Clinical Cancer Research

Homologous recombination (HR) deficiency underlies a range of cancers, and PARP inhibitors (PARPi) targeting this vulnerability have demonstrated clinical efficacy in BRCA1/2-mutant tumors. However, resistance mechanisms and tolerability issues limit their long-term clinical effectiveness. Here, we characterize RP-3467, a novel, selective small-molecule inhibitor targeting the ATPase domain of DNA polymerase theta (Pol{theta}), the key mediator of the error-prone microhomology-mediated end joining (MMEJ) pathway. We demonstrate that RP-3467 potently suppresses Pol{theta} enzymatic activity, abrogates MMEJ, and disrupts mitotic DNA repair in HR-deficient (HRD) models, inducing synthetic lethality. In preclinical studies, we show that RP-3467 synergizes strongly with PARPi (olaparib and rucaparib), achieving sustained tumor regression across multiple xenograft and patient-derived xenograft (PDX) models without added systemic toxicity. Mechanistically, we find that RP-3467 increases mitotic DNA damage in vivo, as evidenced by elevated micronuclei and CIP2A foci. RP-3467 was evaluated in a phase 1 clinical trial (NCT06560632) and has shown preliminary signs of clinical activity. A heavily pretreated germline BRCA1-mutant ovarian cancer patient achieved a confirmed partial response to RP-3467 plus olaparib (RECIST v1.1), with retrospective profiling revealing a TP53BP1 loss-of-function mutation previously linked to both PARPi resistance and POLQ dependency. Although broader clinical validation is needed, these early findings suggest that Pol{theta} inhibition has the potential to address PARPi resistance and offer therapeutic benefit in HRD cancers.

Purpose To assess whether follow-up MRI, biopsy status, and timing of cribriform disease detection predict active surveillance (AS) failure in a contemporary transatlantic cohort. Materials and Methods. Retrospective MRI-driven AS cohort of 619 men at two academic centers (UK/USA), 2012-2024. Primary outcome: AS failure (disease-specific mortality, metastasis, grade group [GG] [&ge;]4 progression, or post-treatment biochemical recurrence). Key exposures: final MRI and biopsy status, timing of cribriform [&ge;]GG2 detection. Cumulative incidence was derived; independent predictors used multivariable Fine-Gray models. Results. Over median follow-up of 6.4 years, 39/619 men (6%) experienced AS failure. Cribriform [&ge;]GG2 was detected in 82/619 (13%) on follow-up biopsy versus 24/619 (4%) at baseline, comparably in baseline GG1 (10%) and non-cribriform GG2 (9%), and only in MRI-visible tumors. Three-year AS failure after final biopsy was 31% in men with cribriform [&ge;]GG2 versus 0-2% with stable or non-cribriform disease; 44% of failures had stable MRI. On Fine-Gray analysis, cribriform [&ge;]GG2 was the only independent predictor of AS failure in re-biopsied men (SHR 30; 95% CI 5.2-167; P<0.001); no type of MRI progression category was independently predictive. Among 595 men cribriform-negative at baseline, 6-year AS failure after a second biopsy was 34% if cribriform [&ge;]GG2 was detected versus 7% if still cribriform-negative. Conclusions. Cribriform status at follow-up biopsy independently predicts AS failure, whereas MRI progression does not. The three-fold higher cribriform prevalence at follow-up likely reflects baseline sampling error. Because newly detected cribriform tumours were exclusively MRI-visible, early repeat biopsy of patients with MRI-visible disease may considerably improve risk stratification.

Purpose: Mifamurtide (liposomal muramyl tripeptide phosphatidylethanolamine [L-MTP-PE] is approved in many countries for osteosarcoma but remains unavailable for routine use in the United States (US). We describe the feasibility and outcomes of patients with osteosarcoma treated with mifamurtide at Memorial Sloan Kettering Cancer Center (MSK) through expanded access (EAP) and compassionate use (SPU) pathways. Methods: Medical records were retrospectively reviewed for patients with completely resected, high-grade osteosarcoma treated with mifamurtide at MSK from 2016-2026 through EAP or SPU protocols. Mifamurtide 2 mg per meter squared was administered intravenously twice weekly for 12 weeks, followed by weekly administration for 24 weeks. Outcomes were evaluated by treatment setting and, for upfront therapy, metastatic status and histologic necrosis. Results: Fifty-seven patients received mifamurtide (median age, 14 years; range, 2-60); 72% had localized and 28% metastatic disease at diagnosis. No serious adverse events were considered possibly or probably related to mifamurtide, and no new grade 3-4 safety signals emerged. Five patients with localized disease and [&ge;]90% necrosis at resection had no events, with 100% progression-free survival (PFS) and overall survival (OS) at median follow-up of 29 months. Among 22 patients with localized disease and <90% necrosis treated upfront, PFS was 82%, 66%, and 59% at 12, 24, and 48 months, respectively; OS was 95% at 12, 24, and 60 months. Among 8 patients with metastatic disease treated upfront, PFS was 73%, 58%, and 39% at 12, 24, and 36 months, respectively; OS was 100%, 86%, and 86%. Among 22 patients treated after relapse, PFS was 34%, 23%, and 17% at 12, 24, and 36 months, respectively; OS was 91%, 70%, and 35% at 12, 24, and 60 months. Conclusions: In this real-world US cohort, mifamurtide was feasible and safe to administer. Despite limitations of the EAP/SPU setting, single-institution administration, and patient heterogeneity, overall survival outcomes were encouraging.

