Patient-derived Pancreatic Ductal Adenocarcinoma Xenografts Research Articles

Abstract C025: A novel pan-RAS/β-catenin inhibitor, ADT-030, produces tumor regression in PDX models of pancreatic cancer

Abstract BACKGROUND: The absence of symptoms often leads to late diagnosis of pancreatic ductal adenocarcinoma (PDAC) limiting treatment options. Tumor cell proliferation and invasion/metastasis are usually driven by KRAS mutations and activation of Wnt/β-catenin pathway components. The recent approval of KRASG12C-specific inhibitors for lung cancer has validated RAS as a druggable target but has had limited application for PDAC given the rarity of this codon in PDAC. In addition, no Wnt/β-catenin inhibitors have been FDA approved. Thus, drugs which inhibit these important targets are urgently needed for PDAC and other cancers. Here, we describe treatment results with ADT-030, a novel dual pan-RAS/β-catenin inhibitor in murine models of PDAC and lung cancer. METHODS: In vitro assays of growth inhibition, colony formation, apoptosis, and cell cycle, along with western blotting for RAS signaling were performed to assess potency and selectivity of ADT-030. Murine 2838c3-Luc KRASG12D PDAC cells were implanted orthotopically into the pancreas of C57BL/6J mice and cells from two PDAC patient-derived xenograft (PDX) models harboring KRASG12D or KRASG12C mutations were implanted subcutaneously (SC) in NSG mice to evaluate the antitumor activity of ADT-030. ADT-030 was administered once daily by gavage 5x/week for 4 weeks. RESULTS: ADT-030 potently inhibited the growth of PDAC cells in vitro harboring the most prevalent KRASG12D mutation as well as those with KRASG12C. Annexin V assay revealed the capacity of ADT-030 to trigger apoptosis in KRAS mutant cells with minimal impact on BxPC3 RASWT cells. Cell cycle analysis using propidium iodide staining revealed that ADT-030 arrested cells in the G2/M phase. Other experiments showed that ADT-030 simultaneously suppressed both MAPK/AKT signaling and β-catenin transcriptional by inhibiting PDE10 and activating cGMP/PKG signaling. ADT-030 caused tumor regression in SC PDAC PDX mouse tumor models harboring either KRASG12D or KRASG12C mutations without discernable toxicity. Inhibition of orthotopic PDAC tumor growth by ADT-030 was dose-dependent, causing up to 80% reduction in tumor volume following a 4-week treatment period. In addition, ADT-030 significantly extended survival and yielded a high percentage of cures after stopping treatment in a murine model of KRAS- mutant lung cancer. CONCLUSION: ADT-030, novel dual pan-RAS/β-catenin inhibitor, showed strong and durable antitumor activity in clinically relevant and aggressive murine models of PDAC and lung cancer. ADT-030 is a promising development candidate for treatment of PDAC and other RAS-driven cancers, particularly given the complex KRAS mutation landscape of many cancers. Citation Format: Dhana Sekhar Reddy Bandi, Ganji P Nagaraju, Jeremy B Foote, Adam B Keeton, Xi Chen, Kristy L Berry, Yulia Y Maxuitenko, Upender Manne, Donald J Buchsbaum, Gary A Piazza, Bassel F El-Rayes. A novel pan-RAS/β-catenin inhibitor, ADT-030, produces tumor regression in PDX models of 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 C025.

Optimizing the Therapeutic Index of sdAb-Based Radiopharmaceuticals Using Pretargeting.

Single-domain antibodies (sdAbs) demonstrate favorable pharmacokinetic profiles for molecular imaging applications. However, their renal excretion and retention are obstacles for applications in targeted radionuclide therapy (TRT). Methods: Using a click-chemistry-based pretargeting approach, we aimed to reduce kidney retention of a fibroblast activation protein α (FAP)-targeted sdAb, 4AH29, for 177Lu-TRT. Key pretargeting parameters (sdAb-injected mass and lag time) were optimized in healthy mice and U87MG (FAP+) xenografts. A TRT study in a pancreatic ductal adenocarcinoma (PDAC) patient-derived xenograft (PDX) model was performed as a pilot study for sdAb-based pretargeting applications. Results: Modification of 4AH29 with trans-cyclooctene (TCO) moieties did not modify the sdAb pharmacokinetic profile. A 200-µg injected mass of 4AH29-TCO and an 8-h lag time for the injection of [177Lu]Lu-DOTA-PEG7-tetrazine resulted in the highest kidney therapeutic index (2.0 ± 0.4), which was 5-fold higher than that of [177Lu]Lu-DOTA-4AH29 (0.4 ± 0.1). FAP expression in the tumor microenvironment was validated in a PDAC PDX model with both immunohistochemistry and PET/CT imaging. Mice treated with the pretargeting high-activity approach (4AH29-TCO + [177Lu]Lu-DOTA-PEG7-tetrazine; 3 × 88 MBq, 1 injection per week for 3 wk) demonstrated prolonged survival compared with the vehicle control and conventionally treated ([177Lu]Lu-DOTA-4AH29; 3 × 37 MBq, 1 injection per week for 3 wk) mice. Mesangial expansion was reported in 7 of 10 mice in the conventional cohort, suggesting treatment-related kidney morphologic changes, but was not observed in the pretargeting cohort. Conclusion: This study validates pretargeting to mitigate sdAbs' kidney retention with no observation of morphologic changes on therapy regimen at early time points. Clinical translation of click-chemistry-based pre-TRT is warranted on the basis of its ability to alleviate toxicities related to biovectors' intrinsic pharmacokinetic profiles. The absence of representative animal models with extensive stroma and high FAP expression on cancer-associated fibroblasts led to a low mean tumor-absorbed dose even with high injected activity and consequently to modest survival benefit in this PDAC PDX.

