Src Inhibitor Research Articles

Anti-Survival Effect of SI306 and Its Derivatives on Human Glioblastoma Cells

Glioblastoma (GBM) is the most common adult brain tumor and, although many efforts have been made to find valid therapies, the onset of resistance is the main cause of recurrence. Therefore, it is crucial to identify and target the molecular mediators responsible for GBM malignancy. In this context, the use of Src inhibitors such as SI306 (C1) and its prodrug (C2) showed promising results, suggesting that SI306 could be the lead compound useful to derivate new anti-GBM drugs. Therefore, a new prodrug of SI306 (C3) was synthesized and tested on CAS-1 and U87 human GBM cells by comparing its effect to that of C1 and C2. All compounds were more effective on CAS-1 than U87 cells, while C2 was the most active on both cell lines. Moreover, the anti-survival effect was associated with a reduction in the expression of epidermal growth factor receptor (EGFR)WT and EGFR-vIII in U87 and CAS-1 cells, respectively. Collectively, our findings demonstrate that all tested compounds are able to counteract GBM survival, further supporting the role of SI306 as progenitor of promising new drugs to treat malignant GBM.

The Androgen Receptor and Its Crosstalk With the Src Kinase During Castrate-Resistant Prostate Cancer Progression.

While the androgen receptor (AR) signalling is the mainstay therapeutic target for metastatic prostate cancers, these tumours will inevitably develop therapy resistance to AR pathway inhibitors suggesting that prostate tumour cells possess the capability to develop mechanisms to bypass their dependency on androgens and/or AR to survive and progress. In many studies, protein kinases such as Src are reported to promote prostate tumour progression. Specifically, the pro-oncogene tyrosine Src kinase regulates prostate cancer cell proliferation, adhesion, invasion, and metastasis. Not only can Src be activated under androgen depletion, low androgen, and supraphysiological androgen conditions, but also through crosstalk with other oncogenic pathways. Reciprocal activations between Src and AR proteins had also been reported. These findings rationalize Src inhibitors to be used to treat castrate-resistant prostate tumours. Although several Src inhibitors had advanced to clinical trials, the failure to observe patient benefits from these studies suggests that further evaluation of the roles of Src in prostate tumours is required. Here, we summarize the interplay between Src and AR signalling during castrate-resistant prostate cancer progression to provide insights on possible approaches to treat prostate cancer patients.

Abstract 3326: Uncovering the molecular mechanisms which predict sensitivity to a novel src kinase inhibitor NXP900 to inform personalized healthcare strategies

Abstract We have previously described the discovery of a highly potent, selective and orally bioavailable Src kinase inhibitor NXP900 with a unique binding mode relative to other Src inhibitors under clinical development or approved (Temps et al 2021). This unique binding mode locks SRC in its native inactive conformation, thereby inhibiting both enzymatic and scaffolding functions. Also, in contrast to other Src inhibitors in the clinic, NXP900 exhibits a unique target selectivity profile with 1000 fold selectivity for Src over Abl kinase. The aim of this current study was to perform broad cancer cell line panel screening to inform disease positioning and identify predictive biomarkers of sensitivity for this novel class of Src kinase inhibitor. We describe the results of extensive cancer cell line panel profiling across a suite of 2-dimensional(D) and 3-D in vitro cell viability and high content phenotypic profiling assays that has revealed clear patterns of cell line sensitivity and insensitivity. Among the most sensitive cell line subtypes are, squamous cell carcinoma, Arid1A mutant ovarian clear cell carcinoma and subtypes of breast luminal A and triple negative breast cancer cell lines. HER2+ breast cancer lines appear insensitive to NXP900 indicating HER2 as a potential driver of resistance to NXP900. Further in-depth bioinformatic analysis of genetic and transcriptomic profiles of sensitive cell lines has identified putative biomarkers to support patient selection and personalized healthcare strategies. These studies demonstrate the identification of cancer cell lines which exhibit high sensitivity to NXP900 and which can be grouped into discrete molecular subtypes based on gene mutation status, transcriptomics and histotype. Our result provide an indication of patient subgroups which may exhibit optimal therapeutic response to NXP900 and provide data to guide further preclinical, biomarker and clinical development. Temps C et al., Cancer Res. 2021 Nov 1;81(21):5438-5450. doi: 10.1158/0008-5472.CAN-21-0613. Citation Format: Neil Carragher, Rebecca Hughes, Valerie Brunton, Asier Unciti-broceta, Iñigo Lanzagorta-Calvillo, Carolin Temps, Enrique Poradosu. Uncovering the molecular mechanisms which predict sensitivity to a novel src kinase inhibitor NXP900 to inform personalized healthcare strategies [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 3326.