Background Immune checkpoint inhibitor (ICI) therapies have revolutionized oncology, but identifying patients who benefit remains a challenge, particularly in populations where ICIs are not the standard of care, including microsatellite stable colorectal cancer (MSS CRC) and rare solid cancers. We evaluated real-world performance of the Immune Profile Score (IPS), a validated DNA- and RNA-based molecular signature, in these cohorts. Methods In an exploratory analysis, we analyzed two real-world cohorts: 1) MSS CRC; and 2) MSS, tumor mutational burden-low rare solid cancers treated with off-label ICI. IPS-High and IPS-Low were calculated using a previously validated threshold. Cox proportional hazards models were fit to demonstrate prognostic utility for real-world overall survival (rwOS). In MSS CRC, time-to-next-treatment (TTNT) on prior non-ICI therapy was compared to rwOS on subsequent ICI therapy. Results In MSS CRC (n=46), IPS-High patients (13%) demonstrated clinically meaningful improvement in rwOS versus IPS-Low (Median OS: not reached vs 7.3 months; HR = 0.22, 90% CI 0.04-1.16). While IPS was not associated with TTNT on the preceding non-ICI line (HR = 1.07, 90% CI 0.60-1.91), it was associated with OS in patients receiving later line ICI (HR 0.21, 90% CI 0.04-1.22). In the rare cancers cohort (n=90; 26 unique malignancies), IPS-High (17.8%) was associated with longer OS to ICI therapy (HR = 0.26, 90% CI 0.11-0.61), even when restricted to histologies represented in both IPS groups (HR = 0.18, 90%, CI 0.05-0.62). Conclusions Preliminary evidence suggests that IPS may serve as a generalizable, pan-cancer biomarker capable of stratifying outcomes for ICI treatment in populations traditionally excluded from immunotherapy benefit.

Background: Immune checkpoint inhibitors (ICI) are efficacious in many solid tumors, but response in glioma is restricted to a small subgroup. The determinants of response and resistance to ICI remain poorly understood. Methods: Here we exploit a syngeneic hypermutated high-grade glioma model with dichotomous response to combined PD-1 and CTLA-4 inhibition to unravel determinants of tumor-infiltrating T-cells driving response. Tumor-infiltrating T-cells from ICI-responsive and non-responsive tumors were analyzed by single-cell RNA and T-cell receptor sequencing and tumor-reactive T-cell receptor clonotypes were functionally validated to characterize their transcriptional phenotypes. We verify our findings in IDH1 wildtype glioblastoma patients treated with neoadjuvant pembrolizumab. Results: ICI response was associated with intratumoral clonal expansion of tumor-reactive cytotoxic T-cells and increased infiltration of CXCR6+ CD4+ tissue resident memory T-cells (Trm). CD4 stem-like memory T-cells in responding tumors demonstrated elevated interferon responses, following trajectories toward clonally expanded Trm, versus trajectories toward exhaustion in non-responsive tumors. In responsive tumors, CD4+ Trm interacted with infiltrating CXCR3+ tumor-reactive and clonally expanded, yet transcriptionally versatile cytotoxic T-cells. Probing the post neoadjuvant ICI high-grade glioma patient tissue dataset, we confirmed increased CXCR6 expression in CD4+ T cells and the association of CD4+ Trm with prolonged overall survival. Conclusion: These findings identify CD4 tissue-resident memory T-cells as determinants of ICI response in IDH1 wildtype high-grade glioma and warrant their further investigation to improve immunotherapy outcomes.

