Vu, Diep

Abstract Background: Cholangiocarcinoma is an aggressive malignancy with limited systemic therapeutics. We have recently identified LCK, a non-receptor tyrosine kinase, as a potential therapeutic target in select subtypes of cholangiocarcinoma. Proteomic profiling in other cancer types has proven beneficial in predicting response to systemic therapies. Herein, we present proteomic profiling of cholangiocarcinoma can predict sensitivity to a novel LCK inhibitor in patient derived xenograft models. Methods: Proteins from 28 unique cholangiocarcinoma patient derived xenograft models (PDX) were isolated, labeled with tandem mass tags (TMT) and then analyzed by liquid chromatography and mass spectroscopy (LC/MS). Phosphopeptides were separately evaluated both by Immobilized Metal Affinity Chromatography and pY-enrichment techniques. Following normalization, the global proteome, serine/threonine phosphoproteome, and tyrosine phosphoproteome were analyzed and a proteomic signature was created for each tumor. These signatures were compared to PDX models with known response profiles to NTRC0652-0, an LCK inhibitor (1 resistant and 1 sensitive). The top 200 differentially abundant proteins (log2FC 2.6) were then utilized to develop a sensitive and a resistant signature. Correlation indices were then calculated across the 28 PDX model library. An additional predicted resistant and predicted sensitive model was expanded. Tumor bearing mice were randomized based on tumor volume to either treatment with NTRC (30mg/kg PO daily), n=5, or vehicle, n=4, (10% Kolliphor, 10% DMSO, 80% Water, 2 mol equivalents HCl). Tumors were measured weekly with calipers and then weighed at the end of the study. Results: In the top 200 differentially abundant proteins that represented the proteomic signature there was over representation of proteins with decreased abundance. There were only 12 proteins with higher abundance in the sensitive model as compared to the resistant. Utilizing the signature developed from the global proteomics data, nine tumors were predicted to be sensitive while 17 were predicted to be resistant to NTRC. PDX115 is a an intrahepatic CCA PDX known to contain an IDH1 mutation and was predicted to be resistant by modeling. The model was resistant to single agent NTRC with no demonstrable effect on tumor growth in vivo. PDX175 is a distal CCA PDX and was predicted to be sensitive by modeling. In vivo growth was inhibited by NTRC as compared to vehicle treated mice following two weeks of treatment (983% vs 435%, p=0.03). Additionally, final tumor weight was significantly lower in NTRC treated mice (0.651 g vs 0.317 g, p=0.04). Conclusion: Cholangiocarcinoma proteomic signatures can be used to predict therapeutic response to a novel LCK inhibitor in preclinical models. Whether or not this could be applied to other treatment modalities in cholangiocarcinoma remains to be determined. Citation Format: Ryan Watkins, Roberto Alva-Ruiz, Caitlin Conboy, Dong-Gi Mun, Erik Jessen, Diep Vu, Jos de Man, Rogier Buijsman, Akhilesh Pandey, Mitesh Borad, Gregory Gores, Rory Smoot. Proteomic profiling of cholangiocarcinoma predicts response to a novel small molecule inhibitor in preclinical models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3094.

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.