Tomlinson, Jennifer L.

Abstract Genes encoding SWI/SNF chromatin remodeling complex subunits are recurrently mutated across human cancers. In cholangiocarcinoma (CCA), AT-rich interaction domain-1A (ARID1A) and polybromo-1 (PBRM1) loss of function mutations occur in approximately 18 and 10% of cases respectively. Yet our mechanistic understanding and ability to therapeutically exploit ARID1A and PBRM1 deficiency are immature. Genetically accurate preclinical models are needed to define the role of ARID1A and PBRM1 and test therapeutic approaches in CCA. Analysis of human CCA tumors showed that loss of function mutations in ARID1A and PBRM1 frequently co-occur with each other and with PI3K/AKT pathway activation. Therefore, we developed novel murine models of cholangiocarcinoma initiated by Arid1a knockout or dual Arid1a/Pbrm1 knockout in the context of AKT activation, employing a surgical biliary transfection method for somatic gene editing with CRISPR/Cas9. Mice with cholangiocyte Arid1a knockout and AKT activation (AAC) or dual Arid1a/Pbrm1 knockout and AKT activation (APAC) formed numerous liver tumors within 8 months. Histologically, tumors were predominantly CCA and mixed HCC/CCA, with a similar spectrum of tumors across AAC and APAC mice. Gene editing with CRISPR/Cas9 was confirmed at the DNA level, and loss of protein expression was confirmed in tumor cells by immunohistochemistry. Similar to human CCA, many tumors were densely infiltrated with cancer-associated fibroblasts and a variable degree of CD45+ immune cells. To generate scalable models for preclinical testing, tumors from AAC and APAC mice were dissociated to create cell lines for in vitro studies and propagated by orthotopic transplantation for in vivo studies. Cell lines and orthotopic tumors were validated and retained the genetic features and histologic characteristics of the parental tumors. Collectively these studies have produced a suite of novel, well-characterized, genetically relevant mouse models of epigenetic dysregulation in CCA due to ARID1A and PBRM1 deficiency, which can be used broadly for discovery research and preclinical testing of novel treatment approaches. Citation Format: Caitlin B. Conboy, Jennifer L. Tomlinson, Ryan D. Watkins, Jayla T. Millender, Danielle M. Carlson, Rory L. Smoot, Keith D. Robertson, Greg J. Gores. Novel murine models of ARID1A and PBRM1-deficient cholangiocarcinoma for preclinical discovery and development [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr A048.

27 Background: Cholangiocarcinoma (CCA) is a devastating cancer with few effective treatment options. We have previously discovered LCK can activate AXL, a TAM receptor tyrosine kinase (RTK), by phosphorylation of AXL-Y866 ( J Hepatol 2022). AXL is known to confer therapeutic resistance to solid cancers. However, the role of AXL in CCA, and the impact of AXL-Y866 activation by LCK still remains to be elucidated. Here, we investigated the significance of AXL expression, signaling, and activation by LCK on Y866. Methods: We first evaluated the expression levels of AXL in CCA and its association with patient outcome using The Cancer Genome Atlas (TCGA) dataset. Next, to evaluate whether AXL inactivation sensitizes CCA cells to gemcitabine and cisplatin (GC), AXL downregulation was achieved via siRNA approach and the selective AXL inhibitor bemcentinib (Bem) using human CCA cell lines, HuCCT1 and RBE, and murine SB-1. We examined 50% inhibitory concentration (IC50) value on GC with or without AXL knockdown (KD) using cell viability assay. Then we assessed the efficacy of the combination of GC and Bem utilizing Calcusyn software and apoptosis by Annexin V assay. I n vivo efficacy was assessed using both a patient derived CCA xenograft and SB-1 a syngeneic model of CCA treated with vehicle, GC, Bem, or the combination. Next, we defined the interactome of wildtype AXL- Y866 compared to AXL-Y866F using biotin proximity labelling methods (BioID), validation of downstream target proteins using Western blot (WB), and a functional study with AXL-/- cells reconstituted with either AXL WT, or AXL Y866F. Results: In TCGA cohorts, AXL transcripts are more abundant than in normal adjacent liver, and higher levels are correlated with worse clinical outcome. In in vitro study, IC50 values of GC decreased after AXL KD. Synergistic effects were observed in the combination. The combination caused increased apoptosis compared to other treatments. In the in vivo studies, the combination suppressed tumor growth and the Ki67 levels, and the phosphorylation levels of PEAK1 decreased in the combination group compared to other groups. In BioID experiments, among the 222 proteins which were detected in both AXL WT and AXL Y866F interactomes, PEAK1 kinase, which localizes to actin cytoskeleton and focal adhesions, was most altered when Y866 was inactivated by the Y866F mutation. In gene ontology analysis, cell-cell adhesion and focal adhesion related proteins were enriched. In WB, we validated that the phosphorylation of PEAK1 decreased after AXL or LCK knockout (KO) and after Bem treatment in above in vivo study. In cell viability assay, reconstitution of AXL KO cells with WT AXL increased the IC50 to GC while reconstitution with AXL-Y866F did not. Conclusions: AXL inhibition sensitizes CCA cells to cytotoxic chemotherapy in preclinical model. LCK-AXL Y866-PEAK1 signaling axis is a potential target for the treatment of CCA.

