Acute myeloid leukemia (AML) is a heterogeneous disease characterized by a broad spectrum of molecular alterations that influence clinical outcomes. TP53 mutations define one of the most lethal subtypes of AML, driving resistance to nearly all available treatment modalities, including venetoclax plus azacitidine (VenAza). Yet, the molecular basis of this resistance, beyond affecting transactivation of BCL-2 family genes, has remained elusive. Here, we demonstrate that VenAza treatment leads to reduced transcriptional upregulation of the p53 signaling pathway in TP53 mutant/deficient AML compared to wild-type AML. Functionally, TP53 mutant/deficient AML exhibits selective failure in apoptosis induction rather than impaired G1 arrest or senescence. Despite inhibition of pro-apoptotic BAX and selective enrichment for MCL-1 in TP53 mutant isogenic AML cells, compensatory upregulation of BIM preserved functional mitochondrial outer membrane permeabilization (MOMP). TP53 mutant primary AML tumors at baseline also had retained capacity for MOMP. Instead, TP53 mutant AML exhibited disruption in caspase-3/7 activation to evade apoptosis after VenAza therapy – decoupling the mitochondrial and executioner phases of apoptosis. Importantly, this “post-MOMP brake” is not a bystander effect but itself a driver of VenAza and chemotherapy resistance in TP53 mutant/deficient AML. This previously unrecognized mechanistic insight shifts the focus from mitochondrial priming to terminal caspase blockade in TP53 mutant AML and opens the door for urgently needed therapeutic strategies that reignite apoptosis at its execution point.

Abstract Introduction: TP53 mutations occur in 30% of relapsed/refractory AML patients and are associated with poor prognosis, therapy resistance, and immune dysfunction. While TP53 mutations have been shown to induce T cell exhaustion and impair adaptive immunity, their impact on innate immune mechanisms, particularly NK cell-mediated cytotoxicity, remains poorly understood. Given the critical role of NK cells in immune surveillance against malignant cells, we investigated how TP53 loss alters AML susceptibility to NK cell killing and explored therapeutic strategies to overcome this resistance. Methods: We established co-culture systems using TP53-wildtype and TP53-inactivated AML cells with NK-92 cells, with validation using primary human NK cells. TP53-inactivated AML cells exhibited significantly reduced susceptibility to NK-92-mediated cytotoxicity, a phenotype confirmed with primary NK cells. To assess whether resistance was mediated by secreted factors, we compared killing in direct co-culture versus conditioned media experiments, revealing that resistance required direct cell-cell contact as conditioned media alone did not impair NK cell killing, suggesting membrane-bound or contact-dependent mechanisms. Results: A genome-wide CRISPR KO screen in TP53-inactivated AML cells was performed to identify genes regulating sensitivity to NK cell-mediated cytotoxicity, revealing several modulators of NK sensitivity, including cIAP1. Functional validation using genetic and pharmacological approaches demonstrated that targeting inhibitor of apoptosis proteins (IAPs) with small-molecule antagonists including birinapant, tolinapant, or PROTAC-based degrader (CST-626) at sublethal doses effectively sensitized TP53-inactivated AML cells to NK cell cytotoxicity. Analysis of NK-activating and inhibiting ligand expression profiles revealed that while TP53 loss altered the expression of multiple NK-interacting ligands, no single ligand was solely responsible for the resistance phenotype, suggesting a broader immune evasion program induced by p53 deficiency that parallels the T cell dysfunction observed in TP53-mutant AML patients. Conclusion: Overall, these results provide a rationale for combination strategies targeting both immune evasion mechanisms and apoptotic resistance in this patient population. Citation Format: Jordan Yong Ming Tan, Chuqi Wang, Wen Kang Soh, Yash Manish Agrawal, En Tong Lim, Fang Qi Lim, Yuhan Wang, Nishtha Chitkara, Xiao Xian Lin, Ahmed M. Mamdouh, Gloryn Chia, Nicholas R.J. Gascoigne, Shruti Bhatt. Functional Dissection of NK Cell Evasion Mechanisms in TP53-Inactivated Acute Myeloid Leukemia [abstract]. In: Proceedings of Frontiers in Cancer Science 2025; 2025 Nov 5-7; Singapore. Philadelphia (PA): AACR; Cancer Res 2026;86(13_Suppl):Abstract nr P09.

TP53 mutations confer treatment resistance across multiple cancers. Mechanisms of therapy resistance, beyond affecting transactivation of BCL-2 family genes, remain a mystery. Here, we report that TP53 mutated AML, triple negative breast cancer, and colorectal cancer escape therapy-induced apoptosis due to inability to activate caspase-3/7, despite having normal mitochondrial outer membrane permeabilization (MOMP) induction. To identify post-MOMP determinants of therapy resistance in TP53 mutated AML, we applied a multiomics approach - whole-genome CRISPR screen, bulk/single-cell RNAseq, and high-throughput drug screen. BIRC5 , encoding survivin, was selectively upregulated in paired hematopoietic stem/multipotent progenitor cells from TP53 mutant AML patients, with further enrichment after venetoclax-azacitidine (VenAza) relapse. Critically, BIRC5 was also upregulated in 17 of 26 TP53 mutant TCGA cancers. Genetic ablation of BIRC5 resensitized TP53 mutated AML to standard therapy by restoring caspase activation, validating therapeutic relevance. Importantly, targeting IAPs and survivin using clinically relevant inhibitors overcame VenAza resistance of TP53 mutant tumors in vivo , achieving sustained AML suppression. Combination with survivin inhibitors also overcame chemotherapy resistance in TP53 deficient solid cancers. Together, we discovered that wild-type TP53 is required in post-MOMP signaling and that BIRC5 dependency is an effective therapeutic target for poor prognosis, TP53 mutated cancers.

