Buckarma, EeeLN H.

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.

Abstract Background: The transcriptional cofactor Yes-associated protein (YAP) is activated in CCA pathophysiology. While canonical regulation by the Hippo pathway resulting in phosphorylation at serine 127 (S127) is inhibitory, SRC-family kinases (SFKs) phosphorylate YAP at tyrosine 357 (Y357), causing activation. Previously, cholangiocytes, hepatic progenitor cells (HPCs), and even mature hepatocytes were suggested as potential cells of origin of CCA, which highlights the plasticity in the liver. However, the role of YAP activation in selecting the cell of origin and lineage commitment during hepatobiliary oncogenesis is unknown. Aim: To determine if YAP Y357 phosphorylation is required for CCA tumorigenesis, we utilized an in vivo model. Unexpectedly, a shift in tumor phenotype to hepatocellular carcinoma (HCC) was identified. Subsequently, we further explored cell signaling and cell of origin contributions to this phenotypic switch. Method: C57BL/6 mice underwent biliary transfection with Sleeping Beauty transposase and transposons encoding myr-AKT and either YAP S127A (YAP-S) or YAP S127A/Y357F (YAP-SY), followed by IL-33 treatment. Tumor nodule count, histologic classification, and liver-to-body ratio were assessed at necropsy after 10 weeks. RNA sequencing and ingenuity pathway analysis (IPA) were performed on HCC and CCA nodules. To determine the cell of origin, tumors were generated in lineage tracing mice (Rosa26 mT/mG mice treated with AAV8-TBG-Cre). Finally, two tumor-derived cell lines with YAP-SY and constitutive AKT expression were isolated. Results: Both YAP-S and YAP-SY models developed similar tumor numbers and bulk. YAP-S mice generated frequent CCA (25.3%) in addition to combined HCC-CCA (cHCC-CCA) tumors (10.7%) and HCC (64%), whereas YAP-SY mice developed predominantly HCC (86.7%), with rare CCA (8.8%) and mixed tumors (4.5%). Transcriptome analysis identified 3,367 differentially expressed genes (DEGs) between HCC from YAP-SY mice and CCA from YAP-S mice. Notably, YAP-regulated genes had decreased expression in the HCC from YAP-SY mice. IPA revealed key upstream regulators, including cytokines and growth factors (e.g., TGFB1, TNF, INFG, CSF2, IL6, IL1B, IL4) and transcription factors (e.g., TP53, JUN, SP1, NFKBIA, STAT3). Lineage tracing demonstrated HCC originated from hepatocytes and CCA from cholangiocytes in both models. Finally, gene expression analysis of novel syngeneic cancer cell lines from YAP-SY mice showed mixed features of both HCC and CCA. Conclusion: In this murine model, we found that sub-maximal YAP activation is associated with increased formation of HCC from hepatocytes, while a higher level of YAP activation due to YAP Y357 phosphorylation is associated with efficient formation of CCA. We identified cytokines and growth factors that may orchestrate this phenotypic switch. Finally, we developed novel syngeneic cancer cell lines with cHCC-CCA characteristics. Citation Format: Muhammed D. Aksu,Jayla T. Millender,Ryan D. Watkins,Jennifer A. Yonkus,EeeLN H. Buckarma,Nathan W. Werneburg,Daniel R. O'Brien,Chantal E. McCabe,Rondell P. Graham,Gregory J. Gores,Rory L. Smoot,Caitlin B. Conboy. YAP tyrosine phosphorylation regulates lineage commitment in liver tumorigenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 1457.

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.