Abstract Background: Cholangiocarcinoma (CCA) is a lethal disease with limited therapeutic options. We have previously discovered LCK driving signaling through AXL, a TAM receptor tyrosine kinase (RTK), by phosphoproteomic analysis of CCA (J Hepatol 2022). AXL mediates acquired drug resistance in solid cancers. However, the exact role of AXL in CCA still remains to be elucidated. Here, we investigated the significance of AXL expression as a potential therapeutic target in CCA and the role of phosphorylated AXL Y866. Methods: We first evaluated the expression levels of AXL in CCA and its associations with patient outcomes using The Cancer Genome Atlas (TCGA) database. Next, to evaluate whether AXL inhibition sensitizes CCA cells to gemcitabine and cisplatin (GemCis) therapy, AXL downregulation was achieved via the siRNA approach and the selective inhibitor bemcentinib. We examined the 50% inhibitory concentration (IC50) value of HuCCT1, a well-characterized CCA cell line, on GemCis therapy with or without AXL knockdown using cell viability assay. Then we assessed the efficacy of the combinatorial therapy of GemCis and bemcentinib utilizing Calcusyn software. Apoptosis was evaluated by Annexin V assay. In vivo efficacy was assessed using an SB-1 (murine CCA cell line) and a syngeneic murine model of CCA treated with vehicle, GemCis, bemcentinib, and the combination of GemCis and bemcentinib. Finally, to investigate the role of AXL Y866 in CCA, we performed BioID to identify and compare the interactomes of AXL WT and AXL Y866F in HuCCT1 cells. Results: In the TCGA cohorts, AXL is significantly expressed in CCA (P < 0.01), and disease-free survival and overall survival in the low AXL expression group are significantly longer than those in the high AXL expression group (P = 0.04, 0.01). In in vitro study, the IC50 value of GemCis was decreased from 685nM to 129nM after AXL knockdown. A synergistic effect was observed with CI = 0.17 and Fa = 0.50 in the combinatorial therapy. The combinatorial therapy caused significantly increased apoptosis compared to GemCis or bemcentinib alone (P < 0.01, P < 0.01). In in vivo study, the combinatorial therapy significantly suppressed the tumor growth compared to GemCis or bemcentinib alone (P = 0.04, 0.01), and the expression levels of Ki67 and the phosphorylation levels of PEAK1 decreased in the combinatorial therapy group compared to other groups in immunostaining examination. In BioID experiments, among the 222 proteins detected in both AXL WT and AXL Y866F interactomes, PEAK1 kinase, which localizes to actin cytoskeleton and focal adhesions, was the kinase most affected by AXL Y866. In gene ontology analysis to characterize the interactome of AXL Y866, cell-cell adhesion and focal adhesion-related proteins were enriched. Conclusions: AXL inhibition sensitizes CCA cells to cytotoxic chemotherapy in the preclinical model. AXL Y866-PEAK1 signaling axis is a potential target for the treatment of CCA. Citation Format: Shohei Takaichi, Dong-Gi Mun, Jennifer L. Tomlinson, Amro M. Abdelrahman, Danielle M. Carlson, Alaa Abou Daher, Nathan W. Werneburg, Xinyan Wu, Akhilesh Pandey, Gregory J. Gores, Rory L. Smoot. Axl inhibition sensitizes cholangiocarcinoma cells to cytotoxic chemotherapy. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4996.

