompetent murine AML models, we found that while tumor-selective DHODH ablation was curative, systemically pharmacologic inhibition of DHODH was profoundly immunosuppressive, as it blunted T-cell proliferation essential for an anti-leukemia response. To overcome this toxicity-efficacy barrier, we developed "DHODHi-ADC", a first-in-class, immune-boosting ADC that achieves leukemia-selective delivery of a potent DHODH inhibitor (DHODH-IN16). The ADC preserves systemic immune function while inducing potent, tumor-intrinsic innate immune activation, a clear distinction from purely cytotoxic ADCs. In humanized AML models, our ADC synergized remarkably with both ICIs and CAR-T cell therapy, resulting in highly effective, durable leukemia clearance, including the eradication of LSCs.Our findings define a novel, therapeutically exploitable immune-metabolic axis where ETC homeostasis controls innate immunity via mtDNA dynamics. Disrupting this homeostasis through C-I hyperactivation provides a powerful and highly translational strategy to sensitize OXPHOS-dependent cancers to immunotherapy. Citation Format: Haojie Dong, Guoyun Kao, Umesh P. Yadav, Lei Zhang, Arshad J. Ansari, Srinivasarao Singireddi, Bei Jia, Jianai Sun, Guohua Wu, Yini Wang, Xin He, Lei Zhang, Zheng Li, Ruiheng Wang, Wei Chen, Meng Liu, Shuaishuai Ge, Yang Li, Whitaker Cohn, Amandeep Salhotra, David B. Sykes, Jie Jin, Jianjun Chen, Guido Marcucci, Shoubao Ma, Hong Zheng, Yong Zhang, Ling Li. Exploiting electron transport chain dynamics to sensitize OXPHOS-dependent cancers to immunotherapy [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 5598.

Abstract Protein arginine methylation regulates several cellular functions, including RNA splicing, translation and DNA damage repair. Protein arginine N-methyltransferase (PRMT) dysregulation is often seen in malignant hematopoiesis. PRMT7, the only type III PRMT, catalyzes monomethyl arginine (MMA) modification, but its role in leukemogenesis is elusive. Re-analysis of a previous genome-wide CRISPR/Cas9 screen revealed PRMT7 to be a crucial negative-regulator of MHC-I, prompting us to ask whether PRMT7 inhibition might promote anti-AML immunity. To assess the potential PRMT7 function in MHC-I presenting, we knocked out PRMT7 in human AML lines including THP-1 and Molm13 and assessed MHC-I levels. Relative to controls, PRMT7 KO remarkably upregulated MHC-I expression in both lines. We also observed MHC-I upregulation was also observed in both cell lines after treatment with the targeted degrader (PRMT7 PROTAC) ex-vivo at relatively low concentrations, while the same dose of either compound spared normal hematopoietic stem/progenitor (CD34+) cells. To confirm MHC-I dynamics in an MLL-AF9 mouse model, we generated a Prmt7 KO MLL-MA9 mouse model from hematopoietic-specific Prmt7 KO mice (Prmt7fl/fl;Vav1-Cre) and observed 2-fold upregulation of H-2Kb expression relative to WT MLL-AF9 cells.Given the critical role of MHC-I in CD8+ T cell activation, we next asked whether PRMT7 deletion would enhance CD8+ T cell responses. We evaluated human T cell killing effects in PRMT7-KO/-WT THP-1 or Molm13 cells cocultured with activated CD8+ T cells derived from healthy donors. Post-coculture, we found that PRMT7 KO AML cells were more susceptible to human T cell-mediated killing. In agreement, PRMT7 KO murine AML cells were more sensitive to mouse T cell-mediated killing using a coculture model of MA9 and syngeneic active CD8+ T cells. Moreover, following PRMT7 PROTAC pretreatment, THP1 cells were more sensitive to human T cells mediated killing in a coculture system of THP1 cells and CD8+ T cells.Next, to assess whether PRMT7 deletion impairs normal hematopoiesis, we analyzed total bone marrow cellularity and lineage frequency in Prmt7 KO (Vav1-Cre+) versus Prmt7-WT (Vav1-Cre-) mice via Cytek full-spectrum flow cytometry. While total BM cellularity was comparable, PRMT7 KO slightly increased the number of CD4+ or CD8+ T cells. These results suggest that although PRMT7 function is likely dispensable for normal hematopoiesis, PRMT7 loss may have a modest effect on T cell proliferation or activation. In future studies, we will confirm whether PRMT7 inhibition promotes anti-tumor T cell activity in-vivo, and whether the underlying mechanism is via MHC-I regulation. Overall, we have shown that PRMT7 depletion or pharmacological inhibition enhances T cell function in part by upregulating MHC-I. These findings suggest that combining PRMT7 inhibitors with immunotherapy could be a promising strategy to overcome AML’s immune-cold properties. Citation Format: Shuaishuai Ge, Kaixiu Luo, Lei Zhang, Meng Liu, Xin He, Guohua Wu, Yang Li, Yadav P. Umesh, Haojie Dong, Shengli Xue, Jian Jin, Ling Li. Targeting PRMT7 elicits anti-AML immunity by promoting MHC-I expression [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 7784.