Cancer Immunotherapy, particularly chimeric antigen receptor (CAR) therapy, has achieved unprecedented success in blood cancers of T/ B-cell origins. However, acute myeloid leukemia (AML) has largely evaded clinical benefit from immunotherapeutic approaches. Current AML CAR-T therapies targeting CD123 and CD33 have failed to exhibit clinical activity and are associated with serious cytopenia, as they share antigen expression with normal HSPCs. In contrast to other AML-associated antigens, CD70, that activates T, B, and NK cells via CD27, is aberrantly expressed in many AML cases compared to normal HSPCs, making it an attractive target. However, CD70-targeting antibody and CAR-T showed limited clinical activity due to antigen loss and heterogeneous expression, suggesting unmet need to identify combination strategies that overcome the limitations of existing immunotherapeutic approaches in AML. To identify upstream regulators of CD70, we performed a genome-wide CRISPR knock out (KO) screen in myeloid leukemia cells using CD70 surface expression as a readout. TP53 emerged as top positive regulator of CD70 expression. We validated this finding in isogenic AML cell lines carrying WT, null and mutant TP53, where TP53 mutation or deletion significantly reduced CD70 expression. Consistently, p53 activation via Nutlin or conventional chemotherapy (cytarabine and daunorubicin) upregulated CD70 levels in TP53-wild-type (WT), but not in TP53-mutant or knockout cells. Analysis of bulk RNA-seq of primary AML samples (BeatAML database) and cell lines (DepMap database) also showed a significantly lower expression of CD70 in TP53-mutant/null cells across different cancer types. To further investigate mechanism of TP53-medited CD70 regulation, we performed chromatin immunoprecipitation sequencing. It revealed p53 binding downstream of the CD70 locus in TP53-WT cells but not null or R248Q mutant. Blocking this site with dCas9-KRAB suppressed CD70 expression, supporting direct transcriptional activation by p53 through enhancer binding. To characterize underlying anti-tumor immune mechanism of CD70-directed therapy, we engineered CD70 overexpression (OE) in the Trp53-/- MLL-AF9 syngeneic AML mouse model. Strikingly, CD70 OE mice exhibited impaired leukemia engraftment and expansion in immunocompetent WT mice. Expansion of CD70 OE compared to EV tumors was also seen in Rag1-/- mice that lack T and B cells, but not in NSG mice, which lack T, B, and NK cells. Moreover, depletion of NK cells but not CD8+ T cells abolished the anti-immune effect of CD70 OE. These data together suggest that AML-associated CD70 activates NK cell-mediated anti-tumor immunity in vivo. Ex vivo assays further confirmed direct involvement of NK-cells in CD70-dependent immunity. Splenic NK cells preferentially killed CD70-OE AML cells, implicating CD70 promotes NK cell-mediated anti-leukemia immunity. To finally explore therapeutic implications of our findings we generated our own CD70-directed CAR-T and TCR-T cells and evaluated CD70 CAR-T cell activity in the context of TP53 status. TP53-WT AML cells were sensitive to CD70 CAR-T-mediated killing, whereas TP53-mutant or KO cells were resistant. Chemical activation of p53 with Nutlin and chemotherapeutic drugs enhanced CD70 expression in cell lines and primary AML cells. A combination of p53 activating therapy, including Nutlin or chemotherapy, selectively synergized with CD70-targeting CAR-T and TCR-T in TP53-WT MV4-11 and MOLM13. Colony formation assay also remarkably showed that Nutlin + CD70 CAR-T combination effectively eliminated AML cells, while many colonies survived either monotherapy. Analysis of primary AML patient samples (n=47) revealed significantly higher CD70 expression in TP53 wild-type samples compared to TP53-mutant/deleted samples following ex vivo chemotherapy exposure. The clinical relevance is further supported by retrospective analysis of a cohort of AML patients, showing significantly better outcomes in patients with wild-type TP53 and high CD70 expression post-chemotherapy. Collectively we show, TP53 status could serve as a predictive biomarker for CD70-targeted immunotherapy, and that combining p53 reactivation agents with CD70 CAR-T represents powerful therapeutic strategy. Our study for the first time identified TP53 as a key transcriptional regulator of CD70 in AML, linking p53 activity to NK cell-mediated immune surveillance and response to CD70-directed immunotherapy.

TP53 mutations are among the worst prognostic factors in acute myeloid leukemia (AML), with affected patients facing relapse-free survival of just five-to-six months compared to TP53 wild-type patients. A major barrier to improving outcomes lies in the dearth of effective therapies, as TP53 mutant patients remain refractory to conventional cytotoxic chemotherapies, targeted therapies, and even allogeneic stem cell transplantation. In this review, we first summarize current clinical strategies and the major setbacks of p53 activators, MDM2/X regulators, and immunotherapy, highlighting the disconnect between promising pre-clinical studies and limited durable clinical responses. We next discuss the mechanisms of therapy resistance in TP53 mutant AML, with specific emphasis on dysfunction in the mitochondrial apoptotic pathway and clonal evolution of TP53 mutant hematopoietic stem cells. We then outline a roadmap for developing tailored therapies that may finally redefine prognosis for this high-risk patient population, including apoptotic activators, cell-cycle modulators, and immune- and metabolic-based therapies. We lastly call attention to new biomarker-driven approaches that can improve patient stratification and optimize identification of responders. By connecting mechanistic understanding with translational insights, this review underscores both the formidable challenges and the emerging opportunities in TP53 mutant AML.

