Aggressive Pancreatic Ductal Adenocarcinoma Research Articles

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 1458: Anti tumoral role of PDX1 on pancreatic ductal adenocarcinoma aggressiveness

Abstract Pancreatic ductal adenocarcinoma (PDAC) represents a very aggressive type of pancreatic cancer (9% survival rate at 5 years). PDX1 is an important transcription factor for embryonic development of the pancreas, endocrine lineage differentiation and the maintenance of mature beta cells. Notably, in PDAC patients, the expression of PDX1 is downregulated in tumor cells compared to adjacent non-tumoral tissue. Interestingly, increasing tumor PDX1 expression has been shown to enhance the overall survival rate in PDAC patients. Therefore, our aim was to analyze the role of PDX1 on tumor aggressiveness of PDAC cells. To induce PDX1 expression, PANC1 cells were treated with BRD7552 (a PDX1 inducer) for 9 days. Overexpression of PDX1 was confirmed by Western Blot analysis and no cytotoxic effects were observed by MTT or Trypan Blue exclusion assays on treated cells compared to control. Wound healing and transwell migration assays showed a significant reduction in the migration rate compared to the control group. A higher proportion of cells in G1 phase was observed in treated cells compared to control by propidium iodide staining followed by flow cytometry assay. Furthermore, the cell confluence assay showed a significant reduction in proliferation rate in treated cells compared to control, but no differences were observed on the proportion of KI67+ and PH3+ cells by immunostaining. To assess the in ovo effects of BRD7552, treated PANC1 cells were implanted onto the chorioallantoic membrane of chick embryos and tumor growth was measured at different stages observing a significant reduction in tumor growth in PANC1 cells compared to control between days 3 and 8 post implantation. No significant differences in morphology or color were observed between treated and control implanted cells. Hematoxylin-eosin staining showed reduced invasion of treated cells into the chorioallantoic membrane compared to control. In conclusion, the overexpression of PDX1 affects cell cycle, reduces proliferation rate and inhibits migratory potential in vitro. Moreover, in an in ovo model this overexpression diminished tumor growth and invasion in PANC1 cells. In conclusion, the overexpression of PDX1 induced by BRD7552 drives pancreatic ductal adenocarcinoma to a less aggressive tumoral phenotype. Citation Format: Jimena Mosna, Federico Garde, Marcelo Stinson, Candela Pastore, Pablo Sanchis, Abel Carcagno. Anti tumoral role of PDX1 on pancreatic ductal adenocarcinoma aggressiveness [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 1458.

Abstract 6927: A refined view of pancreatic cancer genomic evolution through single-nucleus DNA sequencing

Abstract Numerous next-generation sequencing studies have provided an overview pancreatic ductal adenocarcinoma (PDAC)’s genomic evolution. But how its genome evolves through treatment and metastasis has not been extensively studied due to lack of samples and technical limitation of bulk sequencing on low-tumor purity samples. Herein we applied targeted single-nucleus DNA sequencing (snDNA-seq) to a cohort of 18 patients, each with >= 2 multiregional/longitudinal samples, totaling 65 samples. These included 5 early-stage resections, 11 stage IV autopsies and 2 biopsy-derived organoids taken pre- and post-treatment. We also developed a set of computational methods for this new datatype to delineate genomic evolution at high resolution. Despite higher sensitivity than bulk, snDNA-seq did not uncover significant subclonal single-nucleotide variations (SNVs) on our panel targeting 253 select genes for PDAC. But it detected a higher frequency of CDKN2A (72% against 30%) and SMAD4 (56% against 32%) alterations, mostly attributed to chromosomal deletions. Many were subclonal or only affected a few hundred base-pairs of the gene, making them elusive to bulk sequencing. Except for one case driven by BRCA2 mutations rather than the canonical KRAS oncogene, other patients had mostly linear phylogenies with more SNVs occurring before the most recent common ancestor (MRCA) of clones, for early/late-stage and longitudinal cases alike. This early fixation of drivers and rapid clonal sweep afterwards likely correlates with the particular aggressiveness of PDAC. Metastatic clones almost always fell towards the end of phylogenies and were shared among spatially separated sites, indicating late metastatic dissemination in molecular evolution time. Metastases to the liver and diaphragm appeared to be more genetically evolved than other sites, likely due to harsher selective pressure cast by the metastatic routes/distal microenvironments. 16 of 18 PDACs were observed to converge towards tumor cell-intrinsic TGF-β unresponsiveness by mutating various components of the pathway. Such convergent evolution usually happened at the primary site, indicating a strong selective advantage of the phenotype in the desmoplastic, nutrient-poor pancreas microenvironment. Continuous evolution was seen through treatment and metastasis, driven by seemingly random genome-scale copy number variations (CNVs) and focal amplification/deletions to genes such as KRAS, CDKN2A, SMAD4, MYC. These insights on PDAC’s genomic evolution inform more precision medicine efforts to come. Early fixation of driver SNVs sculpts a largely homogeneous disease that could be uniformly targeted. But special care should be taken to obviate resistance mechanisms conferred by continuously evolving CNV events- compared to SNVs, these mechanisms provide faster remodeling of the genome, and thus faster generation of new phenotypes and adaptation. Citation Format: Haochen Zhang, Palash Sashittal, Elias-Ramzey Karnoub, Akhil Jakatdar, Shigeaki Umeda, Nicolas Lecomte, Jungeui Hong, Katelyn Mullen, Akimasa Hayashi, Caitlin A. McIntyre, Benjamin J. Raphael, Christine A. Iacobuzio-Donahue. A refined view of pancreatic cancer genomic evolution through single-nucleus DNA sequencing [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 6927.