AbstractBackground and ObjectiveCurrently, computed tomography‐guided transthoracic biopsy (CTTB) is the most accurate diagnostic approach for pulmonary nodules suspected of malignancy. Traditional bronchoscopy has shown suboptimal diagnostic sensitivity, but the emergence of robotic‐assisted bronchoscopy (RAB) has the potential to improve diagnostic accuracy, maximize diagnostic yield and complete mediastinal and hilar staging in a single procedure. We aim to assess the efficacy and diagnostic performance of RAB compared to CTTB for diagnosing pulmonary nodules suspected of lung cancer.MethodsA multicenter retrospective review of consecutive patients who underwent RAB and CTTB for evaluating pulmonary nodules from January 2019 to March 2021 at Mayo Clinic Florida and Mayo Clinic Rochester, United States. Clinical and demographic information, nodule characteristics, outcomes and complications were compared between RAB and CTTB.ResultsA total of 225 patients were included: 113 in the RAB group and 112 in the CTTB group. Overall diagnostic yield was 87.6% for RAB and 88.4% for CTTB. For malignant disease, RAB had a sensitivity of 82.1% and a specificity of 100%, CTTB had a sensitivity of 88.5% and a specificity of 100%. Complication rate was significantly higher for CTTB compared to RAB (17% vs. 4.4%; p = 0.002).ConclusionRAB, when available, can be as accurate as CTTB for sampling pulmonary nodules with similar or reduced complications and should be considered as a means for nodule biopsy, particularly when mediastinal staging is also clinically warranted.

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.

463 Background: Molecularly targeted therapy is gaining traction in cholangiocarcinoma with the first FDA approval of a targeted agent in 2020, the FGFR inhibitor pemigatinib. Yet response rates and efficacy are modest, and most cholangiocarcinoma patients still lack targeted therapy options. The hippo pathway effector, YAP, is an oncogene that is activated in the majority of cholangiocarcinoma, yet attempts to develop YAP inhibitors have not yet been clinically successful. Recently, we described a novel mechanism of YAP regulation, via tyrosine phosphorylation by the Src-family kinase LCK. Therefore we hypothesized that LCK inhibition by may be therapeutic in cholangiocarcinoma through suppression of YAP activity. Methods: NTRC 0652-0 is a novel tyrosine kinase inhibitor with specificity for LCK. In vitro pharmacodynamics were defined. In cholangiocarcinoma cells, specificity for LCK inhibition was determined by mass spectrometry-based unbiased analysis of the tyrosine phosphoproteome following treatment with NTRC 0652-0 or genetic deletion of LCK. YAP phosphorylation and cotranscriptional activity, cell viability, and apoptosis were assessed. A panel of eight cholangiocarcinoma patient derived organoids (PDO) were characterized and tested for sensitivity to NTRC 0652-0. Two patient-derived xenograft (PDX) models bearing FGFR2-rearrangements were utilized for in vivo assessment of pharmacokinetics, toxicity, and efficacy. Results: NTRC 0652-0 demonstrated selectivity for LCK inhibition in vitro and in cholangiocarcinoma cells. NTRC 0652-0 treatment led to YAP inhibition and apoptotic cell death in cholangiocarcinoma cell lines, associated with inhibition of MCL1 expression. PDOs demonstrated variable sensitivity to NTRC 0652-0, correlated with basal YAP tyrosine phosphorylation and drug-induced suppression of YAP co-transcriptional activity. FGFR2-altered cholangiocarcinoma was also identified as a subset with enrichment of YAP target genes. Cells from an FGFR2-altered PDX were sensitive to NTRC 0652-0 despite being primarily resistant to pemigatinib. In two PDX models of FGFR2-altered cholangiocarcinoma, daily oral treatment with NTRC 0652-0 inhibited YAP tyrosine phosphorylation, and resulted in stable plasma and tumor drug levels, acceptable toxicity, and significantly decreased tumor growth. Conclusions: A novel LCK inhibitor, NTRC 0652-0, reduced YAP signaling and demonstrated preclinical efficacy in multiple patient-derived models of cholangiocarcinoma. LCK is a novel therapeutic target in cholangiocarcinoma, and YAP activation or FGFR2-alteration are potential biomarkers for response.