Abstract 6094: SRC inhibitor saracatinib enhances efficacy of PD-1/PD-L1 immune checkpoint blockade in non-small cell lung cancer

Abstract Background: Although immune checkpoint inhibitors (ICI) included PD-1/PD-L1 have demonstrated durable therapeutic responses in some cases, a significant portion of patients with cancer remain nonresponsive (e.g. only ~20% with anti-PD-1 in lung cancer). Substantial limitations in the rate of response underscore the need for suitable combination therapies. SRC family kinases is overexpressed or activated in multiple human malignancies, as a candidate target in regulating antitumor immunity. Saracatinib, an orally bioavailable multi-kinase inhibitor, has demonstrated clinical efficacy in CML. Here, we proved saracatinib as SRC inhibitor that were capable of increasing the antitumor immunity in non-small lung cancer. Method: Cytotoxicity assays were used to determined antitumor activities of saracatinib in non-small lung cancer cell lines. The expression of PD-L1 expression in saracatinib 1 treated cell lines was investigated by western blots, quantitative PCR and flow cytometry. The effect of saracatinib in antitumor immunity alone or in combination with immunotherapy was also determined in vitro and in vivo. RNAi, overexpression, and gene expression analyses were used to define underlying mechanisms. Result: We found that SRC inhibitor saracatinib robustly decreased expression of PD-L1 in non-small lung cancer cells and animal models. The observed inhibitory effect was time- and concentration-dependent. Furthermore, our results showed that saracatinib and anti-PD-L1 combined to improve T-cell-mediated killing of tumor cells in vitro and in vivo, which were associated with activation of CD8+ T cytotoxic cell included expression of IFN-γ and granzyme-B. Prolonged survival was also observed in mice treated with saracatinib in combination with PD-L1 blockade. Conclusion: Our in vivo and in vitro data demonstrated that SRC inhibitor saracatinib enhance T-cell-mediated anti-tumor immune responses in non-small lung cancer. We also provide evidence that support the combination of saracatinib and anti-PD-1/PD-L1 as a potential combination therapeutic approach to increase the efficacy of immunotherapy in non-small lung cancer. Citation Format: Fei-Teng Lu, Fan Luo, Miao-Zhen Qiu, Jia-Xin Cao, Qiu-Yun Luo, Da-Jun Yang, Hong-Yun Zhao. SRC inhibitor saracatinib enhances efficacy of PD-1/PD-L1 immune checkpoint blockade in non-small cell lung cancer [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 6094.

Abstract 3245: Genes associated with Src-Met driven resistance of head & neck cancers to EGFR-PI3K cotargeting

Abstract Background: In recent years, oncology research has led to the discovery of targeted therapies aimed at specific genetic abnormalities unique to cancer cells. In particular, the overexpression of epidermal growth factor receptor (EGFR) and constitutive activation of the phosphoinositide-3-kinase/protein kinase B (PI3K/AKT) pathway are common aberrations in head and neck cancer. However, intrinsic and acquired resistance are continuously posing challenges to success. Previously, we reported that the combination of EGFR inhibitor erlotinib with PI3K inhibitor BKM120 had synergistic effects that induced efficient apoptosis in a panel of head and neck cancer cell lines, but some cells were still resistant. The purpose of the current study is to understand the mechanism of resistance to EGFR and PI3-K co-targeting. Hypothesis: Src-Met signaling confers apoptosis resistance to EGFR and PI3K cotargeting by regulating downstream genes expresiion. Methods: Apoptosis was measured by annexin V-PE staining. Western blotting and qPCR were used for measuring the expression of proteins and mRNA, respectively. RNASeq was conducted from RNA samples treated with (Erlotinib + BKM120) ± dasatinib (Src inhibitor)/crizotinib(Met inhibitor). Results: Biochemical studies revealed that JHU022 cells, which were resistant to the combination treatment, had an overactivation of c-Met receptor tyrosine kinase that was regulated by Src kinases. Pharmacological or genetic inactivation of both Met or Src kinase sensitized this cell line to apoptosis induced by EGFR and PI3K co-targeting. RNA-Seq analysis identified that a total of 291 genes were modulated (2-folds) by both crizotinib and dasatinib. Pathway enrichment suggested that many of these genes were associated with cell cycle, DNA replication, and cellular response to DNA damage. CPA4 was identified as the most downregulated gene and GDF15 as one of the top upregulated genes. Conclusions: SRC-Met signaling confers resistant to EGFR and PI3K cotargeting by modulating genes associated with cell cycle, DNA replication, and cellular response to DNA damage. Citation Format: Adeoluwa A. Adeluola, A.R.M. Ruhul Amin. Genes associated with Src-Met driven resistance of head & neck cancers to EGFR-PI3K cotargeting [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 3245.

