搜索结果：Cell death discovery

Mast cells contribute to the pathology of various diseases, in particular allergic conditions. Therefore, it is essential to develop strategies that efficiently prevent their harmful effects under such circumstances. Here, we sought to evaluate the possibility of cell death induction as a potential means of selectively depleting mast cells. Previous work has suggested that mast cells are sensitive to regimes that target their acidic secretory granules, and the aim of this study was therefore to identify novel anti-mast cell compounds that act via a granule-mediated pathway. To this end, we evaluated trifluoperazine, an antipsychotic drug known to present lysosomotropic properties. We demonstrate that trifluoperazine is cytotoxic for mast cells, whereas multiple other cell types were resistant. Trifluoperazine induced mainly apoptotic cell death in mast cells. Further, our data indicate that trifluoperazine acts on mast cells by inducing secretory granule permeabilization. In support of this, trifluoperazine caused granule deacidification, accompanied by cytosolic acidification as well as translocation of tryptase from the secretory granules into the cytosol. Trifluoperazine-induced cell death and subsequent DNA degradation were profoundly abrogated when granule acidification was inhibited by the V-ATPase inhibitor bafilomycin A1, suggesting that the granule acidity has a key role in the cell death mechanism. Moreover, mast cell death in response to trifluoperazine was largely caspase-independent, whereas serine protease activity was shown to promote apoptosis-like vs. necrosis-like cell death. Overall, these findings introduce trifluoperazine as a novel anti-mast cell agent that induces cell death through granule permeabilization. Trifluoperazine may thus be evaluated for therapeutic intervention to ameliorate mast cell-mediated detrimental effects.

Natural killer (NK) cell-based therapies are under assessment for the treatment of various cancers due to their intrinsic ability to distinguish between malignant and healthy cells in an allogeneic context, enabling off-the-shelf manufacturing possibilities. However, cryopreservation reduces both the recovery and function of NK cells, thereby limiting their therapeutic feasibility. In this study, we evaluated three cryoprotectants (CryoStor 10; ZKCELL FM-01; FBS + DMSO) for the cryopreservation of NK cells. Post-thaw viability, ATP levels, and cytotoxicity were assessed and found to have persistent differences between cryopreserved and fresh cells. Transmission electron microscopy, flow cytometry, and Western blot analysis revealed a complex mode of cell death in cryopreserved cells, which could be partially mitigated by adding some death inhibitors. We further investigated the effects of centrifugation on thawed cells, identifying lysosomal stability as a key determinant of cell death. Pretreatment with low-dose LLOMe prior to cryopreservation induced stress granule formation, stabilizing lysosomes and improving cell recovery rates without compromising effector functional capacity. These findings offer new insights for optimizing NK cell cryopreservation and facilitating their clinical application.

Cancer cells are more susceptible to oxidative damage due to their reliance on tightly regulated redox homeostasis. Sulfiredoxin (Srx) is an antioxidant enzyme that restores hyperoxidized peroxiredoxins and contributes to cellular redox regulation. Previously, we demonstrated that pharmacological inhibition of Srx induces preferential death of cancer cells and tumor regression by weakening antioxidant defenses. In this study, we investigated the anticancer efficacy of LMT328, a more potent Srx inhibitor developed through molecular modeling. LMT328 exhibited greater efficacy than the previously reported inhibitor J14 in inhibiting cellular Srx activity, elevating intracellular oxidative stress, inducing mitochondrial damage, and triggering apoptotic cell death in cancer cells. These effects were attenuated by ectopic Srx expression or antioxidant treatment, supporting that LMT328 exerts its cytotoxic effects through oxidative stress resulting from Srx inhibition. Notably, LMT328 induced greater oxidative stress, mitochondrial damage, and cytotoxicity in tumorigenic T80H cells compared with nontumorigenic T80 cells. In a xenograft model, LMT328 significantly suppressed tumor growth with minimal toxicity. Collectively, our findings demonstrate that LMT328 disrupts Srx-dependent redox homeostasis, leading to oxidative stress-associated mitochondrial damage and cancer cell death, and suggest that targeting Srx may represent a promising strategy for redox-based cancer therapy.

