Cancer Discovery

Oncogenic KRAS mutations exhibit a striking tissue-restricted tropism, occurring with high frequency in pancreatic, colorectal, and lung adenocarcinomas while remaining rare in other lineages. The molecular basis for why these specific tissues are uniquely permissive to KRAS transformation, and how this context shapes therapeutic vulnerabilities, remains poorly defined. Here, we utilized CRISPR-mediated genome engineering to generate endogenous, conditional KRAS-mutant isogenic cell line models across three primary permissive lineages (lung, colon, and pancreas) and the non-permissive breast lineage. Integrated genome-wide CRISPR fitness screens and comparative transcriptome analyses revealed that KRAS-driven synthetic lethal (SL) dependencies are profoundly shaped by their tissue of origin. Strikingly, we observed minimal overlap in SL hits across lineages, with only three genes shared among the permissive lines, suggesting that the KRAS oncogene operates through divergent, context-specific genetic networks. Mechanistically, we show that KRAS activation induces a universal MYC-driven metabolic signature, but the specific machinery required to sustain this state is lineage-restricted. We identified a dependency on the diphthamide synthesis pathway to maintain translational fidelity amidst a KRAS-induced hyper-translational state. These findings demonstrate that even when driven by the same oncogene, tumors exhibit distinct regulatory landscapes and unique genetic vulnerabilities. Our results provide a framework for developing lineage-aware therapeutic strategies, moving beyond universal KRAS inhibition toward targeted interventions tailored to a tumors specific tissue context. SIGNIFICANCE STATEMENTWhile KRAS mutations drive a significant portion of human malignancies, their prevalence is strikingly restricted to specific lineages, namely pancreatic, colorectal, and lung tissues. This tissue-restricted tropism suggests that oncogenic KRAS does not operate in a vacuum but requires a permissive, tissue-specific molecular landscape to sustain tumorigenesis. By integrating comparative transcriptome analyses with functional genomics across four isogenic lineages, we demonstrate that KRAS synthetic lethal dependencies are not universal but are hardwired to the cell of origin. This work establishes a framework for tissue lineage-aware oncology, shifting treatment paradigms from targeting the KRAS mutation alone to targeting the specific genetic networks, defined by the tissue of origin, that sustain KRAS-driven growth.

Complex karyotype sarcomas (CKS) are heterogeneous mesenchymal malignancies that typically lack recurrent actionable oncogenic drivers and remain therapeutically challenging. Loss of ATRX is a recurrent feature of CKS and defines a particularly high-risk subgroup. ATRX loss is also associated with activation of the alternative lengthening of telomeres (ALT) pathway, and ALT-positive sarcomas have been linked to poor clinical outcomes. However, the molecular underpinnings underlying ALT-status-dependent differences in CKS, as well as the therapeutic vulnerabilities associated with ALT, remain poorly defined. By integrating C-circle-based ALT detection across 776 sarcoma samples with multi-modal sequencing of five CKS subtypes, we find that ALT activity is associated with enriched hallmarks of genomic instability. ALT-positive transcriptomes are dominated by a coordinated DNA damage response and mitotic program, in contrast to oncogenic signaling pathways that drive TERT activation in ALT-negative tumors. Long-read sequencing reveals telomere repeat clusters and telomere-mediated healing at structural breakpoints in ALT-positive tumors. These events also occur on extrachromosomal DNA (ecDNA), linking ALT activity to ecDNA biology. Together, our findings position ALT status as an important stratifying feature of CKS and identify ALT-associated transcriptional programs as potential therapeutic targets.

