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.

Herpesviruses, such as Epstein-Barr virus (EBV), are thought to potentially play a significant role in multiple disease processes, including neoplasia, multiple sclerosis (MS), and more recently, Alzheimer’s disease (AD). Animal models remain vital tools for understanding these diseases and developing therapeutics. Callitrichine herpesvirus 3 (CalHV-3) was identified in the early 2000s in the common marmoset ( Callithrix jacchus ). Although phylogenetically related to human EBV, the biological similarities between CalHV-3 and EBV have not been thoroughly characterized. Over 450 marmosets from five biomedical research colonies in the United States were screened for CalHV-3 using droplet digital PCR (ddPCR). Peripheral blood mononuclear cells (PBMCs) were magnetically separated to determine viral loads in B-cell enriched and B-cell depleted populations. A CalHV-3 infected cell line was reactivated to determine gene expression profiles using quantitative-Reverse Transcription PCR (q-RT-PCR). Archived cases of lymphoma in the marmoset were immunophenotyped by immunohistochemistry (IHC). In the neoplastic tissue, CalHV-3 viral loads were measured by ddPCR, and viral transcripts were visualized using RNAscope. The prevalence of CalHV-3 in these research colonies ranged from 19-63%. The virus was detected longitudinally in PBMCs and saliva. Infected marmosets had CalHV-3 viral loads enriched in B-cells. All cases of B-cell lymphoma in the marmoset were positive for CalHV-3 DNA, with transcripts of EBV latent and lytic gene homologs detected in neoplastic tissue. Like EBV, CalHV-3 is characterized by persistent infection, shedding in saliva, B-cell tropism, latent and lytic gene expression profiles, and lymphomagenesis in a subset of infected animals. These results further suggest that CalHV-3 in the common marmoset may serve as a translational model of EBV infection and associated diseases.

Melanoma plasticity, driven by phenotype state switching, underlies clinically relevant traits such as metastasis and therapy resistance. As melanoma progression is thought to recapitulate aspects of neural crest cell (NCC) development, understanding embryonic melanocyte specification and lineage fate decisions of closely related NCCs may illuminate the pathways co-opted during disease evolution. Here, we use a mouse model to isolate and sequence Dopachrome tautomerase (Dct) expressing NCCs, the precursors of melanocytes, at two key developmental stages. We classify these lineages and devise a Developmental Gene Module (DGM) scoring system to interrogate lineage state switching in melanoma samples. In bulk transcriptomes, activation of DGMs representing embryonic Schwann Cell Precursors (SCPs)-multipotent stem cells-in patient tumors predicts poor response to immune checkpoint inhibitors (ICI). Co-activation of SCP and Mesenchymal-like (Mes.) modules further correlates with resistance to MAPK inhibitors. Notably, single-cell analyses reveal that melanoma cells can simultaneously express multiple DGMs, forming "hybrid" states. Cells in a hybrid Neural/SCP state are enriched in early metastasis and ICI-resistant tumors and are insensitive to inflammatory stimuli. We demonstrate that targeting Hdac2, a histone deacetylase associated with this Neural/SCP hybrid state, promotes a mesenchymal-like state switch, remodels the tumor microenvironment, and sensitizes melanoma cells to TNFα and tumors to ICI therapy. Our methodology thus reveals dynamic patterns of lineage state switching correlated with melanoma tumor evolution to drive insight into new therapeutic targets.

Background/objective: Mucosal melanoma (MM) is a poorly responsive, rare and aggressive subtype with few cases having targetable recurrent driver mutations, although Ras/MAPK and PI3K/AKT/mTOR signaling pathway activations are common. Eventual tumor evasion of targeted therapy continues to limit treatment success. Adequate models are necessary to address therapeutic resistance. The relatively greater incidence of naturally occurring MM in dogs, as well as its comparable clinical and pathological characteristics to human MM, represents an opportunity for study as a human MM patient surrogate. Resistance-promoting crosstalk between Ras/MAPK and PI3K/AKT/mTOR signaling under trametinib inhibition of MEK was studied in canine MM. Emphasis was placed on the suppressive effect of trametinib on cell cycle entry and its potential role in drug resistance. Methods: D-type cyclins were investigated following trametinib treatment of five MM cell lines exhibiting differential drug sensitivities. Signaling pathway activation, proliferation, survival, cell death, and cell cycle were analyzed in the context of D-type cyclin expression. Cyclin D2 expression was manipulated using siRNA knockdown or inducible recombinant overexpression. Results: Trametinib diminished cyclin D1 in all cell lines. While relatively trametinib-resistant MM cells exhibited capacity to upregulate cyclin D2, which promoted proliferation, sensitive MM cells lacked similar cyclin D2 compensation. Inhibition of the compensatory cyclin D2 in resistant cells conferred sensitivity. Induced cyclin D2 overexpression in otherwise trametinib-sensitive MM cells promoted survival. Upregulated PI3K/AKT/mTOR signaling under trametinib treatment was suppressed by mTORC1/2 inhibition, which similarly diminished cyclin D2 response. Conclusions: The compensatory switch from preferential reliance on cyclin D1 to D2 plays a role in MM resistance to MEK inhibition.