Abstract Introduction: Cholangiocarcinoma (CCA) is a highly lethal, and biologically heterogenous malignancy arising from the biliary tree. With only moderate responses to cytotoxic chemotherapy and despite the development of targeted therapies, treatment success has been hindered by primary or acquired resistance. New therapeutic strategies are necessary to improve outcomes in these patients. NXP800 is a heat shock factor 1 (HSF1) small-molecule inhibitor with activity previously noted in ovarian cancers with ARID1A mutations. ARID1A mutations are common, well-known players in cholangiocarcinoma. In preclinical exploratory studies, we examined treatment responses in validated cholangiocarcinoma patient derived xenograft (PDX) models. Methods: In our preliminary studies we evaluated response to HSF1 inhibition in a single, ARID1A wildtype PDX model. Xenografts were expanded into the flank of NOD/SCID mice and treatment begun when tumors averaged 120mm3. 16 tumor bearing mice were randomized and 8 mice were grouped into both the treatment and control arms. Mice were then treated with either NXP800 (35 mg/kg), in the treatment arm, or vehicle, in the control arm, via oral gavage 5 days on, 2 days off, for 28 days. Tumor volume and animal weight were collected twice weekly. Results: After screening of our Human CCA PDX library of 57 patients, we identified an established and histologically validated PDX from a patient with intrahepatic cholangiocarcinoma obtained at surgical resection. Molecular characterization of the tumor identified NRAS Q61 mutation, FGFR1 and FGFR2 overexpression, androgen receptor overexpression, microsatellite stability, and a total tumor mutational burden of 2.6. RNA sequencing was completed and an evaluation of a 21-gene signature of YAP activity had identified this PDX as having an elevated activity signature compared to a cohort of 27 additional PDX tumors. Treatment with NXP800 was associated with a statistically significant decrease in tumor size compared to control tumor bearing mice, with a mean difference of the delta in tumor volume (852.6 mm3; 95% CI: 321 - 1384; P-value = 0.0057). Conclusion: NXP800, a HSF1 inhibitor, demonstrated significant therapeutic activity in a cholangiocarcinoma PDX. Further studies are needed and being performed to determine the role of HSF1 inhibition in human cholangiocarcinoma. Citation Format: Danielle M. Carlson, Amro Abdelrahman, Alessandro Fogliati, Isaac Lynch, Jennifer Tomlinson, Mark Truty, Rory Smoot. Inhibition of HSF1 demonstrates therapeutic efficacy in preclinical models of cholangiocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2237.

Abstract Background: Cholangiocarcinoma (CCA) is a heterogeneous malignancy arising from the biliary epithelium. Its diverse molecular landscape and aggressive biology render many anti-cancer therapies ineffective. Nanovesicle technology provides an opportunity for therapeutic inhibition of oncogenic targets that have been previously classified as undruggable. EpCAM is an epithelial-specific, transmembrane glycoprotein with increased expression in human and murine CCA which can be used for nanovesicle targeting. As a proof of concept study, we designed and validated a novel strategy to direct therapeutic milk-derived nanovesicles (tMNVs) to CCA tumors. Methods: tMNVs were decorated with RNA nanoparticles containing a validated aptamer (EpDT3) against EpCAM conjugated to a cholesterol-triethylene-glycol (TEG) scaffold containing an Alexa647 fluorophore. Human and murine CCA cell lines were treated with aptamer directed tMNVs and assessed for nanovesicle uptake by fluorescent microscopy. CCA tumor tissue, derived from orthotopic implantation of a syngeneic CCA cell line, SB1, into a C57BL/6 mouse, was collected and treated with either aptamer-directed or bare tMNVs ex vivo, and compared with adjacent normal liver tissue. Flow cytometry was utilized to characterize tMNVs absorption profile. C57BL/6 mice who had previously undergone SB1 orthotopic and flank implantation were treated with aptamer-directed tMNVs by tail-vein injection and subsequently euthanized. Tissue was collected for