Medulloblastoma (MB) is the most common childhood brain cancer, with Group 3 (G3) as the most aggressive subgroup, being prone to relapse and treatment resistance. A small subset of stem-like cells contributes to this recurrence, but the mechanisms behind their transformation are not fully understood. In this study, we employed therapeutically relevant in vitro and in vivo chemoradiotherapy (CRT) models of G3 MB and discovered a significant activation of SRC kinase following CRT treatment, while other kinases such as AKT and ERK were unaffected. Remarkably, SRC activation was exclusive to G3 MB cells and was absent in the less aggressive Sonic Hedgehog and Group 4 MB, as well as in normal brain cells. SRC activation in CRT-treated G3 MB cell and tumors corresponded with increased stemness, as evidenced by elevated levels of stemness factors SOX2, NOTCH1, OCT4, Nanog and phosphorylated STAT3, alongside a reduction in the differentiation marker βIII-tubulin/TUBB3. Conversely, SRC knockout or pharmacological inhibition promoted differentiation and reduced aggressiveness in CRT-resistant G3 MB cells, which could be rescued by re-expression of SRC in SRC knockout cells. Additionally, SRC inhibition significantly reduced the viability of CRT-treated G3 MB cells by inducing both apoptosis and necroptosis, while sparing the proliferation and stem-like properties of normal neural stem cells, indicating a promising toxicity profile. Importantly, in a therapeutically relevant orthotopic G3 MB model, administration of the re-purposed blood-brain-barrier permeable SRC inhibitor, Saracatinib, in conjunction with CRT, significantly reduced tumor burden and improved animal survival compared to CRT treatment alone without any neurotoxic side effects. Overall, our results underscore the pivotal role of SRC in enhancing stemness and aggressive behavior in CRT-resistant recurrent G3 MB. Targeting SRC not only promotes cell death through apoptosis and necroptosis but also encourages differentiation, positioning it as a promising therapeutic target for rapid clinical interventions.

Radiotherapy kills cancer cells by inducing DNA damage, but adaptive responses that buffer injury and limit immunogenic signaling remain incompletely understood. Although ionizing radiation can activate cytosolic DNA sensing and immunogenic cell death, the tumor-intrinsic regulators linking these processes to radioresistance are not well defined. Here, we show that XRRA1 is a stress-adaptive determinant of radioresponse identified by integrating discovery proteomics into chronic myeloid leukemia with clinical tissue validation and functional studies across multiple tumor models. In peripheral-blood proteomes, XRRA1 was quantitatively reduced in chronic myeloid leukemia yet associated with a favorable prognosis and with networks enriched for RNA regulation, apoptosis, and DNA-repair biology. In human biopsy specimens, XRRA1 protein abundance was increased in radiation-exposed tissues, and in cultured cells, ionizing radiation induced XRRA1 more strongly and persistently in normal cells than in cancer cells. Silencing XRRA1 had little effect on basal growth but markedly enhanced radiation-induced loss of viability, apoptosis, spheroid disruption, and clonogenic failure, while increasing γH2AX- and DNA-PK-associated damage signaling and linking XRRA1 to non-homologous end joining factors. XRRA1 depletion also amplified cGAS-STING-TBK1-IRF3 activation, interferon-stimulated gene expression, extracellular ATP release, and cell-surface calreticulin exposure. These findings identify XRRA1 as a molecular brake that limits the conversion of radiation-induced DNA damage into immunogenic stress responses. XRRA1, therefore, represents a candidate biomarker of radioadaptive stress and a potential target for radiosensitization and radiotherapy-immunotherapy combinations.

In graft-versus-host disease (GVHD), Ca2+ signals in alloreactive T cells are carefully controlled to determine whether cells survive or thrive, although how this is accomplished during GVHD remains poorly defined. We demonstrate that EZH2, a chromatin-modifying enzyme, promotes alloreactive T-cell survival in GVHD by acting as a Ca2+ signaling brake to limit excessive intracellular Ca2+ responses. Ezh2 loss led to the upregulation of gene programs that promote effector differentiation in activated T cells, coincident with enhanced intracellular Ca2+ responses that ultimately caused massive cell death. Conditional deletion of Stim1 (required for cytosolic Ca2+ entry) led to "synthetic rescue" of Ezh2-null T cells by protecting them from cell death without interfering with effector differentiation, resulting in severe GVHD. Interestingly, Stim1 expression was unaffected by EZH2, whereas the expression of the endoplasmic reticulum Ca2+ release channel inositol 1,4,5-trisphosphate receptor 2 (Itpr2) was suppressed by EZH2. Notably, EZH2 and Ca2+ signals served mutually opposing roles in controlling the expression of genes in chimeric antigen receptor (CAR) T cells. Inhibiting Ca²⁺ signaling restored EZH2 function in CAR-T cells, significantly improving their antitumor activity. Our findings reveal the interdependent roles of EZH2 and Ca2+ signals in coordinating antigen-activated T-cell responses that mediate alloimmunity and tumor immunity.

