KRAS-mutant Pancreatic Ductal Adenocarcinoma Research Articles

Endosomal Trafficking Bypassed by the RAB5B-CD109 Interplay Promotes Axonogenesis in KRAS-Mutant Pancreatic Cancer.

Perineural invasion (PNI) represents a unique biological feature associated with poor prognosis in pancreatic ductal adenocarcinoma (PDAC), especially in the presence of KRAS mutations. Extracellular vesicle (EV)-packaged circular RNAs (circRNAs) function as essential mediators of tumor microenvironment communication, triggering PDAC cell invasion and distant metastasis. However, the regulatory mechanisms of EV-packaged circRNAs in the PNI of KRAS-mutant PDAC have not yet been elucidated. Herein, a KRASG12D mutation-responsive EV-packaged circRNA, circPNIT, which positively correlated with PNI in PDAC patients is identified. Functionally, KRASG12D PDAC-derived EV-packaged circPNIT promoted axonogenesis and PNI both in vitro and in vivo. Mechanistically, the circPNIT-mediated Rab5B-CD109 interplay bypassed traditional endosomal trafficking to anchor Rab5B to the lipid rafts of multivesicular bodies and packaged circPNIT into CD109+ EVs. Subsequently, CD109+ EVs delivered circPNIT to neurons by binding to TRPV1 and facilitating DSCAML1 transcription-induced axonogenesis, which in turn enhanced the PNI by activating the GFRα1/RET pathway. Importantly, circPNIT-loaded CD109+ EVs are established to dramatically promote PNI in a KRASG12D/+ Trp53R172H/+ Pdx-1-Cre mouse model. Collectively, the findings highlight the mechanism underlying how EV-packaged circRNAs mediate the PNI of KRAS-mutant PDAC cells through the Rab5B endosomal bypass, identifying circPNIT as an effective target for the treatment of neuro-metastatic PDAC.

Abstract PR-15: Identifying synergistic combinations with KRAS inhibition in PDAC

Abstract Background: To date, targeted therapies have largely failed in eliciting sustained responses in advanced pancreatic ductal adenocarcinoma (PDAC). KRAS-targeted therapies have the potential to drastically transform clinical management. Emerging data indicate that only a subset of patients respond, and that acquired resistance is common. Here, we used a combination of in vivo CRISPR activation (CRISPRa) screening and in vitro/preclinical models to identify synergistic combinations with KRAS inhibition (KRASi) in pancreatic and lung cancer. Material and Methods: We used our CRISPRa-competent PPKS (P53(F/F), Kras(LSL-G12D/+), R26(LSL-SAM)) model of adenocarcinoma to conduct targeted screening for drivers of resistance to MRTX133. Our library consisted of ∼450 frequently mutated and amplified putative oncogenes across human adenocarcinoma based on TCGA data. Mice were treated with 1e8 TU of lentiviral library through nasal instillation. After 6 weeks, treatment was initiated with 3 mg/kg MRTX1133 daily, 5 days/week (i.p.). Mice were sacrificed after 4 weeks of treatment and the guide distribution determined through bioinformatic analysis. Genes statistically significantly enriched (FDR<0.05) in the treatment group were determined. Cell lines with acquired resistance to MRTX1133 derived from the KPC PDAC model and the KPP lung adenocarcinoma (LUAD) model were generated using long-term, low-dose treatment. Resistance was confirmed with MTT drug response assays. In vitro combinatorial drug response was determined using a 72h, 96-well MTT assay, and clonogenic response using a 2 week, 384-well FACS assay. Mice were treated with 20 mg/kg MRTX1133 and/or 20mg/kg Temuterkib once daily, 7 days/week (i.p.) for 2 weeks. Results: We performed targeted in vivo CRISPRa screening for drivers of resistance to KRASi in an autochthons model of lung adenocarcinoma (LUAD) using a library targeting 452 genes commonly amplified or mutated in cancer. We identified a set of genes that were specifically enriched in at least 50% of tumors treated with MRTX1133, including MAPK1/ERK2. We also generated 16 cell lines with acquired resistance to MRTX1133, derived from KPC PDAC or KPP LUAD tumors. Transcriptomic analysis identified a large set of genes differentially expressed in cell with acquired resistance relative to controls. GSEA indicated that reactivation of the MAPK cascade as a common trait in resistant cells. To test if ERK inhibition synergizes with KRASi, we quantified the combinatorial response to MRTX1133 and Temuterkib, a specific inhibitor of ERK1 and ERK2, in a set of KPC and KPP derived cell lines, revealing significant synergy and reduced clonogenic potential in a majority of cell lines. A majority of cell lines with acquired resistance to MRTX1133 remained sensitive to Temuterkib. Finally, we tested combinatorial treatment of MRTX1133 and Temuterkib in a syngenetic subcutaneous model of PDAC, which showed at minimum additive effect. In conclusion we have identified ERK inhibition as a promising combination with KRAS inhibition in KRAS-mutant PDAC and LUAD. Citation Format: Fredrik I Thege, Amber Hoskins, Sonja M Woermann, Anirban Maitra. Identifying synergistic combinations with KRAS inhibition in PDAC [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr PR-15.

