Pdx1-Cre Mice Research Articles

Semaphorin 3D promotes pancreatic ductal adenocarcinoma progression and metastasis through macrophage reprogramming.

Axon guidance molecules are frequently altered in pancreatic ductal adenocarcinoma (PDA) and influence PDA progression. However, the molecular mechanism remained unclear. Using genetically engineered mouse models to examine semaphorin 3D (SEMA3D), we identified a dual role for tumor- and nerve-derived SEMA3D in the malignant transformation of pancreatic epithelial cells and invasive PDA development. Pancreatic-specific knockout of the SEMA3D gene from the KRASG12D and TP53R172H mutation knock-in, PDX1-Cre(KPC) mouse model demonstrated delayed tumor initiation, prolonged survival, absence of metastasis, and reduced M2 macrophage expression. Mechanistically, tumor- and nerve-derived SEMA3D indirectly reprograms macrophages through KRASMUT-dependent ARF6 signaling in PDA cells, resulting in increased lactate production, which is sensed by GPCR132 on macrophages to stimulate protumorigenic M2 polarization. Multiplex immunohistochemistry demonstrated increased M2-polarized macrophages proximal to nerves in SEMA3D-expressing human PDA tissue. This study suggests that altered SEMA3D expression leads to an acquisition of cancer-promoting functions, and nerve-derived SEMA3D is "hijacked" by PDA cells to support growth and metastasis in a KRASMUT-dependent manner.

Pancreatic STAT5 activation promotes KrasG12D-induced and inflammation-induced acinar-to-ductal metaplasia and pancreatic cancer

ObjectivePancreatic ductal adenocarcinoma (PDAC) is a highly lethal malignancy because it is often diagnosed at a late-stage. Signal transducer and activator of transcription 5 (STAT5) is a transcription factor implicated...

Loss of p53 and SMAD4 induces adenosquamous subtype pancreatic cancer in the absence of an oncogenic KRAS mutation

Pancreatic cancer is associated with an oncogenic KRAS mutation in approximately 90% of cases. However, a non-negligible proportion of pancreatic cancer cases harbor wild-type KRAS (KRAS-WT). This study establishes genetically engineered mouse models that develop spontaneous pancreatic cancer in the context of KRAS-WT. The Trp53loxP/loxP;Smad4loxP/loxP;Pdx1-Cre (PPSSC) mouse model harbors KRAS-WT and loss of Trp53/Smad4. The Trp53loxP/loxP;Tgfbr2loxP/loxP;Pdx1-Cre (PPTTC) mouse model harbors KRAS-WT and loss of Trp53/Tgfbr2. We identify that either Trp53/Smad4 loss or Trp53/Tgfbr2 loss can induce spontaneous pancreatic tumor formation in the absence of an oncogenic KRAS mutation. The Trp53/Smad4 loss and Trp53/Tgfbr2 loss mouse models exhibit distinct pancreatic tumor histological features, as compared to oncogenic KRAS-driven mouse models. Furthermore, KRAS-WT pancreatic tumors with Trp53/Smad4 loss reveal unique histological features of pancreatic adenosquamous carcinoma (PASC). Single-cell RNA sequencing (scRNA-seq) analysis reveals the distinct tumor immune microenvironment landscape of KRAS-WT (PPSSC) pancreatic tumors as compared with that of oncogenic KRAS-driven pancreatic tumors.

Prolactin receptor potentiates chemotherapy through miRNAs-induced G6PD/TKT inhibition in pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive malignancies, with a notoriously dismal prognosis. As a competitive inhibitor of DNA synthesis, gemcitabine is the cornerstone drug for treating PDAC at all stages. The therapeutic effect of gemcitabine, however, is often hindered by drug resistance, and the underlying mechanisms remain largely unknown. It is unclear whether their response to chemotherapeutics is regulated by endocrine regulators, despite the association between PDAC risk and endocrine deregulation. Here, we show that prolactin receptor (PRLR) synergizes with gemcitabine in both invitro and invivo treatment of PDAC. Interestingly, PRLR promotes the expression of miR-4763-3p and miR-3663-5p, two novel miRNAs whose functions are unknown. Furthermore, the analysis of transcriptome sequencing data of tumors from lactating mouse models enriches the PPP pathway, a multifunctional metabolic pathway. In addition to providing energy, the PPP pathway mainly provides a variety of raw materials for anabolism. We demonstrate that two key enzymes of the pentose phosphate pathway (PPP), G6PD and TKT, are directly targeted by miR-4763-3p and miR-3663-5p. Notably, miR-4763-3p and miR-3663-5p diminish the nucleotide synthesis of the PPP pathway, thereby increasing gemcitabine sensitivity. As a result, PRLR harnesses these two miRNAs to suppress PPP and nucleotide synthesis, subsequently elevating the gemcitabine sensitivity of PDAC cells. Also, PDAC tissues and tumors from LSL-KrasG12D/+, LSL-Trp53R172H/+, and PDX1-cre (KPC) mice exhibit downregulation of PRLR. Bisulfite sequencing of PDAC tissues revealed that PRLR downregulation is due to epigenetic methylation. In this study, we show for the first time that the endocrine receptor PRLR improves the effects of gemcitabine by boosting two new miRNAs that block the PPP pathway and nucleotide synthesis by inhibiting two essential enzymes concurrently. The PRLR-miRNAs-PPP axis may serve as a possible therapeutic target to supplement chemotherapy advantages in PDAC.