Reversible drug-tolerant persister states are emerging as key drivers of limited therapeutic durability, offering a complementary non-genetic perspective distinct from traditional models of acquired resistance. This is of particular interest in lung adenocarcinoma where EGFR tyrosine kinase inhibitors (TKIs) elicit dramatic responses, yet residual surviving cells persist and ultimately seed relapse. To define mechanisms that enable survival during this earliest residual-disease phase, we focused on the drug-tolerant persister population that remains after EGFR TKI exposure and can later give rise to outgrowth. Initial observations of elevated transcript levels of PRKCA, which encodes PKC, in established TKI-resistant models, together with markedly delayed tumor relapse following PKC suppression in vivo, nominated PKC as a candidate regulator of the persister-to-relapse transition. Genetic ablation of PRKCA or its inhibition with enzastaurin reduced residual survival and outgrowth after TKI exposure, indicating that PKC functions as an early dependency of drug-tolerant persisters rather than as a general mediator of acquired resistance. Mechanistically, PKC was required for persister-associated EMT, migratory capacity, and robust induction of ALDH1A1, the latter constraining oxidative stress and enhancing persister survival. Functionally, PKC was specifically necessary for survival of a rare, pre-existing CD44High stem-like subpopulation that exhibited marked plasticity and ultimately seeded persistence. Together, these data identify a PKC-dependent EMT/stemness/ROS pathway as a critical survival program in EGFR TKI-tolerant persister cells and support therapeutic strategies aimed at eliminating residual disease to prolong clinical responses.

Brain metastasis (BM) in renal cell carcinoma (RCC) remains poorly understood and often resistant to immune checkpoint inhibitors. We generated a large single-nucleus RNA-seq data of RCC BM, profiling 14 BM samples alongside matched extracranial metastases and primary tumors. Tumor cells in BM displayed neuronal infiltration, neural-like adaptation, and marked remodeling of the microenvironment, including expansion of immunosuppressive myeloid cells and depletion of antigen-presenting dendritic cells. Tumor, immune, and stromal cells exhibited metabolic rewiring characterized by fatty-acid metabolism, oxidative phosphorylation, and MYC-driven programs. CD8 T cells showed terminal exhaustion and impaired proliferative capacity, and tertiary lymphoid structures were absent. Spatial profiling of 12 BM samples (13,128 cells) validated key cellular interactions, while ligand-receptor analysis revealed immunoregulatory circuits between tumor, stromal, and immune cells. These findings define BM-specific adaptations that promote immune evasion and resistance, revealing therapeutic vulnerabilities in RCC BM. SIGNIFICANCESingle-nucleus RNA-sequencing profiling reveals tumor, immune, and metabolic adaptations in renal cell carcinoma (RCC) brain metastases, including neuroglial remodelling and immunosuppressive niche formation. These findings identify immune evasion mechanisms that could contribute to therapeutic resistance, providing new avenues for site-specific therapeutic interventions to improve treatment efficacy and outcomes in patients with RCC BM.

The epidemiological surge of early-onset colorectal cancer (EOCRC) is characterized by accelerated biological kinetics and disproportionately high rates of systemic relapse following curative-intent surgery. Because standard anatomical staging (TNM) lacks the resolution to accurately capture the intrinsic regenerative capacity of microscopic residual disease, we investigated the transcriptomic architecture of post-surgical failure in a strictly defined, curative-intent clinical pan-cohort. Unbiased transcriptomic profiling of the localized (M0) discovery sub-cohort identified IGF2 as the most significantly upregulated correlate of metachronous relapse. High-resolution isoform analysis revealed that this transcriptional output is predominantly driven by the embryonic (P4) and placental (P5) promoters. Systematic allele-specific expression (ASE) analysis supported widespread biallelic IGF2 expression consistent with relaxation of imprinting-domain control. This signal was not restricted to relapsing tumors, suggesting a recurrence-independent oncofetal baseline across the EOCRC spectrum. Because this foundational epigenetic unlocking is functionally insufficient on its own to execute systemic metastasis, we distilled the additional transcriptional plasticity required for dissemination into an internally derived and bootstrap-stabilized 5-gene recurrence-risk module Multivariable analysis across the combined pan-cohort supported an independent association between the high-risk module and systemic relapse (p < 0.001), capturing prognostic dimensions completely unresolved by classical pathological covariates and baseline staging. Ultimately, our findings reframe EOCRC aggressiveness as the product of a dual-hit architecture. This framework resolves the clinical paradox of widespread IGF2 LOI co-existing with heterogeneous outcomes, offering a biologically grounded basis for molecular risk stratification beyond anatomical boundaries.

