Genes In Pancreatic Ductal Adenocarcinoma Research Articles

Abstract B084: Comparative functional analysis of TP53 alleles in pancreatic ductal adenocarcinoma in vivo

Abstract TP53 is the second most mutated gene in pancreatic ductal adenocarcinoma (PDAC), bearing both truncal loss-of-function and missense point mutations. TP53 encodes a stress-induced transcription factor (p53) capable of activating tumor suppressive pathways mediating cell cycle arrest, apoptosis, senescence, and metabolic reprogramming. Despite high mutational frequency, how TP53 mutants function to suppress pancreatic tumorigenesis in vivo remains unclear. Previous studies indicate that the most frequent point mutations observed in human PDAC (TP53 R175H and R273H; mouse homologues Trp53 R172H and R270H) exhibit both dominant-negative (e.g., response to radiation in leukemia cells) and gain-of-function (e.g., increased liver metastasis in advanced PDAC models) roles compared to p53 null cells in various contexts. To rigorously explore the differential functions of p53 point and deletion mutants in PDAC progression, we performed an isogenic comparative analysis of Trp53 alleles in vivo using Mosaic Analysis with Double Markers (MADM) in mice. MADM uses stochastic mitotic recombination to induce two genotypically distinct daughter cells – created at 1-to-1 ratios – expressing different Trp53 variants dependent on the genotype of the mouse (+/+ vs. -/-, R172H/R172H vs. +/+, R172H/R172H vs. -/-, R270H/R270H vs. +/+, R270H/R270H vs. -/-) and simultaneously label them with unique genetically encoded fluorescent markers (TdTomato vs. GFP). We integrated MADM into a faithful Kras-driven model of pancreatic tumorigenesis (KC: Kras LSL-G12D/+ ; Pdx1-Cre) and used fluorescence microscopy to trace subclonal populations with various Trp53 alleles within the same mouse. By analyzing the ratio of TdTomato+ to GFP+ cells, we studied the functional differences between alleles in driving tumor initiation and expansion at various stages of progression. Strikingly, we found that Trp53 -/- cells exhibited greater expansion in preinvasive pancreatic intraepithelial neoplasias (PanINs) compared to not only Trp53 +/+ cells but also cells harboring either point mutation (Trp53 R172H/R172H or Trp53 R270H/R270H ). While Trp53 R270H/R270H cells predominated over Trp53 +/+ in PanINs, Trp53 R172H/R172H cells were surprisingly indistinguishable from Trp53 +/+ cells, suggesting that p53R172H retains tumor suppressive properties in early PDAC progression. Conversely, both Trp53 R172H/R172H and Trp53 R270H/R270H cells progressed to advanced PDAC with comparable PDAC incidence and survival as Trp53 -/- , arguing that Trp53 R172H is a separation-of-function mutant concordant with a distinct transcriptional profile relative to Trp53 -/- tumor cells. Together, these studies offer the strongest evidence to date for stage-specific functional differences between TP53 mutant variants in cancer progression in vivo and provide a faithful MADM-based PDAC model to define molecular mechanisms (through ongoing transcriptomic, epigenomic, and metabolomic studies) that govern functional differences amongst these diverse alleles. Citation Format: Sherry S. Agabiti, Andy Tang, Timothy Nottoli, Mandar D. Muzumdar. Comparative functional analysis of TP53 alleles in pancreatic ductal adenocarcinoma in vivo [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 B084.

Genetics and biology of pancreatic ductal adenocarcinoma

Abstract Pancreatic ductal adenocarcinoma (PDAC), also known as pancreatic ductal adenocarcinoma, is the most prevalent type of pancreatic tumor, predominantly impacting the exocrine portion of the pancreas. Individuals diagnosed with PDAC face a grim prognosis due to its highly malignant nature. By the year 2030, it is projected to emerge as the second leading cause of cancer-related fatalities. PDAC is known for its high degree of genomic instability. This review offers a summary of the frequently mutated genes in PDAC, as well as the morphological features, molecular profiles, available treatment options, and ongoing research in the field of PDAC.

SAMD1 suppresses epithelial-mesenchymal transition pathways in pancreatic ductal adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) poses a significant threat due to its tendency to evade early detection, frequent metastasis, and the subsequent challenges in devising effective treatments. Processes that govern epithelial-mesenchymal transition (EMT) in PDAC hold promise for advancing novel therapeutic strategies. SAMD1 (SAM domain-containing protein 1) is a CpG island-binding protein that plays a pivotal role in the repression of its target genes. Here, we revealed that SAMD1 acts as a repressor of genes associated with EMT. Upon deletion of SAMD1 in PDAC cells, we observed significantly increased migration rates. SAMD1 exerts its effects by binding to specific genomic targets, including CDH2, encoding N-cadherin, which emerged as a driver of enhanced migration upon SAMD1 knockout. Furthermore, we discovered the FBXO11-containing E3 ubiquitin ligase complex as an interactor and negative regulator of SAMD1, which inhibits SAMD1 chromatin-binding genome-wide. High FBXO11 expression in PDAC is associated with poor prognosis and increased expression of EMT-related genes, underlining an antagonistic relationship between SAMD1 and FBXO11. In summary, our findings provide insights into the regulation of EMT-related genes in PDAC, shedding light on the intricate role of SAMD1 and its interplay with FBXO11 in this cancer type.