Abstract 7465: Leukemia inhibitory factor creates an immune suppressive tumor microenvironment and fosters tumor growth in KRAS-driven pancreatic ductal adenocarcinoma

Abstract Immune checkpoint blockade revolutionized cancer treatments by activating anti-tumor immunity. Pancreatic ductal adenocarcinoma (PDA) patients have not benefited from these advances due to an immune suppressive tumor microenvironment. Identifying PDA cell-encoded factors that drive immune evasion is necessary to unlock new immunologically active treatment options in PDA. We have recently demonstrated that the cytokine Leukemia inhibitory factor (LIF) is highly upregulated in PDA compared to the normal healthy pancreas. LIF is known for its immune suppressive qualities, but data on LIF in PDA is currently limited. We generated a conditional Lif flox (L) allele and crossed it into the well-established KrasLSL-G12D/+, Trp53flox/flox, Pdx1-Cre (KPC) mouse model. LKPC mice (n=29) had longer overall survival (69 days) than control KPC mice (n=29) (59 days), (p<0.05). LKPC mice harbored significantly smaller tumors with an average tumor weight of 0.6g compared to KPC control animals with an average tumor weight of 1.0g (p<0.005). Immunohistochemical evaluation of tumor proliferation measured by Ki-67 positivity in LKPC and KPC tumors at endpoint showed comparable results. Tumor-derived cell lines from KPC and LKPC mice showed no growth differences in vitro nor when reimplanted subcutaneously (sc) in immunodeficient mice. In contrast, we observed a significant decrease in tumor growth and weight in LKPC sc tumors (p<0.05) compared to the KPC sc tumors in syngeneic mice. LKPC sc tumors had significantly fewer neutrophils and more macrophages, particularly MHC II-high macrophages, known for their anti-tumor qualities when compared to KPC sc tumors using flow cytometry. A trend towards more CD8+ T-cells in sc LKPC tumors was also observed. Our genetically engineered mouse model confirmed this finding by immunohistochemical evaluation of tumors at endpoint, which demonstrated a significant increase of CD8+ T-cells in the LKPC animals compared to the KPC controls. In summary, we find that cancer-cell-expressed LIF augments tumor growth and shortens overall survival by changing the immune tumor microenvironment, nominating leukemia inhibitory factor depletion as a promising therapeutic immune target in KRAS-driven pancreatic cancer. Citation Format: Lisa Miller-Phillips, Imene Boukhalfa Hanafi, Alexis J. Combes, Shabana Bano, Lisiena Hysenaj, Jeroen P. Roose, Adam Olshen, Ashley Kiemen, Sarah Umetsu, Man-Tzu Wang, Eric A. Collisson. Leukemia inhibitory factor creates an immune suppressive tumor microenvironment and fosters tumor growth in KRAS-driven pancreatic ductal adenocarcinoma [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 7465.

Progesterone receptor potentiates macropinocytosis through CDC42 in pancreatic ductal adenocarcinoma

Endocrine receptors play an essential role in tumor metabolic reprogramming and represent a promising therapeutic avenue in pancreatic ductal adenocarcinoma (PDAC). PDAC is characterized by a nutrient-deprived microenvironment. To meet their ascendant energy demands, cancer cells can internalize extracellular proteins via macropinocytosis. However, the roles of endocrine receptors in macropinocytosis are not clear. In this study, we found that progesterone receptor (PGR), a steroid-responsive nuclear receptor, is highly expressed in PDAC tissues obtained from both patients and transgenic LSL-KrasG12D/+; LSL-Trp53R172H/+; PDX1-cre (KPC) mice. Moreover, PGR knockdown restrained PDAC cell survival and tumor growth both in vitro and in vivo. Genetic and pharmacological PGR inhibition resulted in a marked attenuation of macropinocytosis in PDAC cells and subcutaneous tumor models, indicating the involvement of this receptor in macropinocytosis regulation. Mechanistically, PGR upregulated CDC42, a critical regulator in macropinocytosis, through PGR-mediated transcriptional activation. These data deepen the understanding of how the endocrine system influences tumor progression via a non-classical pathway and provide a novel therapeutic option for patients with PDAC.

Dichotomous role of the serine/threonine kinase MAP4K4 in pancreatic ductal adenocarcinoma onset and metastasis through control of AKT and ERK pathways.

