Tomlinson, Jennifer

ABSTRACT The limited efficacy of current therapies against cholangiocarcinoma (CCA) necessitates the development of novel treatment strategies. Src family kinases (SFKs) contribute significantly to tumor progression and resistance in CCA. Therefore, we investigated the novel, first-in-class SFK ‘OFF’ inhibitor NXP900 in diverse preclinical CCA models, including those with acquired resistance. This study evaluated the therapeutic effects of NXP900 and detailed adaptive molecular responses to SFK inhibitor therapy. We also aimed to identify biomarkers predictive of drug sensitivity using integrated multiomic profiling and develop strategies to overcome resistance. NXP900 inhibited YAP activity through direct inhibition of tyrosine phosphorylation and indirect activation of the Hippo pathway via LATS. These effects were associated with decreased tumor cell viability in CCA cell lines and several in vivo models. Notably, IDH -mutant patient-derived xenograft CCA models were particularly sensitive to NXP900. NXP900 also synergized with gemcitabine/cisplatin chemotherapy, enhancing antitumor efficacy in both in vitro and in vivo models. Multiomic analyses combining transcriptomics, global proteomics, and phosphoproteomics identified molecular features associated with primary response and acquired resistance. IL13RA-AKT signaling was upregulated in resistant models; NXP900 sensitivity could be restored with AKT or IL13RA2 inhibition. Together, these findings demonstrate the therapeutic potential of NXP900 as a novel YAP inhibitor in CCA and support further investigation in a clinical trial. Graphical Abstract

Lacking effective therapeutics, cholangiocarcinoma (CCA) remains a deadly malignancy of the biliary tract. The Hippo pathway effector protein Yes-associated protein (YAP) is implicated in CCA pathogenesis and chemotherapeutic resistance; however, the oncogenic mechanisms underlying YAP regulation remain incompletely understood. An enhanced understanding of YAP and its role in CCA may uncover novel therapeutic targets and better define resistance pathways. Human CCA cells and murine syngeneic CCA models were utilized to explore the molecular relationship of YAP and protein tyrosine phosphatase 1B (PTP1B). Previous work in CCA has demonstrated that YAP interacts with multiple protein tyrosine phosphatases, including SHP2 and PTP1B. We observed that PTP1B pharmacologic inhibition was associated with increased cell proliferation and YAP target gene expression, while genetically enforced overexpression of PTP1B was associated with a decrease in YAP activation. Treatment of CCA cells in vitro and syngeneic, orthotopically implanted CCA murine tumors in vivo with standard cytotoxic chemotherapy, gemcitabine/cisplatin, had enhanced efficacy in the setting of PTP1B overexpression. These findings demonstrate that pYAPY357 can be modulated through protein tyrosine 1B phosphatase activity, and reducing pYAPY357 through enhanced phosphatase levels can sensitize CCA to chemotherapy.

