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Pancreatic ductal adenocarcinoma (PDAC), the most common histological subtype of pancreatic cancer, has one of the highest case fatality rates of all known solid malignancies. Over the past decade, several landmark studies have established mutations in KRAS and TP53 as the predominant drivers of PDAC pathogenesis and therapeutic resistance, though treatment options for PDACs and other tumors with these mutations remain extremely limited. Hampered by late tumor discovery and diagnosis, clinicians are often faced with using aggressive and non-specific chemotherapies to treat advanced disease. Clinically meaningful responses to targeted therapy are often limited to the minority of patients with susceptible PDACs, and immunotherapies have routinely encountered roadblocks in effective activation of tumor-infiltrating immune cells. Alternative RNA splicing (ARS) has recently gained traction in the PDAC literature as a field from which we may better understand and treat complex mechanisms of PDAC initiation, progression, and therapeutic resistance. Here, we review PDAC pathogenesis as it relates to fundamental ARS biology, with an extension to implications for PDAC patient clinical management.

Keratin 17 (K17), an oncofetal intermediate filament protein, is one of the most abundantly expressed proteins in pancreatic ductal adenocarcinomas (PDACs) of the most aggressive molecular subtype. The mechanistic roles of this protein in malignancy, however, are largely unexplored. Here we show that K17 expression and disassembly enhances tumor growth and metastatic potential and shortens survival. Using mass spectrometry in K17 isolated from patient’s tumors, we identified a hotspot phosphorylation site in serines 10-13. Site-mutagenesis revealed that phosphorylation of this hotspot is sufficient to disassemble K17 and promote its nuclear translocation. In silico and pharmacologic inhibition studies uncovered the role of the PKC/MEK/RSK pathway in the phosphorylation and disassembly of K17. Murine models bearing tumors expressing phosphomimetic mutations at the serine hotspot displayed enhanced metastases, compared to mice bearing tumors expressing wild-type K17 or phosphorylation-resistant K17. Lastly, we found that detergent-soluble nuclear K17 promotes the expression of metastasis promoting genes in both patient and murine tumors. These results suggest that phosphorylation at specific serines is sufficient to promote pancreatic cancer metastasis and shorter survival, and that these sites could provide novel, druggable therapeutic domains to enhance PDAC patient survival.

Purpose: Molecular subtyping for pancreatic cancer has made substantial progress in recent years, facilitating the optimization of existing therapeutic approaches to improve clinical outcomes in pancreatic cancer. With advances in treatment combinations and choices, it is becoming increasingly important to determine ways to place patients on the best therapies upfront. Although various molecular subtyping systems for pancreatic cancer have been proposed, consensus regarding proposed subtypes, as well as their relative clinical utility, remains largely unknown and presents a natural barrier to wider clinical adoption. Experimental Design: We assess three major subtype classification schemas in the context of results from two clinical trials and by meta-analysis of publicly available expression data to assess statistical criteria of subtype robustness and overall clinical relevance. We then developed a single-sample classifier (SSC) using penalized logistic regression based on the most robust and replicable schema. Results: We demonstrate that a tumor-intrinsic two-subtype schema is most robust, replicable, and clinically relevant. We developed Purity Independent Subtyping of Tumors (PurIST), a SSC with robust and highly replicable performance on a wide range of platforms and sample types. We show that PurIST subtypes have meaningful associations with patient prognosis and have significant implications for treatment response to FOLIFIRNOX. Conclusions: The flexibility and utility of PurIST on low-input samples such as tumor biopsies allows it to be used at the time of diagnosis to facilitate the choice of effective therapies for patients with pancreatic ductal adenocarcinoma and should be considered in the context of future clinical trials.

