Src Kinase Research Articles

The CD4 transmembrane GGXXG and juxtamembrane (C/F)CV+C motifs mediate pMHCII-specific signaling independently of CD4-LCK interactions

CD4+ T cell activation is driven by five-module receptor complexes. The T cell receptor (TCR) is the receptor module that binds composite surfaces of peptide antigens embedded within MHCII molecules (pMHCII). It associates with three signaling modules (CD3γε, CD3δε, and CD3ζζ) to form TCR-CD3 complexes. CD4 is the coreceptor module. It reciprocally associates with TCR-CD3-pMHCII assemblies on the outside of a CD4+ T cells and with the Src kinase, LCK, on the inside. Previously, we reported that the CD4 transmembrane GGXXG and cytoplasmic juxtamembrane (C/F)CV+C motifs found in eutherian (placental mammal) CD4 have constituent residues that evolved under purifying selection (Lee et al., 2022). Expressing mutants of these motifs together in T cell hybridomas increased CD4-LCK association but reduced CD3ζ, ZAP70, and PLCγ1 phosphorylation levels, as well as IL-2 production, in response to agonist pMHCII. Because these mutants preferentially localized CD4-LCK pairs to non-raft membrane fractions, one explanation for our results was that they impaired proximal signaling by sequestering LCK away from TCR-CD3. An alternative hypothesis is that the mutations directly impacted signaling because the motifs normally play an LCK-independent role in signaling. The goal of this study was to discriminate between these possibilities. Using T cell hybridomas, our results indicate that: intracellular CD4-LCK interactions are not necessary for pMHCII-specific signal initiation; the GGXXG and (C/F)CV+C motifs are key determinants of CD4-mediated pMHCII-specific signal amplification; the GGXXG and (C/F)CV+C motifs exert their functions independently of direct CD4-LCK association. These data provide a mechanistic explanation for why residues within these motifs are under purifying selection in jawed vertebrates. The results are also important to consider for biomimetic engineering of synthetic receptors.

EGFR Inhibition by Erlotinib Rescues Desmosome Ultrastructure and Keratin Anchorage and Protects against Pemphigus Vulgaris IgG–Induced Acantholysis in Human Epidermis

Pemphigus is a severe blistering disease caused by autoantibodies primarily against the desmosomal cadherins desmoglein (DSG)1 and DSG3 which impair desmosome integrity. Especially for the acute phase, additional treatment options allowing to reduce corticosteroids would fulfill an unmet medical need. Here, we provide evidence that epidermal growth factor receptor (EGFR) inhibition by erlotinib ameliorates pemphigus vulgaris immunoglobulin G (PV-IgG) -induced acantholysis in intact human epidermis. PV-IgG caused phosphorylation of EGFR (Y845) and SRC in human epidermis. In line with that, a phosphotyrosine kinome analysis revealed a robust response associated with EGFR and SRC family kinase signaling in response to PV-IgG but not pemphigus foliaceus autoantibodies. Erlotinib inhibited PV-IgG-induced epidermal blistering and EGFR phosphorylation, loss of desmosomes as well as ultrastructural alterations of desmosome size, plaque symmetry, keratin filament insertion and restored the desmosome midline considered as hallmark of mature desmosomes. Erlotinib enhanced both single molecule DSG3 binding frequency and strength and delayed DSG3 fluorescence recovery supporting that EGFR inhibition increases DSG3 availability and cytoskeletal anchorage. Our data indicate that EGFR is a promising target for pemphigus therapy due to its link to several signaling pathways known to be involved in pemphigus pathogenesis.

Enhancement of NETosis by ACE2-cross-reactive anti-SARS-CoV-2 RBD antibodies in patients with COVID-19.

High levels of neutrophil extracellular trap (NET) formation or NETosis and autoantibodies are related to poor prognosis and disease severity of COVID-19 patients. Human angiotensin-converting enzyme 2 (ACE2) cross-reactive anti-severe acute respiratory syndrome coronavirus 2 spike protein receptor-binding domain (SARS-CoV-2 RBD) antibodies (CR Abs) have been reported as one of the sources of anti-ACE2 autoantibodies. However, the pathological implications of CR Abs in NET formation remain unknown. In this study, we first assessed the presence of CR Abs in the sera of COVID-19 patients with different severity by serological analysis. Sera and purified IgG from CR Abs positive COVID-19 patients as well as a mouse monoclonal Ab (mAb 127) that can recognize both ACE2 and the RBD were tested for their influence on NETosis and the possible mechanisms involved were studied. An association between CR Abs levels and the severity of COVID-19 in 120 patients was found. The CR Abs-positive sera and IgG from severe COVID-19 patients and mAb 127 significantly activated human leukocytes and triggered NETosis, in the presence of RBD. This NETosis, triggered by the coexistence of CR Abs and RBD, activated thrombus-related cells but was abolished when the interaction between CR Abs and ACE2 or Fc receptors was disrupted. We also revealed that CR Abs-induced NETosis was suppressed in the presence of recombinant ACE2 or the Src family kinase inhibitor, dasatinib. Furthermore, we found that COVID-19 vaccination not only reduced COVID-19 severity but also prevented the production of CR Abs after SARS-CoV-2 infection. Our findings provide possible pathogenic effects of CR Abs in exacerbating COVID-19 by enhancing NETosis, highlighting ACE2 and dasatinib as potential treatments, and supporting the benefit of vaccination in reducing disease severity and CR Abs production in COVID-19 patients.

