PDAC Model Research Articles

Inhibiting NR5A2 targets stemness in pancreatic cancer by disrupting SOX2/MYC signaling and restoring chemosensitivity

BackgroundPancreatic ductal adenocarcinoma (PDAC) is a profoundly aggressive and fatal cancer. One of the key factors defining its aggressiveness and resilience against chemotherapy is the existence of cancer stem cells (CSCs). The important task of discovering upstream regulators of stemness that are amenable for targeting in PDAC is essential for the advancement of more potent therapeutic approaches. In this study, we sought to elucidate the function of the nuclear receptor subfamily 5, group A, member 2 (NR5A2) in the context of pancreatic CSCs.MethodsWe modeled human PDAC using primary PDAC cells and CSC-enriched sphere cultures. NR5A2 was genetically silenced or inhibited with Cpd3. Assays included RNA-seq, sphere/colony formation, cell viability/toxicity, real-time PCR, western blot, immunofluorescence, ChIP, CUT&Tag, XF Analysis, lactate production, and in vivo tumorigenicity assays. PDAC models from 18 patients were treated with Cpd3-loaded nanocarriers.ResultsOur findings demonstrate that NR5A2 plays a dual role in PDAC. In differentiated cancer cells, NR5A2 promotes cell proliferation by inhibiting CDKN1A. On the other hand, in the CSC population, NR5A2 enhances stemness by upregulating SOX2 through direct binding to its promotor/enhancer region. Additionally, NR5A2 suppresses MYC, leading to the activation of the mitochondrial biogenesis factor PPARGC1A and a shift in metabolism towards oxidative phosphorylation, which is a crucial feature of stemness in PDAC. Importantly, our study shows that the specific NR5A2 inhibitor, Cpd3, sensitizes a significant fraction of PDAC models derived from 18 patients to standard chemotherapy. This treatment approach results in durable remissions and long-term survival. Furthermore, we demonstrate that the expression levels of NR5A2/SOX2 can predict the response to treatment.ConclusionsThe findings of our study highlight the cell context-dependent effects of NR5A2 in PDAC. We have identified a novel pharmacological strategy to modulate SOX2 and MYC levels, which disrupts stemness and prevents relapse in this deadly disease. These insights provide valuable information for the development of targeted therapies for PDAC, offering new hope for improved patient outcomes.Graphical A Schematic illustration of the role of NR5A2 in cancer stem cells versus differentiated cancer cells, along with the action of the NR5A2 inhibitor Cpd3. B Overall survival of tumor-bearing mice following allocated treatment. A total of 18 PDX models were treated using a 2 x 1 x 1 approach (two animals per model per treatment); n=36 per group (illustration created with biorender.com).

Talniflumate abrogates mucin immune suppressive barrier improving efficacy of gemcitabine and nab-paclitaxel treatment in pancreatic cancer

BackgroundPancreatic ductal adenocarcinoma (PDAC) is a lethal disease. This is due to its aggressive course, late diagnosis and its intrinsic drugs resistance. The complexity of the tumor, in terms of cell components and heterogeneity, has led to the approval of few therapies with limited efficacy. The study of the early stages of carcinogenesis provides the opportunity for the identification of actionable pathways that underpin therapeutic resistance.MethodsWe analyzed 43 Intraductal papillary mucinous neoplasms (IPMN) (12 Low-grade and 31 High-grade) by Spatial Transcriptomics. Mouse and human pancreatic cancer organoids and T cells interaction platforms were established to test the role of mucins expression on T cells activity. Syngeneic mouse model of PDAC was used to explore the impact of mucins downregulation on standard therapy efficacy.ResultsSpatial transcriptomics showed that mucin O-glycosylation pathway is increased in the progression from low-grade to high-grade IPMN. We identified GCNT3, a master regulator of mucins expression, as an actionable target of this pathway by talniflumate. We showed that talniflumate impaired mucins expression increasing T cell activation and recognition using both mouse and human organoid interaction platforms. In vivo experiments showed that talniflumate was able to increase the efficacy of the chemotherapy by boosting immune infiltration.ConclusionsFinally, we demonstrated that combination of talniflumate, an anti-inflammatory drug, with chemotherapy effectively improves anti-tumor effect in PDAC.

Vitamin B6 Competition in the Tumor Microenvironment Hampers Antitumor Functions of NK Cells.