CD47 has gained significant attention as a critical anti-phagocytic molecule and therapeutic target for blood cancer immunotherapy. However, the CD47-targeting monoclonal antibody magrolimab recently failed to meet its primary endpoint in Phase III ENHANCE trial in myeloid malignancies. This outcome highlights an urgent need to identify novel regulatory mechanisms for CD47 or to explore entirely new “Don't eat me signal” to overcome existing therapeutic limitations and optimize macrophage-mediated tumor clearance. To search for potential regulators of CD47, we performed genome-wide CRISPR knock-out (KO) screens in AML cell lines (MOLM13 and U937). We identified 53 positive and 25 negative hits based on Pneg/pos<0.05 that consistently influenced CD47 levels in both cell lines. Notably, these genes were predominantly enriched in O-glycosylation, fucosylation and N-glycosylation pathways. To further investigate the role of O-glycosylation and fucosylation in regulating CD47, we carried out CRISPR-mediated genetic deletion of 13 genes. Deletion of Core 1 O-glycosylation-modifying genes C1GALT1 and C1GALT1C1, or the heavily O-glycosylated protein CD43, increased cell surface CD47, and binding to SIRPa-Fc recombinant protein, establishing these targets as negatively regulators of CD47. In contrast, deletion of SLC35C1, regulator of fucosylation, decreased cell surface CD47 and reduced binding to SIRPa-Fc recombinant protein, implying as a positive regulator of CD47. To further validate, we utilized O-glycosylation inhibitors that resulted in upregulation of CD47 levels while the SLC35C1 inhibitor downregulated CD47 in a dosage and time-dependent manner. Glycan analysis revealed that CD47 lacks O-glycosylation sites, hence we hypothesized that O-glycosylation indirectly regulates CD47 expression via CD43. Supportive to this, despite higher CD47 expression, disruption of O-glycosylation (via C1GALT1/C1GAT1C1 KO) or CD43 KO increased macrophage phagocytosis (~3-fold on average, Padj < 0.0001). Further investigation revealed that the CD43-blocking antibody MEM59 and O-glycosylation inhibitors enhanced phagocytosis in AML (MOLM13 and MV4-11), and lymphoma cells (Raji) as well as AML primary tumors. These findings strongly highlight highly O-glycosylated CD43 as a NOVEL “don't eat me” immune checkpoint in hematological tumors. We next investigated the underlying mechanism of CD43-dependent “don't eat me” signaling. Classic activation of ADAM10/17 using PMA induced CD43 shedding in MOLM13 cells, with more pronounced effects in O-glycosylation-deficient cells. ADAM10 inhibition, but not ADAM17 inhibition, effectively prevented CD43 shedding and restored CD43 levels in O-glycosylation-deficient cells. This suggested that CD43 in AML is susceptible to ADAM10-mediated proteolytic cleavage, particularly under O-glycosylation-deficient conditions. We engineered SIGLEC1/7 knockout THP1 cells to test these lectins as potential CD43 receptors on macrophages. SIGLEC1/7 deficiency failed to abolish CD43-mediated anti-phagocytic activity, indicating that CD43 signals through alternative macrophage receptors. Negatively charged sialic acids have been proposed to prevent phagocytosis through electrostatic repulsion. However, CD43 deletion or blockade had minimal impact on AML cell zeta potential, ruling out electrostatic repulsion as the primary mechanism. We finally evaluated the validity of CD43 as a putative immune checkpoint for AML patients. Transcriptomic data from BeatAML and TCGA cohorts revealed significantly elevated CD43 expression on leukemic blasts compared to normal counterpart. CD43 overexpression positively correlated with immunosuppressive M2 macrophage signatures while negatively correlating with anti-tumor immune cell signatures. To isolate tumor-intrinsic CD43 effects from immune cell contribution, we performed Kaplan-Meier survival analysis on BeatAML patients with high tumor purity (>60%), demonstrating that elevated CD43 expression was associated with significantly worse overall survival. These findings for the first time suggest that CD43 on AML blasts promotes an immunosuppressive tumor microenvironment and contributes to poor patient outcomes. Collectively, our work identifies O-glycosylated CD43 as a novel “don't eat me” immune checkpoint that operates independently of CD47, providing a promising therapeutic target to enhance macrophage-mediated tumor clearance in hematologic malignancies.

Acute myeloid leukemia (AML) frequently develops resistance to chemotherapy, posing significant challenges to treatment. The bone marrow microenvironment is crucial in mediating drug resistance in hematological malignancies, though the underlying mechanisms remain poorly understood. In this study, we demonstrate that macrophages contribute to resistance against cytarabine (AraC) in AML, as evidenced by the increased sensitivity to AraC following macrophage depletion in murine AML models. In vitro experiments further reveal that this resistance is partly mediated by soluble factors secreted by macrophages. A whole-genome CRISPR screen identified deoxycytidine kinase (DCK) as a key target of these soluble factors in AML cells. Metabolic profiling pinpointed deoxycytidine (dC) as the primary soluble factor secreted by macrophages that inhibits DCK activity, thereby driving AraC resistance. Additionally, we found that macrophages express high levels of SAM and HD domain-containing protein 1 (SAMHD1), an enzyme in dC biogenesis, suggesting that macrophages are a significant source of dC in vivo. Collectively, our findings uncover a novel mechanism of AraC resistance in AML mediated by macrophage-derived dC and propose that targeting pyrimidine metabolism in macrophages could be a potential strategy to overcome this resistance. Chuqi Wang, Yuhan Wang, Camillo Benetti, Karanpreet Bhatia, Xiao Xian Lin, Shruti Bhatt. Macrophage-Derived Deoxycytidine Drives Cytarabine Resistance in Acute Myeloid Leukemia [abstract]. In: Proceedings of Frontiers in Cancer Science 2024; 2024 Nov 13-15; Singapore. Philadelphia (PA): AACR; Cancer Res 2025;85(15_Suppl):Abstract nr P37.

