Abstract Inactivating mutations in TP53 and ATRX are key alterations defining the genomic landscape of uterine leiomyosarcoma (uLMS), creating chronic replication stress and increasing dependency on Ataxia telangiectasia and Rad3-related kinase (ATR) signaling for survival. ATR-CHK1 signaling limits the efficacy of standard-of-care gemcitabine by facilitating the stabilization of stalled replication forks and preventing lethal DNA strand breaks in checkpoint-deficient cells. Here, we show that ATR inhibitors (elimusertib, ceralasertib, berzosertib) preferentially synergize with gemcitabine over docetaxel or doxorubicin, across a panel of TP53-mutant/ATRX-wildtype uLMS models. When simultaneously administered, gemcitabine/elimusertib demonstrated enhanced synergy and efficacy compared to standard-of-care gemcitabine/docetaxel. This combination benefit was absent in a TP53-wildtype uterine sarcoma control line, highlighting TP53 mutation status as a critical predictive biomarker. Combined gemcitabine/ATRi treatment demonstrated reciprocal potency enhancement at low-nanomolar concentrations and significantly increased apoptotic populations compared to monotherapy. Gemcitabine monotherapy induced CHK1 (S345) phosphorylation and a corresponding S-phase arrest. Elimusertib abrogated this checkpoint, transforming protective S-phase arrest into a cytotoxic response characterized by a marked increase in the sub-G1 population. This transition was driven by the inhibition of gemcitabine-induced CHK1 phosphorylation, resulting in marked accumulation of unrepaired DNA damage (γH2AX) and the induction of apoptosis as evidenced by cleaved caspase-3 expression. These findings demonstrate that elimusertib strongly potentiates gemcitabine-induced damage to kill TP53-mutant uLMS in vitro, outperforming standard gemcitabine/docetaxel when concurrently administered. This combination strategy broadens the clinical utility of ATR inhibitors to uLMS harboring the prevalent TP53-mutant/ATRX-wildtype genotype and supports the clinical evaluation of gemcitabine/ATRi combinations as a novel therapy for uLMS.
Abstract Pancreatic ductal adenocarcinoma (PDAC) displays profound metabolic plasticity that sustains rapid proliferation through heightened demands for nucleotide biosynthesis. Here, we characterize HOSU-53 (JBZ-001), a dihydroorotate dehydrogenase (DHODH) inhibitor demonstrating potent antitumor activity in preclinical PDAC models and define the metabolic determinants of sensitivity and resistance. In HOSU-53-sensitive PDAC cells, DHODH inhibition induces G₂/M cell cycle arrest and triggers apoptotic and ferroptotic cell death. In contrast, resistant cells evade metabolic stress by activating compensatory nucleoside transport and macropinocytosis pathways. Integrated functional and genetic analyses reveal that nucleoside salvage via the equilibrative nucleoside transporter SLC29A1 constitutes a dominant resistance mechanism, enabling uptake of extracellular nucleosides to bypass de novo pyrimidine synthesis. Unbiased metabolomic profiling demonstrates that combined inhibition of DHODH and SLC29A1 precipitates mitochondrial dysfunction, oxidative stress, lipid remodeling, and amino acid depletion, culminating in metabolic collapse and energetic failure. Consistent with these findings, dual inhibition markedly suppresses tumor growth and metastatic burden in xenograft and syngeneic orthotopic PDAC models while reprogramming the tumor microenvironment toward a pro inflammatory, immune activated state. Furthermore, co targeting DHODH and SLC29A1 synergizes with anti–PD 1 immune checkpoint blockade, enhancing immunotherapeutic efficacy. Together, these results establish dual targeting of de novo and salvage nucleotide metabolism as a compelling strategy to overcome metabolic resistance, remodel tumor–immune interactions and potentiate therapeutic responses in PDAC
Abstract Multiple myeloma (MM), the second most common hematologic malignancy, remains incurable despite major therapeutic advances, highlighting an urgent need for novel therapeutic strategies. PCNA is involved in DNA replication and repair, chromatin remodeling, and cell-cycle progression, and increased PCNA expression has been associated with aggressive MM phenotypes and poor clinical outcomes. Cancer cells express a post-translationally modified cancer-associated isoform of PCNA (caPCNA), potentially providing a selective therapeutic target. We evaluated the anti-myeloma activity of the novel caPCNA inhibitor AOH1996 alone and in combination with established anti-myeloma agents in vitro, in vivo, and ex vivo. AOH1996 inhibited growth of multiple MM cell lines in a dose-dependent manner, induced G2-phase accumulation and apoptosis, and reduced sphere formation and colony formation capacity. Combination treatment with bortezomib, venetoclax, melphalan, or cyclophosphamide enhanced anti-myeloma activity relative to single-agent treatment. AOH1996 treatment down-regulated the expression of multiple tumor-associated proteins and inflammatory and pro-survival cytokines. miRNA analysis demonstrated suppression of several oncogenic miRNAs along with upregulation of tumor-suppressive miRNA miR-145. Forced expression of miR-145 inhibited MM cell growth and enhanced sensitivity to AOH1996, implicating miR-145 as a mechanistic mediator of PCNA inhibition’s anti-tumor effects. In xenograft models, AOH1996 significantly enhanced the anti-tumor activity of bortezomib and venetoclax without apparent toxicity. Ex vivo studies demonstrated selective activity against primary MM cells and CD138+ plasma cells with minimal effects on normal PBMCs. Collectively, these findings support caPCNA as a promising therapeutic target in MM and provide strong preclinical rationale for further clinical investigation of AOH1996-based therapeutic strategies.
