The incidence of pancreatic ductal adenocarcinoma (PDAC) is increasing but clinical outcomes remain poor and new treatments are required. IGF2BP3 is an oncofetal m6A-reading RNA regulatory protein whose expression is commonly observed in PDAC and which may represent an important therapeutic target. IGF2BP3 protein is expressed in >95% of primary tumours whilst RNA expression ranges from 3 to 3000-fold above normal epithelium. siRNA knockdown reveals IGF2BP3 to strongly support expression of gene programs related to DNA replication, cell cycle and apoptosis, TNF signalling and epithelial-mesenchymal transition. Direct sequencing of native RNA further reveals IGF2BP3-mediated enhancement of 863 RNA isoforms with suppression of 389 isoforms. m6A, m5C and pseudouridine (ψ) RNA modifications were seen in 97% of transcripts within the PDAC transcriptome and enriched within MYC gene targets. IGF2BP3 regulated the pattern of RNA modification with a substantial impact on m5C modifications of miRNA and scRNA, and genes associated with regulation of TNF signalling. Knockdown of IGF2BP3 expression in primary tumour organoid cultures suppressed proliferation and elicited apoptosis, indicating a critical requirement for IGF2BP3 within malignant stem cells. These data show that IGF2BP3 is expressed consistently in PDAC, plays a dominant role in regulation of RNA splicing and modification, and supports cancer stem cell proliferation and survival. IGF2BP3 therefore occupies a critical position within the RNA regulon of PDAC and represents an important therapeutic target in this tumour of unmet need.

Abstract Background: Pancreatic ductal adenocarcinoma (PDAC) poses significant challenges in treatment due to its complex interaction with the tumour microenvironment (TME). Fibroblasts, a crucial component of the TME, influence tumour progression and therapeutic responses. This study explores the diversity of cancer-associated fibroblast (CAF) populations in PDAC tumours versus adjacent normal fibroblasts (ANFs), focussing on myofibroblasts (myCAFs) and inflammatory fibroblasts (iCAFs). These fibroblast subtypes exhibit distinct characteristics and localisation within the tumour. The research examines the impact of CAFs and ANFs on immune cell behaviour, particularly in relation to adhesion and migration. Methods: Tumour and juxta-tumoral tissue from patients were digested into single cell suspensions. Cells were stained with a 22-plex antibody panel by flow cytometry. Pseudo-emperipolesis assays were assessed by co-culture of lymphocytes with CAF and ANF fibroblasts. Furthermore, these were performed with or without stimulation of fibroblasts with by TGF-b or TNF-a/IFN-g, to promote myCAF or iCAF phenotypes respectively. Gene expression analysis of tumour-derived fibroblast subtypes was assessed by single-cell RNA sequencing using the 10X Genomics platform. The transcriptional profile of spatially resolved fibroblasts which were proximal or distant to tumour epithelium was determined by the NanoString GeoMx Digital Spatial Profiler (DSP) platform. Results: Flow cytometry analysis revealed that iCAFs predominated within the tumour stroma compared to myCAFs, although iCAF populations were less prevalent within ANF populations. Upon stimulation with inflammatory cytokines (TNF-α/IFN-γ), CAFs promoted lymphocyte migration in contrast to ANFs. However, TGF-β stimulation of CAF downregulated the migration of lymphocytes. As such, TGF-β or TNF-α/IFN-γ stimulation exhibited antagonistic effects on lymphocyte adhesion and migration in CAFs. Tumour-proximal fibroblasts exhibited myCAFs characteristics expressing α-SMA and PDPN, as well as ECM molecules such as collagen and integrins, suggesting a barrier to lymphocyte migration. Conversely, distal fibroblasts displayed an inflammatory profile, expressing cytokines and chemokines including CCL2, CXCL9, CXCL12, IL6, IL11 and LIF, thereby contributing to the inflammatory milieu within the tumour microenvironment. Additionally, iCAFs demonstrated higher transcription levels of hyaluronan synthase (Has1) and fibulin 2 (FBLN2) which are crucial for ECM formation and remodelling. This study identifies a differential role for iCAFs and myCAFs in the modulate of lymphocyte adhesion and migration within the PDAC microenvironment, highlighting the complex interplay between fibroblast subtypes and immune cell dynamics. Citation Format: Fouzia Zayou, Hayden Pearce, Wayne Croft, Samantha Nicol, Sandra Margielewska-Davies, Sarah Powell-Brett, Rachel Brown, Jianmin Zuo, Keith Roberts, Helen M. McGettrick, Paul Moss. Modulation of lymphocyte adhesion and migration by iCAF and myCAF fibroblasts in PDAC [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr A030.