Abstract 6167: Spatial transcriptomic analysis identifies pancreatic cancer cell surface markers for imaging and therapy

Abstract Pancreatic cysts and precancer lesions have been reported in up to 50% of adults; however, few of the precancer lesions develop into aggressive pancreatic ductal adenocarcinoma (PDAC). Strategies for the differentiation of precancers and PDAC are needed. Recently proposed molecular imaging targets to accomplish this goal include integrin αvβ6 and fibroblast activation protein (FAP). To discover and quantitatively evaluate potential imaging and therapeutic targets, we performed Visium spatial transcriptomic (ST) sequencing on human surgical sections of normal tissue adjacent to precancers, intraductal papillary mucinous neoplasms and PDAC. All human tissues were collected in accordance with the Stanford Institutional Review Board. The resulting data were correlated with immunohistochemistry (IHC) and combined with analysis with publicly available spatial and single cell PDAC datasets to create a quantitative PDAC cancer surfaceome score. Pseudotime and receiver operating characteristic (ROC) analyses were also performed. We found that clusters with high cancer surfacesome scores were spatially correlated with a cellular morphology consistent with cancer. In the cancer cluster, the expression of surface markers CLDN4, GPRC5A, TSPAN8, and CEACAM5 was enhanced more than 13-fold compared with the normal pancreas, while FAP and ITGB6 were differentially expressed by less than 4-fold. Within cancer clusters, the fraction of spots overexpressing CLDN4, S100P, ITGB6, MUC5AC, TFF1, CEACAM5, and FAP was 88%, 87%, 78%, 66%, 59%, 47%, and 38%, respectively. The Pearson’s correlation across space and expression intensity between CLDN4, S100P, GPRC5A, TSPAN8, MUC1 and TFF1 was greater than 0.6 for all combinations. The correlation of mRNA encoding ITGB6 and FAP with cancer surfaceome markers was smaller, e.g. 0.37 and 0.01 versus S100P, respectively. The area under the curve (AUC) for the differentiation of PDAC from normal or precancerous tissue for CLDN4, S100P, MUC5AC, TFF1, CEACAM5, and TSPAN8 was 0.9, 0.5, 0.3, 0.5, 0.8, and 0.4, respectively. From pseudotime trajectory analysis, we found that MUC5AC and TFF1 increased at a precancerous pseudotime whereas CLDN4 increased in time and space in spots corresponding to a PDAC-specific surfaceome score. This work demonstrates the feasibility of using spatial transcriptomics and IHC in human pancreatic cancer samples to discover and compare the expression of cell surface markers that can be employed in imaging and therapeutic protocols. Citation Format: James Wang, Aris J. Kare, Martn K. Schneider, Jai Woong Seo, Andrei Iagaru, Gregory W. Charville, Walter G. Park, Katherine W. Ferrara. Spatial transcriptomic analysis identifies pancreatic cancer cell surface markers for imaging and therapy [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 6167.

Abstract 3939: Ror2, a novel key regulator driving cell fate decisions throughout pancreatic tumor progression