Abstract C027: Efficacy advantages of a novel pan-RAS inhibitor, ADT-1004, over mutant- specific KRAS inhibitors for suppressing pancreatic tumor growth in murine models

Abstract Background/Objective: Prognosis remains grim for localized or metastasized pancreatic ductal adenocarcinoma (PDAC) with 5-year survival rates of 3-16%. More than 90% of PDAC cases harbor KRAS mutations. KRASG12C inhibitors recently developed have limited use for PDAC because only 3% of cases express this mutation. We developed a novel pan-RAS inhibitor, ADT-1004 and evaluated antitumor activity in tumor models established from murine or human PDAC cell lines or patient-derived xenografts (PDX). ADT-1004 is an orally bioavailable prodrug of ADT-007, a highly potent and selective pan-RAS inhibitor (doi.org/10.1101/2023.05.17.541233). Methods: Murine PDAC cells harboring the KRASG12D mutation (KPC-Luc or 2838C3-Luc) were orthotopically implanted into the pancreas of C57BL/6J mice. Four PDX PDAC tumors harboring KRAS mutations were implanted orthotopically in NSG mice. To assess potential to overcome RAS inhibitor resistance, parental KRASG12C MIA PaCa-2 PDAC cells and a resistant derivative of MIA PaCa-2 cells were implanted subcutaneously. To assess selectivity, mice were implanted with RASWT BxPC-3 cells SC. Mice were treated orally with ADT-1004 (40mg/kg) 5x/week for 4 weeks. Sotorasib and adagrasib KRASG12C inhibitors were administered at the same dose, route, and schedule as ADT-1004. Results: ADT-007 potently and selectively inhibited the growth of human and mouse PDAC cell lines in vitro. For example, the IC50 value of ADT-007 for KRASG12C MIA PaCa-2 PDAC cells was 2 nM compared to 2500 nM for RASWT BxPC-3 PDAC cells. Growth inhibition by ADT-007 was dependent on activated RAS and associated with reduced GTP-RAS levels and MAPK/AKT signaling. ADT-1004 was well tolerated in mice at doses up to 175 mg/kg bid orally with sustained plasma levels of ADT-007 exceeding growth IC50 values. ADT-1004 displayed robust antitumor activity in mouse tumor models using human- or mouse-derived PDAC cell lines with G12D, G12V, G12C, or G13Q KRAS mutations, causing selective inhibitory effects on ERK phosphorylation in tumors but not in normal tissues. ADT-1004 also inhibited growth of KRASG12C mutant MIA PaCa-2 tumors to a comparable extent as sotorasib and adagrasib. Sotorasib and adagrasib were ineffective against xenografted tumors of resistant cells derived from MIA PaCa-2 cells, while ADT-1004 strongly inhibited tumor growth. Target selectivity of ADT-1004 was confirmed in vivo, as ADT-1004 did not affect the growth of RASWT BxPC-3 tumors. Conclusions: ADT-1004 inhibited tumor growth of KRAS mutant PDAC tumors by blocking activated RAS to disrupt downstream MAPK/AKT signaling. Side-by-side comparisons reveal antitumor efficacy advantages of ADT-1004 over mutant-specific KRAS inhibitors. These results highlight the promise of ADT-1004 for treatment of PDAC and other RAS-driven cancers by addressing the complex genetic landscape of many cancers and limitations from resistance to mutant specific KRAS inhibitions. 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. Efficacy advantages of a novel pan-RAS inhibitor, ADT-1004, over mutant- specific KRAS inhibitors for suppressing pancreatic tumor growth in murine models [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 C027.

Abstract C026: Concurrent inhibition of the RAS ERK-MAPK pathway and PIKfyve as a therapeutic strategy for pancreatic cancer

Abstract Pancreatic ductal adenocarcinoma (PDAC) is characterized clinically by poor survival and mechanistically by KRAS- and autophagy-dependent growth. We and others previously demonstrated that inhibition of KRAS signaling via downstream inhibition of the RAF-MEK-ERK pathway enhanced autophagic flux and dependency of PDAC on autophagy. Furthermore, we demonstrated that concurrent treatment with the nonspecific autophagy inhibitor chloroquine (CQ) and ERK MAPK inhibitors synergistically blocked PDAC growth. These findings provided rationale for our initiation of Phase I/II clinical trials evaluating the combination of MEKi (binimetinib; NCT4132505) or ERKi (LY3214996; NCT04386057) with HCQ in PDAC. However, a limitation of this approach is that CQ/HCQ is limited in terms of specificity and potency. Thus, we aimed to identify alternative anti-autophagy strategies. We performed a CRISPR-Cas9 loss- of-function screen in PDAC cell lines that identified the lipid kinase PIKfyve as a growth- promoting gene. PIKfyve is a lipid kinase that is essential for the generation of PI(3,5)P2 and critical for the dynamic regulation of lysosomal recycling. Because PIKfyve has been implicated in regulation of autophagy in other cellular contexts, we evaluated the effects of PIKfyve inhibition on growth and autophagic flux in PDAC cell lines and tumors. We demonstrated that PIKfyve inhibition by the small molecule apilimod resulted in durable growth suppression, with much greater potency than CQ treatment. PIKfyve inhibition potently reduced autophagy as indicated by decreased autophagic flux and the robust accumulation of autophagy-related proteins. Additionally, PIKfyve inhibition resulted in the accumulation of LAMP1 positive vacuoles that exhibited impaired cargo degradation, likely due to a lack of required acidity. Next, we hypothesized that PIKfye inhibition would sensitize PDAC cells to RAS pathway inhibition. We demonstrated that PIKfyve inhibition blocked the compensatory increases in autophagic flux associated both with MEK inhibition and with direct RAS inhibition. Accordingly, combined inhibition of PIKfyve and either the RAS ERK-MAPK pathway or RAS itself showed robust growth suppression across a panel of KRAS-mutant PDAC models. Growth suppression was due, in part, to potentiated cell cycle arrest and induction of apoptosis following loss of IAP proteins. These findings implicate PIKfyve as an effective anti-autophagy target when paired with RAS or ERK-MAPK pathway inhibition in pre-clinical models of PDAC. Citation Format: Jonathan M DeLiberty, Mallory K Roach, Clint A Stalnecker, Ryan Robb, Elyse G Schechter, Noah L Pieper, Runying Yang, Scott Bang, Khalilah E Taylor, Kristina Drizyte-Miller, John P Morris IV, Channing J Der, Adrienne D Cox, Kirsten L Bryant. Concurrent inhibition of the RAS ERK-MAPK pathway and PIKfyve as a therapeutic strategy for 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 C026.