Insights into gemcitabine resistance in pancreatic cancer: association with metabolic reprogramming and TP53 pathogenicity in patient derived xenografts

BackgroundWith poor prognosis and high mortality, pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal malignancies. Standard of care therapies for PDAC have included gemcitabine for the past three decades, although resistance often develops within weeks of chemotherapy initiation through an array of possible mechanisms.MethodsWe reanalyzed publicly available RNA-seq gene expression profiles of 28 PDAC patient-derived xenograft (PDX) models before and after a 21-day gemcitabine treatment using our validated analysis pipeline to identify molecular markers of intrinsic and acquired resistance.ResultsUsing normalized RNA-seq quantification measurements, we first identified oxidative phosphorylation and interferon alpha pathways as the two most enriched cancer hallmark gene sets in the baseline gene expression profile associated with intrinsic gemcitabine resistance and sensitivity, respectively. Furthermore, we discovered strong correlations between drug-induced expression changes in glycolysis and oxidative phosphorylation genes and response to gemcitabine, which suggests that these pathways may be associated with acquired gemcitabine resistance mechanisms. Thus, we developed prediction models using baseline gene expression profiles in those pathways and validated them in another dataset of 12 PDAC models from Novartis. We also developed prediction models based on drug-induced expression changes in genes from the Molecular Signatures Database (MSigDB)’s curated 50 cancer hallmark gene sets. Finally, pathogenic TP53 mutations correlated with treatment resistance.ConclusionOur results demonstrate that concurrent upregulation of both glycolysis and oxidative phosphorylation pathways occurs in vivo in PDAC PDXs following gemcitabine treatment and that pathogenic TP53 status had association with gemcitabine resistance in these models. Our findings may elucidate the molecular basis for gemcitabine resistance and provide insights for effective drug combination in PDAC chemotherapy.

N6-methyladenosine modified TGFB2 triggers lipid metabolism reprogramming to confer pancreatic ductal adenocarcinoma gemcitabine resistance

Gemcitabine resistance is a major obstacle to the effectiveness of chemotherapy in pancreatic ductal adenocarcinoma (PDAC). Therefore, new strategies are needed to sensitize cancer cells to gemcitabine. Here, we constructed gemcitabine-resistant PDAC cells and analyzed them with RNA-sequence. Employing an integrated approach involving bioinformatic analyses from multiple databases, TGFB2 is identified as a crucial gene in gemcitabine-resistant PDAC and is significantly associated with poor gemcitabine therapeutic response. The patient-derived xenograft (PDX) model further substantiates the gradual upregulation of TGFB2 expression during gemcitabine-induced resistance. Silencing TGFB2 expression can enhance the chemosensitivity of gemcitabine against PDAC. Mechanistically, TGFB2, post-transcriptionally stabilized by METTL14-mediated m6A modification, can promote lipid accumulation and the enhanced triglyceride accumulation drives gemcitabine resistance by lipidomic profiling. TGFB2 upregulates the lipogenesis regulator sterol regulatory element binding factor 1 (SREBF1) and its downstream lipogenic enzymes via PI3K-AKT signaling. Moreover, SREBF1 is responsible for TGFB2-mediated lipogenesis to promote gemcitabine resistance in PDAC. Importantly, TGFB2 inhibitor imperatorin combined with gemcitabine shows synergistic effects in gemcitabine-resistant PDAC PDX model. This study sheds new light on an avenue to mitigate PDAC gemcitabine resistance by targeting TGFB2 and lipid metabolism and develops the potential of imperatorin as a promising chemosensitizer in clinical translation.

Abstract 2079: Tumor-derived fibrinogen promotes pancreatic ductal adenocarcinoma progression