MAP4K4 is a serine/threonine kinase of the STE20 family involved in the regulation of actin cytoskeleton dynamics and cell motility. It has been proposed as a target of angiogenesis and inhibitors show potential in cardioprotection. MAP4K4 also mediates cell invasion in vitro, is overexpressed in various types of cancer, and is associated with poor patient prognosis. Recently, MAP4K4 has been shown to be overexpressed in pancreatic cancer, but its role in tumour initiation, progression, and metastasis is unknown. Here, using the KrasG12D Trp53R172H Pdx1-Cre (KPC) mouse model of pancreatic ductal adenocarcinoma (PDAC), we show that deletion of Map4k4 drives tumour initiation and progression. Moreover, we report that the acceleration of tumour onset is also associated with an overactivation of ERK and AKT, two major downstream effectors of KRAS, in vitro and in vivo. In contrast to the accelerated tumour onset caused by loss of MAP4K4, we observed a reduction in metastatic burden with both the KPC model and in an intraperitoneal transplant assay indicating a major role of MAP4K4 in metastatic seeding. In summary, our study sheds light on the dichotomous role of MAP4K4 in the initiation of PDAC onset, progression, and metastatic dissemination. It also identifies MAP4K4 as a possible druggable target against pancreatic cancer spread, but with the caveat that targeting MAP4K4 might accelerate early tumorigenesis. © 2024 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Abstract B095: Leukemia inhibitory factor fosters tumor growth in KRAS-driven pancreatic ductal adenocarcinoma

Abstract While inflammation is a known and well-validated risk factor for developing pancreatic ductal adenocarcinoma (PDA), it remains unclear how Leukemia inhibitory factor (LIF), a cytokine of the IL-6 class family, influences tumor initiation and progression in PDA. LIF is highly upregulated in PDA when compared to the normal healthy pancreas. Using a human single-cell RNAseq PDA data set, we identified pancreatic cancer cells as the main source of LIF, followed by fibroblasts and stellate cells. After confirming that Pdx1-expressing cells represent the majority of Lif-expressing cells in the pancreas, we crossed our newly developed Lif flox/flox allele into the well-established KrasLSL-G12D/+, Trp53flox/flox, Pdx1-Cre (KPC) mouse model. Lif flox/flox, Pdx1-Cre (LC) mice present with normal pancreas histology, mouse weight, and mouse size at 12 months, indicating that Lif is dispensable for mouse pancreas development and function. Lif flox/flox, Trp53flox/flox, Pdx1-Cre (LPC) mice also showed no differences in pancreas histology when compared to normal Lif-expressing PC mice. Lif flox/floxKPC (LKPC) mice develop PDA tumors. Cell lines from LKPC tumors (n=4) presented with undetectable Lif expression and no LIF protein secretion. Control KPC tumor cell lines (n=4) showed Lif expression and LIF secretion, confirming the successful deletion of Lif in our newly developed mouse model. LKPC mice (n=21) showed a significant increase in disease-specific overall survival at 72 days when compared to control KPC mice (n=26) at 62 days (p<0.05). When mice reached endpoint, LKPC mice presented with significantly smaller tumors with an average tumor weight of 0.7g compared to KPC control animals with an average tumor weight of 1.3g (p<0.05). In summary, we find that cancer-cell expressed LIF augments tumor growth and shortens disease-specific overall survival, nominating leukemia inhibitory factor depletion as a promising therapeutic target in KRAS-driven pancreatic cancer. Citation Format: Lisa Miller-Phillips, Imene Boukhalfa Hanafi, Sarah Umetsu, Adam Olshen, Ritu Roy, Ashley Kiemen, Man-Tzu Wang, Eric Collisson. Leukemia inhibitory factor fosters tumor growth in KRAS-driven pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr B095.

A novel anti-galectin-9 immunotherapy limits the early progression of pancreatic neoplastic lesions in transgenic mice.

Pancreatic adenocarcinoma (PDAC) is a devastating disease with an urgent need for therapeutic innovation. Immune checkpoint inhibition has shown promise in a variety of solid tumors, but most clinical trials have failed to demonstrate clinical efficacy in PDAC. This low efficacy is partly explained by a highly immunosuppressive microenvironment, which dampens anti-tumor immunity through the recruitment or induction of immunosuppressive cells, particularly regulatory T cells (Tregs). In this context, our laboratory has developed a novel immunotherapeutic strategy aimed at inhibiting the suppressive activity of Tregs, based on a patented (EP3152234B1) monoclonal antibody (mAb) targeting galectin-9 (LGALS9). CD4+ conventional T cells (TCD4 or Tconv), Treg ratio, and LGALS9 expression were analyzed by immunohistochemistry (IHC) and cytometry in blood and pancreas of K-rasLSL.G12D/+;Pdx-1-Cre (KC) and K-rasWildType (WT);Pdx1-Cre (WT) mice aged 4-13 months. Pancreatic intraepithelial neoplasm (PanIN) progression and grade were quantified using FIJI software and validated by pathologists. The anti-galectin-9 mAb was validated for its use in mice on isolated murine C57BL/6 Treg by immunofluorescence staining and cytometry. Its specificity and functionality were validated in proliferation assays on rLGALS9-immunosuppressed murine Tconv and in suppression assays between murine Treg and Tconv. Finally, 2-month-old KC mice were treated with anti-LGALS9 and compared to WT mice for peripheral and infiltrating TCD4, Treg, and PanIN progression. IHC and cytometry revealed a significant increase in LGALS9 expression and Treg levels in the blood and pancreas of KC mice proportional to the stages of precancerous lesions. Although present in WT mice, LGALS9 is expressed at a basal level with low and restricted expression that increases slightly over time, while Treg cells are few in number in their circulation and even absent from the pancreas over time. Using our anti-LGALS9 mAb in mice, it is shown that (i) murine Treg express LGALS9, (ii) the mAb could target and inhibit recombinant murine LGALS9, and (iii) neutralize murine Treg suppressive activity. Finally, the anti-LGALS9 mAb in KC mice reduced (i) LGALS9 expression in pancreatic cancer cells, (ii) the Treg ratio, and (iii) the total surface area and grade of PanIN. We demonstrate for the first time that an anti-LGALS9 antibody, by specifically targeting endogenous LGALS9 tumor and exogenous LGALS9 produced by Treg, was able to limit the progression of pancreatic neoplastic lesions in mice, opening up new prospects for its use as an immunotherapeutic tool in PDAC.