Abstract 2263: The role of E-Cadherin mediated cell division in promoting collective colon cancer cell migration

Abstract Collective cell migration is the cell cohort migrates toward the direction. Other than single cell migration, collective cell migration requires cell-cell connection with E-cadherin, however, E-cadherin is the epithelial marker in epithelial mesenchymal transition and acts as tumor suppressor function. Therefore, the role of E-cadherin in collective cell migration is still under investigated. In the collected cells, cell migration and cell division are coordinated for the cohort motility, the mechanism is still not clear. This study is to investigate the role of E-cadherin orientation in cell division dependent collective cell migration. Colorectal cancer cell line HT29 with epithelial morphology were used and treated with Cetuximab and Dasatinib to inhibit EGFR and Src activity. Cells were also incubated on fibronectin to monitor the matrix dependent environment. The polarity of E-cadherin distribution and cell contact inhibition were defined and detected by Immunofluorescence. The molecular expression of p-FAK, CDK4, and cell proliferation marker, Ki67 were revealed by Immunofluorescence confocal microscopy and Western blot. The collective cell migration is analyzed and compared by wound healing and transwell assay. In the results, the polarized distribution of E-cadherin in leading cells which revealed pFAK expression on the cell membrane was associated with Ki67 and CDK4 positive proliferated cells. When cells were grown on fibronectin, the proliferated cells in the leading were decreased. When cells were treated with cetuximab, the E-cadherin distribution were not changed and had fewer effect on cell migration. However, dasatinib treatment can modulate E-cadherin orientation and suppress cell proliferation and cell migration. These results suggested modulate E-cadherin orientation such as Src inhibition can be the potential treatment strategy in colon cancer Citation Format: Yu-Lin Li, Yu-Cheng Weng, Viriya Adhiguna Winarso, Yi-Wen Lu, Hui Min Koo, Xiang-Ling Hou, Kai-Yu Tseng, Wei-Ting Chao. The role of E-Cadherin mediated cell division in promoting collective colon cancer cell migration [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 2263.

Abstract 355: Molecular characterization of BRAF V600E CRC in response to Src-targeted therapy

Abstract Oncogenic mutations in B-type Raf Kinase (BRAF) (particularly V600E) are present in 7-10% of metastatic colorectal cancer (mCRC), conferring resistance to conventional therapies and a poor prognosis. Src Family Kinases (SFKs) play a relevant role in CRC, where Src activation is associated with an aggressive phenotype and a worse prognosis; and could be involved in chemoresistance mechanisms. Dasatinib is a potent Src inhibitor with demonstrated anti-proliferative activity, although deeper knowledge is needed. Thus, we aimed to explore the role of Src in BRAF V600E CRC and to investigate this tumor subgroup in response to Dasatinib. SRC activation was evaluated by Western Blot. We generated Knock-Out (KO) Src cell lines by CRISPR/Cas9 system and stable Src overexpression (OE) by lentivirus. Cell viability was analysed by crystal violet and MTS assay. Phospho-kinase proteins screening was performed using the Proteome Profiler Human Phospho-Kinase Array kit. Expression levels of Src and phospho-Src (p-Src) displayed an inverse correlation with IC50 values for Dasatinib in a panel of 7 CRC cell lines including BRAF V600E (HT29, COLO201, RKO, LS411N) and BRAF WT (DIFI, HCA7 and LIM1215), and these doses were able to inhibit Src activation in all cell lines. Focusing on HT29 cells, inhibition of p-Src occurs in a dose-dependent manner and in shortened periods of treatment. Dasatinib showed a significant inhibition of proliferation and migration on HT29. Moreover, stable Src-OE HT29 cells showed higher clonogenicity, migration and proliferation capacities, as well as higher resistance to Dasatinib. Conversely, HT29 Src KO cells were more sensitive. In line with those results, silencing Src in other BRAF V600E cells (RKO and LS411N) also resulted in a remarkable reduction in clonogenicity and migration abilities. Xenograft mouse model of SRC depletion showed that the growth of SRC KO tumors was significantly slower than WT tumors. Phospho-kinase array quantification in basal and Src-OE HT29 cells showed up- and downregulated proteins related to classical tumorigenic signaling pathways such as MAPK (JNK1/2, MSK), PI3K/Akt (Akt, TOR, GSK3), and others. Further studies to explore resistance mechanisms mediated by Src/SFKs are required. To conclude, lower p-Src expression at baseline is related to a higher resistance to Dasatinib in all CRC cell lines. Dasatinib showed a significant anti-proliferative activity in a subset of CRC cell lines and regulates the activity of Src in a time and dose-dependent manner. Modified expression of Src (OE and KO) in BRAF V600E CRC cells significantly regulates clonogenicity, proliferation and migration abilities in vitro. Src overexpression is responsible of the acquired resistance to Dasatinib and regulates several pathways that could be involved in the tumorigenesis of BRAF V600E CRC, although further studies to explore resistance mechanisms mediated by Src/SFKs are required. Citation Format: Beatriz Rubio Cuesta, Beatriz Gil Calderón, Patricia LLamas Granda, Jacinto Sarmentero, Carlos Carretero Puche, María del Carmen Riesco, Rocío García Carbonero, Beatriz Soldevilla. Molecular characterization of BRAF V600E CRC in response to Src-targeted therapy [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 355.