Abstract Background: Hypercoagulability is a common clinical manifestation in patients with cancers, especially in pancreatic ductal adenocarcinoma (PDAC). Over 20% of PDAC patients with advanced-stage and worse prognosis undergo a venous thromboembolism (VTE) incidence. Fibrinogen (FG) may contribute to VTE due to its high expression in advanced PDAC tumors. Here, we set to determine the biological roles of tumor-derived FG in PDAC. In addition, our previous study showed that Von Willebrand Factor (VWF) A2 domain can effectively bind to FG and reduce platelet clot formation. Therefore, the functional roles of A2 on FG in PDAC were also studied. Methods: The correlation between survival rate and FG expression in PDAC was analyzed using RNAseq profiles of 149 PDAC patient samples from the TCGA database. PDAC subtyping of Baylor College of Medicine (BCM) PDAC patient-derived xenograft (PDX) cohort was performed using RNAseq data according to Moffitt’s criteria. The FG (three chains FGA, FGB, FGG) overexpression (OE) and knock-out (KO) PDAC cell lines were established. The gene expression and biological functions of FG were performed in PDX and cell lines using IHC and immunoblot assays. The FG effect on PDAC progression in vivo was studied in mouse models. Results: TCGA analysis showed that elevated tumor-derived FG gene expression is significantly correlated with worse overall survival and disease-free survival rates in PDAC patients. PDX lines RNAseq analysis clearly separated BCM PDAC PDXs into two subtypes: basal-like and classical PDACs. Basal-like PDXs showed significantly stronger FG staining than that in classical ones. Compared to the classical subtype, the basal-like subtype showed significantly upregulated genes that are associated with the epithelial-mesenchymal transition (EMT), IL6/JAK/STAT3, and TNFα signaling pathways. Accordingly, basal-like subtype PDAC exposed to IL6 or TNFα significantly increased FG and FG receptor expression with activated STAT3 and AKT pathways and elevated EMT markers, whereas classical subtype cells have minimal changes. In addition, we found that the FG-OE cells exhibit enhanced cell proliferation and migration properties whereas FG-KO cells reduced the effects. Consistent with in vitro data, FG-OE PDAC showed significantly higher tumor burdens than that of control PDAC in both orthotopic and subcutaneous PDAC mouse models. Furthermore, A2 protein treatment can effectively block the FG function and dramatically inactivate the FG-induced EMT process and AKT signaling pathway. Conclusions: Tumor-derived fibrinogen is upregulated in a basal-like subtype of PDAC cells and may contribute to tumor growth and metastasis by activating aberrant signaling pathways and promoting EMT. Our findings imply that elevated fibrinogen in PDAC could contribute to tumor progression and the A2 protein which target to fibrinogen may be a promising therapy for basal-like subtype PDAC. Citation Format: Dongliang Liu, Lisa Brubaker, Yichi Niu, Emily LaPlante, George Van Buren, Aleksandar Milosavljevic, Changyi Chen, Chenghang Zong, Miguel Cruz, Qizhi Yao. Tumor-derived fibrinogen promotes pancreatic ductal adenocarcinoma progression [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 2079.

Abstract 4737: The combination of the BET inhibitor JQ1 and gemcitabine induces regressions in gemcitabine resistant patient-derived xenograft models of pancreatic cancer

Abstract Efforts to develop targeted therapies for drug resistant tumors have been relatively unsuccessful. The 5-year survival for patients with advanced pancreatic cancer is ~3%. Gemcitabine is usually combined with nab-paclitaxel as a standard of care for treatment of locally advanced or metastatic pancreatic ductal adenocarcinoma (PDAC). However, no chemotherapeutic regimens thus far evaluated have been curative. We developed the first PDAC patient-derived xenograft (PDX) models in which resistance against gemcitabine was acquired in vivo. We used RNA-seq and ingenuity pathway analysis to compare expression profiles of paired parent vs gemcitabine resistant PDX models. This comparison suggested a previously unreported mechanism for gemcitabine resistance, involving aberrations of cholesterol biosynthesis and lipid metabolism pathways. We also made the unexpected observation that gemcitabine-resistant PDAC models derived from primary tumors have high levels of 3-hydroxy-3-methylglutaryl-CoA synthase 2 (HMGCS2), an enzyme essential to cholesterol biosynthesis and rate limiting in ketogenesis. Notably, the BET bromodomain inhibitor JQ1 decreases HMGCS2 levels; and BET inhibitors + gemcitabine exert synergistic cytotoxicity in gemcitabine-resistant PDAC cells. We determined that the BET bromodomain inhibitor JQ1 + gemcitabine induces regressions of two in vivo gemcitabine-resistant tumor models which we developed directly from primary human tumor tissue. These mechanism-based studies suggest that inhibiting HMGCS2 represents a novel approach to therapy for PDAC. We propose that combining gemcitabine with agents that decrease levels of HMGCS2 comprises effective treatment for patients with drug resistant PDAC. Citation Format: Aubrey L. Miller, Samuel C. Fehling, Eric Josh Brown, Eric O. Heard, Tracy L. Gamblin, Rebecca B. Vance, Karina J. Yoon. The combination of the BET inhibitor JQ1 and gemcitabine induces regressions in gemcitabine resistant patient-derived xenograft models of 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 4737.