Pancreatic ductal adenocarcinoma (PDAC) includes two molecular subtypes, of which the basal-like subtype is associated with the shortest survival and is highly resistant to chemotherapy. The basal-like subtype is defined by a 25-gene signature; however, the role of these genes in promoting tumor aggression remains unexplored. Here, we set out to uncover the mechanisms of chemoresistance and explore targeted therapies for this subtype. We focused on studying an oncofetal antigen, keratin 17 (K17), which is the most overexpressed hallmark gene of the basal-like PDAC. We manipulated the expression of K17 and found that in multiple in vitro and in vivo models of PDAC, spanning human and murine PDAC cells and orthotopic xenografts, K17 expression resulted in a greater than two-fold increase in resistance to Gemcitabine (Gem) and 5-fluorouracil, the major chemotherapeutic agents in standard-of-care treatments. To uncover the mechanisms associated with K17-induced chemoresistance, we performed unbiased metabolomic studies in isogenic PDAC cell lines and found that compared to control cells, K17 increases intracellular levels of deoxycytidine (dC) by four-fold that promote Gem (dC analogue) resistance. Based on previous findings that K17 enters nucleus to regulate gene expression, we explored whether K17 triggers metabolic reprogramming at the transcriptional level and found that enzymes involved in pyrimidine biosynthesis are positively correlated with K17 expression in PDAC cells. Given that it is still poorly understood how K17 regulates gene expression, we performed domain-prediction analyses. We discovered and validated a novel chromatin remodeling domain on K17 that is required for metabolic reprogramming. We are now performing ChIP-Seq and RNA-Seq to understand how this domain alters pyrimidine biosynthesis. To identify small molecules that could target K17-expressing PDACs potentially by disrupting metabolic reprograming, we performed an unbiased high-throughput drug screen and found that Podophyllotoxin (PPT), a microtubule inhibitor, significantly and selectively killed K17-positive compared to K17-negative PDAC cells. In the clinic, another microtubule inhibitor, Paclitaxel (PTX), is used in combination with Gem as a first line chemotherapy. Surprisingly, when combined with Gem, PPT but not PTX, was synergistic in inhibiting the viability of K17-expressing PDAC cells and enhanced survival of mice bearing K17-expressing PDACs. Currently, we are exploring the role of PPT in regulating pyrimidine biosynthesis. In summary, we identified a novel pathway of chemoresistance and a compound that could result in developing a biomarker-based personalized therapy. Citation Format: Chun-Hao Pan, Yuka Otsuka, BanuPriya Sridharan, Melissa Woo, Cindy V. Leiton, Sruthi Babu, Mariana Torrente Gonçalves, Ryan R. Kawalerski, Ji Dong K. Bai, Richard A. Moffitt, Jiang Zhao, David K. Chang, Andrew V. Biankin, Tim Duong, Pankaj K. Singh, Louis Scampavia, Timothy Spicer, Kenneth R. Shroyer, Luisa F. Escobar-Hoyos. Targeting a novel rewired pathway of nucleotide metabolism that drives chemoresistance in the most lethal molecular subtype of pancreatic cancer [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2020 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2020;80(22 Suppl):Abstract nr PO-023.

CLINICAL NEED & OBJECTIVES: There is an unmet need for the discovery and development of novel therapeutic approaches for PDAC. Molecular subtyping, however, has recently uncovered potential biomarkers that, subject to further investigation, may be druggable to extend PDAC patient survival. Expression of Keratin 17 (K17), an oncofetal intermediate filament (IF) protein and hallmark of the most aggressive subtype, associates negatively with survival and drives chemotherapy resistance in PDAC. This suggests a functional role of K17 in PDAC, though the underlying mechanisms are unknown. Nuclear localization, however, has been suggested as a part of the overarching processes through which K17 promotes tumor growth. In related keratins, post-translational modifications (PTM) such as phosphorylation drive IF dissociation (solubilization) and can contribute to migration and metastatic dissemination. Thus, we hypothesized that K17 may harbor PTM- and/or solubilization-dependent oncogenic functions that could reveal new PDAC therapeutic options. METHODS & RESULTS: Using isogenic murine PDAC models, we found that K17 expression enhances tumor growth and metastatic potential. Furthermore, K17 solubility was associated with shorter survival, independent of total K17 expression or cell proliferation in both murine and human PDACs. To identify K17 PTMs potentially driving solubilization, we performed liquid chromatography mass spectrometry to sequence K17 isolated from patient PDACs and identified phosphorylation of a conserved N-terminal region (Ser 10-13) as the predominant PTMs. To determine if Ser 10-13 phosphorylation regulates K17 solubility, we generated mutants that mimic K17 Ser 10-13 phosphorylation and found that this was sufficient to solubilize K17 and promote nuclear localization. Building on these findings, we used kinase inhibitors to show that the PKC-MEK-RSK signaling pathway is involved in phosphorylation of K17 Ser 10-13. To assess the impact of K17 Ser 10-13 phosphorylation on tumors, nude mice were implanted orthotopically with isogenic human PDAC cell lines expressing either wild type (WT), loss of function (LOF) Ser 10-13 mutants, or Ser 10-13 phosphorylation gain of function (GOF) mutants. Compared to tumors expressing WT K17, GOF tumors resulted in increased metastases, and LOF mutants abrogated these characteristics. CONCLUSION: These results suggest that enhanced K17 solubility by phosphorylation at Ser 10-13 is sufficient to impact PDAC metastasis and survival and reveal novel druggable sites on K17. These results motivate future studies to uncover K17 phosphorylation targeting approaches for PDAC. If successful, they will be coupled with the identification of PDAC patients by tumor K17 expression, providing the basis of a potential precision medicine strategy. Citation Format: Ryan R. Kawalerski, Mariana T. Gonçalves, Lucia Roa-Peña, Cindy V. Leiton, Chun-Hao Pan, Luke A. Torre-Healy, Taryn Boyle, Natasha T. Snider, Kenneth R. Shroyer, Luisa F. Escobar-Hoyos. Solubilization of keratin 17 promotes the metastatic dissemination of the most lethal form of pancreatic cancer [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2020 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2020;80(22 Suppl):Abstract nr PO-050.