Abstract IA-06: Elucidation of metabolic resistance mechanisms to RAS inhibition

Abstract Pancreatic ductal adenocarcinoma (PDAC) is characterized by KRAS- and autophagy-dependent growth. We previously determined that concurrent inhibition of autophagy, using the lysosomal inhibitor chloroquine (CQ), and of ERK, using a small molecule ERK inhibitor (ERKi), synergistically suppressed the growth of pancreatic ductal adenocarcinoma (PDAC) cell lines and patient xenograft-derived (PDX) organoids in vitro and PDX tumors in vivo. Our findings provided the rationale for our initiation of Phase I and Phase I/II clinical trials evaluating the combination of MEKi (binimetinib; NCT04132505) or ERKi (LY3214996; NCT04386057) with hydroxychloroquine (HCQ) in PDAC. We have extended these findings and determined that the combined inhibition of KRASG12C or KRASG12D and autophagy was effective in KRAS-mutant cancers. Our ongoing studies are centered on developing additional combinations for targeting metabolic resistance mechanisms to KRAS or ERK MAPK targeted therapies. First, we performed a CRISPR/Cas-9 mediated genetic loss-of-function screen in the presence of CQ to determine additional sensitizers to autophagy inhibition. Top sensitizers included multiple facilitators of the DNA damage response, mTOR pathway components, and genes involved in the upstream regulation of the autophagy pathway. Additionally, we identified PIKfyve, a lipid kinase critical for the recycling dynamics of lysosomes, as an essential autophagy-related gene in PDAC cells. PIKfyve inhibition with apilimod resulted in a potent reduction of autophagic flux and growth. Importantly, PIKfyve inhibition blocked the compensatory increases in autophagic flux associated both with MEK inhibition and with direct RAS inhibition. Accordingly, combined inhibition of PIKfyve and either the RAS ERK-MAPK pathway or RAS itself showed robust growth suppression across a panel of KRAS-mutant PDAC models. Growth suppression was due, in part, to potentiated cell cycle arrest and induction of apoptosis following loss of IAP proteins. We conclude that concurrent inhibition of RAS and PIKfyve is a synergistic, cytotoxic combination that may represent a novel therapeutic strategy for PDAC. In a parallel line of investigation, we determined that autophagic signaling is temporally regulated following RAS pathway inhibition, peaking at 48-72 hours, and returning to basal levels by 7 days. Interestingly, we found that as autophagic upregulation subsides, another nutrient scavenging pathway, macropinocytosis, increases. We hypothesize that further dissection of the dynamic regulation of autophagy and macropinocytosis will improve current anti-nutrient scavenging treatment strategies. We conclude that concurrent suppression of multiple metabolic processes, to block compensatory rebound activities, will be needed for effective PDAC treatment. Citation Format: Jonathan M. DeLiberty, Ryan Robb, Mallory K. Roach, Sarah E. Ackermann, Runying Yang, Noah L. Pieper, Khalilah E. Taylor, Scott Bang, Elisa Baldelli, Emanuel F. Petricoin III, John P. Morris IV, Clint A. Stalnecker, Kirsten L. Bryant. Elucidation of metabolic resistance mechanisms to RAS 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 IA-06.

The molecular mechanism underlying KRAS regulation on STK31 expression in pancreatic ductal adenocarcinoma.

KRAS gene mutations are common in pancreatic ductal adenocarcinoma (PDAC), but targeting mutant KRAS is still challenging. Here, an endoribonuclease-prepared small interfering RNA (esiRNA) library was used to screen new kinases that play critical roles in PDAC driven by KRAS gene mutations, and serine/threonine kinase 31 (STK31) was identified and characterized as a potential therapeutic target for KRAS-mutant PDAC. Our results showed that STK31 was upregulated in KRAS-mutant PDAC patients with poor survival and highly expressed in PDAC cell lines with KRASG12D mutation. Inhibition of STK31 in KRAS-mutant cell lines significantly reduced PDAC cell growth invitro and hindered tumor growth invivo. Gain and loss of function experiments revealed that STK31 is a downstream target of KRAS in PDAC. A pharmacological inhibition assay showed MAPK/ERK signaling involved in STK31 regulation. The further mechanistic study validated that c-Jun, regulated by KRAS/MAPK signaling, directly modulates the transcription level of STK31 by binding to its promoter region. Through RNA sequencing, we found that the cell cycle regulators CCNB1 and CDC25C are downstream targets of STK31. Taken together, our results indicate that STK31, which is the downstream target of the KRAS/MAPK/ERK/c-Jun signaling pathway in KRAS-mutant PDAC, promotes PDAC cell growth by modulating the expression of the cell cycle regulators CCNB1 and CDC25C.

