Src Family Kinase Inhibitor Research Articles

Abstract 101: JAK-independent STAT5 signaling as a node of therapeutic vulnerability in glioblastoma

Abstract Glioblastoma (GBM), the most common primary brain tumor in adults, comprises all WHO Grade IV gliomas of astrocytic origin. Despite extensive characterization of the molecular and genetic features of GBM, there remains a paucity of viable treatment options, and this disease confers a dismal 5% 5-year survival rate. Genomic amplification of epidermal growth factor receptor (EGFR) occurs in 40-60% of primary GBM. Half of all EGFR-amplified cases of GBM also harbor EGFRvIII, a constitutively active, truncated variant of EGFR. EGFR and EGFRvIII transduce oncogenic signals via cytoplasmic mediators, including the JAK/STAT signal transduction pathway. The oncogenic role of STAT3 has been well-studied in CNS malignancies; STAT3 has been found to induce the expression of target genes involved in the proliferative and invasive phenotypes of GBM. However, little is known about the mechanism and role of STAT5 activation in GBM progression. Microenvironmental cues and EGFRvIII signaling, lost in adherent cell culture, are required for STAT5 activation, making STAT5 difficult to study by conventional cell culture methods. Our recent data from patient-derived xenograft (PDX) cultures demonstrates that activation of STAT5 is important in GBM cell invasion and survival. Further, STAT5 activation is present predominantly at the invasive periphery of the tumor. In this study, we seek to characterize the mechanism and role of STAT5 activation downstream of EGFRvIII in GBM. RNA sequencing of PDX tumors expressing stable shRNA knockdown of STAT3 or STAT5 indicate both redundant and distinct transcriptional changes induced by STAT3 and STAT5 in GBM. We also find that while STAT3 activation is dependent upon JAK1/2, STAT5 activation is JAK-independent downstream of EGFRvIII in GBM. Depletion of JAK1/2 expression by siRNA oligonucleotides and pharmacologic inhibition of JAK1/2 suppressed STAT3 activation but not STAT5. In fact, targeting JAK2 with kinase inhibitor WP1066 suppressed STAT3 activation, but increased activation of STAT5 in GBM cells expressing EGFR/EGFRvIII. Inhibition of Src family kinases (SFK), a family of upstream signaling effectors of the JAK/STAT pathway, attenuates both STAT3 and STAT5 activation. These results suggest that activation of STAT5 may provide a therapeutic escape mechanism to JAK-STAT3 inhibition in GBM, necessitating dual STAT3 and STAT5 inhibition to successfully inhibit the distinct oncogenic signals transmitted by STAT3 and STAT5 to promote GBM cell survival and resistance. Citation Format: Mylan Blomquist, Serdar Tuncali, Christopher Sereduk, Jean Kloss, Joseph Loftus, Nhan Tran. JAK-independent STAT5 signaling as a node of therapeutic vulnerability in glioblastoma [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 101.

AXL/CDCP1/SRC axis confers acquired resistance to osimertinib in lung cancer

Osimertinib, a third-generation EGFR-TKI, has nowadays been applied to non-small cell lung cancer harboring activated EGFR mutation with or without T790M, but ultimately develop resistance to this drug. Here we report a novel mechanism of acquired resistance to osimertinib and the reversal of which could improve the clinical outcomes. In osimertinib-resistant lung cancer cell lines harboring T790M mutation that we established, expression of multiple EGFR family proteins and MET was markedly reduced, whereas expression of AXL, CDCP1 and SRC was augmented along with activation of AKT. Surprisingly, AXL or CDCP1 expression was induced by osimertinib in a time-dependent manner up to 3 months. Silencing of CDCP1 or AXL restored the sensitivity to osimertinib with reduced activation of SRC and AKT. Furthermore, silencing of both CDCP1 and AXL increased the sensitivity to osimertinib. Either silencing of SRC or dasatinib, a SRC family kinase (SFK) inhibitor, suppressed AKT phosphorylation and cell growth. Increased expression of AXL and CDCP1 was observed in refractory tumor samples from patients with lung cancer treated with osimertinib. Together, this study suggests that AXL/SFK/AKT and CDCP1/SFK/AKT signaling pathways play some roles in acquired osimertinib resistance of non-small cell lung cancer.

