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Introduction: Pancreatic cancer remains a challenging disease, with an overall cumulative 5-year survival rate less than 5%. Metastases are responsible for over 90% of cancer-related deaths. Despite significant progress made in cancer drug development, our understanding of the mechanisms driving metastatic outgrowth remains limited. Systematic dissection of the molecular mechanisms underlying metastatic progression is necessary for the development of new therapeutic strategies. Method: To identify the important genes driving metastatic outgrowth, we performed a novel in vivo cDNA screening using a high metastatic-capacity line (M-4964Liv) and a low metastatic-capacity line (D-4964Liv), which we derived from in vivo passaging of a cell line from a KPC mouse. MBD3 regulated MYC stability was examined by cycloheximide chase assay and further confirmed by polymerase chain reaction and Western blotting. Phenotypic effect of MBD3 was evaluated by sphere assay in 3D and intrasplenic model of liver metastasis in vivo. Results: Methyl-CpG binding domain protein 3 (MBD3) was identified in our screen. We initially evaluated MBD3 with pancreatosphere formation assays. In doing so, we showed that MBD3 overexpression leads to significant augmentation of sphere formation, while knockdown abrogates sphere-forming capacity. We subsequently validated MBD3 in vivo with liver metastases assays, through both overexpression and knockdown, as being a facilitator of metastatic outgrowth. In addition, we observed high expression of MBD3 across a panel of pancreatic cancer cell lines that readily form liver metastases upon splenic injection. To understand how MBD3 may lead to metastases, we used a cycloheximide chase assay and determined that MBD3 overexpression increases MYC stability. With knockout of MBD3 using CRISPR/Cas9, we demonstrated that MBD3 mediates MYC stabilization by increasing the expression of phospho-MYC at Ser-62 (stable form of MYC). In the absence of MBD3, we observed an increase in phospho-MYC at Thr-58 expression (unstable form of MYC). Consequently, we observed a decrease in expression of CDK7, a downstream target of MYC, after MBD3 knockdown. CDK7 is a known master regulator of cyclin-dependent kinases, and loss of CDK7 drives cells to quiescence. Intriguingly, downregulation of MYC expression, through destabilization with MBD3 knockdown, led to activation of negative feedback pathway as observed with an increase in phospho-GSK3β at Ser-9, which inactivates GSK3β and thereby decreases MYC ubiquitination. Conclusion: MBD3 leads to stabilization of MYC in pancreatic cancer cell lines, which results in an increase in the ability to form liver metastases. Further dissection of this previously undescribed MBD-mediated mechanism may lead to new therapeutic targeting of MYC for patients with pancreatic cancer. Citation Format: Alok Ranjan, Surajit Sinha, Reed Ayabe, Michael Wach, Samantha Ruff, Shreya Gupta, James McDonald, Kirsten Remmert, Imani Alexander, Jonathan Hernandez. MBD3 stabilizes MYC, leading to metastatic outgrowth of pancreatic cancer in the liver [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 C44.

Introduction: Although liver metastasis is the major cause of pancreatic cancer mortality, the mechanisms by which KRAS-driven pancreatic tumor cells initiate metastatic outgrowth in the liver are unknown. High-throughput next-generation sequencing studies have revealed that driver mutations and copy number alterations are highly concordant between matched primary tumors and metastases in pancreatic cancer. This suggests that the mechanisms leading to the metastatic outgrowth and colonization involve the cooperation of many other cellular factors in addition to driver mutations. We hypothesize that in the absence of metastasis-specific driver mutations, the initial adaptation and outgrowth steps of metastasis are driven by metabolic reprogramming, which is induced or activated in vivo under physiologic conditions of the target organ like nutrient deprivation. Method: To identify the metabolic drivers, a retroviral cDNA library was generated from a highly metastatic pancreatic cell line (M-4964Liv) derived from a KPC mouse that forms macroscopic liver lesions upon splenic injection in mice. The cDNA library was then transduced into dormant pancreatic KPC cells (D-4964Liv) cells that do not give liver lesions upon splenic injection in mice. This methodology will allow all the cDNAs from the metastatic cells to express in the dormant cells, but only those cDNAs that can trigger the dormant cells to outgrow will give rise to a metastatic liver lesion. Results: The screening strategy led to the identification of the translational regulator general control of amino-acid synthesis 1-like 1 (GCN1). We show that activation of GCN1 at physiologic glutamine activates the master regulator of metabolic homeostasis ATF4, oncogenic mTORC1 signaling, and pluripotency factor SOX2. Consistent with this, knockdown of GCN1 not only inhibits ATF4, mTORC1, and expression of Sox2 but dramatically abrogates liver metastasis in immunocompetent mice upon intrasplenic injection, suggesting that GCN1-ATF4 signaling is indispensable for metastatic outgrowth. Mechanistic insights reveal that the GCN1-ATF4 signaling pathway is governed by a competitive inhibitor, IMPACT, which is induced at physiologic glutamine and inhibits GCN1 function. Surprisingly, the expression of IMPACT is lost in highly metastatic pancreatic cell lines, leading to activation of the GCN1-ATF4 pathway and mTORC1 at physiologic glutamine, while overexpression of IMPACT effectively inhibits the pathway and reduces metastatic outgrowth of pancreatic cancer cells in the liver in immunocompetent mice. Conclusions: The loss of expression of IMPACT leading to GCN1-dependent activation of ATF4 and mTORC1 at physiologic levels of glutamine provides metabolic plasticity during the initial steps of metastatic outgrowth. Thus, inhibitors targeting the GCN1-ATF4 pathway or mTORC1 will greatly benefit pancreatic adenocarcinoma patients by inhibiting the outgrowth of their metastatic disease. Citation Format: Surajit Sinha, Reed I Ayabe, Alok Ranjan, Michael Wach, Samantha Ruff, Kirsten Remmert, Elke Van Beek, Imani Alexander, James D. McDonald, Shreya Gupta, Eileen Li, Jonathan Hernandez. IMPACT inhibits metastatic outgrowth in pancreatic cancer by restraining GCN1-ATF4 signaling [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 A50.