Supplementary report addressing the cell line authentication and Mycoplasma testing

Source data for all immunostaining images in this paper

Durable therapeutic efficacy remains a major barrier to improving outcomes for patients with pancreatic ductal adenocarcinoma (PDAC). An immunosuppressive tumor microenvironment (TME) is a hallmark of PDAC and has been demonstrated to be a dominant driver of therapeutic resistance. The aberrant glycan CA19-9 is prevalent in PDAC and drives tumor progression, but the paracrine mechanisms by which it contributes to TME remodeling are unknown. To address this, we mapped TME changes and performed functional analyses using a genetically engineered mouse model (GEMM) harboring KrasG12D mutation and inducible CA19-9 expression. Elevation of CA19-9 led to expansion of antigen-presenting cancer associated fibroblasts (apCAFs) and regulatory T cells (Tregs), which can drive immunosuppression. Antibody blockade of CA19-9 resulted in significant restoration of normal histology and decreased apCAF and Treg populations. We dissected the paracrine signaling mechanisms that drive this TME remodeling in vitro using mouse and human organoid mono- and co-culture models as well as in vivo using GEMMs and syngeneic orthotopic transplantation models. CA19-9 induced IL1a and TGFb expression, reprogramming pancreatic mesothelial cells into apCAFs in vitro, which in turn directly ligated naïve Cd4+ T cells resulting in Treg differentiation in co-cultures. Antibody blockade of IL1a and TGFb in mice led to reduced apCAF and Treg differentiation. We previously reported that CA19-9 modification of the secreted Fbln3 protein increased Egfr engagement and now find that the induction of IL1a and TGFb expression by CA19-9 is dependent on Fbln3 hyperactivation of EGFR signaling. Genetic depletion of Fbln3 led to reduced tumor progression and increased Cd8+ T cell infiltration in mice. Together these findings identify a previously unknown signaling axis driving immunosuppressive phenotypes in PDAC, uncovering multiple potential nodes to relieve the immunosuppressive pressures within the PDAC TME.

The seminal discovery that the epidermal growth factor receptor (EGFR) possesses intrinsic EGF-stimulated tyrosine kinase activity that serves as a mitogenic signalling mechanism was dependent on two separate lines of research that intersected in the late 1970s and early 1980s, namely studies of retroviral transforming proteins and the identification of growth factor receptor proteins. This historical review outlines the events that ultimately led to the intersection of these two strands of research, resulting from the twin discoveries that many growth factor receptors, including EGFR, are ligand-activated receptor tyrosine kinases (RTKs), and that many retroviral transforming proteins are dysregulated tyrosine kinases that usurp RTK signalling pathways to drive continuous cell proliferation. A French translation of this abstract is available in the supplementary material. This article is part of the discussion meeting issue 'Epidermal growth factor receptor after 40 years'.

e16575 Background: PIN1 is a phosphorylation-dependent prolyl isomerase that regulates oncogenic pathways, including cancer stemness, epithelial–mesenchymal transition, immune modulation, and cholesterol biosynthesis. While mechanistic studies implicate PIN1 in aggressiveness across cancer types, its clinical relevance in UC remains unclear. Methods: The Tempus Lens platform was used to identify a real-world cohort of patients (pts) with UC that underwent DNA (Tempus xT) and RNA (Tempus xR) sequencing (N = 4,886). Short variants and copy number changes are reported in our analysis. RNA data were quantified as transcripts per million (TPM) and reported as log2(TPM+1). Pts were classified as PIN1 high (PIN1-h, N = 1,157) or low (PIN1-l, N = 1,211) based on top and bottom mRNA expression quartiles. Single-sample gene set enrichment analysis (ssGSEA) was used to characterize enrichment in MSigDB Hallmark and cholesterol related metabolic pathways. Real-world progression-free survival (rwPFS) and overall survival (rwOS) were measured from initiation of first line (1L) therapy and sample collection date respectively. Propensity score weighting was used for balancing hyperlipidemia history and biopsy site. Hazard ratios (HR) were calculated using multivariable Cox regression. Median rwOS estimated using Kaplan–Meier curves and compared using log-rank tests. Results: The cohort was composed of pts with bladder (83%) and upper tract (17.2%) UC. Of pts with documented staging, 62.9% were metastatic and 16% had a history of hyperlipidemia. PIN1-h tumors were enriched in pathways suggesting increased lipid biosynthesis such as cholesterol regulation ( q < 0.001), glycerolipid metabolism (q < 0.001) and SREBF signaling (q < 0.004) as well as in oncogenic pathways (WNT/Beta Catenin, Notch, p53, KRAS). Immune-signatures (IL6, JAK STAT3 , IFNa, IFNγ) showed decreased enrichment in PIN1-h. Somatic alterations differed, with PIN1-h tumors having higher prevalence of tumor suppressor gene alterations TP53 (68% vs 54%) and RB1 (23% vs 16%) and lower rates of alterations in FGFR3 (11% vs 18%) and ARID1A (18% vs 27%), consistent with aggressive disease biology. rwPFS was shorter in pts with PIN1-h vs PIN1-l tumors when treated with 1L chemotherapy (HR 3.01, 95% CI 1.43-6.33; p = 0.004) but not 1L immunotherapy. Paradoxically, pts with PIN1-h tumors showed a modest but statistically significant improvement in rwOS (HR 0.85, 95% CI 0.74–0.98; p = 0.030). Median rwOS was longer for PIN1-h vs PIN1-l (12.1 vs 15.5 months, p = 0.026). Conclusions: PIN1 expression identifies a distinct subset of UC characterized by cholesterol pathway enrichment and aggressive genomic features. While PIN1-h tumors show shorter rwPFS on 1L chemotherapy, they exhibit longer rwOS overall. These opposing outcomes highlight PIN1 as a complex, potentially treatment-specific prognostic biomarker.