KMT2A-rearranged (KMT2A-r) acute myeloid leukemia (AML) is an aggressive AML subtype characterized by 11q23 chromosomal rearrangements involving KMT2A gene and clinically associated with poor prognosis. Herein, we show that HDAC8 is upregulated in KMT2A-r AML and high HDAC8 is associated with poor overall survival in KMT2A-r AML patients. Using a KMT2A::MLLT3 mouse model, we demonstrate that both genetic knockout and pharmacological inhibition of HDAC8 significantly delayed leukemia progression, prolonged survival and reduced disease recurrence. Mechanistically, HDAC8 inhibition downregulates STAT3-MYC axis independent of TP53 status across AML genetic subtypes. Biochemical assays revealed that HDAC8 binds directly to STAT3, promoting its deacetylation and stabilization, while HDAC8-selective inhibitor (HDAC8i) treatment results in increased STAT3 acetylation and subsequent STAT3 degradation which in turn downregulates MYC. Given that STAT3-MYC signaling promotes cell survival and Venetoclax resistance, we show that HDAC8i exhibits synergistic anti-leukemia activity with Venetoclax in primary AML cells regardless of TP53 status. Combination of HDAC8i and Venetoclax synergistically reduced leukemia burden and significantly prolonged survival in both KMT2A::MLLT3 AML and patient-derived xenograft models. This study highlights the regulatory function of HDAC8 on STAT3-MYC and provides the proof-of-principle for targeting HDAC8 in combination with Venetoclax for the treatment of KMT2A-r AML.

Venetoclax (VEN), when combined with hypomethylating agents such as azacitidine (AZA), is a critical FDA-approved AML therapy for older patients unfit for intensive chemotherapy. Despite its efficacy, resistance can emerge, and response duration is short, representing an unmet clinical need. The presence of TP53 mutations in 5–10% of newly diagnosed AML cases directly associate with VEN resistance (VEN-R), although underlying mechanisms are not well understood. Aberrant purine metabolism has been seen in cells tolerant to VEN treatment, but the driver gene(s) or their functional relevance remain elusive. Recently, employing a dynamic BH3-priming CRISPR screen, we identified ADSS2, an enzyme functioning in AMP biosynthesis, as a key modulator of VEN sensitivity; ADSS2 deletion re-sensitized VEN- and MCL1i-resistant AML cells by suppressing AMPK signaling (He X et al., ASH 2023). Recently, we evaluated ADSS2 expression in two VEN-R TP53 loss-of-function Molm13 cell lines: TP53-knockout (KO) and R248Q/− isogenic cells. Both models exhibited elevated ADSS2 expressions relative to TP53 wild-type counterparts. Moreover, ADSS2 KO combined with low-dose VEN induced robust apoptosis in both models, highlighting that high ADSS2 activity promotes TP53-mutant AML phenotypes. Herein, to investigate the mechanism of ADSS2 upregulation, we first screened out 31 candidate transcription factors (TFs) associated with ADSS2 transcription through the TFBIND database. Further correlation analysis between TF and ADSS2 expression in the Beat AML cohort, followed by shRNA-mediated knockdown plus ChIP/qPCR validation, identified c-Myc as a critical regulator of ADSS2. Notably, c-Myc levels were significantly elevated in TP53-KO and TP53 R248Q/− Molm13 isogenic cells relative to respective parental counterparts. To track c-Myc-ADSS2-high cell emergence and their relationship to TP53 mutation at single-cell resolution, we utilized Genotyping of Transcriptomes (GoT) approach, which overlays TP53 mutational status onto single-cell RNA-seq data. We applied this approach to a paired TP53 mutant (c.742C>T, p.R248Q) patient-derived xenograft (PDX) sample prior- and after VEN/AZA treatment. Briefly, NSGS mice were engrafted with PDX blasts and treated with either V/A (VEN: 100mg/kg/i.g./q.d., 5 times/week, AZA: 3mg/kg/i.p./t.i.w) or vehicle until resistance developed and then bone marrow (BM) PDX cells were harvested for analysis. The frequency of genotyped blasts in either drug-naïve or post-treatment groups was approximately 83.2%, consistent with reported genotyping efficiency. Comparison of drug-naïve with resistant PDX cells revealed markedly increased ADSS2 and c-Myc expression in the resistant state. Genotype overlay on transcriptome maps showed that TP53-mutant cells expressed significantly higher levels of ADSS2 and c-Myc than did TP53-wild-type cells in drug-naïve samples. Notably, at relapse, the TP53-mut population expanded from 37.5% to 63.2%, contributing to c-Myc and ADSS2 upregulation in resistant samples. These results suggest that ADSS2 upregulation in resistant cells is associated with clonal selection of pre-existing TP53-mutant cells exhibiting elevated c-Myc and ADSS2 levels. We next assessed the therapeutic potential of Cmpd3, our newly developed ADSS2 inhibitor, as monotherapy or combined with V/A in NSGS mice engrafted with a TP53-mutant AML PDX. After confirming engraftment, we treated mice 3 weeks with vehicle, Cmpd3 (100 mg/kg, intraperitoneally, once daily, five times per week), V/A, or Cmpd3 plus V/A. Analysis of BM samples revealed that combination therapy significantly decreased the leukemic burden compared to either monotherapy, indicating a synergistic anti-leukemic effect in this TP53-mutant AML model. We also assessed the impact of Cmpd3 on normal hematopoiesis in WT C57BL/6 mice. Mice treated for 4 weeks with Cmpd3 showed no significant changes in organ (kidney, liver, spleen) histology, body weight, or HSPC frequency, and only modestly decreased numbers of BM B and T cells, indicating relative safety of Cmpd3. Collectively, our study reveals ADSS2 to be a contributor to TP53-associated VEN resistance and a promising target to restore drug sensitivity in this high-risk AML subtype.