Background: Relapsed and refractory osteosarcoma (OS) remains a major therapeutic challenge, with fewer than 20% of patients surviving beyond 3 years. Increasing evidence indicates that MYC amplification/overexpression is associated with inferior survival. Small molecule inhibitors and immunotherapies have limited single-agent efficacy in pediatric solid tumors. Using syngeneic cell lines derived from p53-driven and MYC-hyperactivated genetically engineered mouse models (GEMMs) of OS, we tested cabozantinib, a multi-tyrosine kinase inhibitor with immunomodulatory properties, with TIGIT immune checkpoint blockade and investigated mechanisms underlying therapeutic response and resistance. Methods: In vitro cabozantinib sensitivity was established in GEMM-derived cell lines. Mice bearing tibial tumors were randomized to vehicle control, cabozantinib, anti-TIGIT antibody, or combination therapy, and tumor growth and survival assessed after a 3-week treatment period. Temporal RNA sequencing was performed at early (8-15 days) and late (18-24 days) time points to characterize transcriptomic changes associated with efficacy. Results: MYC-hyperactivated cell lines were more resistant to cabozantinib in vitro than p53-driven lines (mean IC50 5.51 vs 0.65 mciroM, p=0.0016). In p53-driven orthotopic models, combination therapy significantly decreased tumor growth and improved survival compared to solvent and cabozantinib alone, while in MYC-hyperactivated models cabozantinib-containing regimens delayed tumor progression relative to control or anti-TIGIT monotherapy, however the addition of anti-TIGIT did not significantly improve survival over cabozantinib alone. Temporal transcriptomics revealed upregulated anti-tumor immune-response pathways and decreased M2 macrophages only with combination treatment in the p53-driven model. In contrast, combination-treated MYC-hyperactivated models demonstrated increased TNF signaling and elevated Cxcl5 and Ccr2 expression, indicative of increased myeloid cell recruitment, and upregulation of extracellular matrix (ECM) remodeling pathways suggest a therapy-induced stress adapted state that propagates treatment resistance over time. Conclusion: New therapies are needed for patients with relapse or refractory OS. By targeting tumor-intrinsic resistance mechanisms and modulating the tumor microenvironment using cabozantinib and anti-TIGIT therapy, improved tumor control and survival was achieved in p53-driven orthotopic OS models. MYC-hyperactivated models were able to overcome therapeutic pressure and employ myeloid recruitment and ECM remodeling programs to achieve treatment resistance. Targeting of these programs should be considered in future studies investigating therapeutic strategies in relapsed and refractory OS.

Background Chemotherapy (CT) de-escalation strategies based on dual HER2 blockade with trastuzumab and pertuzumab (HP) have shown promising efficacy in HER2-positive (HER2+) early breast cancer (EBC), but biomarkers to guide patient selection are lacking. The Ataraxis Breast platform (ATX) uses artificial intelligence (AI) models integrating hematoxylin-eosin (H&E) whole-slide images and clinical variables to predict pathological complete response (pCR) and long-term outcomes. We evaluated ATX in the phase II PHERGain (NCT03161353) and PHERGain-2 (NCT04733118) trials. Methods The ATX NEO model was assessed for pCR prediction in PHERGain (n=306 evaluable patients) and PHERGain-2 (n=383 evaluable patients). The ATX RISK model was evaluated for prognosis in PHERGain (n=309 evaluable patients). Prespecified cutoffs classified patients into low-, medium-, and high-probability groups for pCR and low- or high-risk groups for recurrence. Associations with pCR were assessed using logistic regression. Prognostic performance was evaluated using Cox proportional hazards models, Kaplan-Meier estimates, and Harrell's C-index. Results Overall pCR rates were 40.2% in PHERGain and 60.3% in PHERGain-2. Continuous NEO scores were significantly associated with pCR in both PHERGain (odds ratio [OR], 1.28; 95% CI, 1.11-1.48; P<0.001) and PHERGain-2 (OR, 1.30; 95% CI, 1.13-1.52; P<0.001). Using prespecified cutoffs, pCR rates increased across low-, medium-, and high-probability groups (19.2%, 41.3%, and 48.3% in PHERGain; 29.5%, 61.6%, and 70.1% in PHERGain-2). In PHERGain, the RISK score was significantly associated with event-free survival (hazard ratio per 1-SD increase, 1.56; 95% CI, 1.17-2.08; P=0.002; C-index, 0.66). Five-year event-free survival was 93.9% in the low-risk group and 83.1% in the high-risk group. Conclusions NEO score was consistently associated with pCR across two independent HER2+ EBC trials, while RISK score provided prognostic information for long-term outcomes in PHERGain. Prespecified score categories identified clinically distinct response and risk groups. These findings support further evaluation of AI-based biomarkers to refine patient selection for CT de-escalation strategies in HER2+ EBC.

