Combined Inhibition Of Src Research Articles

BCL2L11 Induction Mediates Sensitivity to Src and MEK1/2 Inhibition in Thyroid Cancer.

Patients with advanced thyroid cancer, including advanced papillary thyroid cancer and anaplastic thyroid cancer (ATC), have low survival rates because of the lack of efficient therapies available that can combat their aggressiveness. A total of 90% of thyroid cancers have identifiable driver mutations, which often are components of the MAPK pathway, including BRAF, RAS, and RET-fusions. In addition, Src is a non-receptor tyrosine kinase that is overexpressed and activated in thyroid cancer, which we and others have shown is a clinically relevant target. We have previously demonstrated that combined inhibition of Src with dasatinib and the MAPK pathway with trametinib synergistically inhibits growth and induces apoptosis in BRAF- and RAS-mutant thyroid cancer cells. Herein, we identified the pro-apoptotic protein BCL2L11 (BIM) as being a key mediator of sensitivity in response to combined dasatinib and trametinib treatment. Specifically, cells that are sensitive to combined dasatinib and trametinib treatment have inhibition of FAK/Src, MEK/ERK, and AKT, resulting in the dramatic upregulation of BIM, while cells that are resistant lack inhibition of AKT and have a dampened induction of BIM. Inhibition of AKT directly sensitizes resistant cells to combined dasatinib and trametinib but will not be clinically feasible. Importantly, targeting BCL-XL with the BH3-mimeitc ABT-263 is sufficient to overcome lack of BIM induction and sensitize resistant cells to combined dasatinib and trametinib treatment. This study provides evidence that combined Src and MEK1/2 inhibition is a promising therapeutic option for patients with advanced thyroid cancer and identifies BIM induction as a potential biomarker of response.

CSIG-07. c-Src PHOSPHORYLATES AND INACTIVATES THE CIC TUMOR SUPPRESSOR PROTEIN IN GLIOBLASTOMA

Abstract Capicua (CIC) is a transcriptional repressor that counteracts activation of genes in response to receptor tyrosine kinase (RTK)/Ras/MEK/ERK signaling. Following activation of RTK, ERK enters the nucleus and serine-phosphorylates CIC, releasing it from its targets to permit gene expression. We recently showed that ERK triggers ubiquitin-mediated degradation of CIC in glioblastoma (GBM). In this study, we examined whether another important downstream effector of RTK/EGFR, the non-receptor tyrosine kinase c-Src, affects CIC repressor function in GBM. We found that c-Src binds and tyrosine phosphorylates CIC on residue 1455 to promote nuclear export of CIC. On the other hand, CIC mutant allele (CIC-Y1455F), that escapes c-Src-mediated tyrosine phosphorylation, remains localized to the nucleus and retains strong repressor function against CIC targets, the oncogenic ETV transcription factors. Furthermore, we show that the orally available Src family kinase inhibitor, dasatinib, which prevents EGF-mediated tyrosine phosphorylation of CIC and attenuates elevated ETV levels, reduces viability of GBM cells and glioma stem cells (GSC), but not of their control cells with undetectable c-Src activity. In fact, GBM cells and GSC expressing the tyrosine-defective CIC mutant (Y1455F) lose sensitivity to dasatinib, further endorsing the effect of dasatinib on Src-mediated tyrosine phosphorylation of CIC. Importantly we show that combined inhibition of Src and MEK/ERK results in greatest stabilization of CIC and attenuation of proliferation compared to either inhibitor alone. These findings elucidate important mechanisms of CIC regulation and provide the rationale to target c-Src alongside ERK pathway inhibitors as a way to fully restore CIC tumor suppressor function in neoplasms such as GBM.

