Abstract The tumor microenvironment exposes cancer cells to mechanical, thermal, hypoxic, and acidic stresses, yet how cells integrate these signals to remodel RNA processing remains poorly understood. Here, we show that ribosomal RNA Processing 1B (RRP1B), previously characterized as a nucleolar ribosome biogenesis factor and metastasis modifier, functions as a broad-spectrum stress sensor that dynamically repositions among the nuclear envelope, nucleolus, and nuclear speckles (NS). Relocalization is governed by multi-site phosphorylation within intrinsically disordered regions (IDRs): phosphomimetic substitutions promote NS-proximal condensate formation in an RNA-dependent manner, while unphosphorylatable substitutions confine RRP1B to the nucleolus. Under stress, the RRP1B interactome shifts globally, with ribosomal processing partners replaced by pre-mRNA splicing components enriched for NS-resident proteins. RNA immunoprecipitation sequencing (RIP-seq) demonstrates that under basal conditions RRP1B associates with long, intron-rich, nuclear periphery-proximal transcripts, whereas heat shock redirects binding toward shorter, exon-dense transcripts enriched for motifs of serine/arginine-rich (SR) proteins near the NS. RRP1B overexpression nearly abolishes cytoplasmic retained intron accumulation and drives preferential export of specific transcript isoforms in a compartment- and temperature-dependent manner, establishing RRP1B as a regulator of RNA localization fidelity rather than transcriptional output. An RRP1B overexpression signature is most highly activated in basal-like and claudin-low breast tumors, and the RRP1B-associated retained intron splicing program correlates with reduced survival in a tumor-grade-dependent manner. These findings reframe RRP1B as a microenvironmentally sensitive regulator of nuclear RNA processing with direct implications for aggressive breast cancer biology.

Cancer stem cells (CSCs) drive metastasis and therapy resistance, yet their behaviour within the complex tumour microenvironment remains poorly understood. Here we use a fluorescent reporter that marks CSCs to show that CSCs and their more differentiated progeny display strikingly different population dynamics during metastatic lung colonization in breast cancer models. CSC expansion is rapidly curtailed early in colonization, suggesting a strong negative feedback mechanism acting selectively on this subpopulation. We showed that CSCs are exceptionally sensitive to local microenvironmental cues such as cell crowding and nutrient availability. They respond earlier and more extensively than their differentiated progeny, thereby coupling tumour growth to resource and space availability. Microenvironmental signals converge on the transcriptional regulatory complex YAP/TAZ/TEAD, with CSC sensitivity arising from elevated signal reception and greater chromatin accessibility at TEAD-regulated enhancers. Targeting upstream inputs to this pathway reversed chemotherapy-induced CSC enrichment in lung metastases, suggesting a potential therapeutic strategy.

Stromal immunosuppressive pathways are key modulators of response to immune checkpoint inhibitors, but the tumor-intrinsic consequences of blocking these pathways remain incompletely defined. We conducted a clinical trial of bintrafusp alfa, a bifunctional PD-L1/TGFβ inhibitor, in small cell lung cancer (SCLC). Among 34 evaluable patients, 18% had partial responses, 20% stable disease, and 62% progressive disease; 38% of progressors met the criteria for hyperprogressive disease (HPD). HPD was also observed across other tumor types (n = 450), in higher frequencies with bintrafusp alfa than PD-(L)1 blockade alone. Blood and tumor profiling showed that HPD correlated with systemic immune suppression and elevated TGFβ signaling. Functional studies demonstrated that tumor-intrinsic TGFβ signaling restrains proliferation in a subset of SCLC; pathway blockade triggers hyperproliferation. External validation across cell lines and tumor samples confirmed a tumor-intrinsic TGFβ-high transcriptional state associated with inferior survival. These findings identify a context-dependent, growth-constraining function of TGFβ and support tumor-intrinsic biomarker guidance while targeting stromal immunosuppressive pathways. SIGNIFICANCE:This study identifies tumor-intrinsic TGFβ signaling as a context-dependent growth restraint in SCLC and a driver of HPD following TGFβ blockade. A reproducible TGFβ-high mesenchymal state is linked to inferior survival, supporting biomarker-guided use of TGFβ-targeted immunotherapy.