Purpose: Existing prognostic models for brain metastases from lung cancer rely on clinical variables and do not capture the biological heterogeneity of host or tumor factors underlying survival outcomes. We evaluated whether MRI-derived radiomic biomarkers improve prognostic stratification beyond clinical variables. We also investigated their biological correlates using matched histopathology. Experimental Design: This retrospective study included 103 patients with brain metastases from lung cancer and matched pre-operative MRI and histopathology. Radiomic features from T1-weighted contrast-enhanced and T2-weighted fluid-attenuated inversion recovery sequences were combined with clinical variables to train Cox proportional hazards, random survival forest, and DeepSurv models for overall survival (OS), CNS progression, and leptomeningeal (LM) progression. Risk groups from an imaging-only model were compared for clinical and histopathologic differences. Results: Imaging features improved prognostic performance beyond clinical variables across all modeling frameworks, with the combined clinical-imaging DeepSurv model achieving the highest concordance index for OS (0.728). Risk groups from the best-performing model for each endpoint differed significantly in OS (median 26.7 vs. 9.1 months, p < 0.001) and LM progression (p = 0.003), but not in CNS progression. Imaging-only risk groups did not differ significantly in any clinical variable, but high-risk patients were less likely to have tumor-infiltrating lymphocytes (46% vs. 77%, p = 0.010) and PD-L1 expression [&ge;] 50% (10% vs. 32%, p = 0.049). Conclusions: MRI radiomic biomarkers provide prognostic information beyond established clinical variables in lung cancer brain metastases and correspond to features of the tumor immune microenvironment, supporting their potential to refine risk stratification.

Background: Cancer-associated fibroblasts (CAFs) contribute to systemic therapy resistance in muscle-invasive bladder cancer (MIBC), but their functional heterogeneity and relevance to curative bladder-preserving radiotherapy, are poorly understood. Methods: Transcriptomic analysis was performed on 279 tumours from BC2001, a phase 3 radiotherapy clinical trial. To study CAF heterogeneity, we integrated bulk RNA-seq, single-cell spatial analysis of multiplex immunofluorescence images and quantification of extracellular matrix (ECM) features in 155 MIBC biopsies. The functional heterogeneity of distinct CAF populations was evaluated by single-nuclear RNA-seq. Spatial interactions between CAF populations and CD8+ T-cells was assessed and the relevance of lymphocytes to radiation responses was evaluated in a CAF-enriched murine bladder cancer model (BBN963). Results: BC2001 patients with CAF-enriched tumours had worse overall survival (HR=1.671, 95% CI 1.221-2.287, Log-rank p=0.0012). CAF abundance and antigen expression was highly heterogenous. Podoplanin (PDPN) was expressed on the majority of CAFs and was associated with inflammatory pathways. Enrichment of CAF gene signatures was associated with a significant increase in CAFs expressing fibroblast activation protein (FAP) (p=0.004) and dense ECM features (p=0.0004). Fifty-three percent of tumours exhibited stromal CD8+ T-cell exclusion with significant enrichment in FAP-dominant neighbourhoods (p<0.001). In vivo, lymphocytes were critical for radiation-induced tumour control, indicating that immune cold or excluded tumours may have limited radiotherapy responses. Conclusion: In MIBC, CAFs are associated with poor radiotherapy outcomes. Multiple mechanisms are deployed by functionally-heterogeneous CAFs, including promotion of chronic inflammation by PDPN+ CAFs and ECM remodelling by FAP+ CAFs which impact CD8+ T-cell distribution and radiation responses.

Purpose: How sarcoma subtypes engage telomerase versus alternative lengthening of telomeres (ALT), and with what consequence, is incompletely defined. Methods: We analyzed 11,792 patients with sarcoma sequenced across 19 centers in AACR Project GENIE v20.0. Telomerase activation was defined as TERT promoter mutation, amplification, or rearrangement, and ATRX/DAXX inactivation served as the ALT proxy, each assessed only where the assay interrogated the locus. Findings were tested for TERT expression in The Cancer Genome Atlas (TCGA), for overall survival (OS) in MSK-IMPACT 50K, and for replication in an independent Foundation Medicine cohort. Mendelian randomization (MR) in FinnGen tested germline telomere length. Results: Telomerase activation (8.8%) and ATRX/DAXX loss (8.0%) were mutually exclusive across subtypes (odds ratio [OR], 0.52; 95% CI, 0.29 to 0.92) and segregated by histology. In MDM2-amplified dedifferentiated liposarcoma (DDLPS), telomerase was activated through TERT amplification (17.2%) or rearrangement (2.7%), never promoter mutation (0/261), and never with ATRX/DAXX loss (0/49; P = .011). TERT-amplified tumors expressed TERT (TCGA; P = .002), and amplification was largely focal. Telomere maintenance alterations were present in 28.0% of DDLPS versus 8.7% of well-differentiated liposarcoma (P < .001) and predicted shorter OS in DDLPS (hazard ratio, 2.04; 95% CI, 1.23 to 3.39; P = .005). ATRX/DAXX loss was more frequent in uterine than extrauterine leiomyosarcoma in GENIE (adjusted OR, 1.96) and in the independent cohort (OR, 1.95; both P < .001). Genetically longer telomeres were associated with liposarcoma (OR per SD, 4.10; 95% CI, 2.15 to 7.81). Conclusion: Sarcoma telomere maintenance is histology-specific and mutually exclusive. In liposarcoma it is acquired at dedifferentiation through focal TERT amplification or ALT and identifies DDLPS with inferior survival.