Evaluation of a novel Pan-RAS/β-Catenin inhibitor, ADT-030, for treatment of pancreatic cancer.

e15084 Background: Pancreatic ductal adenocarcinoma (PDAC) remains a formidable challenge due to its late diagnosis and limited treatment options. KRAS mutations and activation of Wnt/β-catenin pathway components that drive aberrant signaling cascades resulting in uncontrolled cellular proliferation and metastasis occur in a majority of PDAC patients. The development of mutant specific KRAS inhibitors has emerged as a promising approach to counter the oncogenic impact of KRAS mutations, although have limited use for PDAC given the complex mutational landscape, while inhibitors of Wnt/β-catenin signaling remain elusive. Novel target-directed drugs that effectively tackle the complex mutational status in KRAS mutant PDAC are urgently needed. A novel pan-RAS/β-catenin inhibitor, ADT-030, was evaluated in mouse tumor models of PDAC harboring KRASG12C or KRASG12D mutations. Methods: PDAC cell lines 2838c3 (KRASG12D), MIA PaCa-2 (KRASG12C), and BxPC-3 (RASWT) were utilized to evaluate in vitro growth inhibitory potency and selectivity of ADT-030. Assays for colony formation, apoptosis, and cell cycle, along with western blotting for RAS signaling were also conducted. A murine model involving 2838c3-Luc KRASG12D PDAC cells injected into the pancreas of C57BL/6J mice was used to evaluate in vivo antitumor activity of ADT-030. ADT-030 was administered orally once daily at dosages of 50, 100, and 150mg/kg body weight, 5x/week for 4 weeks. Tumor growth was monitored by bioluminescence imaging using an IVIS Xenogen system. Body weights were measured twice weekly. Pancreatic tumors were collected and weighed at the end of treatment. Results: ADT-030 produced potent inhibition of proliferation of human and murine PDAC cells harboring KRASG12D and KRASG12C mutations. Annexin V assay revealed the capacity of ADT-030 to trigger apoptosis, specifically in KRAS mutant cells with minimal impact on BxPC3 RASWT cells. Cell cycle analysis using Propidium Iodide staining revealed the ability of ADT-030 to arrest cells in the G2/M phase of the cell cycle. Western blot for detecting the key RAS signaling molecule P-ERK1/2 showed decreased levels following ADT-030 treatment. Other experiments revealed that ADT-030 and related analogs simultaneously suppress both MAPK/AKT signaling and β-catenin transcriptional activity (doi: 10.1186/s13048-022-01050-9). In an orthotopic xenograft model in which 2838c3-Luc cells were implanted in the pancreas, oral administration of 50, 100, and 150mg/kg of ADT-030 was well tolerated and inhibited tumor growth by 64.2%, 72.8%, and 80%, respectively, after 4 weeks of treatment. Conclusions: These compelling results position ADT-030 as a novel dual pan-RAS/β-catenin inhibitor for combating the challenges posed by complex mutations in PDAC and other RAS driven cancers and support its clinical evaluation as a valuable therapeutic alternative to mutant-specific KRAS inhibitors.

Abstract 7223: A triplet therapeutic strategy targeting MEK, BCL-XL, and EGFR, for KRAS-mutant pancreatic ductal adenocarcinoma

Abstract KRAS mutations drive over 90% of pancreatic cancers, but KRAS has been proven difficult to target. Downstream of KRAS, MEK inhibition in combination with BCL-XL inhibition is synthetically lethal and an effective treatment for gynecologic, but not colorectal malignancies. Like colorectal cancer, pancreatic cancer often exhibits aberrant HER/EGFR activity. We hypothesized that a triplicate regimen targeting MEK, BCL-XL, and EGFR/HER would be effective for pancreatic cancer. Trametinib (MEK inhibitor), Afatinib (pan-EGFR/HER inhibitor), and Navitoclax (BCL-XL inhibitor); or DT2216 (a BCL-XL/BCL-1 proteolysis targeting chimera, PROTAC) were explored using various combinatorial strategies. These drug combinations were tested using four primary human pancreatic cancer cell lines using live cell imaging (Incucyte) and an in vivo murine model. Group comparisons were analyzed using one-way ANOVAs, and adjusted p-values <0.05 were considered significant. We observed that no single-agent treatments were effective. MEK inhibition plus anti-BCL-XL (Trametinib + Navitoclax or Trametinib + DT2216) demonstrated efficacy in one of the four cell lines. We noted synergistic effects on reduced cell growth and increased cell death when EGFR/HER inhibition was included by the administration of Afatinib. The triplet combination resulted in synergistic effects in three of four cell lines with Navitoclax and four of four cell lines with DT2216 (q<0.05). This effect was confirmed with DT2216 inhibition of BCL-XL in an in-vivo murine model. In conclusion, our study shows that a combinatory strategy targeting MEK, BCL-XL, and pan-EGFR/HER receptor kinase shows significant synergistic effects in pancreatic cancer. Clinical trials that assess the efficacy and safety of this strategy for patients with KRAS-mutant pancreatic cancer are warranted. Our data also suggests that novel KRAS inhibitors as monotherapy are likely to fail. Citation Format: Gerik W. Tushoski-Alemán, Alexandra J. Crespin, Chibeze J. Oguejiofor, Jordan A. McKean, Kelly M. Herremans, Thomas J. George, Carmen J. Allegra, Steven J. Hughes, Song Han. A triplet therapeutic strategy targeting MEK, BCL-XL, and EGFR, for KRAS-mutant pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7223.