A microprotein N1DARP encoded by LINC00261 promotes Notch1 intracellular domain (N1ICD) degradation via disrupting USP10-N1ICD interaction to inhibit chemoresistance in Notch1-hyperactivated pancreatic cancer

The extensively activated Notch signaling pathway in pancreatic cancer cells is important in carcinogenesis, chemoresistance, and recurrence. Targeting this pathway is a promising therapeutic strategy for pancreatic cancer; however, few successful approaches have been reported, and currently used molecular inhibitors of this pathway exhibit limited clinical benefits. In this study, we identified a previously uncharacterized microprotein, Notch1 degradation-associated regulatory polypeptide (N1DARP), encoded by LINC00261. N1DARP knockout accelerated tumor progression and enhanced stem cell properties in pancreatic cancer organoids and LSL-Kras, LSL-Trp53, and Pdx1-Cre (KPC) mice. Mechanistically, N1DARP suppressed canonical and non-canonical Notch1 pathways by competitively disrupting the interaction between N1ICD and ubiquitin-specific peptidase 10 (USP10), thereby promoting K11- and K48-linked polyubiquitination of N1ICD. To evaluate the therapeutic potential of N1DARP, we designed a cell-penetrating stapled peptide, SAH-mAH2-5, with a helical structure similar to that of N1DARP that confers remarkable physicochemical stability. SAH-mAH2-5 interacted with and promoted the proteasome-mediated degradation of N1ICD. SAH-mAH2-5 injection provided substantial therapeutic benefits with limited off-target and systemic adverse effects in Notch1-activated pancreatic cancer models. Taken together, these findings confirm that N1DARP acts as a tumor suppressor and chemosensitizer by regulating USP10-Notch1 oncogenic signaling, and suggest a promising therapeutic strategy targeting the N1DARP–N1ICD interaction in Notch1-activated pancreatic cancer.

A Ketogenic Diet in Combination with Gemcitabine Mitigates Pancreatic Cancer-Associated Cachexia in Male and Female KPC Mice.

Cancer-associated cachexia (CAC) is a critical contributor to pancreatic ductal adenocarcinoma (PDAC) mortality. Thus, there is an urgent need for new strategies to mitigate PDAC-associated cachexia; and the exploration of dietary interventions is a critical component. We previously observed that a ketogenic diet (KD) combined with gemcitabine enhances overall survival in the autochthonous LSL-KrasG12D/+; LSL-Trp53 R172H/+; Pdx1-Cre (KPC) mouse model. In this study, we investigated the effect and cellular mechanisms of a KD in combination with gemcitabine on the maintenance of skeletal muscle mass in KPC mice. For this purpose, male and female pancreatic tumor-bearing KPC mice were allocated to a control diet (CD), a KD, a CD + gemcitabine (CG), or a KD + gemcitabine (KG) group. We observed that a KD or a KG-mitigated muscle strength declined over time and presented higher gastrocnemius weights compared CD-fed mice. Mechanistically, we observed sex-dependent effects of KG treatment, including the inhibition of autophagy, and increased phosphorylation levels of eIF2α in KG-treated KPC mice when compared to CG-treated mice. Our data suggest that a KG results in preservation of skeletal muscle mass. Additional research is warranted to explore whether this diet-treatment combination can be clinically effective in combating CAC in PDAC patients.