T cell-based therapies hold immense promise, but their production remains time-consuming and technically complex. Here, we present a protocol that streamlines the activation and lentiviral transduction of primary human T cells with artificial receptors. We describe steps for T cell isolation, lentivirus production, and the simultaneous activation/transduction of T cells. By eliminating magnetic T cell activation beads, sequential steps, and lengthy spinoculation, this protocol significantly enhances efficiency, scalability, and accessibility for research and therapeutic applications.

Despite the remarkable success of targeted therapy, the emergence of acquired resistance remains a major obstacle to cure acute myeloid leukemia (AML) patients. While existing studies have predominantly focused on genetic heterogeneity as a driver for therapy resistance, recent evidence suggests that non-genetic persister state is equally important, but such studies are lacking in AML. Here, we define the temporal transcriptome and chromatin landscape along with lineage trajectory of individual genetically identical AML persister clones to existing anti-AML therapeutics. To model persister state in AML, we utilized watermelon library (Oren et al., 2021), that expresses lineage barcode with inducible Histone-2B-mCherry to trace clonal evolution and cell division. We first evaluated whether slow-cycling, quiescent persisters emerge in AML lines after exposure to standard therapy. Our data showed the emergence of slow cycling persisters with cytarabine (AraC) in all 4 cell lines, and gilteritinib (GLT) in MOLM-13 and MV4-11 cells while BH3 mimetics (venetoclax (VEN), S63845 and navitoclax) had fast-cycling persisters. Integrating proliferative-index profiles with cell cycle profiles depicted extensive heterogeneity in AML persisters imposed by drug target rather than genetic state. Drug holiday experiments exhibited a reversible drug tolerance state in both slow and fast-cycling persisters. Longitudinal drug sensitivity and BH3 profiling assays showed that persisters emerged with adaptation to reduced mitochondrial priming that reversed after drug holiday. However, transitioning from persister to stable resistant cells resulted in a sustained loss in apoptotic priming, mirroring observations made in PDX models and primary tumors. To understand trajectory of persister emergence and underlying mechanisms, we performed single-cell multiomics on drug-naïve and persister populations (day 9) in MV4-11 (GLT), and OCI-AML2 (VEN and AraC). We expanded 10,000 unique barcoded cells and administered the drug treatment to derive persisters. Using lineage barcode analysis, we report significant reduction in the dominant lineage of day 0 and survival of multiple small lineages indicating an acquired state of persistence than selection of pre-existing resistant clone in both the lines. Further trajectory analysis defined three fates of the lineages as: expanding (log2FC>1), shrinking (log2FC <1) and stable. AraC persisters showed higher expanding and stable lineages, while VEN and GLT persisters resulted in higher shrinking and stable clones, revealing heterogeneity in lineage expansion. We next asked whether persisters resulting from different therapies arised from the same parental lineage. To test this, we performed lineage tracing on VEN and AraC persisters in OCI-AML2 and compared surviving lineages at Day 9 vs Day 0 cells. This analysis identified 52 parental lineages that were able to produce both VEN and AraC persisters in AML2, providing evidence of branched evolution of persisters from the same parental clone. We finally defined transcriptional and epigenetic heterogeneity in baseline and persister cells by performing UMAP on single-cell RNAseq and ATACseq data. We identified persisters to all 3 drugs clusters distinctly from their drug naïve counterparts at both transcriptome and chromatin accessibility. Next, we investigated differentially expressed pathways in persisters and observed higher inflammatory response pathways such as TNFα signaling in GLT, enrichment of MYC targets and oxidative phosphorylation in VEN and high mTORC1 signalling in AraC persisters. We observed higher chromatin accessibility in GLT and VEN whereas condensed chromatin in AraC persisters. We next asked which motifs are highly enriched in each persister and observed the enrichment of various KLF and SP family of motifs in each persister driving multiple gene signatures in each persister type. We also observed enrichment of LSC-primed and cDC like cells in GLT persisters but with AraC and VEN persisters a uniform pattern of all states was observed, indicated stemness may not be common mechanism to persister evolution. Collectively, our integrated single-cell multiomics and lineage tracing approach reveals that AML persisters emerge through stochastic drug-specific, non-genetic adaptive mechanisms characterized by distinct transcriptional and chromatin remodeling programs rather than deterministic Darwinian selection of pre-existing resistant cells.