Combined KRAS-MAPK pathway inhibitors and HER2-directed drug conjugate is efficacious in pancreatic cancer

Targeting the mitogen-activated protein kinase (MAPK) cascade in pancreatic ductal adenocarcinoma (PDAC) remains clinically unsuccessful. We aim to develop a MAPK inhibitor-based therapeutic combination with strong preclinical efficacy. Utilizing a reverse-phase protein array, we observe rapid phospho-activation of human epidermal growth factor receptor 2 (HER2) in PDAC cells upon pharmacological MAPK inhibition. Mechanistically, MAPK inhibitors lead to swift proteasomal degradation of dual-specificity phosphatase 6 (DUSP6). The carboxy terminus of HER2, containing a TEY motif also present in extracellular signal-regulated kinase 1/2 (ERK1/2), facilitates binding with DUSP6, enhancing its phosphatase activity to dephosphorylate HER2. In the presence of MAPK inhibitors, DUSP6 dissociates from the protective effect of the RING E3 ligase tripartite motif containing 21, resulting in its degradation. In PDAC patient-derived xenograft (PDX) models, combining ERK and HER inhibitors slows tumour growth and requires cytotoxic chemotherapy to achieve tumour regression. Alternatively, MAPK inhibitors with trastuzumab deruxtecan, an anti-HER2 antibody conjugated with cytotoxic chemotherapy, lead to sustained tumour regression in most tested PDXs without causing noticeable toxicity. Additionally, KRAS inhibitors also activate HER2, supporting testing the combination of KRAS inhibitors and trastuzumab deruxtecan in PDAC. This study identifies a rational and promising therapeutic combination for clinical testing in PDAC patients.

Physico-chemical characterization of the tumour microenvironment of pancreatic ductal adenocarcinoma

Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive lethal malignancy that accounts for more than 90% of pancreatic cancer diagnoses. Our research is focused on the physico-chemical properties of the tumour microenvironment (TME), including its tumoural extracellular matrix (tECM), as they may have an important impact on the success of cancer therapies. PDAC xenografts and their decellularized tECM offer a great material source for research in terms of biomimicry with the original human tumour. Our aim was to evaluate and quantify the physico-chemical properties of the PDAC TME. Both cellularized (native TME) and decellularized (tECM) patient-derived PDAC xenografts were analyzed. A factorial design of experiments identified an optimal combination of factors for effective xenograft decellularization. Our results provide a complete advance in our understanding of the PDAC TME and its corresponding stroma, showing that it presents an interconnected porous architecture with very low permeability and small pores due to the contractility of the cellular components. This fact provides a potential therapeutic strategy based on the therapeutic agent size.

Dual-Hit Strategy for Therapeutic Targeting of Pancreatic Cancer in Patient-Derived Xenograft Tumors.

Paracrine activation of pro-fibrotic hedgehog (HH) signaling in pancreatic ductal adenocarcinoma (PDAC) results in stromal amplification that compromises tumor drug delivery, efficacy, and patient survival. Interdiction of HH-mediated tumor-stroma crosstalk with smoothened (SMO) inhibitors (SHHi) "primes" PDAC patient-derived xenograft (PDX) tumors for increased drug delivery by transiently increasing vascular patency/permeability, and thereby macromolecule delivery. However, patient tumor isolates vary in their responsiveness, and responders show co-induction of epithelial-mesenchymal transition (EMT). We aimed to identify the signal derangements responsible for EMT induction and reverse them and devise approaches to stratify SHHi-responsive tumors noninvasively based on clinically-quantifiable parameters. Animals underwent diffusion-weighted magnetic resonance (DW-MR) imaging for measurement of intratumor diffusivity. In parallel, tissue-level deposition of nanoparticle probes was quantified as a marker of vascular permeability/perfusion. Transcriptomic and bioinformatic analysis was employed to investigate SHHi-induced gene reprogramming and identify key "nodes" responsible for EMT induction. Multiple patient tumor isolates responded to short-term SHH inhibitor exposure with increased vascular patency and permeability, with proportionate increases in tumor diffusivity. Nonresponding PDXs did not. SHHi-treated tumors showed elevated FGF drive and distinctly higher nuclear localization of fibroblast growth factor receptor (FGFR1) in EMT-polarized tumor cells. Pan-FGFR inhibitor NVP-BGJ398 (Infigratinib) reversed the SHHi-induced EMT marker expression and nuclear FGFR1 accumulation without compromising the enhanced permeability effect. This dual-hit strategy of SMO and FGFR inhibition provides a clinically-translatable approach to compromise the profound impermeability of PDAC tumors. Furthermore, clinical deployment of DW-MR imaging could fulfill the essential clinical-translational requirement for patient stratification.

Abstract C060: Characterization of a collection of patient-derived xenograft models for pancreatic ductal adenocarcinoma translational research

Abstract To conduct successful translational research for pancreatic ductal adenocarcinoma (PDAC), it is imperative to have preclinical models that accurately reflect the intricate molecular, genomic, and histological features of the disease. Patient-derived xenografts (PDXs) have emerged as invaluable platforms for studying tumor biology and personalized therapeutic intervention. Here we present a collection of 32 PDAC PDX models established and fully characterized in our institution. This PDAC PDX collection was generated with tissue from both primary and metastatic lesions, comprehensively representing the disease spectrum. Genomic profiling using next-generation sequencing revealed a remarkable concordance between the PDX samples and their original patient tumor tissue. Notably, identifying common driver mutations associated with PDAC further validates the clinical relevance of this PDX collection. Moreover, histopathologic examination confirmed the similarity between PDX tumor samples and their original patient tissue samples. Interestingly, we also optimized a protocol to generate tumoroids from PDAC PDXs. Leveraging the broad spectrum of disease represented in our collection will facilitate addressing critical research questions, identifying novel therapeutic targets, and ultimately improving patient outcomes of PDAC. Citation Format: Mariana Yáñez, Enrique Javier Arenas, Teresa Macarulla, Tian Tian, Joaquín Arribas, Sofía Llorente, Carles Fabregat, Marta Escorihuela, Florian Castet, Jessica Querol, Maria Teresa Salcedo. Characterization of a collection of patient-derived xenograft models for pancreatic ductal adenocarcinoma translational research [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 C060.