We report here on a novel pro-leukemogenic role of FMS-like tyrosine kinase 3-internal tandem duplication (FLT3-ITD) that interferes with microRNAs (miRNAs) biogenesis in acute myeloid leukemia (AML) blasts. We showed that FLT3-ITD interferes with the canonical biogenesis of intron-hosted miRNAs such as miR-126, by phosphorylating SPRED1 protein and inhibiting the "gatekeeper" Exportin 5 (XPO5)/RAN-GTP complex that regulates the nucleus-to-cytoplasm transport of pre-miRNAs for completion of maturation into mature miRNAs. Of note, despite the blockage of "canonical" miRNA biogenesis, miR-155 remains upregulated in FLT3-ITD+ AML blasts, suggesting activation of alternative mechanisms of miRNA biogenesis that circumvent the XPO5/RAN-GTP blockage. MiR-155, a BIC-155 long noncoding (lnc) RNA-hosted oncogenic miRNA, has previously been implicated in FLT3-ITD+ AML blast hyperproliferation. We showed that FLT3-ITD upregulates miR-155 by inhibiting DDX3X, a protein implicated in the splicing of lncRNAs, via p-AKT. Inhibition of DDX3X increases unspliced BIC-155 that is then shuttled by NXF1 from the nucleus to the cytoplasm, where it is processed into mature miR-155 by cytoplasmic DROSHA, thereby bypassing the XPO5/RAN-GTP blockage via "non-canonical" mechanisms of miRNA biogenesis.

Protein arginine methylation regulates several cellular functions, including RNA splicing, translation and DNA damage repair. Protein arginine N-methyltransferase (PRMT) dysregulation is often seen in malignant hematopoiesis. PRMT7, the only type III PRMT, catalyzes monomethyl arginine (MMA) modification, but its role in leukemogenesis is elusive. Re-analysis of a previous genome-wide CRISPR/Cas9 screen revealed PRMT7 to be a crucial negative-regulator of MHC-I, prompting us to ask whether PRMT7 inhibition might promote anti-AML immunity. Our analysis of human TCGA AML dataset as well as normal healthy dataset (GTEX) revealed that AML cases exhibited higher PRMT7 expression than did those from healthy donors. Among cytogenetic subgroups, PRMT7 levels were significantly higher in KMT2A-rearranged (MLL-r) AML. Moreover, correlation analysis across combined datasets (MDACC, BeatAML and TCGA) demonstrated that PRMT7 levels negatively correlate with expression of MHC-I associated genes, including HLA-A (r = -0.33, p<0.001), HLA-C (r = -0.37, p<0.001), B2M (r = -0.45, p<0.001) and NLRC5 (r = -0.25, p<0.001), but not MHC-II genes, such as HLA-DR, HLA-DQ, HLA-DP and CIITA. PRMT7 levels were also negatively correlated with Cytotoxic T Lymphocyte (CTL) scores in the same combined datasets, highlighting its immunosuppressive role in leukemogenesis. To assess the potential PRMT7 function in MHC-I presenting, we knocked out PRMT7 by electroporating human AML lines including THP-1 and Molm13 with ribonucleoprotein (RNP) complexes formed by Cas9 protein and anti-PRMT7 sgRNA and assessed MHC-I levels. Relative to controls, PRMT7 KO remarkably upregulated MHC-I (HLA-A/B/C) expression in both lines. We also observed MHC-I upregulation was also observed in both cell lines after treatment with the PRMT7 inhibitor SGC3027 or administration of a targeted degrader (PRMT7 PROTAC) ex-vivo at relatively low concentrations, while the same dose of either compound spared normal hematopoietic stem/progenitor (CD34+) cells. To confirm MHC-I dynamics in an MLL-AF9 mouse model, we generated a Prmt7 KO mouse model by introducing MLL-AF9 retrovirus into Kit+ cells from hematopoietic-specific Prmt7 KO mice (Prmt7fl/fl;Vav1-Cre) and observed 2-fold upregulation of H-2Kb expression, based on antibody staining and flow cytometry analyses, relative to WT MLL-AF9 cells. Given the critical role of MHC-I in CD8+ T cell activation, we next asked whether PRMT7 deletion would enhance antigen-specific CD8+ T cell responses. To do so, we evaluated human T cell killing effects in PRMT7-KO/-WT THP-1 or Molm13 cells cocultured with activated CD8+ T cells derived from healthy donors. Post-coculture, we calculated T cell killing effects by flow cytometry using Count-bright absolute counting beads and found that PRMT7 KO AML cells were more susceptible to human T cell-mediated killing. In agreement, we confirmed that relative to PRMT7-WT murine AML cells, PRMT7 KO murine AML cells were more sensitive to mouse T cell-mediated killing using a coculture model of MA9 and syngeneic active CD8+ T cells. Moreover, following PRMT7 PROTAC pretreatment, THP1 cells were more sensitive to human T cells mediated killing in a coculture system of THP1 cells and CD8+ T cells. Next, to assess whether PRMT7 deletion impairs normal hematopoiesis, we analyzed total bone marrow cellularity and lineage frequency in Prmt7 KO (Vav1-Cre+) versus Prmt7-WT (Vav1-Cre-) mice via Cytek full-spectrum flow cytometry. While total BM cellularity was comparable, PRMT7 KO slightly increased the number of CD4⁺ or CD8⁺ T cells. Further analysis revealed increased frequencies of CD25⁺CD4⁺ T and CD69⁺CD8⁺ T cells. These results suggest that although PRMT7 function is likely dispensable for normal hematopoiesis, PRMT7 loss may have a modest effect on T cell proliferation or activation. In future studies, we will confirm whether PRMT7 inhibition promotes anti-tumor T cell activity in-vivo, and whether the underlying mechanism is via MHC-I regulation. Overall, we have shown that PRMT7 depletion or pharmacological inhibition enhances T cell function in part by upregulating MHC-I. These findings suggest that combining PRMT7 inhibitors with immunotherapy could be a promising strategy to overcome AML's immune-cold properties.