ATM deficiency is frequently observed in castration-resistant prostate cancer (CRPC). However, effective therapeutic vulnerabilities associated with this genetic alteration remain poorly defined. This study aimed to identify synthetic lethal strategies that selectively target ATM-deficient prostate cancer cells. An unbiased small-molecule compound screening was performed to identify agents exhibiting selective cytotoxicity in ATM-deficient prostate cancer cells. Candidate vulnerabilities were validated across multiple prostate cancer cell lines with genetic depletion or restoration of ATM. Mechanistic studies were conducted using molecular and biochemical assays to assess DNA damage, replication stress, and PARylation dynamics. In vivo efficacy was evaluated using ATM-deficient xenograft tumor models. ATM-deficient prostate cancer cells exhibited marked sensitivity to pharmacological inhibition of poly (ADP-ribose) glycohydrolase (PARG) using with PDD00017273, an effect that was consistently observed across multiple cell lines and partially restored by ATM re-expression. Mechanistically, PARG inhibition induced persistent PARylation in ATM-deficient cells, not via canonical DNA double-strand break signaling, but via misincorporated ribonucleotides processed by topoisomerase 1 during DNA replication. This replication-associated PARylation resulted in severe replication stress, checkpoint activation, and accumulation of DNA double-strand breaks, ultimately leading to cell death. This cytotoxic mechanism is distinct from classical PAR-dependent cell death pathways, including NAD⁺ depletion and parthanatos. In vivo, PARG inhibition significantly suppressed the growth of ATM-deficient xenograft tumors cells. This study identifies PARG inhibition as a previously unrecognized synthetic lethal vulnerability in ATM-deficient prostate cancer. These findings establish a mechanistic link between ATM loss, aberrant ribonucleotide processing, and replication-associated PARylation, supporting the clinical development of PARG inhibitors as a precision therapeutic strategy for ATM-deficient prostate cancer and potentially other malignancies harboring ATM deficiency.

Hepatocellular carcinoma (HCC) is one of the most prevalent malignancies worldwide. The therapeutic efficacy of conventional chemotherapeutic agents such as doxorubicin (DOX) is limited by dose-dependent toxicity and the development of drug resistance. Combination strategies incorporating bioactive natural products may enhance anticancer efficacy while enabling dose reduction. The present study aimed to evaluate the potential synergistic cytotoxicity of combining Fenugreek aqueous extract (FAE) with (DOX) against the HepG2 cell line. Phytochemical characterization was performed using UHPLC-QTOF-MS/MS Profiling and HPLC. Cell viability and selectivity were assessed using the SRB assay. Apoptosis, necrosis, autophagy, and cell cycle distribution were analysed by flow cytometry and Western blotting. Drug-drug interaction was evaluated using the Chou-Talalay method. Molecular docking was performed to explore potential interactions between selected FAE constituents and apoptosis- and autophagy-related protein targets. FAE enhanced DOX's cytotoxicity on HepG2 cells, with the interaction ranging from synergistic to additive depending on the concentration ratio. The DOX/FAE combination enhanced cell death through sub-G1 arrest and augmented apoptotic, necrotic, and autophagic responses compared with monotherapies. Western blot analysis demonstrated modulation of the Bax/Bcl-2 ratio and increased LC3-II expression. Docking simulations suggested favourable binding of selected steroidal saponins to Bcl-2 and LC3 proteins. These findings indicate that FAE potentiates DOX-induced cytotoxicity in vitro through modulation of multiple regulated cell death pathways. While the results support the possibility of this combination as a dose-modulating strategy, further validation in additional HCC models and in vivo systems is required.