The DNA damage response (DDR) is critical for pancreatic ductal adenocarcinoma (PDAC) development and therapeutic responses, including to genotoxic agents. While epigenetic modulators have been shown to contribute to the DDR, how chromatin regulation dictates responses to DNA damage in PDAC remains incompletely understood. Here, we identify Class I histone deacetylases (HDACs) as critical regulators of the DDR. HDAC1/2 direct the genomic distribution of H3K27ac, ensuring sufficient BRD4 and RNA polymerase II (Pol II) occupancy at DDR gene promoters. HDAC inhibition by entinostat shifts the balance of H3K27 acetylation preferentially toward intergenic regions, diverting BRD4 and Pol II from promoters, thereby suppressing DDR gene expression. In line with this, HDAC inhibition heightens DNA damage and sensitizes PDAC to diverse DNA-damaging and DDR-targeting agents. Since the clinical development of HDAC inhibitors has been limited by systemic toxicity, we developed bottlebrush prodrug (BPD) nanoparticles for tumor-selective entinostat delivery. Entinostat-BPD achieved tumor-specific HDAC inhibition while displaying potent efficacy and reduced systemic toxicity. These findings reveal an HDAC-dependent DDR vulnerability and offer combinational and precision targeting strategies to facilitate clinical translation and improve PDAC patient outcomes.

Supplementary Figure S1. Luminal urothelial cells show higher PIN1 expression levels. Supplementary Figure S2. PIN1 overexpression in normal immortalized SV-HUC-1 and RT4 huBLCA cells promotes cell proliferation, cell motility, invasion, migration and urothelium clearance. Supplementary Figure S3. PIN1 positively regulates T24 and 5637 cell proliferation and spheroid formation in vitro. Supplementary Figure S4. PIN1 positively regulates T24 and 5637 cell invasion, migration, cell motility and urothelium clearance in vitro. Supplementary Figure S5. Tumor masses derived from PIN1-KO T24, and 5637 cells show declining urothelial cell layers, and PIN1 promotes T24 cell tumor growth in vivo. Supplementary Figure S6. PIN1 promotes 5637 cell orthotopic tumor growth in vivo. Supplementary Figure S7. Treatment with the PIN1 specific inhibitor, sulfopin, lowers T24 cell proliferation and negatively regulates 5637 and T24 cell spheroid formation, cell motility, cell invasion and migration in vitro. Supplementary Figure S8. Sulfopin administration reduces urothelial cell layers in tumor masses derived from T24 and 5637 control cells and suppresses T24 cell tumor growth in vivo. Supplementary Figure S9. MB49 cells instilled through the urethra metastasize to distant organs, lung, and kidney. Supplementary Figure S10. Reducing Pin1 expression in organoids derived from primary mouse urothelial cells shows lower cell proliferation. Supplementary Figure S11. PIN1 positively affects cell cholesterol levels and regulates cholesterol metabolism–related gene signatures. Supplementary Figure S12. SREBP2, a crucial transcriptional factor, associates with PIN1 and promotes huBLCA cell proliferation. Supplementary Figure S13. Ablation of Pin1 in mouse MB49 bladder cancer cells reduces the cholesterol level and expression levels of SREBP2 and its downstream targets, HMGCS1 and MVD. Supplementary Figure S14. PIN1 enhances the DNA-binding activity of the SREBP2 transcription factor. Supplementary Figure S15. TNFα promotes JNK phosphorylation and activation, and the interaction between PIN1 and SREBP2. Supplementary Figure S16. JNK-mediated phosphorylation of Ser455 in SREBP2 is required for the interaction between PIN1 and SREBP2 in T24 cells. Supplementary Figure S17. PIN1 inhibition in SREBP2 S455A mutant re-expressing huBLCA cells slightly reduced the SREBP2 expression level compared to that of SREBP2 S455A mutant re-expressing cells. Supplementary Figure S18. The sulfopin PIN1 inhibitor treatment in SREBP2 S455A mutant re-expressing cells slightly reduced the cholesterol biosynthesis, bladder cancer cell proliferation and migration in vitro. Supplementary Figure S19. PIN1 knockout in SREBP2 S455A mutant re-expressing cells slightly reduced the cholesterol biosynthesis, bladder cancer cell proliferation and migration in vitro. Supplementary Figure S20. PIN1 knockout in SREBP2 S455A mutant re-expressing cells slightly reduced the 5637-cell tumor growth in vivo. Supplementary Fig. S21. The cholesterol biosynthesis pathway is affected by the combination therapy of sulfopin plus simvastatin.