BackgroundCancer-associated fibroblasts (CAF) are major regulators of the tumor microenvironment (TME), yet the pathways governing CAF function remain incompletely understood. Type 2 deiodinase (DIO2, D2), which converts thyroxine (T4) to the active thyroid hormone triiodothyronine (T3), has not been systematically investigated in CAF across different solid tumors. MethodsWe analyzed DIO2 expression using single-cell RNA sequencing (scRNA-seq) from primary thyroid tumors, a large integrated scRNASeq thyroid cancer atlas, and publicly available scRNA-seq datasets representing six additional solid tumor types. Bulk RNA sequencing data from the Oncology Research Information Exchange Network (ORIEN) database were used to examine correlations among DIO2, CAF subtype markers, and overall survival (OS). A genetically engineered TPO-CreERT2/BrafV600E/Trp53-deficient (TBP) mouse model was evaluated for CAF-specific Dio2 expression and pharmacologic inhibition of D2 activity. TBP mice were crossed with Dio2-/- (Dio2 KO) mice to investigate the role of Dio2 in cancer development and progression. ResultsDIO2 expression was highly restricted to CAF and was largely absent from epithelial-derived cancer, immune, and endothelial cells. Across thyroid cancer and six additional malignancies, DIO2 was consistently detected in CAF, demonstrating that CAF-specific DIO2 expression is a conserved feature of multiple tumor types. Within thyroid cancer, DIO2 was enriched in myofibroblastic CAF (myCAF) compared with inflammatory CAF (iCAF), and bulk transcriptomic analyses demonstrated stronger correlations between DIO2 expression and canonical myCAF markers than iCAF markers across numerous cancers. Elevated DIO2 expression was associated with worse OS in pancreatic and bladder cancers, paralleling the adverse prognostic impact of increased myCAF abundance. In contrast, thyroid cancer demonstrated an inverse association attributable to loss of epithelial DIO2 expression during tumor dedifferentiation. The TBP mouse model recapitulated human disease, with Dio2 expression restricted to CAF, robust stromal fibrosis, and measurable D2 enzymatic activity that was suppressed by cefuroxime and lenvatinib. Genetic loss of Dio2 in the TME resulted in significantly reduced tumor growth. Conclusions DIO2 is a conserved marker of CAF, and myCAF, across multiple solid tumors and is associated with adverse clinical outcomes in some cancers. The TBP mouse provides a robust preclinical model to define the mechanistic role of DIO2 in CAF biology and to evaluate DIO2-directed therapeutic strategies targeting the tumor microenvironment.

Background: Giant cell tumor of bone (GCTB) is an intermediate osteolytic neoplasm characterized by a high rate of local recurrence. The tumor comprises two main cell populations: neoplastic stromal cells and osteoclast-like giant cells. Current adjuvant therapies primarily target osteoclast-like giant cells but have limited activity against the neoplastic stromal cells that drive tumor growth and recurrence. Here, we sought to identify actionable vulnerabilities in GCTB stromal cells by profiling druggable receptor tyrosine kinases (RTKs) and associated signaling pathways and evaluating their pharmacological inhibition in patient-derived in vitro models. Methods: RTK activation and downstream signaling were assessed using phospho-protein arrays and western blotting in therapy-naive and denosumab-treated GCTB patient samples and in patient-derived GCTB cell lines, representing clinically relevant models of the neoplastic component. Tyrosine kinase inhibitors (TKIs) targeting activated pathways were evaluated in patient-derived GCTB cell lines cultured under both two-dimensional (2D) and three-dimensional (3D) conditions. Effects on cell viability, proliferation, cell cycle distribution, and cell death were assessed using MTT and XTT assays, flow cytometry, live-cell imaging, and confocal microscopy. Results: Several activated RTKs, including EGFR, IGF-IR, InsR, PDGFR, and PDGFR{beta}, together with downstream signaling proteins MEK1/2 and ERK1/2, were identified as potential therapeutic targets. Among the inhibitors tested, trametinib, a MEK1/2 inhibitor, and axitinib, a multi-target inhibitor of VEGFR1-3, PDGFR{beta}, and c-Kit, demonstrated the strongest antiproliferative and cytotoxic effects, markedly reducing cell viability and proliferation while promoting cell death in vitro. Importantly, trametinib showed selective activity against GCTB stromal cells compared with healthy controls, suggesting a potential therapeutic window. Conclusions: This study identifies trametinib and axitinib as promising therapeutic candidates for targeting the neoplastic component of GCTB. Our findings provide a rationale for further preclinical and clinical evaluation of RTK-directed therapies to complement current treatments and specifically target the neoplastic GCTB stromal cells underlying tumor persistence and recurrence.