Bloom syndrome protein (BLM), a RecQ family DNA helicase, is consistently overexpressed in multiple malignancies, yet its therapeutic potential remains largely unexplored. Herein, we focused on targeting the BLM promoter G-quadruplex (BLM-G4) to inhibit the BLM signaling pathway. We first characterized the parallel BLM-G4 in the BLM promoter region. Subsequently, it is shown for the first time that BLM-G4 recruits phosphorylated signal transducer and activator of transcription 1 (pSTAT1) to activate BLM expression. Importantly, two natural alkaloids, berberine (BER) and coptisine (COP), compete with STAT1 for binding to BLM-G4, thereby significantly suppressing BLM expression in colon cancer cells. The BER/COP-BLM-G4 complex structures were determined using nuclear magnetic resonance experiments, which provide valuable insights for the rational design of next-generation BLM-G4-targeting ligands. Beyond BLM regulation, the conjoint analysis of genome-wide STAT1-CUT&Tag-seq, G4-CUT&Tag-seq, and COP-RNA-seq demonstrated STAT1 as a general G4-binding transcription factor and COP as a pan-genomic G4 stabilizer. Furthermore, BER/COP exhibited a pronounced synergistic effect with olaparib in inducing colon cancer cell death by disrupting DNA repair pathways and intensifying DNA damage. Collectively, our findings reveal a novel epigenetic mechanism of BLM gene upregulation mediated by BLM-G4-STAT1 interaction and suggest that the combination therapy of G4 stabilizers with poly(ADP) ribose polymerase (PARP) inhibitors is a promising strategy for treating complex cancers.

Cisplatin remains a first-line chemotherapeutic agent in the treatment of oral squamous cell carcinoma (OSCC). However, the efficacy of cisplatin is frequently compromised by the development of drug resistance. This review systematically examines the multidimensional mechanisms underlying cisplatin resistance in OSCC and the corresponding strategies to overcome this resistance. Mechanisms of chemoresistance involve complex, multi-layered molecular networks, encompassing dysregulation of key gene expression and signaling pathways, epigenetic remodeling, metabolic reprogramming, evasion of regulated cell death, acquisition of epithelial-mesenchymal transition (EMT) and cancer stem cell (CSC) properties, as well as the formation of an immunosuppressive tumor microenvironment (TME). In response to these challenges, multimodal combinatorial approaches are being developed, including small-molecule inhibitors targeting specific resistance nodes, nanotechnology-based targeted drug delivery systems, combination therapies with immune checkpoint inhibitors, and interventions targeting metabolic vulnerabilities. Furthermore, emerging technologies are enabling more precise strategies: patient-derived organoids provide a platform for individualized drug sensitivity testing; single-cell sequencing allows for dissection of cellular heterogeneity within resistant populations and the interactions of these populations with the microenvironment; and artificial intelligence (AI) aids in predictive model building and drug discovery by integrating multi-omics data. In summary, a comprehensive understanding of the systems biology of cisplatin resistance, integrated with novel research paradigms such as nanotechnology, immunotherapy, metabolic targeting, organoid models, single-cell technologies, and AI, will be pivotal for developing personalized combination therapies to ultimately overcome cisplatin resistance in OSCC.

Background: Cancer has emerged as the primary cause of death worldwide in recent years. Current cancer treatment strategies require improvement, creating a pressing need for the development of novel therapeutic agents. This study investigated the anticancer effects of a series of newly synthesized tri- and difluoromethylated spiro[5.5]trienone compounds and evaluated the antitumor efficacy of a lead compound, 3s. Methods: The methyl thiazolyl tetrazolium (MTT) assay was used to assess the effect of the trienone compounds on the growth of cancer cells. Cell cycle distribution and intracellular reactive oxygen species (ROS) levels were analyzed by flow cytometry. Protein expression was examined by Western blot. A mouse xenograft model was utilized to test the anticancer effects and toxicity of 3s in vivo. Results: All 21 tri- and difluoromethylated spiro[5.5]trienones exhibited inhibitory effects on the growth of cancer cells. Among them, compound 3s showed the strongest inhibitory effect. It induced cell cycle arrest at the G2/M phase and promoted apoptosis. Mechanistically, 3s activated JNK and ERK signaling and elevated intracellular ROS levels. Furthermore, in a mouse xenograft model, 3s significantly inhibited tumor growth with minimal toxicity. Conclusions: Compound 3s exhibits potent anticancer efficacy both in vitro and in vivo. The discovery of 3s offers new potential for cancer therapy.

Cancer remains one of the leading causes of human death worldwide. Small extracellular vesicles (sEVs) are nanoscale particles that play important roles in intercellular communication in cancer and represent a valuable source for cancer biomarker discovery. Tissue-derived small extracellular vesicles (Ti-sEVs) are a specific subtype of sEVs and have attracted increasing attention as an emerging research area in cancer diagnostics and therapeutics. Recent progress has highlighted the distinct advantages of Ti-sEVs, including greater cancer specificity, minimal exogenous contamination, enhanced potential for cancer biomarker discovery and understanding tumour microenvironment (TME) dynamics. This review summarises recent advancements in the preparation, isolation, and characterization of Ti-sEVs. It further discusses current knowledge of their biological functions within the TME and critically evaluates their emerging application in cancer diagnosis and prognosis. Ti-sEVs offer unique opportunities for cancer biomarker discovery and translational research. However, key challenges remain, including tissue processing variability, methodological standardization, and technical barriers to clinical implementation. Addressing these issues will be essential to realize the full potential of Ti-sEV-based diagnostics and therapeutics and to guide future research in this rapidly evolving field.