TP53-inactivated (TP53-mutant or deficient) acute myeloid leukemia (AML) is a biologically and clinically aggressive subtype associated with profound treatment resistance, immune evasion, and dismal outcomes. Notably, AML patients with TP53 mutations have limited benefit from standard induction chemotherapy or from recently approved venetoclax-based combination regimens. The consistent failure of these therapies to induce durable remissions underscores the urgent need for alternative treatment strategies. Given the central role of T cells and natural killer (NK) cells in mediating anti-leukemic immunity, developing immunotherapeutic approaches that can overcome immune resistance in TP53-inactivated AML represents a promising avenue to improve outcomes for this high-risk patient population. To test the hypothesis that TP53 inactivation confers resistance to T/NK cell cytotoxicity in AML, we engineered TP53-wildtype (WT), knockout (KO), or mutant isogenic AML cells engineered to express a model antigen (LMP2A) with primary human LMP2A-targeted TCR-T/CAR-T cells, or NK-92 and primary human NK cells as a model for NK cytotoxicity. TP53-inactivated AML cells displayed marked resistance to both LMP2A-targeted TCR-T/CAR-T, as well as to NK-92 and primary NK cell-mediated cytotoxicity compared to their TP53 WT counterparts. Further comparison among isogenic MOLM-13 cells carrying one of six different TP53-hotspot mutants (R248Q, M237I, Y220C, R175H, R273H, R282W) identified the R248Q mutant as the most resistant to immune killing. In addition to impaired cytotoxicity, TP53-inactivated AML cells suppressed T cell proliferation, activation, and cytokine secretion, while promoting an exhausted T cell phenotype. To uncover the mechanisms driving immune resistance in TP53-inactivated AML, we performed functional genomic screening by performing unbiased genome-wide CRISPR KO screens in TP53-inactivated and WT isogenic MOLM-13 cells under LMP2A TCR-T cell or NK-92-mediated immune pressure. Subsequent integration of CRISPR screen data with bulk transcriptomics and total proteomic profiling conducted on MOLM-13 isogenic cells with TP53-WT, R248Q mutation, or KO identified key regulators of immune susceptibility. Notably, inhibitors of apoptosis (IAPs: BIRC2/cIAP1, BIRC3/cIAP2, XIAP) and components of the death-inducing signaling complex (DISC)—comprising FADD, caspase-8, and cFLIP—emerged as top hits, revealing extrinsic apoptosis as a key dependency of TP53-inactivated AML. Targeting IAPs enhances this apoptotic pathway, while inhibition of caspase activity with emricasan, alone or in combination with IAP antagonists, shifts cell death toward immunogenic necroptosis, thereby potentiating immune killing.Functional validation using targeted individual CRISPR KO and pharmacological inhibitors confirmed that antagonizing IAPs restored T and NK cell–mediated killing of TP53-inactivated AML. We found that IAP inhibitors birinapant and tolinapant, as well as IAP PROTAC-degrader CST-626, potently sensitized TP53-inactivated AML to CAR-T/TCR-T and NK-92-mediated cytotoxicity. Strikingly, emricasan, a clinically advanced caspase inhibitor, was also able to sensitize TP53-inactivated AML cells to TCR-T, CAR-T, and NK-mediated cytotoxicity. Moreover, combining emricasan with IAP antagonists synergistically enhanced immune-mediated killing in TP53-inactivated AML. Mechanistically, emricasan inhibits the formation of the caspase-8/cFLIP heterodimer and subsequent DISC complex, thereby promoting non-apoptotic, immunogenic cell death in conjunction with IAP antagonists. This approach restored immune killing and amplified effector T/NK cell responses against TP53-inactivated AML. Collectively, TP53 inactivation drives immune evasion in AML by rewiring death receptor signaling and inducing T cell dysfunction. We employ integrated multiomics and functional assays to identify the IAPs, as well as the DISC—comprising FADD, caspase-8, and cFLIP—as critical, targetable checkpoints to modulate immune effector–mediated killing in TP53-inactivated AML. Pharmacological inhibition of the caspase 8-cFLIP heterodimer with emricasan alone, or combined with IAP antagonists, effectively overcomes this checkpoint and sensitizes AML cells to immune cytotoxicity. These findings establish a strong preclinical rationale for combining cell death–promoting agents with T or NK cell–based immunotherapies to overcome immune resistance in TP53-mutated high-risk AML subset.

Acute myeloid leukemia (AML) is a heterogeneous disease characterized by a broad spectrum of molecular alterations that influence clinical outcomes. TP53 mutations define one of the most lethal subtypes of acute myeloid leukemia (AML), driving resistance to nearly all available treatment modalities, including venetoclax plus azacitidine (VenAza). Yet, the molecular basis of this resistance, beyond affecting transactivation of BCL-2 family genes, has remained elusive. Here, we demonstrate that VenAza treatment leads to reduced transcriptional upregulation of the p53 signaling pathway in TP53 mutant/deficient AML compared to wild-type AML. Functionally, TP53 mutant/deficient AML exhibits selective failure in apoptosis induction rather than impaired G1 arrest or senescence. Despite inhibition of pro-apoptotic BAX and selective enrichment for MCL-1 in TP53 mutant isogenic AML cells, compensatory upregulation of BIM preserved functional mitochondrial outer membrane permeabilization (MOMP). TP53 mutant primary AML tumors at baseline also had retained capacity for MOMP. Instead, TP53 mutant AML exhibited disruption in caspase-3/7 activation to evade apoptosis after VenAza therapy - decoupling the mitochondrial and executioner phases of apoptosis. Importantly, this "post-MOMP brake" is not a bystander effect but itself a driver of VenAza and chemotherapy resistance in TP53 mutant/deficient AML. This previously unrecognized mechanistic insight shifts the focus from mitochondrial priming to terminal caspase blockade in TP53 mutant AML and opens the door for urgently needed therapeutic strategies that reignite apoptosis at its execution point.