Cholangiocarcinoma (CCA) is a highly aggressive form of liver cancer and is an increasing cause of cancer‐related death worldwide. Despite its increasing incidence globally and alarming mortality, treatment options for CCA have largely remained unchanged, stressing the importance of developing new effective therapies. YAP activation is common in CCA, and its major transcriptional signaling partners are the TEAD proteins. CA3 is a small‐molecule YAP‐TEAD disrupter discovered utilizing a TEAD reporter assay. Utilizing CCA, gastric cancer cell lines, and patient‐derived xenograft models (PDX), we demonstrate that CA3 is effective in inducing cell death and delaying tumor growth in both FGFR2 fusion and wild‐type models. CA3 was associated with on‐target decreases in YAP‐TEAD target gene expression, TEAD reporter activity, and overall TEAD levels. Hippo pathway signaling was not altered as there was no change in YAP phosphorylation status in the cells exposed to CA3. RNA sequencing of gastric cancer and CCA models demonstrated upregulation of an androgen receptor–mediated transcriptional program following exposure to CA3 in five unique models tested. Consistent with this upstream regulator analysis, CA3 exposure in CCA cells was associated with increased AR protein levels, and combinatorial therapy with CA3 and androgen receptor blockade was associated with increased cancer cell death. CA3 behaves functionally as a YAP‐TEAD disrupter in the models tested and demonstrated therapeutic efficacy. Exposure to CA3 was associated with compensatory androgen receptor signaling and dual inhibition improved the therapeutic effect.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) is an aggressive and lethal malignancy. Surgical resection is the only curative modality combined with neoadjuvant chemotherapy to improve survival. Given the limitations of traditional responses such as cross-sectional imaging (CT/MRI) or tumor markers, carbohydrate antigen 19-9 (CA19-9), the 2023 National Comprehensive Cancer Network guidelines included 18F-fluorodeoxyglucose (FDG)-PET as an adjunct to assess response to neoadjuvant chemotherapy. There are common misconceptions on the metabolic activity (tumor avidity) in PDAC so we aimed to describe the baseline characteristics and use of FDG-PET in a cohort of treatment-naive patients with PDAC. STUDY DESIGN: A single-center retrospective study was conducted capturing all biopsy-proven, treatment-naive patients with PDAC who underwent either baseline FDG-PET/CT or FDG-PET/MRI imaging between 2008 and 2023. Baseline FDG-PET characteristics were collected, including primary tumors’ maximum standardized uptake value defined as metabolic activity (FDG uptake) of tumor compared with surrounding pancreatic parenchymal background, and the identification of extrapancreatic metastatic disease. RESULTS: We identified 1,095 treatment-naive patients with PDAC who underwent baseline FDG-PET imaging at diagnosis. CA19-9 was elevated in 76% of patients. Overall, 96.3% (1,054) of patients had FDG-avid tumors with a median maximum standardized uptake value of 6.4. FDG-PET also identified suspicious extrapancreatic metastatic lesions in 50% of patients, with a higher proportion (p < 0.001) in PET/MRI (59.9%) vs PET/CT (44.3%). After controlling for CA19-9 elevation, PET/MRI was superior in detection of extrapancreatic lesions compared with PET/CT. CONCLUSIONS: FDG-PET has significant use in PDAC as a baseline imaging modality earlier neoadjuvant therapy given the majority of tumors are FDG-avid. FDG-PET can identify additional extrapancreatic suspicious lesions allowing for optimal initial staging, with PET/MRI having increased sensitivity over PET/CT.

Abstract Dysfunctional liver regeneration following surgical resection remains a major cause of postoperative mortality and has no therapeutic options. Without targeted therapies, the current treatment paradigm relies on supportive therapy until homeostasis can be achieved. Pharmacologic acceleration of regeneration represents an alternative therapeutic avenue. Therefore, we aimed to generate a small molecule inhibitor that could accelerate liver regeneration with an emphasis on diseased models, which represent a significant portion of patients who require surgical resection and are often not studied. Utilizing a clinically approved small molecule inhibitor as a parent compound, standard medicinal chemistry approaches were utilized to generate a small molecule inhibitor targeting serine/threonine kinase 4/3 (MST1/2) with reduced off-target effects. This compound, mCLC846, was then applied to preclinical models of murine partial hepatectomy, which included models of diet-induced metabolic dysfunction-associated steatohepatitis (MASH). mCLC846 demonstrated on target inhibition of MST1/2 and reduced epidermal growth factor receptor inhibition. The inhibitory effects resulted in restored pancreatic beta-cell function and survival under diabetogenic conditions. Liver-specific cell-line exposure resulted in Yes-associated protein activation. Oral delivery of mCLC846 perioperatively resulted in accelerated murine liver regeneration and improved survival in diet-induced MASH models. Bulk transcriptional analysis of regenerating liver remnants suggested that mCLC846 enhanced the normal regenerative pathways and induced them following liver resection. Overall, pharmacological acceleration of liver regeneration with mCLC846 was feasible, had an acceptable therapeutic index, and provided a survival benefit in models of diet-induced MASH.

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.

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.