Bosutinib Stimulates Macrophage Survival, Phagocytosis, and Intracellular Killing of Bacteria.

Host-acting compounds are emerging as potential alternatives to combating antibiotic resistance. Here, we show that bosutinib, an FDA-approved chemotherapeutic for treating chronic myelogenous leukemia, does not possess any antibiotic activity but enhances macrophage responses to bacterial infection. In vitro, bosutinib stimulates murine and human macrophages to kill bacteria more effectively. In a murine wound infection with vancomycin-resistant Enterococcus faecalis, a single intraperitoneal bosutinib injection or multiple topical applications on the wound reduce the bacterial load by approximately 10-fold, which is abolished by macrophage depletion. Mechanistically, bosutinib stimulates macrophage phagocytosis of bacteria by upregulating surface expression of bacterial uptake markers Dectin-1 and CD14 and promoting actin remodeling. Bosutinib also stimulates bacterial killing by elevating the intracellular levels of reactive oxygen species. Moreover, bosutinib drives NF-κB activation, which protects infected macrophages from dying. Other Src kinase inhibitors such as DMAT and tirbanibulin also upregulate expression of bacterial uptake markers in macrophages and enhance intracellular bacterial killing. Finally, cotreatment with bosutinib and mitoxantrone, another chemotherapeutic in clinical use, results in an additive effect on bacterial clearance in vitro and in vivo. These results show that bosutinib stimulates macrophage clearance of bacterial infections through multiple mechanisms and could be used to boost the host innate immunity to combat drug-resistant bacterial infections.

FAK-LINC01089 negative regulatory loop controls chemoresistance and progression of small cell lung cancer.

The focal adhesion kinase (FAK) tyrosine kinase is activated and upregulated in multiple cancer types including small cell lung cancer (SCLC). However, FAK inhibitors have shown limited efficacy in clinical trials for cancer treatment. With the aim of identifying potential therapeutic strategies to inhibit FAK for cancer treatment, we investigated long non-coding RNAs (lncRNAs) that potentially regulate FAK in SCLC. In this study, we identified a long non-coding RNA LINC01089 that binds and inhibits FAK phosphorylation (activation). Expression analysis revealed that LINC01089 was downregulated in SCLC tissues and negatively correlated with chemoresistance and survival in SCLC patients. Functionally, LINC01089 inhibited chemoresistance and progression of SCLC in vitro and in vivo. Mechanistically, LINC01089 inhibits FAK activation by blocking binding with Src and talin kinases, while FAK negatively regulates LINC01089 transcription by activating the ERK signaling pathway to recruit the REST transcription factor. Furthermore, LINC01089-FAK axis mediates the expression of drug resist-related genes by modulating YBX1 phosphorylation, leading to drug resistance in SCLC. Intriguingly, the FAK-LINC01089 interaction depends on the co-occurrence of the novel FAK variant and the non-conserved region of LINC01089 in primates. In Conclusion, our results indicated that LINC01089 may serve as a novel high-efficiency FAK inhibitor and the FAK-LINC01089 axis represents a valuable prognostic biomarker and potential therapeutic target in SCLC.

PDIA2 has a dual function in promoting androgen deprivation therapy induced venous thrombosis events and castrate resistant prostate cancer progression.

Androgen deprivation therapy (ADT) is the first line of treatment for metastatic prostate cancer (PCa) that effectively delays the tumor progression. However, it also increases the risk of venous thrombosis event (VTE) in patients, a leading cause of mortality. How a pro-thrombotic cascade is induced by ADT remains poorly understood. Here, we report that protein disulfide isomerase A2 (PDIA2) is upregulated in PCa cells to promote VTE formation and enhance PCa cells resistant to ADT. Using various in vitro and in vivo models, we demonstrated a dual function of PDIA2 that enhances tumor-mediated pro-coagulation activity via tumor-derived extracellular vehicles (EVs). It also stimulates PCa cell proliferation, colony formation, and xenograft growth androgen-independently. Mechanistically, PDIA2 activates the tissue factor (TF) on EVs through its isomerase activity, which subsequently triggers a pro-thrombotic cascade in the blood. Additionally, TF-containing EVs can activate the Src kinase inside PCa cells to enhance the AR signaling ligand independently. Androgen deprivation does not alter PDIA2 expression in PCa cells but enhances PDIA2 translocation to the cell membrane and EVs via suppressing the clathrin-dependent endocytic process. Co-recruitment of AR and FOXA1 to the PDIA2 promoter is required for PDIA2 transcription under androgen-deprived conditions. Importantly, blocking PDIA2 isomerase activity suppresses the pro-coagulation activity of patient plasma, PCa cell, and xenograft samples as well as castrate-resistant PCa xenograft growth. These results demonstrate that PDIA2 promotes VTE and tumor progression via activating TF from tumor-derived EVs. They rationalize pharmacological inhibition of PDIA2 to suppress ADT-induced VTE and castrate-resistant tumor progression.