The interlacing anticancer effect of pharmacologic ascorbate, chloroquine, and resveratrol.

Currently, a diagnosis with KRAS mutant pancreatic ductal adenocarcinoma (PDAC) means a death warrant, so finding efficient therapeutic options is a pressing issue. Here, we presented that pharmacologic ascorbate, chloroquine and resveratrol co-treatment exerted a synergistic cytotoxic effect on PDAC cell lines. The observed synergistic cytotoxicity was a general feature in all investigated cancer cell lines independent of the KRAS mutational status and seems to be independent of the autophagy inhibitory effect of chloroquine. Furthermore, it seems that apoptosis and necroptosis are also not likely to play any role in the cytotoxicity of chloroquine. Both pharmacologic ascorbate and resveratrol caused double-strand DNA breaks accompanied by cell cycle arrest. It seems resveratrol-induced cytotoxicity is independent of reactive oxygen species (ROS) generation and accompanied by a significant elevation of caspase-3/7 activity, while pharmacologic ascorbate-induced cytotoxicity shows strong ROS dependence but proved to be caspase-independent. Our results are particularly important since ascorbate and resveratrol are natural compounds without significant harmful effects on normal cells, and chloroquine is a known antimalarial drug that can easily be repurposed.

Concurrent inhibition of pBADS99 synergistically improves MEK inhibitor efficacy in KRASG12D-mutant pancreatic ductal adenocarcinoma

Therapeutic targeting of KRAS-mutant pancreatic ductal adenocarcinoma (PDAC) has remained a significant challenge in clinical oncology. Direct targeting of KRAS has proven difficult, and inhibition of the KRAS effectors have shown limited success due to compensatory activation of survival pathways. Being a core downstream effector of the KRAS-driven p44/42 MAPK and PI3K/AKT pathways governing intrinsic apoptosis, BAD phosphorylation emerges as a promising therapeutic target. Herein, a positive association of the pBADS99/BAD ratio with higher disease stage and worse overall survival of PDAC was observed. Homology-directed repair of BAD to BADS99A or small molecule inhibition of BADS99 phosphorylation by NCK significantly reduced PDAC cell viability by promoting cell cycle arrest and apoptosis. NCK also abrogated the growth of preformed colonies of PDAC cells in 3D culture. Furthermore, high-throughput screening with an oncology drug library to identify potential combinations revealed a strong synergistic effect between NCK and MEK inhibitors in PDAC cells harboring either wild-type or mutant-KRAS. Mechanistically, both mutant-KRAS and MEK inhibition increased the phosphorylation of BADS99 in PDAC cells, an effect abrogated by NCK. Combined pBADS99-MEK inhibition demonstrated strong synergy in reducing cell viability, enhancing apoptosis, and achieving xenograft stasis in KRAS-mutant PDAC. In conclusion, the inhibition of BADS99 phosphorylation enhances the efficacy of MEK inhibition, and their combined inhibition represents a mechanistically based and potentially effective therapeutic strategy for the treatment of KRAS-mutant PDAC.

Abstract B103: Development and characterisation of KRASmulti inhibitors for the treatment of KRAS mutant pancreatic ductal adenocarcinoma

Abstract Pancreatic ductal adenocarcinoma (PDAC) is predicted to become the second most common cause of cancer related death within the next decade. Late detection and limited therapeutic options drive the dismal prognosis for PDAC patients. Activating mutations within the KRAS gene occur within the vast majority (>85%) of PDAC tumors. KRAS mutations occur at an early stage within PDAC development but remain necessary for continued tumor growth. Clinical responses observed in patients with KRASG12C mutant PDAC treated with selective KRASG12C inhibitors have driven drug development programmes that can address other, more frequently occurring KRAS alleles. Here we report the characterization of a KRASmulti inhibitor, BI-2493, that potently inhibits the proliferation of mutant KRAS PDAC cancer cell lines in vitro. The DMPK properties of the compound have been optimized so that oral administration resulted in regression of KRAS mutant PDAC xenograft models. These data support the continued development of compounds capable of addressing the most prevalent KRAS mutant alleles in PDAC with the potential to provide a therapeutic option to patients in an indication with a high unmet medical need. Citation Format: Antonio Tedeschi, Otmar Schaaf, Michael Gmachl, Lorenz Herdeis, Francesca Rocchetti, Johannes Popow, Fiorella Schischlik, Daniel Gerlach, Jesse Lipp, Joachim Bröker, Andreas Mantoulidis, Chris Smethurst, Dirk Kessler, Jark Boettcher, Tobias Wunberg, Valeria Santoro, Alex Waterson, Jason Phan, Andrew Little, Jason Abbott, Qi Sun, Stephen Fesik, Darryl Mcconnell, Mark A. Pearson, Norbert Kraut, Dorothea Rudolph. Development and characterisation of KRASmulti inhibitors for the treatment of KRAS mutant pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr B103.