Abstract Introduction: The effector protein of the Hippo pathway, Yes-associated protein (YAP), functions as a transcriptional coactivator and has been implicated in the biology of cholangiocarcinoma (CCA). Although the Hippo cascade consists of serine kinases, which act to sequester YAP in the cytosol, we have recently demonstrated the importance of tyrosine phosphorylation in regulating YAP localization and function in CCA. Furthermore, previous evaluation of clinical samples of CCA demonstrated a high frequency of mutations in tyrosine phosphatases. Evaluation of YAP-interacting proteins identified several tyrosine phosphatases, including SHP2. Herein, we describe the ability to modulate YAPY357 phosphorylation status, localization, and activity through manipulation of SHP2 levels and activity. Methods: The cholangiocarcinoma cell lines HuCCT-1 and KMCH were utilized. SHP2 activity was modulated utilizing the inhibitors NSC87877 and SHP099 (Selleckchem). SHP2 levels were modulated utilizing knockout via a doxycycline-inducible CRISPR/Cas9 system. Enforced expression of SHP2 was explored utilizing catalytically active and dead constructs. Tyrosine phosphorylation was assessed via immunoblot. Transcriptional activity was assessed by qRT-PCR for YAP target genes. YAP sub-cellular localization was assessed by immunofluorescence. Proliferation was evaluated by MTS assay utilizing SHP2 inhibitors and the Src-family kinase inhibitor dasatinib (Selleckchem). Results: The HuCCT-1 and KMCH cell lines were noted to have unique p-YAPY357 profiles (HuCCT-1 [p-YAPY357 high] KMCH [p-YAPY357 low]), and these profiles correlated with SHP2 levels and YAP subcellular localization with HuCCT1 cells demonstrating low levels of SHP2 and prominent nuclear YAP localization, while KMCH cells demonstrated high levels of SHP2 and predominately cytoplasmic YAP. In both cell lines, pharmacologic inhibition of SHP2 was accompanied by an increase in p-YAPY357 levels and YAP cotranscriptional activity (as noted by increased expression of cognate YAP target genes). In KMCH cells SHP2 inhibition shifted YAP from the cytoplasm to the nucleus, and these findings were confirmed by CRISPR/Cas9-mediated deletion of SHP2. In contrast, enforced expression of catalytically active SHP2 in HuCCT1 cells was associated with a decrease in p-YAPY357 levels and a shift of YAP from the nucleus to the cytoplasm. As predicted, based on an increase in YAP activity and nuclear localization, SHP2 inhibition was associated with an increase in cellular proliferation in both cell lines. Incubation of the cell lines with dasatinib was associated with decreased proliferation, which was partially rescued by pretreatment with an SHP2 inhibitor. Conclusions: The tyrosine phosphatase SHP2 can regulate YAPY357 phosphorylation, transcriptional coactivity, and subcellular localization in CCA. Inhibition of SHP2 activity protects cells from the effects of Src kinase inhibition on cellular proliferation and caspase activation. Citation Format: EeeLN Buckarma, Nathan Werneburg, Ayano Kabashima, Gregory Gores, Rory Smoot. The tyrosine phosphatase SHP2 regulates YAPY357 phosphorylation, subcellular localization, and transcriptional coactivity in cholangiocarcinoma [abstract]. In: Proceedings of the AACR Special Conference on the Hippo Pathway: Signaling, Cancer, and Beyond; 2019 May 8-11; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(8_Suppl):Abstract nr A17.

Abstract The Hippo pathway effector Yes-associated protein (YAP) is localized to the nucleus and transcriptionally active in a number of tumor types, including a majority of human cholangiocarcinomas. YAP activity has been linked to chemotherapy resistance and has been shown to rescue KRAS and BRAF inhibition in RAS/RAF-driven cancers; however, the underlying mechanisms of YAP-mediated chemoresistance have yet to be elucidated. Herein, we report that the tyrosine phosphatase SHP2 directly regulates the activity of YAP by dephosphorylating pYAPY357 even in the setting of RAS/RAF mutations, and that diminished SHP2 phosphatase activity is associated with chemoresistance in cholangiocarcinomas. A screen for YAP-interacting tyrosine phosphatases identified SHP2, and characterization of cholangiocarcinomas cell lines demonstrated an inverse relationship between SHP2 levels and pYAPY357. Human sequencing data demonstrated lower SHP2 levels in cholangiocarcinomas tumors as compared with normal liver. Cell lines with low SHP2 expression and higher levels of pYAPY357 were resistant to gemcitabine and cisplatin. In cholangiocarcinomas cells with high levels of SHP2, pharmacologic inhibition or genetic deletion of SHP2 increased YAPY357 phosphorylation and expression of YAP target genes, including the antiapoptotic regulator MCL1, imparting resistance to gemcitabine and cisplatin. In vivo evaluation of chemotherapy sensitivity demonstrated significant resistance in xenografts with genetic deletion of SHP2, which could be overcome by utilizing an MCL1 inhibitor. Implications: These findings demonstrate a role for SHP2 in regulating YAP activity and chemosensitivity, and suggest that decreased phosphatase activity may be a mechanism of chemoresistance in cholangiocarcinoma via a MCL1-mediated mechanism.