Morphological Changes Induced by TKS4 Deficiency Can Be Reversed by EZH2 Inhibition in Colorectal Carcinoma Cells.

The scaffold protein tyrosine kinase substrate 4 (TKS4) undergoes tyrosine phosphorylation by the epidermal growth factor receptor (EGFR) pathway via Src kinase. The TKS4 deficiency in humans is responsible for the manifestation of a genetic disorder known as Frank-Ter Haar syndrome (FTHS). Based on our earlier investigation, the absence of TKS4 triggers migration, invasion, and epithelial-mesenchymal transition (EMT)-like phenomena while concurrently suppressing cell proliferation in HCT116 colorectal carcinoma cells. This indicates that TKS4 may play a unique role in the progression of cancer. In this study, we demonstrated that the enhancer of zeste homolog 2 (EZH2) and the histone methyltransferase of polycomb repressive complex 2 (PRC2) are involved in the migration, invasion, and EMT-like changes in TKS4-deficient cells (KO). EZH2 is responsible for the maintenance of the trimethylated lysine 27 on histone H3 (H3K27me3). We performed transcriptome sequencing, chromatin immunoprecipitation, protein and RNA quantitative studies, cell mobility, invasion, and proliferation studies combined with/without the EZH2 activity inhibitor 3-deazanoplanocine (DZNep). We detected an elevation of global H3K27me3 levels in the TKS4 KO cells, which could be reduced with treatment with DZNep, an EZH2 inhibitor. Inhibition of EZH2 activity reversed the phenotypic effects of the knockout of TKS4, reducing the migration speed and wound healing capacity of the cells as well as decreasing the invasion capacity, while the decrease in cell proliferation became stronger. In addition, inhibition of EZH2 activity also reversed most epithelial and mesenchymal markers. We investigated the wider impact of TKS4 deletion on the gene expression profile of colorectal cancer cells using transcriptome sequencing of wild-type and TKS4 knockout cells, particularly before and after treatment with DZNep. Additionally, we observed changes in the expression of several protein-coding genes and long non-coding RNAs that showed a recovery in expression levels following EZH2 inhibition. Our results indicate that the removal of TKS4 causes a notable disruption in the gene expression pattern, leading to the disruption of several signal transduction pathways. Inhibiting the activity of EZH2 can restore most of these transcriptomics and phenotypic effects in colorectal carcinoma cells.

Abstract CT194: N-myristoylation inhibitor zelenirstat: New mechanistic insights and efficacy signals from a first in human phase I study

Abstract Introduction. Zelenirstat (PCLX-001) is a small molecule inhibitor of N-myristoylation, a process that involves addition of the fatty acid myristate to over 200 proteins by two N-myristoyltransferases (NMT1 and 2). These include Src family kinases and c-Abl. Inhibition of N-myristoylation by zelenirstat leads to the degradation of non-myristoylated proteins and cancer cell death. We conducted a phase I trial to evaluate the safety, tolerability, and maximally tolerated dose (MTD) of zelenirstat in patients with refractory cancer. Methods. Differential mass spectrometry was performed in NMT1 and NMT2 CRISPR/Cas9 KOs or zelenirstat-treated HAP1 cells to identify N-myristoylation-regulated proteins. Fully consented patients with advanced solid malignancies or refractory B-cell lymphomas were administered escalating doses of zelenirstat in 28-day cycles until progression or dose-limiting toxicity (DLT). Results. Proteomic analysis of cells with genetic or pharmacological NMT inhibition was performed to gain insights into NMT substrates and function. In addition to the rapid degradation of SFKs, which disrupts the pro-survival signaling of RTKs, we identified multiple respiratory complex I proteins as the most degraded, including NDUFAF4. NMT1 KO and zelenirstat treatment disrupted complex I leading to oxidative phosphorylation (OXPHOS) inhibition, which is essential for both cancer stem cell survival and metastasis. Continuous once-daily zelenirstat at 20 mg to 210 mg was well tolerated, with no dose-limiting toxicities in 24 patients up to and including 210 mg. Gastrointestinal DLTs were observed in the 280 mg cohort, establishing 210 mg as the MTD. Oral absorption was rapid, and pharmacokinetics were suitable for once daily dosing. Seven patients had stable disease as best response, including pancreatic, ovarian and colon cancer patients; one colon cancer patient with 5 prior lines of therapy continues 210 mg beyond 11 cycles with reduction in carcinoembryonic antigen (CEA) and tumor dimensions. Kaplan-Meier analysis revealed longer progression-free survival (log-rank p=0.033) and overall survival (p=0.026) in the patients treated at 210 mg (n=7) when compared with patients treated in lower dose cohorts (n=17). Conclusion. Zelenirstat has a unique dual impact on growth signaling and OXPHOS. Having previously demonstrated pre-clinical efficacy in hematologic cancers in vitro and in vivo, we now demonstrate the therapeutic potential of zelenirstat in patients with refractory advanced solid malignancies, warranting further clinical evaluation in these indications. Citation Format: Luc Gerard Berthiaume, Erwan Beauchamp, Jay Gamma, Rony Pain, Morris A. Kostiuk, Christopher R. Cromwell, Eman W. Moussa, Olivier Julien, Basil P. Hubbard, John Kuruvilla, Laurie H. Sehn, Jennifer Spratlin, Rahima Jamal, Randeep Sangha, John R. Mackey. N-myristoylation inhibitor zelenirstat: New mechanistic insights and efficacy signals from a first in human phase I study [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr CT194.