MICAL2 Is a Super Enhancer Associated Gene that Promotes Pancreatic Cancer Growth and Metastasis.

Pancreatic ductal adenocarcinoma (PDAC) remains one of the deadliest solid cancers and thus identifying more effective therapies is a major unmet need. In this study we characterized the super enhancer (SE) landscape of human PDAC to identify novel, potentially targetable, drivers of the disease. Our analysis revealed that MICAL2 is a super enhancer-associated gene in human PDAC. MICAL2 is a flavin monooxygenase that induces actin depolymerization and indirectly promotes SRF transcription by modulating the availability of serum response factor coactivators myocardin related transcription factors (MRTF-A and MRTF-B). We found that MICAL2 is overexpressed in PDAC and correlates with poor patient prognosis. Transcriptional analysis revealed that MICAL2 upregulates KRAS and EMT signaling pathways, contributing to tumor growth and metastasis. In loss and gain of function experiments in human and mouse PDAC cells, we observed that MICAL2 promotes both ERK1/2 and AKT activation. Consistent with its role in actin depolymerization and KRAS signaling, loss of MICAL2 expression also inhibited macropinocytosis. Through in vitro phenotypic analyses, we show that MICAL2, MRTF-A and MRTF-B influence PDAC cell proliferation, migration and promote cell cycle progression. Importantly, we demonstrate that MICAL2 is essential for in vivo tumor growth and metastasis. Interestingly, we find that MRTF-B, but not MRTF-A, phenocopies MICAL2-driven phenotypes in vivo . This study highlights the multiple ways in which MICAL2 impacts PDAC biology and suggests that its inhibition may impede PDAC progression. Our results provide a foundation for future investigations into the role of MICAL2 in PDAC and its potential as a target for therapeutic intervention.

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.

Liquid biopsy in patients with metastatic pancreatic cancer using an optimized targeted sequencing approach: Data from the pancreatic cancer registry PanDaDETECT.

e16303 Background: Metastatic pancreatic ductal adenocarcinoma (PDAC) conveys a dismal prognosis, and biomarkers for clinical diagnostics and treatment monitoring are urgently needed. Detection of circulating tumor DNA (ctDNA) using liquid biopsy (LB) has been shown to correlate with treatment response in other tumor entities. Here, we evaluated the feasibility of LB by targeted next generation sequencing (NGS) in metastatic PDAC, using an NGS approach optimized for PDAC. Methods: Patients from our PanDaDETECT registry with metastatic PDAC were included. Plasma samples were collected at initial diagnosis and every three months during palliative treatment. Targeted NGS of plasma samples was performed using a small, custom-generated gene panel optimized for LB provided by Qiagen (QiaSeq Targeted DNA Panel), covering the four most frequently mutated genes in PDAC with full exon coverage (KRAS, TP53, SMAD4 and CDKN2A). Sequencing was performed on the Illumina NextSeq 550 platform. LB results were compared to matched tissue NGS data from tumor samples. Results were correlated with the serological biomarker Ca 19-9 and radiographic response data. All patients provided written informed consent. The responsible ethics committee approved the study. Results: From 2020 to 2022, 57 patients were included and underwent plasma NGS at least at initial diagnosis. For 52 of these patients a tissue NGS result was available, and in 49 patients (94,2%), at least one of the four genes covered by our LB panel was mutated in the tumor tissue. In 35 patients, our LB approach detected ctDNA in the plasma sample at initial diagnosis (61,4%). Comparing tissue and plasma sequencing data at initial diagnosis, our LB approach detected ctDNA in 32 out of 49 patients who had a known mutation in tissue NGS in at least one of the four genes covered by the LB panel (65,3%). For 22 patients with positive ctDNA detection in plasma NGS, follow-up plasma samples after three months of treatment were available. In this subgroup, detection of ctDNA showed a superior correlation with radiographic response assessed by RECIST criteria than the serological biomarker Ca 19-9. In patients with a partial response or stable disease as defined by RECIST, ctDNA levels decreased markedly, while remaining elevated in patients showing disease progression. For 11 patients, plasma NGS at 6 months of treatment or later was performed, the results at these later time points correlated well with clinical and radiographic response in 9 out of 11 patients. Conclusions: Our LB approach optimized for PDAC is feasible and shows a good correlation with serological and radiographic response. Our optimized NGS panel covering only four genes is able to detect ctDNA in a majority of PDAC patients. LB by targeted NGS has the potential to detect a broad spectrum of relevant mutations in PDAC in an automatable and cost-effective matter.