In vivo label-free optical signatures of chemotherapy response in human pancreatic ductal adenocarcinoma patient-derived xenografts

Pancreatic cancer is a devastating disease often detected at later stages, necessitating swift and effective chemotherapy treatment. However, chemoresistance is common and its mechanisms are poorly understood. Here, label-free multi-modal nonlinear optical microscopy was applied to study microstructural and functional features of pancreatic tumors in vivo to monitor inter- and intra-tumor heterogeneity and treatment response. Patient-derived xenografts with human pancreatic ductal adenocarcinoma were implanted into mice and characterized over five weeks of intraperitoneal chemotherapy (FIRINOX or Gem/NabP) with known responsiveness/resistance. Resistant and responsive tumors exhibited a similar initial metabolic response, but by week 5 the resistant tumor deviated significantly from the responsive tumor, indicating that a representative response may take up to five weeks to appear. This biphasic metabolic response in a chemoresistant tumor reveals the possibility of intra-tumor spatiotemporal heterogeneity of drug responsiveness. These results, though limited by small sample size, suggest the possibility for further work characterizing chemoresistance mechanisms using nonlinear optical microscopy.

Species-Deconvolved Proteomics for In Situ Investigation of Tumor-Stroma Interactions after Treatment of Pancreatic Cancer Patient-Derived Xenografts with Combined Gemcitabine and Paclitaxel.

Tumor-stroma interactions are critical in pancreatic ductal adenocarcinoma (PDAC) progression and therapeutics. Patient-derived xenograft (PDX) models recapitulate tumor-stroma interactions, but the conventional antibody-based immunoassay is inadequate to discriminate tumor and stromal proteins. Here, we describe a species-deconvolved proteomics approach embedded in IonStar that can unambiguously quantify the tumor (human-derived) and stromal (mouse-derived) proteins in PDX samples, enabling unbiased investigation of tumor and stromal proteomes with excellent quantitative reproducibility. With this strategy, we studied tumor-stroma interactions in PDAC PDXs that responded differently to Gemcitabine combined with nab-Paclitaxel (GEM+PTX) treatment. By analyzing 48 PDX animals 24 h/192 h after treatment with/without GEM+PTX, we quantified 7262 species-specific proteins under stringent cutoff criteria, with high reproducibility. For the PDX sensitive to GEM+PTX, the drug-dysregulated proteins in tumor cells were involved in suppressed oxidative phosphorylation and the TCA cycle, and in the stroma, inhibition of glycolytic activity was predominant, suggesting a relieved reverse Warburg effect by the treatment. In GEM+PTX-resistant PDXs, protein changes suggested extracellular matrix deposition and activation of tumor cell proliferation. Key findings were validated by immunohistochemistry (IHC). Overall, this approach provides a species-deconvolved proteomic platform that could advance cancer therapeutic studies by enabling unbiased exploration of tumor-stroma interactions in the large number of PDX samples required for such investigations.

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 > 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 979: Development of a methylation-based classifier to identify pancreatic adenocarcinoma subtype

Abstract Background: Pancreatic ductal adenocarcinoma (PDAC) demonstrates variable prognosis and response to first-line FOLFIRINOX therapy depending on molecular subtype; patients with classical tumors showed a greater response compared to basal-like tumors. Identifying molecular subtype may inform treatment approaches, but current subtyping schemas rely on tumor biopsies which are invasive and can be technically challenging. Blood based epigenetic assays are commercially available, and identification of subtype-associated epigenetic modifications may provide a path toward blood-based PDAC molecular subtyping. We analyzed methylation levels and developed a subtype classifier using methylation data on PDAC tumors. Using this classifier, we then developed a methylation-specific qPCR assay to subtype PDAC tumors by analyzing methylation at particular sites in tumor DNA which may now be further adapted to more facile liquid biopsy approaches. Methods: Using methylation data (n=150) from TCGA PAAD, we applied a random forest (RF) model followed by a k-Top Scoring Pairs (k-TSP) algorithm to develop a methylation site-based classifier that allows prediction of subtype by comparing methylation levels between paired sites. The classifier was validated on two independent datasets, including ICGC (n=82) and patient-derived xenografts (PDXs) (n=21). We designed methylation-specific qPCR primers for these sites and validated their specificity with fully methylated or fully unmethylated DNA. Using genomic DNA extracted from 10 PDAC PDXs of known molecular subtype, we obtained methylation levels at sites identified by the classifier via a SYBR Green-based qPCR assay. We then used our classifier to predict subtype of the PDX tumors. Results: We validated our RF-kTSP methylation subtype classifier on two independent cohorts and predicted subtype using 20 methylation sites. In ICGC, we found balanced accuracy=0.94, AUROC=0.98, AU-PR=0.89. In PDX PDAC tumors, we found balanced accuracy=1, AUROC=1, AU-PR=1. We designed 14/20 primers for qPCR and confirmed specificity for methylated DNA. Using 14/20 methylation sites, we achieved 80% correct subtype prediction of 10 PDX tumors (5/5 basal-like, 3/5 classical). Conclusion: We have developed a methylation-based classifier that accurately and replicably predicts PDAC subtype. We continue to develop a qPCR assay for PDAC subtype that so far has 80% accuracy using 14/20 probes. We are completing validation of the remaining 6 primers and correlating methylation levels at each site with those obtained from EPIC array methylation analysis. The long-term goal is to apply our complete set of primers in a digital droplet PCR assay that will be amenable for subtyping using circulating tumor DNA (ctDNA). Citation Format: Zachary Schrank, Des Weighill, Hannah Thel, Hannah Trembath, Ashley Morrison, Jen Jen Yeh. Development of a methylation-based classifier to identify pancreatic adenocarcinoma subtype [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 979.