Combined analysis of T cell activation and T cell-mediated cytotoxicity by imaging cytometry

Immunotherapies for the treatment of cancer have spurred the development of new drugs that seek to harness the ability of T cells to recognize and kill malignant cells. There is a substantial need to evaluate how these experimental drugs influence T cell functional outputs in co-culture systems that contain cancerous cells. We describe an imaging cytometry-based platform that can simultaneously quantify activated T cells and the capacity of these T cells to kill cancer cells. Our platform was developed using the Nur77-GFP reporter system because GFP expression provides a direct readout of T cell activation that is induced by T cell antigen receptor (TCR) signaling. We combined the Nur77-GFP reporter system with a cancer cell line that displays a TCR-specific antigen and evaluated the relationship between T cell activation and cancer cell death. We demonstrate that imaging cytometry can be used to quantify the number of activated cytotoxic CD8+ T cells (CTLs) and the capacity of these CTLs to recognize and kill adherent MC38 cancer cells. We tested whether this platform could evaluate heterogenous lymphocyte populations by quantifying the proportion of antigen-specific activated T cells in co-cultures that contain unresponsive lymphocytes. The effects of a SRC family kinase inhibitor on CTL activation and MC38 cell death were also determined. Our findings demonstrate that the Nur77-GFP reporter system can be used to evaluate the effects of diverse treatment conditions on T cell-cancer co-cultures in a microtiter plate-based format by imaging cytometry. We anticipate the combined analysis of T cell activation with T cell-mediated cancer cell death can be used to rapidly assess immuno-oncology drug candidates and T cell-based therapeutics.

27-Hydroxycholesterol induces expression of zonula occludens-1 in monocytic cells via multiple kinases pathways

Zonula occludens (ZO)-1, a tight-junction protein (TJP), is expressed in dendritic cells (DCs) but not in monocytes, and 27-hydroxycholesterol (27OHChol) drives the differentiation of monocytes into DCs. Because the effects of 27OHChol on ZO-1 are not yet clearly defined, we investigated whether 27OHChol induces expression of the TJP. The treatment of human THP-1 monocytic cells with 27OHChol resulted in the elevated transcript levels of ZO-1 but not of ZO-2 or -3. 27OHChol increased the total amount of ZO-1 protein in the cells as well as its level on the cells surface. Cholesterol, however, did not influence expression of ZO-1. And, the expression of ZO-1 protein was mediated by endoplasmic reticulum (ER)-to-Golgi body transport system. Pharmacological kinase inhibition with LY294002 (a PI3K inhibitor), U0126 (a MEK/ERK inhibitor), or PP2 (a Src family kinase inhibitor) resulted in impaired ZO-1 expression at both transcript and protein levels. Drugs that are reported to suppress DC differentiation also inhibited 27OHChol-mediated expression and the localization of ZO-1, indicating the coincidence of ZO-1 upregulation and DC differentiation. These results suggest that ZO-1 is differentially expressed while monocytes differentiate into DCs in the presence of 27OHChol via pathways in which distinct signaling molecules are involved.

Targeting the FAK-Src Complex in Desmoplastic Small Round Cell Tumors, Ewing Sarcoma, and Rhabdomyosarcoma

Desmoplastic small round cell tumors (DSRCTs), Ewing sarcoma (ES), and alveolar and embryonal rhabdomyosarcoma (ARMS and ERMS) are malignant sarcomas typically occurring at young age, with a poor prognosis in the metastatic setting. New treatment options are necessary. Src family kinase inhibitor dasatinib single-agent treatment has been investigated in a phase 2 study in patients with advanced sarcomas including ES and RMS but failed as a single agent in these subtypes. Since previous studies demonstrated high FAK and Src activities in RMS and ES tissue and cell lines, and dasatinib treatment was shown to upregulate activated FAK, we hypothesized that FAK-Src combination treatment could potentially be an interesting treatment option for these tumor types. We examined the effects of targeting the FAK-Src complex by addressing (p)FAK and (p)Src expressions in tumor sections of DSRCT (n = 13), ES (n = 68), ARMS (n = 21), and ERMS (n = 39) and by determining the antitumor effects of single and combined treatment with FAK inhibitor defactinib and multikinase (Abl/SFK) inhibitor dasatinib in vitro on cell lines of each subtype. In vivo effects were assessed in DSRCT and ERMS models. Concurrent pFAK and pSrc expressions (H-score >50) were observed in DSRCT (67%), ES (6%), ARMS (35%), and ERMS (19%) samples. Defactinib treatment decreased pFAK expression and reduced cell viability in all subtypes. Dasatinib treatment decreased pSrc expression and cell viability in each subtype. Combination treatment led to a complete reduction in pFAK and pSrc in each cell line and showed enhanced cell viability reduction, drug synergy, DNA damage induction, and a trend toward higher apoptosis induction in DSRCT, ERMS, and ARMS but not in ES cells. These promising in vitro results unfortunately do not translate into promising in vivo results as we did not observe a significant effect on tumor volume in vivo, and the combination did not show superior effects compared to dasatinib single-agent treatment.

Ephrin B Activate Src Family Kinases in Fibroblasts Inducing Stromal Remodeling in Prostate Cancer.