Acute myeloid leukemia (AML) remains limited by high relapse rates and a scarcity of biomarker-directed therapies, underscoring the need to identify actionable vulnerabilities and mechanisms of therapeutic resistance. O6-methylguanine-DNA methyltransferase (MGMT), a DNA repair enzyme that directly reverses mutagenic O6-alkylguanine lesions, is epigenetically silenced in multiple cancers. Although MGMT silencing is predictive of temozolomide (TMZ) response in glioblastoma with significant survival advantage, prior clinical trials of TMZ in AML have shown only modest responses. Therefore, the prevalence and therapeutic relevance of MGMT silencing, as well as genetic factors that modify its therapeutic response in AML, remain to be comprehensively investigated. Here, we first profiled MGMT status across a diverse cohort of 23 de novo and 16 relapsed/refractory (R/R) primary AMLs using an integrated analysis of MGMT mRNA expression, promoter methylation, and protein expression. We found that approximately 25-30% of AMLs harbor MGMT silencing, in contrast to consistent MGMT expression in healthy CD34+ hematopoietic stem and progenitor cells. We further identified frequent loss of mismatch repair (MMR) proteins in these AML cohorts, a known resistance mechanism against TMZ in glioblastoma. Using CRISPR knockout screening and genetically engineered AML models, we found that MMR loss also drives both upfront and acquired TMZ resistance in MGMT-silenced AML. These findings identify MMR deficiency as an important and previously underappreciated genetic contributor to the modest responses observed in prior trials of TMZ in AML. To therapeutically exploit MGMT silencing while bypassing genetic determinants of resistance, including MMR deficiency, we evaluated clinically and preclinically characterized MGMT-dependent DNA-alkylating agents and identified a fluoroethylating analog of TMZ, N3-(2-fluoroethyl) imidazotetrazine (KL50), with pronounced and selective activity in MGMT-silenced AML. KL50 retained antileukemic activity irrespective of MMR status, significantly prolonging survival in humanized MISTRG6 mice harboring primary AML patient-derived xenografts. Mechanistically, KL50 induced DNA damage through the time-dependent formation of interstrand DNA crosslinks, bypassing MMR dependence and triggering a replication stress response dominated by ATR signaling. Pharmacologic ATR inhibition synergized with KL50, producing marked antileukemic activity and significantly extending survival across AML models without compromising hematologic safety. Together, these findings establish MGMT silencing as a prevalent and therapeutically actionable biomarker in AML, define MMR status as a key determinant of TMZ response in AML but not KL50 sensitivity, and provide a translational rationale for combining low-dose O6-fluoroethylating imidazotetrazines with ATR inhibitors to target both de novo and R/R AML across diverse genetic backgrounds.

The dynamic reprogramming of the bone marrow (BM) microenvironment during metastatic progression is poorly defined. Through single-cell profiling of BM samples spanning benign conditions, high-risk bone metastatic potential localized prostate cancer (HMP) and established bone metastatic prostate cancer (PCa), we uncovered a primary tumor-to-BM signaling axis that orchestrates niche formation through neutrophil-mediated remodeling. Tumor-associated neutrophils (TANs) emerge during the HMP stage, persist throughout metastatic progression, correlate with poor patient prognosis, and exhibit direct immunosuppressive activity that contributes to a BM environment permissive to metastatic colonization. Functional and genetic studies showed that tumor-associated systemic IGF1 reprogrammed BM neutrophils through IGF1R-MAPK/ERK-KLF4 signaling, with IL-1{beta}, reactive oxygen species and nitric oxide contributing to TAN-mediated T cell suppression. Tumor-cell Igf1 perturbation, IGF1R blockade and neutrophil-specific Igf1r deletion attenuated TAN-associated BM remodeling, and reduced subsequent bone metastatic progression. In bone-metastasis models, combined IGF1R and PD-1 blockade suppressed bone-metastatic progression. Translationally, our investigator-initiated clinical trial (NCT06866548) of teprotumumab plus tislelizumab in patients with established metastatic castration-resistant prostate cancer (mCRPC) provided initial safety data and exploratory clinical observations. Together, these findings identify IGF1R-dependent neutrophil reprogramming as a functionally important component of the evolving PCa bone metastatic microenvironment and provide a rationale for therapeutic targeting of IGF1R signaling together with immune checkpoint blockade.

