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Cancer-associated Fibroblasts (CAFs) have emerged as critical regulators of anti-tumour immunity, with both beneficial and detrimental properties that remain poorly characterised. To investigate this, we performed single-cell and spatial transcriptomic analysis, comparing head & neck squamous cell carcinoma (HNSCC) subgroups, which although heterogenous, can be considered broadly immune-hot and immune-cold (human papillomavirus [HPV]+ve and HPV-ve tumours respectively). This identified six fibroblast subpopulations, including two with immunomodulatory gene expression profiles ( IL-11 +  inflammatory [i]CAF and CCL19  + fibroblastic reticular cell [FRC]-like). IL-11 +  iCAF were spatially associated with inflammatory monocytes and regulated in vitro through synergistic activation of canonical NF-κB signalling by IL-1β and TNF-α. FRC-like were enriched in immune-hot HPV+ve tumours, associated with CD4 + T-cells and B-cells in tertiary lymphoid structures and regulated through non-canonical NF-κB signalling via lymphotoxin. Pan-cancer analysis revealed several ‘iCAF’ subgroups present in both normal and cancer tissues; IL11 +  iCAF were found in cancers from the gastrointestinal (GI) tract and transcriptomically distinct from iCAFs previously described in pancreatic and breast cancers with greater inflammatory properties; FRC-like fibroblasts were present at low frequencies in all tumour types, and were associated with significantly better survival in patients receiving checkpoint immunotherapy. This work clarifies and expands current literature on immunomodulatory CAFs, highlighting links with important immunological niches.

Immunoglobulin heavy chain (IgH) variable region exons are assembled from V H , D and J H gene segments in developing B lymphocytes. Within the 2.7-megabase mouse Igh locus, V(D)J recombination is regulated to ensure specific and diverse antibody repertoires. Here we report in mice a key Igh V(D)J recombination regulatory region, termed intergenic control region 1 (IGCR1), which lies between the V H and D clusters. Functionally, IGCR1 uses CTCF looping/insulator factor-binding elements and, correspondingly, mediates Igh loops containing distant enhancers. IGCR1 promotes normal B-cell development and balances antibody repertoires by inhibiting transcription and rearrangement of D H -proximal V H gene segments and promoting rearrangement of distal V H segments. IGCR1 maintains ordered and lineage-specific V H (D)J H recombination by suppressing V H joining to D segments not joined to J H segments, and V H to DJ H joins in thymocytes, respectively. IGCR1 is also required for feedback regulation and allelic exclusion of proximal V H -to-DJ H recombination. Our studies elucidate a long-sought Igh V(D)J recombination control region and indicate a new role for the generally expressed CTCF protein. Enforcing order in heavy chains The variable region of the immunoglobulin heavy chain (IgH) is generated by selecting one segment each from the V H , D and J H loci. Many studies have proposed the existence of a control region between the V H and D loci that would regulate V H -to-D recombination, but its identity has been elusive. Frederick Alt and colleagues have now located this regulatory sequence, IGCR1 (intergenic control region 1), and show that it uses CTCF sites to promote looping and interaction with distal enhancer elements. It also suppresses other events that would disrupt the ordered rearrangement of the V H , D and J H loci.

Chronic hepatitis B virus (HBV) infection is characterized by the presence of high circulating levels of non-infectious lipoprotein-like HBV surface antigen (HBsAg) particles thought to contribute to chronic immune dysfunction in patients. Lipid and metabolomic analysis of humanized livers from immunodeficient chimeric mice (uPA/SCID) revealed that HBV infection dysregulates several lipid metabolic pathways. Small molecule inhibitors of lipid biosynthetic pathway enzymes acetyl-CoA carboxylase (ACC), fatty acid synthase, and subtilisin kexin isozyme-1/site-1 protease in HBV-infected HepG2-NTCP cells demonstrated potent and selective reduction of extracellular HBsAg. However, a liver-targeted ACC inhibitor did not show antiviral activity in HBV-infected liver chimeric mice, despite evidence of on-target engagement. Our study suggests that while HBsAg production may be dependent on hepatic de novo lipogenesis in vitro, this may be overcome by extrahepatic sources (such as lipolysis or diet) in vivo. Thus, a combination of agents targeting more than one lipid metabolic pathway may be necessary to reduce HBsAg levels in patients with chronic HBV infection.

Chronic hepatitis B virus (HBV) infection is characterized by the presence of high circulating levels of non-infectious lipoprotein-like HBV surface antigen (HBsAg) particles thought to contribute to chronic immune dysfunction in patients. Lipid and metabolomic analysis of humanized livers from immunodeficient chimeric mice (uPA/SCID) revealed that HBV infection dysregulates several lipid metabolic pathways. Small molecule inhibitors of lipid biosynthetic pathway enzymes acetyl-CoA carboxylase (ACC), fatty acid synthase, and subtilisin kexin isozyme-1/site-1 protease in HBV-infected HepG2-NTCP cells demonstrated potent and selective reduction of extracellular HBsAg. However, a liver-targeted ACC inhibitor did not show antiviral activity in HBV-infected liver chimeric mice, despite evidence of on-target engagement. Our study suggests that while HBsAg production may be dependent on hepatic de novo lipogenesis in vitro , this may be overcome by extrahepatic sources (such as lipolysis or diet) in vivo . Thus, a combination of agents targeting more than one lipid metabolic pathway may be necessary to reduce HBsAg levels in patients with chronic HBV infection.