The nutrient competition among the different tumor microenvironment components drives tumor growth, immune tolerance, and therapeutic resistance. PDAC cells demand a high amount of VB6, thus competitively causing NK-cell dysfunction. Supplying VB6 with blocking VB6-dependent one-carbon metabolism amplifies the NK-cell antitumor immunity and inhibits tumor growth in PDAC models. This article is featured in Selected Articles from This Issue, p. 5.

The Lin28b/Wnt5a axis drives pancreas cancer through crosstalk between cancer associated fibroblasts and tumor epithelium

Bidirectional signal transduction between tumor epithelial cells and tumor microenvironment (TME) is important for tumor development. Here we show that Lin28b/let-7 pathway is indispensable for modulating the expression of Wnt5a in tumor epithelium, which could be secreted and then up-regulates Lin28b in cancer-associated fibroblasts (CAFs). Moreover, we demonstrate that Lin28b in CAFs promoted growth of PDAC by inducing cytokine PCSK9’s production. Using an orthotopic mouse model of PDAC, we find that depletion of Lin28b in CAFs reduced tumor weight, highlighting the importance of Lin28b in PDAC stroma. Thus, our study shows that the Lin28b-Wnt5a axis plays a critical role in bidirectional crosstalk between pancreatic tumor epithelium and TME and results in a pro-‍tumorigenic contexture.

Immunization with a multi-antigen targeted DNA vaccine eliminates chemoresistant pancreatic cancer by disrupting tumor-stromal cell crosstalk

BackgroundPancreatic ductal adenocarcinoma (PDAC) is characterised by limited responses to chemoimmunotherapy attributed to highly desmoplastic tumor microenvironment. Disrupting the tumor-stromal cell crosstalk is considered as an improved PDAC treatment strategy, whereas little progress has been made due to poor understanding of its underlying mechanism. Here, we examined the cellular role of melanoma associated antigen A isoforms (MAGEA) in regulating tumor-stromal crosstalk mediated chemoresistance.MethodsWe used clinical samples to explore the correlation between MAGEA expression and patient prognosis in multiple cancers. We utilized cancer cell lines, patient derived organoids and orthotopic PDAC model to examine the function of MAGEA in chemoresistance. We performed biochemical, proteome profiler array and transcriptional analysis to uncover a mechanism that governs tumor-stromal crosstalk. We developed a multi-MAGEA antigen targeted DNA vaccine and tested its effect on PDAC tumor growth.ResultsWe establish MAGEA as a regulator of the tumor-stromal crosstalk in PDAC. We provide strong clinical evidence indicating that high MAGEA expression, including MAGEA2, MAGEA3 and MAGEA10, correlates with worse chemotherapeutic response and poor prognosis in multiple cancers, while their expression is up-regulated in chemoresistant PDAC patient derived organoids and cancer cell lines. Mechanistically, MAGEA2 prohibits gemcitabine-induced JNK-c-Jun-p53 mediated cancer cell apoptosis, while gemcitabine stimulated pancreatic stellate cells secretes GDF15 to further enhance the gemcitabine resistance of MAGEA2 expressing cells by activating GFRAL-RET mediated Akt and ERK1/2 dependent survival pathway. Strikingly, immunization with a DNA vaccine that targeting multiple MAGEA antigens, including MAGEA2, MAGEA3 and MAGEA10, elicits robust immune responses against the growth of gemcitabine resistant tumors.ConclusionsThese findings suggest that targeting MAGEA-mediated paracrine regulation of chemoresistance by immunotherapy can be an improved pancreatic cancer treatment strategy.

Conditional Dependency of LP-184 on Prostaglandin Reductase 1 is Synthetic Lethal in Pancreatic Cancers with DNA Damage Repair Deficiencies.

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

B-233 Chromatin Accessibility Uncovers KRAS-driven FOSL2 Promoting Pancreatic Ductal Adenocarcinoma Progression Via Upregulating CCL28