ABSTRACT:We previously demonstrated that reduced intrinsic electron transport chain (ETC) activity predicts and promotes sensitivity to the B-cell lymphoma 2 (BCL-2) antagonist, venetoclax (Ven), in multiple myeloma (MM). Heme, an iron-containing prosthetic group and metabolite, is fundamental to maintaining ETC activity. Interrogation of the cyclin D1 group 2 subgroup of MM from the Relating Clinical Outcomes in MM to Personal Assessment of Genetic Profile (CoMMpass) trial (NCT01454297), which can be used as a proxy for Ven-sensitive MM (VS MM), shows reduced expression of the conserved heme biosynthesis pathway gene signature. Consistent with this, we identified that VS MM exhibits reduced heme biosynthesis and curiously elevated hemin (oxidized heme) uptake. Supplementation with hemin or protoporphyrin IX (heme lacking iron) promotes Ven resistance, whereas targeting ferrochetalase, the penultimate enzyme involved in heme biosynthesis, increases Ven sensitivity in cell lines and primary MM cells. Mechanistically, heme-mediated activation of prosurvival rapidly accelerated fibrosarcoma-rat sarcoma virus-mitogen-activated protein kinase (MEK) signaling and metabolic rewiring, increasing de novo purine synthesis, were found to contribute to heme-induced Ven resistance. Cotargeting BCL-2 and myeloid cell leukemia-1 suppresses heme-induced Ven resistance. Interrogation of the Multiple Myeloma Research Foundation CoMMpass study of patients shows increased purine and pyrimidine biosynthesis to corelate with poor progression-free survival and overall survival. Elevated heme and purine biosynthesis gene signatures were also observed in matched relapse refractory MM, underscoring the relevance of heme metabolism in therapy-refractory MM. Overall, our findings reveal, for the first time, a role for extrinsic heme, a physiologically relevant metabolite, in modulating proximity to the apoptotic threshold with translational implications for BCL-2 antagonism in MM therapy.

Increasing evidence highlights macrophages as critical players in tumorigenesis and therapeutic resistance in both solid and liquid tumors. While macrophages in solid cancers are largely pro-tumor and associated with poor prognosis, their contribution to acute myeloid leukemia (AML) pathogenesis—a deadly cancer of myeloid origin—remains controversial. To elucidate precise role of macrophages in AML, we analyzed AML patient dataset (BeatAML) and revealed that high macrophage gene signatures correlated with poor survival outcomes (n=57). We demonstrated that macrophages promote chemoresistance to standard-of-care chemotherapy drug cytarabine (AraC) in AML, as evidenced by the increased sensitivity to AraC following macrophage depletion in AML patient-derived xenograft (PDX) models and mouse syngeneic leukemia models. To identify direct contribution of macrophages in mediating chemoresistance, we conducted in vitro coculture experiments and drug screen (n=31 drugs) with bone-marrow derived macrophage conditioned media (BMDM-CM). In vitro BMDM-CM fractionation experiments revealed that the resistance is primarily mediated by soluble factors (<3kDa) secreted by macrophages. Functional genetic screening using genome-wide CRISPR Knock-out studies identified deoxycytidine kinase (DCK) as a critical target of soluble factors. Metabolite profiling pinpointed deoxycytidine (dC), a pyrimidine metabolite, as the primary soluble factor secreted by macrophages that inhibited DCK activity in AML cells to drive AraC resistance. Intriguingly, single-cell sequencing of healthy and AML samples, and cancer-wide Dependency Map (DepMap) analysis identified that SAM and HD domain-containing protein 1 (SAMHD1) causes dC accumulation. Inhibition of SAMHD1 and dihydroorotate dehydrogenase (DHODH) blocked dC biogenesis and reversed AraC resistance mediated by macrophages in vitro. Further, single cell RNA sequencing analysis of paired AML patient samples revealed an enrichment of SAMHD1high macrophages/monocytes in patient bone marrow after AraC-based chemotherapies. Since SAMHD1 is an interferon-stimulated gene, we hypothesize that the inflammatory state in the bone marrow microenvironment could modulate SAMHD1 expression. Consistent with this hypothesis, the inflammatory level is indeed correlated with SAMHD1 expression in primary AML samples according to BeatAML patient database, implying that inflammation might give rise to AraC resistance by modulating SAMHD1 in immune microenvironment. Abstract Collectively, our findings uncover a novel mechanism of AraC resistance in AML mediated by macrophage-derived dC. We propose that targeting dC metabolism in macrophages could be a potential strategy to overcome chemoresistance and improve outcomes for AML patients. Chuqi Wang, Yuhan Wang, Camillo Benetti, Karanpreet Bhatia, Xiao Xian Lin, Petra Hyroššová, Edward Ayoub, Jakub Rohlena, Katerina Rohlenova, Michael Andreeff, Shruti Bhatt. SAMHD1high macrophages drive resistance to cytarabine in acute myeloid leukemia (AML) by pyrimidine metabolite deoxycytidine (dC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 6381.

