Xenograft Model Of Pancreatic Ductal Adenocarcinoma Research Articles

Unveiling the 4-aminoquinoline derivatives as potent agents against pancreatic ductal adenocarcinoma (PDAC) cell lines

Common antimalarials such as artemisinins, chloroquine and their derivatives also possess potent anti-inflamantory, antiviral and anticancer properties. In the search for new therapeutics to combat difficult-to-treat pancreatic carcinomas, we unveiled that 4-aminoquinoline derivatives, with significant antiplasmodial properties and a great safety profile in vivo, have remarkable anticancer activity against pancreatic ductal adenocarcinoma (PDAC) and considerable efficacy in the xenograft model in vivo. The aim of the present study was to further investigate anticancer properties of these compounds in a drug-repurposing manner. The compounds showed profound cytotoxic effects at nanomolar to low micromolar concentration in 2D cultured cells (in vitro) and in the zebrafish PDAC xenograft model (in vivo). A deeper insight into their mechanisms of cytotoxic action showed these compounds induce apoptosis while increasing reactive oxygen species levels along with autophagy inhibition. Additional investigation of the autophagy modulation proved that tested quinoline derivatives cause P62 and LC3-II accumulation in PDAC cells alongside lysosomal alkalinization. Further, in vivo toxicity studies in the zebrafish model showed low toxicity without developmental side effects of the investigated 4-aminoquinolines, while the applied compounds effectively inhibited tumor growth and prevented the metastasis of xenografted pancreatic cells. Taken together, these results highlight the 4-aminoquinolines as privileged structures that ought to be investigated further for potential application in pancreatic carcinoma treatment.

Abstract C033: Targeting PIKfyve-driven lipid metabolism in pancreatic cancer

Abstract Pancreatic ductal adenocarcinoma (PDAC) subsists in a nutrient-deregulated microenvironment, making it particularly susceptible to treatments that interfere with cancer metabolism. For example, PDAC utilizes and is dependent on high levels of autophagy and other lysosomal processes including macropinocytosis. Although targeting these pathways has shown potential in preclinical studies, progress has been hampered by the challenge of identifying and characterizing favorable targets for drug development. In our study, we aimed to characterize PIKfyve, a lipid kinase integral to lysosomal functioning, as a novel and targetable vulnerability in PDAC. We first confirmed that PIKfyve inhibition disrupted autophagic flux and disrupted lysosomal function. Next, to understand the metabolic effects of PIKfyve inhibition in PDAC cells, we employed a metabolism-focused CRISPR screen. Through this, we identified that PIKfyve inhibition increases PDAC cells’ dependency on de novo fatty acid synthesis through genes such as FASN and ACACA. Accordingly, PIKfyve inhibition triggered an increase in SREBP1-driven transcriptional and metabolic signatures favoring de novo fatty acid synthesis through increasing expression of genes such as FASN and ACACA. Further, employing targeted lipidomics, we determined that PIKfyve inhibition triggered an SREBP1-dependent accumulation of long-chain sphingolipids and that knockout of serine palmitoyl transferase subunits SPTLC1 and SPTLC2 sensitized PDAC cells to PIKfyve inhibition. Taken together, PIKfyve inhibition disrupts PDAC lipid homeostasis such that PDAC cells upregulate de novo synthesis of sphingolipids as a compensatory mechanism, suggesting that co-targeting de novo fatty acid synthesis and PIKfyve would result in synergistic effects. Given the importance of KRAS-MAPK signaling in controlling PDAC metabolic homeostasis, we hypothesized that the KRAS-MAPK signaling pathway is a primary driver of de novo lipid synthesis, specifically enhancing FASN and ACACA levels. Utilizing chromatin immunoprecipitation sequencing, we determined that KRAS or MEK inhibition reduces MYC binding to FASN and ACACA promoters. In line with this observation, KRAS or MEK perturbation decreased FASN and ACACA transcript and protein levels, suggesting that KRAS or MEK inhibition may sensitize PDAC cells to PIKfyve inhibition. Indeed, the simultaneous targeting of PIKfyve and KRAS-MAPK resulted in the elimination of tumor burden in a syngeneic orthotopic model and tumor regression in a xenograft model of PDAC. Taken together, these studies suggest that disrupting lipid metabolism through PIKfyve inhibition induces synthetic lethality in conjunction with KRAS-MAPK-directed therapies for PDAC. Citation Format: Caleb Cheng, Jasmine P Wisniewski, Sydney Peters, Jing Hu, Rahul Mannan, Bailey Jackson, Rüya Pakkan, Pietro Morlacchi, Brian Magnuson, Tongchen He, Rupam Bhattacharyya, Yuanyuan Qiao, Costas A Lyssiotis, Arul M Chinnaiyan. Targeting PIKfyve-driven lipid metabolism in pancreatic cancer [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 C033.