Background: Galectins (Gal's) are a family of carbohydrate-binding proteins that are known to support the tumour microenvironment through their immunosuppressive activity and ability to promote metastasis. As such they are attractive therapeutic targets, but little is known about the cellular expression pattern of galectins within the tumour and its neighbouring stromal microenvironment. Here we investigated the cellular expression pattern of Gals within pancreatic ductal adenocarcinoma (PDAC). Methods: Galectin gene and protein expression were analysed by scRNAseq (n=4) and immunofluorescence imaging (n=19) in fibroblasts and epithelial cells of pancreatic biopsies from PDAC patients. Galectin surface expression was also assessed on tumour adjacent normal fibroblasts and cancer associated primary fibroblasts from PDAC biopsies using flow cytometry. Results: scRNAseq revealed higher Gal-1 expression in fibroblasts and higher Gal-3 and -4 expression in epithelial cells. Both podoplanin (PDPN+, stromal/fibroblast) cells and EpCAM+ epithelial cells expressed Gal-1 protein, with highest expression seen in the stromal compartment. By contrast, significantly more Gal-3 and -4 protein was expressed in ductal cells expressing either EpCAM or PDPN, when compared to the stroma. Ductal Gal-4 cellular expression negatively correlated with ductal Gal-1, but not Gal-3 expression. Higher ductal cellular expression of Gal-1 correlated with smaller tumour size and better patient survival. Conclusions: In summary, the intricate interplay and cell-specific expression patterns of galectins within the PDAC tissue, particularly the inverse correlation between Gal-1 and Gal-4 in ducts and its significant association with patient survival, highlights the complex molecular landscape underlying PDAC and provides valuable insights for future therapeutic interventions.

Abstract Background: Pancreatic ductal adenocarcinoma (PDAC) is highly aggressive malignancy characterised by its abundant extracellular matrix and diverse stromal cell components such as cancer associated fibroblasts (CAFs). CAFs play a pivotal role in shaping the tumour microenvironment and influencing the behaviour of immune cells, particularly T cells, through various mechanisms. The impact of CAFs on T cells is multifaceted as they can exert both pro- and anti-tumoral effects, influencing the delicate balance between immunosurveillance and immune evasion. In this study, we examined the phenotype and function of T cells when co-cultured with CAFs and Adjacent-normal fibroblasts (ANF) isolated from primary PDAC tissue following surgical resection. Methods: Tumour and juxta-tumoral tissue from patients underwent digestion into a single cell suspension. Subsequently, the isolated fibroblasts were cultured and stained with a 22-plex antibody panel designed for flow cytometry. Additional functional assays involved co-culture of CAFs and ANFs with unstimulated or anti-CD3/CD28 stimulated allogeneic T cells. Cell proliferation rates were quantified through the CFSE assay, while the T cells were simultaneously stained to distinguish CD4+ and CD8+ T cells subpopulations. Additionally, the expression of key co-stimulatory molecules: CD69, NKG2D and DNAM-1 and the co-inhibitory molecule: PD-1 were assessed, providing a thorough examination of the immune dynamics within the tumour microenvironment. Results: Flow Cytometry analysis revealed a distinct expression profile in CAFs compared to ANFs, with CAFs expressing higher levels of FAP and CD29. The CFSE assay further revealed that CAFs induced T cell proliferation of resting T cells with no significant increase in stimulated T cells. Notably, this co-culture led to a highly differentiated phenotype in both unstimulated and stimulated CD4+ and CD8+ T cells, characterised by elevated expression of activating markers (CD69 and NKG2D) and the inhibitory molecule PD-1. These findings suggest a dynamic interplay between fibroblasts and T cells in the tumour microenvironment, influencing both the proliferation and phenotypic aspects of T cell behaviour. Citation Format: Fouzia Zayou, Hayden Pearce, Samantha Nicol, Sandra Margielewska-Davies, Sarah Powell-Brett, Rachel Brown, Jianmin Zuo, Keith Roberts, Helen M. McGettrick, Paul Moss. Cancer-associated fibroblasts modulate T cell killing activity & phenotype in 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 1604.