The tumor microenvironment (TME) programs cancer cells to influence therapeutic responses. Macrophages residing in TME switch from pro-phagocytic to tumor-promoting and immunosuppressive phenotypes as cancer develops. While these pro-tumor functions of macrophages are associated with poor outcomes, the underlying mechanisms by which bone-marrow (BM)-associated macrophages fuel myeloid malignancy and their precise contribution to relapse remain undissected. Here, we show expansion of monocyte/macrophage population in leukemia patients post-chemotherapy relapse, and spatial proximity of macrophages to leukemia blasts in the BM niche. This proximity proved functionally consequential—depletion of macrophages delayed leukemia relapse post cytarabine (AraC), a frontline chemotherapy, in patient-derived xenografts (PDX) and syngeneic leukemia models. Mechanistically, a pyrimidine metabolite, deoxycytidine (dC), secreted by BM macrophages, is taken up by leukemia cells to directly inhibit deoxycytidine kinase (DCK) to hamper AraC activation and subsequent resistance in a cell non-autonomous manner. Diagnosis AML patients exhibited significantly higher circulating dC levels than healthy donors, and dC levels further increased following chemotherapy. SAMHD1, which catalyzes deoxynucleoside triphosphates (dNTPs) into deoxynucleoside, was highly abundant in macrophages and mediated dC accumulation. Blockade of dC production in mouse and human macrophages via genetic and pharmacological inhibition of SAMHD1 or DHODH , a critical enzyme in pyrimidine synthesis, restored AraC sensitivity. Combination with DHODH inhibitors significantly delayed AraC relapse in human PDX and mouse syngeneic AML models. Collectively, we identify a metabolic immune–leukemia crosstalk in which SAMHD1 high macrophages mediates chemoresistance by secreting pyrimidine metabolites and propose macrophage metabolic reprogramming as a tractable strategy to overcome TME-driven chemoresistance in myeloid leukemia. ### Competing Interest Statement The authors have declared no competing interest.

Supplemental Figure 12 shows targeted agents cluster by mechanism of action.

Metabolic enzymes are key mediators of the metabolic fate of a drug determining pharmacokinetic, pharmacodynamics, and therapeutic effect of the drug. Human Cytochrome P450 (CYP450) enzymes are essential for the metabolism of drugs and endogenous compounds. CYP enzymes mediated metabolism occurs in multiple sites, of which liver and intestine are the primary sites. CYP450 enzymes, distributed into four major families, metabolize over 70% of clinically approved drugs in humans. Drug metabolism is achieved through Phase-I reactions (oxidation, reduction hydrolysis, and hydroxylation), Phase II reactions (conjugation), or both. Drugs and chemicals serve as inducers or inhibitors of various isozymes. Besides, genes encoding CYP450 enzymes are highly variable, resulting in genetic polymorphisms that impact the therapeutic efficacy of drugs. This chapter summarizes the classes and functions of cytochrome P450 enzymes, biochemistry, polymorphism, and factors that contribute to interindividual variations in drug responses.

Supplemental Figure 2 shows RNA-seq signature of drug-resistant AML PDX models.

Background Despite recent advances, the reasons for discrepancies between ELN risk assessment and clinical outcomes in acute myeloid leukemia (AML) remain poorly understood. This creates an opportunity to explore additional predictive biomarkers using functional phenotypic analysis. Most cancer therapies induce cell death by activating the mitochondrial apoptotic pathway. Therefore, measuring the apoptotic threshold maintained between pro-survival proteins (BCL-2, BCL-XL, and MCL-1) and pro-apoptotic proteins through BH3 profiling (BP) and dynamic BH3 profiling (DBP) can provide valuable information beyond known genetic risk factors in acute leukemia. We propose that variations in apoptosis thresholds, assessed through BP and DBP, can offer insights into the risk, therapeutic response, treatment options, and outcomes of acute leukemia. Assessing patient-specific dependencies on BCL-2 family proteins can guide the selection of BH3 mimetic and chemotherapy combinations for potential therapies. Methods We exposed blasts from bone marrow aspirates or peripheral blood to various pro-apoptotic BH3 peptides and measured mitochondrial outer membrane permeabilization using BP. We examined pro-survival BCL-2 family members (BCL-2, BCL-XL, and MCL-1) and their sensitivity to BAD, HRK, and MS-1 peptides to identify factors influencing differential chemosensitivity to BH3-mimetics in these malignancies. We evaluated 30 pediatric and 43 adult AML patients, and 70 pediatric and six adult ALL patients. The samples included 96 at diagnosis, 46 at relapse, and seven during treatment. For DBP, nine pediatric AML tumors were treated with cytarabine, daunorubicin, etoposide, and fludarabine, while 12 T-ALL samples were treated with nelarabine, vincristine, dasatinib, asparaginase, and dexamethasone. We analyzed delta priming to assess chemotherapy response in vitro and conducted bulk RNA-seq of pediatric AML samples to understand differential gene expression using DESeq2 version 1.42.0. Our analysis aimed to comprehensively understand acute leukemia. Results We present an initial analysis of functional BH3 profiling on 149 primary tumor samples (73 AML, 29 T-ALL, and 47 B-ALL cases) with complete clinical and molecular details from three cancer centers in Singapore. Pediatric favorable-risk AML demonstrated significantly higher susceptibility to apoptosis than non-favorable-risk AML (p<0.05), measured by cytochrome c release when exposed to BIM peptide (mean 60% ± 20% for favorable risk vs. 27% ± 15% for non-favorable risk). Adult AML showed higher mitochondrial priming in favorable risk group compared to intermediate/adverse risk group, indicating apoptotic sensitivities correlates with ELN risk. Pediatric AML (N=30) exhibited greater priming than adults (N=43) (p<0.001), potentially explaining functional basis of improved survival outcomes in children vs adults. Direct mitochondrial sensitivity to venetoclax in lymphoblasts was higher in T-ALL (n=29) than in B-ALL (n=47). Lymphoblasts showed greater direct mitochondrial sensitivity to navitoclax, suggesting the potential use of dual inhibition of BCL2 and BCL-XL in ALL. Ex vivo apoptotic priming was enhanced after etoposide treatment in myeloblasts of 50% of pediatric AML cases refractory to standard induction. Differential gene signatures derived from RNAseq identified pathways associated with low and high apoptotic sensitivity in pediatric AML patients. Conclusions This study demonstrates the potential of integrating mitochondrial priming phenotypes with genetic data to refine AML risk assessment. Our approach facilitates the evaluation of treatment sensitivity at the cellular level to address limitations in current ELN risk stratification where favorable-risk patients may relapse, and some adverse-risk patients achieve good outcomes. Incorporating functional BH3 profiling into diagnostic assessments could enhance contemporary risk stratification strategies for both pediatric and adult AML patients. The consistent sensitivity of lymphoblasts and myeloblasts to BCL-2 inhibition across age groups and lineages suggests the broad applicability of our approach. By integrating cellular vulnerabilities to anti-apoptotic proteins into clinical assessments, we may significantly improve treatment decision-making and risk stratification in acute leukemia.