Clinical Need and Objectives: Our previous reports suggest that keratin 17 (K17), an intermediate filament (IF), is an oncoprotein and biomarker of the most aggressive and treatment-resistant form of pancreatic ductal adenocarcinoma (PDAC). Importantly, K17 transitions from its “insoluble” IF form to a “soluble” form that can regulate cell cycle progression and gene expression, though the mechanisms that mediate K17 solubilization are yet unknown. Determining these mechanisms may uncover new keratin 17-specific PDAC therapeutic vulnerabilities. Methods and Results: We found that in human and murine PDAC models, the soluble fraction of K17 exists in the full-length form (flK17) and as a caspase-cleaved peptide (ccK17), each with potential roles as biomarkers and/or functional drivers of PDAC aggression. In a PDAC tumor mouse model and human samples, we show, independent of total K17 protein amount, that the percent of soluble and ccK17 in tumor cells is negatively prognostic. We sequenced both the soluble and cytoskeletal insoluble forms of K17 from primary PDACs using liquid chromatography mass spectrometry (LC-MS) and identified that K17 is phosphorylated on several N-terminal serine residues, some of which identify a semiconserved IF motif that may regulate caspase cleavage and solubilization. Serine phosphatase inhibition in vitro demonstrated that phosphorylation is necessary to trigger K17 solubilization followed by nuclear translocation. Mutagenesis of N-terminal serine sites demonstrated that phosphorylation of three serine residues is sufficient to solubilize K17 from its filamentous state, and that phosphorylation of K17 by a PKC-RSK signaling pathway is likely to induce solubilization. In vitro and in vivo experiments are ongoing to address the functional role of this mechanism in regulating PDAC pathogenesis. Conclusion: Phosphorylation is an important regulator of K17 dynamics in PDAC, promoting solubilization and, in some cases, caspase cleavage. Our results suggest that soluble K17 may have a functional role in promoting tumor aggression. Inhibiting RSK or PKC kinases that phosphorylate K17 could impact tumor growth by blocking K17 solubilization/cleavage, and this line of research could accelerate the development of more effective treatments, coupled to soluble K17 testing as a predictive biomarker for PDAC. Citation Format: Ryan R. Kawalerski, Lucia Roa-Pena, Luke A. Torre-Healy, Taryn Boyle, Cindy V. Leiton, Natasha T. Snider, Kenneth R. Shroyer, Luisa F. Escobar-Hoyos. Oncogenic mechanism of soluble keratin 17 offers potential therapeutic vulnerability in pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2019 Sept 6-9; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2019;79(24 Suppl):Abstract nr C26.