Metastatic castration-resistant prostate cancer (mCRPC) remains lethal as adaptive resistance to standard-of-care therapy develops, often driven by AR splice variants alongside transcriptional and translational reprogramming. To identify strategies capable of overcoming these mechanisms, we performed an unbiased high-throughput screen of 2,480 mechanistically annotated compounds across advanced prostate cancer models. Exportin-1 (XPO1)-mediated nuclear export emerged as a critical dependency, and matrix-based combination screening uncovered robust synergy between inhibitors of XPO1 and the translation initiation factor EIF4A1. Dual inhibition induced coordinated disruption of oncogenic protein networks, including AR/AR-V7, triggering apoptosis and suppressing cell-cycle and metabolic programs. These effects extended to genetically diverse patient-derived organoids and in vivo xenografts at low doses, approximately 8-fold (Eltanexor) and 12-fold (Zotatifin) below established human single-agent regimens. Together, these findings reveal concurrent control of nuclear export and protein translation as a therapeutic vulnerability in mCRPC, providing a strong rationale for clinical evaluation of XPO1-EIF4A1 co-inhibition to overcome AR-driven resistance. STATEMENT OF SIGNIFICANCEUnbiased combinatorial screening reveals co-inhibition of nuclear export and translation initiation as a vulnerability in metastatic castration-resistant prostate cancer. Dual targeting of XPO1 and EIF4A1 drives synergistic collapse of oncogenic protein networks, including AR/AR-V7 signaling, to overcome key resistance mechanisms and induce potent antitumor responses across heterogeneous models. Notably, these effects are achieved at substantially reduced doses using clinically tractable agents, defining a mechanistically grounded therapeutic strategy poised for rapid clinical translation.

Tertiary lymphoid structures (TLS) predict benefit from immune checkpoint inhibitors (CPIs), yet mature, germinal-center-rich TLS are infrequent in solid tumors by histological review. Here, using 38-plex MIBI spatial proteomics across 165 lymphoid structures from 14 NSCLC resections, we establish a continuum of TLS maturity using high dimensional compositional, spatial, and molecular features. We demonstrate that histologically-defined lymphoid aggregates (LA) comprise a heterogeneous class of structures, which span this continuum of maturity. We identify a subset of lymphoid aggregates that harbor follicular dendritic cell networks, T follicular helper cells, and activated B cell states characteristic of mature TLS, yet are not readily distinguished from other LA structures in our histological review. We developed a novel digital pathology classifier to identify mature LAs in CPI trials, and demonstrate in a retrospective analysis of Atezolizumab in advanced NSCLC that the inclusion of mature LAs greatly expands the biomarker-eligible population while maintaining strong predicted benefit. Together, these data redefine the biological spectrum of tumor-associated lymphoid aggregates and provide a framework for implementing maturity-informed TLS biomarker strategies.

Centrosomes are key microtubule-organizing centers required for accurate spindle assembly and chromosome segregation, and their dysfunction in cancer creates therapeutic vulnerabilities. Prior work identified a synthetic lethal interaction between TRIM37 overexpression and Polo-like kinase 4 inhibition (PLK4i) in 17q23-amplified tumors, motivating the clinical development of centrosome-depleting PLK4 inhibitors. However, the broader determinants of sensitivity and resistance to PLK4 inhibition remain poorly defined. Using genome-wide CRISPR-Cas9 screening, we identify multiple genetic suppressors of sensitivity to centrosome depletion, including loss of PPP6C as a general escape mechanism, mediated by enhanced activation of Aurora kinase A (AURKA) on the spindle. This process requires NuMA, which scaffolds robust acentrosomal spindle assembly, and operates independently of the TRIM37-regulated pathway that restores pericentriolar material (PCM) foci to reconstitute microtubule-organizing center activity. We further show that centrosome depletion creates a dependence on the AURKA-TPX2 axis for spindle assembly, such that modulation of this pathway shapes cellular responses to PLK4 inhibition. Loss of PPP6C elevates AURKA activity and confers resistance, whereas disruption of the AURKA-TPX2 axis sensitizes cells to centrosome depletion. Together, these findings reveal how centrosome depletion rewires mitotic organization, rendering cells dependent on distinct adaptive spindle assembly pathways.