Abstract Trastuzumab deruxtecan (T-DXd) has demonstrated clinical efficacy in advanced HER2-low to -ultralow breast cancer. We employed a mouse model of triple-negative breast cancer in the difficult-to-treat brain metastasis setting to determine T-DXd efficacy and investigate mechanism(s). T-DXd (10 and 3 mg/kg) was compared to an identical ADC except HER2-nonbinding (control) in a hematogenous model of HER2-ultralow brain-tropic triple-negative breast cancer, MDA-MB-231-BR7 (231-BR). The roles of HER2 and the neonatal Fc receptor (FcRn) were determined by CRISPR-Cas9 deletion and shRNA knockdown, respectively. 231-BR cells exhibited HER2 0+ staining, with occasional membrane expression. T-DXd at 10 mg/kg reduced 231-BR brain metastases by 49% (P=0.002), while a dose reduction showed a 30% (ns) metastasis reduction, each compared to the control-ADC. HER2-specific and -nonspecific ADC uptake into metastases was accompanied by a 23-47% tumor cell Ki-67 decline, and 5-fold increase in tumor apoptosis. A HER2 CRISPR-Cas9 knockout 231-BR formed multiple brain metastases but was insensitive to T-DXd in vivo. 231-BR tumor cells expressed FcRn, potentially enabling nonspecific ADC uptake. Analysis of human tumors showed TNBC tumors expressed FcRn mRNA. T-DXd failed to inhibit brain metastases in two 231-BR FcRn knockdown clones compared to a 231-BR clone transduced with a scrambled shRNA where T-DXd inhibited brain metastases (67% reduction, P=0.01). Despite ultralow HER2 expression, the HER2 binding capacity of T-DXd is a significant contributor to its efficacy. Breast cancers variably express FcRn, suggesting that nonspecific tumor cell uptake of T-DXd via FcRn is also a potential mechanism of T-DXd action.
Abstract Hormone receptor-positive (HR+)/HER2-negative metastatic breast cancer that progresses on CDK4/6 inhibitors (CDK4/6i) and endocrine therapy (ET) faces a major therapeutic challenge, and chemotherapy is frequently used despite its modest clinical benefit. Paclitaxel is commonly used in this setting; however, intrinsic and acquired resistance substantially limit the depth and durability of response. Polo-like kinase 1 (PLK1) is a key mitotic regulator implicated in resistance to taxanes and ET/CDK4/6i. We evaluated whether PLK1 inhibition with onvansertib enhances paclitaxel activity in HR+ breast cancer models with intrinsic or acquired ET/CDK4/6i resistance, testing across six cell lines and eight patient-derived xenograft models, including paclitaxel-resistant tumors. The combination synergistically inhibited cell viability in vitro and improved antitumor efficacy in xenograft models. Notably, the combination induced tumor regression across all paclitaxel-resistant models, with complete responses in more than half of treated animals in three of five models, and deep, durable regressions in paclitaxel-sensitive models, markedly outperforming either agent alone. Mechanistically, the combination exacerbated mitotic stress, disrupted spindle architecture, suppressed proliferative and cell-cycle-associated transcriptional programs, and promoted proteasome-dependent c-MYC degradation, resulting in enhanced apoptotic cell death in vitro and in vivo. Importantly, genetic modulation of c-MYC expression altered the apoptotic response, establishing c-MYC downregulation as a key mediator of the observed antitumor efficacy. Collectively, these findings support PLK1 inhibition as a mechanistically rational strategy to potentiate taxane efficacy and overcome resistance in advanced HR+/HER2-negative breast cancer.