High throughput screening identifies dasatinib as synergistic with trametinib in low grade serous ovarian carcinoma

BackgroundLow grade serous ovarian carcinoma (LGSOC) is a distinct histotype of ovarian cancer characterised high levels of intrinsic chemoresistance, highlighting the urgent need for new treatments. High throughput screening in clinically-informative cell-based models represents an attractive strategy for identifying candidate treatment options for prioritisation in clinical studies. MethodsWe performed a high throughput drug screen of 1610 agents across a panel of 6 LGSOC cell lines (3 RAS/RAF-mutant, 3 RAS/RAF-wildtype) to identify novel candidate therapeutic approaches. Validation comprised dose-response analysis across 9 LGSOC models and 5 high grade serous comparator lines. Results16 hits of 1610 screened compounds were prioritised for validation based on >50% reduction in nuclei counts in over half of screened cell lines at 1000 nM concentration. 11 compounds passed validation, and the four agents of greatest interest (dasatinib, tyrosine kinase inhibitor; disulfiram, aldehyde dehydrogenase inhibitor; carfilzomib, proteasome inhibitor; romidepsin, histone deacetylase inhibitor) underwent synergy profiling with the recently approved MEK inhibitor trametinib. Disulfiram demonstrated excellent selectivity for LGSOC versus high grade serous ovarian carcinoma comparator lines (P = 0.003 for IC50 comparison), while the tyrosine kinase inhibitor dasatinib demonstrated favourable synergy with trametinib across multiple LGSOC models (maximum zero interaction potency synergy score 46.9). The novel, highly selective Src family kinase (SFK) inhibitor NXP900 demonstrated a similar trametinib synergy profile to dasatinib, suggesting that SFK inhibition is the likely driver of synergy. ConclusionDasatinib and other SFK inhibitors represent novel candidate treatments for LGSOC and demonstrate synergy with trametinib. Disulfiram represents an additional treatment strategy worthy of investigation.

Homocysteine-induced sustained GluN2A NMDA receptor stimulation leads to mitochondrial ROS generation and neurotoxicity

Homocysteine, a sulfur-containing amino acid derived from methionine metabolism, is a known agonist of N-methyl-D-aspartate receptor (NMDAR) and is involved in neurotoxicity. Our previous findings showed that neuronal exposure to elevated homocysteine levels leads to sustained low-level increase in intracellular Ca2+, which is dependent on GluN2A subunit-containing NMDAR (GluN2A-NMDAR) stimulation. These studies further showed a role of ERK MAPK in homocysteine-GluN2A-NMDAR-mediated neuronal death. However, the intracellular mechanisms associated with such sustained GluN2A-NMDAR stimulation and subsequent Ca2+ influx have remained unexplored. Using live-cell imaging with Fluo3-AM and biochemical approaches, we show that homocysteine-GluN2A NMDAR-induced initial Ca2+ influx triggers sequential phosphorylation and subsequent activation of the proline rich tyrosine kinase 2 (Pyk2) and Src family kinases, which inturn phosphorylates GluN2A-Tyr1325 residue of GluN2A-NMDARs to maintain channel activity. The continuity of this cycle of events leads to sustained Ca2+ influx through GluN2A-NMDAR. Our findings also show that lack of activation of the regulatory tyrosine phosphatase STEP, which can limit Pyk2 and Src family kinase activity further contributes to the maintenance of this cycle. Additional studies using live-cell imaging of neurons expressing a redox-sensitive GFP targeted to the mitochondrial matrix show that treatment with homocysteine leads to a progressive increase in mitochondrial reactive oxygen species generation, which is dependent on GluN2A-NMDAR-mediated sustained ERK MAPK activation. This later findingdemonstrates a novel role of GluN2A-NMDAR in homocysteine-induced mitochondrial ROS generation and highlights the role of ERK MAPK as the intermediary signaling pathway between GluN2A-NMDAR stimulation and mitochondrial reactive oxygen species generation.

HIF-1α-dependent upregulation of angiogenic factors by mechanical stimulation in retinal pigment epithelial cells.

Mechanical stimulation as a mimic of drusen formation in the eye increases the expression of angiogenic factors in retinal pigment epithelial (RPE) cells, but the underlying molecular mechanisms remain unclear. We investigated and characterized the effects of mechanical stimulation on the expression of angiogenic factors in RPE cells both in vitro and in a mouse model. Mechanical stimulation increased the expression of vascular endothelial growth factor (VEGF, encoded by VEGFA) and other angiogenesis-related genes in cultured RPE1 cells. The presence of hypoxia-inducible factor 1α (HIF-1α, encoded by HIF1A) was also increased, and both knockdown of HIF-1α and treatment with the HIF-1α inhibitor CAY10585 attenuated the effect of mechanical stimulation on angiogenesis factor gene expression. Signaling by the tyrosine kinase SRC and p38 mitogen-activated protein kinase was involved in HIF-1α activation and consequent angiogenesis-related gene expression induced by mechanical stimulation. Our results suggest that SRC-p38 and HIF-1α signaling are involved in the upregulation of angiogenic factors in RPE cells by mechanical stimulation. Such in vivo suppression of upregulated expression of angiogenesis-related genes by pharmacological inhibitors of HIF-1α suggests a new potential approach to the treatment of age-related macular degeneration.