Abstract C046: Targeting wild-type IDH1 sensitizes homologous recombination proficient pancreatic cancer to PARP inhibition

Abstract Introduction: Pancreatic ductal adenocarcinoma (PDAC) is a leading cause of cancer-related mortality in the United States. While Poly (ADP-ribose) polymerase (PARP) inhibitors show promise for PDAC with Homologous Recombination Deficiency (HRD), this mutation is present in less than 10% of cases. Our research focuses on exploiting a vulnerability in the remaining 90% of HR proficient (HRP) tumors. We identified that wild-type isocitrate dehydrogenase 1 (IDH1) plays a critical role in HR repair. By inhibiting IDH1, we discovered suppression of the homologous recombination (HR) pathway, leading to compromised genomic integrity. This renders cancer cells more susceptible to PARP inhibitor therapy, offering a novel therapeutic approach for HRP PDAC. Methods:We assessed the impact of IDH1 inhibition on HR pathway activity using Western blotting to measure HR repair protein levels and HR reporter assays. The effect of IDH1 inhibition (Ivosidenib) combined with PARP inhibition (Olaparib) on in vitro growth inhibition was assessed through drug combination assays. Additionally, we investigated the molecular mechanisms underlying the synergistic effects of the drug combination. To further evaluate the therapeutic potential of IDH1 inhibition in combination with PARP inhibitors, we examined tumor growth in xenograft models of PDAC in athymic nude mice and survival studies were performed in C57BL/6J mice transplanted with orthotopic murine pancreatic cancer. Results:Wild-type IDH1 blockade induces a BRCA-like phenotype by decreasing α-ketoglutarate (αKG) levels and causing histone hypermethylation. This leads to decreased HR repair efficiency in HRP pancreatic cancer cells (MiaPaCa-2 and Panc-1), resulting in a strong synergistic effect when combined with the DNA-damaging agent Olaparib. The combination treatment significantly reduced cell survival in both short and long-term assays. Furthermore, we discovered that these compounds complemented each other in the DNA damage response, leading to increased γ-H2AX (DNA damage marker) and apoptosis. In vivo studies using murine PDAC models demonstrated that the combination of ivosidenib and olaparib synergistically enhanced anti-tumor activity compared to either single agent alone. Conclusion:These data provide novel insight into the mechanisms underlying in the context of HRP cancer susceptibility to a dual treatment approach involving an IDH1 inhibitor and a PARP inhibitor. This approach holds promise for precision medicine in pancreatic cancer treatment, although further pre- clinical studies are needed for validation and refinement. Citation Format: Mehrdad Zarei, Omid Hajihassani, Hallie J. Graor, Soubhi Tahhan, Alexander W. Loftus, Faith Nakazzi, Semmer A. Ali, Parnian Naji, Luke D. Rothermel, Jonothan R. Brody, Jordan M. Winter. Targeting wild-type IDH1 sensitizes homologous recombination proficient pancreatic cancer to PARP inhibition [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 C046.

CCK Receptor Inhibition Reduces Pancreatic Tumor Fibrosis and Promotes Nanoparticle Delivery.

The poor prognosis for pancreatic ductal adenocarcinoma (PDAC) patients is due in part to the highly fibrotic nature of the tumors that impedes delivery of therapeutics, including nanoparticles (NPs). Our prior studies demonstrated that proglumide, a cholecystokinin receptor (CCKR) antagonist, reduced fibrosis pervading PanIN lesions in mice. Here, we further detail how the reduced fibrosis elicited by proglumide achieves the normalization of the desmoplastic tumor microenvironment (TME) and improves nanoparticle uptake. One week following the orthotopic injection of PDAC cells, mice were randomized to normal or proglumide-treated water for 3-6 weeks. Tumors were analyzed ex vivo for fibrosis, vascularity, stellate cell activation, vascular patency, and nanoparticle distribution. The histological staining and three-dimensional imaging of tumors each indicated a reduction in stromal collagen in proglumide-treated mice. Proglumide treatment increased tumor vascularity and decreased the activation of cancer-associated fibroblasts (CAFs). Additionally, PANC-1 cells with the shRNA-mediated knockdown of the CCK2 receptor showed an even greater reduction in collagen, indicating the CCK2 receptors on tumor cells contribute to the desmoplastic TME. Proglumide-mediated reduction in fibrosis also led to functional changes in the TME as evidenced by the enhanced intra-tumoral distribution of small (<12 nm) Rhodamine-loaded nanoparticles. The documented in vivo, tumor cell-intrinsic anti-fibrotic effects of CCK2R blockade in both an immunocompetent syngeneic murine PDAC model as well as a human PDAC xenograft model demonstrates that CCK2R antagonists, such as proglumide, can improve the delivery of nano-encapsulated therapeutics or imaging agents to pancreatic tumors.

Abstract 7125: Inhibition of IDH1 impairs homologous recombination and induces sensitivity to PARP inhibitor in pancreatic cancer

Abstract Introduction: Pancreatic ductal adenocarcinoma (PDAC) is a major cause of cancer-related deaths in the United States. While Poly (ADP-ribose) polymerase (PARP) inhibitors hold promise in treating PDAC with Homologous Recombination Deficiency (HRD). However, HRD occurs in only 10% of cases, leaving 90% with intact Homologous Recombination Proficiency (HRP). Our research highlights the key role of wild-type isocitrate dehydrogenase 1 (IDH1) in maintaining cellular function. We found that inhibiting IDH1 increases DNA damage, suppressing the DNA repair pathway crucial for genomic integrity. This makes cancer cells susceptible to PARP inhibitors, offering a new PDAC treatment approach. Methods: The effect of IDH1 inhibition (Ivosidenib) combined with PARP inhibition (Olaparib) on in vitro growth inhibition were assessed through Trypan blue and PicoGreen in drug combination assays. Additionally, we investigated the molecular mechanism underlying the synergistic effects of drug combination. To further evaluate the therapeutic potential of IDH1 inhibition in combination with PARP inhibitors, we examined tumor growth in xenograft models of PDAC in athymic nude mice. Results: Our data report that, mechanistically, IDH1 inhibition decreased expression of HR DNA repair pathway molecules, such as BRCA1 and BRCA2, and leading to decreased HR repair efficiency in pancreatic cancer cells. Short-term cell viability data demonstrated that targeting IDH1 with ivosidenib when combined with DNA-damaging agent (Olaparib) had a synergistic effect in HRP cell lines (MiaPaCa-2 and Panc-1) with a positive synergy score and Bliss score greater than 1. Additionally, we assessed long-term cell survival using colony formation assays, which yielded a dramatic reduction in cell survival for both Panc1 and MiaPaCa-2 cells when olaparib was combined with ivosidenib, as compared to single-agent controls. Consequently, this reduction increased the levels of apoptosis induced by olaparib. Furthermore, we discovered that these two compounds complemented each other in DNA damage response, leading to an increase in the DNA damage biomarker γ-H2AX. In vivo studies using xenograft models demonstrated that the combination of ivosidenib and olaparib synergistically enhanced anti-tumor activity as compared to either single-agent used alone. Conclusion: Our data provides insights into the mechanisms underlying the susceptibility of PDAC tumor growth to a dual treatment approach involving an IDH1 inhibitor and a PARP inhibitor, particularly in the context of HRP cancer. This highlights the promise of precision medicine for pancreatic cancer treatment, though further pre-clinical studies are needed for validation and refinement. Citation Format: Mehrdad Zarei, Omid Hajihassani, Hallie J. Graor, Alexander W. Loftus, Soubhi Tahhan, Faith Nakazzi, Parnian Naji, Luke D. Rothermel, Jonothan R. Brody, Jordan M. Winter. Inhibition of IDH1 impairs homologous recombination and induces sensitivity to PARP inhibitor in pancreatic cancer [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 7125.