Clinical Trial Protocol, IRB amendments and DSMB recommendations

Interferon (IFN) signaling plays a pivotal role in orchestrating antitumor immunity and shaping the response to immune checkpoint blockade (ICB). Although genetic alterations that impair the IFN pathway have been reported, such events are relatively rare, suggesting a potential contribution of epigenetic dysregulation. Here, we identified a RUNX2-mediated epigenetic mechanism that disrupts the type I interferon (IFN-I) signaling pathway in osteosarcoma (OS), thereby limiting the efficacy of ICB. Development of an algorithm to assess the association of 1,425 transcription factors with IFN pathway activation in human OS tumors enabled identification of RUNX2 as a potential negative regulator of IFN signaling. RUNX2 depletion in OS cells activated the IFNB1-driven IFN-I response. Mechanistically, RUNX2 formed a transcriptional repressor complex with NCOR1 and HDAC3 that reduced H3K9 acetylation at the enhancers of key IFN-I genes, leading to their downregulation. Inhibition of the RUNX2-NCOR1-HDAC3 complex enhanced IFN-I signaling, with cGAS, STING, and IFNB1 being required for the induction of interferon-stimulated genes and tumor suppression. Paradoxically, reactivation of IFN-I signaling also upregulated immune checkpoint molecules PD-L1 and PD-L2. Combination treatment with a selective HDAC3 inhibitor and anti-PD-1 antibody led to durable tumor regression in syngeneic OS mouse models, accompanied by increased cytotoxic T cell infiltration. These findings reveal a mechanistic link between RUNX2-driven epigenetic repression and impaired antitumor immunity via the cGAS-STING-IFN-I axis and suggest a rational combinatorial strategy to overcome OS resistance to ICB.

Trial Schema; Gating strategy; KEY RESOURCES table

Protein and RNA acetylation are crucial for development and cancer progression. NAT10 is the only known acetyltransferase responsible for N4-acetylcytidine (ac4C) modification of RNA. However, the mechanism by which NAT10 contributes to cancer progression remains unclear. Here, we show that NAT10 interacts with a mechanosensitive, metastasis-susceptibility protein complex at the nuclear pore. Loss of NAT10's acetylation activity significantly reduces lung metastasis in both allograft and genetically engineered mouse models of breast cancer. Unexpectedly, upon NAT10 knockout, loss of ac4C modification in chromatin-associated tRNAs disrupts p300/CBP function, resulting in genome-wide chromatin reorganization and altered expression of genes that recruit metastasis-promoting myeloid cells to the tumor microenvironment. These findings highlight a role for NAT10 in regulating enhancer activity in metastatic tumor cells and reveal its impact on tumor-immune interactions that contribute to metastatic progression.

Metastasis remains a major cause of cancer mortality. This study, expanding upon previous findings in the MMTV-PyMT model, investigated four independent mouse models, representing luminal (MMTV-PyMT, MMTV-Myc), HER2-amplified (MMTV-Her2), and triple-negative (C3(1)TAg) breast cancer subtypes. Consistent with previous results, limited evidence for metastasis-associated somatic point mutations was found for all models. We also found that oncogenic drivers significantly influenced the number and size of metastasis-specific copy number variations (MSCNVs), but common driver-independent MSCNVs were rare. Furthermore, analyzing a cohort with varying genetic backgrounds while maintaining a constant oncogenic driver (PyMT) revealed that genetic background profoundly impacts MSCNVs. Transcriptome analysis demonstrated that oncogenic drivers strongly shaped metastasis-specific gene expression (MSGE), with each driver exhibiting distinct expression profiles. In contrast, MSGE in the PyMT-F1 cohort was more variable across strains. Despite the diversity of MSCNV and MSGE, functional analysis revealed that both mechanisms converge on the modulation of key cellular processes, including immune responses, metabolism, and extracellular matrix interactions. These findings emphasize the complex interplay between oncogenic drivers and genetic background in shaping the genomic and transcriptional landscapes of metastatic lesions.

This is supplementary Table 2.

