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Pancreatic ductal adenocarcinoma (PDAC) presents a formidable clinical challenge due to its intricate microenvironment characterized by desmoplasia and complex tumor-stroma interactions. Conventional models hinder studying cellular crosstalk for therapeutic development. To recapitulate key features of PDAC masses, this study creates a novel sea-and-island PDAC tumor construct (s&i PTC). The s&i PTC consists of 3D-printed islands of human PDAC cells positioned within an interstitial extracellular matrix (ECM) populated by human cancer-associated fibroblasts (CAFs). This design closely mimics the in vivo desmoplastic architecture and nutrient-poor conditions. The model enables studying dynamic tumor-stroma crosstalk and signaling reciprocity, revealing both known and yet-to-be-discovered multicellular metabolic adaptations. Using the model, we discovered the orchestrated dynamic alterations of CAFs under nutrient stress, resembling critical in vivo human tumor niches, such as the secretion of pro-tumoral inflammatory factors. Additionally, nutrient scarcity induces dynamic alterations in the ECM composition and exacerbates poor cancer cell differentiation-features well-established in PDAC progression. Proteomic analysis unveiled the enrichment of proteins associated with aggressive tumor behavior and ECM remodeling in response to poor nutritional conditions, mimicking the metabolic stresses experienced by avascular pancreatic tumor cores. Importantly, the model's relevance to patient outcomes is evident through an inverse correlation between biomarker expression patterns in the s&i PTCs and PDAC patient survival rates. Key findings include upregulated MMPs and key ECM proteins (such as collagen 11 and TGFβ) under nutrient-avid conditions, known to be regulated by CAFs, alongside the concomitant reduction in E-cadherin expression associated with a poorly differentiated PDAC state under nutrient deprivation. Furthermore, elevated levels of hyaluronic acid (HA) and integrins in response to nutrient deprivation underscore the model's fidelity to the PDAC microenvironment. We also observed increased IL-6 and reduced α-SMA expression under poor nutritional conditions, suggesting a transition of CAFs from myofibroblastic to inflammatory phenotypes under a nutrient stress akin to in vivo niches. In conclusion, the s&i PTC represents a significant advancement in engineering clinically relevant 3D models of PDAC masses. It offers a promising platform for elucidating tumor-stroma interactions and guiding future therapeutic strategies to improve patient outcomes., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:Kaiming Ye reports was provided by Binghamton University. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Advancing age is a negative prognostic factor for cutaneous melanoma. However, the role of extracellular vesicles (EVs) within the melanoma tumor microenvironment (TME) has remained unexplored in the context of aging. While the size and morphology of the EVs isolated from young vs. aged fibroblasts remained unaltered, the contents of the protein cargo were changed. Aging reduced the expression of the tetraspanin CD9 in both the dermal fibroblasts and released EVs. CD9 is a crucial regulator of EV cargo sorting. Modulating the CD9 expression in fibroblasts was sufficient to alter its levels in EVs. Mass spectrometry analysis of EVs released by CD9 knockdown (KD) vs. control cells revealed a significant increase in angiopoietin-like protein 2 (ANGPTL2), an angiogenesis promoter. Analysis of primary endothelial cells confirmed increased sprouting under CD9 KD conditions. Together, our data indicate that aged EVs play an important role in promoting a tumor-permissive microenvironment., Competing Interests: Declaration of interests A.T.W. is on the boards of reGAIN Therapeutics and the Melanoma Research Foundation and the scientific advisory committee of the V Foundation. D.J.Z. reports grant funding (paid to Johns Hopkins University) from Roche/Genentech., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Small-cell lung cancer (SCLC) is the most lethal type of lung cancer. Paradoxically, this tumor displays an initial exquisite response to chemotherapy; however, at relapse, the tumor is highly resistant to subsequent available therapies. Here, we report that the expression of three prime repair exonuclease 1 (TREX1) is strongly induced in chemoresistant SCLCs. Assay for transposase-accessible chromatin using sequencing and chromatin immunoprecipitation sequencing revealed a significant increase in chromatin accessibility and transcriptional activity of TREX1 gene locus in chemoresistant SCLCs. Analyses of human SCLC tumors and patient-derived xenografts (PDX) also showed an increase in TREX1 expression in postchemotherapy samples. TREX1 depletion caused the activation of cyclic GMP-AMP synthase stimulator of interferon gene pathway due to cytoplasmic accumulation of damage-associated double-stranded DNA, inducing immunogenicity and enhancing the sensitivity of drug-resistant cells to chemotherapy. These findings suggest TREX1 upregulation may partially contribute to the survival of resistant cells, and its inhibition may represent a promising therapeutic strategy to enhance antitumor immunity and potentiate the efficacy of chemotherapy and/or immunotherapy in chemoresistant SCLCs. Significance: In this study, we show that targeting TREX1 induces an innate immune response and resensitizes SCLC cells to chemotherapy, representing a promising novel target for "immunologically" cold tumors, such as SCLC., (©2024 The Authors; Published by the American Association for Cancer Research.)

Hypothyroidism is commonly detected in patients with medulloblastoma (MB). However, whether thyroid hormone (TH) contributes to MB pathogenicity remains undetermined. Here, we find that TH plays a critical role in promoting tumor cell differentiation. Reduction in TH levels frees the TH receptor, TRα1, to bind to EZH2 and repress expression of NeuroD1, a transcription factor that drives tumor cell differentiation. Increased TH reverses EZH2-mediated repression of NeuroD1 by abrogating the binding of EZH2 and TRα1, thereby stimulating tumor cell differentiation and reducing MB growth. Importantly, TH-induced differentiation of tumor cells is not restricted by the molecular subgroup of MB, suggesting that TH can be used to broadly treat MB subgroups. These findings establish an unprecedented association between TH signaling and MB pathogenicity, providing solid evidence for TH as a promising modality for MB treatment., Competing Interests: Declaration of interests A patent related to this work has been filed by Fox Chase Cancer Center (Yang Y.J. and Yang Z.J.)., (Copyright © 2024 Elsevier Inc. All rights reserved.)

PARP inhibitor (PARPi) therapy has transformed outcomes for patients with homologous recombination DNA repair (HRR) deficient ovarian cancers, for example those with BRCA1 or BRCA2 gene defects. Unfortunately, PARPi resistance is common. Multiple resistance mechanisms have been described, including secondary mutations that restore the HR gene reading frame. BRCA1 splice isoforms △11 and △11q can contribute to PARPi resistance by splicing out the mutation-containing exon, producing truncated, partially functional proteins. However, the clinical impacts and underlying drivers of BRCA1 exon skipping are not fully understood.We analyzed nine ovarian and breast cancer patient derived xenografts (PDX) with BRCA1 exon 11 frameshift mutations for exon skipping and therapy response, including a matched PDX pair derived from a patient pre- and post-chemotherapy/PARPi. BRCA1 exon 11 skipping was elevated in PARPi resistant PDX tumors. Two independent PDX models acquired secondary BRCA1 splice site mutations (SSMs) that drive exon skipping, confirmed using qRT-PCR, RNA sequencing, immunoblotting and minigene modelling. CRISPR/Cas9-mediated disruption of splicing functionally validated exon skipping as a mechanism of PARPi resistance. SSMs were also enriched in post-PARPi ovarian cancer patient cohorts from the ARIEL2 and ARIEL4 clinical trials.Few PARPi resistance mechanisms have been confirmed in the clinical setting. While secondary/reversion mutations typically restore a gene's reading frame, we have identified secondary mutations in patient cohorts that hijack splice sites to enhance mutation-containing exon skipping, resulting in the overexpression of BRCA1 hypomorphs, which in turn promote PARPi resistance. Thus, BRCA1 SSMs can and should be clinically monitored, along with frame-restoring secondary mutations., (© 2024. The Author(s).)