biodistribution analyses by fluorescent microscopy. The experiment was repeated in NOD-scid mice following orthotopic implantation of patient derived xenograft (PDX) CCA tumor. Results: Both human and murine CCA cells treated with aptamer-directed tMNVs demonstrated high fluorescent signal consistent with tMNV absorption within 12 hours of application. Flow cytometry analysis showed aptamer-directed tMNVs were absorbed at a higher proportion by CCA tumors than bare tMNVs ex vivo. Aptamer-directed tMNVs also had better absorption by CCA tumors compared to adjacent normal liver tissue. Following treatment with aptamer-directed or bare tMNVs in vivo, fluorescent microscopy demonstrated that aptamer-directed tMNVs were significantly better absorbed in the orthotopic SB1 tumors, followed by the subcutaneous tumors. Minimal fluorescent signal was noted in the normal adjacent liver. Orthotopically implanted PDX tumors also demonstrated high fluorescent signals following intravenous treatment with aptamer-directed tMNVs. Conclusions: Utilizing a novel targeting strategy, we were able to design tMNVs capable of reliably and specifically targeting CCA in preclinical models. This work is foundational to the future application of nanovesicle technology in the CCA treatment paradigm. Citation Format: Mincheng Yu, Jennifer L. Tomlinson, Emilien J. Loeuillard, Ryan D. Watkins, Caitlin B. Conboy, Shohei Takaichi, Nathan W. Werneburg, Roberto Alva-Ruiz, Amro Abdelrahman, Danielle M. Carlson, Jingchun Yang, Sumera I. Ilyas, Gregory J. Gores, Tushar Patel, Rory L. Smoot. Novel strategy for aptamer-directed nanovesicle targeting in cholangiocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 814.

591 Background: Cholangiocarcinoma (CCA) is a lethal disease with limited therapeutic options. We have demonstrated the interaction of Src-family kinase LCK with AXL, a TAM receptor tyrosine kinase (RTK), by phosphoproteomic analysis in CCA ( J Hepatol 2022). AXL is reported to act as a mechanism of acquired drug resistance in solid cancers. However, the role of AXL in CCA remains to be elucidated. Here, we investigated the significance of AXL expression as a potential therapeutic target in CCA. Methods: We first evaluated the expression levels of AXL in CCA and the associations with patient outcome using publicly available data from The Cancer Genome Atlas (TCGA). Next, to evaluate whether AXL inactivation sensitizes CCA cells to gemcitabine and cisplatin (GemCis) therapy as a standard therapy. AXL downregulation was achieved via siRNA approach and the selective inhibitor bemcentinib. We examined 50% inhibitory concentration (IC50) value of HuCCT1, a well characterized CCA cell line, on GemCis therapy with and without AXL knockdown using CellTiter-Glo cell viability assay. Then we assessed the efficacy of the combinatorial therapy of GemCis and bemcentinib utilizing Calcusyn software. Finally, apoptosis was evaluated by Annexin V assay. In vivo efficacy was assessed using a SB-1 (murine CCA cell line) and syngeneic murine model of CCA in C57Bl/6J mice treated with vehicle, GemCis, bemcentinib, and the combination of GemCis and bemcentinib. Results: In the TCGA cohorts, AXL is significantly expressed in CCA (P < 0.01), and disease-free survival and overall survival in low AXL expression group are significantly longer than those in high AXL expression group (P = 0.04, 0.01). In in vitro study, IC50 value of GemCis was decreased from 685nM to 129nM after AXL knockdown. Synergy effect was observed with the value of CI = 0.17 and Fa = 0.50 in the combinatorial therapy. The combinatorial therapy caused significantly increased apoptosis compared to GemCis or bemcentinib alone (P < 0.01, P < 0.01). In in vivo study, the combinatorial therapy significantly suppressed the tumor growth compared to GemCis or bemcentinib alone (P = 0.04, 0.01). Conclusions: Targeting AXL sensitizes CCA cell lines and preclinical models to standard of care GemCis combinatorial therapy. Our study suggests that the addition of AXL targeted therapy to cytotoxic chemotherapy can increase the response in CCA patients and is a promising combination.