Positive feedback regulation between glycolysis and histone lactylation drives oncogenesis in pancreatic ductal adenocarcinoma

BackgroundMetabolic reprogramming and epigenetic alterations contribute to the aggressiveness of pancreatic ductal adenocarcinoma (PDAC). Lactate-dependent histone modification is a new type of histone mark, which links glycolysis metabolite to the epigenetic process of lactylation. However, the role of histone lactylation in PDAC remains unclear.MethodsThe level of histone lactylation in PDAC was identified by western blot and immunohistochemistry, and its relationship with the overall survival was evaluated using a Kaplan-Meier survival plot. The participation of histone lactylation in the growth and progression of PDAC was confirmed through inhibition of histone lactylation by glycolysis inhibitors or lactate dehydrogenase A (LDHA) knockdown both in vitro and in vivo. The potential writers and erasers of histone lactylation in PDAC were identified by western blot and functional experiments. The potential target genes of H3K18 lactylation (H3K18la) were screened by CUT&Tag and RNA-seq analyses. The candidate target genes TTK protein kinase (TTK) and BUB1 mitotic checkpoint serine/threonine kinase B (BUB1B) were validated through ChIP-qPCR, RT-qPCR and western blot analyses. Next, the effects of these two genes in PDAC were confirmed by knockdown or overexpression. The interaction between TTK and LDHA was identified by Co-IP assay.ResultsHistone lactylation, especially H3K18la level was elevated in PDAC, and the high level of H3K18la was associated with poor prognosis. The suppression of glycolytic activity by different kinds of inhibitors or LDHA knockdown contributed to the anti-tumor effects of PDAC in vitro and in vivo. E1A binding protein p300 (P300) and histone deacetylase 2 were the potential writer and eraser of histone lactylation in PDAC cells, respectively. H3K18la was enriched at the promoters and activated the transcription of mitotic checkpoint regulators TTK and BUB1B. Interestingly, TTK and BUB1B could elevate the expression of P300 which in turn increased glycolysis. Moreover, TTK phosphorylated LDHA at tyrosine 239 (Y239) and activated LDHA, and subsequently upregulated lactate and H3K18la levels.ConclusionsThe glycolysis-H3K18la-TTK/BUB1B positive feedback loop exacerbates dysfunction in PDAC. These findings delivered a new exploration and significant inter-relationship between lactate metabolic reprogramming and epigenetic regulation, which might pave the way toward novel lactylation treatment strategies in PDAC therapy.

Abstract 5928: Structure-activity relationships for the pan-quadruplex experimental drug QN-302 and two analogs probed with comparative transcriptome profiling and molecular modeling

Abstract The tetrasubstituted naphthalene diimide compound QN-302 binds to G-quadruplex DNA structures. It inhibits the transcription of cancer-related genes in pancreatic ductal adenocarcinoma (PDAC) cells, shows high potency in PDAC cells in vitro and in PDAC animal models. It is currently in Phase 1 clinical evaluation as an anticancer drug for advanced, metastatic solid tumors. A study of structure-activity relationships of QN-302 and two related analogs (CM03 and SOP1247) is reported here. These have been probed using comparisons of transcriptional profiles from whole-genome RNA-seq analyses, together with qualitative molecular modelling. Compounds CM03 and SOP1247 differ by the presence of a methoxy substituent in the former: these two have closely similar transcriptional profiles, whereas that of QN-302, although showing effects in the same cancer-related pathways, highlights the down regulation of distinct genes, for example in the hedgehog pathway. The distinct pattern of genes affected by QN-302 is hypothesized to contribute to its superior potency compared to CM03 and SOP1247. Its superior ability to stabilize quadruplex structures has been attributed to its benzyl-pyrrolidine substituent fitting into and filling most of the space in a quadruplex groove compared to the hydrogen atom in CM03 or the methoxy group in SOP1247. Citation Format: Ahmed A. Ahmed, Shozeb Haider, Tariq Arshad, Stephen Neidle. Structure-activity relationships for the pan-quadruplex experimental drug QN-302 and two analogs probed with comparative transcriptome profiling and molecular modeling [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 5928.

Abstract 3350: The pan-quadruplex drug QN-302 targets up-regulated genes in pancreatic cancer and in other cancer types and correlate with poor patient prognosis

Abstract We have previously disclosed (Ahmed et al, ACS Med Chem Lett, 2020) a novel small molecule G-quadruplex binding compound, QN-302, that is highly potent in pancreatic ductal adenocarcinoma (PDAC) cells and in several in vivo models for this disease. QN-302 was invented at University College London UK and has been subsequently developed by Qualigen Therapeutics Inc. It was cleared by the FDA in August 2023 for clinical evaluation and is currently in a Phase 1 clinical trial at select oncology centers in the USA. Genes that are significantly down-regulated by QN-302 are mostly up-regulated genes in human PDAC. We report here that some of these genes are also up regulated in other cancer types, whose expression frequently correlates with poor patient prognosis. We have used human cancer data from https://www.proteinatlas.org which contains pathology information based on mRNA and protein expression data from 17 different forms of human cancer, together with millions of in-house generated immunohistochemically stained tissue sections, images and Kaplan-Meier plots showing the correlation between mRNA expression of each human protein gene and cancer patient survival. We have revisited the RNA-seq data for QN-302 in PDAC MIA-PACA2 cells exposed to QN-302 for 24 hrs. (GSE151741 at https://www.ncbi.nlm.nih.gov/geo/) in detail and now identify a small group of eleven down-regulated genes, all with >3-fold expression changes. Interestingly all these genes and their gene products have been previously identified as playing a role in human PDAC (they also have varying roles in other cancer types). Some have been validated as anticancer targets. 10/11 of these genes (https://www.proteinatlas.org/) have been previously reported as being over-expressed in significant fractions of large (>150) groups of human cancer patients from The Cancer Genome Atlas (TCGA) project. Members of the PDAC susceptible gene panel are mostly disfavored in prostate, lung and breast cancer. However, the patient data does not discriminate between cancer sub-types nor does it show resistant data. They are especially favored in colorectal, liver and renal cancers, suggesting that QN-302 may have anticancer effects in these cancers as well as in PDAC. We also do not exclude the strong possibility that each cancer type also has its own group of G-quadruplex containing genes that are sensitive to QN-302. Citation Format: Stephen Neidle, Ahmed A. Ahmed, Tariq Arshad. The pan-quadruplex drug QN-302 targets up-regulated genes in pancreatic cancer and in other cancer types and correlate with poor patient prognosis [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 3350.