Simple SummaryCarcinoma associated fibroblasts (CAF) play a critical role in the tumor microenvironment (TME) of prostate cancer (PCa). Ephrin receptors (Eph) and ligands (EFN) have been implicated in distinct types of cancers. Alterations on EphB receptors are frequently found in PCa, but the role of ligands (EFNB1, EFNB2, EFNB3) activation in prostate fibroblasts and consequent effects on PCa is not known. We found increased EFNB ligands in fibroblasts isolated from PCa tissues. In this study, we assessed the effects of elevated stromal EFNB ligands on PCa tumor growth. Increased EFNB1 and EFNB3 expression transformed normal fibroblasts into CAF phenotypes through activation of Src family kinases. The secretome of EFNB-expressing CAF increased PCa cell proliferation and promoted TME remodeling. Overall, EFNB activation in CAF may participate in PCa progression via the release of soluble factors that modulate the surrounding tumor environment, which, in turn, promote prostate tumor growth and invasion.Through stromal-epithelial interactions, carcinoma associated fibroblasts (CAF) play a critical role in tumor growth and progression. Activation of erythrophoyetin-producing human hepatocellular (Eph) receptors has been implicated in cancer. Eph receptor interactions with Ephrin ligands lead to bidirectional signals in the recipient and effector cells. The consequences of continuous reverse Ephrin signaling activation in fibroblasts on prostate cancer (PCa) is unknown. When compared to benign prostate fibroblast, CAF displayed higher expression of Ephrin B1, B2, and B3 ligands (EFNB1, EFNB2, and EFNB3). In this study, we found that continuous activation of EFNB1 and EFNB3 in a benign human prostate stromal cell line (BHPrS1) increased the expression of CAF markers and induced a CAF phenotype. BHPrS1EFNB1 and BHPrS1EFNB3 displayed a pro-tumorigenic secretome with multiple effects on neovascularization, collagen deposition, and cancer cell proliferation, overall increasing tumorigenicity of a premalignant prostate epithelial cell line BPH1 and PCa cell line LNCaP, both in vitro and in vivo. Inhibition of Src family kinases (SFK) in BHPrS1EFNB1 and BHPrS1EFNB3 suppressed EFNB-induced ɑ-SMA (Alpha-smooth muscle actin) and TN-C (Tenascin-C) in vitro. Our study suggests that acquisition of CAF characteristics via SFK activation in response to increased EFNB ligands could promote carcinogenesis via modulation of TME in PCa.

Radotinib inhibits multiple myeloma cell proliferation via suppression of STAT3 signaling.

Multiple myeloma (MM) is a hematological cancer causing from accumulated abnormal plasma cells. STAT3 overexpression in MM appears to be mediated by a variety of factors, and it may be associated with an adverse prognosis and play a role in microenvironment-dependent treatment resistance. Unfortunately, MM remains an incurable disease, as relapse is very common. Therefore, there is urgent need to develop new treatment options for MM. Radotinib is a novel anti-cancer drug, currently approved in South Korea for the treatment of chronic myeloid leukemia patients. It is an oral, multitargeted inhibitor of receptor tyrosine kinases, including BCR-ABL, c-KIT, PDGFR, and Src family kinases. However, little is known about the effects of radotinib on multiple myeloma cells. However, little is known about the effects of radotinib on multiple myeloma cells. But even tinip almost not known about the impact of multiple myeloma cells. Moreover, nothing is known about how it affects STAT3 and JAK2. In this study, we analyzed the effect of radotinib on multiple myeloma cells. Herein, Moreover, nothing is known about how it. Moreover, not all is known about how the affects STAT3 and JAK2. We investigated the effect of radotinib on the STAT3 signaling pathway in MM cells, including several MM cell lines and mouse models. So we investigated the effect of radotinib on MM cells, including several MM cell lines and mouse models. Interestingly, radotinib induced apoptosis, and inhibited cell proliferation in MM cells including RPMI-8226, MM.1S, U266B1, and IM-9 cells. Moreover, radotinib treatment significantly increased the number Annexin V-positive cells and G0/G1-phase cells. In addition, radotinib treatment in various MM cells strongly suppressed the activity and expression of STAT3 and JAK2 proteins. We also observed that diverse proteins related to the STAT3 signaling pathway, including c-Myc, Bcl-xL, Mcl-1, cyclin D1 and cyclin D3, were powerfully inhibited by radotinib treatment in MM cells. Furthermore, radotinib significantly suppressed MM cell growth in a xenograft animal model using IM-9 cells. In conclusion, radotinib may play an important role as a candidate agent for MM treatment.