IL-36 cytokines are emerging as beneficial in immunity against pathogens and cancers but can also be detrimental when dysregulated in autoimmune and autoinflammatory conditions. Interest in targeting IL-36 activity for therapeutic purposes is rapidly growing, yet many unknowns about the functions of these cytokines remain. Thus, the availability of robust research tools is essential for both fundamental basic science and pre-clinical studies to fully access outcomes of any manipulation of the system. For this purpose, a floxed Il1rl2 , the gene encoding the IL-36 receptor, mouse strain was developed to facilitate the generation of conditional knockout mice. The targeted locus was engineered to contain an inverted mCherry reporter sequence that upon Cre-mediated recombination will be flipped and expressed under the control of the endogenous Il1rl2 promoter. This feature can be used to confirm knockout in individual cells but also as a reporter to determine which cells express the IL-36 receptor IL-1RL2. The locus was confirmed to function as intended and further used to demonstrate the expression of IL-1RL2 in barrier tissues. Il1rl2 expression was detected in leukocytes in all barrier tissues. Interestingly, strong expression was observed in epithelial cells at locations in direct contact with the environment such as the skin, oral mucosa, the esophagus, and the upper airways, but almost absent from epithelial cells at more inward facing sites, including lung alveoli, the small intestine, and the colon. These findings suggest specialized functions of IL-1RL2 in outward facing epithelial tissues and cells. The generated mouse model should prove valuable in defining such functions and may also facilitate basic and translational research.

Severe influenza A virus (IAV) infections can result in hyper-inflammation, lung injury and acute respiratory distress syndrome 1-5 (ARDS), for which there are no effective pharmacological therapies. Necroptosis is an attractive entry point for therapeutic intervention in ARDS and related inflammatory conditions because it drives pathogenic lung inflammation and lethality during severe IAV infection 6-8 and can potentially be targeted by receptor interacting protein kinase 3 (RIPK3) inhibitors. Here we show that a newly developed RIPK3 inhibitor, UH15-38, potently and selectively blocked IAV-triggered necroptosis in alveolar epithelial cells in vivo. UH15-38 ameliorated lung inflammation and prevented mortality following infection with laboratory-adapted and pandemic strains of IAV, without compromising antiviral adaptive immune responses or impeding viral clearance. UH15-38 displayed robust therapeutic efficacy even when administered late in the course of infection, suggesting that RIPK3 blockade may provide clinical benefit in patients with IAV-driven ARDS and other hyper-inflammatory pathologies., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

RAC1 P29S is the third most prevalent hotspot mutation in sun-exposed melanoma. RAC1 alterations in cancer are correlated with poor prognosis, resistance to standard chemotherapy, and insensitivity to targeted inhibitors. Although RAC1 P29S mutations in melanoma and RAC1 alterations in several other cancers are increasingly evident, the RAC1-driven biological mechanisms contributing to tumorigenesis remain unclear. Lack of rigorous signaling analysis has prevented identification of alternative therapeutic targets for RAC1 P29S -harboring melanomas. To investigate the RAC1 P29S -driven effect on downstream molecular signaling pathways, we generated an inducible RAC1 P29S expression melanocytic cell line and performed RNA-sequencing (RNA-seq) coupled with multiplexed kinase inhibitor beads and mass spectrometry (MIBs/MS) to establish enriched pathways from the genomic to proteomic level. Our proteogenomic analysis identified CDK9 as a potential new and specific target in RAC1 P29S -mutant melanoma cells. In vitro, CDK9 inhibition impeded the proliferation of in RAC1 P29S -mutant melanoma cells and increased surface expression of PD-L1 and MHC Class I proteins. In vivo, combining CDK9 inhibition with anti-PD-1 immune checkpoint blockade significantly inhibited tumor growth only in melanomas that expressed the RAC1 P29S mutation. Collectively, these results establish CDK9 as a novel target in RAC1-driven melanoma that can further sensitize the tumor to anti-PD-1 immunotherapy., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

Activating innate immunity in cancer cells through cytoplasmic nucleic acid sensing pathways, a phenomenon known as "viral mimicry," has emerged as an effective strategy to convert immunologically "cold" tumors into "hot." Through a curated CRISPR-based screen of RNA helicases, we identified DExD/H-box helicase 9 (DHX9) as a potent repressor of double-stranded RNA (dsRNA) in small cell lung cancers (SCLC). Depletion of DHX9 induced accumulation of cytoplasmic dsRNA and triggered tumor-intrinsic innate immunity. Intriguingly, ablating DHX9 also induced aberrant accumulation of R-loops, which resulted in an increase of DNA damage-derived cytoplasmic DNA and replication stress in SCLCs. In vivo, DHX9 deletion promoted a decrease in tumor growth while inducing a more immunogenic tumor microenvironment, invigorating responsiveness to immune-checkpoint blockade. These findings suggest that DHX9 is a crucial repressor of tumor-intrinsic innate immunity and replication stress, representing a promising target for SCLC and other "cold" tumors in which genomic instability contributes to pathology., Significance: One promising strategy to trigger an immune response within tumors and enhance immunotherapy efficacy is by inducing endogenous "virus-mimetic" nucleic acid accumulation. Here, we identify DHX9 as a viral-mimicry-inducing factor involved in the suppression of double-stranded RNAs and R-loops and propose DHX9 as a novel target to enhance antitumor immunity. See related commentary by Chiappinelli, p. 389. This article is featured in Selected Articles from This Issue, p. 384., (©2024 The Authors; Published by the American Association for Cancer Research.)

Purpose: Dedifferentiated liposarcoma (DDL) and leiomyosarcoma (LMS) are two common subtypes of soft-tissue sarcoma, a rare group of diseases for which new treatments are needed. Chemotherapy remains the standard option for advanced disease. Targeting cyclin-dependent kinase 4 and 6 (CDK4/6) in DDL and mTOR in LMS is of biologic interest. When combined, the CDK4 inhibitor ribociclib and the mTOR inhibitor everolimus have shown synergistic growth inhibition in multiple tumor models, suggesting that this combination could be beneficial in patients., Patients and Methods: This was a single arm, open label, multicenter phase II study of the combination of ribociclib and everolimus. Patients were enrolled into one of two cohorts: DDL or LMS with intact Rb. The primary endpoint was progression-free rate (PFR) at 16 weeks. Secondary endpoints included progression-free survival (PFS) and overall survival, safety and biomarker analyses., Results: In the DDL cohort, 33.3% [95% confidence interval (CI), 15.6%-55.3%] of patients were progression-free at 16 weeks. Median PFS in this cohort was 15.4 weeks (95% CI, 8-36 weeks) with 2 partial responses. In the LMS cohort the PFR at 16 weeks was 29.2% (95% CI, 12.6%-51.1%). Median PFS in this cohort was 15.7 weeks (95% CI, 7.7-NA). Most common toxicities included fatigue (66.7%), anorexia (43.8%), and hyperglycemia (43.8%). Concordance between Rb testing methodologies was poor., Conclusions: The combination of ribociclib and everolimus demonstrates activity in DDL with prolonged stable disease (≥16 weeks) meeting the primary endpoint. Notably partial responses were observed. The primary endpoint was not reached in the LMS cohort. The combination was well tolerated with expected side effects., (©2023 American Association for Cancer Research.)