Abstract Introduction: Reprogramming of pancreas cell fate drives development of pancreatic ductal adenocarcinoma (PDAC). Acinar cells, the most probable origin of pancreatic cancer, undergo a rapid cell identity switch towards a duct-like phenotype upon KrasG12D expression and when combined with pancreatitis or the loss of acinar differentiation factors. Metaplastic and dysplastic duct-like cells are heterogeneous with a proportion acquiring features reminiscent of gastric lineages. While some gastric signatures are maintained in the classical PDAC subtype, they are eroded in the more aggressive, basal-like PDAC. Since subtype identity has a major impact on prognosis and therapeutic targetability, druggable targets that regulate cellular reprogramming in pancreatic cancer can be exploited to increase sensitivity to therapy. Methods: To elucidate mechanisms responsible for early reprogramming, pancreatic tissue of mice with conditional KrasG12D expression and loss of Pdx1 was analyzed by single-nucleus ATAC-seq. Expression of identified target genes was studied in precancerous lesions and PDAC by using multiplex RNAscope and IHC staining. Computational analyses of publicly available sequencing data were used to establish correlation to PDAC subtype identity. Genes were functionally studied in PDAC cell lines. Results: By performing snATAC-seq and RNA-seq of early transformed pancreatic tissue, we discovered that acinar cells with the combined expression of KrasG12D and loss of Pdx1 activate expression of a gastric metaplastic gene signature accompanied by elevated levels of the receptor kinase Ror2. Ror2 is also highly expressed in distinct subpopulations of metaplastic and dysplastic cells, associated with a gastric neck cell phenotype and enhanced proliferative capacity. In contrast, Ror2Low lesions are characterized by a gastric pit cell-like and a senescent phenotype. Genetic ablation of Ror2 resulted in a shift in lesion identity, enriching those with a pit cell and senescent identity. In PDAC, we found that Ror2 anti-correlates with a similar gastric pit cell phenotype that is maintained in the classical subtype PDAC but is strongly associated with the more aggressive basal-like subtype. Overexpression of ROR2 in human PDAC cell lines with a classical differentiation induced loss of the classical gene signature as well as epithelial-to-mesenchymal transition. Moreover, ROR2 enforces a strong dependency on AKT signaling, causing increased vulnerability of ROR2-expressing cells to AKT inhibition, but increased resistance to the KRAS inhibitor MRTX1133. Conclusions: We discovered Ror2 as a critical determinant of precancerous lesion as well as PDAC subtype identity. Its role in driving an aggressive PDAC phenotype that is inherently resistant to Kras inhibition suggests that inhibiting this receptor tyrosine kinase will enhance sensitivity to the new generation of targeted therapies. Citation Format: Simone Benitz, Alexander Steep, Malak Nasser, Jonathan Preall, Ujjwal Mahajan, Ian Loveless, Holly McQuithey, Erick Davis, Hui-Ju Wen, Daniel Long, Thomas Metzler, Samuel Zwernik, Daniel Salas-Escabillas, Ling Huang, Nina Steele, Ivonne Regel, Filip Bednar, Howard Crawford. Ror2, a novel key regulator driving cell fate decisions throughout pancreatic tumor progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3939.

Abstract 6818: Obesity-mediated perineural invasion promotes PDAC tumorigenesis

Abstract Obesity is a modifiable host factor that has been reported to increase the risk of pancreatic ductal adenocarcinoma (PDAC) by ~60% and the mortality by two-fold, however, the exact mechanisms by which obesity enhances PDAC aggressiveness is incompletely understood. Given that nearly 74% of U.S. adults are overweight or obese (US Center for Disease Control), resolving the complex association between obesity and PDAC may unveil new targets for prevention and/or intervention to decrease incidence and improve outcomes. The prevailing notion is that obesity-induced tumor in situ factors (i.e., oxidative, hypoxic, nutrient, and biochemical) play crucial roles in enhancing cancer cell fitness and survival. However, these concepts have yet to lead to the identification of any actionable target in patients, with obesity-related cancer in general and PDAC in particular. Prior studies and our preliminary data show the presence of a distinctive tumor immune profile in PDAC models under conditions of obesity, which encompasses immune suppressive myeloid and type 2 immune cells, namely M2-like tumor-associated macrophages (TAMs), and myeloid-derived suppressor cells (MDSCs), that inhibit effector CD8+ T cell expansion and function enabling PDAC progression. However, the genesis of this ‘type 2 immune cell’ dominant tumor-immune microenvironment (TIME) of obesity-driven PDAC remains to be fully understood. This led us to take a holistic approach utilizing multiple genetically engineered mouse models (GEMMs), pharmacological inhibitors, transcriptomic analysis (RNA-seq, spatial transcriptomic, and scRNA-seq), fresh PDAC patient samples, and imaging technologies (confocal microscopy) to understand the molecular mechanisms. Our novel mechanistic study using some of the above-mentioned tools shows that obesity-driven PDAC has significantly higher perineural invasion (PNI) that enhances PDAC progression. We found that sympathetic nerve terminals (of the PNI) via neurotransmitter release control the expression of integral cytokines that cause a dramatic remodeling of the TIME. Specifically, the PNI-mediated neuro-adrenergic signaling activates β2-adrenergic receptor (β2-AR) on PDAC cells, leading to increased tumor cell production/proliferation and secretion of the ‘alarmins’ IL-33 and IL-1β. Importantly, we showed that mitigation of adrenergic signaling using two separate approaches: (i) pharmacological antagonists of β2-ARs (β-blockers), and (ii) the use of a thermoneutral model leads to substantial abrogation of obesity-mediated tumor progression. These, and other key data raise the provocative question that reducing adrenergic signaling-dependent IL-33 expression and release favor a pro-inflammatory TIME and significantly increase patient survival in obesity-driven PDAC. Citation Format: Shyamananda Singh Mayengbam, Aftab Alam, Sharon Senchanthisai, Irmak Bektas, Prasenjit Dey. Obesity-mediated perineural invasion promotes PDAC tumorigenesis [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 6818.