Pancreatic ductal adenocarcinoma has a poor clinical outcome and responses to immunotherapy are suboptimal. Stromal fibroblasts are a dominant but heterogenous population within the tumor microenvironment and therapeutic targeting of stromal subsets may have therapeutic utility. Here, we combine spatial transcriptomics and scRNA-Seq datasets to define the transcriptome of tumor-proximal and tumor-distal cancer-associated fibroblasts (CAFs) and link this to clinical outcome. Tumor-proximal fibroblasts comprise large populations of myofibroblasts, strongly expressed podoplanin, and were enriched for Wnt ligand signaling. In contrast, inflammatory CAFs were dominant within tumor-distal subsets and expressed complement components and the Wnt-inhibitor SFRP2. Poor clinical outcome was correlated with elevated HIF-1α and podoplanin expression whilst expression of inflammatory and complement genes was predictive of extended survival. These findings demonstrate the extreme transcriptional heterogeneity of CAFs and its determination by apposition to tumor. Selective targeting of tumor-proximal subsets, potentially combined with HIF-1α inhibition and immune stimulation, may offer a multi-modal therapeutic approach for this disease.

Abstract Background: Resistance of pancreatic ductal adenocarcinoma (PDAC) to current treatment regimens contributes to the dismal prognosis for patients diagnosed with the disease. Accumulated evidence indicates that metabolic alterations may play a role in chemo-resistance. In this project, we investigated the role of pyruvate dehydrogenase Alpha1 (PDHA1) in chemo-resistance. Methods: Wild type and Gemcitabine-resistant pancreatic cancer cell lines MIA PaCa-2, HPAF-II, and UN-KPC-961 were used. Mitochondrial respiration, ATP production and glycolytic activities were measured by seahorse. PDHA1 was overexpressed or its activity inhibited by over-expressing PDHA1 kinase in these cells and cancer cell survival was measured by MTT and cancer stemness measured by Western and RT-PCR. PDHA1 level was measured in human and mouse PDAC tissues. Results: Seahorse analysis showed a significantly lower mitochondrial activity, ATP production, and increased glycolytic activity and lactate production in gemcitabine-resistant compared to non- resistant pancreatic cancer cells. PDHA1 mRNA and protein levels were less in gemcitabine-resistant cancer cells compared to non-resistant cells. PDHA1 levels were less in human PDAC tissues from patients and mice who developed resistance to chemotherapy. PDHA1 over-expression in gemcitabine-resistant cancer cells reversed resistance to chemotherapy, decreased cancer cell survival and decreased expression of cancer stemness markers. Whereas, PDHA1 inhibition by over-expressing PDHA1 kinase, in non-resistant cancer cells, induced resistance to chemotherapy, increased cancer cell survival and increased expression of cancer stemness markers. We found that inhibition of histone deacetylase (HDAC) (class 1 and 2) increased transcription level of PDHA1 and reversed chemo-resistance. Conclusion: We show that PDHA1 is a regulator of chemo-resistance in pancreatic cancer cells. PDHA1is a novel target for treating chemo-resistant PDAC. Citation Format: Fouzia Zayou, Chintan Chheda, Adrian Lim, Omer H. Elmadbouh, Stephen J. Pandol, Mouad Edderkaoui. Pyruvate dehydrogenase mediates chemo-resistance in pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2403.