Abstract C003: RAMP 205: A phase 1b/2a study of gemcitabine, nab-paclitaxel, avutometinib, and defactinib in untreated metastatic pancreatic ductal adenocarcinoma

Abstract Background: Pancreatic ductal adenocarcinoma (PDAC) accounts for ~90% of pancreatic cancer. Surgery is the only curative treatment. Unfortunately, most patients are diagnosed with advanced disease and are not candidates for resection. Combination chemotherapy approaches have provided incremental improvements to PDAC outcomes. However, impact on survival for patients with inoperable disease remains modest, with a 2-year survival rate of less than 7%. Activating mutations in the KRAS oncogene are a key initiating event in ~80-90% of PDAC. Permanent activation of KRAS drives tumor cell proliferation and survival, tumor microenvironment modulation, and metabolic alterations in PDAC. Patients with KRAS mutant PDAC have worse outcomes compared to those with KRAS wild-type PDAC. In addition, hyperactivation of focal adhesion kinase (FAK) has been correlated with high stromal density, resulting in therapeutic resistance and poor survival. Avutometinib is a first-in-class oral RAF/MEK clamp that potently inhibits MEK kinase activity while also blocking the compensatory reactivation of MEK by upstream RAF. Defactinib is a selective inhibitor of focal adhesion kinase (FAK), which has been shown to mediate resistance to multiple anticancer agents. Preclinical data have shown that the combination of avutometinib + FAK inhibition (FAKi) + chemotherapy (gemcitabine + paclitaxel as a surrogate for nab-paclitaxel) induced tumor regression in a autochthonous KRAS/TP53 mutant PDAC mouse model. Prior clinical experience of defactinib + pembrolizumab + chemotherapy was safe, showed responses, decreased stromal density, and increased CD8+ T cell infiltration in patients with PDAC (NCT02546531). Methods: RAMP 205 is a multi-center, open-label, single arm Phase 1b/2a study of gemcitabine and nab-paclitaxel in combination with avutometinib and defactinib in patients with previously untreated metastatic PDAC (NCT05669482). The study is being conducted in 2 parts: Parts A (dose evaluation) and Part B (dose expansion). The starting dose is 2.4 mg avutometinib orally (PO) twice weekly + 200 mg defactinib PO twice per day, in combination with standard once weekly gemcitabine/nab-paclitaxel (QW IV). All study drugs are dosed 3 weeks on, 1 week off. Following determination of the Recommended Phase 2 Dose (RP2D) in Part A, Part B will evaluate efficacy of the RP2D via objective response rate (primary endpoint). Mandatory tumor biopsies and blood samples will be collected for correlative studies, including circulating tumor DNA, molecular profiling and pharmacodynamic markers. Eligible patients must have histologic or cytologic evidence of metastatic PDAC and measurable disease according to RECIST v1.1. Patients with prior or concomitant treatment for metastatic PDAC or prior treatment with RAS/MAPK or FAK inhibitors are not eligible. The study will enroll ~35 patients with 6-12 patients in Part A, and 23 patients in Part B. Citation Format: Kian-Haut Lim, Manuel Hidalgo, Mark H. O'Hara, Kristen R. Spencer, Ignacio Garrido-Laguna, David G. DeNardo, Vijeta Bhambhani, Gloria Patrick, Yaofeng Cheng, Silvia Coma, Jonathan A. Pachter, Louis J. Denis. RAMP 205: A phase 1b/2a study of gemcitabine, nab-paclitaxel, avutometinib, and defactinib in untreated metastatic pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr C003.

The suppressive efficacy of THZ1 depends on KRAS mutation subtype and is associated with super-enhancer activity and the PI3K/AKT/mTOR signalling in pancreatic ductal adenocarcinoma: A hypothesis-generating study.

Inhibition of CDK7, a potent transcription regulator, may bring new hope for treating pancreatic ductal adenocarcinoma (PDAC), which is featured by large genetic heterogeneity and abundant KRAS mutations. This investigation aimed at exploring the discrepant efficacies of THZ1, a small-molecule covalent CDK7 inhibitor, on PDACs with different KRAS mutations and the underlying mechanisms. Associations of CDK7 expression with survival by KRAS mutations were first assessed. Effects of THZ1 on PDAC by different KRAS mutations were then investigated in vitro and in vivo. Moreover, the effects of THZ1 on gene transcription and phosphorylation of RNA polymerase II (RNAPOLII) in different KRAS mutant PDACs were assessed, and the effect of THZ1 on super-enhancer activity was evaluated using chromatin immunoprecipitation sequencing. Lastly, the effects of THZ1 on the binding of H3K27ac to PIK3CA and on the PI3K/AKT/mTOR signalling were analysed. High CDK7 expression was significantly linked to worse survival within PDAC patients carrying KRAS-G12V mutation but not in those with KRAS-G12D mutation. The apoptosis-inducing effect of THZ1 was markedly stronger in KRAS-G12V PDAC than KRAS-G12D cancer. THZ1 significantly inhibited the growth of xenograft tumour with KRAS-G12V mutation, and the inhibition was markedly stronger than for KRAS-G12D tumour. In mini-cell-derived xenograft (CDX) models, THZ1 significantly suppressed KRAS-G12V PDAC but not KRAS-G12D cancer. THZ1 significantly suppressed the phosphorylation of RNAPOLII, and this effect was stronger in KRAS-G12V PDAC (especially at ser5). KRAS-G12V PDAC had more H3K27ac-binding super-enhancers, and the inhibition of THZ1 on super-enhancer activity was also stronger in KRAS-G12V PDAC. Furthermore, THZ1 significantly weakened the binding of H3K27ac to PIK3CA in KRAS-G12V PDAC. THZ1 significantly suppressed the PI3K/AKT/mTOR pathway and its downstream markers, and this effect was stronger in KRAS-G12V cells. In this hypothesis-generating study, THZ1 might selectively inhibit certain PDACs with KRAS-G12V mutation more potently compared with some other PDACs with KRAS-G12D mutation, which might be associated with its effect on super-enhancer activity and the PI3K/AKT/mTOR signalling. Our findings might offer novel key clues for the precise management of PDAC and important evidence for future targeted trial design. THZ1 had a stronger effect on PDAC-bearing KRAS-G12V mutation than G12D mutation. Suppressive effect of THZ1 on phosphorylation of RNAPOLII was stronger in KRAS-G12V than KRAS-G12D PDAC. Inhibition of THZ1 on super-enhancer activity and H3K27ac binding to PIK3CA was stronger in KRAS-G12V PDAC. Suppressive effect of THZ1 on PI3K/AKT/mTOR pathway was stronger in KRAS-G12V PDAC.