295-LB: Pancreatic Peroxisomes Are Required for Physiological Regulation of Insulin Secretion and Maintenance of Normal Glucose Tolerance in Male Mice

The regulation of glucose-stimulated insulin secretion (GSIS) is central to maintenance of glucose disposal. The role of lipid storage and lipid oxidation to regulate pancreatic islet β-cell insulin secretion is not completely understood. Peroxisomes are enriched in insulin-producing β-cells, but their contribution to β-cell function and maturity is lacking. Therefore, we generated a novel model of peroxisomal dysfunction to test the hypothesis that peroxisomal lipid metabolism is required to prevent a hyperlipidemic environment that interferes with β-cell function and maturity. Pex5 plays a critical role in importing the majority of peroxisomal proteins into the peroxisomal matrix; thus, deletion of the Pex5 gene creates a cellular environment with reductions in function of this organelle. In this study, we bred Pex5 mice on a C57BL/6J background containing ‘floxed’ alleles to Pdx1-cre mice to generate offspring with a pancreas-wide deficiency in Pex5 expression and abundance. Glucose tolerance was impaired by 12 weeks in male mice (n=20; AUC p<0.05). Surprisingly, GSIS was elevated in Pex5Pdx1-/- mice compared to littermate controls both in vivo (n=15; AUC p<0.05) and ex vivo in isolated islets as measured by perifusion analysis (n=12; AUC p<0.01). We further found that a subset of β-cells from Pex5Pdx1-/- mice expressed the de-differentiation marker Aldh1a3, while a subset of β-cells showed reductions in MafA and Nkx6.1 staining compared to controls. Perilipin-1 staining showed increased presence of lipid droplets in pancreatic tissue of Pex5Pdx1-/- mice versus controls. Thus, pancreatic deletion of peroxisomal function results in increased lipid storage in pancreatic tissue that is correlated with exaggerated insulin secretion. Collectively, these data are in support of a model whereby peroxisomal function supports β-cell insulin secretion and maintenance of the fully differentiated state. Disclosure S. Burke: None. S. Ghosh: None. D. Burk: None. R. Noland: None. J. Collier: None.

Abstract LB299: Identification of key regulators for nuclear protein export inhibitor and chemotherapy combination in pancreatic cancer using spatial genomics approach

Abstract Background: All patients with pancreatic ductal adenocarcinoma (PDAC) experience disease progression after gemcitabine-nab-paclitaxel (GemPac) treatment with a median time to progression of just under 6 months. There is an urgent need for more effective combination therapies for this highly recalcitrant disease. Previously we showed that the selective inhibitor of nuclear export (SINE) selinexor (Sel) against XPO1 in combination with GemPac enhanced the survival of LSL-Kras G12D/+; Trp53 fl/+; Pdx1-Cre (KPC) mice. The Sel-GemPac combination also showed evaluable response in PDAC patients in a Phase I study. Here we identified key regulatory molecules involved in the synergy of the Sel-GemPac combination, utilizing KPC mouse tumors and patient biopsy samples. We also evaluated the efficacy of this combination in a Phase II study. Methods: The activity of Sel-GemPac was evaluated in KPC mouse derived 2D cell line and 3D spheroid models. RNA-seq was performed to evaluate transcriptional differences associated with synergy. The identified differentially expressed genes (DEG) were validated in vitro through siRNA mediated silencing, qPCR, cytotoxicity, and related assays. The molecular mechanism of synergy was also evaluated in KPC mice tumors and in biopsies from patients enrolled in the Phase I portion of the trial using spatial transcriptomic and proteomic analysis. The efficacy of Sel-Gem was evaluated in a Phase II study in patients with metastatic PDAC (NCT02178436). Results: In a KPC tumor derived cell line, Sel inhibited cell growth and suppressed spheroid formation. Sel synergized with GemPac (CI<1) leading to superior inhibition of cell proliferation and suppressed long term growth (colonies). GSEA of RNA-seq showed inhibition of protein transport mechanism after treatment. In spatial transcriptomic analysis, marked suppression of stroma supporting markers were observed in tumor and stromal compartments of KPC tumors. In the treated cancer and stromal cells, 197 and 42 genes were differentially expressed respectively out of a total 11,650 genes, affecting 16 pathways. The top upregulated genes included Mt2, Nrn1, Gpihbp1 and Pals2 and the top downregulated genes include Chil3, Pglyrp1, Ear10 and Prap1. In the Phase II portion of the trial, 4 patients were given Sel (60 mg oral) with Gem (1000 mg/m2 i.v.) on days 1, 8, and 15 of the 28-day cycle. One patient from Phase II showed stable reduction of tumor lesion from 38.3 × 33.2 cm (before) to 33.2 × 26.9 cm (after). The best response based on RECIST 1.1 was minus 25%. This patient had an overall survival of 16 months. In patient biopsies, spatial transcriptomic analysis of malignant ductal tissues, detected 346 and 487 DEG between a responder and a non-responder at baseline, affecting more than 29 pathways. Most important the top DEG included S100P, B2M, CXCL5 and HP. Conclusions: Collectively, our studies show that XPO1 may be a valid therapeutic target in PDAC and Sel-GemPac is a combination warranting further investigation. Citation Format: Md. Hafiz Uddin, Sahar F. Bannoura, Husain Y. Khan, Amro Aboukameel, Yiwei Li, Ibrahim Azar, Eliza W. Bael, Miguel Tobon, Donald Weaver, Steve Kim, Tanya Odisho, Amr Mohammed, Gregory Dyson, Rafic Beydoun, Ramzi M. Mohammad, Herbert Chen, Bassel El-Rayes, Philip A. Philip, Mohammad N. Al-Hallak, Asfar S. Azmi. Identification of key regulators for nuclear protein export inhibitor and chemotherapy combination in pancreatic cancer using spatial genomics approach [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB299.