Design, Synthesis and Biological Evaluation of 7-Azaindole Analogues as Novel Antiproliferative Agent and Tyrosine Protein Kinase SRC Inhibitors

Design, Synthesis and Biological Evaluation of 7-Azaindole Analogues as Novel Antiproliferative Agent and Tyrosine Protein Kinase SRC Inhibitors

Multi-target mechanism of polyherbal extract to treat diabetic foot ulcer based on network pharmacology and molecular docking

Introduction: Diabetic foot ulcer (DFU) potentially leads to loss of function, infections, hospitalization, lower-extremity amputation, and even death. The potential therapeutic efficacy of a polyherbal candidate named TIP-Heal was identified for treating DFU. TIP-Heal, which stands for Tinospora crispa, Isotoma longiflora, and Piper betle L var nigra, consists of extracts from these three herbs in a ratio of 2:1:1. The Indonesian population commonly uses these herbs due to their wound-healing properties. It is our interest to analyse the mechanism of the polyherbal extract using network pharmacology and molecular docking. Methods: This study uses network pharmacology and molecular docking methods to analyze the multi-target mechanism of active compounds in TIP-Heal extract for DFU treatment. The proteins targeted by the bioactive chemical present in TIP-Heal and DFU were identified within a particular dataset with the keyword "homo sapiens." The identified target proteins were assessed using gene ontology (GO) analysis, the Kyoto Encyclopaedia of Gene and Genomes (KEGG) pathways, protein-protein interactions (PPIs), and molecular docking. Results: The critical proteins obtained were AKT serine/threonine kinase 1 (AKT1), caspase-3 (CASP3), epidermal growth factor receptor (EGFR), proto-oncogene tyrosine-protein kinase Src (SRC) and matrix metalloproteinase-9 (MMP-9). Several compounds, namely PubChem (Compound Identifier=CID: 5319898), 3-epiursolic acid, palmitic acid, and alpha-linolenic acid showed great potential as viable candidates to facilitate the healing process of DFU. Conclusion: The findings of this study indicate that the TIP-Heal extract has the potential to be used as a natural herbal treatment for DFUs with the involvement of AKT1, CASP3, EFGR, and SRC proteins.

Isoalantolactone exerts anti-melanoma effects via inhibiting PI3K/AKT/mTOR and STAT3 signaling in cell and mouse models.

Although the anti-cancer activity of isoalantolactone (IATL) has been extensively studied, the anti-melanoma effects of IATL are still unknown. Here, we have investigated the anti-melanoma effects and mechanism of action of IATL. MTT and crystal violet staining assays were performed to detect the inhibitory effect of IATL on melanoma cell viability. Apoptosis and cell cycle arrest induced by IATL were examined using flow cytometry. The molecular mechanism of IATL was explored by Western blotting, confocal microscope analysis, molecular docking, and cellular thermal shift assay (CETSA). A B16F10 allograft mouse model was constructed to determine the anti-melanoma effects of IATL in vivo. The results showed that IATL exerted anti-melanoma effects in vitro and in vivo. IATL induced cytoprotective autophagy in melanoma cells by inhibiting the PI3K/AKT/mTOR signaling. Moreover, IATL inhibited STAT3 activation both in melanoma cells and allograft tumors not only by binding to the SH2 domain of STAT3 but also by suppressing the activity of its upstream kinase Src. These findings demonstrate that IATL exerts anti-melanoma effects via inhibiting the STAT3 and PI3K/AKT/mTOR signaling pathways, and provides a pharmacological basis for developing IATL as a novel phytotherapeutic agent for treating melanoma clinically.

Abstract 614: Src family kinase inhibition demonstrates antitumor activity in vitro and in patient-derived xenograft models of human cholangiocarcinoma