Background & Aims: Autophagy plays roles in esophageal pathologies both benign and malignant. Here, we aim to define the role of autophagy in esophageal epithelial homeostasis., Methods: We generated tamoxifen-inducible, squamous epithelial-specific Atg7 (autophagy related 7) conditional knockout mice to evaluate effects on esophageal homeostasis and response to the carcinogen 4-nitroquinoline 1-oxide (4NQO) using histologic and biochemical analyses. We fluorescence-activated cell sorted esophageal basal cells based on fluorescence of the autophagic vesicle (AV)-identifying dye Cyto-ID and then subjected these cells to transmission electron microscopy, image flow cytometry, three-dimensional organoid assays, RNA sequencing, and cell cycle analysis. Three-dimensional organoids were subjected to passaging, single-cell RNA sequencing, cell cycle analysis, and immunostaining., Results: Genetic autophagy inhibition in squamous epithelium resulted in increased proliferation of esophageal basal cells under homeostatic conditions and also was associated with significant weight loss in mice treated with 4NQO that further displayed perturbed epithelial tissue architecture. Esophageal basal cells with high AV level (Cyto-ID High ) displayed limited organoid formation capability on initial plating but passaged more efficiently than their counterparts with low AV level (Cyto-ID Low ). RNA sequencing suggested increased autophagy in Cyto-ID High esophageal basal cells along with decreased cell cycle progression, the latter of which was confirmed by cell cycle analysis. Single-cell RNA sequencing of three-dimensional organoids generated by Cyto-ID Low and Cyto-ID High cells identified expansion of 3 cell populations and enrichment of G2/M-associated genes in the Cyto-ID High group. Ki67 expression was also increased in organoids generated by Cyto-ID High cells, including in basal cells localized beyond the outermost cell layer., Conclusions: Autophagy contributes to maintenance of the esophageal proliferation-differentiation gradient. Esophageal basal cells with high AV level exhibit limited proliferation and generate three-dimensional organoids with enhanced self-renewal capacity., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Anaplastic large cell lymphoma (ALCL), a subgroup of mature T-cell neoplasms with an aggressive clinical course, is characterized by elevated expression of CD30 and anaplastic cytology. To achieve a comprehensive understanding of the molecular characteristics of ALCL pathology and to identify therapeutic vulnerabilities, we applied genome-wide CRISPR library screenings to both anaplastic lymphoma kinase positive (ALK+) and primary cutaneous (pC) ALK- ALCLs and identified an unexpected role of the interleukin-1R (IL-1R) inflammatory pathway in supporting the viability of pC ALK- ALCL. Importantly, this pathway is activated by IL-1α in an autocrine manner, which is essential for the induction and maintenance of protumorigenic inflammatory responses in pC-ALCL cell lines and primary cases. Hyperactivation of the IL-1R pathway is promoted by the A20 loss-of-function mutation in the pC-ALCL lines we analyze and is regulated by the nonproteolytic protein ubiquitination network. Furthermore, the IL-1R pathway promotes JAK-STAT3 signaling activation in ALCLs lacking STAT3 gain-of-function mutation or ALK translocation and enhances the sensitivity of JAK inhibitors in these tumors in vitro and in vivo. Finally, the JAK2/IRAK1 dual inhibitor, pacritinib, exhibited strong activities against pC ALK- ALCL, where the IL-1R pathway is hyperactivated in the cell line and xenograft mouse model. Thus, our studies revealed critical insights into the essential roles of the IL-1R pathway in pC-ALCL and provided opportunities for developing novel therapeutic strategies.

The greater efficacy of DNA-damaging drugs for pancreatic adenocarcinoma (PDAC) relies on targeting cancer-specific vulnerabilities while sparing normal organs and tissues due to their inherent toxicities. We tested LP-184, a novel acylfulvene analog, for its activity in preclinical models of PDAC carrying mutations in the DNA damage repair (DDR) pathways. Cytotoxicity of LP-184 is solely dependent on prostaglandin reductase 1 (PTGR1), so that PTGR1 expression robustly correlates with LP-184 cytotoxicity in vitro and in vivo. Low-passage patient-derived PDAC xenografts with DDR deficiencies treated ex vivo are more sensitive to LP-184 compared with DDR-proficient tumors. Additional in vivo testing of PDAC xenografts for their sensitivity to LP-184 demonstrates marked tumor growth inhibition in models harboring pathogenic mutations in ATR, BRCA1, and BRCA2. Depletion of PTGR1, however, completely abrogates the antitumor effect of LP-184. Testing combinatorial strategies for LP-184 aimed at deregulation of nucleotide excision repair proteins ERCC3 and ERCC4 established synergy. Our results provide valuable biomarkers for clinical testing of LP-184 in a large subset of genetically defined characterized refractory carcinomas. High PTGR1 expression and deleterious DDR mutations are present in approximately one third of PDAC making these patients ideal candidates for clinical trials of LP-184., (©2023 American Association for Cancer Research.)

Periodontal disease is a multifactorial, bacterially induced inflammatory condition characterized by the progressive destruction of periodontal tissues. The successful nonsurgical treatment of periodontitis requires multifunctional technologies offering antibacterial therapies and promotion of bone regeneration simultaneously. For the first time, in this study, an injectable piezoelectric hydrogel (PiezoGEL) was developed after combining gelatin methacryloyl (GelMA) with biocompatible piezoelectric fillers of barium titanate (BTO) that produce electrical charges when stimulated by biomechanical vibrations (e.g., mastication, movements). We harnessed the benefits of hydrogels (injectable, light curable, conforms to pocket spaces, biocompatible) with the bioactive effects of piezoelectric charges. A thorough biomaterial characterization confirmed piezoelectric fillers' successful integration with the hydrogel, photopolymerizability, injectability for clinical use, and electrical charge generation to enable bioactive effects (antibacterial and bone tissue regeneration). PiezoGEL showed significant reductions in pathogenic biofilm biomass (∼41%), metabolic activity (∼75%), and the number of viable cells (∼2-3 log) compared to hydrogels without BTO fillers in vitro. Molecular analysis related the antibacterial effects to be associated with reduced cell adhesion (downregulation of porP and fimA ) and increased oxidative stress (upregulation of oxyR ) genes. Moreover, PiezoGEL significantly enhanced bone marrow stem cell (BMSC) viability and osteogenic differentiation by upregulating RUNX2, COL1A1 , and ALP . In vivo, PiezoGEL effectively reduced periodontal inflammation and increased bone tissue regeneration compared to control groups in a mice model. Findings from this study suggest PiezoGEL to be a promising and novel therapeutic candidate for the treatment of periodontal disease nonsurgically.

Esophageal squamous cell carcinoma (ESCC) is one of the most aggressive forms of human malignancy, often displaying limited therapeutic response. Here, we examine the non-steroidal anti-inflammatory drug diclofenac (DCF) as a novel therapeutic agent in ESCC using complementary in vitro and in vivo models. DCF selectively reduced viability of human ESCC cell lines TE11, KYSE150, and KYSE410 as compared with normal primary or immortalized esophageal keratinocytes. Apoptosis and altered cell cycle profiles were documented in DCF-treated TE11 and KYSE 150. In DCF-treated TE11, RNA-Sequencing identified differentially expressed genes and Ingenuity Pathway Analysis predicted alterations in pathways associated with cellular metabolism and p53 signaling. Downregulation of proteins associated with glycolysis was documented in DCF-treated TE11 and KYSE150. In response to DCF, TE11 cells further displayed reduced levels of ATP, pyruvate, and lactate. Evidence of mitochondrial depolarization and superoxide production was induced by DCF in TE11 and KYSE150. In DCF-treated TE11, the superoxide scavenger MitoTempo improved viability, supporting a role for mitochondrial reactive oxygen species in DCF-mediated toxicity. DCF treatment resulted in increased expression of p53 in TE11 and KYSE150. p53 was further identified as a mediator of DCF-mediated toxicity in TE11 as genetic depletion of p53 partially limited apoptosis in response to DCF. Consistent with the anticancer activity of DCF in vitro, the drug significantly decreased tumor burdene in syngeneic ESCC xenograft tumors and 4-nitroquinoline 1-oxide-mediated ESCC lesions in vivo. These preclinical findings identify DCF as an experimental therapeutic that should be explored further in ESCC., (© The Author(s) 2023. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Studies on the microbiome of oral squamous cell carcinoma (OSCC) have been limited to 16S rRNA gene sequencing. Here, laser microdissection coupled with brute-force, deep metatranscriptome sequencing was employed to simultaneously characterize the microbiome and host transcriptomes and predict their interaction in OSCC. The analysis involved 20 HPV16/18-negative OSCC tumor/adjacent normal tissue pairs (TT and ANT) along with deep tongue scrapings from 20 matched healthy controls (HC). Standard bioinformatic tools coupled with in-house algorithms were used to map, analyze, and integrate microbial and host data. Host transcriptome analysis identified enrichment of known cancer-related gene sets, not only in TT versus ANT and HC, but also in the ANT versus HC contrast, consistent with field cancerization. Microbial analysis identified a low abundance yet transcriptionally active, unique multi-kingdom microbiome in OSCC tissues predominated by bacteria and bacteriophages. HC showed a different taxonomic profile yet shared major microbial enzyme classes and pathways with TT/ANT, consistent with functional redundancy. Key taxa enriched in TT/ANT compared with HC were Cutibacterium acnes , Malassezia restricta , Human Herpes Virus 6B, and bacteriophage Yuavirus. Functionally, hyaluronate lyase was overexpressed by C. acnes in TT/ANT. Microbiome-host data integration revealed that OSCC-enriched taxa were associated with upregulation of proliferation-related pathways. In a preliminary in vitro validation experiment, infection of SCC25 oral cancer cells with C. acnes resulted in upregulation of MYC expression. The study provides a new insight into potential mechanisms by which the microbiome can contribute to oral carcinogenesis, which can be validated in future experimental studies., Significance: Studies have shown that a distinct microbiome is associated with OSCC, but how the microbiome functions within the tumor interacts with the host cells remains unclear. By simultaneously characterizing the microbial and host transcriptomes in OSCC and control tissues, the study provides novel insights into microbiome-host interactions in OSCC which can be validated in future mechanistic studies., (© 2023 The Authors; Published by the American Association for Cancer Research.)