Long non-coding RNAs (lncRNAs) are increasingly recognised as effectors of oncogenic signalling, yet the transcriptional programmes through which driver mutations regulate lncRNA expression remain poorly defined. Here we identify LINC00941 as a direct transcriptional target of FOSL1, an AP-1 transcription factor downstream of the KRAS-MAPK pathway, establishing the first FOSL1-regulated lncRNA in lung adenocarcinoma (LUAD). LINC00941 is significantly upregulated in LUAD across multiple independent cohorts, and its depletion via siRNAs, shRNAs, and antisense oligonucleotides (ASOs) induces proliferative arrest and stress-induced premature senescence, accompanied by transcriptomic suppression of cell cycle and DNA damage response (DDR) genes. Mechanistically, LINC00941 operates through a dual-compartment mechanism engaging two STAR-family RNA-binding proteins in distinct subcellular contexts. In the nucleus, LINC00941 binds SAM68 through its 700-1300 nucleotide region and shields it from proteasomemediated degradation, thereby sustaining SAM68-dependent PARP1 activation and DDR competency; RNF123 is identified as a candidate E3 ligase mediating SAM68 turnover in the absence of LINC00941. In the cytoplasm, LINC00941 sequesters QKI, preventing its nuclear translocation; LINC00941 depletion releases QKI to the nucleus, driving alternative splicing dysregulation including validated NUMB exon 12 exclusion, and QKI co-depletion rescues the anti-proliferative phenotype both in vitro and in xenograft models. Multi-cohort survival analysis across three independent LUAD datasets (n=649) identifies LINC00941 as an independent prognostic factor for poor overall survival. Gymnotic ASO-mediated targeting of LINC00941 significantly suppresses xenograft tumour growth without systemic toxicity, providing preclinical proof-of-concept for therapeutic tractability. Together, these findings establish LINC00941 as a compartment-specific oncogenic scaffold within the KRAS-FOSL1 transcriptional axis and a tractable therapeutic target in LUAD.

Cancer cachexia is a wasting syndrome that remodels the anatomy of the patient. How this remodeling unfolds across tissues, whether it defines distinct disease states, and how these states relate to underlying biology remain unknown. We used longitudinal computed tomography imaging from 4,516 patients to quantify evolution of muscle, adipose, and organs during cachexia. Across two independent institutional cohorts, unsupervised analysis identified three reproducible anatomical subtypes of cachexia, including an inflammatory Type A marked by progressive hepatosplenic enlargement and inferior survival, a Type B dominated by visceral organ atrophy, and a mild Type C. These anatomical subtypes were associated with distinct serological signatures and reflected in molecular phenotypes in tumors and non-cancerous liver tissue, establishing cachexia as discrete anatomical disease states that link whole-body remodeling to systemic and tissue-level biology. This anatomy-first framework for cachexia classification provides a foundation for future patient stratification and development of subtype-specific anti-cachexia therapies.

Clear cell renal cell carcinoma (ccRCC) progresses along two predominant evolutionary trajectories, defined by PBRM1 ([~]40%) or BAP1 ([~]15%) mutations on a VHL-inactivated background. They have distinct patterns of evolutionary tempo and mode, and vastly different clinical outcomes, yet the underlying genotype-specific molecular phenotypic programmes are unknown. We established a patient-derived preclinical model biobank that captures the genetic diversity of ccRCC. Through integrative analyses of preclinical models and tumour bulk and single cell profiling, we identified transcriptional and epigenetic changes specific to PBRM1- and BAP1-driven ccRCC. Modelling PBRM1 loss in vitro demonstrates that it reinforces renal lineage identity and maintains progenitor-like cell state. In contrast, BAP1 loss drives inflammatory signalling and chromosomal instability. These insights reconcile the distinct evolutionary modes (branched versus punctuated), tempo (slow versus fast) and clinical outcomes associated with PBRM1 and BAP1 mutations, respectively, establishing a framework for patient stratification and genotype-directed therapeutic development.