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.

For older AML populations ineligible for intensive chemotherapy and allogeneic stem cell transplantation, combining the BH3 mimetic venetoclax (VEN) with hypomethylating agents has emerged as first-line therapy. Despite early responses, resistance to VEN, marked by decreased mitochondrial-apoptotic-priming (“BH3 priming”), emerges over time. Protein arginine N-methyltransferases (PRMTs), which frequently exhibit aberrant activity in malignant hematopoiesis, reportedly regulate RNA splicing. Recent reports reveal that disruption of the splicing machinery increases AML cell sensitivity to VEN. Hence, we asked if modulating PRMT activity would enhance VEN responses. Indeed, datamining of published CRISPR screen results revealed synthetic lethal interactions between loss of individual PRMTs (2,6,7,8, or 9) and VEN treatment. To precisely identify PRMTs whose loss enhances sensitivity to mitochondrial apoptosis, we performed loss-of-function studies (based on pharmacological inhibitors or shRNAs) targeting individual PRMTs in VEN-resistant AML patient-derived-xenograft (PDX) cells and Molm13 (Molm13/R) cells using dynamic BH3 priming as readout. In these models, cells were rendered VEN-resistant via in-vivo or ex-vivo administration. The top hit in the BH3 priming assay was PRMT9, a recently defined PRMT (PMID: 38413714). Similar results were obtained using an isogenic Molm13 line resistant to VEN with TP53 R248Q heterozygosity (a gift from PMID: 35026842). Next, we examined PRMT9 function in Molm13/R cells transduced with a doxycycline (dox)-inducible PRMT9-shRNA. PRMT9 KD by dox treatment induced modest apoptosis in resistant cells (shCtrl 4.5%, shPRMT9 13.3%, p=0.02), while combining PRMT9 KD with VEN at clinically achievable levels (IC25 1µM) induced robust apoptosis (VEN monotherapy 14.6%, combination 70.7%, p<0.001). Viability assessment showed that PRMT9 KD decreased VEN IC50 from 3.9µM to 0.2µM. We then verified PRMT9 function in an MLL-AF9/FLT3-ITD double-hit Mx1-Cre/Prmt9f/f (MA9/FLT3-ITD/Prmt9-cKO) mouse model. Unlike the MA9 single-hit model, the double-hit model exhibited poor responsiveness to VEN plus azacitidine (V/A) treatment, due to MCL1 upregulation. We next transplanted double-hit Prmt9-cKO or control leukemic cells into NSG mice and treated them with PIPC to induce PRMT9-KO or with V/A simultaneously. Prmt9 targeting combined with V/A treatment almost completely eliminated leukemia burden, and mice treated with both survived the entire observation period. We then tested anti-AML effects of our in-house PRMT9 inhibitor (P9i) combined with VEN in VEN-relapsed samples (n=4). P9i alone induced modest apoptosis, while the P9i/VEN combination (at their IC25s) significantly increased apoptosis. We evaluated P9i as part of combination therapy in vivo using the double-hit model. Leukemic mice were divided into four groups and treated for 3 weeks with: 1) vehicle, 2) P9i (100mg/kg, IP, twice a day, 5 times/week), 3) VEN/AZA (V/A: VEN, 100 mg/kg, orally, daily; AZA, 3 mg/kg, IP, 3 times/week) or 4) the P9i/VEN/AZA combination. Notably, combination treatment significantly extended survival relative to control or monotherapy groups. Mechanistically, Molm13/R cell transcriptome analysis revealed that PRMT9-KD significantly increased aberrant splicing of some transcripts whose loss exhibits synthetic lethality with VEN treatment, such as ALG13. ALG13 encodes the protein-forming uridine diphosphate-N-acetylglucosamine (UDP-GlcNAc) transferase, which is crucial for catalyzing protein asparagine (N)-linked glycosylation and maturation of proteins such as ABCC1 (PMID: 20535133) Interestingly, targeting ABCC1 reportedly reverses VEN resistance (PMID: 37726279). PRMT9 inhibition remarkably decreased ALG13 protein levels; ALG13 knockout (KO) phenocopied PRMT9 targeting-induced sensitivity to mitochondrial apoptosis. PRMT9 inhibition also significantly repressed protein translation, decreasing levels of short-lived proteins. Notably, MCL1 protein expression, which contributes to VEN resistance, remarkably decreased after PRMT9 inhibition, as evidenced by shifts in polysome profiling seen following PRMT9 KD. In summary, our study indicates that targeting PRMT9 can overcome VEN resistance in AML, likely through splicing modulation and translation inhibition.