Background & Aims: Aberrant DNA methylation is frequent in colorectal cancer (CRC), but underlying mechanisms and pathologic consequences are poorly understood., Methods: We disrupted active DNA demethylation genes Tet1 and/or Tdg from Apc Min mice and characterized the methylome and transcriptome of colonic adenomas. Data were compared to human colonic adenocarcinomas (COAD) in The Cancer Genome Atlas., Results: There were increased numbers of small intestinal adenomas in Apc Min mice expressing the Tdg N151A allele, whereas Tet1-deficient and Tet1/Tdg N151A -double heterozygous Apc Min colonic adenomas were larger with features of erosion and invasion. We detected reduction in global DNA hypomethylation in colonic adenomas from Tet1- and Tdg-mutant Apc Min mice and hypermethylation of CpG islands in Tet1-mutant Apc Min adenomas. Up-regulation of inflammatory, immune, and interferon response genes was present in Tet1- and Tdg-mutant colonic adenomas compared to control Apc Min adenomas. This up-regulation was also seen in murine colonic organoids and human CRC lines infected with lentiviruses expressing TET1 or TDG short hairpin RNA. A 127-gene inflammatory signature separated colonic adenocarcinomas into 4 groups, closely aligned with their microsatellite or chromosomal instability and characterized by different levels of DNA methylation and DNMT1 expression that anticorrelated with TET1 expression. Tumors with the CpG island methylator phenotype (CIMP) had concerted high DNMT1/low TET1 expression. TET1 or TDG knockdown in CRC lines enhanced killing by natural killer cells., Conclusions: Our findings reveal a novel epigenetic regulation, linked to the type of genomic instability, by which TET1/TDG-mediated DNA demethylation decreases methylation levels and inflammatory/interferon/immune responses. CIMP in CRC is triggered by an imbalance of methylating activities over demethylating activities. These mice represent a model of CIMP CRC., (Copyright © 2023 AGA Institute. Published by Elsevier Inc. All rights reserved.)

Nearly all adults are infected with one or more herpes viruses. The most common are herpes simplex virus (HSV)-1 and HSV-2, which upon reactivation can cause painful skin and mucosal erosions. Patients who are immune compromised often experience frequent, atypical, or chronic lesions and thus a greatly diminished QOL. Pellino1 is a ubiquitin ligase involved in IL-1 and toll-like receptor signaling; however, the role of Pellino1 in skin immunity against HSV is unknown. In this study, using the mouse-flank HSV-1 skin infection model, we show that Pellino1 has several critical functions during active viral replication. Peli1 ‒/‒ mice succumb more than wild-type mice to systemic disease and develop larger zosteriform skin lesions along affected dermatomes. In Pellino1-deficient mice, the virus spread extensively through the epidermis and follicular infundibulum into sebaceous glands where sebocytes were found positive for the virus. The latter did not appear to involve a shift in how the virus migrated through the nervous system. Immunohistochemistry revealed delayed recruitment of myeloid and T cells to the infected epidermis in Peli1 ‒/‒ mice. This was associated with decreased expression of the cytokine mRNAs Il1a, Il36b and 2610528A11Rik; the latter also known as Gpr15l. In conclusion, Pellino1 plays important roles in restricting viral dissemination, and the involved pathways may represent novel therapeutic targets in patients with frequent or chronic HSV infections., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

SHP1, a tyrosine phosphatase, negatively regulates B-cell receptor (BCR) signaling. Ibrutinib selectively inhibits BTK and has been approved for the treatment of several types of B-cell lymphomas, but not yet in diffuse large B-cell lymphoma (DLBCL). A phase 3 clinical trial of ibrutinib-containing regimen has been completed to evaluate its activity in subtypes or subsets of DLBCL patients. Although the subtype of activated B-cell like (ABC) DLBCL is characterized by chronic active BCR signaling, only a fraction of ABC-DLBCL patients seem to benefit from ibrutinib-containing regimen. New alternative predictive biomarkers are needed to identify patients who better respond. We investigated if SHP1 plays a role in defining the level of the BCR activity and impacts the response to ibrutinib. A meta-analysis revealed that lack of SHP1 protein expression as well as SHP1 promoter hypermethylation is strongly associated with NHL including DLBCL. On a tissue microarray of 95 DLBCL samples, no substantial difference in SHP1 expression was found between the GCB and non-GCB subtypes of DLBCL. However, we identified a strong reverse correlation between SHP1 expression and promoter methylation suggesting that promoter hypermethylation is responsible for SHP1 loss. SHP1 knockout in BCR-dependent GCB and ABC cell lines increased BCR signaling activities and sensitize lymphoma cells to the action of ibrutinib. Rescue of SHP1 in the knockout clones, on the other hand, restored BCR signaling and ibrutinib resistance. Further, pharmacological inhibition of SHP1 in both cell lines and patient-derived primary cells demonstrate that SHP1 inhibition synergized with ibrutinib in suppressing tumor cell growth. Thus, SHP1 loss may serve as an alternative biomarker to cell-of-origin to identify patients who potentially benefit from ibrutinib treatment. Our results further suggest that reducing SHP1 pharmacologically may represent a new strategy to augment tumor response to BCR-directed therapies. Schematic diagram summarizing the major findings. Left panel. When SHP1 is present and functional, it negatively regulates the activity of the BCR pathway. Right pane. When SHP1 is diminished or lost, cells depend more on the increased BCR signaling and making them vulnerable to BTK inhibitor, ibrutinib. Diagram was generated using BioRender., (© 2022. The Author(s), under exclusive licence to Springer Nature Limited.)

Interferon-gamma (IFNG) has long been regarded as the flag-bearer for the anti-cancer immunosurveillance mechanisms. However, relatively recent studies have suggested a dual role of IFNG, albeit there is no direct experimental evidence for its potential pro-tumor functions. Here we provide in vivo evidence that treatment of mouse melanoma cell lines with Ifng enhances their tumorigenicity and metastasis in lung colonization allograft assays performed in immunocompetent syngeneic host mice, but not in immunocompromised host mice. We also show that this enhancement is dependent on downstream signaling via Stat1 but not Stat3, suggesting an oncogenic function of Stat1 in melanoma. The experimental results suggest that melanoma cell-specific Ifng signaling modulates the tumor microenvironment and its pro-tumorigenic effects are partially dependent on the γδ T cells, as Ifng-enhanced tumorigenesis was inhibited in the TCR-δ knockout mice. Overall, these results show that Ifng signaling may have tumor-promoting effects in melanoma by modulating the immune cell composition of the tumor microenvironment., (© 2022. The Author(s), under exclusive licence to Springer Nature Limited.)