Abstract 369: Gemcitabine nab-paclitaxel combination with DMAPT in PDAC cachexia

Abstract Background: Cachexia is the progressive, unintentional wasting of muscle and adipose tissue caused by tumors. Cancer cachexia affects >80% of patients with pancreatic ductal adenocarcinoma (PDAC). Certain chemotherapy regimens cause debilitation in patients and muscle and fat wasting in mouse models. Gemcitabine with nab-paclitaxel (GemNP) is a first line treatment for PDAC. Whether GemNP promotes cachexia is unknown. Muscle wasting in cancer cachexia has been functionally linked to NF-κB, which can be induced by chemotherapies and is active in PDAC tumors. Among other functions, NF-κB induces expression of Interleukin-6 (IL-6), a major mediator of cancer cachexia. DMAPT is a small molecule NF-κB inhibitor. In prior work, DMAPT plus Gem reduced tumor burden in a genetic model of pancreatic cancer and separately, prevented muscle wasting in a genetic model of mammary cancer. Therefore, the purpose of our study was to determine whether GemNP promotes cachexia and whether DMAPT could reduce tumor burden and/or cachexia in PDAC, potentially through reducing IL-6 expression. Methods: In our in vitro model, conditioned media (CM) from KPC pancreatic cancer cells derived from the KrasG12D;Trp53R172H;Pdx1-Cre mouse, induces wasting of C2C12 myotubes. Thus, myotubes were treated with 50% KPC-CM or control CM and supplemented with 0, 5, or 10uM DMAPT for 48hrs. For in vivo modeling of PDAC cachexia, 12-week-old male C57BL/6J mice were orthotopically implanted with 5x104 KPC cells or KPC cells deleted for IL6 (KPC-IL6KO) cells; controls underwent sham surgery. Starting on day 4, every 6 days tumor-bearing mice were given 120 mg/kg gemcitabine and 10 mg/kg nab-paclitaxel (GemNP) for four rounds, then gemcitabine only for subsequent rounds, with or without 100mg/kg DMAPT (by gavage 5 days/week). Controls received vehicle only. Results: DMAPT prevented atrophy in KPC-CM treated myotubes. In vivo, GemNP increased median survival, to 23.5d in KPC + GemNP mice versus 17d in untreated KPC mice (P<0.001). GemNP + DMAPT increased survival (25d) compared to untreated KPC (P<0.001), but was similar to KPC + GemNP alone (P=0.693). There were no differences in tumor mass at euthanasia. All KPC groups lost significant body weight and fat mass compared to sham controls. In mice with KPC IL-6KO tumors, GemNP increased survival, with median survival of 39d in KPC-IL6KO + GemNP mice compared with 30d in untreated KPC-IL6KO (P<0.001). GemNP + DMAPT increased survival (35 d in KPC IL-6KO) compared to untreated KPC-IL6KO (P<0.001), but was similar to KPC IL-6KO + GemNP (P=0.276). There was a trend for GemNP + DMAPT to have smaller KPC-IL6KO tumors compared to GemNP (p=0.055). Conclusion: Regardless of IL-6 expression from tumor cells, treatment with GemNP improved survival in orthotopic PDAC without exacerbating cachexia. DMAPT did not extend survival above GemNP alone. Despite promising in vitro data and prior published work, DMAPT was not efficacious in reducing tumor growth or treating PDAC cachexia in this model. Citation Format: Brittany Counts, Sephora Jean, Reggie Wang, Teresa Zimmers. Gemcitabine nab-paclitaxel combination with DMAPT in PDAC cachexia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 369.

SDCBP promotes pancreatic cancer progression by preventing YAP1 from β-TrCP-mediated proteasomal degradation

ObjectivePancreatic ductal adenocarcinoma (PDAC) is a highly lethal tumour with limited treatment options. Here, we identified syndecan binding protein (SDCBP), also known as syntenin1, as a novel targetable factor in...