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.

652O Preliminary clinical activity of RMC-6236, a first-in-class, RAS-selective, tri-complex RAS-MULTI(ON) inhibitor in patients with KRAS mutant pancreatic ductal adenocarcinoma (PDAC) and non-small cell lung cancer (NSCLC)

652O Preliminary clinical activity of RMC-6236, a first-in-class, RAS-selective, tri-complex RAS-MULTI(ON) inhibitor in patients with KRAS mutant pancreatic ductal adenocarcinoma (PDAC) and non-small cell lung cancer (NSCLC)

DNA methylation-associated allelic inactivation regulates Keratin 19 gene expression during pancreatic development and carcinogenesis.

Within the pancreas, Keratin 19 (KRT19) labels the ductal lineage and is a determinant of pancreatic ductal adenocarcinoma (PDAC). To investigate KRT19 expression dynamics, we developed a human pluripotent stem cell (PSC)-based KRT19-mCherry reporter system in different genetic backgrounds to monitor KRT19 expression from its endogenous gene locus. A differentiation protocol to generate mature pancreatic duct-like organoids was applied. While KRT19/mCherry expression became evident at the early endoderm stage, mCherry signal was present in nearly all cells at the pancreatic endoderm (PE) and pancreatic progenitor (PP) stages. Interestingly, despite homogenous KRT19 expression, mCherry positivity dropped to 50% after ductal maturation, indicating a permanent switch from biallelic to monoallelic expression. DNA methylation profiling separated the distinct differentiation intermediates, with site-specific DNA methylation patterns occurring at the KRT19 locus during ductal maturation. Accordingly, the monoallelic switch was partially reverted upon treatment with a DNA-methyltransferase inhibitor. In human PDAC cohorts, high KRT19 levels correlate with low locus methylation and decreased survival. At the same time, activation of oncogenic KRASG12D signalling in our reporter system reversed monoallelic back to biallelic KRT19 expression in pancreatic duct-like organoids. Allelic reactivation was also detected in single-cell transcriptomes of human PDACs, which further revealed a positive correlation between KRT19 and KRAS expression. Accordingly, KRAS mutant PDACs had higher KRT19 mRNA but lower KRT19 gene locus DNA methylation than wildtype counterparts. KRT19 protein was additionally detected in plasma of PDAC patients, with higher concentrations correlating with shorter progression-free survival in gemcitabine/nabPaclitaxel-treated and opposing trends in FOLFIRINOX-treated patients. Apart from being an important pancreatic ductal lineage marker, KRT19 appears tightly controlled via a switch from biallelic to monoallelic expression during ductal lineage entry and is aberrantly expressed after oncogenic KRASG12D expression, indicating a role in PDAC development and malignancy. Soluble KRT19 might serve as a relevant biomarker to stratify treatment. © 2023 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Mutant KRAS Mediates circARFGEF2 Biogenesis to Promote Lymphatic Metastasis of Pancreatic Ductal Adenocarcinoma.

Increased splicing-mediated biogenesis of circARFGEF2 in KRAS-mutant pancreatic ductal adenocarcinoma activates JAK2-STAT3 signaling and triggers lymph node metastasis, suggesting circARFGEF2 could be a therapeutic target to inhibit pancreatic cancer progression.

Structural Manipulations of Marine Natural Products Inspire a New Library of 3-Amino-1,2,4-Triazine PDK Inhibitors Endowed with Antitumor Activity in Pancreatic Ductal Adenocarcinoma

Pancreatic ductal adenocarcinoma (PDAC) is one of the main aggressive types of cancer, characterized by late prognosis and drug resistance. Among the main factors sustaining PDAC progression, the alteration of cell metabolism has emerged to have a key role in PDAC cell proliferation, invasion, and resistance to standard chemotherapeutic agents. Taking into account all these factors and the urgency in evaluating novel options to treat PDAC, in the present work we reported the synthesis of a new series of indolyl-7-azaindolyl triazine compounds inspired by marine bis-indolyl alkaloids. We first assessed the ability of the new triazine compounds to inhibit the enzymatic activity of pyruvate dehydrogenase kinases (PDKs). The results showed that most of derivatives totally inhibit PDK1 and PDK4. Molecular docking analysis was executed to predict the possible binding mode of these derivatives using ligand-based homology modeling technique. Evaluation of the capability of new triazines to inhibit the cell growth in 2D and 3D KRAS-wild-type (BxPC-3) and KRAS-mutant (PSN-1) PDAC cell line, was carried out. The results showed the capacity of the new derivatives to reduce cell growth with a major selectivity against KRAS-mutant PDAC PSN-1 on both cell models. These data demonstrated that the new triazine derivatives target PDK1 enzymatic activity and exhibit cytotoxic effects on 2D and 3D PDAC cell models, thus encouraging further structure manipulation for analogs development against PDAC.