Abstract Background Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal human cancer with 5-year survival rate less than 10%. Pancreatic cancer remains a malignancy of poor prognosis and effective therapies are lacking, therefore, studying the pathogenesis and identifying more therapeutic targets are urgently needed for PDAC treatment. The epigenetic mechanisms of PDAC progression are poorly understood. This study aims to identify key transcription factor (TF) by multiomics sequencing to explore the molecular mechanisms of TF with critical roles in PDAC. Methods we used ATAC-seq, H3K27ac ChIP-seq, and RNA-seq to characterized the epigenetic landscape by genetically engineered mouse models (GEMMs) of PDAC with or without KRAS and/or TP53 mutations. Survival of FOSL2 was assessed using Kaplan-Meier method and multivariate Cox regression analysis for patients with PDAC. We performed Cleavage Under Targets and Tagmentation (CUT&Tag) to study the potential targets of FOSL2. CCK8, transwell migration and invasion, RT-qPCR, western blot, IHC, ChIP-qPCR, dual-luciferase reporter, and xenograft model assays were used to explore the functions and underlying mechanisms of FOSL2 in PDAC progression. Results We found that the epigenetic changes were involved in some immuno-suppressed signaling with PDAC progression. Also, we identified a critical regulator FOSL2 which showed enriched open chromatin, H3K27ac signal, and higher RNA expression in KC and KPC than WT. The high expression of FOSL2 was associated with poor prognosis of patients with PDAC. Single-cell RNA-seq data showed that FOSL2 expression was very low in T, B, and acinar cells, and enriched in endothelial, ductal cells and upregulated in cancerous ductal cells compared with normal pancreatic ductal cells. functionally, FOSL2 can not only promote the proliferation, migration and invasion of pancreatic cancer, but also inhibit tumor immunity such as reducing the infiltration of CD8+T cells and increasing the infiltration of regulatory T (Treg) cells.In terms of mechanism, we found that CCL28 was a key target gene of FOSL2 by CUT&Tag sequencing. We confirmed that FOSL2 can regulate the expression of CCL28 by knockdown or overexpression of FOSL2 in vitro. ChIP-qPCR and dual luciferase reporter assays confirmed that FOSL2 activated the transcription of CCL28 by binding to the upstream of CCL28. In vivo mouse assay showed that FOSL2 promoted tumor growth and Treg cell recruitment through CCL28. We also explored the mechanism by which FOSL2 was upregulated in pancreatic cancer and found that KRAS mutation mediated FOSL2 transcription and subsequent CCL28 expression through the MAPK/ERK pathway, which implicated an immuno-suppressed regulatory axis of KRAS/MAPK-FOSL2-CCL28-Treg cells in PDAC development. Conclusion We described the changes of epigenetic landscape during PDAC progression and found that KRAS mutation promoted FOSL2 expression, which recruited more Treg cells by upregulating CCL28 and ultimately led to immunosuppression in pancreatic cancer. Targeting CCL28 with CCL28 antibody can reduce the invasion of Treg cells, which provides a new way to overcome the Treg cell-mediated tumor immune escape in patients with PDAC.

IL-12 Treatment Attenuates Muscle Wasting Via Regulation Of MCP-1 In A Murine Model Of PDAC

IL-12 Treatment Attenuates Muscle Wasting Via Regulation Of MCP-1 In A Murine Model Of PDAC

KRASG12D inhibition reprograms the microenvironment of early and advanced pancreatic cancer to promote FAS-mediated killing by CD8+ T cells

The KRASG12D mutation is present in nearly half of pancreatic adenocarcinomas (PDAC). We investigated the effects of inhibiting the KRASG12D mutant protein with MRTX1133, a non-covalent small molecule inhibitor of KRASG12D, on early and advanced PDAC and its influence on the tumor microenvironment. Employing 16 different models of KRASG12D-driven PDAC, we demonstrate that MRTX1133 reverses early PDAC growth, increases intratumoral CD8+ effector Tcells, decreases myeloid infiltration, and reprograms cancer-associated fibroblasts. MRTX1133 leads to regression of both established PanINs and advanced PDAC. Regression of advanced PDAC requires CD8+ Tcells and immune checkpoint blockade (ICB) synergizes with MRTX1133 to eradicate PDAC and prolong overall survival. Mechanistically, inhibition of KRASG12D in advanced PDAC and human patient derived organoids induces FAS expression in cancer cells and facilitates CD8+ Tcell-mediated death. Collectively, this study provides a rationale for a synergistic combination of MRTX1133 with ICB in clinical trials.

Whole-body bioluminescence imaging of T-cell response in PDAC models.