Current anticancer therapies cannot eliminate all cancer cells, which hijack normal arginine methylation as a means to promote their maintenance via unknown mechanisms. Here we show that targeting protein arginine N -methyltransferase 9 (PRMT9), whose activities are elevated in blasts and leukemia stem cells (LSCs) from patients with acute myeloid leukemia (AML), eliminates disease via cancer-intrinsic mechanisms and cancer-extrinsic type I interferon (IFN)-associated immunity. PRMT9 ablation in AML cells decreased the arginine methylation of regulators of RNA translation and the DNA damage response, suppressing cell survival. Notably, PRMT9 inhibition promoted DNA damage and activated cyclic GMP-AMP synthase, which underlies the type I IFN response. Genetically activating cyclic GMP-AMP synthase in AML cells blocked leukemogenesis. We also report synergy of a PRMT9 inhibitor with anti-programmed cell death protein 1 in eradicating AML. Overall, we conclude that PRMT9 functions in survival and immune evasion of both LSCs and non-LSCs; targeting PRMT9 may represent a potential anticancer strategy.

Inv(16) [inv(16)(p13q22) or t(16;16)(p13.1;q22)], a common recurrent chromosomal translocation in acute myeloid leukemia (AML), creates CBFb-MYH11 (CM) leukemogenic fusion gene by fusing core binding factor CBFb with smooth muscle myosin heavy chain gene MYH11. Despite the relatively favorable prognosis of inv(16) patients, only about 50-60% achieve long-term survival with standard chemotherapy, highlighting the need for novel therapies to achieve cure. We previously reported that CM fusion protein physically interacts with HDAC8 and enhances its activity, which is critical for CM-driven leukemogenesis. We also presented CM expression increases DNA damage and impairs homologous recombination-directed repair through novel HDAC8-mediated mechanisms. HDAC8 interacts with BRCA1, BCLAF1, and U2AF1 as part of the RNA splicing machinery, critical for efficient DNA damage repair. HDAC8 deacetylates U2AF1, thereby disrupting the recruitment of BRCA1/BCLAF1 to RNA splicing machinery. Accordingly, CM expression, through enhanced HDAC8 activity, leads to dysregulated RNA splicing and defective DNA repair. Our analysis of the TARGET dataset revealed higher HDAC8 expression in AML samples compared to healthy controls (HL) and high HDAC8 (top 25%) is significantly correlated with increased DDR signature score (GO:00006974) in both TARGET and Beat AML datasets. We postulated that HDAC8-high malignant cells, including CM-AML, may exhibit increased sensitivity to splicing modulators or PARP inhibitors (PARPi). First, we evaluated the effects of H3B-8800 (H3B; an orally bioavailable small molecule inhibitor for SF3B complex) and PARPi (Olaparib or Talazoparib) in primary murine CM-AML cells. Compared to normal bone marrow (BM) cells, CM-AML cells showed significantly increased sensitivity to H3B (IC50: 55.21 ± 9.585 vs. 432.0 ± 21.49 nM; p<0.0001) and Olaparib (IC50: 4.394 ± 1.008 vs. 57.67 ± 2.358 µM; p<0.0001). Similar results were obtained in primary human inv(16) AML samples. Compared to HL-CD34+, inv(16)-CD34+ cells were significantly more sensitive to H3B (IC50: 40.45 ± 12.54 vs. 270.7 ± 18.04 nM; p<0.0001), Olaparib (IC50: 3.251 ± 1.498 vs. 28.04 ± 4.827 µM; p=0.0004) and Talazoparib (IC50: 130.4 ± 27.57 vs. 2681 ± 487.9 nM; p<0.0001). To test in vivo effects, cohorts of CM-AML mice were generated and given H3B (8 mg/kg), Talazoparib (0.25 mg/kg), or vehicle (0.5% methylcellulose) by daily oral gavage for 10 days. Compared with vehicle, H3B or Talazoparib group showed significantly reduced splenic disease burden (H3B: 0.253 ± 0.0214 g vs. 0.422 ± 0.0734 g; p=0.0401; Tala: 0.143 ± 0.033 g vs. 0.422 ± 0.0734 g; p=0.0051), decreased ckit+ blasts in BM (H3B: 7.825 ± 2.08% vs. 32.22 ± 2.689%; p<0.0001; Tala: 18.5 ± 2.422% vs. 32.22 ± 2.689%; p=0.0042), decreased ckit+ blasts in spleen (H3B: 9.4 ± 1.118% vs. 52.84 ± 5.326%; p<0.0001; Tala: 24.09 ± 6.973% vs. 52.84 ± 5.326%; p=0.0115) with prolonged survival (H3B: 95 vs. 68 days; p=0.0003; Tala: 95.5 vs. 68 days; p=0.0007). In support of our hypothesis, analysis of Beat AML dataset showed that Olaparib-sensitive patients (IC50<4 μM) are enriched for HDAC8-high (top 50%) expression. In addition, HDAC8 overexpression (HDAC8-OE) in several cell lines representing different disease subtypes (MV4-11, HEL, MDS-L) were more sensitive to H3B, Olaparib, and Talazoparib compared to empty vector (EV) control. To assess in vivo effects, MV4-11-luciferase cells transduced with EV or HDAC8-OE vector were transplanted into NSGS mice (i.v. 0.5x106 cells/mouse, no radiation) and given H3B (8 mg/kg), Talazoparib (0.25 mg/kg), or vehicle (0.5% methylcellulose) by daily oral gavage for 10 days. Bioluminescence imaging showed a significant reduction in tumor burden in both H3B and Talazoparib treated groups compared to vehicle. MV4-11-Luci-HDAC8 cells were selectively more sensitive to H3B (HDAC8 vs. EV; p<0.0001) or Talazoparib (HDAC8 vs. EV; p=0.001) treatment with reduced tumor burden compared to MV4-11-Luci-EV cells. Compared to MV4-11-Luci-EV mice, significantly prolonged survival was seen for MV4-11-Luci-HDAC8 mice treated with H3B (HDAC8: 39 vs. EV: 31 days; p=0.0001) or Talazoparib (HDAC8: 36 vs. EV: 29 days; p=0.0002). Overall, these results indicate that both splicing modulator and PARPi can effectively target HDAC8-high AML cells, including inv(16) AML, offering novel therapeutic avenues for inv(16) AML and AML with aberrant high-HDAC8 activity.