Abstract 5341: Mechanisms driving resistance in Group 3 and recurrent/disseminated medulloblastoma

Abstract Background: Medulloblastoma (MB) is the most common malignant brain tumor in children and accounts for 20% of childhood brain tumors. Among the 4 molecular MB subtypes, Group 3 and 4 MBs have the worst prognosis. The biology of recurrence and dissemination in MB is less understood. Overexpression of platelet-derived growth factor receptor (PDGFR) has been associated with metastatic MB, and PDGFR activity promotes MB migration. PDGFR-induced migration is regulated by Src kinase. Src kinase activity is high in MB and Src kinase inhibition restricts tumor growth. Group 3 MB demonstrates high PDGFRA expression. Aim: Our objective was to evaluate if molecular inhibition of PDGFR or Src kinase will control metastatic progression/dissemination in Group 3 MB. Methods: We treated a panel of 4 patient-derived MB cell lines with dasatinib, which can inhibit BCRABL, c-KIT, PDGFR and Src kinase. Dasatinib dose required to inhibit c-KIT and PDGFR is higher (50-100 nM) than that needed for Src inhibition (1-10 nM). Results: Proliferation assay with dasatinib (1-10000nM) performed on all 4 cell lines did not demonstrate any inhibition of proliferation except at 10000nM dose. We treated all 4 MB cell lines with higher dose range of dasatinib (625-10000nM). One cell line did not respond to dasatinib, remaining cell lines demonstrated proliferation inhibition over 13 days between 625-5000nM. There were 2 interesting observations. Firstly, cell line which required lowest dose (625nM) was a recurrent MB, while paradoxically, its paired parental cell line (non-recurrent/treatment-naïve) responded to proliferation inhibition only at higher 5000nM dose. IC50 of parental cell line was 38-times higher than recurrent cell line. We postulated that recurrent MB may be responsive to dasatinib. However, dasatinib-treated recurrent cells demonstrated minimal changes in cell cycle phases (IC50 day 4 dose 187.4nM) and apoptotic cell death (187.4nM and 293.2nM) compared to DMSO-treated control. Since Group 3 MB demonstrates high PDGFRA expression, we employed a patient-derived orthotopic xenograft (PDOX) model of Group 3 MB to investigate the in-vivo response to dasatinib. The 2nd interesting observation was that dasatinib-treated mice exhibited earlier neuro-symptoms, more rapid tumor growth and clinical deterioration compared to control mice. However, overall animal survival time was not statistically significant between treated versus untreated groups (p=0.24 Log-rank test). Conclusions: Recurrent cells were more sensitive to dasatinib in-vitro compared to paired parental cells. In-vivo, mice treated with dasatinib developed neuro-symptoms faster than controls. It is currently not known if recurrent MB cells demonstrating early dasatinib-sensitivity to proliferation inhibition, escaping cell death, can formulate an in-vivo mechanism to drive more rapid tumor progression/dissemination as observed in our Group 3 MB Model. Citation Format: Shiying Huang, Jie-Ling Pan, Qi Lin, YuChen Du, Jack Su, Angela Major, M. Tarek Elghetany, Kam-Man Hui, Xiaonan Li, Wan-Yee Teo. Mechanisms driving resistance in Group 3 and recurrent/disseminated medulloblastoma [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 5341.

Verbascoside potentiates the effect of tyrosine kinase inhibitors on the induction of apoptosis and oxidative stress via the Abl-mediated MAPK signalling pathway in chronic myeloid leukaemia.