The location of T-cells during tumor progression and treatment provides crucial information in predicting the response in vivo. Here, we investigated, using our bioluminescent, dual color, T-cell reporter mouse, termed TbiLuc, T-cell location and function during murine PDAC tumor growth and checkpoint blockade treatment with anti-PD-1 and anti-CTLA-4. Using this model, we could visualize T-cell location and function in the tumor and the surrounding tumor microenvironment longitudinally. We used murine PDAC clones that formed in vivo tumors with either high T-cell infiltration (immunologically 'hot') or low T-cell infiltration (immunologically 'cold'). Differences in total T-cell bioluminescence could be seen between the 'hot' and 'cold' tumors in the TbiLuc mice. During checkpoint blockade treatment we could see in the tumor-draining lymph nodes an increase in bioluminescence on day 7 after treatment. In the current work, we showed that the TbiLuc mice can be used to monitor T-cell location and function during tumor growth and treatment.

Krüppel-like factor 10 modulates stem cell phenotypes of pancreatic adenocarcinoma by transcriptionally regulating notch receptors

BackgroundPancreatic adenocarcinoma (PDAC) is well known for its rapid distant metastasis and local destructive behavior. Loss of Krüppel-like factor 10 (KLF10) contributes to distant migration of PDAC. The role of KLF10 in modulating tumorigenesis and stem cell phenotypes of PDAC is unclear.MethodsAdditional depletion of KLF10 in KC (LSL: KrasG12D; Pdx1-Cre) mice, a spontaneous murine PDAC model, was established to evaluate tumorigenesis. Tumor specimens of PDAC patients were immune-stained of KLF10 to correlate with local recurrence after curative resection. Conditional overexpressing KLF10 in MiaPaCa and stably depleting KLF10 in Panc-1 (Panc-1-pLKO-shKLF10) cells were established for evaluating sphere formation, stem cell markers expression and tumor growth. The signal pathways modulated by KLF10 for PDAC stem cell phenotypes were disclosed by microarray analysis and validated by western blot, qRT-PCR, luciferase reporter assay. Candidate targets to reverse PDAC tumor growth were demonstrated in murine model.ResultsKLF10, deficient in two-thirds of 105 patients with resected pancreatic PDAC, was associated with rapid local recurrence and large tumor size. Additional KLF10 depletion in KC mice accelerated progression from pancreatic intraepithelial neoplasia to PDAC. Increased sphere formation, expression of stem cell markers, and tumor growth were observed in Panc-1-pLKO-shKLF10 compared with vector control. Genetically or pharmacologically overexpression of KLF10 reversed the stem cell phenotypes induced by KLF10 depletion. Ingenuity pathway analysis and gene set enrichment analysis showed that Notch signaling molecules, including Notch receptors 3 and 4, were over-expressed in Panc-1-pLKO-shKLF10. KLF10 transcriptionally suppressed Notch-3 and -4 by competing with E74-like ETS transcription factor 3, a positive regulator, for promoter binding. Downregulation of Notch signaling, either genetically or pharmacologically, ameliorated the stem cell phenotypes of Panc-1-pLKO-shKLF10. The combination of metformin, which upregulated KLF10 expression via phosphorylating AMPK, and evodiamine, a non-toxic Notch-3 methylation stimulator, delayed tumor growth of PDAC with KLF10 deficiency in mice without prominent toxicity.ConclusionsThese results demonstrated a novel signaling pathway by which KLF10 modulates stem cell phenotypes in PDAC through transcriptionally regulating Notch signaling pathway. The elevation of KLF10 and suppression of Notch signaling may jointly reduce PDAC tumorigenesis and malignant progression.

Phase 1b study of batiraxcept (AVB-S6-500, BT) plus gemcitabine (G) and nab-paclitaxel (NP) as first-line treatment (1L) for advanced pancreatic adenocarcinoma (PDAC).