The commonly used therapeutic management of PC involves androgen deprivation therapy (ADT) followed by treatment with AR signaling inhibitors (ARSI). However, nearly all patients develop drug-resistant disease, with a median progression-free survival of less than 2 years in chemotherapy-naïve men. Acetyl-coenzyme A (acetyl-CoA) is a central metabolic signaling molecule with key roles in biosynthetic processes and cancer signaling. In signaling, acetyl-CoA serves as the acetyl donor for acetylation, a critical post-translational modification. Acetylation affects the androgen receptor (AR) both directly and indirectly increasing expression of AR dependent genes. Our studies reveal that PC cells respond to the treatment with ARSI by increasing expression of ATP-citrate lyase (ACLY), a major enzyme responsible for cytosolic acetyl-CoA synthesis, and up-regulation of acetyl-CoA intracellular levels. Inhibition of ACLY results in a significant suppression of ligand-dependent and -independent routes of AR activation. Accordingly, the addition of exogenous acetyl-CoA, or its precursor acetate, augments AR transcriptional activity and diminishes the anti-AR activity of ARSI. Taken together, our findings suggest that PC cells respond to antiandrogens by increasing activity of the acetyl-coA pathway in order to reinstate AR signaling.

The role of store-operated Ca 2+ entry (SOCE) in melanoma metastasis is highly controversial. To address this, we here examined UV-dependent metastasis, revealing a critical role for SOCE suppression in melanoma progression. UV-induced cholesterol biosynthesis was critical for UV-induced SOCE suppression and subsequent metastasis, although SOCE suppression alone was both necessary and sufficient for metastasis to occur. Further, SOCE suppression was responsible for UV-dependent differences in gene expression associated with both increased invasion and reduced glucose metabolism. Functional analyses further established that increased glucose uptake leads to a metabolic shift towards biosynthetic pathways critical for melanoma metastasis. Finally, examination of fresh surgically isolated human melanoma explants revealed cholesterol biosynthesis-dependent reduced SOCE. Invasiveness could be reversed with either cholesterol biosynthesis inhibitors or pharmacological SOCE potentiation. Collectively, we provide evidence that, contrary to current thinking, Ca 2+ signals can block invasive behavior, and suppression of these signals promotes invasion and metastasis., (© 2022 The Authors.)

Although Ras/mitogen-activated protein kinase (MAPK) signaling is activated in most human cancers, attempts to target this pathway using kinase-active site inhibitors have not typically led to durable clinical benefit. To address this shortcoming, we sought to test the feasibility of an alternative targeting strategy, focused on the ERK2 substrate binding domains, D and DEF binding pocket (DBP). Disabling the ERK2-DBP domain in mice caused baseline erythrocytosis. Consequently, we investigated the role of the ERK2-D and -DBP domains in disease, using a JAK2-dependent model of polycythemia vera (PV). Of note, inactivation of the ERK2-DBP domain promoted the progression of disease from PV to myelofibrosis, suggesting that the ERK2-DBP domain normally opposes progression. ERK2-DBP inactivation also prevented oncogenic JAK2 kinase (JAK2V617F) from promoting oncogene-induced senescence in vitro. The ERK2-DBP mutation attenuated JAK2-mediated oncogene-induced senescence by preventing the physical interaction of ERK2 with the transcription factor Egr1. Because inactivation of the ERK2-DBP created a functional ERK2 kinase limited to binding substrates through its D domain, these data suggested that the D domain substrates were responsible for promoting oncogene-induced progenitor growth and tumor progression and that pharmacologic targeting of the ERK2-D domain may attenuate cancer cell growth. Indeed, pharmacologic agents targeting the ERK2-D domain were effective in attenuating the growth of JAK2-dependent myeloproliferative neoplasm cell lines. Taken together, these data indicate that the ERK-D and -DBP domains can play distinct roles in the progression of neoplasms and that the D domain has the potential to be a potent therapeutic target in Ras/MAPK-dependent cancers., (© 2022 by The American Society of Hematology.)

The lack of tumor infiltration by CD8 + T cells is associated with poor patient response to anti-PD-1 therapy. Understanding how tumor infiltration is regulated is key to improving treatment efficacy. Here, we report that phosphorylation of HRS, a pivotal component of the ESCRT complex involved in exosome biogenesis, restricts tumor infiltration of cytolytic CD8 + T cells. Following ERK-mediated phosphorylation, HRS interacts with and mediates the selective loading of PD-L1 to exosomes, which inhibits the migration of CD8 + T cells into tumors. In tissue samples from patients with melanoma, CD8 + T cells are excluded from the regions where tumor cells contain high levels of phosphorylated HRS. In murine tumor models, overexpression of phosphorylated HRS increases resistance to anti-PD-1 treatment, whereas inhibition of HRS phosphorylation enhances treatment efficacy. Our study reveals a mechanism by which phosphorylation of HRS in tumor cells regulates anti-tumor immunity by inducing PD-L1 + immunosuppressive exosomes, and suggests HRS phosphorylation blockade as a potential strategy to improve the efficacy of cancer immunotherapy., (© 2022. The Author(s).)

Because loss of the NF2 tumor suppressor gene results in p21-activated kinase (Pak) activation, PAK inhibitors hold promise for the treatment of NF2-deficient tumors. To test this possibility, we asked if loss of Pak2, a highly expressed group I PAK member, affects the development of malignant mesothelioma in Nf2;Cdkn2a-deficient (NC) mice and the growth properties of NC mesothelioma cells in culture. In vivo, deletion of Pak2 resulted in a markedly decreased incidence and delayed onset of both pleural and peritoneal malignant mesotheliomas in NC mice. In vitro, Pak2 deletion decreased malignant mesothelioma cell viability, migration, clonogenicity, and spheroid formation. RNA-sequencing analysis demonstrated downregulated expression of Hedgehog and Wnt pathway genes in NC;Pak2-/- mesothelioma cells versus NC;Pak2+/+ mesothelioma cells. Targeting of the Hedgehog signaling component Gli1 or its target gene Myc inhibited cell viability and spheroid formation in NC;P+/+ mesothelioma cells. Kinome profiling uncovered kinase changes indicative of EMT in NC;Pak2-/- mesothelioma cells, suggesting that Pak2-deficient malignant mesotheliomas can adapt by reprogramming their kinome in the absence of Pak activity. The identification of such compensatory pathways offers opportunities for rational combination therapies to circumvent resistance to anti-PAK drugs., Implications: We provide evidence supporting a role for PAK inhibitors in treating NF2-deficient tumors. NF2-deficient tumors lacking Pak2 eventually adapt by kinome reprogramming, presenting opportunities for combination therapies to bypass anti-PAK drug resistance., (©2022 American Association for Cancer Research.)

We investigated the contributions of platelet microRNAs (miRNAs) to the rate of growth and regulation of gene expression in primary ectopic tumors using mouse models. We previously identified an inhibitory role for platelets in solid tumor growth, mediated by tumor infiltration of platelet microvesicles (microparticles) which are enriched in platelet-derived miRNAs. To investigate the specific roles of platelet miRNAs in tumor growth models, we implanted pancreatic ductal adenocarcinoma cells as a bolus into mice with megakaryocyte-/platelet-specific depletion of mature miRNAs. We observed an ~50% increase in the rate of growth of ectopic primary tumors in these mice compared to controls including at early stages, associated with reduced apoptosis in the tumors, in particular in tumor cells associated with platelet microvesicles-which were depleted of platelet-enriched miRNAs-demonstrating a specific role for platelet miRNAs in modulation of primary tumor growth. Differential expression RNA sequencing of tumor cells isolated from advanced primary tumors revealed a broad cohort of mRNAs modulated in the tumor cells as a function of host platelet miRNAs. Altered genes comprised 548 up-regulated transcripts and 43 down-regulated transcripts, mostly mRNAs altogether spanning a variety of growth signaling pathways-notably pathways related to epithelial-mesenchymal transition-in tumor cells from platelet miRNA-deleted mice compared with those from control mice. Tumors in platelet miRNA-depleted mice showed more sarcomatoid growth and more advanced tumor grade, indicating roles for host platelet miRNAs in tumor plasticity. We further validated increased protein expression of selected genes associated with increased cognate mRNAs in the tumors due to platelet miRNA depletion in the host animals, providing proof of principle of widespread effects of platelet miRNAs on tumor cell functional gene expression in primary tumors in vivo. Together, these data demonstrate that platelet-derived miRNAs modulate solid tumor growth in vivo by broad-spectrum restructuring of the tumor cell transcriptome., Competing Interests: The authors have declared that no competing interests exist.