Combined analysis of T cell activation and T cell-mediated cytotoxicity by imaging cytometry

Abstract Immunotherapies have transformed the treatment of cancer through the development of drugs that allow T cells to recognize and kill malignant cells. With this has come a need to evaluate how these drugs affect T cell function. We developed an imaging cytometry-based method that can simultaneously assess T cell activation and the functional ability of these activated T cells to kill cancer cells. We used the Nur77-GFP reporter, which expresses GFP upon T cell receptor signaling, as a readout of T cell activation. We combined this reporter system with an MC38 colon adenocarcinoma cell line expressing a TCR-specific antigen that is used as a model tumor antigen (TA). We show that in T cell-cancer co-culture systems, we are able to quantify the number of activated cytotoxic CD8+ T cells and their ability to recognize and kill cancer cells by imaging cytometry. We tested the sensitivity of this platform by quantifying the proportion of antigen-specific activated T cells in co-cultures with varying ratios of unresponsive T cells, mimicking heterogenous lymphocyte populations. To test our system in a pharmacological context, we evaluated the effects of a SRC family kinase inhibitor on T cell activation and cancer cell death. In each case, we were able to quantify the proportion of activated T cells and the extent of TA-specific cancer cell death. Our findings show that the Nur77-GFP reporter system combined with imaging cytometry is a fast and reproducible system to evaluate how different treatment conditions influence TA recognition by T cells using T cell-cancer co-cultures. The simultaneous analysis of T cell activation with T cell-mediated cancer cell death can be used to rapidly assess immuno-oncology drug candidates and T cell-based therapeutics. Supported by The Elsa U. Pardee Foundation (AHC), the V Foundation Scholar Award (AHC), the Cancer Biology Training Program T32CA009676 (MKC), the Pharmacological Sciences Training Program T32GM140223 (CES), and a Charles W. Edmunds Pharmacology Fellowship (YZ). This study was also supported by resources provided by the U-M Flow Cytometry Core and the U-M Rogel Cancer Center (P30CA046592).

Stimulation of the EP3 receptor causes lung oedema by activation of TRPC6 in pulmonary endothelial cells.

Prostaglandin E2 (PGE2) increases pulmonary vascular permeability by activation of the PGE2 receptor 3 (EP3), which may explain adverse pulmonary effects of the EP1/EP3 receptor agonist sulprostone in patients. In addition, PGE2 contributes to pulmonary oedema in response to platelet-activating factor (PAF). PAF increases endothelial permeability by recruiting the cation channel transient receptor potential canonical 6 (TRPC6) to endothelial caveolae via acid sphingomyelinase (ASMase). Yet, the roles of PGE2 and EP3 in this pathway are unknown. We hypothesised that EP3 receptor activation may increase pulmonary vascular permeability by activation of TRPC6, and thus, synergise with ASMase-mediated TRPC6 recruitment in PAF-induced lung oedema. In isolated lungs, we measured increases in endothelial calcium (ΔCa2+) or lung weight (Δweight), and endothelial caveolar TRPC6 abundance as well as phosphorylation. PAF-induced ΔCa2+ and Δweight were attenuated in EP3-deficient mice. Sulprostone replicated PAF-induced ΔCa2+ and Δweight which were blocked by pharmacological/genetic inhibition of TRPC6, ASMase or Src-family kinases (SrcFK). PAF, but not sulprostone, increased TRPC6 abundance in endothelial caveolae. Immunoprecipitation revealed PAF- and sulprostone-induced tyrosine-phosphorylation of TRPC6 that was prevented by inhibition of phospholipase C (PLC) or SrcFK. PLC inhibition also blocked sulprostone-induced ΔCa2+ and Δweight, as did inhibition of SrcFK or inhibitory G-protein (Gi) signalling. EP3 activation triggers pulmonary oedema via Gi-dependent activation of PLC and subsequent SrcFK-dependent tyrosine phosphorylation of TRPC6. In PAF-induced lung oedema, this TRPC6 activation coincides with ASMase-dependent caveolar recruitment of TRPC6, resulting in rapid endothelial Ca2+ influx and barrier failure.

Dysregulated Erythroid Mg2+ Efflux in Type 2 Diabetes.