Abstract 2618: BCG017, a bispecific ADC targeting PTK7 and EGFR exhibits anti-tumor efficacy in PDX models

Abstract EGFR is a favored target in various cancer therapies. However, EGFR-based treatments have been challenged by drug resistance and cytotoxicity. Targeting EGFR with an antibody-drug conjugate (ADC) is a promising new therapeutic strategy to address the drug resistance, due to the potent killing effects of the payload. Furthermore, we hypothesized that development of a bispecific ADC (bsADC) targeting EGFR and another tumor-associated antigen (TAA) might improve the tumor selectivity, thereby limiting the on-target off-tumor toxicity. One such TAA candidate is PTK7, which belongs to the same receptor tyrosine kinase family as EGFR, and is co-expressed with EGFR in multiple types of solid tumors, including lung, esophageal, head and neck, and colon cancers. Herein, we developed a fully human anti-human PTK7 EGFR bsAb using our proprietary common light chain RenLite® mouse platform and knobs-into-holes technology. The anti-PTK7 EGFR bsAb demonstrates good purity by SEC-HPLC, and can cross-react with human and cynomolgus monkey antigens with favorable affinity. The PTK7 x EGFR bsAb demonstrated higher binding to various types of cancer cell lines, as well as enhanced internalization in vitro compared with PTK7 parental antibody. Next, the bsAb was conjugated with monomethyl auristatin E (MMAE) to generate an anti-PTK7 x EGFR bsADC, BCG017, with a drug-to-antibody ratio of approximately 4. BCG017 demonstrated superior anti-tumor activity compared to benchmarks and its PTK7 parental ADC in cell-derived non-small-cell lung cancer (NSCLC) xenografts, and showed enhanced anti-tumoractivity when compared to benchmark ADCs and parental ADCs in patient-derived breast and pancreatic ductal adenocarcinoma xenograft models. These results suggest that BCG017 have the potential to be a novel therapeutic alternative for PTK7 and EGFR co-expressing tumors. Citation Format: Sufei Yao, Chengzhang Shang, Zhuolin Li, Gao An, Chaoshe Guo, W. Frank An, Yi Yang. BCG017, a bispecific ADC targeting PTK7 and EGFR exhibits anti-tumor efficacy in PDX models [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 2618.

Abstract 4267: Inflammation mediated expression of integrin αvβ3 potentiates KRAS driven PDAC oncogenesis

Abstract Understanding how pancreatic epithelial cells respond to inflammation and why some become tumorigenic and aggressive could identify new methods of early detection and novel therapeutic targets to control disease progression. Here, we leveraged an in vivo model in which mice with pancreas-specific expression of mutant Kras progress from pre-malignant intraepithelial neoplasias (PanINs) to pancreatic ductal adenocarcinoma (PDAC) following caerulein-induced chronic pancreatitis. During the transition from PanINs to PDAC, we observe two classes of neoplastic lesions: classically-described PanIN tubular structures that are highly organized and previously uncharacterized atypical highly disorganized lesions that are marked by robust integrin αvβ3 expression, observations which are consistent with human pancreatitis and PDAC. In vitro, we report that immune-, stroma-, and tumor cell-derived inflammatory cytokines induce β3 expression in a STAT3-dependent manner leading to increased integrin αvβ3. Considering that we previously established αvβ3 as a driver of cancer cell stress tolerance, drug resistance, and a stem-like phenotype, the acquisition of αvβ3 during chronic inflammation may promote tumorigenesis. Indeed, using the spontaneous tumor mouse model and xenograft models of human PDAC, we find that cells with an inability to upregulate β3 during tumor initiation fail to form tumors. These findings implicate integrin αvβ3 as a critical driver of inflammation-mediated pancreatic tumorigenesis. Citation Format: Ryan M. Shepard, Alejandro D. Campos, Zachary M. Ortega, Hiromi I. Wettersten, Tami Von Schalscha, Sara M. Weis, David A. Cheresh. Inflammation mediated expression of integrin αvβ3 potentiates KRAS driven PDAC oncogenesis [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 4267.