BackgroundPreclinical models recapitulating the metastatic phenotypes are essential for developing the next-generation therapies for metastatic prostate cancer (mPC). We aimed to establish a cohort of clinically relevant mPC models, particularly androgen receptor positive (AR+) bone metastasis models, from LuCaP patient-derived xenografts (PDX) that reflect the heterogeneity and complexity of mPC.MethodsPDX tumors were dissociated into single cells, modified to express luciferase, and were inoculated into NSG mice via intracardiac injection. The progression of metastases was monitored by bioluminescent imaging. Histological phenotypes of metastases were characterized by immunohistochemistry and immunofluorescence staining. Castration responses were further investigated in two AR-positive models.ResultsOur PDX-derived metastasis (PDM) model collection comprises three AR+ adenocarcinomas (ARPC) and one AR- neuroendocrine carcinoma (NEPC). All ARPC models developed bone metastases with either an osteoblastic, osteolytic, or mixed phenotype, while the NEPC model mainly developed brain metastasis. Different mechanisms of castration resistance were observed in two AR+ PDM models with distinct genotypes, such as combined loss of TP53 and RB1 in one model and expression of AR splice variant 7 (AR-V7) expression in another model. Intriguingly, the castration-resistant tumors displayed inter- and intra-tumor as well as organ-specific heterogeneity in lineage specification.ConclusionGenetically diverse PDM models provide a clinically relevant system for biomarker identification and personalized medicine in metastatic castration-resistant prostate cancer.

PAX3/7 fusion-negative rhabdomyosarcoma (FN-RMS) is a childhood mesodermal lineage malignancy with a poor prognosis for metastatic or relapsed cases. Limited understanding of advanced FN-RMS is partially attributed to the absence of sequential invasion and dissemination events and the challenge in studying cell behavior, using, for example, non-invasive intravital microscopy (IVM), in currently used xenograft models. Here, we developed an orthotopic tongue xenograft model of FN-RMS to study cell behavior and the molecular basis of invasion and metastasis using IVM. FN-RMS cells are retained in the tongue and invade locally into muscle mysial spaces and vascular lumen, with evidence of hematogenous dissemination to the lungs and lymphatic dissemination to lymph nodes. Using IVM of tongue xenografts reveals shifts in cellular phenotype, migration to blood and lymphatic vessels, and lymphatic intravasation. Insight from this model into tumor invasion and metastasis at the tissue, cellular, and subcellular level can guide new therapeutic avenues for advanced FN-RMS.

Supplementary Figure Legends 1-3 from Deletion of the Proline-Rich Region of the Murine Metastasis Susceptibility Gene Brd4 Promotes Epithelial-to-Mesenchymal Transition- and Stem Cell-Like Conversion

Supplementary Figure Legend 7 from Metastatic Growth from Dormant Cells Induced by a Col-I–Enriched Fibrotic Environment

Supplementary Methods, Figure Legends

Supplementary Figure 2 from Deletion of the Proline-Rich Region of the Murine Metastasis Susceptibility Gene Brd4 Promotes Epithelial-to-Mesenchymal Transition- and Stem Cell-Like Conversion