Abstract Pancreatic ductal adenocarcinoma (PDA) is one of the deadliest malignancies, characterized by a dense, fibrotic, and immune-excluded tumor microenvironment (TME) that resists current therapies. Emerging evidence implicates the Oncostatin M (OSM)-Oncostatin M receptor (OSMR) signaling axis, a member of the IL-6 cytokine family, as a key driver of PDA progression through regulation of tumor-stroma-immune interactions. My research aims to define how OSM-OSMR signaling orchestrates the immune-suppressive microenvironment in PDA and to evaluate whether its inhibition can reprogram tumors toward immune responsiveness.Spatial transcriptomics analysis of treatment-naïve human PDA revealed high OSMR expression associated with cancer-associated fibroblast (CAF) activation, stromal stiffness, and enrichment of macrophage and monocyte gene signatures, alongside reduced CD8+ T-cell infiltration. Integrated transcriptomic analyses identified OSMR-associated genes enriched for NF-κB, JAK-STAT, and IFN-γ pathways. Functional studies showed that OSM-OSMR signaling promotes PDA cell proliferation, migration, and invasion, while driving CAF activation and polarization of monocytes toward immunosuppressive CD163+/CD206+ macrophages. In ex vivo human PDA slice cultures, recombinant OSM enhanced proliferation and STAT3 phosphorylation, effects reversed by OSM or OSMR neutralizing antibodies.To dissect the underlying mechanisms, I employed CRISPR-engineered OSMR knockout and rescue PDA cell lines, OSMR-overexpressing CAFs, and 3D co-cultures with monocyte-derived macrophages, integrated with single-cell RNA-seq, phospho-proteomics, and spatial transcriptomics. Parallel translational studies will test OSM/OSMR-neutralizing antibodies in patient-derived organoids, tumor slice cultures, and genetically engineered mouse models (GEMMs), alone or in combination with FDA-approved chemotherapies. These studies will assess the effects of OSM-OSMR signaling on tumor growth, stromal remodeling, immune infiltration, and metastasis.By defining how OSM-OSMR signaling sustains PDA’s fibrotic and immune-suppressive TME and establishing its therapeutic vulnerability, this work aims to develop rational strategies that reprogram the TME, enhance immunotherapy responsiveness, and improve outcomes for patients with pancreatic cancer. Citation Format: Yuan Sui, Tony Hunter. Targeting the OSM-OSMR signaling axis to reprogram the tumor microenvironment and overcome immune suppression in pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 7950.

We present a genome assembly from a specimen of Ectobius pallidus (tawny cockroach; Arthropoda; Insecta; Blattodea; Ectobiidae). The assembly contains two haplotypes with total lengths of 2,087.55 megabases and 2,124.67 megabases, respectively. Most of haplotype 1 (98.55%) is scaffolded into 11 chromosomal pseudomolecules, while haplotype 2 is assembled to scaffold level. The mitochondrial genome has also been assembled and is 15.75 kilobases in length.