Background HPV status is a key determinant of prognosis and treatment response in head and neck squamous cell carcinoma (HNSCC). To investigate how HPV influences the tumor-immune-stromal landscape, we performed high-dimensional spatial profiling, including its impact on spatial organization, tertiary lymphoid structures (TLSs), and spatially organized cellular neighborhoods. Methods Tumor biopsies from HNSCC patients ( n  = 16; 7 HPV-positive, 9 HPV-negative) were stained with a multiplex immunofluorescence (mIF) panel focused on immune profiling. A deep learning-based analysis pipeline enabled the identification and phenotypic state profiling of 14 cell types. Tissues were segmented into four distinct tumor regions, and spatial neighborhoods and TLSs were identified and analyzed for differential cellular composition, activation states, and spatial interactions between HPV-positive and HPV-negative tumors. Results HPV-positive and HPV-negative tumors differ in their tumor microenvironment (TME) composition, tumor cell state and spatial organization. The TME of HPV-positive tumors exhibited a greater abundance of activated lymphocytes, B- and T-cell-enriched spatial neighborhoods, and PD-1–PD-L1 interactions within the tumor area, whereas HPV-negative tumors were dominated by fibroblast- and macrophage-rich niches. T - cells in HPV-positive tumors showed greater activation across neighborhoods and areas, while in HPV-negative tumors T - cells demonstrated enrichment of exhaustion and terminal differentiation markers such as PD-1 and CD57. HPV-positive tumor cells had increased IDO1, HLA-DR, and Ki67 positivity, whereas HPV-negative tumor cells were more frequently CD44 positive, reflecting a more stem-like phenotype. Importantly, TLSs in HPV-positive tumors were located closer to the tumor area and enriched in activated immune cells, including ICOS + CD4 T - cells, memory T - cells, and CD21 + B- cells. In contrast, TLSs in HPV-negative tumors were more distant and enriched for immunosuppressive populations such as PD-1 + /PD-L1 + Tregs and macrophages. Conclusions HPV status defines distinct spatial immune architectures in HNSCC. HPV-positive tumors harbor immune-activating TLSs and cellular neighborhoods that support antitumor immunity, whereas HPV-negative tumors exhibit suppressive niches and stromal dominance. These findings highlight TLSs, particularly their proximity and composition, as key features of the HPV-stratified TME and potential biomarkers for immunotherapy response. Graphical abstract

SignificanceInhibitors of bromodomain and extraterminal domain family proteins (BETi) have generated considerable excitement and are in clinical trials for treatment of several cancers. Cancers treated with targeted therapies eventually become resistant, yet molecular mechanisms underlying resistance to BETi are poorly understood. To discover novel molecular mechanisms mediating resistance to BETi, we performed a shRNA-based genetic screen. We found that loss of tripartite motif-containing protein 33 (TRIM33), a chromatin-associated E3 ubiquitin ligase, confers resistance to BETi. TRIM33 loss diminished BETi-mediated reduction in MYC expression and enhanced TGF-β signaling. Notably, inhibition of TGF-β signaling increased sensitivity of cells to the antiproliferative effects of BETi. In particular, a TGF-β receptor inhibitor potentiated growth suppression by BETi, suggesting a clinically viable strategy for combination therapy. Bromodomain and extraterminal domain protein inhibitors (BETi) hold great promise as a novel class of cancer therapeutics. Because acquired resistance typically limits durable responses to targeted therapies, it is important to understand mechanisms by which tumor cells adapt to BETi. Here, through pooled shRNA screening of colorectal cancer cells, we identified tripartite motif-containing protein 33 (TRIM33) as a factor promoting sensitivity to BETi. We demonstrate that loss of TRIM33 reprograms cancer cells to a more resistant state through at least two mechanisms. TRIM33 silencing attenuates down-regulation of MYC in response to BETi. Moreover, loss of TRIM33 enhances TGF-β receptor expression and signaling, and blocking TGF-β receptor activity potentiates the antiproliferative effect of BETi. These results describe a mechanism for BETi resistance and suggest that combining inhibition of TGF-β signaling with BET bromodomain inhibition may offer new therapeutic benefits.

Regulated expression of miRNAs influences development in a wide variety of contexts. We report here that miR290-5p (100049710) and miR292-5p (100049711) are induced at the pre-B stage of murine B cell development and that they influence assembly of the Igκ light chain gene (243469) by contributing to the activation of germline Igκ transcription (κGT). We found that upon forced over-expression of miR290-5p/292-5p in Abelson Murine Leukemia Virus (AMuLV) transformed pro-B cells, two known activators of κGT, E2A (21423) and NF-κB (19697), show increased chromosomal binding to the kappa intronic enhancer. Conversely, knockdown of miR290-5p/292-5p in AMuLV pro-B cells blunts drug-induced activation of κGT. Furthermore, miR290-5p/292-5p knockdown also diminishes κGT activation, but not Rag1/2 (19373, 19374) expression, in an IL-7 dependent primary pro-B cell culture system. In addition, we identified a deficiency in κGT induction in miR290 cluster knockout mice. We hypothesize that increased expression of miR290-5p and miR292-5p contributes to the induction of κGT at the pre-B stage of B cell development through increased binding of NF-κB and E2A to kappa locus regulatory sequences.