Prostate cancer displays substantial clinical and histopathological heterogeneity which is not fully captured by conventional Gleason grading. To resolve the spatial and phenotypic complexity of the prostate tumor microenvironment, we performed imaging mass cytometry using a prostate-tailored 34-plex antibody panel on a clinically annotated tissue microarray cohort of 195 patients of primary stage disease after radical prostatectomy (523 regions of interest; 2.19 million cells). We identified 34 distinct cell types spanning epithelial, endothelial, stromal and immune compartments, and further organized into 18 epithelial-dominated, cancer associated fibroblast-dominated, and immune-rich spatial niches. Within the epithelial compartment, we detected an ERGp53 luminal population whose abundance is independently associated with poor overall and progression-free survival. In the stroma, we defined extracellular matrix remodeling-related cancer associated fibroblast and smooth muscle cell lineages, including a periglandular CD105high niche with strong stromal-immune connectivity that is selectively associated with worse clinical outcome. Finally, cumulative immune niche burden correlated with histological inflammation and stratifies for worse patient survival. Together, these data provide a spatially resolved single-cell atlas of primary PCa and reveal stromal-immune-epithelial niches with prognostic relevance beyond Gleason grade.

Ductal carcinoma in situ (DCIS) exhibits substantial heterogeneity in its risk of progression to invasive breast cancer, yet the cellular and molecular determinants of high-risk lesions remain incompletely defined. Using spatially resolved single-cell transcriptomic and epigenomic profiling of 43 patient-derived DCIS and DCIS/invasive ductal carcinoma (IDC) samples, we delineate cellular programs, spatial organization, and epigenetic regulatory mechanisms associated with invasive potential. We identify an epithelial population with stemness features within luminal hormone-responsive (LumHR) cells that progressively expands from benign tissue to DCIS and IDC, and is strongly associated with invasive progression and recurrence-linked transcriptional programs. Spatial mapping reveals discrete DCIS niches enriched for stem-like LumHR cells, characterized by elevated CEACAM6 expression and enhanced ligand-receptor interactions, including CEACAM6-EGFR signaling between epithelial and stromal compartments, including cancer-associated fibroblasts, macrophages (APOC1-positive) and perivascular cells. These niches define a microenvironmental context that supports stemness and invasive potential. Epigenomic analyses implicate FOXA1 as a key regulator of these stem-like transcriptional states. Pharmacologic disruption of FOXA1-regulatory network using LSD1 inhibition suppresses stemness-associated transcriptional programs in vitro and significantly restrains tumor growth in vivo. Collectively, these findings define high-risk DCIS as a stemness-driven disease embedded within specialized microenvironments, and identify associated regulatory networks as candidate biomarkers and therapeutic vulnerabilities.

An inflammatory bone marrow microenvironment is increasingly recognized as critical in myeloid disease evolution, yet how stromal inflammation interfaces with adaptive immunity remains poorly defined. Here, we show that stromal pyroptosis drives mutation-specific myeloid expansion by coordinating monocytic remodeling and CD4 T-cell activation. Genetic ablation of gasdermin D in the bone marrow stroma suppressed stromal pyroptosis and attenuated Tet2-deficient myeloid expansion. Tet2 deficiency skewed monocyte and macrophage differentiation toward an activated, antigen-presenting state that interacted with pyroptotic stromal cells to promote expansion of a distinct CD4 T-cell population. These cells expressed canonical T follicular helper markers (Bcl6, Cxcr5, Il21, and Cd40l) together with interferon-responsive and tissue-interaction programs, consistent with an inflammation-adapted TFH-like state. CD40L produced by these cells reinforced the expansion of Tet2-deficient monocytes and macrophages, establishing a feed-forward stromal-immune circuit. Disruption of this axis through stromal gasdermin D deficiency or CD40L blockade attenuated myeloid expansion in vivo. Consistent with these findings, patients with isolated TET2 loss-of-function mutations exhibited CD4 T-cell skewing and CD40L+ T-cell-rich tertiary lymphoid structures in the bone marrow. Together, these data identify a pyroptosis-dependent stromal-immune axis that links early myeloid inflammation to maladaptive remodeling of adaptive immunity and reveals a context-dependent therapeutic vulnerability in Tet2-deficient hematopoiesis.