575 Background: Cholangiocarcinoma (CCA) is a lethal hepatobiliary adenocarcinoma with increasing incidence and poor outcomes. Treatment success has been hindered by resistance, and new therapeutic strategies are necessary. The Src-family tyrosine kinase, LCK, activates Yes-associated protein (YAP), a known oncogene in CCA. While YAP has been historically difficult to target, we recently described LCK inhibition as a potential therapeutic option in CCA. We probed the LCK interactome employing biotinylation site identification technology (BioSITe) to discover molecular co-dependencies in the LCK-YAP axis. Methods: LCK-deficient HuCCT1, wild-type HuCCT1, and wild-type RBE cells were transfected with pEF-LCK-TurboID and used to reveal protein-protein interactions. Biotinylated peptides were enriched using anti-biotin antibodies followed by mass spectrometry analysis. Co-immunoprecipitation (Co-IP) was used to confirm interactions between LCK and identified interactors. Phosphoproteome profiling of human CCA tumors and patient derived xenografts was conducted to identify active kinase signatures in CCA. Tyrosine phosphorylated peptides were enriched using anti-phosphotyrosine antibody beads and analyzed by high-resolution mass spectrometery/liquid chromatography. Immunoblot analysis of HuCCT1 and LCK-deficient HuCCT1 cells was used to map the mechanistic interaction between LCK and epidermal growth factor receptor, EGFR. Inhibitors of EGFR and LCK, afatinib and NTRC 0652-0, respectively, were tested in vitro on human and murine CCA cell lines. Cell viability was determined with CellTiter-Glo; Calcusyn software was used to determine synergistic drug effects. Results: We discovered intracellular interactors of LCK in CCA using BioSITe. We identified both known and novel LCK interactors, including EGFR. Given that EGFR functions as a molecular driver of tumorigenesis and is increasingly recognized as a biomarker of resistance in a variety of tumors, we performed a phosphoproteomic evaluation of CCA tumors in which activated EGFR was identified by the presence of EGFR-Y1092, Y1197, and Y1172 phosphorylation. EGFR phosphorylation decreased under conditions of genetic LCK knock out and inhibition by NTRC 0652-0. Co-IP confirmed the interaction of EGFR with LCK. EGFR-Y1092 phosphorylation was downregulated with genetic knockout of LCK by immunoblot. Finally, EGFR inhibition was evaluated as a therapeutic strategy utilizing afatinib. Effects on cell viability were noted in both human and murine CCA cells. Afatinib and NTRC 0652-0 demonstrated a synergistic effect in vitro, with calculated combination indices of 0.007 – 0.790. Conclusions: A novel interaction between EGFR and LCK in CCA was identified using an unbiased proximity-dependent biotinylation technique. Dual blockade of LCK and EGFR is synergistic in CCA and may be a viable therapeutic approach for patients.

Disrupted liver regeneration following hepatectomy represents an “undruggable” clinical challenge associated with poor patient outcomes. Yes-associated protein (YAP), a transcriptional coactivator that is repressed by the Hippo pathway, is instrumental in liver regeneration. We have previously described an alternative, Hippo-independent mechanism of YAP activation mediated by downregulation of protein tyrosine phosphatase nonreceptor type 11 (PTPN11, also known as SHP2) inhibition. Herein, we examined the effects of YAP activation with a selective SHP1/SHP2 inhibitor, NSC-87877, on liver regeneration in murine partial hepatectomy models. In our studies, NSC-87877 led to accelerated hepatocyte proliferation, improved liver regeneration, and decreased markers of injury following partial hepatectomy. The effects of NSC-87877 were lost in mice with hepatocyte-specific Yap/Taz deletion, and this demonstrated dependence on these molecules for the enhanced regenerative response. Furthermore, administration of NSC-87877 to murine models of nonalcoholic steatohepatitis was associated with improved survival and decreased markers of injury after hepatectomy. Evaluation of transcriptomic changes in the context of NSC-87877 administration revealed reduction in fibrotic signaling and augmentation of cell cycle signaling. Cytoprotective changes included downregulation of Nr4a1 , an apoptosis inducer. Collectively, the data suggest that SHP2 inhibition induces a pro-proliferative and cytoprotective enhancement of liver regeneration dependent on YAP.