Abstract Background: Cholangiocarcinoma (CCA) is a lethal and heterogenous malignancy of the biliary tree characterized by perineural invasion. About 85% of CCA show activation of YAP signaling, which promotes proliferation, anti-apoptosis, and therapeutic resistance. YAP signaling is mediated by Src family kinases (SFK) by phosphorylation of YAP (pYAPy357). NXP900 is a first-in-class, highly selective, SFK inhibitor with a novel mechanism of action which locks SFK in its closed and inhibited conformation, in contrast to other Src inhibitors such as dasatinib. A phase 1a study of NXP900 in patients with advanced solid tumors was recently initiated. Here, we examined treatment responses of CCA to NXP900 in vitro and in vivo. Methods: Cell viability was determined in seven CCA cell lines by CellTiter-Glo. Cell death and apoptosis were assayed by PI/Hoechst and Caspase-Glo 3/7 assay. CalcuSyn software was used to determine synergistic drug effects. Immunoblot analysis, RT-PCR, and IF staining of YAP localization were used to evaluate YAP knockdown. Clones of human CCA cell lines resistant to NXP900 were generated through escalating exposure to NXP900 over 6 months. Five patient-derived xenograft (PDX) tumors were expanded into flanks of NOD/SCID mice. Tumor bearing mice were randomized 1:1 with 5 mice per arm and treated with vehicle or NXP900 (40 mg/kg) once daily for up to 4 weeks. To determine drivers of sensitivity and resistance, predicted sensitivity scores based on tumor growth were linked to multi-omics (RNA seq and [phospho-]proteomics) of our PDX models. Results: All cell lines were sensitive to NXP900 with IC50 values between 7nM-15µM; IC50’s of resistant clones were 1000 times higher. NXP900 induced more cell death compared to vehicle, which appeared to be through apoptosis. NXP900 inhibited pSrc, decreased pYAPy357, and upregulated inactive pYAPs127. Correspondingly, decreased YAP target gene (Cyr61, NUAK, and CTGF) levels and a nuclear-to-cytoplasmic translocation were observed. NXP900/GemCis combination therapy increased cell death demonstrated a synergistic effect (Combination Indices <1) at all concentrations. In our PDX models, treatment was associated with a significant decrease in tumor growth in 3 models (mean fold change 3.1 vs. 14.2; 1.0 vs. 7.6; 1.4 vs. 2.6). The major resistant signature was the TRK signaling network, involved in nerve growth factor binding activity, while drivers of sensitivity included the SRC network. Conclusion: NXP900 demonstrated therapeutic activity in vitro and in human PDX models. We are currently performing multi-omic approaches in NXP900 sensitive and resistant cell lines to unravel determinants of activity and resistance. Additional in vivo studies will be performed to determine effects of NXP900/GemCis and NXP900/anti-PDL-1 combination therapy. Citation Format: Hendrien Kuipers, Jennifer L. Tomlinson, Danielle M. Carlson, Amro M. Abdelrahman, Erik Jessen, Jack W. Sample, Nathan W. Werneburg, Hannah E. Stumpf, Mark J. Truty, Sumera I. Ilyas, Gregory J. Gores, Rory L. Smoot. Src family kinase inhibition demonstrates antitumor activity in vitro and in patient-derived xenograft models of human cholangiocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 614.

Abstract 3017: Lack of MCPIP1 enzymatic activity leads to neoplastic transformation of normal kidney cells

Abstract Introduction: The MCPIP1 protein (Monocyte Chemotactic Protein-1 Induced Protein) is involved in the negative regulation of inflammation due to its RNase activity. A growing number of publications suggest that the MCPIP1 protein may influence the development of cancer by regulation of factors involved in angiogenesis, proliferation and cell death. We believe, that MCPIP1 may be a potential tumor suppressor. Our recent results show that lack of enzymatic activity of MCPIP1 leads to increased tumor growth, angiogenesis and metastasis. The main aim of our research is to study the importance of MCPIP1 for neoplastic transformation of normal, epithelial kidney cells, as well as acquirement of stemness features. Material and Methods: To examine the effect of MCPIP1 in normal epithelial cell lines TCMK-1, RPTEC-Tert1 and HK-2 were transduced to overexpress mutated, inactive form of MCPIP1 (pLIX D141N) with proper control (pLIX PURO). We analyzed cells clonogenicity, as well as the levels of CSCs markers by western blot, NGS and qPCR. Next, cells were injected subcutaneously into NOD-SCID mice to check if mutation of MCPIP1 will induce tumor growth in vivo. Mice blood was analyzed for the presence of circulating tumor cells (CTCs), and secretion of cytokines by Proteome Profiler assay. Tumors were evaluated by multiple IHC stainings, as well as western blot and qPCR. Results and Discussion: We have shown that the mutation of MCPIP1 increases the clonogenicity of kidney cells. Moreover, pLIX D141N cells were characterized by higher levels of the c-Met receptor, vimentin, Twist, CD44 and c-Myc, which are the markers of epithelial to mesenchymal transition and cancer stemness. The obtained results suggest that the loss of the MCPIP1 protein causes the acquisition of the characteristics of neoplastic cells. Next, we checked whether MCPIP1 mutation in normal cells predisposes them to proliferate when administered to mice. We found that, D141N mutation caused the growth of large tumors with higher expression of Cd44, Klf4 and Myc and number of CTCs, while the control cells injected into mice developed only local fibrosis with high expression of aSMA. pLIX D141N tumors secreted CXCL16 into mice plasma, and were characterized by increased phosphorylation of c-Met, Src kinase and c-Myc. Conclusions: We believe that the MCPIP1 protein may be a marker of tumor initiation and play a key role in neoplastic transformation by regulating the changes in cell phenotype and levels of CSCs markers. This study was supported by National Science Center grants no. 2022/47/B/NZ5/02724, 2022/45/B/NZ5/01973 and MNS 15/2021. Citation Format: Paulina Marona, Iga Piasecka, Judyta Gorka, Oliwia Kwapisz, Rafal Myrczek, Ewelina Pospiech, Jolanta Jura, Katarzyna Miekus. Lack of MCPIP1 enzymatic activity leads to neoplastic transformation of normal kidney cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3017.