Hyperglycemia is associated with decreased Mg2+ content in red blood cells (RBC), but mechanisms remain unclear. We characterized the regulation of Mg2+ efflux by glucose in ex vivo human RBC. We observed that hemoglobin A1C (HbA1C) values correlated with Na+-dependent Mg2+ efflux (Na+/Mg2+ exchange) and inversely correlated with cellular Mg content. Treatment of cells with 50 mM D-glucose, but not with sorbitol, lowered total cellular Mg (2.2 ± 0.1 to 2.0 ± 0.1 mM, p < 0.01) and enhanced Na+/Mg2+ exchange activity [0.60 ± 0.09 to 1.12 ± 0.09 mmol/1013 cell × h (flux units, FU), p < 0.05]. In contrast, incubation with selective Src family kinase inhibitors PP2 or SU6656 reduced glucose-stimulated exchange activation (p < 0.01). Na+/Mg2+ exchange activity was also higher in RBC from individuals with type 2 diabetes (T2D, 1.19 ± 0.13 FU) than from non-diabetic individuals (0.58 ± 0.05 FU, p < 0.01). Increased Na+/Mg2+ exchange activity in RBC from T2D subjects was associated with lower intracellular Mg content. Similarly increased exchange activity was evident in RBC from the diabetic db/db mouse model as compared to its non-diabetic control (p < 0.03). Extracellular exposure of intact RBC from T2D subjects to recombinant peptidyl-N-glycosidase F (PNGase F) reduced Na+/Mg2+ exchange activity from 0.98 ± 0.14 to 0.59 ± 0.13 FU (p < 0.05) and increased baseline intracellular Mg content (1.8 ± 0.1 mM) to normal values (2.1 ± 0.1 mM, p < 0.05). These data suggest that the reduced RBC Mg content of T2D RBC reflects enhanced RBC Na+/Mg2+ exchange subject to regulation by Src family kinases and by the N-glycosylation state of one or more membrane proteins. The data extend our understanding of dysregulated RBC Mg2+ homeostasis in T2D.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

Fyn and argonaute 2 participate in maternal-mRNA degradation during mouse oocyte maturation

ABSTRACT Fertilization triggers physiological degradation of maternal-mRNAs, which are then replaced by embryonic transcripts. Ample evidence suggests that Argonaut 2 (AGO2) is a possible post-fertilization regulator of maternal-mRNAs degradation; but its role in degradation of maternal-mRNAs during oocyte maturation remains obscure. Fyn, a member of the Src family kinases (SFKs), and an essential factor in oocyte maturation, was reported to inhibit AGO2 activity in oligodendrocytes. Our aim was to examine the role of Fyn and AGO2 in degradation of maternal-mRNAs during oocyte maturation by either suppressing their activity with SU6656 – an SFKs inhibitor; or by microinjecting DN-Fyn RNA for suppression of Fyn and BCl-137 for suppression of AGO2. Batches of fifteen mouse oocytes or embryos were analyzed by qPCR to measure the expression level of nine maternal-mRNAs that were selected for their known role in oocyte growth, maturation and early embryogenesis. We found that Fyn/SFKs are involved in maintaining the stability of at least four pre-transcribed mRNAs in oocytes at the germinal vesicle (GV) stage, whereas AGO2 had no role at this stage. During in-vivo oocyte maturation, eight maternal-mRNAs were significantly degraded. Inhibition of AGO2 prevented the degreadation of at least five maternal-mRNAs, whereas inhibition of Fyn/SFK prevented degradation of at least five Fyn maternal-mRNAs and two SFKs maternal-mRNAs; pointing at their role in promoting the physiological degradation which occurs during in-vivo oocyte maturation. Our findings imply the involvement of Fyn/SFKs in stabilization of maternal-mRNA at the GV stage and the involvement of Fyn, SFKs and AGO2 in degradation of maternal mRNAs during oocyte maturation.

A Soluble PrPC Derivative and Membrane-Anchored PrPC in Extracellular Vesicles Attenuate Innate Immunity by Engaging the NMDA-R/LRP1 Receptor Complex.

Nonpathogenic cellular prion protein (PrPC) demonstrates anti-inflammatory activity; however, the responsible mechanisms are incompletely defined. PrPC exists as a GPI-anchored membrane protein in diverse cells; however, PrPC may be released from cells by ADAM proteases or when packaged into extracellular vesicles (EVs). In this study, we show that a soluble derivative of PrPC (S-PrP) counteracts inflammatory responses triggered by pattern recognition receptors in macrophages, including TLR2, TLR4, TLR7, TLR9, NOD1, and NOD2. S-PrP also significantly attenuates the toxicity of LPS in mice. The response of macrophages to S-PrP is mediated by a receptor assembly that includes the N-methyl-d-aspartate receptor (NMDA-R) and low-density lipoprotein receptor-related protein-1 (LRP1). PrPC was identified in EVs isolated from human plasma. These EVs replicated the activity of S-PrP, inhibiting cytokine expression and IκBα phosphorylation in LPS-treated macrophages. The effects of plasma EVs on LPS-treated macrophages were blocked by PrPC-specific Ab, by antagonists of LRP1 and the NMDA-R, by deleting Lrp1 in macrophages, and by inhibiting Src family kinases. Phosphatidylinositol-specific phospholipase C dissociated the LPS-regulatory activity from EVs, rendering the EVs inactive as LPS inhibitors. The LPS-regulatory activity that was lost from phosphatidylinositol-specific phospholipase C-treated EVs was recovered in solution. Collectively, these results demonstrate that GPI-anchored PrPC is the essential EV component required for the observed immune regulatory activity of human plasma EVs. S-PrP and EV-associated PrPC regulate innate immunity by engaging the NMDA-R/LRP1 receptor system in macrophages. The scope of pattern recognition receptors antagonized by S-PrP suggests that released forms of PrPC may have broad anti-inflammatory activity.