Impaired cell proliferation causes fibrotic changes in tissues, leading to loss of function. Although exocyst component 5 (Exoc5), a central component of the eight-protein exocyst complex, regulates the targeting and docking of intracellular vesicles which are essential for cell proliferation, its role in tissue regeneration remains to be defined. Here, we investigated the role of Exoc5 in the repair of kidney injury induced by ischemia-reperfusion (I/R) using proximal tubule cell (PTC)-specific Exoc5 knockout (Exoc5KO) mice generated by crossing Exoc5f/f with PEPCK-cre mice. Exoc5KO and wild-type (Exoc5WT) mice were subjected to either bilateral kidney I/R or sham surgery. I/R induced functional and structural kidney damage in both Exoc5KO and Exoc5WT mice, as evidenced by increased plasma creatinine and BUN, decreased glomerular filtration rate, and histological damage. Kidney function and structure gradually improved in both Exoc5KO and Exoc5WT mice over time; however, neither group fully recovered normal function, and the recovery was less pronounced in Exoc5KO than in Exoc5WT mice. Twenty-one days after I/R, Exoc5KO mice showed greater collagen deposition and α-smooth muscle actin (α-SMA) and vimentin expression compared to Exoc5WT mice, whereas E-cadherin expression was lower. Post-I/R PTC proliferation in Exoc5KO mice was significantly lower than in Exoc5WT mice. In contrast, post-I/R induction of paired box 2 (Pax2) was greater in Exoc5KO than in Exoc5WT mice. In HK-2 cells, a human PTC line, Exoc5 downregulation by siRNA increased Pax2 expression and further increased N-cadherin, phosphorylated-Smad3 (p-Smad3), and α-SMA expression compared to control cells following TGF-β treatment. Collectively, these findings indicate that loss of Exoc5 impairs PTC regeneration and exacerbates fibrosis in the injured kidney, suggesting its therapeutic potential in preventing the transition from acute kidney injury (AKI) to chronic kidney disease (CKD).

Ferroptosis, an iron-dependent form of programmed cell death driven by lipid peroxidation, represents a new potential therapeutic target in cancer. However, emerging evidence indicates that hepatocellular carcinoma (HCC) frequently exhibits resistance to ferroptosis induction, while the underlying molecular mechanism is poorly understood. Here, we found that aldo-keto reductase family 1 member C3 (AKR1C3), a protein highly expressed in ferroptosis-resistant HCC cells, negatively regulates ferroptosis in an enzyme-independent manner. Mechanistically, AKR1C3 promotes ubiquitin-proteasomal degradation of the transferrin receptor (TFRC), which is indispensable for cellular iron uptake. AKR1C3 knockdown restores TFRC expression, increases the level of labile iron pool, and sensitizes HCC cells to ferroptosis. Furthermore, AKR1C3 acts as a scaffolding protein to promote the degradation of TFRC and reduce iron uptake by promoting nuclear export of Beta-transducin repeats-containing proteins (β-TrCP) and its binding to TFRC. Notably, AKR1C3 is upregulated in NRF2-driven sorafenib-resistant HCC, and its inhibition reversed ferroptosis and sorafenib resistance. Our work uncovers AKR1C3 suppresses ferroptosis in HCC by promoting β-TrCP-mediated TFRC degradation, positioning AKR1C3 as a promising therapeutic target to enhance ferroptosis-based anticancer strategies.

Quiescent prostate cancer (PCa) cells that survive therapy can later reactivate and drive tumor recurrence and metastasis. Here, we identify a strategy to eliminate these cells during their vulnerable reactivation phase. We show that guttiferone K (GUTK), a bioactive compound isolated from Garcinia yunnanensis Hu, selectively eradicates reactivating quiescent PCa cells by inducing mitochondrial apoptosis through caspase activation and loss of mitochondrial membrane potential (ΔΨm). Mechanistically, GUTK suppresses Aurora A recovery and stabilizes SOD2 protein, thereby promoting mitochondrial dysfunction and apoptosis. SOD2 enhances, whereas Aurora A overexpression attenuates, GUTK-induced cell death. In orthotopic and xenograft prostate tumor models, GUTK combined with docetaxel significantly inhibits tumor growth and suppresses post-chemotherapy recurrence without evident toxicity. These findings identify GUTK as a potential therapeutic agent targeting reactivating quiescent PCa cells and highlight the Aurora A-SOD2 axis as a promising pathway for preventing PCa recurrence.