Connectivity mapping-based identification of pharmacological inhibitor targeting HDAC6 in aggressive pancreatic ductal adenocarcinoma

Pancreatic ductal adenocarcinoma (PDAC) remains highly lethal due to limited therapeutic options and expensive/burdensome drug discovery processes. Utilizing genomic-data-driven Connectivity Mapping (CMAP) to identify a drug closer to real-world PC targeting may improve pancreatic cancer (PC) patient outcomes. Initially, we mapped CMAP data to gene expression from 106 PC patients, identifying nine negatively connected drugs. These drugs were further narrowed down using a similar analysis for PC cell lines, human tumoroids, and patient-derived xenografts datasets, where ISOX emerged as the most potent agent to target PC. We used human and mouse syngeneic PC cells, human and mouse tumoroids, and in vivo mice to assess the ability of ISOX alone and in combination with 5FU to inhibit tumor growth. Global transcriptomic and pathway analysis of the ISOX-LINCS signature identified HDAC 6/cMyc as the target axis for ISOX. Specifically, we discovered that genetic and pharmacological targeting of HDAC 6 affected non-histone protein cMyc acetylation, leading to cMyc instability, thereby disrupting PC growth and metastasis by affecting cancer stemness. Finally, KrasG12D harboring tumoroids and mice responded effectively against ISOX and 5FU treatment by enhancing survival and controlling metastasis incidence. Overall, our data validate ISOX as a new drug to treat advanced PC patients without toxicity to normal cells. Our study supports the clinical utility of ISOX along with 5FU in future PC clinical trials.

Limited Role of the Apparent Diffusion Coefficient (ADC) for Tumor Grade and Overall Survival in Resectable Pancreatic Ductal Adenocarcinoma.

This study evaluated the relationship between apparent diffusion coefficient (ADC) values in pancreatic ductal adenocarcinoma (PDAC) and tumor grades based on WHO, Adsay, and Kalimuthu classifications, using whole-mount pancreatectomy specimens. If glandular formation plays a key role in the degree of diffusion restriction, diffusion-weighted imaging could facilitate non-invasive grading of PDAC. A freehand region of interest (ROI) was drawn along tumor borders on the preoperative ADC map in each tumor-containing slice. Resection specimens were retrospectively graded according to WHO, Adsay, and Kalimuthu classifications and correlated with overall survival and the 10th percentile of whole-volume ADC values. Findings from 40 patients (23 male, median age 67) showed no correlation between ADC p10 values and WHO differentiation (p = 0.050), Adsay grade (p = 0.955), or Kalimuthu patterns (p = 0.117). There was no association between ADC p10 and overall survival (p = 0.082) and other clinicopathological variables. Survival was significantly lower for poor tumor differentiation (p = 0.046) and non-glandular Kalimuthu patterns (p = 0.016) and there was a trend towards inferior survival for Adsay G3 (p = 0.090) after correction for age, tumor location, and stage. Preoperative ADC measurements for determining PDAC aggressiveness had limited clinical utility, as there was no correlation with histological parameters or overall survival in resectable PDAC.

Nutrient scavenging-fueled growth in pancreatic cancer depends on caveolae-mediated endocytosis under nutrient-deprived conditions.

Pancreatic ductal adenocarcinoma (PDAC) is characterized by its nutrient-scavenging ability, crucial for tumor progression. Here, we investigated the roles of caveolae-mediated endocytosis (CME) in PDAC progression. Analysis of patient data across diverse datasets revealed a strong association of high caveolin-1 (Cav-1) expression with higher histologic grade, the most aggressive PDAC molecular subtypes, and worse clinical outcomes. Cav-1 loss markedly promoted longer overall and tumor-free survival in a genetically engineered mouse model. Cav-1-deficient tumor cell lines exhibited significantly reduced proliferation, particularly under low nutrient conditions. Supplementing cells with albumin rescued the growth of Cav-1-proficient PDAC cells, but not in Cav-1-deficient PDAC cells under low glutamine conditions. In addition, Cav-1 depletion led to significant metabolic defects, including decreased glycolytic and mitochondrial metabolism, and downstream protein translation signaling pathways. These findings highlight the crucial role of Cav-1 and CME in fueling pancreatic tumorigenesis, sustaining tumor growth, and promoting survival through nutrient scavenging.