Abstract 6513: Discovery and characterization of a KRASG12C/D/V degrader with potent anti-tumor activity in KRASmut-driven preclinical models

Abstract KRAS mutations are one of the most common oncogenic drivers for multiple cancers. KRASG12X mutations are identified in approximately 25% of lung cancer, 24% of colorectal (CRC), and 79% of pancreatic ductal adenocarcinoma (PDAC). Although the approval of Sotorasib in 2021 by the FDA partially addressed the needs of patients with KRASG12C mutant, other mutations, such as G12D and G12V, still lack effective target therapies. Here, we report RD0255359, a proteolysis targeting chimera (PROTAC) KRASG12C/D/V degrader with highly potent cell viability and KRASG12C/D/V degradation ability. In vitro, RD0255359 exhibited a remarkable KRAS degradation property in NCI-H358 (G12C), AGS (G12D), and SW620 (G12V) cell lines at concentrations below 10 nM. RD0255359 suppressed the proliferation of these cells with nanomolar IC50 without affecting the viability of KRASWT or KRAS-independent cell lines. Meanwhile, using protease inhibitor MG132 or corresponding ligand pretreatment and VHLKD-AGS cell experiment confirmed that RD0255359-induced KRAS degradation was VHL and UPS dependent. In vivo, weekly IV injection of 1-10 mg/kg RD0255359 led to tumor growth suppression or tumor regression in PK-59 PDAC xenograft model. The in vivo PK/PD demonstrated that durable KRAS degradation could be achieved with once- weekly IV injection. Notably, after injection, the drug can distribute into tumor tissue rapidly and abundantly, and maintaining a high concentration till the next administration. Taken together, RD0255359 is a highly potent and selective KRASG12C/D/V degrader with favorable PK properties. It exhibits a promising therapeutic index in preclinical studies. Citation Format: Zhengqing Li, Renqi Xu, Xiaofeng Yang, Zhengyao Zou, Xiaoqiang Kong, Haibo Chen, Yongqing Sun, Ying Zhao, Jing Guo, Cheng Yang, Han Han, Xiaoyun Liu, Hong Lan, Lieming Ding, Quan Zhou, Hao Wu, Jiabing Wang. Discovery and characterization of a KRASG12C/D/V degrader with potent anti-tumor activity in KRASmut-driven preclinical models [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 6513.

Abstract 1392: Neutrophil extracellular trap (NET)-induced pancreatic tumor metastasis requires integrin-linked kinase (ILK) signaling

Abstract Neutrophil extracellular traps (NETs) are an important contributor to the immunosuppressive tumor microenvironment and facilitate metastasis of solid cancers. NETs have been shown to stimulate breast and colon cancer cell metastasis through a mechanism potentially involving an interaction between integrin-linked kinase (ILK) and the cell surface receptor coiled-coil domain containing protein 25 (CCDC25). Here, we have interrogated whether ILK is required for NET-induced metastasis in pancreatic ductal adenocarcinoma (PDAC), an inflammation-driven tumor that is difficult to treat. Evaluation of the circulating neutrophil to lymphocyte ratio (NLR) in the blood samples of patients presenting with metastatic PDAC showed that a higher baseline NLR is associated with significantly shorter overall patient survival. Western blot and immunofluorescence analyses demonstrated that human PDAC cell lines, patient-derived xenograft cells, cells derived from the KPCY genetic model of PDAC and patient-derived organoids co-express CCDC25 and ILK. Co-immunoprecipitation analyses showed that CCDC25 and ILK associate in both mouse and human PDAC cells, and that exposure of the cells to NETs results in an increased amount of ILK associated with CCDC25. Exposure of human and KPCY mouse PDAC cells to NETs increases phosphorylation of GSK3-β and ERK, increases activation of Rac1 and Cdc42 and upregulates markers of epithelial-to-mesenchymal transition (EMT), including ZEB1 and Snail, in a time and concentration-dependent manner. Furthermore, time-lapse imaging using Incucyte-based wounding assays showed that exposure to NETs induces pseudopodia-like protrusions, migration and invasion by these cells. RNAi-mediated suppression of ILK expression or inhibition of ILK activity using a highly selective inhibitor of ILK, QLT-0267, significantly inhibits the NET-induced increase in GSK3-β phosphorylation, Rac1/Cdc42 activation, migration and invasion of PDAC cells. In vivo, co-localization of CCDC25 and ILK is observed in tumors and metastases from KPCY mouse and MIA PaCa-2 human PDAC xenograft models stained by multispectral immunofluorescence and analyzed by confocal microscopy. Tumors and metastases show the presence of infiltrating neutrophils and NETs as indicated by staining for myeloperoxidase and citrullinated histone H3. Importantly, doxycycline-inducible shRNA-mediated suppression of ILK expression by luciferase-positive PDAC cells following injection of these cells through the tail vein of mice administered LPS intranasally results in a significant reduction in metastasis as measured by bioluminescence-based imaging, compared to control animals. Collectively, these data identify NET-induced ILK signaling as an important contributor to PDAC progression and suggest the potential for targeting this axis to prevent PDAC progression and metastasis. Citation Format: Paul C. McDonald, Shannon J. Awrey, Hossein Tavakoli, Rebekah Carroll, Zachary J. Gerbec, Joanna M. Karasinska, David F. Schaeffer, Daniel J. Renouf, Shoukat Dedhar. Neutrophil extracellular trap (NET)-induced pancreatic tumor metastasis requires integrin-linked kinase (ILK) signaling [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 1392.