Inhibition of the SLC35B2–TPST2 Axis of Tyrosine Sulfation Attenuates the Growth and Metastasis of Pancreatic Ductal Adenocarcinom

Pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of cancer deaths in the United States. Tyrosine sulfation, catalyzed by the tyrosylprotein sulfotransferase 2 (TPST2), is a post-translational modification essential for protein-protein interactions and cellular functions. Solute carrier family 35 member B (SLC35B2) is a key transporter that transports the universal sulfate donor 3'-phosphoadenosine 5'-phosphosulfate into the Golgi apparatus where the protein sulfation occurs. The goal of this study was to determine whether and how the SLC35B2-TPST2 axis of tyrosine sulfation plays a role in PDAC. Gene expression was analyzed in PDAC patients and mice. Human PDAC MIA PaCa-2 and PANC-1 cells were used for invitro studies. TPST2-deficient MIA PaCa-2 cells were generated to assess xenograft tumor growth invivo. Mouse PDAC cells derived from the KrasLSL-G12D/+;Tp53L/+;Pdx1-Cre (KPC) mice were used to generate Tpst2 knockout KPC cells to evaluate tumor growth and metastasis invivo. High expressions of SLC35B2 and TPST2 were correlated with poor PDAC patient survival. Knocking down SLC35B2 or TPST2, or pharmacologicically inhibiting sulfation, resulted in the inhibition of PDAC cell proliferation and migration invitro. TPST2-deficient MIA PaCa-2 cells showed inhibited xenograft tumor growth. Orthotopic inoculation of Tpst2 knockout KPC cells in mice showed inhibition of primary tumor growth, local invasion, and metastasis. Mechanistically, the integrin β4 was found to be a novel substrate of TPST2. Inhibition of sulfation destabilizes integrin β4 protein, which may have accounted for the suppression of metastasis. Targeting the SLC35B2-TPST2 axis of tyrosine sulfation may represent a novel approach for therapeutic intervention of PDAC.

Cystatin B increases autophagic flux by sustaining proteolytic activity of cathepsin B and fuels glycolysis in pancreatic cancer: CSTB orchestrates autophagy and glycolysis in PDAC.

Both autophagy and glycolysis are essential for pancreatic ductal adenocarcinoma (PDAC) survival due to desmoplasia. We investigated whether targeting a hub gene which participates in both processes could be an efficient strategy for PDAC treatment. The expression pattern of glycolysis signatures (GS) and autophagy signatures (AS) and their correlation with cystatin B (CSTB) in PDAC were analysed. It was discovered how CSTB affected the growth, glycolysis, and autophagy of PDAC cells. We assessed competitive binding to cathepsin B (CTSB) between CSTB and cystatin C (CSTC) via immunoprecipitation (IP) and immunofluorescence (IF). Chromatin immunoprecipitation quantitative polymerase chain reaction (ChIP-qPCR) and luciferase reporter gene assays were used to unveil the mechanism underlying CSTB upregulation. The expression pattern of CSTB was examined in clinical samples and KrasG12D/+, Trp53R172H/+, Pdx1-Cre (KPC) mice. GS and AS were enriched and closely associated in PDAC tissues. CSTB increased autophagic flux and provided substrates for glycolysis. CSTB knockdown attenuated the proliferation of PDAC cells and patient-derived xenografts. The liquid chromatography-tandem mass spectrometry assay indicated CSTB interacted with CTSB and contributed to the proteolytic activity of CTSB in lysosomes. IF and IP assays demonstrated that CSTB competed with CSTC to bind to CTSB. Mutation of the key sites of CSTB abolished the interaction between CSTB and CTSB. CSTB was highly expressed in PDAC due to H3K27acetylation and SP1 expression. High expression of CSTB in PDAC was observed in tissue microarray and patients' serum samples. Our work demonstrated the tumorigenic roles of autophagy and glycolysis in PDAC. CSTB is a key role in orchestrating these processes to ensure energy supply of PDAC cells.

A ketogenic diet in combination with gemcitabine increases survival in pancreatic cancer KPC mice.

Pancreatic ductal adenocarcinoma (PDAC) continues to be a major health problem. A ketogenic diet (KD), characterized by a very low carbohydrate and high fat composition, has gained attention for its anti-tumor potential. We evaluated the effect and mechanisms of feeding a strict KD alone or in combination with gemcitabine in the autochthonous LSL-KrasG12D/+; LSL-Trp53 R172H/+; Pdx1-Cre (KPC) mouse model. For this purpose, both male and female pancreatic tumor-bearing KPC mice were allocated to a control diet (CD; %kcal: 70% carb, 14% protein, 16% fat), a KD (%kcal: 14% protein, 1% carb, 85% fat), a CD + gemcitabine (CG), or a KD + gemcitabine (KG) group. Mice fed a KD alone or in combination with gemcitabine showed significantly increased blood β-hydroxybutyrate levels compared to mice fed a CD or CG. KPC mice fed a KG had a significant increase in overall median survival compared to KPC mice fed a CD (increased overall median survival by 42%). Interestingly, when the data was disaggregated by sex, the effect of a KG was significant in female KPC mice (60% increase in median overall survival), but not in male KPC mice (28% increase in median overall survival). Mechanistically, the enhanced survival response to a KD combined with gemcitabine was multifactorial, including inhibition of ERK and AKT pathways, regulation of fatty acid metabolism and the modulation of the gut microbiota. In summary, a KD in combination with gemcitabine appears beneficial as a treatment strategy in PDAC in KPC mice, deserving further clinical evaluation.