Src Family Kinases Inhibition Ameliorates Hypoxic-Ischemic Brain Injury in Immature Rats.

Hypoxic-ischemic (HI) injury is one of the initial factors contributing to neonatal brain injury. Src family kinases (SFKs) are considered to act as molecular hubs for N-methyl-d-aspartate receptor (NMDAR) regulation and participate in the HI injury process. The objectives of this study were to evaluate the levels of phospho-Src (p-Src), the relationship between NMDARs and SFKs, and the effects of SFK inhibition on an immature rat HI brain injury model. The model was induced in 3-day-old Sprague–Dawley rats using the Rice-Vannucci model operation. The level of p-Src was evaluated using Western blotting. The association of NMDARs with SFKs was detected using Western blotting and coimmunoprecipitation. After intraperitoneal injection of 4-amino-5-(4-chlorophenyl)-7-(t-butyl) pyrazolo [3,4-d] pyrimidine (PP2), an SFK-selective inhibitor, neuropathological changes were observed by performing H&E and immunofluorescence staining, and the neurological functions were assessed using the following behavioral tests: modified neurological severity score, open field test, and Morris water maze test. The levels of p-Src first decreased at 0 h after injury, increased at 2 h after injury, and continuously decreased from 6 h to 3 days. Along with the increased p-Src levels observed at 2 h after injury, the phosphorylation of NMDAR subunit NR2B at tyrosine 1472 was increased. Following the administration of PP2, the increased p-Src and NMDAR-2B levels detected at 2 h after injury were decreased, and tissue injury and myelin basic protein expression were improved at 7 days after injury. The PP2 intervention improved the performance of injured rats on behavioral tests. In conclusion, we determined the patterns of p-Src expression after HI brain injury in immature rats and showed a relationship with the activated NMDA receptor. The inhibition of p-Src ameliorates neuropathological changes and damages neurological functions induced by HI injury.

Abstract P076: Novel mechanisms of acquired TKI resistance in ROS1+ NSCLC

Abstract ROS1 tyrosine kinase inhibitors (TKIs) entrectinib and crizotinib have significantly improved outcomes for patients with ROS1 gene fusion positive (ROS1+) lung adenocarcinoma. However, drug resistance inevitably develops, leading to disease progression. Approximately one-third of patients resistant to ROS1 TKIs demonstrate on-target ROS1 kinase domain (KD) mutations (Dziadziuszko et al. ESMO 2019), but the mechanism of resistance in the majority of patients remain unknown or poorly characterized. The purpose of this study was to model and characterize mechanisms of acquired ROS1 TKI resistance. To investigate resistance to ROS1 TKIs, we used patient-derived cell lines CUTO28 (TPM3-ROS1) and CUTO37 (CD74-ROS1) to generate drug-resistant derivatives to entrectinib (CUTO28-ER, CUTO37-ER) or crizotinib (CUTO28-CR,CUTO37-CR) in vitro. We utilized several techniques to probe the mechanisms driving resistance, including DNA and RNA sequencing, fluorescence in-situ hybridization (FISH), cell proliferation assays, and western blotting. DNA sequencing of the ROS1 KD in all resistant cell lines did not reveal any mutations. CUTO28-ER cells displayed sensitivity to MET-selective TKIs in cell proliferation assays. Consistent with MET dependency, MAPK and Akt pathways were inhibited by treatment with a MET-selective, but not a ROS1-selective, TKI. Western blot demonstrated MET overexpression and interphase FISH confirmed MET gene amplification (MET:CEP7 ratio 4.2) compared to parental cells (MET:CEP7 ratio 1.0). Notably, metaphase FISH revealed extrachromosomal DNA (ecDNA) amplification of MET. CUTO28-CR cells displayed sensitivity to Src family kinase (SFK) TKIs, and addition of pan-HER TKI afatinib resulted in synergistic inhibition of proliferation and downstream MAPK signaling. CUTO37-ER cells displayed sensitivity via proliferation and signaling to pan-HER inhibitor afatinib, and phosphorylation of EGFR Y845 consistent with Src-mediated activation of EGFR. Parental CUTO28 and CUTO37 cells were not sensitive to SFK or HER inhibition. Interphase FISH revealed modest EGFR gene amplification in CUTO28-CR cells—(EGFR:CEP7 ratio 2.1) compared to parental (EGFR:CEP7 ratio 1.0)—but not in CUTO37-ER or CUTO37-CR cells. HER2 gene amplification was not identified in any of the cell lines. Stimulation with epidermal growth factor (EGF) markedly enhanced MAPK signaling in CUTO28-CR, CUTO37-ER, and CUTO37-CR cells to a greater extent than in corresponding parental cells, consistent with EGFR or HER2 dependence. In conclusion, we demonstrated novel mechanisms of resistance to ROS1 TKIs entrectinib and crizotinib. These include ecDNA amplification of MET as an alternate oncogene driver and Src/EGFR axis as a driver of bypass signaling. Combination strategies with existing TKIs could be explored in patients with ROS1 TKI resistance. The discovery of ecDNA MET amplification is particularly intriguing; ecDNA is associated with more aggressive cancers and next generation sequencing analyses do not typically involve amplicon reconstruction to detect ecDNA. Citation Format: Logan C. Tyler, Anh T. Le, Hala Nijmeh, Liming Bao, Kristen Turner, Jason Christianson, Robert C. Doebele. Novel mechanisms of acquired TKI resistance in ROS1+ NSCLC [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P076.