Clinical significance of germline mutations in <i>BRCA1/2</i> gene in pancreatic ductal adenocarcinoma (literature review)

Pancreatic ductal adenocarcinoma occupies a leading position in mortality among other malignant neoplasms. The five-year survival of patients is less than 9 %. Risk of a tumor development increases with a mutation in the BRCA gene, however, the effect of this mutation on the clinical course of the disease has been poorly studied and is of certain scientific and practical interest. A review of the world literature on the clinical significance of germline mutations – the risks of malignancy, the choice of treatment tactics – is presented.

Abstract C098: Impact of KRAS mutations and co-mutations on clinical outcomes in pancreatic ductal adenocarcinoma

Abstract Background KRAS is the most frequently mutated gene in pancreatic ductal adenocarcinoma (PDAC). Over the past decade, targeting KRAS has emerged as a promising approach for PDAC treatment. However, the associations between KRAS mutations status and comutation landscape with clinical outcomes remain understudied. We investigated the impact of KRAS mutant allelic status and genomic co-mutations with clinical outcomes. Methods 803 patients with PDAC who underwent CLIA somatic tumor mutation testing at MD Anderson Cancer Center (MDACC) from 1997 to 2023 were identified after an IRB approval. Tumor genomic alterations, demographics, clinical, molecular, and pathological data were extracted from electronic medical records and tumor registry through the Palantir Foundry platform. An external cohort from PanCAN’s Know Your Tumor® dataset with 408 patients served as the validation cohort. Overall Survival (OS) was calculated from the date of initial diagnosis to death or last follow-up. Pearson’s chi-squared tests (or Fisher’s exact tests) were used to determine the association between groups. The Kaplan-Meier method was used to estimate the median OS. Results 703 patients had KRAS gene included in the mutation testing at MDACC from 1997 to 2023. KRAS was the top mutated gene (82%), followed by TP53 (69%), then CDKN2A (20%). In KRAS mutant patients, G12D was the most common variant (39%), followed by G12V (31%), then G12R (14%), and Q61 (6%). In patients tested with multi-gene panels, a co-mutation analysis revealed that the most common co-occurring mutations with KRAS mutation was TP53 (75%), followed by CDKN2A (22%), SMAD4 (14%) and ARID1A (10%). The median follow-up was 41 months and median OS was 19 months in the overall population. KRAS mutation status and subtypes was prognostic of OS (p<0.001). Patients with KRAS wildtype had median OS of 38 months and KRASG12R had median OS of 34 months (HR:1.0, 95% CI 0.71-1.5, p=0.88), while patients with KRASQ61 had worst median OS of 20 months (HR: 1.9, 95% CI 1.2-3.0, p=0.006) and KRASG12D had median OS of 22 months (HR: 1.7, 95% CI 1.3-2.3, p<0.001). Similar findings were observed in patients with stage IV disease (n=302), KRASG12D remained associated with significantly worse OS (HR= 1.7, 95% CI 1.1-2.6, p=0.009). There was a significant enrichment of KRASG12D mutation in metastatic disease compared to localized disease (stage I-III) (OR: 1.7, 95% CI 1.2-2.4, p=0.001) and enrichment of KRASG12R mutation in well and moderately differentiated tumors versus poorly differentiated tumors (OR: 1.7, 95% CI 1.05-2.99, p=0.04). In the external cohort (PanCAN’s Know Your Tumor® dataset), similar findings were observed. KRASG12R was associated with longer median OS (32 months), while KRASQ61 (16 months, HR: 2.6, 95% CI 0.88-7.8, p=0.02) and KRASG12D (23 months, HR: 1.68, 95% CI 1.06-2.65, p=0.04) were associated with significantly worse median OS. Conclusion Allele specific associations of KRAS mutation with clinical outcomes were identified in PDAC. We found better OS with G12R and worse OS with G12D and Q61. Citation Format: Abdelrahman Yousef, Mahmoud Yousef, Saikat Chowdhury, Kawther Abdilleh, Mark Knafl, Paul Edelkamp, Kristin Alfaro-Munoz, Ray Chacko, Jennifer Peterson, Brandon Smaglo, Robert Wolff, Shubham Pant, Michael Lee, Jason Willis, Michael Overman, Sudheer Doss, Lynn Matrisian, Mark Hurd, Rebecca Snyder, Matthew Katz, Huamin Wang, Anirban Maitra, John Paul Shen, Dan Zhao. Impact of KRAS mutations and co-mutations on clinical outcomes in 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 C098.