Nutrient restriction-activated Fra-2 promotes tumor progression via IGF1R in miR-15a downmodulated pancreatic ductal adenocarcinoma

Pancreatic ductal adenocarcinoma (PDAC) is a lethal disease, characterized by an intense desmoplastic reaction that compresses blood vessels and limits nutrient supplies. PDAC aggressiveness largely relies on its extraordinary capability to thrive and progress in a challenging tumor microenvironment. Dysregulation of the onco-suppressor miR-15a has been extensively documented in PDAC. Here, we identified the transcription factor Fos-related antigen-2 (Fra-2) as a miR-15a target mediating the adaptive mechanism of PDAC to nutrient deprivation. We report that the IGF1 signaling pathway was enhanced in nutrient deprived PDAC cells and that Fra-2 and IGF1R were significantly overexpressed in miR-15a downmodulated PDAC patients. Mechanistically, we discovered that miR-15a repressed IGF1R expression via Fra-2 targeting. In miR-15a-low context, IGF1R hyperactivated mTOR, modulated the autophagic flux and sustained PDAC growth in nutrient deprivation. In a genetic mouse model, Mir15aKO PDAC showed Fra-2 and Igf1r upregulation and mTOR activation in response to diet restriction. Consistently, nutrient restriction improved the efficacy of IGF1R inhibition in a Fra-2 dependent manner. Overall, our results point to a crucial role of Fra-2 in the cellular stress response due to nutrient restriction typical of pancreatic cancer and support IGF1R as a promising and vulnerable target in miR-15a downmodulated PDAC.

Innate and adaptive immune-directed tumour microenvironment in pancreatic ductal adenocarcinoma.

One of the most deadly and aggressive cancers in the world, pancreatic ductal adenocarcinoma (PDAC), typically manifests at an advanced stage. PDAC is becoming more common, and by the year 2030, it is expected to overtake lung cancer as the second greatest cause of cancer-related death. The poor prognosis can be attributed to a number of factors, including difficulties in early identification, a poor probability of curative radical resection, limited response to chemotherapy and radiotherapy, and its immunotherapy resistance. Furthermore, an extensive desmoplastic stroma that surrounds PDAC forms a mechanical barrier that prevents vascularization and promotes poor immune cell penetration. Phenotypic heterogeneity, drug resistance, and immunosuppressive tumor microenvironment are the main causes of PDAC aggressiveness. There is a complex and dynamic interaction between tumor cells in PDAC with stromal cells within the tumour immune microenvironment. The immune suppressive microenvironment that promotes PDAC aggressiveness is contributed by a range of cellular and humoral factors, which itself are modulated by the cancer. In this review, we describe the role of innate and adaptive immune cells, complex tumor microenvironment in PDAC, humoral factors, innate immune-mediated therapeutic advances, and recent clinical trials in PDAC.

Abstract C090: Elucidation of misp53-driven subtype specification and functions in pancreatic cancer