Proto-oncogene tyrosine-protein kinase SRC (Src) inhibition in microglia relieves neuroinflammation in neuropathic pain mouse models

ABSTRACT Chronic neuroinflammation is an important factor in the development of neuropathic pain (NP). Excess microglia activation releases a mass of pro-inflammatory cytokines during neuroinflammation process, leading to a constant painful irritation of the sensory nerve. Src belongs to a non-receptor tyrosine kinase associated with sarcoma, whereas the role of Src in neuropathic pain is controversial. We designed to testify the inflammation-regulatory role of Src in the lipopolysaccharide (LPS)-induced BV2 microglia line and the mouse model of neuropathic pain by partial sciatic nerve ligation (PNL). In BV2 microglia, Src expression was inhibited using a Src family kinase inhibitor PP2 after LPS induced inflammatory response. In vivo, the neuropathic pain in mice was induced by PNL surgery and then treated with PP2. The neuroinflammation level in vitro was detected by enzyme-linked immunosorbent assay (ELISA), immunofluorescence (IF), trans-well and Western blotting (WB) assays, in vivo was examined in PNL mice using immunohistochemistry (IHC) and IF. Finally, mechanical allodynia and thermal hyperalgesia assays were used to access the functional evaluation. Inhibition of Src was decreased microglial inflammation and migration after LPS stimuli. Mechanistically, the expression of nuclear factor kappa B (NF-κB) pathway decreased after Src inhibition. The data in vivo showed that the decrease expression of Src reduced neuroinflammation and the amount of microglia in spinal dorsal horn (SDH), the mechanical allodynia of mice thereby attenuated after Src inhibition. These results indicated that the inhibition of Src took a protective effect in neuropathic pain mouse models via reducing microglia-induced neuroinflammation.

Evidence of off-target effects of bosutinib that promote retinoic acid-induced differentiation of non-APL AML cells

ABSTRACT In the present study, we determined the effects of the Src family kinase (SFK) inhibitor, Bosutinib, and the engineered loss of the Lyn SFK on all-trans retinoic acid-induced leukemic cell differentiation. Retinoic acid (RA) is an embryonic morphogen and dietary factor that demonstrates chemotherapeutic efficacy in inducing differentiation of a non-APL AML cell model, the HL-60 human myeloblastic (FAB-M2) leukemia cell line, via activation of a novel signalsome containing an ensemble of signaling molecules that drive differentiation. Bosutinib is an inhibitor of SFKs used to treat myeloid leukemias where prominent high expression of SFKs, in particular Lyn, has been observed. Using either Bosutinib or loss of Lyn expression due to shRNA promoted RA-induced phenotypic differentiation, G0 arrest, and respiratory burst (functional differentiation) of HL-60 cells. Signaling events putatively seminal to RA-induced differentiation, the expression of Fgr, Cbl, Slp-76 and Vav, and the phosphorylation of c-Raf (pS259), Vav (p-tyr), and Slp76 (p-tyr) were not inhibited by Bosutinib or loss of Lyn. Nor was RA-induced upregulation of p-tyr phosphorylation of p47phox, a member of the NADPH complex that produces ROS, a putative phosphorylation dependent signaling regulator. Surprisingly, Bosutinib still works in the absence of Lyn to enhance RA-induced differentiation and neither compromised RA-induced expression, nor phosphorylation of signaling molecules that drive differentiation. These findings suggested there is a novel, off-target, Lyn-independent effect of Bosutinib that is of therapeutic significance to differentiation therapy.