Anti-proliferative and apoptotic effect of cannabinoids on human pancreatic ductal adenocarcinoma xenograft in BALB/c nude mice model

Human pancreatic ductal adenocarcinoma (PDAC) is a highly malignant and lethal tumor of the exocrine pancreas. Cannabinoids extracted from the hemp plant Cannabis sativa have been suggested as a potential therapeutic agent in several human tumors. However, the anti–tumor effect of cannabinoids on human PDAC is not entirely clarified. In this study, the anti–proliferative and apoptotic effect of cannabinoid solution (THC:CBD at 1:6) at a dose of 1, 5, and 10 mg/kg body weight compared to the negative control (sesame oil) and positive control (5-fluorouracil) was investigated in human PDAC xenograft nude mice model. The findings showed that cannabinoids significantly decreased the mitotic cells and mitotic/apoptotic ratio, meanwhile dramatically increased the apoptotic cells. Parallelly, cannabinoids significantly downregulated Ki-67 and PCNA expression levels. Interestingly, cannabinoids upregulated BAX, BAX/BCL-2 ratio, and Caspase-3, meanwhile, downregulated BCL-2 expression level and could not change Caspase-8 expression level. These findings suggest that cannabinoid solution (THC:CBD at 1:6) could inhibit proliferation and induce apoptosis in human PDAC xenograft models. Cannabinoids, including THC:CBD, should be further studied for use as the potent PDCA therapeutic agent in humans.

A Cancer-Specific Monoclonal Antibody against Podocalyxin Exerted Antitumor Activities in Pancreatic Cancer Xenografts.

Podocalyxin (PODXL) overexpression is associated with poor clinical outcomes in various tumors. PODXL is involved in tumor malignant progression through the promotion of invasiveness and metastasis. Therefore, PODXL is considered a promising target of monoclonal antibody (mAb)-based therapy. However, PODXL also plays an essential role in normal cells, such as vascular and lymphatic endothelial cells. Therefore, cancer specificity or selectivity is required to reduce adverse effects on normal cells. Here, we developed an anti-PODXL cancer-specific mAb (CasMab), PcMab-6 (IgG1, kappa), by immunizing mice with a soluble PODXL ectodomain derived from a glioblastoma LN229 cell. PcMab-6 reacted with the PODXL-positive LN229 cells but not with PODXL-knockout LN229 cells in flow cytometry. Importantly, PcMab-6 recognized pancreatic ductal adenocarcinoma (PDAC) cell lines (MIA PaCa-2, Capan-2, and PK-45H) but did not react with normal lymphatic endothelial cells (LECs). In contrast, one of the non-CasMabs, PcMab-47, showed high reactivity to both the PDAC cell lines and LECs. Next, we engineered PcMab-6 into a mouse IgG2a-type (PcMab-6-mG2a) and a humanized IgG1-type (humPcMab-6) mAb and further produced the core fucose-deficient types (PcMab-6-mG2a-f and humPcMab-6-f, respectively) to potentiate the antibody-dependent cellular cytotoxicity (ADCC). Both PcMab-6-mG2a-f and humPcMab-6-f exerted ADCC and complement-dependent cellular cytotoxicity in the presence of effector cells and complements, respectively. In the PDAC xenograft model, both PcMab-6-mG2a-f and humPcMab-6-f exhibited potent antitumor effects. These results indicated that humPcMab-6-f could apply to antibody-based therapy against PODXL-expressing pancreatic cancers.

Tetrandrine synergizes with MAPK inhibitors in treating KRAS-mutant pancreatic ductal adenocarcinoma via collaboratively modulating the TRAIL-death receptor axis

Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive and lethal malignancies lacking effective therapies. KRAS mutations that occur in over 90% of PDAC are major oncogenic drivers of PDAC. The MAPK signaling pathway plays a central role in KRAS-driven oncogenic signaling. However, pharmacological inhibitors of the MAPK pathway are poorly responded in KRAS-mutant PDAC, raising a compelling need to understand the mechanism behind and to seek new therapeutic solutions. Herein, we perform a screen utilizing a library composed of 800 naturally-derived bioactive compounds to identify natural products that are able to sensitize KRAS-mutant PDAC cells to the MAPK inhibition. We discover that tetrandrine, a natural bisbenzylisoquinoline alkaloid, shows a synergistic effect with MAPK inhibitors in PDAC cells and xenograft models. Mechanistically, pharmacological inhibition of the MAPK pathway exhibits a double-edged impact on the TRAIL-death receptor axis, transcriptionally upregulating TRAIL yet downregulating its agonistic receptors DR4 and DR5, which may explain the limited therapeutic outcomes of MAPK inhibitors in KRAS-mutant PDAC. Of great interest, tetrandrine stabilizes DR4/DR5 protein via impairing ubiquitination-mediated protein degradation, thereby allowing a synergy with MAPK inhibition in inducing apoptosis in KRAS-mutant PDAC. Our findings identify a new combinatorial approach for treating KRAS-mutant PDAC and highlight the role of TRAIL-DR4/DR5 axis in dictating the therapeutic outcome in KRAS-mutant PDAC.