The immune escape signature predicts the prognosis and immunotherapy sensitivity for pancreatic ductal adenocarcinoma.

BackgroundPancreatic ductal adenocarcinoma (PDAC) is one of the deadliest malignancies worldwide. Immune escape is considered to be a reason for immunotherapy failure in PDAC. In this study, we explored the correlation between immune escape-related genes and the prognosis of PDAC patients.Methods1163 PDAC patients from four public databases, including The Cancer Genome Atlas (TCGA), International Cancer Genome Consortium (ICGC), Array-express, and Gene Expression Omnibus (GEO), were included in our study. Cox regression analysis was used to identify the 182 immune genes which were significantly associated with overall survival (OS). And then we established an immune escape-related gene prognosis index (IEGPI) score using several datasets as the training cohort and validated it using the validation cohort. Kaplan-Meier (KM) and Cox regression analysis were used to detect the relationship of IEGPI score with OS. We further explored the relationship between the IEGPI and immune indexes. And the prediction value of response for immunotherapy in Tumor Immune Dysfunction and Exclusion (TIDE) dataset.ResultsWe establish an IEGPI score based on 27 immune escape genes which were significantly related to the prognosis of OS in PDAC patients. Patients in the high-IEGPI group had a significantly worse overall survival rate compared with that in the low-IEGPI groups by KM curves and cox-regression. 5 of the 32 cancer types in TCGA could be significantly distinguished in survival rates through the low- and high-IEGPI groups. Moreover, the correlation between the IEGPI score was negatively correlated with an immune score in several datasets. And higher IEGPI better recurrence-free survival (RFS) and OS in the patients after patients were treated with both PD-1 and CTLA4 in the public datasets (P<0.05). Intriguingly, by using RT-PCR, we verified that the gene of PTPN2, CEP55, and JAK2 were all higher in the BxPC-3 and PANC-1 than HPDE5 cells. Lastly, we found that the IEGPI score was higher in K-rasLSL.G12D/+, p53LSL.R172H/+, Pdx1Cre (KPC) mice model with anti-PD-L1 than that without anti-PD-L1.ConclusionUsing the immune escape-related genes, our study established and validated an IEGPI score in PDAC patients from the public dataset. IEGPI score has the potential to serve as a prognostic marker and as a tool for selecting tumor patients suitable for immunotherapy in clinical practice.

Abstract 4231: Hypoxia reduction in tandem with anti-angiogenic therapy remodels the pancreatic tumor microenvironment

Abstract Pancreatic cancer patients remain largely unresponsive to single agent immune checkpoint blockade (ICB) therapy. This refractory state originates from PDAC’s unique immunosuppressive microenvironment that is densely populated by suppressive myeloid cells, exhibits poor vascularity, and is highly hypoxic. We previously showed that the hypoxia-activated prodrug TH-302 (Evofosfamide) reduces intratumoral hypoxia through a tissue remodeling process, initiates tumor vasculature reorganization, and sensitizes aggressive, spontaneous murine models of prostate cancer to ICB. In a clinical trial testing the combination of Evofosfamide with cytotoxic T-lymphocyte-associated protein (CTLA-4) blockade (NCT03098160) a subset of metastatic, immune checkpoint blockade refractory patients showed prolonged progression free survival. While these studies highlight hypoxia reduction as therapeutically tractable, we lack complete understanding of the contribution of the tumor vasculature to hypoxia reduction therapy, as well as the downstream consequences of hypoxia reduction on the cellular composition of the tumor microenvironment and the responsiveness to immunotherapy. We used a transplantable, orthotopic pancreatic tumor model derived from Kras+/LSL-G12D; Trp53+/LSL-R172H; Pdx1-Cre mice and a syngeneic prostate tumor model to study tumor responses to hypoxia-reduction, anti-angiogenic therapy, and combination immunotherapy. We find that Evofosfamide with anti-VEGFR-2 (DC101) significantly extends mouse survival. Combination therapy reduces intratumoral hypoxia, improves vessel integrity, and increases intratumoral DNA damage. In response to the improved metabolic microenvironment, CD8 T cells gain enhanced effector function and lose expression of exhaustion-associated features. Like other anti-angiogenic regimens, combination Evofosfamide and DC101 leads to an increased frequency of PD-L1 expressing cells within the tumor, however blockade of PD-1 failed to prolong survival. Bulk-tumor RNA sequencing and tumor infiltrating lymphocyte analysis implicates immature myeloid cells as mediators of resistance. Re-education of the infiltrating myeloid stroma with CD40 agonist could improve responses to hypoxia-reducing, anti-angiogenic therapy. Put together, these data indicate that targeted hypoxic reduction with anti-angiogenic therapy remodels the pancreatic tumor microenvironment. In this setting, CD40 agonist over PD-1 blockade provides an additive benefit in prolonging mouse survival. Citation Format: Arthur J. Liu, Michael A. Curran. Hypoxia reduction in tandem with anti-angiogenic therapy remodels the pancreatic tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 4231.