Abstract 3870: TNF-MK2 signaling drives protective autophagy following MAPK pathway inhibition in pancreatic cancer

Abstract Targeting the oncogenic KRAS-RAF-MEK-ERK (MAPK) pathway has remain unsuccessful in the clinic for patients with pancreatic ductal adenocarcinoma (PDAC), a highly lethal cancer with few effective treatment options. To identify novel, effective therapeutic strategies, we investigated the impact of MEK and ERK inhibitors on production of inflammatory cytokines in PDAC cells. We found that both MEK and ERK inhibitors dramatically upregulated production of TNF, leading to activation of multiple TNF signaling pathways which include the pro-death and pro-survival NF-kB and p38.MK2 cascades. Silencing of TNF receptor 1 (TNFR1) abrogated the pro-apoptotic effect of MAPK inhibitors, suggesting that pro-death effect driven by TNF signaling is required for the therapeutic effect of MAPK inhibitors. However, targeting the NF-kB and MK2 pathways greatly augment the suppressive effect of MAPK inhibitors. Notably, the mechanisms via which MK2 promotes PDAC cell survival are mediated through phospho-activation of Heat shock protein 27 (Hsp27) and Beclin1, a critical mediator of autophagy. Intriguingly, silencing of TNFR1 abrogated induction of proactive autophagy following MAPK inhibition, strongly suggesting autocrine signaling as the inciting event of autophagy. Mechanistically, we showed that TNF signaling upregulate unfolded protein response (UPR) in PDAC cells, which is required for autophagy induction. Targeting MK2 abrogated transcription of key UPR genes and blocks subsequent autophagy. The combination of MK2 inhibitor ATI-450 and ERK inhibitor ulixertinib was more effective in curbing the growth of PDAC patient-derived xenograft in vivo and prolonged the survival of autochthonous PDAC mice (KPC model). Overall, our study provided novel insights on the mechanisms that drive protective autophagy following MAPK pathway inhibition and a rationale and feasible therapeutic combination that can be tested in clinical trials for PDAC patients. Citation Format: Iftikhar Ali Khawar, Qing Wei, Timothy Hung-Po Chen, Lin Lin, Patrick M. Grierson, Kian-Huat Lim. TNF-MK2 signaling drives protective autophagy following MAPK pathway inhibition in pancreatic cancer. [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 3870.

Pharmacological targeting of the receptor ALK inhibits tumorigenicity and overcomes chemoresistance in pancreatic ductal adenocarcinoma

Pancreatic ductal adenocarcinoma (PDAC) is an extremely aggressive disease characterized by its metastatic potential and chemoresistance. These traits are partially attributable to the highly tumorigenic pancreatic cancer stem cells (PaCSCs). Interestingly, these cells show unique features in order to sustain their identity and functionality, some of them amenable for therapeutic intervention. Screening of phospho-receptor tyrosine kinases revealed that PaCSCs harbored increased activation of anaplastic lymphoma kinase (ALK). We subsequently demonstrated that oncogenic ALK signaling contributes to tumorigenicity in PDAC patient-derived xenografts (PDXs) by promoting stemness through ligand-dependent activation. Indeed, the ALK ligands midkine (MDK) or pleiotrophin (PTN) increased self-renewal, clonogenicity and CSC frequency in several in vitro local and metastatic PDX models. Conversely, treatment with the clinically-approved ALK inhibitors Crizotinib and Ensartinib decreased PaCSC content and functionality in vitro and in vivo, by inducing cell death. Strikingly, ALK inhibitors sensitized chemoresistant PaCSCs to Gemcitabine, as the most used chemotherapeutic agent for PDAC treatment. Consequently, ALK inhibition delayed tumor relapse after chemotherapy in vivo by effectively decreasing the content of PaCSCs. In summary, our results demonstrate that targeting the MDK/PTN-ALK axis with clinically-approved inhibitors impairs in vivo tumorigenicity and chemoresistance in PDAC suggesting a new treatment approach to improve the long-term survival of PDAC patients.