Abstract B114: Mutational "lineage traces" of an acinar cell-of-origin in human pancreatic ductal adenocarcinoma

Abstract A clinical challenge of pancreatic ductal adenocarcinoma (PDAC) is that most cases are detected late, after intractable metastasis is well underway. This also poses a challenge to our basic understanding of PDAC development, as the early steps of neoplasia are separated by a gulf of years or decades from accessible biospecimens and cell lines. Although mouse genetic models of PDAC have helped to fill in this gap, key questions remain incompletely answered. For example, what is the cell of origin of human PDAC – and does the answer to this question actually matter at a clinical level? In mouse, PDAC can be induced from both acinar and duct cells, with the former apparently more susceptible to transformation while the latter appears harder to transform, but capable of generating more aggressive tumors. Meanwhile, human PDAC has been divided into two major subtypes based on genomic data, classical and basal-like, the latter of which appears to be more aggressive. Is there a relationship between cell-of-origin, tumor subtype and clinical outcomes? This question requires the ability to directly infer cell-of-origin in human tumors, an approach to which we report here. Studies in human non-pancreatic cancers have identified somatic “passenger” (non-selected) mutations in several highly-expressed genes of the normal organ – for example, mutations in the ALB (albumin) gene in hepatocellular carcinoma, or SFTP genes (surfactant proteins) in lung adenocarcinoma. These might indicate a mutational process occurring in normal cells, prior to transformation, and constituting a “lineage mark” for the cell of origin – however, as these genes are also highly-transcribed in the cancer cells themselves, their mutations may arise after tumorigenesis is underway. This problem does not apply to acinar genes and PDAC: we and others have established that an initiating event in Kras-induced transformation of acinar cells is complete downregulation of acinar-specific gene products. Therefore, if a mechanism of transcription-induced mutation applies in the pancreas, we would expect it to leave a trace in acinar-derived PDAC. We have completed a large-scale analysis (n>2,000) of human whole genome sequencing data, across several carcinoma types including pancreas and liver, focused on unbiased detection of tissue-specific mutations similar to those previously found in the ALB and SFTP genes. Remarkably, we find robust enrichment of mutations in acinar-specific genes in PDAC, affecting approximately 25% of tumors. This constitutes the first in vivo genetic evidence for an acinar cell-of-origin in human pancreatic cancer, and opens the door to comparative genomic, transcriptomic and outcomes analyses of acinar-derived tumors. These are underway, and the results will be described in our presentation. Citation Format: Charles Murtaugh. Mutational "lineage traces" of an acinar cell-of-origin in human 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 B114.

Abstract A111: Impact of KRAS mutations and co-mutations on clinical outcomes in Pancreatic Ductal Adenocarcinoma

Abstract KRAS is the most frequently mutated gene in pancreatic ductal adenocarcinoma (PDAC). Over the past decade, targeting KRAS has emerged as a promising approach for PDAC treatment. However, the associations between KRAS mutations status and comutation landscape with clinical outcomes remain understudied. We investigated the impact of KRAS mutant allelic status and genomic co-mutations with clinical outcomes. 803 patients with PDAC who underwent CLIA somatic tumor mutation testing at MDACC from 1997 to 2023 were identified after an IRB approval. Tumor genomic alterations, demographics, clinical, molecular, and pathological data were extracted from electronic medical records and tumor registry through the Palantir Foundry platform. An external cohort from PanCAN’s Know Your Tumor® dataset with 408 patients served as the validation cohort. Overall Survival (OS) was calculated from the date of initial diagnosis to death or last follow-up. Pearson’s chi-squared tests (or Fisher’s exact tests) were used to determine the association between groups. The Kaplan-Meier method was used to estimate the median OS. 703 patients had KRAS gene included in the mutation testing at MDACC from 1997 to 2023. KRAS was the top mutated gene (82%), followed by TP53 (69%), then CDKN2A (20%). In KRAS mutant patients, G12D was the most common variant (39%), followed by G12V (31%), then G12R (14%), and Q61 (6%). In patients tested with multi-gene panels, a co-mutation analysis revealed that the most common co-occurring mutations with KRAS mutation was TP53 (75%), followed by CDKN2A (22%), SMAD4 (14%) and ARID1A (10%). The median follow-up was 41 months and median OS was 19 months in the overall population. KRAS mutation status and subtypes was prognostic of OS (p<0.001). Patients with KRAS wildtype had median OS of 38 months and KRASG12R had median OS of 34 months (HR:1.0, 95% CI 0.71-1.5, p=0.88), while patients with KRASQ61 had worst median OS of 20 months (HR: 1.9, 95% CI 1.2-3.0, p=0.006) and KRASG12D had median OS of 22 months (HR: 1.7, 95% CI 1.3-2.3, p<0.001). Similar findings were observed in patients with stage IV disease (n=302), KRASG12D remained associated with significantly worse OS (HR= 1.7, 95% CI 1.1-2.6, p=0.009). There was a significant enrichment of KRASG12D mutation in metastatic disease compared to localized disease (stage I-III) (OR: 1.7, 95% CI 1.2-2.4, p=0.001) and enrichment of KRASG12R mutation in well and moderately differentiated tumors versus poorly differentiated tumors (OR: 1.7, 95% CI 1.05-2.99, p=0.04). In the external cohort (PanCAN’s Know Your Tumor® dataset), similar findings were observed. KRASG12R was associated with longer median OS (32 months), while KRASQ61 (16 months, HR: 2.6, 95% CI 0.88-7.8, p=0.02) and KRASG12D (23 months, HR: 1.68, 95% CI 1.06-2.65, p=0.04) were associated with significantly worse median OS. Allele specific associations of KRAS mutation with clinical outcomes were identified in PDAC. We found better OS with G12R and worse OS with G12D and Q61. Citation Format: Abdelrahman Yousef, Mahmoud Yousef, Saikat Chowdhury, Kawther Abdilleh, Mark Knafl, Paul Edelkamp, Kristin Alfaro-Munoz, Ray Chacko, Jennifer Peterson, Brandon Smaglo, Robert Wolff, Shubham Pant, Michael Lee, Jason Willis, Michael Overman, Sudheer Doss, Lynn Matrisian, Mark Hurd, Rebecca Snyder, Matthew Harold Katz, Huamin Wang, Anirban Maitra, John Paul Shen, Dan Zhao. Impact of KRAS mutations and co-mutations on clinical outcomes in Pancreatic Ductal Adenocarcinoma [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr A111.

