Schmitt, Daniel M.

Abstract Pancreatic ductal adenocarcinoma (PDAC) is a deadly malignancy with a dismal 5-year survival rate, highlighting the urgent need to improve drug response and overcome primary or acquired (secondary) resistance to conventional chemotherapy. Glycogen synthase kinase 3β (GSK-3β) is a target in PDAC and the GSK-3 inhibitor, elraglusib, has been shown to improve chemosensitivity of PDAC and survival in preclinical cancer models. In addition, data from phase II clinical trials in patients with previously untreated metastatic pancreatic cancer is demonstrating preliminary evidence of clinical benefit in patients receiving a combination of gemcitabine/nab-paclitaxel. While this early evidence is highly encouraging, the biological determinants of elraglusib response are unknown. Using genome wide CRISPR/Cas9 library screening, we identify aurora kinase A (AURKA), an oncogenic kinase with major roles in mitosis, as a critical regulator for elraglusib sensitivity. Elraglusib treatment activates AURKA by inducing a G2/M cell cycle arrest. We show that knocking out AURKA reduces the phosphorylation of polo like kinase 1 (PLK1), arresting cells in mitosis, and leading to mitotic catastrophe and significant apoptosis after elraglusib treatment. Strikingly, the AURKA inhibitor, alisertib, works synergistically with elraglusib to inhibit PDAC cell line growth and induce apoptosis. In summary, our results demonstrate that targeting AURKA is an effective approach to sensitize PDAC tumors to elraglusib. Citation Format: Li Ding, Easton Maeder, Cheng Zhang, Taylor Weiskittel, Daniel Schmitt, Andrew Mazar, Hu Li, Daniel Billadeau. Genome wide CRISPR/Cas9 library screening identifies aurora kinase A as a regulator of elraglusib sensitivity in pancreatic cancer [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 4658.

Abstract Purpose: Pancreatic ductal adenocarcinoma (PDAC) is a predominantly fatal common malignancy with inadequate treatment options. Glycogen synthase kinase 3β (GSK-3β) is an emerging target in human malignancies including PDAC. Experimental Design: Pancreatic cancer cell lines and patient-derived xenografts were treated with a novel GSK-3 inhibitor 9-ING-41 alone or in combination with chemotherapy. Activation of the DNA damage response pathway and S-phase arrest induced by gemcitabine were assessed in pancreatic tumor cells with pharmacologic inhibition or siRNA depletion of GSK-3 kinases by immunoblotting, flow cytometry, and immunofluorescence. Results: 9-ING-41 treatment significantly increased pancreatic tumor cell killing when combined with chemotherapy. Inhibition of GSK-3 by 9-ING-41 prevented gemcitabine-induced S-phase arrest suggesting an impact on the ATR-mediated DNA damage response. Both 9-ING-41 and siRNA depletion of GSK-3 kinases impaired the activation of ATR leading to the phosphorylation and activation of Chk1. Mechanistically, depletion or knockdown of GSK-3 kinases resulted in the degradation of the ATR-interacting protein TopBP1, thus limiting the activation of ATR in response to single-strand DNA damage. Conclusions: These data identify a previously unknown role for GSK-3 kinases in the regulation of the TopBP1/ATR/Chk1 DNA damage response pathway. The data also support the inclusion of patients with PDAC in clinical studies of 9-ING-41 alone and in combination with gemcitabine.

Abstract Glycogen synthase kinase-3 (GSK-3) is overexpressed and accumulates in the nucleus in several human malignancies including pancreatic cancer. We recently demonstrated that GSK-3 inhibition sensitizes pancreatic cancer cells to chemotherapy by abrogating the TopBP1/ATR-mediated DNA damage response. Specifically, GSK-3 kinase activity is required to stabilize TopBP1 protein. Herein, we investigated the mechanism by which GSK-3 phosphorylation of TopBP1 prevents its degradation. Interestingly, GSK-3 inhibition not only decreased the total levels of TopBP1, but also reduced the ability of TopBP1 to bind chromatin following gemcitabine treatment. Mutation of a putative GSK-3 phosphorylation site in TopBP1 (S860A) resulted in decreased TopBP1 stability and chromatin recruitment, whereas a phosphomimetic mutant (S860E) had the opposite effect. Furthermore, we found enhanced TopBP1 ubiquitination after GSK-3 inhibition as well as an increased interaction between TopBP1 and hHYD, an ubiquitin ligase that has been shown to ubiquitylate TopBP1 resulting in its degradation. Significantly, the S860A mutation showed a stronger interaction with hHYD compared to WT or the S860E mutant of TopBP1. In conclusion, our study identifies GSK-3 phosphorylation of TopBP1 as a mechanism to prevent the interaction with and ultimate ubiquitylation-mediated degradation by hHYD. Citation Format: Li Ding, Kaely R. Roeck, Olga Alekhina, Daniel M. Schmitt, Daniel D. Billadeau. Glycogen synthase kinase-3 inhibition impairs DNA damage response through ubiquitin ligase hHYD-mediated TopBP1 degradation [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4138.