Nestin, a class IV intermediate filament protein, is generally considered as a putative marker of neural stem and progenitor cells in the central nervous system. Glioma is a common type of adult brain tumors, and glioblastoma (GBM) represents the most aggressive form of glioma. Here, we report that Nestin expression is significantly upregulated in human GBM, compared with other types of glioma. Nestin knockdown or deletion in U251 cells and tumor cells from GBM patients derived xenografts resulted in G 2 -M arrest, finally leading to apoptosis in tumor cells. Using proximity-dependent biotin identification method, we identified βII-tubulin as an interacting protein of Nestin in U251 cells. Nestin stabilized βII-tubulin in U251 cells through physical interaction. Knockdown of Nestin or βII-tubulin disrupted spindle morphology in tumor cells. Our studies further revealed that Nestin deficiency in U251 cells and GBM PDX cells repressed tumor growth upon transplantation. Finally, we found that Nestin deficiency sensitized GBM cells to microtubule-destabilizing drugs such as vinblastine and vincristine. Our studies demonstrate the essential functions and underlying mechanisms of Nestin in the growth and drug response of GBM cells. IMPLICATIONS: Through interaction with βII-tubulin, Nestin facilitates cell-cycle progression and spindle assembly of tumor cells in glioblastoma., (©2021 American Association for Cancer Research.)

Astrocytes, a major glial cell type in the brain, play a critical role in supporting the progression of medulloblastoma (MB), the most common malignant pediatric brain tumor. Through lineage tracing analyses and single-cell RNA sequencing, we demonstrate that astrocytes are predominantly derived from the transdifferentiation of tumor cells in relapsed MB (but not in primary MB), although MB cells are generally believed to be neuronal-lineage committed. Such transdifferentiation of MB cells relies on Sox9, a transcription factor critical for gliogenesis. Our studies further reveal that bone morphogenetic proteins (BMPs) stimulate the transdifferentiation of MB cells by inducing the phosphorylation of Sox9. Pharmacological inhibition of BMP signaling represses MB cell transdifferentiation into astrocytes and suppresses tumor relapse. Our studies establish the distinct cellular sources of astrocytes in primary and relapsed MB and provide an avenue to prevent and treat MB relapse by targeting tumor cell transdifferentiation., Competing Interests: Disclosures: The authors declare no competing interests exist., (© 2021 Guo et al.)

To better understand the etiology of inflammatory breast cancer (IBC) and identify potential therapies, we studied genomic alterations in IBC patients. Targeted, next-generation sequencing (NGS) was performed on cell-free DNA (cfDNA) ( n = 33) and paired DNA from tumor tissues ( n = 29) from 32 IBC patients. We confirmed complementarity between cfDNA and tumor tissue genetic profiles. We found a high incidence of germline variants in IBC patients that could be associated with an increased risk of developing the disease. Furthermore, 31% of IBC patients showed deficiencies in the homologous recombination repair (HRR) pathway (BRCA1, BRCA2, PALB2, RAD51C, ATM, BARD1) making them sensitive to poly (ADP-ribose) polymerase (PARP) inhibitors. We also characterized the tumor-infiltrating lymphocytes (TILs) in tumor tissue biopsies by studying several markers (CD4, CD8, FoxP3, CD20, PD-1, and PD-L1) through immunohistochemistry (IHC) staining. In 7 of 24 (29%) patients, tumor biopsies were positive for PD-L1 and PD-1 expression on TILs, making them sensitive to PD-1/PD-L1 blocking therapies. Our results provide a rationale for considering PARP inhibitors and PD-1/PDL1 blocking immunotherapy in qualifying IBC patients.

The transcription factor ThPOK (encoded by the Zbtb7b gene) controls homeostasis and differentiation of mature helper T cells, while opposing their differentiation to CD4 + intraepithelial lymphocytes (IELs) in the intestinal mucosa. Thus CD4 IEL differentiation requires ThPOK transcriptional repression via reactivation of the ThPOK transcriptional silencer element (Sil ThPOK ). In the present study, we describe a new autoregulatory loop whereby ThPOK binds to the Sil ThPOK to maintain its own long-term expression in CD4 T cells. Disruption of this loop in vivo prevents persistent ThPOK expression, leads to genome-wide changes in chromatin accessibility and derepresses the colonic regulatory T (T reg ) cell gene expression signature. This promotes selective differentiation of naive CD4 T cells into GITR lo PD-1 lo CD25 lo (Triple lo ) T reg cells and conversion to CD4 + IELs in the gut, thereby providing dominant protection from colitis. Hence, the ThPOK autoregulatory loop represents a key mechanism to physiologically control ThPOK expression and T cell differentiation in the gut, with potential therapeutic relevance., (© 2021. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Cystathionine beta-synthase (CBS) is a key enzyme of the trans-sulfuration pathway that converts homocysteine to cystathionine. Loss of CBS activity due to mutation results in CBS deficiency, an inborn error of metabolism characterized by extreme elevation of plasma total homocysteine (tHcy). C57BL6 mice containing either a homozygous null mutation in the cystathionine β-synthase (Cbs -/- ) gene or an inactive human CBS protein (Tg-G307S Cbs -/- ) are born in mendelian numbers, but the vast majority die between 18 and 21 days of age due to liver failure. However, adult Cbs null mice that express a hypomorphic allele of human CBS as a transgene (Tg-I278T Cbs -/- ) show almost no neonatal lethality despite having serum tHcy levels similar to mice with no CBS activity. Here, we characterize liver and serum metabolites in neonatal Cbs +/- , Tg-G307S Cbs -/- , and Tg-I278T Cbs -/- mice at 6, 10, and 17 days of age to understand this difference. In serum, we observe similar elevations in tHcy in both Tg-G307S Cbs -/- and Tg-I278T Cbs -/- compared to control animals, but methionine is much more severely elevated in Tg-G307S Cbs -/- mice. Large scale metabolomic analysis of liver tissue confirms that both methionine and methionine-sulfoxide are significantly more elevated in Tg-G307S Cbs -/- animals, along with significant differences in several other metabolites including hexoses, amino acids, other amines, lipids, and carboxylic acids. Our data are consistent with a model that the neonatal lethality observed in CBS-null mice is driven by excess methionine resulting in increased stress on a variety of related pathways including the urea cycle, TCA cycle, gluconeogenesis, and phosphatidylcholine biosynthesis., (© 2021 Federation of American Societies for Experimental Biology.)

Ferroptosis is associated with lipid hydroperoxides generated by the oxidation of polyunsaturated acyl chains. Lipid hydroperoxides are reduced by glutathione peroxidase 4 (GPX4) and GPX4 inhibitors induce ferroptosis. However, the therapeutic potential of triggering ferroptosis in cancer cells with polyunsaturated fatty acids is unknown. Here, we identify conjugated linoleates including α-eleostearic acid (αESA) as ferroptosis inducers. αESA does not alter GPX4 activity but is incorporated into cellular lipids and promotes lipid peroxidation and cell death in diverse cancer cell types. αESA-triggered death is mediated by acyl-CoA synthetase long-chain isoform 1, which promotes αESA incorporation into neutral lipids including triacylglycerols. Interfering with triacylglycerol biosynthesis suppresses ferroptosis triggered by αESA but not by GPX4 inhibition. Oral administration of tung oil, naturally rich in αESA, to mice limits tumor growth and metastasis with transcriptional changes consistent with ferroptosis. Overall, these findings illuminate a potential approach to ferroptosis, complementary to GPX4 inhibition.