e16258 Background: PDAC is a deadly aggressive cancer characterized by up to 80% tumor stroma enriched by macrophages, fibroblasts, and collagen deposition that influences resistance to therapy. Tyrosine kinase receptor AXL and ligand expression of growth arrest specific protein 6 (GAS6) produce tumor associated macrophages and fibroblasts and correlate with poor prognosis. GAS6 blockade partially reverses epithelial-to-mesenchymal transition of tumor and supports NK cell activation, inhibiting metastasis. As a novel decoy FC fusion protein, batiraxcept (BT) binds to GAS6 and demonstrates highly potent and specific AXL inhibition. Preclinical studies showed a relationship between AXL signaling and response to DNA damage in PDAC models. Methods: This dose escalation study tested BT 15 mg/kg on day 1 and 15 in combination with G 1000 mg/m2 and NP 125 mg/m2 days 1, 8, 15 of a 28-day cycle. Primary endpoints are safety and objective response rate (ORR) using RECIST v1.1; key secondary endpoints are progression free survival (PFS) and overall survival (OS); pharmacokinetic data were collected to determine exposure-PFS response (E-R) relationship. Results: As of January 17, 2023, 21 patients (pts) received BT/G/NP of which 10 (48%) were male and 3 (14%) received prior neoadjuvant/adjuvant therapy. BT-related AEs > 10% pts included fatigue (n=7, 33%), diarrhea (n=4, 19%), infusion related reaction and neutropenia (n=3, 14% each). Grade ≥ 3 BT-related AE occurred in 6 pts (29%). BT administration reduced GAS6 to non-quantifiable levels until treatment ended. 18 pts (86%) discontinued treatment, primarily due to toxicity (n=2) and progressive disease (n=13). Median (m) BT/G/NP treatment duration was 13.1 weeks (range 0.1─74.7). ORR is 29%; mPFS and mOS were 5.4 mo (2.1, 5.6) and 12.3 mo (4.11─NE) respectively. BT efficacy results are described by a minimally efficacious concentration (MEC) of 14.5 mg/L identified by E-R modeling. Conclusions: BT/G/NP combination was well tolerated, and the BT safety profile is consistent with prior trials. Patients with BT trough > MEC had longer PFS and OS. In addition, in these patients OS appears longer than historical data of 8.5 months. Due to increased stroma seen in PDAC, a higher BT MEC may be required to maximize efficacy. Higher BT dose levels in combination with G/NP are under study. Clinical trial information: NCT04983407 . [Table: see text]

CheMo4METPANC: A phase 2 study with combination chemotherapy (gemcitabine and nab-paclitaxel), chemokine (C-X-C) Motif receptor 4 inhibitor (motixafortide), and immune checkpoint blockade (cemiplimab) in metastatic treatment-naïve pancreas adenocarcinoma.

TPS4200 Background: Metastatic pancreatic ductal adenocarcinoma (mPDAC) is a uniformly fatal disease for which treatments result in limited benefit. Failure of immune checkpoint blockade is attributed to multiple immunosuppressive pathways within the tumor microenvironment. The C-X-C motif chemokine receptor 4 (CXCR4)/C-X-C motif chemokine ligand 12 (CXCL12) axis results in exclusion of anti-tumor immune cells. Preclinical studies demonstrated that simultaneous CXCR4 inhibition (CXCR4i) and anti-programmed cell death 1 (aPD1) resulted in tumor stabilization. We extended these findings by testing multiple combinations of CXCR4i, aPD1, and gemcitabine in the KPC mouse model of PDAC. Mice treated with gemcitabine, CXCR4i, and aPD1 (triple therapy) experienced a survival benefit compared to mice treated with either gemcitabine alone, or with CXCR4i/aPD1. Tumors from mice treated with triple therapy demonstrated increased apoptosis and a favorable tumor immune microenvironment (TIME). Motixafortide with pembrolizumab, fluorouracil, and nanoliposomal irinotecan has shown encouraging results in the second-line setting in mPDAC with a confirmed ORR of 13.2% and progression free survival (PFS) of 3.8 months (m). The goal of this first-in-man trial is to test preliminary safety and efficacy of Motixafortide (CXCR4i), Cemiplimab (aPD1), Gemcitabine, and Nab-paclitaxel (MCGN) in treatment naïve mPDAC. Methods: This is an open label, multicenter, investigator-initiated simon-2-stage phase 2 clinical trial in mPDAC testing MCGN. The study includes a six-patient safety run-in cohort and an additional 4 patients comprising a pilot efficacy signal seeking study (N=10). If ≥3 of the 10 patients within the pilot stage were to experience a partial response (PR) by RECIST criteria within 16 weeks, the combination would be considered promising and an expansion cohort of an additional 30 patients was planned. On 09/21/22, we amended the study to forego the planned open-label expansion cohort (N=30) and transition directly to a randomized phase 2 trial testing MCGN compared toGemcitabine and Nab-paclitaxel (GN) alone (N=102), after completion of the pilot phase of the study. The primary endpoint is PFS. The study has 80% power to detect an improvement in PFS from 6 to 9.2 m (HR 0.65) with a one-sided alpha of 0.20. One interim analysis for futility is planned when 50% of the PFS events are observed. Secondary objectives include ORR, disease control rate, duration of clinical benefit and OS. Required (pilot portion) and optional (randomized portion) paired tumor biopsies will undergo exploratory analysis including interrogation of the TIME. This trial was started in September 2020 and has enrolled 10 patients to the pilot stage as of 02/2023. Clinical trial information: NCT04543071 .