Clonal evolution of cancer results in intratumor heterogeneity (ITH), making treatment and cure challenging. Single-cell sequencing has advanced our understanding of ITH, but tracing subclonal evolution using mutational profiles of cells is limited by scale and noise. Moreover, available tumor progression tree inference methods usually offer a single tree to explain the progression of a tumor, and do not inform about alternative evolutionary scenarios. In this paper we introduce the partition function for a tumor progression tree, to assess the reliability of any proposed subclonal structure in a single-cell sequenced tumor. Given that tumor progression can be represented as a perfect phylogeny, the partition function for a given set of cells, R, and a given mutation, ρ , is defined as the likelihood that ρ is present exclusively in R. As such, the partition function evaluates whether R forms a subclone with ρ as a possible subclonal driver, which is especially useful if the cells of R are biologically or clinically significant, e.g., have aggressive growth, therapy resistance, or metastatic potential. We also present an algorithm to estimate the partition function and provide theoretical proofs that our algorithm asymptotically approaches the ground truth. In addition, we present empirical evidence of its accuracy on simulated data, as well as its practical value in analyzing the progression history of real tumors. The implementation is available at: https://github.com/algo-cancer/Partition-Function/ .

Tumor dissemination is increasingly recognized to begin early in tumor development. Although most of these early disseminated cells are cleared, some survive and persist below clinical detection, acting as reservoirs for metastatic relapse. Metastatic tumor cells often rely on interactions with local stromal cells to support their colonization. In this study, we propose that pericyte-tumor cell interactions promote dormancy induction in the early metastatic lung, enhancing disseminated tumor cell (DTC) persistence. Extravital imaging demonstrated that DTCs interact with pericytes upon extravasation into the lung. Co-culture experiments were used to assess DTC fate after pericyte contact and revealed that transient contact with pericytes reduced the proliferation of metastatic 4T1 breast cancer cells but had no effect on non-metastatic 67NR cells. In vivo , transient pericyte contact resulted in higher lung metastatic burden, driven by small, non-proliferative lesions (<6 cells), 10 days after intracardiac injection. These lesions exhibited reduced KI67 staining and EdU incorporation compared to those from monocultured cells. We further observed that primary lung pericytes transferred lyso-phospholipids (lyso-PLs) specifically to metastatic 4T1 cells through direct contact. Gene expression analysis indicated that transient pericyte contact activated pathways related to syncytium formation in metastatic cells. In normal physiology, pericytes act in a syncytium to regulate blood flow via mechanosensitive channels in response to blood pressure changes. We hypothesize that tumor cells exploit these mechanosensitive responses to trigger lyso-PL transfer from pericytes. Supporting this, calcium imaging showed higher calcium activity in pericytes co-cultured with 4T1 cells, and calcium channel inhibitors significantly reduced lyso-PL transfer. Pharmacological activation of pericyte calcium channels induced lyso-PL release, which was subsequently taken up by tumor cells. Conditioned medium from activated pericytes, containing free lyso-PLs, recapitulated the reduced proliferation observed in transient co-culture. Finally, we found our pericyte-induced dormancy signature to be associated with tumor dormancy and distant metastasis free survival latency in breast cancer patients. Together, these findings suggest that early DTCs may exploit pericyte signaling mechanisms to enter dormancy, facilitating their persistence at metastatic sites and contributing to future relapse.

This file contains supplementary figure 1-9.

Cancer stem cells (CSCs) are key drivers of metastasis and therapy resistance but have been challenging to visualize and study in situ . Using a fluorescent CSC reporter, we observed very different population dynamics for CSCs and nonCSCs during metastatic lung colonization in breast cancer models. CSC expansive self-renewal drives early lesion formation before switching to a maintenance mode of balanced self-renewal and differentiation, whereupon nonCSC proliferation takes over as the main driver of metastatic expansion. Mechanistic analyses showed that CSCs are hyper-responsive to microenvironmental cues such as cell crowding and nutrient availability, suggesting a novel role for CSCs as sensors and early responders to fluctuating local conditions in the tumor. Incoming signals converge on YAP/TAZ/TEAD, with heightened CSC sensitivity and response supported by elevated receptor expression and increased chromatin accessibility around enhancers with TEAD binding sites. Targeting inputs to the YAP/TAZ/TEAD node reversed chemotherapy-induced enrichment of CSCs in lung metastases. Highlights:Different population dynamics for breast cancer stem cells (CSCs) and their differentiated progeny in early metastatic colonizationCSCs are hyper-responsive to microenvironmental cues and serve as sensors of local conditions for the tumorMany microenvironmental inputs converge on YAP/TAZ to regulate self-renewal vs differentiation fate decisions in the CSCTargeting YAP/TAZ input pathways blocks chemotherapy-induced enrichment of CSCs.