Abstract Background: Cancer of the biliary tract, cholangiocarcinoma (CCA), is increasing in incidence and has limited treatment options. In an attempt to further understand signaling pathways driving oncogenesis and impacting response to therapy we, and others, have identified altered activation of the transcriptional co-activator, Yes- associated protein (YAP) in CCA. Canonical regulatory pathways impacting YAP consist of serine kinases; however, more recently we have demonstrated a central role for tyrosine phosphorylation in regulating YAP function in CCA. Herein we explore the role of tyrosine phosphatases in regulating YAP tyrosine phosphorylation. Methods: In silico analysis of the TCGA cholangiocarcinoma dataset was undertaken specifically evaluating tyrosine phosphatase levels. Molecular studies utilized the human CCA cell lines HuCCT-1 and KMCH as well as the murine CCA cell line SB1 (expressing a flag-tagged YAP). Baseline tyrosine phosphatase levels were assessed by RT-PCR and immunoblot. YAP-interacting phosphatases were identified by co-immunoprecipitation. Tyrosine phosphorylation was inhibited utilizing the multi-kinase inhibitor dasatinib. The pan-tyrosine phosphatase inhibitor sodium orthovanadate (Na3VO4) and the selective SHP1/2 inhibitor NSC87877 were utilized. Tyrosine phosphorylation was assessed by immunoblot. YAP transcriptional activity was evaluated by RT-PCR. Apoptosis was evaluated by caspase 3/7 assay. Results: Multiple tyrosine phosphatases were noted to be expressed at lower levels in tumor versus normal adjacent liver in the TCGA RNAseq dataset. YAP-interacting tyrosine phosphatases identified by co-IP included PTPN1, 11, 23, and PTPRK. Profiling of tyrosine phosphatase levels by RT-PCR and immunoblot demonstrated higher levels in KMCH cells compared to HuCCT-1; notably the YAP-interacting phosphatase PTPN11 (SHP2) was elevated. Consistent with the anticipated function of the phosphatases, immunoblot demonstrated lower levels of tyrosine phosphorylated YAP (p-YAPY357) in KMCH cells compared to HuCCT-1. The role of SHP2 was further probed by incubation of KMCH and HuCCT-1 cells with NSC87877 which was associated with an increase in p-YAPY357 levels and YAP co-transcriptional activity. Conversely, incubation of HuCCT-1 cells with dasatinib rapidly decreased p-YAPY357 levels; and this effect was diminished by pre-incubation with either Na3VO4or NSC87877. The effect on YAP tyrosine phosphorylation paralleled effects on apoptosis; incubation of HuCCT-1 cells with dasatinib lead to an apoptotic response as measured by caspase 3/7 assay, which was eliminated by pre-incubation with Na3VO4 or NSC87877. Conclusions: Inhibition of the tyrosine phosphatase SHP2 is associated with elevated p-YAPY357 levels, elevated YAP co-transcriptional activity, and a blunted therapeutic response to dasatinib in cholangiocarcinoma cell lines. Citation Format: EeeLN Buckarma, Nathan Werneburg, Ayano Niibe, Gregory Gores, Rory Smoot. Tyrosine phosphatase activity is required for response to Src kinase inhibition in cholangiocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3449.