P-335 AUM001 potentiates the anti-tumor activity of gemcitabine in an AsPC-1 PDAC model

P-335 AUM001 potentiates the anti-tumor activity of gemcitabine in an AsPC-1 PDAC model

Acid sphingomyelinase expression is associated with survival in resectable pancreatic ductal adenocarcinoma

Pancreatic adenocarcinoma (PDAC) is one of the most common cancers worldwide. Unfortunately, the prognosis of PDAC is rather poor, and for instance, in the USA, over 47,000 people die because of pancreatic cancer annually. Here, we demonstrate that high expression of acid sphingomyelinase in PDAC strongly correlates with long-term survival of patients, as revealed by the analysis of two independent data sources. The positive effects of acid sphingomyelinase expression on long-term survival of PDAC patients were independent of patient demographics as well as tumor grade, lymph node involvement, perineural invasion, tumor stage, lymphovascular invasion, and adjuvant therapy. We also demonstrate that genetic deficiency or pharmacological inhibition of the acid sphingomyelinase promotes tumor growth in an orthotopic mouse model of PDAC. This is mirrored by a poorer pathologic response, as defined by the College of American Pathologists (CAP) score for pancreatic cancer, to neoadjuvant therapy of patients co-treated with functional inhibitors of the acid sphingomyelinase, in particular tricyclic antidepressants and selective serotonin reuptake inhibitors, in a retrospective analysis. Our data indicate expression of the acid sphingomyelinase in PDAC as a prognostic marker for tumor progression. They further suggest that the use of functional inhibitors of the acid sphingomyelinase, at least of tricyclic antidepressants and selective serotonin reuptake inhibitors in patients with PDAC, is contra-indicated. Finally, our data also suggest a potential novel treatment of PDAC patients with recombinant acid sphingomyelinase.Key messagesPancreatic ductal adenocarcinoma (PDAC) is a common tumor with poor prognosis.Expression of acid sphingomyelinase (ASM) determines outcome of PDAC.Genetic deficiency or pharmacologic inhibition of ASM promotes tumor growth in a mouse model.Inhibition of ASM during neoadjuvant treatment for PDAC correlates with worse pathology.ASM expression is a prognostic marker and potential target in PDAC.

The phospholipid transporter PITPNC1 links KRAS to MYC to prevent autophagy in lung and pancreatic cancer

BackgroundThe discovery of functionally relevant KRAS effectors in lung and pancreatic ductal adenocarcinoma (LUAD and PDAC) may yield novel molecular targets or mechanisms amenable to inhibition strategies. Phospholipids availability has been appreciated as a mechanism to modulate KRAS oncogenic potential. Thus, phospholipid transporters may play a functional role in KRAS-driven oncogenesis. Here, we identified and systematically studied the phospholipid transporter PITPNC1 and its controlled network in LUAD and PDAC.MethodsGenetic modulation of KRAS expression as well as pharmacological inhibition of canonical effectors was completed. PITPNC1 genetic depletion was performed in in vitro and in vivo LUAD and PDAC models. PITPNC1-deficient cells were RNA sequenced, and Gene Ontology and enrichment analyses were applied to the output data. Protein-based biochemical and subcellular localization assays were run to investigate PITPNC1-regulated pathways. A drug repurposing approach was used to predict surrogate PITPNC1 inhibitors that were tested in combination with KRASG12C inhibitors in 2D, 3D, and in vivo models.ResultsPITPNC1 was increased in human LUAD and PDAC, and associated with poor patients’ survival. PITPNC1 was regulated by KRAS through MEK1/2 and JNK1/2. Functional experiments showed PITPNC1 requirement for cell proliferation, cell cycle progression and tumour growth. Furthermore, PITPNC1 overexpression enhanced lung colonization and liver metastasis. PITPNC1 regulated a transcriptional signature which highly overlapped with that of KRAS, and controlled mTOR localization via enhanced MYC protein stability to prevent autophagy. JAK2 inhibitors were predicted as putative PITPNC1 inhibitors with antiproliferative effect and their combination with KRASG12C inhibitors elicited a substantial anti-tumour effect in LUAD and PDAC.ConclusionsOur data highlight the functional and clinical relevance of PITPNC1 in LUAD and PDAC. Moreover, PITPNC1 constitutes a new mechanism linking KRAS to MYC, and controls a druggable transcriptional network for combinatorial treatments.Graphical