OBJECTIVE:To investigate the anti-tumor effect of a newly-developed dual inhibitor (APCS-540) of glycogen synthase kinase 3 beta (GSK3B) and histone deacetylases (HDACs) in ovarian cancer cells.METHODS:The effects of APCS-540 on cancer cell proliferation, migration, invasion and cancer stemness were investigated in vitro in human (KURAMOCHI, OVCA420, OVSAHO) and mouse (BR-Luc, ID8, MOSE-HRas-Myc) ovarian cancer cells. Cisplatin-sensitive (A2780) and cisplatin-resistant (A2780cis) cell lines were used to evaluate APCS-540's effect on chemoresistance. The immunocompetent syngeneic mouse model BR-Luc was used to test the effect of APCS-540 on ovarian cancer progression and survival.RESULTS:APCS-540 showed significant anti-tumor effects in vitro in both human and mouse ovarian cancer cells. Importantly, APCS-540 demonstrated marked cytotoxicity against cisplatin-resistant cancer cells and reversed cisplatin-resistance when used in combination with platinum. APCS-540 significantly decreased cancer cell invasion. A significant 66% increase in survival was observed in mice treated with APCS-540 compared to control mice.CONCLUSION:Dual inhibition of GSK3B and HDACs via APCS-540 showed potent anti-tumor activity in vitro and in vivo, suggesting that APCS-540 may provide a novel treatment option for ovarian cancer, including the platinum-resistant disease.

Abstract Background: Resistance to chemotherapy is a major cause of the failure of treatments in many cancers including pancreatic cancer. Accumulated evidence indicates that mitochondrial metabolic alterations are associated with cancer cells growth and proliferation. In this project, we investigated the role of mitochondrial bioenergetics in drug resistance. Methods: Human pancreatic cancer cell lines MIA PaCa-2 and HPAF-II and mouse pancreatic cancer cell line UN-KPC-961 were treated with increased doses of chemotherapy drug Gemcitabine for 4 months until developing resistance. Mitochondrial proteins, cancer stemness markers and metabolic enzymes were measured by Western and RT-PCR. Cell survival was measured by MTT assay. Mitochondrial bioenergetics were measured by Seahorse Assay. Results: We found that gemcitabine resistant (GR) cells grow at a much slower rate than wild type (WT) cancer cells; however, they express a significantly higher amount of cancer stemness markers such as Nanog, Oct4 and Sox2. They also expressed higher level of pro-cancer cytokines such as IL-6 and IL-4. Seahorse analysis showed a significantly lower oxygen consumption rate (OCR) in GR cells compared to WT cells. OCR did not change much in GR cells when applying inhibitors of the mitochondrial respiratory chain complex. GR cells produced lower level of ATP and lower spare respiratory capacity. However, GR cells expressed the same amount of mitochondrial proteins such as TOM40, TOM70 and COX4 suggesting the same mass of mitochondria. Pyruvate dehydrogenase-E1 Alpha1 (PDHA1) expression level was significantly lower in GR cells compared to WT cells. PDHA1 inhibition induced WT cells to develop a partial resistance to Gemcitabine; whereas, PDH1 overexpression in GR cells made these cells less resistant to Gemcitabine. Conclusion: We showed for the first time the difference in mitochondrial bioenergetics between WT and GR cells and showed that GR cells grow independently of the mitochondrial energy production machinery. Furthermore, we identified PDHA1 as a critical mediator of cancer drug resistance. Citation Format: Fouzia Zayou, Chintan Chheda, Stephen Pandol, Mouad Edderkaoui. Mitochondrial bioenergetics mediate chemo-resistance of cancer cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6343.