Our aims are to uncover the molecular mechanisms through which keratin 17 (K17), a prognostic biomarker, drives tumor aggression and to target these mechanisms to provide more effective treatment for pancreatic ductal adenocarcinoma (PDAC). In murine orthotopic xenografts, we found that K17-positve PDACs survive for a shorter interval than controls. Prompted by previous reports that post-translational modifications (PTMs) regulate intermediate filament dynamics, we established in vitro that phosphorylated K17 detaches from the cytoskeleton and enters the nucleus, where it promotes tumor growth by targeting tumor suppressor proteins, including p27, for nuclear export and degradation. To further understand the events that control K17 solubilization, we sequenced K17 from primary PDACs by liquid chromatography-mass spectrometry and identified serine sites within the N-terminus that are phosphorylated only in soluble K17. Furthermore, phosphorylation is required to maintain K17 solubility and soluble K17 accumulates in the nucleus of PDAC cells. By an unbiased screen of 80 small-molecule kinase inhibitors in PDAC, we determined that SYK kinase inhibitors, already in clinical trials for other malignancies, abrogated K17 solubilization. Prompted by our finding that K17 serves as a nuclear shuttle of p27, we identified two amino acid sequences in K17 that have similar polarity to sequences that are used by cyclins to dock to p27. Point mutations in two of these domain key residues blocked K17-mediated degradation of nuclear p27, and we identified similar effects in the background of wild-type and oncogenic KrasG12D PDAC cells. Current studies are under way to find additional protein and RNA targets for potential therapeutic intervention. Using patient-derived organoids, human and murine PDAC cells, we determined that K17-expressing PDAC cells are more than twice as resistant as isogenic K17-negative cells to gemcitabine (Gem) and 5-fluorouracil (5-FU), two key components of current chemotherapeutic regimens. By unbiased liquid chromatography-coupled tandem mass spectrometry metabolomics, RNA-sequencing analyses (TCGA), and in vivo magnetic resonance spectroscopy, we found that K17 induces metabolic reprogramming by increasing glycolysis and pyrimidine biosynthesis, pathways that have been linked to chemoresistance. We are extending this work to determine if disruption of K17-mediated metabolic rewiring by small-molecule inhibitors will resensitize tumor cells to pyrimidine analogues. In conclusion, K17 undergoes key post-translational modifications that enable solubilization and nuclear translocation, the targeting of tumor suppressor proteins, and enhanced pyrimidine biosynthesis to drive chemoresistance. Uncovering these mechanisms could ultimately lead to the identification of novel approaches to target the oncogenic functions of K17, and thereby, to enable the development of more effective treatment options for PDAC. Citation Format: Kenneth R. Shroyer, Luisa Escobar-Hoyos, Cindy Leiton, Chun-Hao Pan, Ryan Kawalerski, Lucia Roa-Peña, Sruthi Babu. Keratin 17 drives tumor aggression and could be targeted for treatment of pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2019 Sept 6-9; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2019;79(24 Suppl):Abstract nr B50.

Pancreatic ductal adenocarcinoma (PDAC) is characterized by two molecular subtypes, of which the basal-like subtype is associated with the worst survival and is highly resistant to the currently available first-line chemotherapy. Our laboratory has identified that keratin 17 (K17) is a novel negative prognostic biomarker, as accurate as molecular subtyping in predicting patient survival. Patient-derived data analysis suggests that K17 expression correlates with increased resistance to chemotherapeutic agents. The goal of this study is to determine the role of K17 in chemoresistance, and to identify novel therapeutic approaches for around 50% of PDAC patients with tumors that express high levels of K17. In multiple in vivo and in vitro models of PDAC, spanning human and murine PDAC cells, patient-derived organoids, and orthotopic xenograft models, we determined that K17 expression causes more than two-fold increase in resistance to gemcitabine (Gem) and 5-fluorouracil (5-FU), key components of the current standard-of-care chemotherapeutic regimens. To uncover the mechanism associated to K17-induced chemoresistance, we performed unbiased metabolomic studies in isogenic PDAC cell lines and found that K17 reprograms several key metabolic pathways. In particular, K17 increases pyrimidine biosynthesis, a pathway has been linked to chemoresistance. Rescue experiments showed that deoxycytidine (dC) was sufficient to promote Gem (dC analogue) resistance in K17-nonexpressing PDAC cells, suggesting that upregulation of pyrimidine synthesis by K17 underlies resistance to chemotherapeutic agents. Through unbiased RNA-sequencing studies, we identified that gene expression of enzymes involved in pyrimidine biosynthesis was increased specifically in high K17-expressing cells. Previous reports from our group and others suggest that nuclear K17 regulates cell-cycle progression and gene expression. Through domain-prediction analyses, we discovered a novel domain on K17 involved in transcriptional regulation that is required for metabolic reprogramming. Currently, we are testing the role of this domain in metabolic reprograming. In addition, are pursuing two approaches to determine the “druggability” of these findings. First, we are testing if interrupting K17-mediated nucleotide metabolism, by means of small-molecule inhibitors, resensitizes tumor cells to pyrimidine analogues. Second, we are validating the results of a large-scale small-molecule inhibitor screen of FDA-approved, pharma-developed tools to identify compounds that target DNA metabolism and transcription in K17-expressing PDAC cells. In summary, we identified a novel and potentially druggable pathway of chemoresistance that could ultimately result in developing novel therapeutic strategies to enhance patient survival. Citation Format: Chun-Hao Pan, Cindy V. Leiton, Lucia Roa-Peña, Ryan R. Kawalerski, Richard A. Moffitt, Jiang Zhao, Timothy Spicer, Peter Bailey, David K. Chang, Andrew Biankin, Tim Duong, Pankaj K. Singh, Kenneth R. Shroyer, Luisa F. Escobar-Hoyos. A novel rewired pathway of nucleotide metabolism drives chemoresistance in pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2019 Sept 6-9; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2019;79(24 Suppl):Abstract nr C39.