Safety, tolerability, and effectiveness of the sodium-glucose cotransporter 2 inhibitor (SGLT2i) dapagliflozin in combination with standard chemotherapy for patients with advanced, inoperable pancreatic adenocarcinoma: a phase 1b observational study

BackgroundPancreatic ductal adenocarcinoma (PDAC) is a lethal malignancy. Thus, there is an urgent need for safe and effective novel therapies. PDAC’s excessive reliance on glucose metabolism for its metabolic needs provides a target for metabolic therapy. Preclinical PDAC models have demonstrated that targeting the sodium-glucose co-transporter-2 (SGLT2) with dapagliflozin may be a novel strategy. Whether dapagliflozin is safe and efficacious in humans with PDAC is unclear.MethodsWe performed a phase 1b observational study (ClinicalTrials.gov ID NCT04542291; registered 09/09/2020) to test the safety and tolerability of dapagliflozin (5 mg p.o./day × 2 weeks escalated to 10 mg p.o./day × 6 weeks) added to standard Gemcitabine and nab-Paclitaxel (GnP) chemotherapy in patients with locally advanced and/or metastatic PDAC. Markers of efficacy including Response Evaluation Criteria in Solid Tumors (RECIST 1.1) response, CT-based volumetric body composition measurements, and plasma chemistries for measuring metabolism and tumor burden were also analyzed.ResultsOf 23 patients who were screened, 15 enrolled. One expired (due to complications from underlying disease), 2 dropped out (did not tolerate GnP chemotherapy) during the first 4 weeks, and 12 completed. There were no unexpected or serious adverse events with dapagliflozin. One patient was told to discontinue dapagliflozin after 6 weeks due to elevated ketones, although there were no clinical signs of ketoacidosis. Dapagliflozin compliance was 99.4%. Plasma glucagon increased significantly. Although abdominal muscle and fat volumes decreased; increased muscle-to-fat ratio correlated with better therapeutic response. After 8 weeks of treatment in the study, partial response (PR) to therapy was seen in 2 patients, stable disease (SD) in 9 patients, and progressive disease (PD) in 1 patient. After dapagliflozin discontinuation (and chemotherapy continuation), an additional 7 patients developed the progressive disease in the subsequent scans measured by increased lesion size as well as the development of new lesions. Quantitative imaging assessment was supported by plasma CA19-9 tumor marker measurements.ConclusionsDapagliflozin is well-tolerated and was associated with high compliance in patients with advanced, inoperable PDAC. Overall favorable changes in tumor response and plasma biomarkers suggest it may have efficacy against PDAC, warranting further investigation.

PGM3 inhibition shows cooperative effects with erastin inducing pancreatic cancer cell death via activation of the unfolded protein response.

Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive cancer with a poor patient prognosis. Remarkably, PDAC is one of the most aggressive and deadly tumor types and is notorious for its resistance to all types of treatment. PDAC resistance is frequently associated with a wide metabolic rewiring and in particular of the glycolytic branch named Hexosamine Biosynthetic Pathway (HBP). Transcriptional and bioinformatics analysis were performed to obtain information about the effect of the HBP inhibition in two cell models of PDAC. Cell count, western blot, HPLC and metabolomics analyses were used to determine the impact of the combined treatment between an HBP's Phosphoglucomutase 3 (PGM3) enzyme inhibitor, named FR054, and erastin (ERA), a recognized ferroptosis inducer, on PDAC cell growth and survival. Here we show that the combined treatment applied to different PDAC cell lines induces a significant decrease in cell proliferation and a concurrent enhancement of cell death. Furthermore, we show that this combined treatment induces Unfolded Protein Response (UPR), NFE2 Like BZIP Transcription Factor 2 (NRF2) activation, a change in cellular redox state, a greater sensitivity to oxidative stress, a major dependence on glutamine metabolism, and finally ferroptosis cell death. Our study discloses that HBP inhibition enhances, via UPR activation, the ERA effect and therefore might be a novel anticancer mechanism to be exploited as PDAC therapy.

Adjustable extracellular matrix rigidity tumor model for studying stiffness dependent pancreatic ductal adenocarcinomas progression and tumor immunosuppression.