Abstract This study examines how mutations in the TP53 gene, specifically gain-of-function (GOF) mutations, impact the subtyping and stratification of pancreatic ductal adenocarcinoma (PDAC). In addition, it investigates the underlying mechanisms as well as the potential therapeutic implications.TP53 mutations/deletions are present in 60-75 % of cases in PDAC. The most common mutations are three specific hotspot missense mutations (misp53) found at codons R273H, R248W, and R175H. Several studies indicate that these misp53 mutants, despite losing their tumor suppressive function, acquire new oncogenic effects promoting disease aggressiveness and resistance to conventional therapies. Recent findings show that misp53 mutants are associated with a poorly differentiated metastatic subtype in PDAC. However, it is unclear how distinct misp53 mutants, such as misp53R273H, affect PDAC progression and therapy resistance and whether different mutants exert distinct functions. In this study, we explore misp53R273H- and misp53R248W-driven subtype-specificity and functions in PDAC aggressiveness by exploiting transcriptomics, genomic binding and regulation, controlled functional pathways and potential therapeutic targets. By combining cell line-based RNA- and ChIP-seq data we report that misp53R273H associates with and fuels basal-like (BL) neoplastic features by a potential regulation of cell cycle progression. Preclinical mouse models underpinned these findings by connecting misp53R273H tumors to metastatic spread. Generation of constitutive knockdown cell lines and corresponding overexpression assays could support these findings by bridging a loss of misp53R273H to the more favorable classical subtype properties. Of note, co-immunoprecipitation coupled to mass spectrometry analysis revealed misp53-specific and common hotspot mutant interaction partners: for instance, proteins of the group of chromatin remodelers or the ubiquitination pathway that could serve as a mechanistic foundation for misp53-specific functional outcomes. Further, implementation of a large-scale drug screen, flow cytometry and protein expression analyses revealed insights in the potential misp53-specific regulated pathways and therapeutic vulnerabilities, which strongly correlate to cell cycle progression and thus to the previous results. Integrating these findings with preclinical mouse studies, patient-derived xenograft cell lines and publicly available patient data sets strengthens the reported results and highlights their translational relevance. In conclusion, this study clearly demonstrates that hotspot misp53 mutants play a critical role in influencing PDAC subtype-specification and disease aggressiveness. In particular, misp53R273H was associated with BL aggressiveness and cell cycle progression. The ultimate aim of the study is to identify misp53 mutant-specific patient stratification to broaden individualized therapy options. Citation Format: Laura Urbach, Lena Wieland, Frederike Penz, Lukas Klein, Jennifer Appelhans, Christof Lenz, Carolin Schneider, Schneider Günter, Ramona Schulz-Heddergott, Volker Ellenrieder, Elisabeth Heßmann, Shiv K. Singh. Elucidation of misp53-driven subtype specification and functions in pancreatic cancer [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 C090.

Abstract C114: Engrailed-1 promotes pancreatic cancer metastasis

Abstract Engrailed-1 (EN1) is a critical homeodomain transcription factor (TF) required for neuronal survival, and EN1 expression has been shown to promote aggressive forms of triple negative breast cancer. Here, we report that EN1 is aberrantly expressed in a subset of pancreatic ductal adenocarcinoma (PDA) patients with poor outcomes. EN1 predominantly repressed its target genes through direct binding to gene enhancers and promoters, implicating a role in the acquisition of mesenchymal cell properties. Gain- and loss-of-function experiments demonstrated that EN1 promoted PDA transformation and metastasis in vitro and in vivo. Our findings nominate the targeting of EN1 and downstream pathways in aggressive PDA. Citation Format: Jihao Xu, Jae-Seok Roe, EunJung Lee, Claudia Tonelli, Tim Somervile, Melissa Yao, Joseph Milazzo, Herve Tiriac, Anna Kolarzyk, Esak Lee, Jeam Grem, Audrey Lazenby, James Grunkemeyer, Michael Hollingsworth, Paul Grandgenett, Alexander Borowsky, Youngkyu Park, Christopher Vakoc, David Tuveson, Chang-il Hwang. Engrailed-1 promotes pancreatic cancer metastasis [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 C114.

Abstract B085: K17-induced pyrimidine biosynthesis drives chemoresistance in PDAC

Abstract Background: The 5-year survival rate of pancreatic cancer is extremely low compared with other cancers, although it has increased to 12% in 2023 in the United States. 80% of pancreatic ductal adenocarcinoma (PDAC) patients are not eligible for surgical procedure, and unfortunately, PDAC is refractory to standard-of-care chemotherapy. Thus, developing novel and more effective treatment strategies for PDAC is urgently needed. We previously reported keratin 17 (K17) as a novel negative prognostic and predictive biomarker, whose overexpression confers the resistance to chemotherapies. Here, we investigated the mechanisms of chemoresistance and tumor-specific vulnerabilities that can be exploited for K17-mediated targeted therapies. Methods: Survival analyses, immunohistochemistry (IHC), and matrix assisted laser desorption/ionization mass spectrometry (MALDI MSI) metabolite imaging were applied in patient derived samples. The K17 stable expression cell line models (KPC and PANC-1) and K17 loss-of-function cell line model (L3.6) were generated, where the analyses were applied including western blots, qRT-PCR, mass spectrometric metabolomics analysis, immunofluorescence imaging, cell viability and proliferation assays. KPC cells with or without K17 expression were orthotopically implanted into the head of the pancreas of c57B6J mice for tumor growth and treatment study. Results: Unbiased metabolomic studies in isogenic PDAC models identified several key metabolic pathways that are upregulated in the presence of K17. We demonstrate that K17 increases pyrimidine biosynthesis, a pathway that drives chemoresistance. Patient dataset analysis revealed that K17 expression and enzymes involved in pyrimidine, but not purine, de novo biosynthesis is associated with shorter patient survival. Furthermore, K17-expressing cells were more sensitive to Brequinar, a specific inhibitor of dihydroorotate dehydrogenase (DHODH), the rate-limiting enzyme in de novo pyrimidine biosynthetic pathway. Targeting DHODH by small interfering RNA or by Brequinar with Gemcitabine synergistically inhibited the viability of K17- positive cells. Importantly, the combination of Gemcitabine and Brequinar significantly inhibited the growth of K17-expressing tumors and extended survival of mice bearing K17-positive tumors. Mechanistically, K17 increased mitochondrial DHODH translocation which made these cells more sensitive to Brequinar. We also found that the coil 2 domain of K17 was crucial for triggering metabolic reprogramming and promoting tumor aggression. Conclusions: Overall, we define a novel connection between K17, nucleotide metabolism, and chemoresistance in PDAC. We report for the first time that K17, a negative biomarker of the most aggressive PDAC subtype, drives chemoresistance by up-regulating de novo pyrimidine biosynthesis, which provides a tumor-specific vulnerability: DHODH. Further study could lead to the development of a DHODH targeted therapy for K17-expressing PDAC. Citation Format: Yinghuan Lyu, Kenneth Shroyer, Chun-Hao Pan, Robert Tseng, Md Afjalus Siraj, Girish Halemirle Rajacharya, Bo Chen, Katie L Donnelly, Michael Horowitz, Carlos Mauricio Mejia Arbelaez, Sumedha Chowdhury, Lyanne Oblein, Natalia Marchenko, Luisa Escobar-Hoyos, Pankaj K. Singh. K17-induced pyrimidine biosynthesis drives chemoresistance in PDAC [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 B085.