Trichothiodystrophy (TTD), a rare, autosomal recessive, multisystem developmental disorder, is characterized by short, brittle hair with "tiger-tailed banding" on polarized microscopy. TTD is caused by variations in 10 genes: 3 nucleotide excision repair/basal transcription factor IIH genes, 4 amino acid charging transfer RNA genes, basal transcription factor IIE, RNF113A, and an RNA-splicing gene (MPLKIP/TTDN1). We performed whole-exome sequencing to identify a candidate gene in Sabinas brittle hair syndrome, a mild form of TTD. We report 5 nonphotosensitive adult patients from 3 unrelated families with a homozygous missense variation in DBR1 (p.D262Y) encoding the RNA lariat-debranching enzyme DBR1, which is involved in the removal of introns from pre-mRNA in the nucleus. Post-UV DNA cell survival was normal, indicating that DBR1 was not involved in nucleotide excision repair/transcription factor IIH. There were reduced levels of DBR1 mRNA and protein. Interacting TTDN1 protein in cells from patients with DBR1 variations was markedly reduced. Genetic analysis suggests an ancient origin of this variation. Thus, Sabinas syndrome is caused by DBR1 variations, further indicating that TTD is a disorder of RNA metabolism (RNAopathy).

Patients with ER-negative breast cancer have the worst prognosis of all breast cancer subtypes, often experiencing rapid recurrence or progression to metastatic disease shortly after diagnosis. Given that metastasis is the primary cause of mortality in most solid tumors, understanding metastatic biology is crucial for effective intervention. Using a mouse systems genetics approach, we previously identified 12 genes associated with metastatic susceptibility. Here, we extend those studies to identify Resf1, a poorly characterized gene, as a novel metastasis susceptibility gene in ER- breast cancer. Resf1 is a large, unstructured protein with an evolutionarily conserved intron-exon structure, but with poor amino acid conservation. CRISPR or gene trap mouse models crossed to the Polyoma Middle-T antigen genetically engineered mouse model (MMTV-PyMT) demonstrated that reduction of Resf1 resulted in a significant increase in tumor growth, a shortened overall survival time, and increased incidence and number of lung metastases, consistent with patient data. Furthermore, an analysis of matched tail and primary tissues revealed loss of the wildtype copy in tumor tissue, consistent with Resf1 being a tumor suppressor. Mechanistic analysis revealed a potential role of Resf1 in transcriptional control through association with compound G4 quadruplexes in expressed sequences, particularly those associated with ribosomal biogenesis. These results suggest that loss of Resf1 enhances tumor progression in ER- breast cancer through multiple alterations in both transcriptional and translational control.

Despite the promising results of immune checkpoint blockade (ICB) therapy, outcomes for patients with brain metastasis (BrM) remain poor. Identifying resistance mechanisms has been hindered by limited access to patient samples and relevant preclinical models. Here, we developed two mouse melanoma BrM models that recapitulate the disparate responses to ICB seen in patients. We demonstrate that these models capture the cellular and molecular complexity of human disease and reveal key factors shaping the tumor microenvironment and influencing ICB response. BR1-responsive tumor cells express inflammatory programs that polarize microglia into reactive states, eliciting robust T cell recruitment. In contrast, BR3-resistant melanoma cells are enriched in neurological programs and exploit tolerance mechanisms to maintain microglia homeostasis and limit T cell infiltration. In humans, BR1 and BR3 expression signatures correlate positively or negatively with T cell infiltration and BrM patient outcomes, respectively. Our study provides clinically relevant models and uncovers mechanistic insights into BrM ICB responses, offering potential biomarkers and therapeutic targets to improve therapy efficacy.