Abstract Targeting the B-cell receptor and phosphatidylinositol 3-kinase/mTOR signaling pathways has shown meaningful, but incomplete, antitumor activity in lymphoma. Glycogen synthase kinase 3 (GSK3) α and β are 2 homologous and functionally overlapping serine/threonine kinases that phosphorylate multiple protein substrates in several key signaling pathways. To date, no agent targeting GSK3 has been approved for lymphoma therapy. We show that lymphoma cells abundantly express GSK3α and GSK3β compared with normal B and T lymphocytes at the messenger RNA and protein levels. Utilizing a new GSK3 inhibitor 9-ING-41 and by genetic deletion of GSK3α and GSK3β genes using CRISPR/CAS9 knockout, GSK3 was demonstrated to be functionally important to lymphoma cell growth and proliferation. GSK3β binds to centrosomes and microtubules, and lymphoma cells treated with 9-ING-41 become arrested in mitotic prophase, supporting the notion that GSK3β is necessary for the progression of mitosis. By analyzing recently published RNA sequencing data on 234 diffuse large B-cell lymphoma patients, we found that higher expression of GSK3α or GSK3β correlates well with shorter overall survival. These data provide rationale for testing GSK3 inhibitors in lymphoma patient trials.

Abstract Glycogen synthase kinase-3 (GSK3) is a ubiquitously expressed serine-threonine protein kinase involved in multiple cellular functions ranging from the control of glycogen metabolism to transcriptional regulation. We have previously demonstrated that GSK-3β is overexpressed in human pancreatic ductal adenocarcinoma (PDAC) and aberrant nuclear accumulation of GSK-3β serves as a hallmark of poorly differentiated PDAC. Moreover, genetic ablation of GSK-3β or GSK-3 inhibition led to PDAC tumor cell killing, identifying GSK-3β as a therapeutic target in PDAC. Herein, we describe a novel GSK-3 inhibitor, 9-ING-41. The effect of 9-ING-41 on tumor cell growth was tested using several PDAC and patient-derived xenograft (PDX) cell lines in vitro and in vivo using an orthotopic pancreatic cancer animal model. Interestingly, 9-ING-41 treatment resulted in the stabilization of cyclin B levels as well as enhanced serine-10 phosphorylated histone H3 (pS10HH3) and consequently M phase arrest and blocked mitotic exit. The prolonged M phase arrest induced mitotic catastrophe in pancreatic cancer cell lines and inhibited in vitro colony formation. An in vivo orthotopic pancreatic cancer animal model study using PDX-derived cell lines demonstrated that 9-ING-41 hindered tumor growth in combination with several standard chemotherapeutic agents. Significantly, we found that 9-ING-41 treatment impaired the DNA damage response pathway activated by Gemcitabine in pancreatic cancer cell lines. In conclusion, our study indicates that GSK-3 plays an important role in pancreatic cancer progression and 9-ING-41 in combination with existing gemcitabine or irinotecan could be a new strategy for treating patients with PDAC. Citation Format: Li Ding, Jin-san Zhang, John R. Dube, Vijay S. Madamsetty, Daniel M. Schmitt, Debabrata Mukhopadhyay, Daniel D. Billadeau. The glycogen synthase kinase-3 inhibitor, 9-ING-41, synergizes with chemotherapy to inhibit pancreatic tumor growth in vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2926.

AbstractAcinar‐to‐ductal metaplasia (ADM) is a reversible epithelial transdifferentiation process that occurs in the pancreas in response to acute inflammation. ADM can rapidly progress towards pre‐malignant pancreatic intraepithelial neoplasia (PanIN) lesions in the presence of mutant KRas and ultimately pancreatic adenocarcinoma (PDAC). In the present work, we elucidate the role and related mechanism of glycogen synthase kinase‐3beta (GSK‐3β) in ADM development using in vitro 3D cultures and genetically engineered mouse models. We show that GSK‐3β promotes TGF‐α‐induced ADM in 3D cultured primary acinar cells, whereas deletion of GSK‐3β attenuates caerulein‐induced ADM formation and PanIN progression in KrasG12D transgenic mice. Furthermore, we demonstrate that GSK‐3β ablation influences ADM formation and PanIN progression by suppressing oncogenic KRas‐driven cell proliferation. Mechanistically, we show that GSK‐3β regulates proliferation by increasing the activation of S6 kinase. Taken together, these results indicate that GSK‐3β participates in early pancreatitis‐induced ADM and thus could be a target for the treatment of chronic pancreatitis and the prevention of PDAC progression. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.