Pancreatic ductal adenocarcinomas (PDAC) is one of the stiffest malignancies with strong solid stresses. Increasing stiffness could alter cellular behavior and trigger internal signaling pathways and is strongly associated with a poor prognosis in PDAC. So far, there has been no report on of an experimental model that can rapidly construct and stably maintain a stiffness gradient dimension in both vitro and in vivo. In this study, a gelatin methacryloyl (GelMA)-based hydrogel was designed for in vitro and in vivo PDAC experiments. The GelMA-based hydrogel has porous, adjustable mechanical properties and excellent in vitro and in vivo biocompatibility. The GelMA-based in vitro 3D culture method can effectively form a gradient and stable extracellular matrix stiffness, affecting cell morphology, cytoskeleton remodeling, and malignant biological behaviors such as proliferation and metastasis. This model is suitable for in vivo studies with long-term maintenance of matrix stiffness and no significant toxicity. High matrix stiffness can significantly promote PDAC progression and tumor immunosuppression. This novel adaptive extracellular matrix rigidity tumor model is an excellent candidate for further development as an in vitro and in vivo biomechanical study model of PDAC or other tumors with strong solid stresses.

Abstract 5069: Cancer immunotherapy based on the RNAi-based PD-L1 inhibitor, MN-siPDL1, demonstrates efficacy in preclinical pancreatic adenocarcinoma

Abstract The recent past has seen impressive progress in the treatment of various malignancies using immunotherapy. One of the most promising approaches involves immune checkpoint inhibition, mainly relying on monoclonal antibodies or small molecules. With specific relevance to pancreatic cancer, however, checkpoint inhibitors are largely ineffective, in part because of poor T cell infiltration, low tumor mutational burden, an immunosuppressive tumor microenvironment, and the presence of a dense desmoplastic stroma. To overcome these challenges, here we describe the preclinical testing of a new checkpoint inhibitor named MN-siPDL1 in combination with gemcitabine in an aggressive model of PDAC. MN-siPDL1 utilizes the RNA interference mechanism (siRNA) to block the synthesis of the critical immune checkpoint mediator, PD-L1 by tumor cells. The siRNA is delivered by a nanoparticle carrier (MN) that is optimized for oligonucleotide delivery to solid tumors. The efficacy and preliminary safety of MN-siPDL1 were tested in an orthotopic syngeneic model of pancreatic ductal adenocarcinoma generated by implanting Hy15549 murine PDAC cells into the pancreas of C57BL/6J mice. Our studies demonstrated that MN-siPDL1 was successfully delivered and effective even in the highly desmoplastic and hypovascular Hy15549 murine model of PDAC, which has been deemed nonresponsive to antibody-based immune checkpoint blockade. Anatomic MRI showed that in the animals treated with MN-siPDL1 plus gemcitabine, tumor growth rates were lower than in the vehicle control. After two weekly treatments with MN-siPDL1 plus gemcitabine, tumor volumes were four times smaller than in untreated animals. Importantly, animal survival was improved dramatically in animals treated with MN-siPDL1 plus gemcitabine compared to all other groups. Among the animals treated with MN-siPDL1 plus gemcitabine, the hazard ratio for overall survival (OS) relative to PBS was 0.08. Interestingly, even in the absence of gemcitabine, MN-siPDL1 as monotherapy improved survival more dramatically than gemcitabine (HR, 0.24 for MN-siPDL1 vs. 0.42 for gemcitabine). Immunohistology on the tumor tissues post-necropsy indicated that the treatment inhibited PD-L1 and induced a cytotoxic immune response. Finally, as an initial measurement of tissue damage due to the treatment, we analyzed major organs by histopathology and saw no differences from the vehicle-treated controls. Considering the aggressive and fibrous nature of the Hy15549 model and its resistance to traditional checkpoint inhibitors, the described RNAi-based therapeutic approach could be promising against PDAC and could make an impact on one of the most intractable cancers which has long evaded the power of modern medicine to deliver long-term survival. Citation Format: Byunghee Yoo, Mozhdeh Sojoodi, Veronica Clavijo Jordan, Pamela Pantazopoulos, Subrata Ghosh, Peter Caravan, Zdravka Medarova. Cancer immunotherapy based on the RNAi-based PD-L1 inhibitor, MN-siPDL1, demonstrates efficacy in preclinical pancreatic adenocarcinoma. [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 5069.