Phase 2 study of elraglusib (9-ING-41), a glycogen synthase kinase-3b inhibitor, in combination with gemcitabine plus nab-paclitaxel (GnP) in patients with previously untreated advanced pancreatic ductal adenocarcinoma (PDAC).

e16289 Background: Despite the increasing incidence and low survival rates in PDAC, treatment options remain somewhat limited. Glycogen synthase kinase-3 (GSK-3) has been known to regulate tumor cell survival and proliferation, suppress apoptosis, and counteract chemotherapy resistance through activation of NF-kB-dependent gene transcription. Elraglusib (9-ING-41), a potent small molecule inhibitor of GSK-3β, has demonstrated inhibition of tumor growth and regression of tumors in patient-derived PDAC xenograft models in combination with gemcitabine (G). This provides the rationale for evaluating the efficacy and safety of elraglusib in combination with GnP in advanced PDAC. Methods: In an open-label, non-randomized, phase 2 study (NCT03678883), pts with previously untreated advanced PDAC received elraglusib 15 mg/kg IV on Days 1 and 4 of each week and nab-paclitaxel (nP) 125 mg/m2 plus G 1000 mg/m2 on Days 1, 8, and 15 in a 28-day cycle. Based on the Simon’s 2-stage design, if &gt; 50% of the first 23 efficacy evaluable (EE) pts achieved a disease control rate (DCR) ≥16 weeks in Stage 1, an additional 37 pts would be enrolled during Stage 2. The primary endpoint was DCR, defined as stable disease for ≥16 weeks, confirmed complete response, or confirmed partial response. Secondary endpoints consisted of objective response rate (ORR), duration of response (DoR), progression-free survival (PFS), overall survival (OS), and treatment-emergent adverse events (TEAEs). Results: 42 pts (ITT) with ECOG 0 to 2 were enrolled in the study (38 received elraglusib at 15 mg/kg and 4 at 9.3 mg/kg); 29 pts with complete first post-baseline tumor assessment comprised the EE population. The median duration of treatment (as of February 4, 2023, cutoff) in the EE was 5.5 months. The DCR was 51.7% (95% CI, 32.5 to 70.6), and the ORR was 37.9% (95% CI, 20.7 to 57.7). Given that Stage 1 criterium of Simon’s 2-stage design was met, the study proceeded to Stage 2. The median PFS and OS were 4.9 months (95% CI, 4.1 to 7.5) and 15.8 months (95% CI, 7.9 to not estimable), respectively, in the EE and 4.9 months (95% CI, 4.1 to 7.5) and 13.7 months (95% CI, 7.9 to 18.1) in the ITT. Grade ≥3 TEAEs occurred in 85.7% of pts in the ITT population. Two pts (4.8%) developed grade ≥3 reversible visible disturbances related to elraglusib and 8 pts (19%) developed grade ≥3 TEAEs (most common being febrile neutropenia and anemia) related to the combination of elraglusib with GnP. All of these were observed at the highest dose of elraglusib (15 mg/kg). Conclusions: Elraglusib in combination with GnP demonstrates early evidence of clinical activity against advanced PDAC and an acceptable safety profile. The study has advanced to randomized phase 2 in pts with metastatic PDAC evaluating the 9.3 mg/kg dose of elraglusib plus GnP compared to GnP alone. Clinical trial information: NCT03678883 .

Abstract 1598: RM-046, a first-in-class, mutant-selective, and oral KRASQ61H(ON) inhibitor that drives tumor regression in preclinical models and validates KRASQ61H as a therapeutic target

Abstract KRASQ61H mutant cancers represent a significant unmet medical need and include common solid tumor histotypes such as non-small cell lung cancer (NSCLC), pancreatic ductal adenocarcinoma (PDAC), and colorectal cancer (CRC). Currently, there are no targeted inhibitors of KRASQ61H in clinical development. Mutant RAS proteins exist predominantly in the GTP-bound active RAS(ON) state, leading to excessive downstream signaling via interaction with effectors such as RAF kinases. The properties of the intrinsic GTPase cycle of KRASQ61H should lead to accumulation of the active ON state. RM-046 is a potent, selective, and oral tri-complex inhibitor of KRASQ61H(ON). RM-046 binds to the abundant intracellular chaperone protein cyclophilin A (CypA) with high affinity to form a binary complex that then non-covalently and selectively engages the active ON state of KRASQ61H. The resulting tri-complex sterically blocks KRASQ61H binding to its effectors, thereby inhibiting its downstream signaling. RM-046 potently suppressed ERK phosphorylation and proliferation in KRASQ61H mutant cancer cells with sub-nanomolar EC50 and IC50 values while displaying selectivity over cancer cells with wildtype KRAS. RM-046 showed no evidence of off-target interactions based on in vitro GTPase, kinome, and safety panels. As a single agent, RM-046 induced dose-dependent, deep, and durable suppression of RAS pathway signaling in preclinical xenograft models in vivo. Daily oral administration of RM-046 demonstrated dose-dependent anti-tumor activity and drove deep tumor regressions across several preclinical xenograft models of human KRASQ61H tumors, including NSCLC and PDAC. All treatment regimens tested with RM-046 were tolerated in vivo. As far as we are aware, RM-046 is the first oral and mutant-selective inhibitor of KRASQ61H(ON). It also represents an example of a non-covalent, mutant-selective tri-complex inhibitor of a KRAS oncogenic driver mutation. Citation Format: Yu C. Yang, Severin Thompson, David Montgomery, Antonio J. Quiñones, Xing Wei, Benjamin Madej, Aidan Tomlinson, Nataliya T. Shifrin, Alexander McNamara, Ethan Ahler, Laura L. McDowell, Michael Flagella, John Setser, Stephanie Chang, Zhican Wang, Zhengping Wang, Jun Huang, Steve Ballmer, Shaong li, Andreas Buckl, Elena Koltun, Adrian Gill, Jingjing Jiang, Mallika Singh, Jacqueline A. Smith. RM-046, a first-in-class, mutant-selective, and oral KRASQ61H(ON) inhibitor that drives tumor regression in preclinical models and validates KRASQ61H as a therapeutic target [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 1598.

HNF1A regulates oxaliplatin resistance in pancreatic cancer by targeting 53BP1.

