An unbiased high-throughput drug screen reveals a potential therapeutic vulnerability in the most lethal molecular subtype of pancreatic cancer

Abstract

Pancreatic ductal adenocarcinoma (PDAC) is predicted to become the second leading cause of cancer-related deaths in the United States by 2020, due in part to innate resistance to widely used chemotherapeutic agents and limited knowledge about key molecular factors that drive tumor aggression. We previously reported a novel negative prognostic biomarker, keratin 17 (K17), whose overexpression in cancer results in shortened patient survival. In this study, we aimed to determine the predictive value of K17 and explore the therapeutic vulnerability in K17-expressing PDAC, using an unbiased high-throughput drug screen. Patient-derived data analysis showed that K17 expression correlates with resistance to Gemcitabine (Gem). In multiple in vitro models of PDAC, spanning human and murine PDAC cells, we determined that the expression of K17 results in a more than twofold increase in resistance to Gem and 5-fluorouracil, key components of current standard-of-care chemotherapeutic regimens. Furthermore, through an unbiased drug screen, we discovered that Podophyllotoxin (PPT), a microtubule inhibitor, showed at least two-fold higher sensitivity in K17expressing compared to K17-negative PDAC cells. In the clinic, another microtubule inhibitor, Paclitaxel (PTX), is used in combination with Gem as a first line chemotherapeutic regimen for pancreatic, breast, lung, and ovarian cancer. Surprisingly, we found that when combined with Gem, PPT but not PTX, was synergistic in inhibiting the viability of K17-expressing PDAC cells. This provides evidence that PPT or its derivatives could potentially be combined with Gem to enhance treatment efficacy for the approximately 50% of PDACs that express high levels of K17. In summary, we reported that K17 is a novel target for developing a biomarker-based personalized treatment for PDAC.3 4 2. Methods 2.1. Predictive-value analyses from patient-derived samples K17 mRNA expression levels of PDAC cases were acquired from the Australian Pancreatic Cancer Genome Initiative (APGI)13. K17 mRNA expression and survival were evaluated in 94 PDAC patients that were treated with adjuvant Gem alone or received no treatment. Based on the established cutoff of the maximum likelihood fit of a Cox proportional hazard model15, we applied the 76th percentile of mRNA expression to categorize patients into high-K17 versus low-K17 groups. Overall survival in high-versus low-K17 mRNA was determined using the Kaplan-Meier method, calculated from the date of diagnosis to the date of death. Patients still alive at the last follow-up were censored. Prognostic and predictive analyses were performed based on the criteria described by Ballman18. Adjusting for potential confounders, a multivariate analysis was performed by Cox proportional hazard regression. Statistical significance was set at p<0.05 and analysis was done using SAS 9.4 (SAS Institute) and GraphPad Prism 7 (Graph Pad Software). Compounds testedGem (purity > 99%), 5-FU (purity > 99%), Podophyllotoxin (PPT, purity > 99%), Taxol (PTX, purity > 95%), Mitoxant...

Disassembly of embryonic keratin filaments promotes pancreatic cancer metastases

“…Prior work has shown that pancreatic cancers expressing high levels of K17 are resistant to commonly deployed first-line chemotherapeutic agents, notably gemcitabine (Wang et al 2011; Aung et al 2018; Pan et al 2020; Yao et al 2020). The finding that K17-phosphorylation at the N-terminus S10-13 is regulated by a PKC/MEK/RSK pathway is important because it suggests that K17 may be sensitive to targeted kinase inhibition.…”

“…In carcinomas of the lung, pancreas, cervix, and other sites, K17 is reexpressed for reasons that are not fully understood (Real et al 1993;Mikami et al 2015;Roa-Pena et al 2019;Roa-Pena et al 2021). In PDAC, K17 expression is a hallmark of the basallike subtype, which is associated with the shortest patient survival and resistance to first-line therapies (Wang et al 2011;Grasso et al 2017;Aung et al 2018;Roa-Pena et al 2019;Pan et al 2020;Roa-Pena et al 2021). Growing evidence, however, suggests that K17 expression contributes to a variety of cancer hallmarks, suggesting its role as a potent oncoprotein (Ide et al 2012;Mikami et al 2015;Wu et al 2017;Liu et al 2018;Wang et al 2019;Liu et al 2020; Baraks et al 2021).…”

Targeting keratin 17-mediated reprogramming of de novo pyrimidine biosynthesis to overcome chemoresistance in pancreatic cancer

“…All experimental procedures were approved by the Institutional Animal Care and Use Committee (IACUC) at Stony Brook University and Yale University and are in accordance with the Guide for the Care and Use of Laboratory Animals from the National Institutes of Health. As previously described 9 , KPC cells with or without K17 expression were harvested during log-phase growth and resuspended in DMEM (Gibco) with Matrigel (Life Sciences) at a ratio of 1:1, to a final of 1,000 cells in a 30 μl volume. Cells were orthotopically implanted into the head of the pancreas of c57B6J mice.…”

“…These findings highlight novel lines of investigation of surrogate biomarkers in molecular subtypes that may aid the development of more effective targeted therapy. We independently reported that keratin 17 (K17), one of the signature genes overexpressed in the Moffitt basal-like subtype 4 , is a novel negative prognostic and predictive biomarker in PDAC [7][8][9] . We also showed that K17 expression drives resistance to Gemcitabine (Gem) and 5-fluorouracil (5-FU) 9 , two key components of the main chemotherapeutic regimens for pancreatic cancer.…”

“…We independently reported that keratin 17 (K17), one of the signature genes overexpressed in the Moffitt basal-like subtype 4 , is a novel negative prognostic and predictive biomarker in PDAC [7][8][9] . We also showed that K17 expression drives resistance to Gemcitabine (Gem) and 5-fluorouracil (5-FU) 9 , two key components of the main chemotherapeutic regimens for pancreatic cancer. Moreover, considering that there are no available therapeutic agents to inhibit K17, we performed a high-throughput drug screen and identified a small-molecule compound that selectively targets K17-expressing PDACs and increases survival of mice with K17-positive PADC 9 .…”