Recently, the BH3 mimetic-Bcl2 inhibitor venetoclax (VEN), has been approved by FDA in 2018 for the treatment of patients with AML in combination with a hypomethylating agent (HMA) such as azacytidine (AZA). This synergy is likely due to HMA-induced mitochondrial vulnerability associated with cytoplasmic RNA metabolism. Nonetheless, most AML patients treated with the combination eventually relapse, possibly due to persistence of tumor cells not dependent on antiapoptotic proteins (Bcl2, Mcl1). There is a pressing need to develop targeted therapeutics sensitizing AML cells to mitochondrial apoptosis, particularly for those patients failed from VEN associated treatment. To achieve the goal, we performed a CRISPR/Cas9 Knockout (KO) screen (using Bim peptide-based dynamic ΔBH3 profiling as read-out) targeting genes (n=534) involved in cytosolic nucleotides metabolism pathways to increase mitochondrial vulnerability in a BH3 mimetic-refractory AML cell line (HEL). That search identified ADSS2, which catalyzes the first committed/rate-limiting step of AMP biosynthesis from IMP, emerged as the top hit, exhibiting highest ΔBH3 priming. Subsequent BH3 priming assays validated that ADSS2 KO significantly enhanced sensitivity to BH3 mimetics across various AML cell lines, including MOLM13, SKM1, OCI-AML3, and HEL cells. ADSS2 KO also led to a noteworthy reduction in the IC50 values of both VEN and the Mcl1 inhibitor S63845 in these cell lines. Additionally, the synergistic effect between sgRNA targeting ADSS2 and VEN or S63845 was also confirmed in Cas9 expressing cells from two AML patient-derived-xenograft (PDX) samples. We next transplanted AML cells (form one PDX samples) with or without ADSS2-KO into NGS mice, then treated mice with VEN. Notably, VEN treatment (100 mg/kg, QDx14, PO) significantly reduced tumor burden in ADSS2-KO AML mice compared to WT AML mice. Additionally, ADSS2 KO combined with VEN treatment group displayed a survival advantage compared to single VEN treatment group. Further mechanistic investigations revealed that ADSS2 KO led to a remarkable depletion of AMP rather than ATP, and a robust repression of AMPK activity in HEL cells. To assess whether AMPK inhibition mimics the effect of ADSS2 KO, AMPKα-deficient (Mx1-Cre/Prkaa1 fl/fl/ Prkaa2 fl/fl) MLL-AF9 (MA9) transformed murine AML cells were assessed. Indeed, AMPKα depletion substantially enhanced VEN treatment efficiency ex-vivo. Notably, enforced overexpression of a constitutively active AMPKa2 (T172D) construct in a VEN sensitive cell line SKM1, promoted cell resistance to mitochondrial apoptosis by VEN or S63845. Moreover, T172D expression can also abolish ADSS2-KO mediated mitochondrial vulnerability to VEN or S63845 treatment, indicating the pivotal role of AMPK signaling in mediating ADSS2 function. There are no reported ADSS2 inhibitors. To explore ADSS inhibitor, we conducted a fragment-based virtual screen of 8,000,000 compounds (MolPort) library and requested the top-ranking hits for ADSS2 catalysis evaluation by ELISA. Notably, the top two hits AS104 and AS71 exhibited the most potent inhibitory effects (AS104 IC50=2.43µM, AS71 IC50=3.85µM). NMR-STD analysis was conducted in a cell-free system, which unequivocally confirmed the physical binding of inhibitors and the ADSS2 protein. To further investigate the interactions between AS71 and AS104 with ADSS2 in a cellular context, both compounds were subjected to a cellular thermal shift assay (CETSA). Notably, treatment with AS71 and AS104 led to substantial shifts in ADSS2 protein thermal stability, providing evidence of their interactions with ADSS2 within the cellular environment. In both HEL cells and a primary VEN-resistant AML samples, treatment with AS71 and AS104 significantly increased ΔBH3 priming. Collectively, our observation revealed that targeting ADSS2 is critical for sensitizing AML cells to BH3 mimetics. We are now conducting preclinical assessments of ADSS2 inhibitors.