Verbascoside (Verb) may exhibit potential antitumour activities in leukaemia. The present study investigated the effect of Verb, in combination with imatinib (IM), dasatinib (Das), lipopolysaccharide (LPS) and TNF, on cell survival, Abl expression, apoptosis, oxidative stress and the MAPK pathway in chronic myeloid leukaemia (CML) cells. Cell viability was determined using the WST-8 assay in K562 and R-K562 cells treated with Verb and/or IM, Das, LPS and TNF. Apoptosis and DNA damage in CML cells was detected by caspase-3 and comet analysis. The protein levels of Abl (Phospho-Tyr412), and total/phosphorylated p38, JNK and ERK in CML cells were analysed using a Colorimetric Cell-Based Assay. Oxidative stress was examined using total antioxidant and oxidant status assays. Treatment with Verb and/or tyrosine kinase inhibitors (TKIs), LPS and TNF resulted in a significant decrease in the Tyr-412 phosphorylation of Abl in K562 and R-K562 cells. In addition, cotreatment with Verb and IM or Das additively induced apoptosis by activating caspase-3 levels in both cell lines. Activation of p38 and JNK can result in growth arrest and cell death, whereas ERK stimulation results in cell division and differentiation. The present study demonstrated that cotreatment with Verb and TKIs suppressed phosphorylated-ERK1/2, whereas the levels of phosphorylated-p-38 and phosphorylated-JNK were significantly elevated by Verb and/or IM, Das, LPS and TNF, thus suggesting that Abl and Src inhibition could be involved in the effects of Verb on MAPK signalling in R-K562 cells. Furthermore, Verb elevated reactive oxygen species levels additively with TKIs in both cell lines by increasing the oxidant capacity and decreasing the antioxidant capacity. In conclusion, anti-leukemic mechanisms of Verb may be mediated by Abl protein and regulation of its downstream p38-MAPK/JNK pathway, caspase-3 and oxidative stress in CML cells.

MODL-12.Ex vivo model identifies druggable vulnerabilities in medulloblastoma

Abstract Medulloblastoma (MB) is the most common malignant brain tumour in children and long-term sequelae in survivors caused by the harsh, non-targeted treatments is common. Current screening protocols for the detection of novel druggable vulnerabilities rely on 2D and 3D cell culture systems using established or primary tumour cells. However, these assays do neither provide the cellular, chemical and biophysical context of the tissue, nor reveal drug effects on the invasive behavior of the tumour cells. To overcome these limitations, we have established ex vivo Organotypic Cerebellum Slice Co-culture (OCSC) models for SHH and group 3 MB tumours, which provide spatio-temporal insights in the growth and dissemination behaviour of the tumour cells. OCSCs represent an excellent translational model for patient sample profiling, drug testing, and microenvironmental or explorative target identification studies in a physiologically relevant tissue context. We found that the AURKB inhibitor Barasertib (AZD-1152), which was ineffective to reduce ONS-76 cell invasion in vitro, effectively reduced tumour volume and proliferation of these SHH tumour cells in the tissue context. The AURKB inhibitor thus displays higher efficacy ex vivo, indicating an impact of the microenvironment on its functional activity. We further validated tumour suppressive activity of AURKB inhibition with Barasertib at low nanomolar concentration or with siRNA-mediated depletion of AURKB in Grp3 MB tumour cells and in primary tumour cells ex vivo, thereby confirming the therapeutic potential of AURKB inhibition against MB progression. We furthermore found that the combination of AURKB inhibition with irradiation or with the SRC inhibitor Dasatinib, lead to a near complete eradication of the tumour cells. Thus, OCSCs are a clinically relevant model where tumour material can be efficiently tested for drug sensitivities, which allowed us to identify AURKB inhibition as an effective treatment against tissue invasive MB.

Mechanisms Causing Acantholysis in Pemphigus-Lessons from Human Skin.

Pemphigus vulgaris (PV) is an autoimmune bullous skin disease caused primarily by autoantibodies (PV-IgG) against the desmosomal adhesion proteins desmoglein (Dsg)1 and Dsg3. PV patient lesions are characterized by flaccid blisters and ultrastructurally by defined hallmarks including a reduction in desmosome number and size, formation of split desmosomes, as well as uncoupling of keratin filaments from desmosomes. The pathophysiology underlying the disease is known to involve several intracellular signaling pathways downstream of PV-IgG binding. Here, we summarize our studies in which we used transmission electron microscopy to characterize the roles of signaling pathways in the pathogenic effects of PV-IgG on desmosome ultrastructure in a human ex vivo skin model. Blister scores revealed inhibition of p38MAPK, ERK and PLC/Ca2+ to be protective in human epidermis. In contrast, inhibition of Src and PKC, which were shown to be protective in cell cultures and murine models, was not effective for human skin explants. The ultrastructural analysis revealed that for preventing skin blistering at least desmosome number (as modulated by ERK) or keratin filament insertion (as modulated by PLC/Ca2+) need to be ameliorated. Other pathways such as p38MAPK regulate desmosome number, size, and keratin insertion indicating that they control desmosome assembly and disassembly on different levels. Taken together, studies in human skin delineate target mechanisms for the treatment of pemphigus patients. In addition, ultrastructural analysis supports defining the specific role of a given signaling molecule in desmosome turnover at ultrastructural level.

SAR Probing of KX2-391 Provided Analogues With Juxtaposed Activity Profile Against Major Oncogenic Kinases.