Non-small cell lung cancer (NSCLC) has limited treatment options. Expression of the RNA-binding protein (RBP) Musashi-2 (MSI2) is elevated in a subset of non-small cell lung cancer (NSCLC) tumors upon progression, and drives NSCLC metastasis. We evaluated the mechanism of MSI2 action in NSCLC to gain therapeutically useful insights. Reverse phase protein array (RPPA) analysis of MSI2-depleted versus control Kras LA1/+ ; Trp53 R172HΔG/+ NSCLC cell lines identified EGFR as a MSI2-regulated protein. MSI2 control of EGFR expression and activity in an NSCLC cell line panel was studied using RT-PCR, Western blots, and RNA immunoprecipitation. Functional consequences of MSI2 depletion were explored for cell growth and response to EGFR-targeting drugs, in vitro and in vivo. Expression relationships were validated using human tissue microarrays. MSI2 depletion significantly reduced EGFR protein expression, phosphorylation, or both. Comparison of protein and mRNA expression indicated a post-transcriptional activity of MSI2 in control of steady state levels of EGFR. RNA immunoprecipitation analysis demonstrated that MSI2 directly binds to EGFR mRNA, and sequence analysis predicted MSI2 binding sites in the murine and human EGFR mRNAs. MSI2 depletion selectively impaired cell proliferation in NSCLC cell lines with activating mutations of EGFR (EGFR mut ). Further, depletion of MSI2 in combination with EGFR inhibitors such as erlotinib, afatinib, and osimertinib selectively reduced the growth of EGFR mut NSCLC cells and xenografts. EGFR and MSI2 were significantly co-expressed in EGFR mut human NSCLCs. These results define MSI2 as a direct regulator of EGFR protein expression, and suggest inhibition of MSI2 could be of clinical value in EGFR mut NSCLC.

Pancreatic ductal adenocarcinoma (PDAC) has a poor 5-year survival rate and lacks effective therapeutics. Therefore, it is of paramount importance to identify new targets. Using multiplex data from patient tissue, three-dimensional coculturing in vitro assays, and orthotopic murine models, we identified Netrin G1 (NetG1) as a promoter of PDAC tumorigenesis. We found that NetG1 + cancer-associated fibroblasts (CAF) support PDAC survival, through a NetG1-mediated effect on glutamate/glutamine metabolism. Also, NetG1 + CAFs are intrinsically immunosuppressive and inhibit natural killer cell-mediated killing of tumor cells. These protumor functions are controlled by a signaling circuit downstream of NetG1, which is comprised of AKT/4E-BP1, p38/FRA1, vesicular glutamate transporter 1, and glutamine synthetase. Finally, blocking NetG1 with a neutralizing antibody stunts in vivo tumorigenesis, suggesting NetG1 as potential target in PDAC. SIGNIFICANCE: This study demonstrates the feasibility of targeting a fibroblastic protein, NetG1, which can limit PDAC tumorigenesis in vivo by reverting the protumorigenic properties of CAFs. Moreover, inhibition of metabolic proteins in CAFs altered their immunosuppressive capacity, linking metabolism with immunomodulatory function. See related commentary by Sherman, p. 230 . This article is highlighted in the In This Issue feature, p. 211 ., (©2020 American Association for Cancer Research.)

Background: Inflammatory breast cancer (IBC) is a rare but aggressive carcinoma characterized by severe erythema and edema of the breast, with many patients presenting in advanced metastatic disease. The "inflammatory" nature is not due to classic immune-mediated inflammation, but instead results from tumor-mediated blockage of dermal lymphatic ducts. Previous work has shown that expression of PD-L1 on tumor cells can suppress T cell activation in triple-negative (TN) non-IBC breast cancer. In the present work, we investigated immune parameters in peripheral blood of metastatic IBC patients to determine whether cellular components of the immune system are altered, thereby contributing to pathogenesis of the disease. These immune parameters were also compared to PD-1 and PD-L1 expression in IBC tumor biopsies., Methods: Flow cytometry-based immune phenotyping was performed using fresh peripheral blood from 14 stage IV IBC patients and compared to 11 healthy age-similar control women. Immunohistochemistry for CD20, CD3, PD-1, and PD-L1 was performed on tumor biopsies of these metastatic IBC patients., Results: IBC patients with Stage IV disease had lymphopenia with significant reductions in circulating T, B, and NK cells. Reductions were observed in all subsets of CD4 + T cells, whereas reductions in CD8 + T cells were more concentrated in memory subsets. Immature cytokine-producing CD56 bright NK cells expressed higher levels of FcγRIIIa and cytolytic granule components, suggesting accelerated maturation to cytolytic CD56 dim cells. Immunohistochemical analysis of tumor biopsies demonstrated moderate to high expression of PD-1 in 18.2% of patients and of PD-L1 in 36.4% of patients. Interestingly, a positive correlation was observed between co-expression levels of PD-L1 and PD-1 in tumor biopsies, and higher expression of PD-L1 in tumor biopsies correlated with higher expression of cytolytic granule components in blood CD4 + T cells and CD56 dim NK cells, and higher numbers of CD8 + effector memory T cells in peripheral blood. PD-1 expression in tumor also correlated with increased infiltration of CD20 + B cells in the tumor., Conclusions: Our results suggest that while lymphocyte populations are severely compromised in stage IV IBC patients, an immune response toward the tumor had occurred in some patients, providing biological rationale to evaluate PD-1/PD-L1 immunotherapies for IBC.

Endometrial carcinoma (EC) is the most common gynecologic malignancy in the United States, with limited effective targeted therapies. Endometrial tumors exhibit frequent alterations in protein kinases, yet only a small fraction of the kinome has been therapeutically explored. To identify kinase therapeutic avenues for EC, we profiled the kinome of endometrial tumors and normal endometrial tissues using Multiplexed Inhibitor Beads and Mass Spectrometry (MIB-MS). Our proteomics analysis identified a network of kinases overexpressed in tumors, including Serine/Arginine-Rich Splicing Factor Kinase 1 (SRPK1). Immunohistochemical (IHC) analysis of endometrial tumors confirmed MIB-MS findings and showed SRPK1 protein levels were highly expressed in endometrioid and uterine serous cancer (USC) histological subtypes. Moreover, querying large-scale genomics studies of EC tumors revealed high expression of SRPK1 correlated with poor survival. Loss-of-function studies targeting SRPK1 in an established USC cell line demonstrated SRPK1 was integral for RNA splicing, as well as cell cycle progression and survival under nutrient deficient conditions. Profiling of USC cells identified a compensatory response to SRPK1 inhibition that involved EGFR and the up-regulation of IGF1R and downstream AKT signaling. Co-targeting SRPK1 and EGFR or IGF1R synergistically enhanced growth inhibition in serous and endometrioid cell lines, representing a promising combination therapy for EC., Competing Interests: Conflict of interest—The authors declare that they have no conflicts of interest with the contents of this article., (© 2020 Kurimchak et al.)