Abstract C022: Heterodimeric bispecific antibodies for highly specific treatment of EGFR(+)HER2(+) pancreatic ductal adenocarcinoma

Abstract Background: EGFR and HER2 are highly expressed in many pancreatic cancers which strongly correlate with poorer prognoses. We tested homodimeric (EGFRxEGFR or HER2xHER2) and heterodimeric (EGFRxHER2) T cell-engaging bispecific antibodies (BsAbs) to direct polyclonal T cells to these antigens on pancreatic tumors. Methods: We used the 2+2 IgG(L)-scFv format to create BsAbs, previously shown to have exceptional anti-tumor properties compared to other structural platforms. These BsAbs utilize two anti-CD3 scFvs attached to the light chains of an IgG to engage T cells while retaining bivalent binding to tumor antigens with both Fab arms. Anti-EGFR was based on the Cetuximab and anti-HER2 on the Trastuzumab amino acid sequences. Using a ‘knob-into-hole’ strategy, we generated an EGFRxHER2 heterodimeric BsAb bearing one Fab specific for EGFR and one for HER2. T cells were tagged with luciferase for tracking by bioluminescence for in vivo trafficking studies. Results: The EGFRxEGFR, HER2xHER2, and EGFRxHER2 BsAbs demonstrate high avidity (KD: 2pM to 300 pM) and T cell-mediated cytotoxicity against several human pancreatic ductal adenocarcinoma (PDAC) cell lines in vitro with EC50s in the picomolar range (0.17pM to 18pM). They mediate potent T cell-mediated antitumor effects against subcutaneous PDAC xenografts and were highly efficient in driving human polyclonal T cells into tumors as measured by bioluminescence. To examine the importance of anti-tumor valency in antitumor effects, EGFR and HER2 BsAbs were heterodimerized with a CD33 control BsAb to generate EGFRxCD33 and HER2xCD33 BsAbs. Both EGFRxCD33 and HER2xCD33 BsAbs showed modest reductions in target avidity by surface plasmon resonance (2.7-fold and 12.6-fold, respectively), cell binding strength by FACS (8.0-fold and 63.6-fold) and cytotoxicity (7.7-fold and 47.2-fold) compared to their homodimeric counterparts and, remarkably, showed no therapeutic efficacy against SW1990 cell line-derived and patient-derived PDAC xenografts, correlating with reduced tumor-infiltrating bioluminescent T cells, when compared to EGFRxHER2 BsAb. When tested against SW1990 with CRISPR-mediated target KOs, only HER2xHER2 BsAb treatment could eliminate EGFR-KO tumors, while EGFRxEGFR and EGFRxHER2 BsAbs were ineffective. Conversely, in HER2-KO SW1990 tumors, while EGFRxEGFR BsAb demonstrated strong tumor control, HER2xHER2 and EGFRxHER2 BsAbs demonstrated only weak activity. Conclusion: EGFR and HER2 were useful targets for driving T cell infiltration and tumor ablation. HER2 was overall more effective as a target when compared to EGFR for pancreatic cancer in vivo. Bivalent Fab binding to tumor was critical for robust anti-tumor effect in vivo even when in vitro differences of monovalent BsAb were less obvious. EGFRxHER2 heterodimeric BsAb exhibited potent antitumor efficacy against EGFR(+)HER2(+) double positive PDAC xenografts, but not single-positive tumors, and could offer a strategy to spare healthy single-positive tissues to reduce ‘on target/off tumor’ side effects. Citation Format: Alan W. Long, Brian Santich, Hongfen Guo, Nai-Kong Cheung. Heterodimeric bispecific antibodies for highly specific treatment of EGFR(+)HER2(+) pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr C022.

Imatinib facilitates gemcitabine sensitivity by targeting epigenetically activated PDGFC signaling in pancreatic cancer

Pancreatic ductal adenocarcinoma (PDAC) is a malignant tumor with poor prognosis. Gemcitabine-based chemotherapy has become one of the main modalities of its management. However, gemcitabine resistance frequently occurs, leading to failure of PDAC therapy. Platelet-derived growth factors (PDGFs) and their receptors play important roles in cancer progression and chemoresistance. We aimed to investigate the biological function and therapeutic significance of platelet-derived growth factor C (PDGFC) in drug-resistant PDAC. Our study showed that PDGFC was abnormally highly expressed in gemcitabine-resistant PDAC. Silencing PDGFC expression can enhance the therapeutic effect of gemcitabine on PDAC. Mechanistically, the transcription of PDGFC is mediated by H3K27 acetylation, and PDGFC promotes gemcitabine resistance by activating the PDGFR-PI3K-AKT signaling pathway. The PDGFR inhibitor imatinib inhibits the PDGFR pathway. Imatinib and gemcitabine have a synergistic effect on the treatment of PDAC, and imatinib can significantly enhance the anti-tumor effect of gemcitabine in a drug-resistant PDAC patient-derived xenograft model. In conclusion, PDGFC is a potential predictor of gemcitabine-resistant PDAC. Imatinib inhibits PDGFR activation to promote gemcitabine sensitivity in PDAC. Combined modality regimen of imatinib and gemcitabine is likely to translate into clinical trial for the treatment of PDGFC-associated gemcitabine-resistant patients.