Dysregulated hepatocyte lipid metabolism is a hallmark of hepatic lipotoxicity and contributes to the pathogenesis of nonalcoholic steatohepatitis (NASH). Acetyl CoA carboxylase (ACC) inhibitors decrease hepatocyte lipotoxicity by inhibiting de novo lipogenesis and concomitantly increasing fatty acid oxidation (FAO), and firsocostat, a liver‐targeted inhibitor of ACC1/2, is under evaluation clinically in patients with NASH. ACC inhibition is associated with improvements in indices of NASH and reduced liver triglyceride (TG) content, but also increased circulating TG in subjects with NASH and preclinical rodent models. Here we evaluated whether enhancing hepatocyte FAO by combining ACC inhibitors with peroxisomal proliferator‐activated receptor (PPAR) or thyroid hormone receptor beta (THRβ) agonists could drive greater liver TG reduction and NASH/antifibrotic efficacy, while ameliorating ACC inhibitor–induced hypertriglyceridemia. In high‐fat diet–fed dyslipidemic rats, the addition of PPAR agonists fenofibrate (Feno), elafibranor (Ela), lanifibranor (Lani), seladelpar (Sela) or saroglitazar (Saro), or the THRb agonist resmetirom (Res), to an analogue of firsocostat (ACCi) prevented ACCi‐induced hypertriglyceridemia. However, only PPARα agonists (Feno and Ela) and Res provided additional liver TG reduction. In the choline‐deficient high‐fat diet rat model of advanced liver fibrosis, neither PPARα (Feno) nor THRβ (Res) agonism augmented the antifibrotic efficacy of ACCi. Conclusion: These data suggest that combination therapies targeting hepatocyte lipid metabolism may have beneficial effects on liver TG reduction; however, they may not be sufficient to drive fibrosis regression. Here we evaluated whether combinations of an acetyl CoA carboxylase inhibitor with agents that modulate hepatocyte lipotoxicity would synergistically decrease liver triglyceride and inhibit fibrosis progression in rodent models. Our data clearly demonstrate that some combinations have synergistically lower liver TG without inhibiting fibrosis progression.

Signal regulatory protein alpha (SIRPα) is the receptor for cluster of differentiation (CD)47, a potent “don't eat me” signal for macrophages. Disruption of CD47‐SIRPα signaling in the presence of prophagocytic signals can lead to enhanced phagocytosis of tumor cells, resulting in a direct antitumor effect; agents targeting this pathway have shown efficacy in non‐Hodgkin lymphoma (NHL) and other tumor types. GS‐0189 is a novel anti‐SIRPα humanized monoclonal antibody. Here we report: (1) clinical safety, preliminary activity, and pharmacokinetics of GS‐0189 as monotherapy and in combination with rituximab from a phase 1 clinical trial in patients with relapsed/refractory NHL (NCT04502706, SRP001); (2) in vitro characterization of GS‐0189 binding to SIRPα; and (3) in vitro phagocytic activity. Clinically, GS‐0189 was well tolerated in patients with relapsed/refractory NHL with evidence of clinical activity in combination with rituximab. Receptor occupancy (RO) of GS‐0189 was highly variable in NHL patients; binding affinity studies showed significantly higher affinity for SIRPα variant 1 than variant 2, consistent with RO in patient and healthy donor samples. In vitro phagocytosis induced by GS‐0189 was also SIRPα variant–dependent. Although clinical development of GS‐0189 was discontinued, the CD47‐SIRPα signaling pathway remains a promising therapeutic target and should continue to be explored.

To what extent might the regulation of translation contribute to differentiation programs, or to the molecular pathogenesis of cancer? Pre-B cells transformed with the viral oncogene v-Abl are suspended in an immortalized, cycling state that mimics leukemias with a BCR-ABL1 translocation, such as Chronic Myelogenous Leukemia (CML) and Acute Lymphoblastic Leukemia (ALL). Inhibition of the oncogenic Abl kinase with imatinib reverses transformation, allowing progression to the next stage of B cell development. We employed a genome-wide polysome profiling assay called Gradient Encoding to investigate the extent and potential contribution of translational regulation to transformation and differentiation in v-Abl-transformed pre-B cells. Over half of the significantly translationally regulated genes did not change significantly at the level of mRNA abundance, revealing biology that might have been missed by measuring changes in transcript abundance alone. We found extensive, gene-specific changes in translation affecting genes with known roles in B cell signaling and differentiation, cancerous transformation, and cytoskeletal reorganization potentially affecting adhesion. These results highlight a major role for gene-specific translational regulation in remodeling the gene expression program in differentiation and malignant transformation.