Genetic alterations in the neuronal development genes are associated with changes of the tumor immune microenvironment in pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) has a poor prognosis and is highly metastatic. Our prior studies have demonstrated the critical role of axon guidance pathway genes in PDAC and the connection between neuronal development and the tumor microenvironment. A recent study newly identified 20 neuronal development genes [disks large homolog 2 (DLG2), neuron-glial-related cell adhesion molecule (NRCAM), neurexin3 (NRXN3), mitogen-activated protein kinase 10 (MAPK10), platelet-derived growth factor D (PDGFD), protein kinase C epsilon (PRKCE), potassium calcium-activated channel subfamily M alpha 1 (KCNMA1), polycystic kidney and hepatic disease 1 (PKHD1), neural cell adhesion molecule 1 (NCAM1), neuregulin-1 (NRG1), zinc finger protein 667 (ZNF667), cystic fibrosis transmembrane conductance regulator (CFTR), acyl-CoA medium-chain synthetase-3 (ACSM3), complement 6 (C6), protein tyrosine phosphatase receptor type M (PTPRM), hypoxia-inducible factor 1 alpha (HIF1A), adenylyl cyclase 5 (ADCY5), adherens junctions-associated protein 1 (AJAP1), neurobeachin (NBEA), sodium voltage-gated channel alpha subunit 9 (SCN9A)] that are associated with perineural invasion and poor prognosis of PDAC. The relationship between genetic alterations in these 20 genes and tumor immune microenvironment (TME) has not previously been investigated. We hence applied the sequential multiplex immunohistochemistry results of biopsy specimens from 63 PDAC patients to investigate this relationship. We found that, except for PTPRM and NBEA, genetic alterations involving these 20 genes are associated with significant changes in the densities of major immune cell subtypes. Except for AJAP1, the copy number loss involving this panel of neuronal development genes is significantly associated with changes in immune cell infiltrates. In contrast, the copy number gain in fewer genes, including NRXN3, ZNF667, ACSM3, C6, ADCY5, SCN9A, and PRKCE, is significantly associated with changes in immune cell infiltrates. Our study suggested that neuronal development genes play a role in modulating TME in a pancreatic cancer setting.

Single cell RNA-seq analysis with a systems biology approach to recognize important differentially expressed genes in pancreatic ductal adenocarcinoma compared to adjacent non-cancerous samples by targeting pancreatic endothelial cells

Pancreatic ductal adenocarcinoma (PDAC) is a cancer that is usually diagnosed at late stages. This highly aggressive tumor is resistant to most therapeutic approaches, necessitating identification of differentially expressed genes to design new therapies. Herein, we have analyzed single cell RNA-seq data with a systems biology approach to identify important differentially expressed genes in PDAC samples compared to adjacent non-cancerous samples. Our approach revealed 1462 DEmRNAs, including 1389 downregulated DEmRNAs (like PRSS1 and CLPS) and 73 upregulated DEmRNAs (like HSPA1A and SOCS3), 27 DElncRNAs, including 26 downregulated DElncRNAs (like LINC00472 and SNHG7) and 1 upregulated DElncRNA (SNHG5). We also listed a number of dysregulated signaling pathways, abnormally expressed genes and aberrant cellular functions in PDAC which can be used as possible biomarkers and therapeutic targets in this type of cancer.

The expression characteristics of transmembrane protein genes in pancreatic ductal adenocarcinoma through comprehensive analysis of bulk and single-cell RNA sequence.