Engrailed-1 Promotes Pancreatic Cancer Metastasis.

Engrailed-1 (EN1) is a critical homeodomain transcription factor (TF) required for neuronal survival, and EN1 expression has been shown to promote aggressive forms of triple negative breast cancer. Here, it is reported that EN1 is aberrantly expressed in a subset of pancreatic ductal adenocarcinoma (PDA) patients with poor outcomes. EN1 predominantly repressed its target genes through direct binding to gene enhancers and promoters, implicating roles in the activation of MAPK pathways and the acquisition of mesenchymal cell properties. Gain- and loss-of-function experiments demonstrated that EN1 promoted PDA transformation and metastasis in vitro and in vivo. The findings nominate the targeting of EN1 and downstream pathways in aggressive PDA.

TRPV6 Channel Is Involved in Pancreatic Ductal Adenocarcinoma Aggressiveness and Resistance to Chemotherapeutics.

Pancreatic ductal adenocarcinoma (PDAC) stands as a highly aggressive and lethal cancer, characterized by a grim prognosis and scarce treatment alternatives. Within this context, TRPV6, a calcium-permeable channel, emerges as a noteworthy candidate due to its overexpression in various cancers, capable of influencing the cell behavior in different cancer entities. Nonetheless, the exact expression pattern and functional significance of TRPV6 in the context of PDAC remains enigmatic. This study scrutinizes the expression of TRPV6 in tissue specimens obtained from 46 PDAC patients across distinct stages and grades. We manipulated TRPV6 expression (knockdown, overexpression) in the human PDAC cell lines Panc-1 and Capan-1. Subsequently, we analyzed its impact on multiple facets, encompassing Ca2+ influx, proliferation, apoptosis, migration, chemoresistance, and tumor growth, both in vitro and in vivo. Notably, the data indicate a direct correlation between TRPV6 expression levels, tumor stage, and grade, establishing a link between TRPV6 and PDAC proliferation in tissue samples. Decreasing TRPV6 expression via knockdown hampered Ca2+ influx, resulting in diminished proliferation and viability in both cell lines, and cell cycle progression in Panc-1. The knockdown simultaneously led to an increase in apoptotic rates and increased the susceptibility of cells to 5-FU and gemcitabine treatments. Moreover, it accelerated migration and promoted collective movement among Panc-1 cells. Conversely, TRPV6 overexpression yielded opposing outcomes in terms of proliferation in Panc-1 and Capan-1, and the migration of Panc-1 cells. Intriguingly, both TRPV6 knockdown and overexpression diminished the process of tumor formation in vivo. This intricate interplay suggests that PDAC aggressiveness relies on a fine-tuned TRPV6 expression, raising its profile as a putative therapeutic target.

ZKSCAN5 activates LAPTM5 expression by recruiting SETD7 to promote metastasis in pancreatic ductal adenocarcinoma.