Alternative splicing (AS) is a key driver of development and a major contributor to species diversity. Accumulating evidence indicates its high activity in various cancers. Here, we identified the spliceosome component SF3B1 as a key regulator of cell fate in hepatocellular carcinoma (HCC), with its expression elevated in HCC tissues/cells versus adjacent non-tumor tissues. Using SF3B1 inhibitor Pladienolide B (Pla B), we found that it suppresses HCC cells' proliferation and induces apoptosis. RNA-Seq revealed Pla B modulates AS events in HCC cells; KEGG analysis indicated it affects the AMPK-mTOR pathway to activate autophagy. In vivo xenograft experiments further demonstrated that the combined treatment of Pla B and cisplatin achieved a more potent inhibitory effect on tumor growth compared to either monotherapy. This combinatorial strategy not only reduced tumor cell proliferation and promoted apoptosis but also enhanced autophagy. Collectively, our findings highlight the potential of combining Pla B with cisplatin as a novel and promising therapeutic approach for the treatment of HCC.

Rheumatoid arthritis (RA) is a progressive autoimmune disorder characterized by aberrant T-cell activation with clinical outcomes often hindered by adverse effects and inadequate remission. While mesenchymal stem cell-derived extracellular vesicles (MSC-EVs) hold promise for the treatment of autoimmune disorders, the mechanisms by which cytokine priming enhances their efficacy remain poorly characterized. In this study, we investigated the immunomodulatory effects of EVs derived from interferon-gamma (IFN-γ)-primed MSCs (IFN-γ-EVs) in a collagen-induced arthritis (CIA) murine model. These results demonstrate that IFN-γ-EVs administration markedly attenuated joint inflammation, bone erosion, and proinflammatory cytokine levels in CIA mice, surpassing the performance of native MSC-EVs and achieving therapeutic parity with methotrexate. IFN-γ-EVs suppress pathogenic T-cell proliferation and activation while promoting the secretion of anti-inflammatory cytokines. Mechanistically, IFN-γ priming upregulates programmed death-ligand 1 (PD-L1) on the EVs; the subsequent genetic ablation of PD-L1 completely abolished these therapeutic benefits, identifying PD-L1 as the pivotal mediator. These findings suggest that IFN-γ priming amplifies the immunoregulatory capacity of MSC-EVs through a PD-L1-dependent pathway, presenting a safe, cell-free therapeutic strategy for RA intervention.

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Differentiation therapy offers a promising approach in acute myeloid leukemia (AML) by overcoming the developmental block that maintains leukemic blasts. Increasing evidence indicates that DNA replication stress can promote differentiation rather than cytotoxicity; however, the metabolic mechanisms linking replication stress to differentiation remain poorly defined. Here, we investigated how perturbations in nucleotide metabolism regulate replication stress-driven differentiation. Using metabolomic and functional analyses in AML cell lines, we show that agents inducing differentiation through replication stress, including 5-aminoimidazole-4-carboxamide ribonucleoside (AICAr), dihydroorotate dehydrogenase (DHODH) inhibition, and low-dose cytarabine, converge on disruption of nucleotide pool balance. Low-dose AICAr induced a pyrimidine-purine imbalance, S phase arrest, and enhanced differentiation, whereas high-dose reduced these effects. Although brequinar and cytarabine altered nucleotide metabolism through distinct mechanisms, differentiation induced by all agents was abolished by supplementation with high levels of ribo- and deoxyribonucleosides, confirming that nucleotide imbalance is a central driver. We further identify ribonucleotide reductase (RNR) as a critical modulator of this process. Replication stress induced context-dependent regulation of RNR subunits, with RRM2 upregulated in p53-mutant U937 cells and the p53-responsive RRM2B isoform predominating in p53-wild-type MOLM-13 cells. Consistent with these differences, RRM2 depletion enhanced differentiation in U937 cells without affecting viability but impaired differentiation and survival in MOLM-13 cells. These findings position nucleotide metabolism as a key regulator of AML differentiation and suggest that combining RNR-targeted and checkpoint-modulating strategies could optimize therapeutic responses.