Pancreatic ductal adenocarcinoma (PDA) is a highly lethal cancer with a low survival rate and significant global mortality impact, having only a 13% 5-year relative survival rate in the US for all stages combined. The interaction between the immunosuppressive tumor microenvironment (TME) in PDA, characterized by limited CD8+ T cell infiltration and an imbalance of cytokines, underscores the need to explore mechanisms underlying the inhibitory immune microenvironment (IME) to improve immunotherapies and adjuvant treatments. Intercellular crosstalk within the PDA TME, driven by secreted proteins, cytokines, chemokines, and their receptors, plays a critical role in maintaining immunosuppression. Our studies highlight Oncostatin M (OSM) and its receptor OSMR as potential regulators of the IME, promoting its shift from “hot” to “cold.” Spatial transcriptomics reveals OSM expression in tumor-associated macrophages (TAM) and OSMR on cancer-associated fibroblasts (CAFs) and PDA cells, which may sustain an immune-suppressive IME, marked by immunosuppressive macrophages and fewer cytotoxic T cells. Monocytes cocultured with PDA tumor cells exhibit enhanced OSM-OSMR responses, affecting macrophage activity. Blocking OSM-OSMR signaling could disrupt this suppression, enhancing immunotherapy effectiveness. We aim to elucidate this immune switch mechanism and test OSM-OSMR blockade in the KPC mouse PDA model to support its potential as a novel adjuvant PDA therapy. This work was in part funded by NIH-P01 (CA265762 Project 2), NIH-R35 (CA242443), a Lustgarden Award for Pancreatic Research, and the NOMIS fundation. Tumor Immunology: Cellular Responses and Tumor Microevironment (TIME)

SIGNIFICANCE:This study provides deeper insights into the regulatory role of the phospho-dependent prolyl isomerase PIN1 in bladder cancer. The identification of the link between PIN1 and SREBP2-mediated transcription and cholesterol biosynthesis offers the potential for developing novel therapeutic strategies for bladder cancer.

Histidine phosphorylation is a non-canonical post-translational modification (PTM), with 1-phosphohistidine (1-pHis) and 3-phosphohistidine (3-pHis) isoforms, that is understudied due to a lack of robust reagents, including high-affinity pHis-specific antibodies. Engineering pHis antibodies is challenging due to the labile nature of its phosphoramidate (P-N) bond. We developed a strategy for in vitro engineering of antibodies for the detection of native 3-pHis targets, in which the rabbit SC44-8 anti-3-pTza mAb is humanized into a scaffold (hSC44) that is suitable for phage display. Six unique Fab phage-displayed hSC44 scaffold libraries were screened for antibodies that bound 3-pHis with higher affinity and had specificity for 3-pHis versus 3-pTza. hSC44.20N32FL, the best engineered antibody, has 10-fold higher affinity for 3-pHis than parental hSC44. Eleven new Fab structures, including the first antibody-pHis peptide structures, together with structural and quantum mechanical calculations, provided molecular insights into 3-pHis and 3-pTza discrimination by hSC44.20N32FL and the increased affinity obtained through engineering. We demonstrated the utility of these high-affinity 3-pHis-specific antibodies for the recognition of pHis proteins in mammalian cells by immunoblotting and immunofluorescence staining. Our work describes a general method for engineering labile PTM-specific antibodies and provides novel antibodies for investigating the role of 3-pHis in cell biology. Histidine phosphorylation is an elusive post-translational modification (PTM) whose role in mammalian cell biology is largely unknown due to the lack of robust tools and methods for its analysis. Here, the authors report the development of antibodies with unprecedented affinity and specificity towards 3-pHis and present the first crystal structures of a pHis peptide in complex with an antibody, showing how these antibodies can be used in standard molecular biology workflows to investigate pHis-dependent biology.

Triple-negative breast cancer (TNBC) is a highly aggressive and metastatic form of breast cancer that lacks an effective targeted therapy. To identify potential therapeutic targets, we investigated the phosphohistidine phosphatase, LHPP, which has been implicated in the development of several types of cancer. However, the full significance of LHPP in cancer progression remains unclear due to our limited understanding of its molecular mechanism. We found that levels of the LHPP phosphohistidine phosphatase were significantly increased in human breast cancer patients compared to normal adjacent tissues, with the highest levels in the TNBC subtype. When LHPP was knocked out in the MDA-MB-231 human TNBC cell line, cell proliferation, wound healing capacity, and invasion were significantly reduced. However, LHPP knockout in TNBC cells did not significantly affect overall phosphohistidine protein levels. Interestingly, LHPP knockout in MDA-MB-231 cells delayed tumor growth and reduced metastasis when orthotopically transplanted into mouse mammary glands. To investigate LHPP’s role in breast cancer progression, we used next-generation sequencing and proximity-labeling proteomics, and found that LHPP regulates gene expression in chemokine-mediated signaling and actin cytoskeleton organization. Depletion of LHPP reduced the presence of tumor-infiltrating macrophages in mouse xenografts. Our results support a tumor promoter role for LHPP phosphohistidine phosphatase in MDA-MB-231TNBC cells and suggest that targeting LHPP phosphatase could be a potential therapeutic strategy for TNBC.