Abstract Background: Small cell lung cancer (SCLC) is the most aggressive type of lung cancer. Current therapies have limited efficacy in SCLC, and despite a highly mutated genome, SCLC is largely unresponsive to immunotherapy. We sought to leverage the immune surveillance triggered by DNA damage in patients with relapsed SCLC using bintrafusp alfa, a bifunctional fusion protein targeting both PD-L1 and TGF-beta. Hyperprogressive disease (HPD), characterized by the rapid acceleration of tumor growth, has been reported in approximately 13% of patients treated with immune checkpoint inhibitors. However, the mechanistic basis is poorly understood. Methods: This is a safety run-in and phase II clinical trial. Cohort 1 enrolled patients with relapsed SCLC across Arms A (bintrafusp alfa 2400 mg q3 wks), B (bintrafusp alfa 2400 mg on D1 plus topotecan 1 mg/m2/day on D1-5 q3 wks), and C (bintrafusp alfa 1200 mg on D1 q2 wks plus temozolomide 200 mg/m2/day on D1-5 q4 wks). The primary endpoint was objective response rate (ORR). Results: Thirty-seven patients enrolled. Grade 3 treatment-related adverse events (TRAE) included transaminitis (27.0%), anemia (24.4%), lymphopenia (10.8%), and maculopapular rash (5.4%). Grade 4 TRAE included thrombocytopenia (8.1%), lymphopenia (5.4%) and transaminitis (5.4%). One patient experienced grade 5 tumor hemorrhage. By RECIST 1.1, partial responses were observed in 5 (13.5%), stable disease in 11 (29.7%), and progressive disease in 21 (56.8%) patients. Importantly, 13 (35.1%) patients developed HPD (≥ 2x increase in tumor growth rate from pre-trial vs on-trial, time to treatment failure ≤ 2 months, and ≥ 50% increase tumor burden). Changes in circulating cell-free DNA (n=20) tumor fraction using DELFI-TF, a mutation-independent, low-coverage whole genome sequencing approach, correlated with treatment responses. Pre-treatment serum from HPD (n=10 vs. 9 non-HPD) patients showed significantly lower levels of inflammatory cytokines (CXCL10, CCL13, CCL8, CCL19, TRAIL) and Granzyme H, and higher levels of anti-inflammatory cytokine IL10. Consistently, pre-treatment tumor transcriptomes of HPD (n=5 vs. 7 non-HPD) patients revealed suppression of IFN-gamma response, allograft rejection, and inflammatory response pathways. Conclusions: Concomitant TGF-beta and PD-L1 blockade is associated with a high frequency of HPD in SCLC patients. cfDNA could be critical for monitoring HPD. Tumor and blood immune signatures may inform the likelihood of HPD, with immune excluded tumors more likely to develop HPD. NCT Number: NCT03554473 Bintrafusp alfa was provided by EMD Serono (CrossRef Funder ID: 10.13039/100004755) Citation Format: Brett Schroeder, Nobuyuki Takahashi, Howard Yang, Renee Donahue, Zachary Skidmore, Alissa Konicki, Michael Nirula, Max Greenberg, George Chrisafis, Yang Zhang, Yo-Ting Tsai, Linda Sciuto, Samantha Nichols, Melissa Abel, Parth Desai, Rajesh Kumar, Christopher Schultz, Danielle Pinkiert, Chante Graham, Ajit Sharma, Justin Malin, Manan Krishnamurthy, Sophie Zhuang, Maxwell Lee, Lorenzo Rinaldi, Jeffrey Schlom, Lalage Wakefield, Anish Thomas. Hyperprogressive disease following bintrafusp alfa and DNA damaging chemotherapy in relapsed small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr CT257.