Tirbanibulin (KX2-391, KX-01), a dual non-ATP (substrate site) Src kinase and tubulin-polymerization inhibitor, demonstrated a universal anti-cancer activity for variety of cancer types. The notion that KX2-391 is a highly selective Src kinase inhibitor have been challenged by recent reports on the activities of this drug against FLT3-ITD mutations in some leukemic cell lines. Therefore, we hypothesized that analogues of KX2-391 may inhibit oncogenic kinases other than Src. A set of 4-aroylaminophenyl-N-benzylacetamides were synthesized and found to be more active against leukemia cell lines compared to solid tumor cell lines. N-(4-(2-(benzylamino)-2-oxoethyl)phenyl)-4-chlorobenzamide (4e) exhibited activities at IC50 0.96 µM, 1.62 µM, 1.90 µM and 4.23 µM against NB4, HL60, MV4-11 and K562 leukemia cell lines, respectively. We found that underlying mechanisms of 4e did not include tubulin polymerization or Src inhibition. Such results interestingly suggested that scaffold-hopping of KX2-391 may change the two main underlying cytotoxic mechanisms (Src and tubulin). Kinase profiling using two methods revealed that 4e significantly reduces the activities of some other potent oncogenic kinases like the MAPK member ERK1/2 (>99%) and it also greatly upregulates the pro-apoptotic c-Jun kinase (84%). This research also underscores the importance of thorough investigation of total kinase activities as part of the structure-activity relationship studies.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

Inhibition of Src but not Syk causes weak reversal of GPVI-mediated platelet aggregation measured by light transmission aggregometry

Src tyrosine kinases and spleen tyrosine kinase (Syk) have recently been shown to contribute to sustained platelet aggregation on collagen under arterial shear. In the present study, we have investigated whether Src and Syk are required for aggregation under minimal shear following activation of glycoprotein VI (GPVI) and have extended this to C-type lectin-like receptor-2 (CLEC-2) which signals through the same pathway. Aggregation was induced by the GPVI ligand collagen-related peptide (CRP) and the CLEC-2 ligand rhodocytin and monitored by light transmission aggregometry (LTA). Aggregation and tyrosine phosphorylation by both receptors were sustained for up to 50 min. The addition of inhibitors of Src, Syk or Bruton’s tyrosine kinase (Btk) at 150 sec, by which time aggregation was maximal, induced rapid loss of tyrosine phosphorylation of their downstream proteins, but only Src kinase inhibition caused a weak (~10%) reversal in light transmission. A similar effect was observed when the inhibitors were combined with apyrase and indomethacin or glycoprotein IIb-IIIa (GPIIb-IIIa) antagonist, eptifibatide. On the other hand, activation of GPIIb-IIIa by GPVI in a diluted platelet suspension, as measured by binding of fluorescein isothiocyanate-labeled antibody specific for the activated GPIIb-IIIa (FITC-PAC1), was reversed on the addition of Src and Syk inhibitors showing that integrin activation is rapidly reversible in the absence of outside-in signals. The results demonstrate that Src but not Syk and Btk contribute to sustained aggregation as monitored by LTA, possibly as a result of inhibition of outside-in signaling from GPIIb-IIIa to the cytoskeleton through a Syk-independent pathway. This is in contrast to the role of Syk in supporting sustained aggregation on collagen under arterial shear.

Src Activation Aggravates Podocyte Injury in Diabetic Nephropathy via Suppression of FUNDC1-Mediated Mitophagy.

Background and purpose: Mitophagy plays a significant role in the progression of diabetic nephropathy (DN), although the regulatory mechanisms remain unclear. Recently, accumulating evidence demonstrated that impaired mitochondrial function and mitophagy are involved in DN. Here, we are aimed to explore the role of c-Src (Src) and FUNDC1-related mitophagy in the development of DN. Methods: The db/db mice were used to establish a DN mice model. The mice accepted PP2 (Src inhibitor) treatment to study the role of Src in DN. Kidney function was measured via biochemical testing. Renal histopathology and morphometric analysis were conducted via hematoxylin-eosin (HE), periodic acid-Schiff (PAS), Masson’s staining, and transmission electron microscopy (TEM). We measured degree of apoptosis in kidney by TUNEL assay. Indices of mitophagy (LC3 and p62) were evaluated by Western blotting and immunofluorescence. Complementary in vitro assays were conducted using human podocytes subjected to high glucose in combination with PP2 treatment or FUNDC1 small interfering RNAs (siRNAs). Flow cytometry was used to detect the apoptotic cells. Mitochondrial function was evaluated by JC-1 staining. Double immunofluorescence labeling of LC3 and TOMM20 used to assess the degree of mitophagy. Results: Increased Src activation was detected in the kidneys of db/db mice, and its expression was positively correlated with mitochondrial damage, podocyte apoptosis, and renal dysfunction. Inhibition of Src activation with PP2 protected against mitochondrial damage and podocyte apoptosis. In vitro experiments in podocytes established that high glucose increased Src activation, promoting FUNDC1 phosphorylation and inhibiting mitophagy. Consistent with the mouse model, inhibiting Src activity protected podocytes against mitochondrial damage. FUNDC1 silencing negated the actions of PP2, indicating that FUNDC1-mediated mitophagy is downstream pathway of Src. Conclusion: In summary, our data indicated that Src is a culprit factor in diabetic renal damage via suppression of FUNDC1-mediated mitophagy, promoting the development of DN.

