Mun, Dong-Gi

Abstract Introduction: Src Family Kinases (SFKs) have a crucial role in tumor proliferation, survival, and metastasis. NXP900 is a highly selective SFK inhibitor with a novel mechanism of action that locks SFKs in a closed, inhibited conformation, providing sustained suppression of catalytic and non-catalytic functions, which is distinct from other SFK inhibitors like dasatinib. A phase I clinical trial of NXP900 in patients with advanced solid tumors is ongoing. Our preclinical studies showed therapeutic efficacy of NXP900 in cholangiocarcinoma (CCA) through decreased YAP, RAF-MAP and PI3K-AKT signaling. Multiomic profiling (RNA-seq, proteomics, phosphoproteomics) of CCA patient-derived xenografts implicated AKT pathway activation in primary NXP900 resistance. NXP900-resistant CCA cell lines generated via prolonged sublethal dose escalation showed a strong IL13RA2-AKT signature on multiomic analysis. Thus, we hypothesized that IL13RA2-AKT signaling was a mechanism of NXP900 resistance. We aimed to define this mechanism and demonstrate that the combinatorial targeting of IL13RA2 or AKT resensitizes to NXP900 treatment. Methods: HuCCT1 or HuCCT1-Resisant (HuCCT1-R) cells were injected bilaterally into the flanks of NOD/SCID mice and were allowed to grow for 3 months prior to tumor collection and analysis. Tumor engraftment and growth between HuCCT1 and HuCCT1-R groups were compared. To delineate the mechanisms for acquired NXP900 resistance, in-vitro treatment studies were performed using siRNAs targeting IL13RA2 as well as combination treatment with capivasertib, a pan-AKT inhibitor. Results: The HuCCT1-R cell line exhibited a higher rate of tumor engraftment and formed significantly larger tumors compared to the parental HuCCT1 cell line, indicating enhanced tumorigenic potential associated with acquired resistance. Silencing of IL13RA2 in HuCCT1-R cells resulted in pronounced reduction of cell proliferation and partially restored sensitivity to NXP900 treatment. Immunoblot analysis demonstrated a corresponding reduction in phosphorylated AKT (Thr308), consistent with suppression of downstream PI3K-AKT signaling. Furthermore, combination treatment of HuCCT1-R cells with the AKT inhibitor capivasertib, and NXP900 led to a significant increase in cell death and a greater inhibition in cell proliferation compared to either agent alone, indicating enhanced responsiveness and improved therapeutic efficacy. Conclusion: These findings confirm the central role of IL13RA2 signaling and the PI3K-AKT pathway in mediating acquired resistance to NXP900, consistent with our previous multiomics analysis. Importantly, this study identifies combination strategies that may be able to overcome NXP900 resistance, supporting the development of combinatorial approaches to restore therapeutic efficacy in resistant tumors. Citation Format: Shelby K. Yee, Hendrien Kuipers, Danielle M. Carlson, Jack W. Sample, Enis Ozmert, Hidemi Nishi, Erik Jessen, Dong-Gi Mun, Aushinie M. Abeynayake, Jennifer L. Tomlinson, Amro M. Abdelrahman, Nathan W. Werneburg, Binbin Li, Mitesh J. Borad, Mark J. Truty, Sumera I. Ilyas, Gregory J. Gores, Rory L. Smoot. Resistance to the SFK inhibitor NXP900 in cholangiocarcinoma is characterized by IL13RA2-AKT signaling and can be overcome by combination therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 7124.

Abstract Introduction: The intricate cellular structure is a constantly evolving environment where cells interact, communicate, and adapt to their surroundings. The fate, role, and actions of each cell are shaped by its specific location within the tissue. Therefore, studying the spatial proteome is crucial for gaining a deeper understanding of physiological or pathological processes. With significant advancements in microscopy and mass spectrometry, it has become feasible to explore the cellular proteome at low or even single cell numbers. However, spatial proteomics is an evolving field with scope for newer sample preparation and data analysis techniques. Our objective was to develop a sample preparation workflow for analyzing proteome from limited cells extracted from formalin fixed paraffin embedded (FFPE) tissue using laser capture microdissection (LCM). Methods: One hundred cells from the FFPE normal colon tissue were extracted by LCM and collected into an AFA-compatible 96-well plate. Decrosslinking was performed at 900C for 50 minutes followed by lysis in a buffer composed of 100 mM triethylammonium bicarbonate and 0.1% n-dodecyl-β-D-maltoside using adaptive focused acoustic (AFA) energetics in scanning mode for 5 minutes. After lysis, the proteins were digested using Trypsin/Lys-C mix using AFA energetics for 1 hour. The resulting peptides were acidified with trifluoroacetic acid and analyzed on a timsTOF Ultra 2 mass spectrometer coupled to a nanoElute 2 liquid chromatography system using a 15 cm (75 μm) IonOpticks column, in DDA-PASEF mode for a total run time of 34 minutes. The data were searched against the UniProt Human Reviewed protein database using MSFragger. Results: We utilized AFA energetics to extract and digest proteins from a limited number of cells isolated from FFPE tissue using LCM. This experiment was conducted with technical replicates to ensure accuracy and reliability. A total of 3,723 proteins were identified across the three replicates, with replicate 1 identifying 3,895 proteins, replicate 2 identifying 3,754 proteins, and replicate 3 identifying 3,519 proteins. In total, we identified 21,005 peptides, with replicate 1 identifying 22,896 peptides, replicate 2 identifying 22,377 peptides, and replicate 3 identifying 17,741 peptides from 100 colon cells. Notably, we observed a 70% overlap in the proteins identified across the three replicates, which highlights the reproducibility of the approach. Conclusion: The entire sample preparation process, after cell collection, was completed within a concise four-hour timeframe, demonstrating the efficiency of this methodology for analyzing protein profiles from small cell populations. Further, this approach is highly adaptable to automation and high-throughput applications. Its adaptability and scalability make it a promising strategy for spatial proteomics applications. Citation Format: Dong-Gi Mun, Kiran Mangalaparthi, Daigo Gunji, Amy J. French, Raghavendra Pasupuleti, Cristine Charlesworth, Sameer Vasantgadkar, DEB BHATTACHARYYA, Akhilesh Pandey. An AFA assisted workflow for mass-spectrometry based proteomics of FFPE samples [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 7657.

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

Abstract Molecular profiling of human primary cell types is essential for understanding human biology. We present a transcriptome and proteome map of 28 primary human cell types. Three major clusters of epithelial, endothelial, and mesenchymal cell types were observed in both the transcriptome and proteome levels along with the discovery of cell type enriched molecules including GRAP and C1orf116. The epithelial cell specific protein C1orf116 was further validated using immunohistochemistry across various human tissues. An exhaustive protein database search considering 39 post-translational modifications (PTMs) revealed novel insights into the PTM landscape including identification of understudied PTMs such as serine O-acetylation and histidine methylation. This also enabled comprehensive characterization of proteins with diverse PTMs. Interestingly, an unexpectedly higher frequency of dioxidation on tryptophan compared to methionine led to the identification of oxidative mitochondria complex subunit proteins. Further, a search strategy accounting for alternative translational start sites, splice junctions and translational readthrough refined genome annotation using proteomic evidence. For example, peptides from translational readthrough including extended sequence of LDHB and MDH1 were detected representing the first peptide-level evidence of these protein readthrough isoforms. Our comprehensive transcriptome and proteome data revealed cell type-specific molecular cues and heterogeneity, offering new insights into disease mechanisms often overlooked by tissue proteomics.