Phosphatidylinositol 3-kinase (PI3K) phosphorylates PI(4,5)P2 to produce PI(3,4,5)P3, thereby activating AKT and other effector proteins. However, whether PI3K has non-PI(3,4,5)P3-related functions critical for tumor development remains unclear. Here, we demonstrate that high glucose induces PI3Kβ binding to O-linked β-D-N-acetylglucosamine (O-GlcNAc) transferase (OGT) in glioblastoma cells, dependent on hexokinase 1 (HK1)-mediated OGT Y889 phosphorylation and subsequent p85α recruitment. Importantly, PI3Kβ functions as a protein kinase, phosphorylating OGT at T985 and enhancing OGT activity and total cellular protein O-GlcNAcylation. Activated OGT O-GlcNAcylates ATP-citrate synthase (ACLY) at T639 and S667, leading to ACLY activation-dependent acetyl-coenzyme A (CoA) production to increase fatty acid levels and histone H3 acetylation for gene transcription. Intervention in PI3Kβ-mediated OGT phosphorylation and ACLY O-GlcNAcylation inhibits glioblastoma cell proliferation and tumor growth in xenografts. These findings underscore the critical role of PI3Kβ in governing protein O-GlcNAcylation, fatty acid metabolism, and chromatin modification through its protein kinase activity and provide instrumental insight into the roles of PI3K in tumor progression.

Pancreatic ductal adenocarcinoma (PDAC) has an atypical, highly stromal tumour microenvironment (TME) that profoundly contributes to its poor prognosis1. Here, to better understand the intercellular signalling between cancer and stromal cells directly in PDAC tumours, we developed a multidimensional proteomic strategy called TMEPro. We applied TMEPro to profile the glycosylated secreted and plasma membrane proteome of 100 human pancreatic tissue samples to a great depth, define cell type origins and identify potential paracrine cross-talk, especially that mediated through tyrosine phosphorylation. Temporal dynamics during pancreatic tumour progression were investigated in a genetically engineered PDAC mouse model. Functionally, we revealed reciprocal signalling between stromal cells and cancer cells mediated by the stromal PDGFR-PTPN11-FOS signalling axis. Furthermore, we examined the generic shedding mechanism of plasma membrane proteins in PDAC tumours and revealed that matrix-metalloprotease-mediated shedding of the AXL receptor tyrosine kinase ectodomain provides an additional dimension of intercellular signalling regulation in the PDAC TME. Importantly, the level of shed AXL has a potential correlation with lymph node metastasis, and inhibition of AXL shedding and its kinase activity showed a substantial synergistic effect in inhibiting cancer cell growth. In summary, we provide TMEPro, a generically applicable clinical functional proteomic strategy, and a comprehensive resource for better understanding the PDAC TME and facilitating the discovery of new diagnostic and therapeutic targets.

Abstract Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal forms of cancer, characterized by a low survival rate and a significant global mortality impact. The interaction between PDAC and the immune system is complex and plays a crucial role in the progression of the disease. PDAC creates an immunosuppressive tumor microenvironment, which hampers the ability of immune cells to effectively infiltrate the tumor and mount an anti-cancer immune response. Additionally, there is an imbalance of cytokines, with an overabundance of immunosuppressive factors and a deficiency in pro-inflammatory cytokines. This hostile immunological landscape contributes to the aggressive nature of PDAC and hinders the success of immunotherapies and adjuvant treatments. One cytokine of particular interest is oncostatin M (OSM), a member of the interleukin-6 (IL-6) family. OSM has been implicated in the regulation of cancer and has been associated with poor overall survival in pancreatic cancer and other malignancies. Similar to other cytokines, OSM is secreted by immune cells and acts as a regulator of the inflammatory microenvironment surrounding the tumor. The levels of OSM in this milieu can potentially influence cancer development and regulation through multiple mechanisms. Despite the growing understanding of cytokines in cancer, research on the role of OSM in PDAC is still limited and requires further investigation to identify potential therapeutic targets. Therefore, there is a critical need to delve deeper into the functions of OSM and its receptor, oncostatin M receptor (OSMR), in the initiation and progression of pancreatic cancer. As such, our goal is to examine the functions of OSM and its receptor (OSMR) in initiating and advancing pancreatic cancer to better comprehend the role of the OSM-OSMR axis in PDAC, both in vivo and in vitro. Citation Format: Yuan Sui, Tony Hunter. Investigating the function of the OSM-OSMR axis in pancreatic ductal adenocarcinoma (PDAC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6984.