Transmembrane (TMEM) protein genes are a class of proteins that spans membranes and function to many physiological processes. However, there is very little known about TMEM gene expression, especially in cancer tissue. Using single-cell and bulk RNA sequence may facilitate the understanding of this poorly characterized protein genes in PDAC. We selected the TMEM family genes through the Human Protein Atlas and characterized their expression by single-cell and bulk transcriptomic datasets. Identification of the key TMEM genes was performed through three machine learning algorithms: LASSO, SVM-RFE and RF-SRC. Then, we established TMEM gene riskscore and estimate its implication in predicting survival and response to systematic therapy. Additionally, we explored the difference and impact of TMEM gene expression in PDAC through immunohistochemistry and cell line research. 5 key TMEM genes (ANO1, TMEM59, TMEM204, TMEM205, TMEM92) were selected based on the single-cell analysis and machine learning survival outcomes. Patients stratified into the high and low-risk groups based on TMEM riskscore, were observed with distinct overall survival in internal and external datasets. Moreover, through bulk RNA-sequence and immunohistochemical staining we verified the protein expression of TMEM genes in PDAC and revealed TMEM92 as an essential regulator of pancreatic cancer cell proliferation, migration, and invasion. Our study on TMEM gene expression and behavior in PDAC has revealed unique characteristics, offering potential for precise therapeutic approaches. Insights into molecular mechanisms expand understanding of PDAC complexity and TMEM gene roles. Such knowledge may inform targeted therapy development, benefiting patients.

Abstract 1328: Building a 3D co-culture model for metastatic pancreatic ductal cancer using Curiochips microphysiological system

Abstract Despite the use of multi-agent conventional chemotherapy regimens, metastatic pancreatic ductal adenocarcinoma (PDAC) remains one of the most lethal solid tumors with a 5-year survival of 9%. PDAC is characterized by a complex tumor microenvironment with multilayered interplay between neoplastic and stromal cells and the natural extracellular matrix (ECM). To date, models that recapitulate the complexity of these features are only partially reproduced ex vivo and lack the capacity to recreate PDAC tumor microenvironment physiology, fluid flow and the ECM architecture. To address these issues, we developed Curiochips MPS platform for metastatic PDAC on-a-chip. To replicate an accurate PDAC complex tumor microenvironment, we investigated human pancreatic cancer cell lines with/without SMAD4 mutation as a test model. SMAD4 is among the most frequently altered genes in PDAC and its mutation accelerates pancreatic cancer cell migration. To tailor cell-ECM interactions and recreate natural biochemical environment of tumors, we examined various ECM conditions. ECM substitutes such as type I collagen and fibrin gel offer mechanical properties to closely mimic the tumor ECM. We accessed the effect of ECM condition on the pancreatic stellate cells (PSC) activation and pancreatic cancer cell migration using PANC-1 (SMAD4-WT) and BxP3 (SMAD4-null) cell lines. Notably, type I collagen induced more PSC activation than fibrin gel. Interestingly BxP3 spheroids migrated toward to the endothelial compartment compared to PANC-1. Angiogenesis of endothelial cells was more prominent in co-culture with BxP3 than PANC-1. Furthermore, we are building the metastatic model using PDAC patient derived organoids and co-culture with primary PSC in different types of ECM. Preliminary analysis showed functional responses such as 3D organoid migration and aggregation, PSC activation and migration controlled by specific properties of EMC. Our data highlight the importance of the ECM composition on PDAC migration, PSC activation and angiogenesis in the context of genetic heterogeneity and presence/absence oncogenic mutation. The 3D co-culture platform, Curiochips offers suitable and tailored solution to investigate tumor-stroma interaction and potentially can be used as a platform for drug discovery and precision oncology to target PDAC desmoplastic tumor microenvironment. Citation Format: Sumin Kim, Yeongmin Choi, Jihye Baek, Bharti Garg, Jan Pencik, Andrew M. Lowy, Herve Tiriac, Sanghee Yoo. Building a 3D co-culture model for metastatic pancreatic ductal cancer using Curiochips microphysiological system [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 1328.

Abstract 3: In vivo CRISPR activation screening identifies tissue-specific oncogene selection