DNA double‑strand break repair is critically involved in oxaliplatin resistance in pancreatic ductal adenocarcinoma (PDAC). Hepatocyte nuclear factor1 homeobox A (HNF1A) has received increased attention regarding its role in cancer progression. The present study explored the role of HNF1A in oxaliplatin resistance in PDAC. The results revealed that HNF1A expression was negatively associated with oxaliplatin chemoresistance in PDAC tissues and cell lines. HNF1A inhibition promoted the proliferation, colony formation and stemness of PDAC cells, and suppressed their apoptosis. Furthermore, HNF1A inhibition switched nonhomologous end joining to homologous recombination, thereby enhancing genomic stability and oxaliplatin resistance. Mechanistically, HNF1A transcriptionally activates p53‑binding protein 1 (53BP1) expression by directly interacting with the 53BP1 promoter region. Upregulation of HNF1A and 53BP1 induced significant inhibition of PDAC growth and oxaliplatin resistance in patient‑derived PDAC xenograft models and orthotopic models. In conclusion, the findings of the present study suggested that HNF1A/53BP1 may be a promising PDAC therapeutic target for overcoming oxaliplatin resistance.

Systemic deuteration of SCID mice using the water-isotopologue deuterium oxide (D2 O) inhibits tumor growth in an orthotopic bioluminescent model of human pancreatic ductal adenocarcinoma.

Since its initial discovery as a natural isotopologue of dihydrogen oxide (1 H2 O), extensive research has focused on the biophysical, biochemical, and pharmacological effects of deuterated water (2 H2 O [D2 O, also referred to as "heavy water"]). Using a panel of cultured human pancreatic ductal adenocarcinoma (PDAC) cells we have profiled (i) D2 O-induced phenotypic antiproliferative and apoptogenic effects, (ii) redox- and proteotoxicity-directed stress response gene expression, and (iii) phosphoprotein-signaling related to endoplasmic reticulum (ER) and MAP-kinase stress response pathways. Differential array analysis revealed early modulation of stress response gene expression in both BxPC-3 and PANC-1 PDAC cells elicited by D2 O (90%; ≤6 h; upregulated: HMOX1, NOS2, CYP2E1, CRYAB, DDIT3, NFKBIA, PTGS1, SOD2, PTGS2; downregulated: RUNX1, MYC, HSPA8, HSPA1A) confirmed by independent RT-qPCR analysis. Immunoblot-analysis revealed rapid (≤6 h) onset of D2 O-induced MAP-kinase signaling (p-JNK, p-p38) together with ER stress response upregulation (p-eIF2α, ATF4, XBP1s, DDIT3/CHOP). Next, we tested the chemotherapeutic efficacy of D2 O-based drinking water supplementation in an orthotopic PDAC model employing firefly luciferase-expressing BxPC-3-FLuc cells in SCID mice. First, feasibility and time course of systemic deuteration (30% D2 O in drinking water; 21 days) were established using time-resolved whole-body proton magnetic resonance imaging and isotope-ratio mass spectrometry-based plasma (D/H)-analysis. D2 O-supplementation suppressed tumor growth by almost 80% with downregulated expression of PCNA, MYC, RUNX1, and HSP70 while increasing tumor levels of DDIT3/CHOP, HO-1, and p-eIF2α. Taken together, these data demonstrate for the first time that pharmacological induction of systemic deuteration significantly reduces orthotopic tumor burden in a murine PDAC xenograft model.

A Preclinical and Phase Ib Study of Palbociclib plus Nab-Paclitaxel in Patients with Metastatic Adenocarcinoma of the Pancreas.

To assess the preclinical efficacy, clinical safety and efficacy, and MTD of palbociclib plus nab-paclitaxel in patients with advanced pancreatic ductal adenocarcinoma (PDAC). Preclinical activity was tested in patient-derived xenograft (PDX) models of PDAC. In the open-label, phase I clinical study, the dose-escalation cohort received oral palbociclib initially at 75 mg/day (range, 50‒125 mg/day; modified 3+3 design; 3/1 schedule); intravenous nab-paclitaxel was administered weekly for 3 weeks/28-day cycle at 100‒125 mg/m2. The modified dose-regimen cohorts received palbociclib 75 mg/day (3/1 schedule or continuously) plus nab-paclitaxel (biweekly 125 or 100 mg/m2, respectively). The prespecified efficacy threshold was 12-month survival probability of ≥65% at the MTD. Palbociclib plus nab-paclitaxel was more effective than gemcitabine plus nab-paclitaxel in three of four PDX models tested; the combination was not inferior to paclitaxel plus gemcitabine. In the clinical trial, 76 patients (80% received prior treatment for advanced disease) were enrolled. Four dose-limiting toxicities were observed [mucositis (n = 1), neutropenia (n = 2), febrile neutropenia (n = 1)]. The MTD was palbociclib 100 mg for 21 of every 28 days and nab-paclitaxel 125 mg/m2 weekly for 3 weeks in a 28-day cycle. Among all patients, the most common all-causality any-grade adverse events were neutropenia (76.3%), asthenia/fatigue (52.6%), nausea (42.1%), and anemia (40.8%). At the MTD (n = 27), the 12-month survival probability was 50% (95% confidence interval, 29.9-67.2). This study showed the tolerability and antitumor activity of palbociclib plus nab-paclitaxel treatment in patients with PDAC; however, the prespecified efficacy threshold was not met. Pfizer Inc (NCT02501902). In this article, the combination of palbociclib, a CDK4/6 inhibitor, and nab-paclitaxel in advanced pancreatic cancer evaluates an important drug combination using translational science. In addition, the work presented combines preclinical and clinical data along with pharmacokinetic and pharmacodynamic assessments to find alternative treatments for this patient population.