Conventional short-read sequencing cannot determine whether co-occurring variants within a cancer gene reside on the same allele (cis) or on opposing alleles (trans), a distinction with direct biological and therapeutic consequences. Trans configurations confirm biallelic tumor suppressor inactivation and inform therapy selection, while cis configurations generate compound oncogenic alleles with enhanced activity. We analyzed 768 patients with prostate, breast, or ovarian cancers in the PROBLEM cohort, using mutational signatures to nominate cryptic genomic instability cases where the causative biallelic event was not apparent from short-read sequencing. Long-read nanopore sequencing resolved 32 of 46 cryptic cases (69.6%), leveraging its unique advantages in direct methylation detection, long insertion resolution, and complex structural variant characterization, confirming trans biallelic inactivation in all resolved tumor suppressor cases. Systematic analysis of 4,496 MiOncoSeq samples identified 17,519 multi-hit gene pairs, of which 78.7% exceeded the 500 bp short-read phasing limit. Long-read phasing further revealed recurrent compound cis oncogenic alleles in NOTCH1, PIK3CA, PDGFRB, and KIT with functionally synergistic activity. Haplotype phasing resolves a systematically overlooked gap in cancer variant interpretation and warrants broader integration into precision oncology workflows. Statement of SignificanceShort-read sequencing cannot resolve whether co-occurring variants within a cancer gene are cis or trans, a distinction critical for clinical interpretation. Long-read nanopore sequencing addresses this gap through direct haplotype phasing, methylation detection, and complex structural variant resolution, confirming biallelic tumor suppressor inactivation and revealing compound cis oncogenic alleles with enhanced activity.

Neoadjuvant chemoradiotherapy (CRT) is standard for locally advanced rectal cancer (LARC), yet many patients retain residual disease. To resolve CRT-associated remodeling of the tumor microenvironment, we generated a multimodal spatial atlas from serial sections of paired pretreatment and post-treatment specimens from 24 patients using Xenium single-cell spatial transcriptomics and PhenoCycler multiplex proteomics, profiling 2.8 million cells; matched Visium HD datasets were generated on adjacent serial sections. Resistance was most strongly associated with fibroblast and myeloid programs adjacent to residual tumor. We identify a periostin (POSTN)-expressing CAF subset selectively enriched around residual tumor cells in non-responders, displaying a myofibroblastic phenotype and activating extracellular matrix remodeling, noncanonical WNT signaling, and immunosuppressive pathways. Tumor cells neighboring POSTN+ CAFs show consistent epithelial-mesenchymal transition signatures. Together, this atlas enables interrogation of CRT-induced spatial remodeling and nominates POSTN+ CAFs as key mediators and targets of CRT resistance, with direct relevance to CRT-based combination strategies.

Chemotherapy and radiation reduce tumor burden but leave behind residual cells that survive via therapy-induced senescence (TIS). These cells constitute a latent reservoir fueling recurrence, yet strategies for their selective elimination are lacking. Here, we identify lysosomal ferrous iron accumulation as a conserved hallmark and actionable vulnerability of TIS tumor cells. Across diverse models, senescent tumor cells exhibit marked hypersensitivity to ferroptosis induction. In breast cancer PDX models, sequential ferroptosis induction following chemotherapy significantly delays recurrence, while dual inhibition of GPX4 and FSP1 produces durable, often complete, eradication of residual tumors without overt toxicity. Mechanistically, activation of the TFEB-HO-1 axis in TIS tumor cells drives ferrous iron accumulation, thereby priming cells for ferroptosis. Together, these findings establish ferrous iron accumulation as a defining feature of TIS and position ferroptosis induction as a potent senolytic strategy to eliminate therapy-refractory residual disease. Statement of significanceSenescent tumor cells remaining after treatment can drive cancer recurrence yet remain poorly understood and therapeutically intractable. Here, we identify lysosomal ferrous iron accumulation as a universal hallmark of therapy-induced senescence and demonstrate that ferroptosis induction functions as an effective senolytic strategy. Our findings provide mechanistic and translational support for the "one-two punch" therapeutic paradigm.