TCR-independent Activation in Presence of a Src-family Kinase Inhibitor Improves CAR-T Cell Product Attributes.

Chimeric antigen receptor expressing T cells (CAR-T cells) have shown remarkable efficacy against some blood cancers and have potential to treat many other human diseases. During CAR-T cell manufacturing, T cells are activated via engagement of the T-cell receptor (TCR); however, persistent TCR engagement can induce unchecked activation, differentiation, and exhaustion, which can negatively affect CAR-T cell product quality and in vivo potency. In addition, T cells may not uniformly respond to TCR-dependent activation (TCRD) contributing to lot-to-lot variability, poor expansion, and manufacturing failures. TCRD also presents challenges during manufacturing of allogeneic CAR-T cells when endogenous TCR is deleted to prevent graft-versus-host disease. Thus, novel strategies to activate T cells may help improve CAR-T cell product attributes and reduce manufacturing failures. In this study, we compared the effect of TCRD and TCR-independent activation (TCRI) on CAR-T cell product attributes. We found that TCRI in presence of a Src-kinase inhibitor significantly improved CAR-T cell expansion and yield without affecting viability and CD4/CD8 ratio. Markers of T-cell activation, exhaustion and differentiation were also reduced in these CAR-T cells compared with CAR-T cells manufactured by TCRD. TCRI did not affect CAR-T cell in vitro potency; however, following co-culture with target cells, CAR-T cells manufactured by TCRI released significantly less inflammatory cytokines compared with CAR-T cells manufactured by TCRD. Together, these data suggest that manufacturing CAR-T cells by TCRI activation in the presence of a Src-kinase inhibitor improves product quality attributes and may help reduce manufacturing failures and improve CAR-T cell safety and efficacy in vivo.

Src family kinases inhibit differentiation of intestinal epithelial cells through the Hippo effector YAP1

ABSTRACTIntestinal cell lineage differentiation is a tightly regulated mechanism that involves several intracellular signaling pathways affecting the expression of a variety of transcription factors, which ultimately regulate cell specific gene expression. Absorptive and goblet cells are the two main epithelial cell types of the intestine. Previous studies from our group using an shRNA knockdown approach have shown that YAP1, one of the main Hippo pathway effectors, inhibits the differentiation of these two cell types. In the present study, we show that YAP1 activity is regulated by Src family kinases (SFKs) in these cells. Inhibition of SFKs led to a sharp reduction in YAP1 expression at the protein level, an increase in CDX2 and the P1 forms of HNF4α and of absorptive and goblet cell differentiation specific markers. Interestingly, in Caco-2/15 cells which express both YAP1 and its paralog TAZ, TAZ was not reduced by the inhibition of SFKs and its specific knockdown rather impaired absorptive cell differentiation indicating that YAP1 and TAZ are not always interchangeable for regulating cell functions.This article has an associated First Person interview with the first author of the paper.

TRPA1-Mediated Src Family Kinases Activity Facilitates Cortical Spreading Depression Susceptibility and Trigeminovascular System Sensitization.

Transient receptor potential ankyrin 1 (TRPA1) plays a role in migraine and is proposed as a promising target for migraine therapy. However, TRPA1-induced signaling in migraine pathogenesis is poorly understood. In this study, we explored the hypothesis that Src family kinases (SFKs) transmit TRPA1 signaling in regulating cortical spreading depression (CSD), calcitonin gene-related peptide (CGRP) release and neuroinflammation. CSD was monitored in mouse brain slices via intrinsic optical imaging, and in rats using electrophysiology. CGRP level and IL-1β gene expression in mouse trigeminal ganglia (TG) was detected using Enzyme-linked Immunosorbent Assay and Quantitative Polymerase Chain Reaction respectively. The results showed a SFKs activator, pYEEI (EPQY(PO3H2)EEEIPIYL), reversed the reduced cortical susceptibility to CSD by an anti-TRPA1 antibody in mouse brain slices. Additionally, the increased cytosolic phosphorylated SFKs at Y416 induced by CSD in rat ipsilateral cerebral cortices was attenuated by pretreatment of the anti-TRPA1 antibody perfused into contralateral ventricles. In mouse TG, a SFKs inhibitor, saracatinib, restored the CGRP release and IL-1β mRNA level increased by a TRPA1 activator, umbellulone. Moreover, umbellulone promoted SFKs phosphorylation, which was reduced by a PKA inhibitor, PKI (14–22) Amide. These data reveal a novel mechanism of migraine pathogenesis by which TRPA1 transmits signaling to SFKs via PKA facilitating CSD susceptibility and trigeminovascular system sensitization.