Use of DNA-alkylating pyrrole-imidazole polyamides for anti-cancer drug sensitivity screening in pancreatic ductal adenocarcinoma.

Activating mutations of the KRAS occurs in >90% of pancreatic ductal adenocarcinoma (PDAC) cases. However, direct pharmacological targeting of the activated KRAS protein has been challenging. We previously reported that KR12, a DNA-alkylating pyrrole-imidazole polyamide designed to recognize the KRAS G12D/V mutation, showed an anti-tumor effect in colorectal cancer. In this study, we evaluated the anti-tumor effect of KR12 in PDAC. KR12 was synthesized by an automated peptide synthesizer PSSM-8 and tested for anti-tumor effect in PDAC mouse models. KR12 inhibited tumor growth in a spontaneous PDAC mouse model, although the anti-tumor activity appeared to be limited in a human PDAC xenograft model. We developed a pyrrole-imidazole polyamide screening process based on the hypothesis that genetic elements otherwise unaffected by KR12 could exert attenuating effects on KRAS-suppression-resistant PDAC. We identified RAD51 as a potential therapeutic target in human PDAC cells. A RAD51 inhibitor showed an inhibitory effect on cell growth and affected the cytotoxic activity of KR12 in PDAC cells. These data suggested that the simultaneous inhibition of RAD51 and mutant KRAS blockage would be an important therapeutic strategy for PDAC.

Antibody-mediated blockade for galectin-3 binding protein in tumor secretome abrogates PDAC metastasis

The major challenges in pancreatic ductal adenocarcinoma (PDAC) management are local or distant metastasis and limited targeted therapeutics to prevent it. To identify a druggable target in tumor secretome and to explore its therapeutic intervention, we performed a liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based proteomic analysis of tumors obtained from a patient-derived xenograft model of PDAC. Galectin-3 binding protein (Gal-3BP) is identified as a highly secreted protein, and its overexpression is further validated in multiple PDAC tumors and primary cells. Knockdown and exogenous treatment of Gal-3BP showed that it is required for PDAC cell proliferation, migration, and invasion. Mechanistically, we revealed that Gal-3BP enhances galectin-3-mediated epidermal growth factor receptor signaling, leading to increased cMyc and epithelial-mesenchymal transition. To explore the clinical impact of these findings, two antibody clones were developed, and they profoundly abrogated the metastasis of PDAC cells invivo. Altogether, our data demonstrate that Gal-3BP is an important therapeutic target in PDAC, and we propose its blockade by antibody as a therapeutic option for suppressing PDAC metastasis.

Abstract 5974: Multimodal molecular imaging detects early reoxygenation induced by hyaluronan depletion in pancreatic cancer model mouse

Abstract [Purpose] In pancreatic ductal adenocarcinoma (PDAC) which is characterized by an intense desmoplastic feature, the extracellular matrix (ECM) can significantly influence the tumor microenvironment (TME). Hyaluronan (HA), a major component of ECM, is associated with elevated tumor pressure, vascular collapse, and poor perfusion in TME, conferring hypoxia. HA expression is also correlated with poor prognosis in the patients with PDAC. PEGylated human hyaluronidase (PEGPH20) enzymatically depletes hyaluronan in tumors. The resultant improvement in vascular patency and blood perfusion is expected to increase the delivery of therapeutic molecules. The aim of this study was to investigate the change in physiologic and metabolic profile of the tumor in response to treatment with PEGPH20 using multi-modal imaging techniques. We also investigated the capability of PEGPH20 to enhance treatment effect of radiation. [Methods] Athymic nude mice were inoculated with BxPC3 (human pancreatic adenocarcinoma) tumor cells transduced with hyaluronan synthase 3 (HAS3) to the right tibial periosteum. BxPC3-HAS3 tumor treated with PEGPH20 or control buffer were scanned with Electron paramagnetic Resonance imaging (EPRI), dynamic contrast enhanced (DCE) MRI, ultra-small superparamagnetic iron oxide (USPIO) MRI, Photoacoustic imaging (PAI), and Hyperpolarized 13C-MRI using [1-13C] pyruvate to evaluate intratumor pO2, intratumor perfusion, blood volume, O2 saturation, and glycolysis, respectively. [Results] EPRI showed significantly increased pO2 in PEGPH20 treated group. DCE-MRI and USPIO-MRI showed improved perfusion/permeability and local blood volume, respectively after PEGPH20 treatment, accounting for the increase in tumor oxygenation. PAI provided the evidence of immediate changes in tumor oxygenation after treatment. Hyperpolarized 13C-MRI using [1-13C] pyruvate suggested the decreased glycolytic flux evaluated by lactate/pyruvate ratio after PEGPH20 treatment. Combination of radiotherapy and PEGPH20 synergistically delayed tumor progression and prolonged the survival. [Conclusions] This study examined the effect of PEGPH20 on TME in PDAC xenograft model by using non-invasive multimodal imaging techniques. In summary, the non-invasive imaging modalities were useful in evaluating the changes in hemodynamics and metabolism in TME induced by modulation of ECM such as PEGPH20 treatment. PEGPH20 enhanced treatment effect of radiation therapy. The results validated the utility of the imaging methods to non-invasively monitor the changes in TME and predicted the radiosensitizing effect of hyaluronan depletion. Citation Format: Yu Saida, Tomohiro Seki, Shun Kishimoto, Jeffrey R. Brender, Gadisetti VR. Chandramouli, Yasunori Otowa, Kota Yamashita, Kazutoshi Yamamoto, Nallathamby Devasahayam, Murali C. Krishna. Multimodal molecular imaging detects early reoxygenation induced by hyaluronan depletion in pancreatic cancer model mouse [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 5974.