Despite advances in targeted therapy and use of molecular/cytogenetic risk stratification to guide treatment, clinical outcomes for patients with acute myeloid leukemia (AML) remain unfavorable. Harnessing the immune system with CAR-T or immune checkpoint inhibitors (ICIs) has been proven successful in other hematological malignancies and solid tumors. The use of these immunotherapies for the AML treatment remains challenging due to lack of selective target antigens or low expression of immune checkpoint proteins. Recently, one strategy proposed to transform AML from immune-cold to immune-hot is to trigger a type-I interferon (IFN-I) response via a dsDNA related cGAS/STING signaling or dsDNA related RIG-I/MDA5 signaling. We recently reported that the dNTP and Ara-CTP (active form of cytarabine [AraC]) hydrolase SAMHD1, whose expression levels are elevated in AML patients relative to normal donors, limits IFN-I and T cell responses in murine AML model ( Blood, 2022, 140 [Supplement 1]: 679-680). We also found that innate immune activation seen following SAMHD1 knockdown is associated with its catalytic activity inhibition in cancer cells, providing a rationale for the development of SAMHD1 inhibitor. To identify SAMHD1 inhibitor, we conducted a virtual screen on 8,000,000 compounds based on SAMHD1 crystal structure (PBD 6TXC). The top 500 hits were then assessed in SAMHD1-WT- or H233A (SAMHD1 loss-of-function mutant) -expressing Molm13 cells using a phenotypic screen for AraC sensitization. Specifically, a SAMHD1 inhibitor should sensitize SAMHD1-WT (S-WT) but not H233A (S-H233A) cells to AraC treatment. Among the top 20 hits from phenotypic screen, our HPLC-based cell-free and cell-based SAMHD1 enzymatic assay revealed Samin27 as the most potent hit in blocking SAMHD1 hydrolysis and promoting AraCTP accumulation. Therefore, we assessed the efficacy of Samin27 combined with AraC in vivo. Samin27 administration (25mg/kg i.p. qd, starting on day 8 for 7 days) enhanced AraC efficacy (50mg/kg i.p. qd, starting 8th day for 5 days) in NSG mice implanted with Molm13 cells, as evidenced by extended survival relative to AraC alone. Moreover, the physical interaction between Samin27 and SAMHD1 protein were confirmed in a Carr-Purcell-Meiboom-Gill (CPMG) nuclear magnetic resonance (NMR) titration assay. RNA-seq results revealed Samin27 ex-vivo treatment remarkably upregulated IFN-I response genes in Molm13 cells, consistent with transcriptome results of SAMHD1-knockdown (S-KD) Molm13 cells. SAMHD1 activity is known to restrict replication of LINE-1 (L1), which is the only autonomously active transposable elements (TE) in mammals; elevated retrotransposition induces DNA damage and promotes cytoplasmic DNA accumulation, resulting in an IFN-I response via cGAS signaling. Next, to explore the mechanism underlying SAMHD1-inhibition induced immunity, we asked if SAMHD1 loss promoted retrotransposition and upregulated TE transcripts in leukemia cells. Through re-analysis of RNA-seq data in Molm13 cells, we found SAMHD1 inhibition upregulated most L1 elements and LTR-containing endogenous retrovirus (ERV) subfamilies. To determine if Samin27 induced immunity via L1 upregulation, we employed 3TC treatment (3TC, the nucleoside reverse transcriptase inhibitor) to block L1 upregulation. 3TC treatment blocked Samin27-mediated IFN-I response genes upregulation. Given that IFN-I response can stimulate innate immune cells (macrophage and T cell) cross-priming, potentially rendering AML cells more susceptible to anti-CD47 monoclonal antibody (aCD47) treatment, we then assessed whether combining a SAMHD1 inhibitor with an aCD47 act synergistically. We first implanted murine MLL-AF9 (MA9) leukemia cells into WT B6 mice. Then we treated leukemic mice with isotype control (Ctrl), anti-CD47 (BE0283 [BioXCell], 10mg/kg, i.p. qd, 3 weeks), Samin27 (25mg/kg i.p. qd, 3 weeks) or combination. Notably, pharmacological inhibition of SAMHD1 remarkably inhibited leukemia progression, decreasing leukemia burden (Fig. A.B). In another cohort, combination treatment extended mouse survival (Fig. C). Collectively, our results may provide a rationale for combining Samin27 with aCD47 against AML population, where single anti-CD47 treatment shows little effects.