EGFR Inhibition in Cardiomyocytes Stabilizes Cardiomyocyte Cohesion in a Murine Model for Arrhythmogenic Cardiomyopathy

The epidermal growth factor receptor (EGFR) is a receptor tyrosine kinase ubiquitously expressed in different tissues, including the heart. EGFR is dysregulated in several kinds of cancer, as well as in heart diseases. Arrhythmogenic cardiomyopathy (ACM) is a familial heart disease and one of the underlying mechanisms is impaired desmosome turnover. The intercalated disc (ICD) not only provides cellular cohesion but can also provide the structural framework of a signaling hub. Thus, stabilizing desmosome integrity, thereby enhancing cardiomyocyte stability, may provide potential new treatment options. In this study, we investigated the role of EGFR inhibition on cardiomyocyte cohesion under physiological as well as under pathophysiological conditions using the cardiomyocyte‐specific plakoglobin knockout (Jup‐/‐) ACM model in which EGFR was upregulated. Dissociation assays in HL‐1 cells and murine cardiac slice cultures showed that EGFR inhibition by erlotinib similar to SRC inhibition enhanced cardiomyocyte cohesion. Immunoprecipitation showed an interaction of EGFR, desmoglein 2 (DSG2) and desmoplakin (DP), indicating that altered EGFR signaling might affect desmosomes. Immunostainings and atomic force microscopy (AFM) revealed enhanced DSG2 localization and binding at the cell borders upon EGFR inhibition. In addition, during desmosome assembly induced by a Ca2+‐switch, DSG2 and DP were augmented at the cell borders after EGFR or SRC inhibition. Our study shows that EGFR inhibition by erlotinib leads to increased DSG2 binding and desmosome assembly, which stabilizes cardiomyocyte cohesion in the ACM mouse model. Because EGFR inhibition by erlotinib is already used in cancer patients, these data reveal a potential new therapeutic strategy in ACM.

P‐cadherin regulates intestinal epithelial repair, but is dispensable for colitis associated colon cancer development

Recurrent chronic mucosal inflammation, which is characteristics for inflammatory bowel diseases (IBD), triggers significant changes in the intestinal epithelial homeostasis. These changes include leakiness of the gut barrier, formation of mucosal wounds and, in most severe cases, oncogenic transformation of colonic epithelium resulting in colitis‐associated colon cancer (CAC). Altered structure and dynamics of epithelial junctions is a hallmark of intestinal inflammation, mediating epithelial barrier injury and repair. P‐cadherin (gene name: CDH3) is an important component of adherens junctions (AJ), which is poorly expressed in normal intestinal epithelium, but could be induced in inflamed and injured mucosa. The goal of this his study was to investigate the roles of P‐cadherin in regulating intestinal inflammation, mucosal repair and CAC.P‐cadherin expression was markedly induced in the colonic epithelium of human IBD patients and CAC tissues. Roles of P‐cadherin in intestinal inflammation and tumorigenesis in vivo were investigated using a mouse strain with total P‐cadherin knockout. Dextran sulfate sodium (DSS)‐induced colitis was utilized to study mucosal inflammation, whereas CAC was established using a classical azoxymetane (AOM)/DSS model. Severity of acute DSS colitis was not affected by P‐cadherin knockout, however, P‐cadherin null mice exhibited faster recovery after DSS withdrawal indicating accelerated repair of injured mucosa. No significant differences in the number and size of colonic tumors was observed in P‐cadherin null and control mice after AOM/DSS induced CAC. Consistently, CRISPR/Cas9‐mediated knockout of P‐cadherin in SK‐CO15 and HCA7 human colonic epithelial cell lines accelerated epithelial wound healing without affecting cell proliferation. The faster migration of P‐cadherin depleted cells was associated with diminished cell‐matrix adhesions and increased cell spreading. Loss of P‐cadherin resulted in activation of Src kinase and the pro‐migratory phenotype of P‐cadherin depleted cell was reversed by pharmacological inhibition of Src. Furthermore, accelerated epithelial wound healing caused by loss of P‐cadherin was prevented by Rac1 inhibition. Our findings highlight P‐cadherin as a negative regulator of intestinal epithelial would heling in vitro and mucosal repair in vivo. By contrast, this AJ protein appears to be dispensable for intestinal epithelial cell proliferation and CAC development.