Purpose: Metastatic pheochromocytoma and paraganglioma (PPGL) are rare neuroendocrine malignancies with limited systemic treatment options. Although inhibition of vascular endothelial growth factor (VEGF) signaling has demonstrated clinical benefit in metastatic PPGL, prospective data evaluating selective VEGF receptor inhibition remain limited. Axitinib is a potent and selective inhibitor of VEGFR-1, -2, and -3 with established activity in several solid tumors. We evaluated the efficacy and safety of axitinib in patients with progressive metastatic PPGL. Methods: Patients with progressive metastatic or unresectable PPGL were enrolled in a prospective, open-label, single-arm, phase II study (NCT03839498). Eligible patients had measurable disease according to RECIST version 1.1 and no prior treatment with a tyrosine kinase inhibitor. Axitinib was administered orally at an initial dose of 5 mg twice daily with protocol-defined dose modifications based on tolerability. The primary endpoints were objective response rate (ORR) and progression-free survival (PFS). Safety was assessed using the National Cancer Institute Common Terminology Criteria for Adverse Events (CTCAE). Results: Nineteen patients were enrolled, and 17 received study treatment. A confirmed partial response was achieved in 35.3% of treated patients. Median progression-free survival was 7.9 months (95% CI, 6.1-16.8 months), median duration of response was 7.4 months, median duration of treatment was 9.5 months, and median overall survival was 29 months. Hypertension, the most common treatment-related adverse event, occurring in 79% of patients, was effectively managed with antihypertensive therapy and dose modification. Other common treatment-related adverse events included fatigue, diarrhea, oral mucositis, and palmar-plantar erythrodysesthesia. No patient permanently discontinued treatment because of treatment-related toxicity. Conclusions: In patients with progressive metastatic PPGL, single-agent axitinib demonstrated clinically meaningful antitumor activity with a manageable safety profile. The observed efficacy compares favorably with sunitinib in this rare disease. The ability to maintain treatment despite predictable VEGF-associated toxicities suggests that axitinib represents a viable therapeutic option for patients with metastatic PPGL.

Head and neck squamous cell carcinoma (HNSCC) is characterized by poor survival and limited benefit from immune checkpoint blockade (ICB), underscoring the need to define targetable mechanisms of immune escape. In a prospective, patient-centered study, we investigated how tumor metabolic reprogramming impairs T cell function and drives ICB resistance. Targeted metabolomics identified two dominant and distinct tumor states: the Warburg phenotype and glutamine deprivation and revealed increased tumor-specific glutamine to glutamate turnover. Integrating spatial metabolic imaging with immunohistochemistry linked these states to immune-cold and immune-excluded tumor architectures. Metabolic states selectively reduced the expression of T cell chemokine receptors highly abundant and prognostic in HNSCC. Accordingly, T cell migration into tumor spheroids was reduced at lower glutamine concentrations. Interfering with glutamine metabolism by pharmacologic inhibition or genetic deletion of glutaminase-1 markedly increased tumor susceptibility to immune cell-mediated cytotoxicity in vitro and ex vivo and sensitized patient-derived tumor fragments (PDTFs) to ICB. This effect extended beyond glutamine restoration and involved disruption of glutathione-dependent ROS defense, impaired tumor proliferation, and increased MHC expression in tumor cells. Consistently, high expression of genes involved in glutamine and glutathione metabolism associated with reduced ICB response in patients. In contrast to HNSCC, other tumor entities including breast cancer show low glutaminase-1 expression. Accordingly, breast cancer PDTFs were not responsive to glutaminase-1 inhibition. Together, these data identify glutaminase-1 as a metabolic checkpoint driving immune escape in HNSCC, and support guided patient stratification for metabolism-targeted immunotherapy, similar to current precision approaches for mutation targeted drugs.

Purpose: Cyclin-dependent kinase 4/6 inhibitors in combination with endocrine therapy have become widely adopted as a first-line treatment approach for hormone receptor-positive (HR+)/human epidermal growth factor receptor 2-negative (HER2-) advanced breast cancer (ABC). Through a collaboration between Novartis and the US Food and Drug Administration, we integrated multimodal data from phase III MONALEESA trials to identify novel prognostic factors for HR+/HER2- ABC. Experimental Design: Baseline clinical and genomics data were collected from patients in the MONALEESA-2, -3, and -7 trials. Circulating tumor DNA (ctDNA) from plasma samples and gene expression in archival tumor biopsies were analyzed. Machine learning models were trained (n = 1,355) on progression-free survival (PFS) using multimodal features via fusion models. In a test set (n = 382), prognostic performances were evaluated using the Kaplan-Meier, Cox, and Weibull models. Results: The late fusion model achieved a prognostic performance (C-index: 0.727, 95% CI: 0.641-0.799) slightly outperforming early fusion (C-index: 0.718, 95% CI: 0.631-0.791) and single-modality models. Model-based risk stratification identified distinct low- and high-risk groups (PFS HR, 0.28; 95% CI: 0.20-0.38). Among the top 40 features for progression risk (18 clinical, 20 gene expression, and 2 ctDNA), these were associated with higher risk: liver involvement, higher histological grades, early relapse; lower STC2 and RBBP8 expressions and higher FBX05 expression; and higher tumor mutational burden and ctDNA purity. Conclusions: Integrating clinical and genomic modalities improved progression risk prediction compared with single-modality models, while highlighting salient features that inform the underlying biology and support hypothesis generation.