Oncogenic transformation alters lipid metabolism to sustain tumor growth. We define a mechanism by which cholesterol metabolism controls the development and differentiation of pancreatic ductal adenocarcinoma (PDAC). Disruption of distal cholesterol biosynthesis by conditional inactivation of the rate-limiting enzyme Nsdhl or treatment with cholesterol-lowering statins switches glandular pancreatic carcinomas to a basal (mesenchymal) phenotype in mouse models driven by Kras G12D expression and homozygous Trp53 loss. Consistently, PDACs in patients receiving statins show enhanced mesenchymal features. Mechanistically, statins and NSDHL loss induce SREBP1 activation, which promotes the expression of Tgfb1, enabling epithelial-mesenchymal transition. Evidence from patient samples in this study suggests that activation of transforming growth factor β signaling and epithelial-mesenchymal transition by cholesterol-lowering statins may promote the basal type of PDAC, conferring poor outcomes in patients., Competing Interests: Declaration of Interests I.A. served as a consultant for Caris Life Sciences, Inc., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Synthetic lethality triggered by PARP inhibitor (PARPi) yields promising therapeutic results. Unfortunately, tumor cells acquire PARPi resistance, which is usually associated with the restoration of homologous recombination, loss of PARP1 expression, and/or loss of DNA double-strand break (DSB) end resection regulation. Here, we identify a constitutive mechanism of resistance to PARPi. We report that the bone marrow microenvironment (BMM) facilitates DSB repair activity in leukemia cells to protect them against PARPi-mediated synthetic lethality. This effect depends on the hypoxia-induced overexpression of transforming growth factor beta receptor (TGFβR) kinase on malignant cells, which is activated by bone marrow stromal cells-derived transforming growth factor beta 1 (TGF-β1). Genetic and/or pharmacological targeting of the TGF-β1-TGFβR kinase axis results in the restoration of the sensitivity of malignant cells to PARPi in BMM and prolongs the survival of leukemia-bearing mice. Our finding may lead to the therapeutic application of the TGFβR inhibitor in patients receiving PARPis., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.)

The Dlx5 homeobox gene was first implicated as an oncogene in a T-ALL mouse model expressing myristoylated (Myr) Akt2. Furthermore, overexpression of Dlx5 was sufficient to drive T-ALL in mice by directly activating Akt and Notch signaling. These findings implied that Akt2 cooperates with Dlx5 in T-cell lymphomagenesis. To test this hypothesis, Lck-Dlx5;Lck-MyrAkt2 transgenic mice were generated. MyrAkt2 synergized with Dlx5 to greatly accelerate and enhance the dissemination of T-lymphomagenesis. RNA-seq analysis performed on lymphomas from Lck-Dlx5;Lck-MyrAkt mice revealed upregulation of genes involved in the Wnt and cholesterol biosynthesis pathways. Combined RNA-seq and ChIP-seq analysis of lymphomas from Lck-Dlx5;Lck-MyrAkt mice demonstrated that β-catenin directly regulates genes involved in sterol regulatory element binding transcription factor 2 (Srebf2)-cholesterol synthesis. These lymphoma cells had high Lef1 levels and were highly sensitive to β-catenin and Srebf2-cholesterol synthesis inhibitors. Similarly, human T-ALL cell lines with activated NOTCH and AKT and elevated LEF1 levels were sensitive to inhibition of β-catenin and cholesterol pathways. Furthermore, LEF1 expression positively correlated with expression of genes involved in the cholesterol synthesis pathway in primary human T-ALL specimens. Together, these data suggest that targeting β-catenin and/or cholesterol biosynthesis, together with AKT, could have therapeutic efficacy in a subset of T-ALL patients.

BRCA1 gene mutations impair homologous recombination (HR) DNA repair, resulting in cellular senescence and embryonic lethality in mice. Therefore, BRCA1-deficient cancers require adaptations that prevent excessive genomic alterations from triggering cell death. RNF168-mediated ubiquitination of γH2AX at K13/15 (ub-H2AX) serves as a recruitment module for the localization of 53BP1 to DNA break sites. Here, we found multiple BRCA1 -mutant cancer cell lines and primary tumors with low levels of RNF168 protein expression. Overexpression of ectopic RNF168 or a ub-H2AX fusion protein induced cell death and delayed BRCA1 -mutant tumor formation. Cell death resulted from the recruitment of 53BP1 to DNA break sites and inhibition of DNA end resection. Strikingly, reintroduction of BRCA1 or 53BP1 depletion restored HR and rescued the ability of cells to maintain RNF168 and ub-H2AX overexpression. Thus, downregulation of RNF168 protein expression is a mechanism for providing BRCA1-null cancer cell lines with a residual level of HR that is essential for viability. Overall, our work identifies loss of RNF168 ubiquitin signaling as a proteomic alteration that supports BRCA1 -mutant carcinogenesis. We propose that restoring RNF168-ub-H2AX signaling, potentially through inhibition of deubiquitinases, could represent a new therapeutic approach. SIGNIFICANCE: This study explores the concept that homologous recombination DNA repair is not an all-or-nothing concept, but a spectrum, and that where a tumor stands on this spectrum may have therapeutic relevance. See related commentary by Wang and Wulf, p. 2720 ., (©2020 American Association for Cancer Research.)

Tumor cells are characterized by unlimited proliferation and perturbed differentiation. Using single-cell RNA sequencing, we demonstrate that tumor cells in medulloblastoma (MB) retain their capacity to differentiate in a similar way as their normal originating cells, cerebellar granule neuron precursors. Once they differentiate, MB cells permanently lose their proliferative capacity and tumorigenic potential. Differentiated MB cells highly express NeuroD1, a helix-loop-helix transcription factor, and forced expression of NeuroD1 promotes the differentiation of MB cells. The expression of NeuroD1 in bulk MB cells is repressed by trimethylation of histone 3 lysine-27 (H3K27me3). Inhibition of the histone lysine methyltransferase EZH2 prevents H3K27 trimethylation, resulting in increased NeuroD1 expression and enhanced differentiation in MB cells, which consequently reduces tumor growth. These studies reveal the mechanisms underlying MB cell differentiation and provide rationales to treat MB (potentially other malignancies) by stimulating tumor cell differentiation., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.)

BRCA1 promotes the DNA end resection and RAD51 loading steps of homologous recombination (HR). Whether these functions can be uncoupled, and whether mutant proteins retaining partial activity can complement one another, is unclear and could affect the severity of BRCA1-associated Fanconi anemia (FA). Here we generated a Brca1 CC mouse with a coiled-coil (CC) domain deletion. Brca1 CC/CC mice are born at low frequencies, and post-natal mice have FA-like abnormalities, including bone marrow failure. Intercrossing with Brca1 Δ11 , which is homozygous lethal, generated Brca1 CC/Δ11 mice at Mendelian frequencies that were indistinguishable from Brca1 +/+ mice. Brca1 CC and Brca1 Δ11 proteins were individually responsible for counteracting 53BP1-RIF1-Shieldin activity and promoting RAD51 loading, respectively. Thus, Brca1 CC and Brca1 Δ11 alleles represent separation-of-function mutations that combine to provide a level of HR sufficient for normal development and hematopoiesis. Because BRCA1 activities can be genetically separated, compound heterozygosity for functional complementary mutations may protect individuals from FA., Competing Interests: Declaration of Interests The authors declare no conflicts of interest., (Copyright © 2020 Elsevier Inc. All rights reserved.)

High-grade serous ovarian carcinoma (HGSOC) is the most lethal gynecological cancer with few effective, targeted therapies. HGSOC tumors exhibit genomic instability with frequent alterations in the protein kinome; however, only a small fraction of the kinome has been therapeutically targeted in HGSOC. Using multiplexed inhibitor beads and mass spectrometry, we mapped the kinome landscape of HGSOC tumors from patients and patient-derived xenograft models. The data revealed a prevalent signature consisting of established HGSOC driver kinases, as well as several kinases previously unexplored in HGSOC. Loss-of-function analysis of these kinases in HGSOC cells indicated MRCKA (also known as CDC42BPA) as a putative therapeutic target. Characterization of the effects of MRCKA knockdown in established HGSOC cell lines demonstrated that MRCKA was integral to signaling that regulated the cell cycle checkpoint, focal adhesion, and actin remodeling, as well as cell migration, proliferation, and survival. Moreover, inhibition of MRCKA using the small-molecule BDP9066 decreased cell proliferation and spheroid formation and induced apoptosis in HGSOC cells, suggesting that MRCKA may be a promising therapeutic target for the treatment of HGSOC., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)