Abstract Background: While the genetic landscapes of pancreatic ductal adenocarcinoma (PDAC) and non-small cell lung cancer (NSCLC) are relatively well described, the contribution of individual genes and transcriptomic programs to progression and metastasis remain incompletely understood. To quantify the tissue-specific selective advantage of transcriptomic drivers of tumor progression and metastasis, we have developed a platform for in vivo CRISPR activation (CRISPRa) oncogene screening. Material and Methods: Our platform consists of an autochthonous CRISPRa-competent mouse tumor model (PPKS; P53(F/F), Kras(LSL-G12D/+), R26(LSL-SAM), coupled with in vivo delivery of Cre/sgRNA lentiviral libraries. To evaluate tissue-specific differences in oncogene selection, we designed a CRISPRa library targeting 450 of the most frequently altered genes across human adenocarcinoma. We next generated lung and pancreas tumors through administration of controlled doses of Cre/sgRNA library in vivo. Tumors and metastatic lesions were isolated and guide enrichment was determined using NGS. We also performed CRISPRa screening on proto-oncogenic (PPKS) pancreatic organoids orthotopically transplanted into syngeneic or immunodeficient mice (NU/J). The results from our CRISPRa screening were confirmed using single-gene activation in vivo and in pancreatic organoids. Results: We found that pancreatic tumors displayed dramatically higher levels of guide selection relative to lung tumors. While, on average, 99.7% of guides were represented in primary lung tumors at survival endpoint, only 40.4% of guides were detected in pancreatic tumors. We identified very strong selection for Egfr-activation in primary lung tumors, followed by MafB, Sox2 and Myc. In contrast, we found near-complete dominance of Myc-activation in pancreatic tumors. Since EGFR and MYC represent two of the most frequently altered genes in NSCLC and PDAC, respectively, this indicates our ability to recapitulate clinically observed oncogene selection. To determine if the selection for Myc-activation in the pancreas was driven by immune response, we performed screening in pancreatic organoids transplanted into immune competent and incompetent mice and found that Myc was uniformly the most highly enriched target. To confirm our findings, we generated Myc-activated proto-oncogenic organoids and autochthonous tumors, and both displayed accelerated tumor formation relative to non-targeted controls. Conclusions: We present one of the first implementations of autochthonous in vivo CRISPR activation screening, allowing for direct observation and quantification of oncogene selection and competition in vivo. Our model faithfully recapitulated oncogene selection observed in patient tumors by identifying EGFR and MYC as dominating oncogenes in the context of KRAS mutant lung and pancreas tumors, respectively. Citation Format: Fredrik Ivar Thege, Sonja M. Woermann, Anirban Maitra. In vivo CRISPR activation screening identifies tissue-specific oncogene selection [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 3.

Increased SPRY1 expression activates NF-κB signaling and promotes pancreatic cancer progression by recruiting neutrophils and macrophages through CXCL12-CXCR4 axis.

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive disease with a high mortality rate, in which about 90% of patients harbor somatic oncogenic point mutations in KRAS. SPRY family genes have been recognized as crucial negative regulators of Ras/Raf/ERK signaling. Here, we investigate the expression and role of SPRY proteins in PDAC. Expression of SPRY genes in human and mice PDAC was analyzed using The Cancer Genome Atlas and Gene Expression Omnibus datasets, and by immunohistochemistry analysis. Gain-of-function, loss-of-function of Spry1 and orthotopic xenograft model were adopted to investigate the function of Spry1 in mice PDAC. Bioinformatics analysis, transwell and flowcytometry analysis were used to identify the effects of SPRY1 on immune cells. Co-immunoprecipitation and K-ras4B G12V overexpression were used to identify molecular mechanism. SPRY1 expression was remarkably increased in PDAC tissues and positively associated with poor prognosis of PDAC patients. SPRY1 knockdown suppressed tumor growth in mice. SPRY1 was found to promote CXCL12 expression and facilitate neutrophil and macrophage infiltration via CXCL12-CXCR4 axis. Pharmacological inhibition of CXCL12-CXCR4 largely abrogated the oncogenic functions of SPRY1 by suppressing neutrophil and macrophage infiltration. Mechanistically, SPRY1 interacted with ubiquitin carboxy-terminal hydrolase L1 to induce activation of nuclear factor κB signaling and ultimately increase CXCL12 expression. Moreover, SPRY1 transcription was dependent on KRAS mutation and was mediated by MAPK-ERK signaling. High expression of SPRY1 can function as an oncogene in PDAC by promoting cancer-associated inflammation. Targeting SPRY1 might be an important approach for designing new strategy of tumor therapy.

Germline mutations in homologous recombination repair genes among Chinese pancreatic ductal adenocarcinoma patients detected using next-generation sequencing.

Genetic testing for pancreatic ductal adenocarcinoma (PDAC) patients is in constant development. However, the status of homologous recombination repair (HRR) genes in unselected Chinese PDAC has not been fully explored. This study aims to characterize the profile of germline mutations in HRR genes in Chinese PDAC patients. A cohort of 256 PDAC patients were enrolled at Zhongshan Hospital Fudan University between 2019 and 2021. Germline DNA was analyzed by next-generation sequencing using a multigene panel of the 21 HRR genes. The germline pathogenic (P)/likely pathogenic (LP) variant rates were 7.0% (18/256) in unselected patients with pancreatic cancer. Among them, 1.6% (4/256) were identified as harboring BRCA2 variants, and 5.5% (14/256) patients carried non-BRCA variants. Variants were detected in eight non-BRCA genes, including ATM (4, 1.6%), PALB2 (4, 1.6%), ATR (1, 0.4%), BRIP1 (1, 0.4%), CHEK2 (1, 0.4%), MRE11 (1, 0.4%), PTEN (1, 0.4%), and STK11 (1, 0.4%). ATM, BRCA2, and PALB2 were the most prevalent variant genes. If only BRCA1/2 was tested, 5.5% of P/LP variants would have been lost. Further, we found that the landscape of P/LP HRR variants in various population cohorts was quite different. However, no significant difference was found in clinical characteristics between germline HRR P/LP carriers and non-carriers. In our study, one case carrying a germline PALB2 variant showed a long-time response to platinum-based chemotherapy and PARP inhibitor. This study comprehensively depicts the prevalence and characteristics of germline HRR mutations in unselected Chinese PDAC patients. Our findings show the clinical utility of a multigene panel may increase the detection of P/LP HRR carriers.