Among the spectrum of mutations driving malignancy, mutation in the TP53 gene is perhaps the most important event leading to therapy resistance. Only ~25% of patients with TP53-mutated acute myeloid leukemia (AML) respond to standard induction therapy, with dismal median survival of 4.2 months. This underscores a pressing need to characterize therapy resistance mechanisms for TP53-mutated AML and to identify novel therapeutic approaches. To investigate TP53 mutation-driven chemoresistance, we generated isogenic AML cells harboring TP53 WT, mutant, or knockout (KO) variants. TP53 WT cells exhibited the highest sensitive to Venetoclax+Azacytidine (VenAza), while TP53-/mut cells displayed varying sensitivities, with TP53-/R248Q cells being the least responsive. This variability suggests that different mutations confer distinct levels of TP53 function loss. TP53-/mut and KO AML cells showed reduced sensitivity to cytarabine, etoposide, and VenAza-induced apoptosis without impairment of G1 arrest or cell cycle. To pinpoint the dysfunction in intrinsic apoptotic signaling allowing TP53 mutant cells to evade apoptosis, we performed Gene Set Enrichment Analysis (GSEA) on RNA-seq data. This analysis demonstrated significant enrichment of p53 signaling pathways in WT cells treated with VenAza, but not in TP53-/R248Q and KO cells. Further we observed that despite the loss of key pro-apoptotic regulators (BAX, PUMA, NOXA), upregulation inactivator protein BIM compensated for proapoptotic stimuli in TP53-/R248Q and KO after VenAza. While mitochondrial outer membrane permeabilization (MOMP) is typically considered a crucial step in apoptosis, BH3 profiling analysis revealed comparable MOMP (priming) at baseline between TP53-mutant and TP53-WT AML primary tumors (n=37) and cell lines. This suggests that MOMP is not the definitive “point-of-no-return” for TP53 mutants. Instead, we found a blockade in executioner caspase-3/7 activity in TP53-/mut and KO AML cells, following VenAza or cytotoxic chemotherapy, contrasting with WT cells. This finding was also recapitulated in TP53 null isogenic solid cancer cell lines, revealing a novel post-MOMP resistance mechanism in TP53-deficient cells across cancer types. To identify targetable dependencies driving caspase blockade in TP53 defective cells, we conducted whole-genome CRISPR knockout screen in WT, TP53-/R248Q, and KO cells following VenAza treatment. Our findings confirmed that knockout of pro-apoptotic genes (BAX, PMAIP1, BCL2L11) conferred resistance across all genotypes. Notably, we discovered a novel functional selective dependence on BIRC5, a member of the inhibitor of apoptosis (IAP) family, in TP53-/R248Q cells exposed to VenAza. Mechanistically, BIRC5 deletion strongly sensitized TP53-mutant/KO cells to VenAza and other chemotherapy drugs by restoring caspase 3/7 activation. RNA-seq and global proteomic analyses of isogenic cells revealed upregulation of IAP family genes in TP53-mutant/KO cells. We validated translational implications of these findings by differential RNA-seq analysis comparing TP53-mutants (n = 63) and WT (n = 668) AML patient samples, and reverse phase protein array (RPPA) in TP53-mutants (n=153) as compared to TP53-WT (n=501) primary tumors. Both analyses identified BIRC5, encoding survivin, as one of the top overexpressed targets in TP53-mutant primary tumors. Extending beyond AML, TCGA data shows BIRC5 upregulation in TP53-mutant tumors across 19/25 cancers, indicating a pan-cancer dependency in p53-inactivated tumors. To uncover targetable vulnerabilities, we conducted high-throughput drug screening (n=250 agents) in isogenic AML cells, with IAP inhibitors emerging as top hits against TP53-mutant/KO cells. In isogenic mice (CDX) models, a combination of VenAza with IAP inhibitor demonstrated strikingly durable leukemia blast inhibition in the bone marrow and spleen, with triple therapy showing superior efficacy in TP53-/R248Q mice compared to WT treated with VenAza alone (median survival 44.5 vs 26 days). Our research unveils a critical post-mitochondrial defect in TP53 mutant cells as a key driver of chemoresistance and shows IAP inhibition can reinstate the apoptotic potential of both cytotoxic chemotherapy and targeted therapies. This insight offers a potential therapeutic target for TP53-mutated cancers, spanning both hematological and solid tumors.