BACKGROUND & AIMS Growth, progression, and drug resistance of pancreatic ductal adenocarcinomas (PDACs) have been associated with increased levels and activity of glycogen synthase kinase 3 beta (GSK3B) and histone deacetylases (HDACs). We designed and synthesized molecules that simultaneously inhibit the activities of both enzymes. We tested the effects of one of these molecules, Metavert, in pancreatic cancer cells and mice with pancreatic tumors. METHODS We tested the ability of Metavert to bind GSK3B and HDACs using surface plasmon resonance. MIA PaCa-2, Bx-PC3, HPAF-II, and HPDE6 cell lines were incubated with different concentrations of Metavert, with or without paclitaxel or gemcitabine, or with other inhibitors of GSK3B and HDACs; cells were analyzed for apoptosis and migration and by immunoblotting, immunofluorescence, and real-time polymerase chain reaction. Krasþ/LSLG12D;Trp53þ/LSLR172H;Pdx-1-Cre (KPC) mice (2 months old) were given injections of Metavert (5 mg/kg, 3 times/week) or vehicle (control). B6.129J mice with tumors grown from UN-KPC961-Luc cells were given injections of Metavert or vehicle. Tumors and metastases were counted and pancreata were analyzed by immunohistochemistry. Glucose metabolism was measured using 13C-glucose tracer and mass spectroscopy and flow cytometry. Cytokine levels in blood samples were measured using multiplexing enzyme-linked immunosorbent assay. RESULTS Metavert significantly reduced survival of PDAC cells but not nontransformed cells; the agent reduced markers of the epithelial-to-mesenchymal transition and stem cells in PDAC cell lines. Cells incubated with Metavert in combination with irradiation and paclitaxel or gemcitabine had reduced survival compared with cells incubated with either agent alone; Metavert increased killing of drug-resistant PDAC cells by paclitaxel and gemcitabine. PDAC cells incubated with Metavert acquired normalized glucose metabolism. Administration of Metavert (alone or in combination with gemcitibine) to KPC mice or mice with syngeneic tumors significantly increased their survival times, slowed tumor growth, prevented tumor metastasis, decreased tumor infiltration by tumor-associated macrophages, and decreased blood levels of cytokines. CONCLUSIONS In studies of PDAC cells and 2 mouse models of PDAC, we found a dual inhibitor of GSK3B and HDACs (Metavert) to induce cancer cell apoptosis, reduce migration and expression of stem cell markers, and slow growth of tumors and metastases. Metavert had synergistic effects with gemcitabine.

Background In diabetic patients, elevated glucose increases the O-GlcNAcylation in the various tissues through upregulation of the hexosamine biosynthesis pathway.Increased O-GlcNAcylation could contribute to a part of diabetic complications such as retinopathy, nephropathy.On the other hand, increased O-GlcNAcylation is a general feature of cancer cells and contributes to various cancer phenotypes, including cell proliferation, survival, invasion, metastasis, energy metabolism, and epigenetics.Recently, it was reported that O-GlcNAcylation, was upregulated in patients with both diabetes and cancer.Furthermore, it was shown that the expression level of FoxM1, which played a key role in carcinogenesis, was increased by O-GlcNAcylation in cancer cells such as prostate cancer cell line.There are many reports about diabetes as the risk factor for carcinogenesis.Aims we examined the molecular mechanism underlying the regulation of FoxM1 function by O-GlcNAcylation in gastric cancer cells.Methods We used human gastric cancer cell lines.We first examined the effect of glucose concentration on the levels of O-GlcNAcylated proteins and FoxM1 expression by Western blot analyses and Immunofluorescence staining.We next determined the levels of O-GlcNAcylated proteins and FoxM1 expression in the presence or absence of O-GlcNAc transferase (OGT) inhibitor, BADGP or O-GlcNAcase (OGA) inhibitor, Thiamet G, by Western blot analyses and IF staining.And we assessed cellular proliferation after Thiamet G or BADGP treatment using Cell Counting Kit(CCK8) to investigate the relationship between proliferation in gastric cancer cells and O-GlcNAcylation.Then We used co immunoprecipitation to detect the relationships between ubquitination of FoxM1 and O-GlcNAcylation.Results The levels of O-GlcNAcylation and FoxM1 expression were increased under high glucose concentration, whereas they were decreased under low glucose concentration in all cancer cell lines we used.The levels of O-GlcNAcylation and FoxM1 expression were upregulated by Thiamet G and downregulated by BADGP in all of the cells.And Elevated O-GlcNAcylation and Upregulation of FoxM1 expression increased cell proliferation in gastric cancer cells.Then ThiametG decreased FoxM1 ubiquitination in FoxM1 trancefected HEK293 Conclusions Our data indicate that O-GlcNAcylation increases FoxM1 protein level without affecting the mRNA level and blockade of proteasomal proteolysis also increases FoxM1 protein level in gastric cancer cells.Previous reports suggest that O-GlcNAcylation hampers proteasome system through modulating activities of ubiquitin ligases or blocking ubiquitination of target proteins.In diabetes patients, elevated O-GlcNAcylation might decrease FoxM1 degradation mediated by proteasome system resulting in upregulation of FoxM1 which promotes cancer development and progression .