Abstract 5333: Pancreatic cancer enhances HER2 signaling through DUSP6 to circumvent therapeutic MAPK inhibition

Abstract Background: Oncogenic KRAS is present in almost all cases of pancreatic ductal adenocarcinoma (PDAC). However, targeting KRAS or its canonical signaling cascades, especially the mitogen-activated protein kinase (MAPK) pathway, remains clinically unsuccessful. Targeting ERK kinases has recently emerged as a promising therapeutic strategy and combinatorial strategies should be developed. Aim: Identify adaptive mechanisms to ERK inhibition that can be co-targeted to achieve effective tumor inhibition in multiple patient-derived xenograft (PDX) models. Methods: Reverse-phase protein array (RPPA) was used in early-passage patient-derived cell lines (PDCLs) to identify potential resistance mechanisms. These were confirmed using RNA interference and overexpression in PDAC cell lines and PDCLs. The promising combinations were tested in 30 early-passaged PDAC PDXs. Results: RPPA showed dramatic downregulation of DUSP4 and DUSP6 phosphatases following MEK and ERK inhibition, which coincided with upregulation of phospho-HER2 and -HER3. Knockdown of DUSP6, but not DUSP4, was sufficient in phosphorylation of HER2. Conversely, overexpression of DUSP6 curbed HER2 and ERK activation. Downregulation of DUSP4 and DUSP6 induced by ulixertinib, an ERK inhibitor now in clinical development, was reversed by bortezomib, suggesting DUSP4 and DUSP6 are proteosomally degraded. Combined ulixertinib plus PI3K inhibitor copanlisib, or pan-HER inhibitor afatinib slowed but did not arrest PDX tumor growth in vivo, and addition of gemcitabine was required to achieve tumor regression or durable growth arrest. Alternatively, ulixertinib or MEK inhibitor (trametinib) in combination with trastuzumab deruxtecan (DS-8201a), an anti-HER2 conjugated topoisomerase I inhibitor, were extremely effective, leading to complete and durable tumor regression for all tested PDX models. We showed that upregulation of HER2 expression following MEK or ERK inhibitor treatment provides a conduit for enhanced internalization of DS-8201a. Conclusions: Our study provided novel mechanistic insight on how PDAC cells evade MAPK inhibition via enhancing HER2 signaling. We demonstrated that the combination of MEK or ERK inhibitor plus DS-8201a is extremely effective, leading to complete tumor regression in multiple PDAC PDX models. This combination should be advanced as a clinical trial for PDAC patients. Keys: DS-8201a, DUSP6, HER2, KRAS, ulixertinib, pancreatic ductal adenocarcinoma Citation Format: Ashenafi Shiferaw Bulle, Kuljeet Seehar, Sapana Bansod, Yali Chen, Chen Hung-Po, Paarth B. Dodhiawala, Lin Li, Vikas Somani, Jacqueline Mudd, Ryan C. Fields, Deborah Knoerzer, Andrea Wang-Gillam, Lim Kian-Huat. Pancreatic cancer enhances HER2 signaling through DUSP6 to circumvent therapeutic MAPK inhibition [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 5333.

Proteolytic pan-RAS Cleavage Leads to Tumor Regression in Patient-derived Pancreatic Cancer Xenografts.

The lack of effective RAS inhibition represents a major unmet medical need in the treatment of pancreatic ductal adenocarcinoma (PDAC). Here, we investigate the anticancer activity of RRSP-DTB, an engineered biologic that cleaves the Switch I of all RAS isoforms, in KRAS-mutant PDAC cell lines and patient-derived xenografts (PDX). We first demonstrate that RRSP-DTB effectively engages RAS and impacts downstream ERK signaling in multiple KRAS-mutant PDAC cell lines inhibiting cell proliferation at picomolar concentrations. We next tested RRSP-DTB in immunodeficient mice bearing KRAS-mutant PDAC PDXs. Treatment with RRSP-DTB led to ≥95% tumor regression after 29 days. Residual tumors exhibited disrupted tissue architecture, increased fibrosis and fewer proliferating cells compared with controls. Intratumoral levels of phospho-ERK were also significantly lower, indicating in vivo target engagement. Importantly, tumors that started to regrow without RRSP-DTB shrank when treatment resumed, demonstrating resistance to RRSP-DTB had not developed. Tracking persistence of the toxin activity following intraperitoneal injection showed that RRSP-DTB is active in sera from immunocompetent mice for at least 1 hour, but absent after 16 hours, justifying use of daily dosing. Overall, we report that RRSP-DTB strongly regresses hard-to-treat KRAS-mutant PDX models of pancreatic cancer, warranting further development of this pan-RAS biologic for the management of RAS-addicted tumors.