Purpose: A minority of patients with bladder cancer derive lasting benefit from immunotherapy, and current biomarkers capture only part of the biology. We evaluated whether tumor-cell MHC class II defines an immune state associated with outcome across disease stages and after PD-1/PD-L1 blockade. Experimental Design: We localized differential MHC-II activity to malignant epithelial cells by single-nucleus and single-cell RNA sequencing and confirmed HLA-DR expression by immunohistochemistry in 122 specimens. Chromatin profiling and IFN-{gamma} stimulation of cell lines and primary tumors supported derivation of an 11-gene tumor-cell program. Associations were tested in 434 patients with non-muscle-invasive disease, 82 receiving neoadjuvant pembrolizumab, and 288 receiving atezolizumab for metastatic disease. Results: Tumor-cell MHC-II was present in approximately one third of bladder cancers, and malignant cells accounted for the differential signal between MHC-II-high and MHC-II-low tumors. The program was inducible by IFN-{gamma} through JAK/STAT signaling. Within luminal non-muscle-invasive disease, MHC-II-low status was associated with progression (HR 3.31, 95% CI 1.37 to 7.99). In PURE-01, the 11-gene program was associated with pathological complete response (52% versus 24%, p = 0.018) and recurrence-free survival (p = 0.0054), retaining an independent association in models including tumor mutational burden and PD-L1. In bladder-primary metastatic disease, program-high status was associated with overall survival in unadjusted analysis (HR 0.62, 95% CI 0.44 to 0.89); the association did not extend to upper tract tumors (interaction p = 0.0018). Conclusions: Tumor-cell MHC-II defines an inducible state associated with outcomes across bladder cancer stages and after checkpoint blockade, supporting prospective evaluation by RNA and immunohistochemistry.

Immune checkpoint blockade (ICB) with ipilimumab and nivolumab is a front-line standard of care in patients with mesothelioma. However, only a minority of patients respond. The tyrosine receptor kinase AXL is a putative ICB resistance factor. We evaluated AXL and programmed death 1 (PD1) inhibition (AXL-PD1) with bemcentinib and pembrolizumab respectively, in a phase IIA clinical trial in patients with ICB naive-relapsed mesothelioma (MIST3, NCT03654833). The study met its primary endpoint with a 12-week disease control rate of 46.2%, 90% confidence interval 29.2-63.8%. Grade 3 or greater adverse events occurred in 53.9% of patients. Multiomic analyses identified tumour-intrinsic and tumour extrinsic features linked to response to AXL-PD1 inhibition. Responders exhibited greater NF2 loss, heightened immune response signalling, and epithelial mesenchymal transition, which was lower in biopsies obtained at the time of acquired drug resistance. Neutrophil- related transcriptional programmes were associated with response to AXL-PD1 inhibition. Neutrophil regulating erythroblast transformation specific (ETS) transcription factors SPI1, ETS2, FLI1 were differentially activated in responding tumours. Gut microbiota rheostat differed between responders and non-responders and positively correlated with neutrophil- associated transcription program, IRF3 and TLR2. In summary, our findings support a hypothesis linking gut microbiota composition, neutrophil- associated tumour biology and response to AXL-PD1 inhibition in mesothelioma.

Although MSI-H/dMMR colorectal cancers and the rarer subset with pathogenic POLE exonuclease domain mutations can benefit from immune checkpoint blockade, MSS colorectal cancer remains largely refractory, raising the question of whether other DNA damage response defects may also confer sensitivity to immunotherapy. Here, we performed a systematic in vivo screen of recurrently altered DNA damage response (DDR) genes to determine whether distinct repair defects modulate response to PD-1 blockade in MSS colorectal cancer. Using a genome-edited murine model in immunocompetent mice, we found that DDR perturbations produced heterogeneous outcomes, indicating that the immunologic consequences of DNA repair deficiency may be pathway-specific. Loss of Brca1, Brca2, Atm, and Fanca enhanced response to anti-PD-1 therapy, whereas Atr and Msh6 deficiency were associated with poor benefit. Brca1/2-deficient tumors showed the strongest regressions, supporting a link between core homologous recombination deficiency and increased immune sensitivity. In an independent cohort of colorectal cancer patients treated with ICB from the MSK-IMPACT Clinical Sequencing Cohort, BRCA1 alterations were associated with improved survival status, while FANCA alterations showed a similar trend. Because ATM is therapeutically targetable, we evaluated pharmacologic ATM inhibition in MSS colorectal cancer. Sustained ATM inhibition induced immune-related transcriptional programs in vitro and, when administered as a pre-treatment in vivo, enhanced the antitumor activity of PD-1 blockade. Together, these findings suggest that selected DDR alterations may represent candidate biomarkers of immunotherapy sensitivity, while ATM inhibition may provide a rational combination strategy to enhance PD-1 blockade in MSS colorectal cancer.