Abstract B034: Determination of KRAS- and ERK-regulated phosphoproteomes in KRAS-mutant cancers

Abstract Direct inhibitors of mutationally activated KRAS are currently under intense preclinical and clinical development, with one KRASG12C mutant-selective inhibitor approved. However, treatment-associated resistance to KRASG12C inhibitors has been reported, with ~60% of relapsed patients acquiring mutations in signaling components both upstream and downstream, and at the level of RAS itself, that led to reactivation of RAF-MEK-ERK and PI3K-AKT effector pathways. Unexpectedly, we found that ectopic expression of constitutively activated MEK1, ERK1 or ERK2, but not AKT1, drove near-complete resistance to direct inhibitors of KRASG12C or KRASG12D in KRAS-mutant pancreatic ductal adenocarcinoma (PDAC). Despite comprehensive studies, how ERK supports KRAS-dependent cancer growth remains poorly understood. Therefore, we focused on delineating the ERK-dependent phosphoproteome. We treated a panel of six KRAS-mutant PDAC cell lines acutely (1 h) and long-term (24 h) with the highly selective ERK1/2 inhibitor SCH772984. Our proteomics analyses identified 5,117 phosphosites on 2,252 proteins significantly dysregulated by ERK inhibition. We first compared our PDAC ERK phosphoproteome with a recent compendium of published direct/indirect ERK substrates identified in non-PDAC cells and found, surprisingly, only 12% overlap. Thus, our dataset substaintially extends the complexity of ERK-regulated phosphoproteins, in part, reflecting a pancreatic cancer selective profile. To elucidate the kinase network that drives our PDAC ERK-dependent phosphoproteome, we utilized our newly described global atlas of kinase-substrate specificities and reported kinase-substrate interaction datasets. This revealed a highly dynamic kinome dominated by ERK at 1 h, then dominated by ERK-regulation of CDK1-6 cell cycle components and RHO GTPase signaling. Next, we established the subcellular distribution of the PDAC ERK phosphoproteome, then evaluated data from DepMap, and found enrichment of nuclear ERK substrates that displayed strong dependencies in PDAC. We also determined the KRASG12C-regulated phosphoproteome in pancreatic, lung, and colorectal cancer cells using the KRASG12C selective inhibitor MRTX1257. We used our global atlas of kinase-motif specificities and ERK-regulated phosphoproteome to determine KRAS-regulated signaling pathways in KRASG12C mutant cancers. We found a high correlation between KRAS- and ERK-regulated phosphorylations, primarily driven by CDK substrates. However, we also noted key differences, principally in DNA damage response pathways not identified in our ERK-regulated phosphoproteome. Finally, we performed a high-throughput drug sensitivity and resistance screen of (DSRT) comprised of >500 clinically actionable drugs in combination with a KRASG12C inhibitor, and found ERK inhibitors as top hits. In summary, our studies establish a comprehensive profile of KRAS-ERK signaling output that may define new therapeutic targets for targeting KRAS- and ERK-dependent cancer growth. Citation Format: Jennifer E. Klomp, Jeff A. Klomp, Nathaniel J. Diehl, Cole A. Edwards, Kristina Drizyte-Miller, Priya S. Hibshman, Runying Yang, Alexis J. Morales, Khalilah E. Taylor, Mariaelena Pierobon, Emanuel F. Petricoin III, Johnson L. Jared, Emily M. Huntsman, Tomer M. Yaron, Markus Vähä-Koskela, Laura E. Herring, Alex W. Prevatte, Natalie K. Barker, Lee M. Graves, Wennerberg Krister, Lewis C. Cantley, James G. Christensen, Adrienne D. Cox, Channing J. Der, Clint A. Stalnecker. Determination of KRAS- and ERK-regulated phosphoproteomes in KRAS-mutant cancers [abstract]. In: Proceedings of the AACR Special Conference: Targeting RAS; 2023 Mar 5-8; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Res 2023;21(5_Suppl):Abstract nr B034.

Abstract 2218: The molecular mechanism underlying KRAS regulation on STK31 expression in pancreatic ductal adenocarcinoma

Abstract KRAS is frequently mutated in pancreatic ductal adenocarcinoma (PDAC). However, targeting mutant KRAS is still challenging. Kinase inhibitors are ideal targeted therapeutics for mutant KRAS-driven cancer. In our previous study, an esiRNA screening was prepared to identify some vital kinases that took part in KRAS mutant-driven pancreatic cancer. The STK31 was identified as a potential therapeutic target in KRAS mutant PDAC. This study aimed to investigate the underlying mechanism of STK31 in KRAS mutant PDAC as well as its therapeutic potential. The results demonstrated that STK31 was upregulated in KRAS mutant PDAC patients with poor survival. STK31 was also highly expressed in PDAC cell lines with KRAS mutant background. Inhibition of STK31 in KRAS mutant cell lines significantly reduced PDAC cell proliferation, colony formation, and migration. Gain and loss of function experiments revealed that STK31 was a downstream target of KRAS in PDAC. Pharmacological inhibition of several signaling pathways involved in KRAS showed MAPK/ERK signaling involved in STK31 regulation. Then, further mechanistic study validated that c-Jun, regulated by KRAS/MAPK/ERK signaling, directly regulated the transcription level of STK31 by binding to its promoter region. In summary, our results show that STK31 promotes PDAC cell proliferation under the control of the KRAS/MAPK/ERK/c-Jun transcriptional axis in PDAC. STK31 may act as a potential target in PDAC treatment. Citation Format: Yuting Liu, Shing Chun Tang, Yangchao Chen. The molecular mechanism underlying KRAS regulation on STK31 expression in pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2218.