Abstract 6496: Assessment of tyrosine kinase inhibitor bypass mechanisms in lung cancer using single cell multiomics, patient derived organoids and rationally designed drug screens

Abstract Lung cancer (LC) is the leading cause of cancer-induced mortality, with 350 patients in the US dying from LC daily, the majority from non-small cell lung cancer (NSCLC). Molecular-targeted therapy such as tyrosine kinase inhibitors (TKI) are effective in first and subsequent lines of therapy against oncogenic drivers (KRAS, EGFR, or ALK fusions, others); however, nearly all treated patients progress eventually and run out of therapeutic options. Key reasons are the lack of predictive biomarkers and LC models that reflect the underlying heterogeneity and different phenotypic cell states among subclones within and between patients. Here, we developed a novel platform for refining the resolution of molecular interrogation to the single cell level, using single cell spatial multiomic (scSpMO), coupled with rational designed drug screens in patient-derived organoids (PDOs) from LC biopsies obtained upon progression. By deriving LC PDOs in epithelial and tumor microenvironment (TME) conditions to faithfully maintain the histological and genetic features of their respective LC tissues, we identified enrichment conditions for LC PDOs guided by tumor mutation variants and activated pathways. PDOs are maintained under the same treatment condition in the clinic (e.g., Lorlatinib) and drug testing is tailored to identify next lines of combined therapy. With six patients enrolled and others in ongoing studies, single cell PDOs (at >80% establishment rate) were used to determine key molecular bypass mechanism for resistance to TKI, including on-target variants, driver bypass, lineage plasticity and acquired resistance. Functional assays for acquired resistance such as PI3K/AKT signaling, Src kinase, BRAF fusion and MET hyperactivity allow the identification of potential novel combined lines of therapy for each patient through the compassionate care program and/or trial participation. Our platform when empowered with datasets of bulk, scRNA-seq and scSpMO signatures of primary human NSCLC, together with PDOs offer the ability to perform analysis of LC phenotypes such as lineage transformation in high-content assays, predict and/or act on resistance to therapy and provides a path for precision medicine-guided immediate impact on patient care. Citation Format: Liqiong Liu, Ann Strange, Schuyler Lee, Bifeng Gao, Daniel Merrick, Tejas Patil, James DeGregori, D Ross Camidge, Sharon R. Pine, Hatim E. Sabaawy. Assessment of tyrosine kinase inhibitor bypass mechanisms in lung cancer using single cell multiomics, patient derived organoids and rationally designed drug screens [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6496.

Abstract 1675: Investigating the role of SRC-family kinases in MPNST tumorigenesis

Abstract Purpose: Malignant peripheral nerve sheath tumor (MPNST), an aggressive soft-tissue sarcoma, is the leading cause of mortality in patients with neurofibromatosis type 1. Over the past two decades, significant advancement has been made in understanding the pathogenesis of MPNST. Still, this progress has not improved overall survival of patients. Therefore, ongoing efforts to identify alterations contributing to the transformation, progression, and metastasis of MPNST are essential for pinpointing targetable oncogenic signaling pathways for this cancer. Previous studies have revealed an increase in YAP/TAZ activity through both LATS1/2-dependent and LATS1/2-independent mechanisms, suggesting that YAP/TAZ hyperactivity may act as a convergence point for MPNST pathogenesis. YES and SRC, two members of the SRC-family kinases (SFKs), were shown to phosphorylate LATS1/2 and YAP/TAZ, and stimulate the transcriptional activity of YAP/TAZ by promoting their nuclear accumulation in hepatocellular carcinoma and colorectal cancer cells. Thus, we have investigated whether YES and SRC are involved in the pathogenesis of MPNSTs. Experimental Design: To assess whether the SFK-YAP/TAZ signaling axis is implicated in the development of MPNST, we have used genetic and pharmacological approaches to investigate the roles of YES and SRC in the proliferation of a subset of human MPNST cell lines, and in the growth of xenografted MPNST tumors in ectopic and orthotopic mouse models. We also performed a phosphoproteome and a transcriptome analysis of YES and SRC-depleted MPNST cells to assess their oncogenic signaling network. Lastly, we evaluated the clinical significance of phospho-SFK and YAP in human MPNST TMAs. Results: Our findings reveal that YES and SRC activity play an essential and redundant role in sustaining the proliferation of human MPNST cell lines. Further, the simultaneous depletion of both kinases impedes tumor growth in xenograft MPNST mouse models. Transcriptomic analyses identified YAP and TAZ as potential effectors of SFKs, as well as other family of transcription factors. Finally, TMA data suggested a correlation between the expressions of YAP, YAP/TAZ, and phospho-SFK and the progression of MPNST. Citation Format: Elamine Zereg, Laure Voisin, Karl Grenier, Mathieu Courcelles, Sungmi Jung, Pierre Thibault, Sylvain Meloche. Investigating the role of SRC-family kinases in MPNST tumorigenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1675.