Supplementary Materials and Methods and Supplementary Figures S1-S10

TGFβs play a central regulatory role in maintaining homeostasis in the adult animal, and dysregulation of TGFβ signaling occurs in many cancers. Consequently, TGFβ pathway antagonists are now in early phase clinical oncology trials, but surprisingly little is known about when and where the TGFβ pathway is activated in the adult animal. We recently generated a TGFβ pathway reporter mouse in which expression of eGFP is driven by an enhancer consisting of 6 repeats of a strong Smad3 binding element (S3x6>GFP reporter), knocked into the ROSA26 locus. Here we present further characterization of the founder line (“Lime” mouse). Whole body fluorescent imaging of an adult mouse highlighted Smad3 activation in the expected tissues, such as the gastrointestinal tract, costal cartilage, and brown adipose tissue among others. Unexpectedly, intercrossing the Lime mouse with a Smad3 germline knockout mouse did not reveal a significant reduction in signal. However, since Smad3 knockout mice survive to adulthood despite having no Smad3, the persistence of signal could reflect compensatory signaling through other Smads, such as Smad1/5 or the Smad3-like Smad2 splice variant, Smad2DelEx3. Alternatively, the reporter may not report with fidelity in the chromatin context of the ROSA26 locus. To address these alternatives, Lime mice were intercrossed with the MMTV-PyVT model of mammary tumorigenesis. Mammary tumors showed high reporter activity, and a mammary tumor cell line (“LimePyVT”) was derived for characterization. Reporter activity in LimePyVT cells was enhanced by TGFβ and reduced by TGFβ receptor kinase inhibitors, but not by a BMP kinase inhibitor, as expected. Reduction in reporter signal with TGFβ receptor kinase inhibitors was only seen with prolonged treatment and 2x daily redosing, likely reflecting both the long half-life of the GFP (1-2 days), and the high sensitivity of the reporter to breakthrough signaling. siRNA knockdown in LimePyVT cells ex vivo confirmed that GFP reporter expression is dependent on Smad3 and Smad4. In other validation experiments, multicolor immunofluorescence showed heterogeneous reporter activity in the normal mammary epithelium, with highest activity in ER+ cells, where TGFβ is most active. Normal mammary epithelial cells were FACS-sorted into GFP-high and GFP-low fractions. RNASeq and pathway analysis showed TGFβ to be the top upstream regulator of the GFP-high cell transcriptome, consistent with the reporter reflecting TGFβ pathway activation. Overall, we believe that the high sensitivity of the reporter and the long GFP half-life likely mean that it will not report accurately on the impact of TGFβ antagonists in vivo. However, this reporter mouse should be a useful tool to assess the cellular location and extent of TGFβ/activin pathway activation during tumor progression, and the transcriptomic consequences. Citation Format: Yuan Yang, Zachary Millman, Christina Stuelten, Madhu Gargesha, Mark Simpson, Howard Yang, Maxwell Lee, Lalage M. Wakefield. Characterization and validation of a Smad3/TGFb pathway reporter mouse for analysis of TGFβ signaling in normal homeostasis and cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2393.

Drug resistance and disease progression are common in multiple myeloma (MM) patients, underscoring the need for new therapeutic combinations. A high-throughput drug screen in 47 MM cell lines and in silico Huber robust regression analysis of drug responses revealed 43 potentially synergistic combinations. We hypothesized that effective combinations would reduce MYC expression and enhance p16 activity. Six combinations cooperatively reduced MYC protein, frequently over-expressed in MM and also cooperatively increased p16 expression, frequently downregulated in MM. Synergistic reductions in viability were observed with top combinations in proteasome inhibitor-resistant and sensitive MM cell lines, while sparing fibroblasts. Three combinations significantly prolonged survival in a transplantable Ras-driven allograft model of advanced MM closely recapitulating high-risk/refractory myeloma in humans and reduced viability of ex vivo treated patient cells. Common genetic pathways similarly downregulated by these combinations promoted cell cycle transition, whereas pathways most upregulated were involved in TGFβ/SMAD signaling. These preclinical data identify potentially useful drug combinations for evaluation in drug-resistant MM and reveal potential mechanisms of combined drug sensitivity.

Supplementary Table 1. Identification of mice used is Western blots and immunoprecipitation experiments (M1-M20) and the mice used for the early time periods upon doxycycline induction.

Extinct lineages of Yersinia pestis , the causative agent of the plague, have been identified in several individuals from Eurasia between 5000 and 2500 years before present (BP). One of these, termed the ‘LNBA lineage’ (Late Neolithic and Bronze Age), has been suggested to have spread into Europe with human groups expanding from the Eurasian steppe. Here, we show that the LNBA plague was spread to Europe’s northwestern periphery by sequencing three Yersinia pestis genomes from Britain, all dating to ~4000 cal BP. Two individuals were from an unusual mass burial context in Charterhouse Warren, Somerset, and one individual was from a single burial under a ring cairn monument in Levens, Cumbria. To our knowledge, this represents the earliest evidence of LNBA plague in Britain documented to date. All three British Yersinia pestis genomes belong to a sublineage previously observed in Bronze Age individuals from Central Europe that had lost the putative virulence factor yapC . This sublineage is later found in Eastern Asia ~3200 cal BP. While the severity of the disease is currently unclear, the wide geographic distribution within a few centuries suggests substantial transmissibility.

Supplementary Figure 3 from Deletion of the Proline-Rich Region of the Murine Metastasis Susceptibility Gene Brd4 Promotes Epithelial-to-Mesenchymal Transition- and Stem Cell-Like Conversion

Supplementary Figure 1 from Deletion of the Proline-Rich Region of the Murine Metastasis Susceptibility Gene Brd4 Promotes Epithelial-to-Mesenchymal Transition- and Stem Cell-Like Conversion

Supplemental Figures S1-S4 + two supp. tables

Supplementary Tables 1-4 from Deletion of the Proline-Rich Region of the Murine Metastasis Susceptibility Gene Brd4 Promotes Epithelial-to-Mesenchymal Transition- and Stem Cell-Like Conversion

The authors have withdrawn their manuscript owing to questions about 2 panels within one figure. Therefore, the authors do not wish this work to be cited as reference for the project. If you have any questions, please contact the corresponding author.