The DNA damage response (DDR) is critical for pancreatic ductal adenocarcinoma (PDAC) development and therapeutic responses, including to genotoxic agents. While epigenetic modulators have been shown to contribute to the DDR, how chromatin regulation dictates responses to DNA damage in PDAC remains incompletely understood. Here, we identify Class I histone deacetylases (HDACs) as critical regulators of the DDR. HDAC1/2 directs the genomic distribution of H3K27ac, ensuring sufficient BRD4 and RNA polymerase II (Pol II) occupancy at DDR gene promoters. HDAC inhibition by entinostat shifts the balance of H3K27 acetylation preferentially towards intergenic regions, diverting BRD4 and Pol II from promoters, thereby suppressing DDR gene expression. In line with this, HDAC inhibition heightens DNA damage and sensitizes PDAC to diverse DNA-damaging and DDR-targeting agents. Since the clinical development of HDAC inhibitors has been limited by systemic toxicity, we developed bottlebrush prodrug (BPD) nanoparticles for tumor-selective entinostat delivery. Entinostat-BPD achieved tumor-specific HDAC inhibition while displaying potent efficacy and reduced systemic toxicity. These findings reveal an HDAC-dependent DDR vulnerability and offer combinational and precision targeting strategies to facilitate clinical translation and improve PDAC patient outcomes. SIGNIFICANCE STATEMENTThe ability of tumor cells to tolerate DNA damage limits the efficacy of many anticancer therapies. Our study reveals that pancreatic cancer cells enforce this resistance by sustaining expression of DNA damage response (DDR) genes through Class I histone deacetylases (HDACs). HDACs maintain genome-wide acetylation patterns required for efficient recruitment of the transcriptional machinery to DDR genes. Pharmacological HDAC inhibition disrupts this process and sensitizes pancreatic cancer cells to diverse DNA-damaging agents. To overcome systemic toxicity that limits translational potential, we further establish a bottlebrush prodrug nanoparticle platform that enables tumor-selective HDAC inhibition. Given the central role of the DDR in cancer, targeting HDAC-mediated DDR regulation through drug combinations and precision delivery may have broad therapeutic relevance across cancer types.

Circadian dysregulation is increasingly linked to prostate cancer (PCa) progression, yet its role in directing DNA damage response (DDR) pathway selection remains poorly understood. Here, we identify circadian cryptochrome 1 (CRY1), a core circadian regulator, as a stage-specific determinant of DDR dependencies. Integrated transcriptomic and CRISPR-based analyses reveal that CRY1 promotes non-homologous end joining (NHEJ) and base excision repair (BER)-associated programs in hormone-sensitive disease (HTS), while driving a switch toward homologous recombination (HR) dependency in castration-resistant prostate cancer (CRPC). Mechanistically, CRY1 couples proliferative signaling to genome maintenance, enabling tumor cells to tolerate genotoxic stress and sustain progression. Notably, loss of CRY1 exposes distinct, context-dependent DDR vulnerabilities, revealing repair plasticity as a targetable actionable feature of disease evolution. These findings position CRY1 as a central regulator of DDR rewiring and support CRY1-directed combination strategies with DDR inhibitors as a rationale to delay or prevent progression to advanced, treatment-resistant PCa.

Clinical poly (ADP)-ribose polymerase (PARP) inhibitors (PARPi) are limited by toxicities associated with inhibition of multiple PARP family proteins and acquired resistance. As PARP1-specific inhibitors, like saruparib (AZD5305), move toward standard-of-care status for BRCA and HR-deficient cancers replacing less specific PARPi, defining mechanisms of intrinsic and acquired resistance is essential for developing effective treatment strategies. Here, we established 5 saruparib-resistant (SR) cell lines from BRCA1-deficient MDA-MB-436 triple negative breast cancer (TNBC) cells using a selection strategy of high-level dosing consistent with clinical exposure, yielding models that are >1,000-fold resistant to saruparib. Whole genome sequencing identified PARP1 catalytic domain mutations in all SR cell lines, and in vitro reconstitution of these PARP1 mutants confirmed them as drivers of saruparib resistance, in contrast to HR restoration as observed in the case of less-selective PARPi. PARP1 mutations also induce altered saruparib-dependent PARP1 trapping and PARylation inhibition. While these mutations render cells highly resistant to saruparib, differential sensitivity to other PARPi was observed and SR cell lines retain, and in some cases, increase, sensitivity to alternative clinical PARPi and DNA damage response (DDR)-targeted therapeutics. Our findings demonstrate that high-intensity selection pressure favors target-site mutation over pathway restoration as a primary escape mechanism from PARP1-selective inhibition. This study provides a first-in-class characterization of saruparib resistance and maps a clear therapeutic path forward. By identifying these specific PARP1 mutations and their collateral DDR vulnerabilities, we provide the molecular framework necessary to monitor and treat patients who progress on next-generation PARP1-selective inhibitors.