Lysosomal-associated transmembrane protein 5 (LAPTM5) has been associated with poor prognosis in cancer patients. Its role in regulating metastasis in pancreatic ductal adenocarcinoma (PDAC), however, remains vague. The study here aimed to expound the metastasis-promoting properties of LAPTM5 in PDAC and the detailed mechanism. LAPTM5 was overexpressed in metastatic PDAC cells and was related to the dismal prognosis of patients in GEO datasets. By using lentiviral vectors harboring short hairpin RNA, we found that LAPTM5 downregulation reduced PDAC cell viability, proliferation, and aggressiveness in vitro and liver metastasis in vivo. Zinc finger with KRAB and SCAN domains 5 (ZKSCAN5) was predicted and verified to mediate LAPTM5 transcription in PDAC cells. Both ZKSCAN5 and SET domains, containing lysine methyltransferase 7 (SETD7) bound to the LAPTM5 promoter, and ZKSCAN5 recruited SETD7 to form a complex promoting LAPTM5 transcription. LAPTM5 knockdown reversed the promoting effect of ZKSCAN5 on the metastasis of PDAC cells. Thus, our findings on the ZKSCAN5/SETD7/LAPTM5 axis provide insights into the underlying mechanism of liver metastasis dissemination in PDAC.

Incorporation of human iPSC-derived stromal cells creates a pancreatic cancer organoid with heterogeneous cancer-associated fibroblasts

The aggressiveness of pancreatic ductal adenocarcinoma (PDAC) is affected by the tumor microenvironment (TME). In this study, to recapitulate the PDAC TME exvivo, we cocultured patient-derived PDAC cells with mesenchymal and vascular endothelial cells derived from human induced pluripotent stem cells (hiPSCs) to create a fused pancreatic cancer organoid (FPCO) in an air-liquid interface. FPCOs were further induced to resemble two distinct aspects of PDAC tissue. Quiescent FPCOs were drug resistant, likely because the TME consisted of abundant extracellular matrix proteins that were secreted from the various types of cancer-associated fibroblasts (CAFs) derived from hiPSCs. Proliferative FPCOs could re-proliferate after anticancer drug treatment, suggesting that this type of FPCO would be useful for studying PDAC recurrence. Thus, we generated PDAC organoids that recapitulate the heterogeneity of PDAC tissue and are a potential platform for screening anticancer drugs.

Clinically impactful metabolic subtypes of pancreatic ductal adenocarcinoma (PDAC).

Background: Pancreatic ductal adenocarcinoma (PDAC) is a lethal disease characterized by a diverse tumor microenvironment. The heterogeneous cellular composition of PDAC makes it challenging to study molecular features of tumor cells using extracts from bulk tumor. The metabolic features in tumor cells from clinical samples are poorly understood, and their impact on clinical outcomes are unknown. Our objective was to identify the metabolic features in the tumor compartment that are most clinically impactful. Methods: A computational deconvolution approach using the DeMixT algorithm was applied to bulk RNASeq data from The Cancer Genome Atlas to determine the proportion of each gene's expression that was attributable to the tumor compartment. A machine learning algorithm designed to identify features most closely associated with survival outcomes was used to identify the most clinically impactful metabolic genes. Results: Two metabolic subtypes (M1 and M2) were identified, based on the pattern of expression of the 26 most important metabolic genes. The M2 phenotype had a significantly worse survival, which was replicated in three external PDAC cohorts. This PDAC subtype was characterized by net glycogen catabolism, accelerated glycolysis, and increased proliferation and cellular migration. Single cell data demonstrated substantial intercellular heterogeneity in the metabolic features that typified this aggressive phenotype. Conclusion: By focusing on features within the tumor compartment, two novel and clinically impactful metabolic subtypes of PDAC were identified. Our study emphasizes the challenges of defining tumor phenotypes in the face of the significant intratumoral heterogeneity that typifies PDAC. Further studies are required to understand the microenvironmental factors that drive the appearance of the metabolic features characteristic of the aggressive M2 PDAC phenotype.

Mutant SF3B1 promotes malignancy in PDAC.

The splicing factor SF3B1 is recurrently mutated in various tumors, including pancreatic ductal adenocarcinoma (PDAC). The impact of the hotspot mutation SF3B1K700E on the PDAC pathogenesis, however, remains elusive. Here, we demonstrate that Sf3b1K700E alone is insufficient to induce malignant transformation of the murine pancreas, but that it increases aggressiveness of PDAC if it co-occurs with mutated KRAS and p53. We further show that Sf3b1K700E already plays a role during early stages of pancreatic tumor progression and reduces the expression of TGF-β1-responsive epithelial-mesenchymal transition (EMT) genes. Moreover, we found that SF3B1K700E confers resistance to TGF-β1-induced cell death in pancreatic organoids and cell lines, partly mediated through aberrant splicing of Map3k7. Overall, our findings demonstrate that SF3B1K700E acts as an oncogenic driver in PDAC, and suggest that it promotes the progression of early stage tumors by impeding the cellular response to tumor suppressive effects of TGF-β.