Inversion of chromosome 16, inv(16) [inv(16)(p13q22) or t(16;16)(p13.1;q22)], is a recurrent chromosomal translocation observed in 5-8% of acute myeloid leukemia (AML) cases. Inv(16) creates a fusion gene CBFb-MYH11 (CM) which impairs hematopoietic differentiation and creates abnormal progenitor populations prone to leukemic transformation. We reported that CM interacts with HDAC8 and enhances its activity, and high HDAC8 activity impaired DNA damage response (DDR) in CM knock-in (KI) hematopoietic stem and progenitor cells (HSPCs) (Zhang, L et al, ASH meeting 2022, https://doi.org/10.1182/blood-2022-160280). The recruitment of BRCA1-mRNA splicing machinery is critical for efficient DDR and the U2 small nuclear RNA auxiliary factor 1 (U2AF1) is required for accurate 3'-splice site selection. Here, we show how U2AF1 is post-translationally regulated following DNA damage and its impact on alternative splicing in CM KI HSPCs upon DNA damage. First, we identified TIP60 as the histone acetyltransferase (HAT) responsible for the increased U2AF1 acetylation induced by ionizing radiation (IR). We detected enhanced interaction between U2AF1 and TIP60 but not with other HATs (GCN5, PCAF, CBP) along with the increased U2AF1 acetylation in response to IR. In addition, TIP60 knockdown (KD) reduced U2AF1 acetylation, while TIP60 expression increased U2AF1 acetylation, indicating that TIP60 mediates the acetylation of U2AF1. To pinpoint the specific lysine residue subjected to TIP60 catalysis, we introduced lysine site-specific mutants U2AF1-HA-K15R, K23R, K39R, K175R, or 4 lysine sites mutant (U2AF1-HA-4mut) with TIP60. Co-immunoprecipitation (anti-HA) followed by western blotting (anti-acetyl-lysine) showed undetectable acetylation of U2AF1-HA-K23R and K175R mutants (similar to U2AF1-HA-4mut), suggesting that K23 and K175 residues are essential for TIP60-mediated acetylation. To investigate the impact of K23 or K175 acetylation on the assembly of the BRCA1-mRNA splicing complex and DDR, we subjected U2AF1-K23R or K175R expressing 32D cells to IR (3.5 Gy). Both U2AF1-K23R and K175R mutants exhibited markedly reduced acetylation levels after IR, confirming that K23 and K175 sites are the acetylation sites responding to DNA damage. Furthermore, the interaction of U2AF1-K23R and K175R with BRCA1 and BCLAF1 was impaired. We observed increased apoptosis in U2AF1-K23R and K175R cells compared to wild-type (WT) U2AF1 cells (K23R vs WT, 51.975±7.867% vs 6.055±0.634%, P=0.0011; K175R vs WT,22.645±3.037% vs 6.055±0.634%, P=0.0017) after KD of endogenous U2AF1 and subjected to IR (3.5 Gy). These results indicate that U2AF1 is acetylated by TIP60 upon DNA damage on K23 and K175 which is critical for cell survival and assembly of the BRCA1-mRNA splicing complex in DDR. Our studies revealed that HDAC8 interacts with U2AF1 and modulates U2AF1 acetylation following DNA damage and acetylation on U2AF1-K23 is important for HDAC8 binding. Consistent with the enhanced HDAC8 activity induced by CM, we show that CM also diminished U2AF1 acetylation and impaired the assembly of the BRCA1-mRNA splicing complex after IR. To examine the consequences in alternative splicing events upon IR, we sorted CM KI (n=5) or control (n=3) Lin-Sca1+Kit+ (LSK) cells and exposed them to IR (2.0 Gy) followed by RNA-seq. We identified a total of 829 splicing events in WT-IR vs WT-NIR (NIR: no IR) and 643 splicing events in CM-IR vs CM-NIR. Specifically, we found that skipped exon (SE) and retained intron (RI) events are selectively reduced in CM vs WT (SE events: 496 vs 653; RI events: 2 vs 26). Gene set enrichment analysis revealed striking differential enrichment in 38 BRCA1-related pathways, including regulation of response to DNA damage stimulus (NES: -1.0816 vs 0.8707), regulation of DNA repair (NES: -1.1557 vs 0.7729), cell cycle checkpoint signaling (NES: -0.9719 vs 0.7895), and recombinational repair (NES: -0.9215 vs 0.7299). Altogether, these studies reveal mechanistic insights into DNA damage induced regulation of post-translational acetylation of U2AF1 splicing factor and provide a mechanistic link to the dysregulated alternative splicing and impaired DNA damage response in inv(16) AML.

Angiogenin (ANG) is the first human tumor-derived angiogenic protein, which can promote angiogenesis and tumor growth. In a previous study, we identified alpha-actinin 2 (ACTN2), a cytoskeletal protein, as a direct interacting protein with angiogenin. However, the interaction between ANG and ACTN2 was not characterized in detail, which may provide information on the molecular mechanisms of ANG functions. In this study, we mapped the accurate binding domain and sites in ANG and ACTN2, respectively. In ANG, the residues from 83 to 105 are the smallest motif that can bind to ACTN2. We then use site mutation analysis to identify the precise binding sites of ANG in the interaction and found that the 101st residue arginine (R101) represents the critical residue involved in the ANG-ACTN2 interaction. In ACTN2, the residues from 383 to 632, containing two spectrin domains in the middle of the rod structure of ACTN2, play an important role in the interaction. Furthermore, we validated the interaction of ACTN2-383-632 to ANG by glutathione-S-transferase (GST) pull-down assay. In functional analysis, overexpressed ACTN2-383-632 could impair tumor cell motility observably, including cell migration and invasion. Meanwhile, ACTN2-383-632 overexpression inhibited tumor cell proliferation and survival as well. These data suggest that an excess expression of ACTN2 segment ACTN2-383-632 can inhibit tumor cell motility and proliferation by interfering with the interaction between ANG and ACTN2, which provides a potential mechanism of ANG action in tumor growth and metastasis.