Targeting Src-Hic-5 Signal Cascade for Preventing Migration of Cholangiocarcinoma Cell HuCCT1.

Cholangiocarcinoma (CCA) is the second most common primary liver cancer with poor prognosis. The deregulation of a lot of oncogenic signaling molecules, such as receptor tyrosine kinases (RTKs), has been found to be associated with CCA progression. However, RTKs-based target therapy showed limited improvement suggesting a need to search for alternative targets for preventing CCA progression. To address this issue, we screened the oncogenic signal molecules upregulated in surgical tissues of CCAs. Interestingly, over-expression of hydrogen peroxide inducible clone-5 (Hic-5) coupled with over-activation of Src, AKT, JNK were observed in 50% of the cholangiocarcinoma with metastatic potential. To investigate whether these molecules may work together to trigger metastatic signaling, their up-and-down relationship was examined in a well-established cholangiocarcinoma cell line, HuCCT1. Src inhibitors PP1 (IC50, 13.4 μM) and dasatinib (IC50, 0.1 μM) significantly decreased both phosphorylated AKT (phosphor-AKT Thr450) and Hic-5 in HuCCT1. In addition, a knockdown of Hic-5 effectively suppressed activation of Src, JNK, and AKT. These implicated a positive cross-talk occurred between Hic-5 and Src for triggering AKT activation. Further, depletion of Hic-5 and inhibition of Src suppressed HuccT1 cell migration in a dose-dependent manner. Remarkably, prior transfection of Hic-5 siRNA for 24 h followed by treatment with PP1 or dasatinib for 24 h resulted in additive suppression of HuCCT1 migration. This suggested that a promising combinatory efficacy can be achieved by depletion of Hic-5 coupled with inhibition of Src. In the future, target therapy against CCA progression by co-targeting Hic-5 and Src may be successfully developed in vivo.

CD55 is upregulated by cAMP/PKA/AKT and modulates human decidualization via Src and ERK pathway and decidualization-related genes.

Decidualization is an essential process for embryo implantation and maintenance of pregnancy, and abnormal decidualization contributed to several pregnancy disorders like a miscarriage. The objective of this study was to explore the regulation and function of CD55 in human decidualization. By immunohistochemical staining, it was found that CD55 expression was higher in first-trimester decidua than in the endometrium. In both primary endometrial stromal cells and immortalized cell line T-hESCs, CD55 was upregulated by induction of in vitro decidualization with medroxyprogesterone acetate (MPA) and 8-Br-cAMP. During decidualization in vitro, CD55was stimulated by 8-Br-cAMP in a time- and concentration-dependent manner, which was reversed by a PKA inhibitor H89 and partially by an AKT activator SC79. Knocking down CD55 expression diminished the expression of decidualization markers prolactin (PRL) and insulin-like growth factor-binding protein 1 (IGFBP1), accompanied by inhibition of Src, aberrant activation of ERK and decreased expression of several decidualization-related genes, including FOXO1, EGFR, and STAT3. Furthermore, the decidua of unexplained miscarriage women and the endometrium of unexplained infertile women both exhibited decreased CD55 expression. Collectively, these findings revealed that 8-Br-cAMP promotes CD55 expression via PKA activation and AKT dephosphorylation, and decreased CD55 impairs decidualization by inactivation of Src, aberrant activation of ERK pathway, and compromised expression of decidualization-related genes, indicating that CD55 deficiency may contribute to the pathogenesis of spontaneous miscarriage and infertility.

Early SRC activation skews cell fate from apoptosis to senescence.

Cells responding to DNA damage implement complex adaptive programs that often culminate in one of two distinct outcomes: apoptosis or senescence. To systematically identify factors driving each response, we analyzed human IMR-90 fibroblasts exposed to increasing doses of the genotoxin etoposide and identified SRC as a key kinase contributing early to this dichotomous decision. SRC was activated by low but not high levels of etoposide. With low DNA damage, SRC-mediated activation of p38 critically promoted expression of cell survival and senescence proteins, while SRC-mediated repression of p53 prevented a rise in proapoptotic proteins. With high DNA damage, failure to activate SRC led to elevation of p53, inhibition of p38, and apoptosis. In mice exposed to DNA damage, pharmacologic inhibition of SRC prevented the accumulation of senescent cells in tissues. We propose that inhibiting SRC could be exploited to favor apoptosis over senescence in tissues to improve health outcomes.