Abstract Despite promising results with immune checkpoint blockade (ICB) therapy, brain metastases (BrM) remain a deadly complication for cancer patients. Understanding the highly specialized brain metastatic tumor microenvironment (BrTME) is crucial for identifying determinants of response, however, progress has been hindered by limited access to patient samples and scarcity of relevant preclinical models. Here, we developed two mouse melanoma BrM models by intracardiac injection of cells derived from our previously established M4-B2905 melanoma mouse cell line. We characterized their mutational landscape and performed single-cell phenotypic and transcriptomic analysis, demonstrating that these models recapitulate the cellular and molecular features of human melanoma BrMs. Comparative and interactome analysis of our models revealed key factors contributing to ICB response and resistance. We found that the responsive model (BR1) was characterized by tumor cells that polarize microglia toward reactive states via inflammatory programs and response to IFN signaling. These microglia express high levels of antigen presentation and immunostimulatory molecules, triggering T cell recruitment and activation and promoting ICB therapy response. In contrast, in the resistant model (BR3), the tumor cells express neurological molecular signatures and ligands that sustain microglia homeostasis. This results in poor T cell infiltration predominantly composed of naïve cells, evading immune reaction and promoting ICB resistance. While we showed that systemic ICB therapy induced BrTME changes in both models, differences between responder and resistant models were maintained, emphasizing the importance of tumor cell-intrinsic programs in dictating the BrTME. We validated the translational relevance of our findings and demonstrated that BR1 signatures positively correlate with T cell infiltration and are associated with improved patient outcomes, whereas BR3 signatures negatively correlate with T cells and are found in patients with worse prognosis. Here we address an important challenge in the field by providing clinically relevant BrM models that recapitulate both the cellular and molecular features of human disease and the variability of ICB response seen in patients. We further reveal mechanistic insights into BrM ICB response and identify potential therapeutic targets to modulate the BrTME. Citation Format: Amelie Daugherty-Lopes, Eva Perez-Guijarro, Vishaka Gopalan, Jessica Rappaport, Quanyi Chen, April Huang, Khiem C. Lam, Sung Chin, Jessica Ebersole, Emily Wu, Gabriel Needle, Isabella Church, George Kyriakopoulos, Shaojun Xie, Yongmei Zaho, Charli Gruen, Antonella Sassano, Romina E Araya, Andres Thorkelsson, Cari Smith, Maxwell P. Lee, Sridhar Hannenhalli, Chi-Ping Day, Glenn Merlino, Romina S. Goldszmid, Shaojun Xie. Dissecting the brain metastatic microenvironment to uncover immune and molecular correlates of response to immunotherapy [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor-body Interactions: The Roles of Micro- and Macroenvironment in Cancer; 2024 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(22_Suppl):Abstract nr A002.

Abstract Background Gastric cancer (GC), a molecularly heterogeneous disease, is the third leading cause of cancer death worldwide. The majority of GC cases worldwide occur in East Asia, predominantly China. Mutational Signature Framework offers an elegant approach to identify mutational processes present in tumors. Methods To identify mutational signature patterns, we conducted whole exome sequencing (WES) analysis in Chinese patients with GC. Mutect2 and MutsigCV were used to identify significantly mutated genes in 175 Chinese GC cases using paired tumor-normal tissues. We investigated mutational signatures using Catalogue of Somatic Mutations in Cancer (COSMIC) Version 2 (V2) and Version 3 (V3). Results We identified 104 mutated genes with P < 0.01. Seven genes (OR6B1, B2M, ELF3, RHOA, RPL22, TP53, ARIDIA) had q < 0.0001, including six previously associated with GC. Mutational signatures (COSMIC-V3) observed include 14 single base substitutions (SBS), one doublet base substitution (DBS) Signature A, and one InDel (ID2). The most frequent SBS signatures (SBS05, SBS01, SBS15, SBS20, SBS40) were also observed in 254 White GC cases from The Cancer Genome Atlas (TCGA) Project. However, SBS01 and SBS20 showed significant differences between Whites vs. All Asians (19.3% vs. 11.3% for SBS 1 (P = 0.012) and 11.4% vs. 5.9% for SBS20 (P = 0.025), respectively). Using COSMIC V2, signatures 6, 15, and 1 were the most frequent in Chinese GC cases. Further, most Chinese GC cases carried multiple signatures. Conclusions This effort represents the most detailed mutational signatures analysis of GC cases from China to date. Results hold promise for new insights in understanding risk and prognosis factors in GC.