Abstract 3913: Molecular mechanism of action and targets of glucocorticoids in lymphoma therapy

Abstract Prednisone is an anti-inflammatory glucocorticoid (GC) that is cytotoxic for normal and malignant B cells and, on this basis, has long been included in combination chemotherapies to treat aggressive B-cell lymphomas. However, the mechanisms by which GCs kill malignant lymphoma cells still largely remains elusive due to the pleiotropic consequences of GC binding to the glucocorticoid receptor (GR; NR3C1), a ligand-induced transcription factor. This prompted us to search for critical direct targets of GC action with the hypothesis that GCs may inhibit key survival pathways in lymphomas. To determine effector genes that increase glucocorticoid toxicity, we performed genome-wide CRISPR-Cas9 screens in the presence and absence of prednisolone. Screens in cell line models of Burkitt lymphoma (BL) and diffuse large B cell lymphoma (DLBCL) revealed strong synergy between GC treatment and inactivation of genes encoding components of the B cell receptor (BCR) signaling pathway (≥2 SD). To identify direct transcriptional GR targets and binding sites genome-wide, we performed the cleavage under targets and release using nuclease (CUT&RUN) assay and integrated this dataset with RNA-seq upon GC treatment. The pairwise comparison analysis (p<0.05) for GR-bound genes across BL and DLBCL cell lines revealed a significant enrichment of negative regulators of BCR/PI3K signaling and BCR-related tumor suppressors (LAPTM5, KLHL14, ARID5B, DDIT4, DAPP1, INPP5D, and CSK). Interestingly, expression of CSK, an inhibitor of all Src-family kinases (SFKs), was repressed while that of other BCR negative regulators were directly activated by binding of GR. Combinatorial deletion of activated genes counteracted the toxicity of GC up to 83.9%. In contrast, depletion of CSK paradoxically increased the cytotoxic effects of GC in BCR-dependent aggressive lymphoma with decreased proximal BCR signaling. To gain further insights on CSK function in aggressive lymphomas, we performed phospho-proteome and ubiquitinome profiling by treating lymphoma models with a small molecule inhibitor of CSK kinase activity (CSKi). The CSKi treatment initially increased constitutive BCR signaling, as expected, but then triggered exuberant ubiquitination of the LYN, HCK and BLK, leading to their proteasomal degradation. Consequently, the CSKi blocked BCR-dependent NF-κB activation in ABC DLBCL models and BCR-dependent PI3 kinase activation in models of GCB DLBCL and BL. In summary, inhibition of oncogenic BCR signaling is a major mode of action for GCs. Furthermore, small molecule inhibition of CSK kinase activity potentiated the effect of GCs on oncogenic BCR signaling and strongly synergized with GCs in killing ABC and GCB DLBCL models in vitro and preventing the growth of ABC and GCB DLBCL and patient-derived xenografts, warranting the development of clinical-grade CSK inhibitors for the treatment of these aggressive cancers. Citation Format: Jaewoo Choi, Michele Ceribelli, James D. Phelan, Björn Häupl, Da Wei Huang, George W. Wright, Tony Hsiao, Vivian Morris, Francesco Ciccarese, Boya Wang, Sean Corcoran, Sebastian Scheich, Xin Yu, Weihong Xu, Yandan Yang, Hong Zhao, Joyce Zhou, Grace Zhang, Jagan Muppidi, Giorgio Ga Inghirami, Thomas Oellerich, Craig J. Thomas, Wyndham H. Wilson, Louis M. Staudt. Molecular mechanism of action and targets of glucocorticoids in lymphoma therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3913.

Abstract 1043: Identification of the novel immune synapse-localized proteome for immuno-oncology using Microscoop-induced targeted photo-biotinylation

Abstract Although tens of immune synapse (IS) protein species are known, there remain many unknown IS-localized protein species. Understanding the proteome of the IS between a target cell and a lymphocyte is crucial for advancing immuno-oncology. The low abundance of ISs and the lack of a definitive enrichment marker have limited efficient proteomic profiling. Here, we used Microscoop™, an innovative system that integrates microscopy, machine learning, and targeted photo-biotinylation to enable precise and spatially specific enrichment of IS proteins, thereby facilitating proteomic discovery of the IS. We utilized Raji B cells as antigen-presenting cells (APCs) and induced IS formation with Jurkat T cells. The system first employed immunofluorescence imaging of CD3, a common IS marker in Jurkat T cells, and used a convolutional neural network-based deep learning algorithm to train the recognition of ISs from CMTPX-stained Raji B cells. Our automated system then successfully achieved spatially-targeted biotin-tagging of proteins at ISs through thousands of fields of view of imaging, deep learning-driven pattern generation, and photochemical labeling. Subsequent streptavidin pull-down and mass spectrometry analysis enabled the identification of IS-specific proteins. Remarkably, our spatial proteomic approach led to the isolation and identification of hundreds of different species at the IS interface, including proteins associated with key components of T-cell receptor (TCR) signaling pathways such as the TCR/CD3 complex, Src and Tec family tyrosine kinases, and pivotal NF-kB signaling proteins. Beyond these, we also identified a significant enrichment of proteins not previously associated with the IS. Our study not only illuminates previously unknown aspects of immune regulation at the IS interface, but also shedded light on proteins potentially important for immuno-oncology. These insights have significant implications for cancer research, particularly in understanding and manipulating immune responses for therapeutic purposes. Citation Format: Weng-Man Chong, Cecile Li, Chun-Kai Huang, Chantal Hoi Yin Cheung, Hsiao-Jen Chang, Chia-Wen Chung, Jung-Chi Liao. Identification of the novel immune synapse-localized proteome for immuno-oncology using Microscoop-induced targeted photo-biotinylation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1043.