Characterization of the Src-regulated kinome identifies SGK1 as a key mediator of Src-induced transformation

Despite significant progress, our understanding of how specific oncogenes transform cells is still limited and likely underestimates the complexity of downstream signalling events. To address this gap, we use mass spectrometry-based chemical proteomics to characterize the global impact of an oncogene on the expressed kinome, and then functionally annotate the regulated kinases. As an example, we identify 63 protein kinases exhibiting altered expression and/or phosphorylation in Src-transformed mammary epithelial cells. An integrated siRNA screen identifies nine kinases, including SGK1, as being essential for Src-induced transformation. Accordingly, we find that Src positively regulates SGK1 expression in triple negative breast cancer cells, which exhibit a prominent signalling network governed by Src family kinases. Furthermore, combined inhibition of Src and SGK1 reduces colony formation and xenograft growth more effectively than either treatment alone. Therefore, this approach not only provides mechanistic insights into oncogenic transformation but also aids the design of improved therapeutic strategies.

Abstract 1820: Overcoming drug resistance in NSCLC with SRC inhibitor

Abstract Lung cancer accounts for about 27 percent of all cancer deaths in the United States. Approximately 85 percent of lung cancers are non-small cell type (NSCLC) and, when treated with both chemotherapy and targeted therapies, have a five-year overall survival rate of approximately 16 percent. These statistics clearly emphasize the need for developing treatment methodologies more effective than the current standard of care. Here, we evaluated the efficacy of combined inhibition of SRC and AXL in NSCLC.Dasatinib, a BCR/abl and src family tyrosine kinase inhibitor (TKI) is a drug that has a potential to be used against both EGFR and KRAS mutated lung cancer patients, alone (phase II clinical trial) and in combination with cetuximab (in phase III clinical trial), osimertinib and erlotinib (both in phase II clinical trials) by exploiting its SRC inhibition capabilities. However, NSCLC patients treated with dasatinib often develop resistance and is the major reason for the low median survival in stage IV patients. Recently, AXL pathway has been shown to be upregulated in Dasatinib resistant cells causing activation of EMT, AKT and survival pathways. It has been suggested that auto-phosphorylation of DDR2 and subsequent activation of SHP2/RAS/AKT pathway as a result of AXL upregulation is one of the major reasons for failure to induce apoptosis in cells during dasatinib resistance. Therefore, we examined whether inhibition of AXL resensitized TKI resistant NSCLC to dasatinib. Our results show that AXL knockdown can resensitize cells to dasatinib. IC50 values decreased significantly following AXL inhibition. We have also demonstrated that nanoparticle-mediated AXL knockdown also produces similar resensitization. The details of the study will be presented. Citation Format: Soumavo Mukherjee, Dhananjay Suresh, Ajit P. Zambre, Anandhi Upendran, Raghuraman Kannan. Overcoming drug resistance in NSCLC with SRC inhibitor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1820.

Src-mediated regulation of the PI3K pathway in advanced papillary and anaplastic thyroid cancer

Advanced stages of papillary and anaplastic thyroid cancer continue to be plagued by a dismal prognosis, which is a result of limited effective therapies for these cancers. Due to the high proportion of thyroid cancers harboring mutations in the MAPK pathway, the MAPK pathway has become a focal point for therapeutic intervention in thyroid cancer. Unfortunately, unlike melanoma, a similar responsiveness to MAPK pathway inhibition has yet to be observed in thyroid cancer patients. To address this issue, we have focused on targeting the non-receptor tyrosine kinase, Src, and we and others have demonstrated that targeting Src results in inhibition of growth, invasion, and migration both in vitro and in vivo, which can be enhanced through the combined inhibition of Src and the MAPK pathway. Therefore, we examined the efficacy of the combination therapy across a panel of thyroid cancer cell lines representing common oncogenic drivers (BRAF, RAS, and PIK3CA). Interestingly, combined inhibition of Src and the MAPK pathway overcomes intrinsic dasatinib resistance in cell lines where both the MAPK and PI3K pathways are inhibited, which we show is likely due to the regulation of the PI3K pathway by Src in these responsive cells. Interestingly, we have mapped downstream phosphorylation of rpS6 as a key biomarker of response, and cells that maintain rpS6 phosphorylation likely represent drug tolerant persisters. Altogether, the combined inhibition of Src and the MAPK pathway holds great promise for improving the overall survival of advanced thyroid cancer patients with BRAF and RAS mutations, and activation of the PI3K pathway and rpS6 phosphorylation represent important biomarkers of response for patients treated with this therapy.

MicroRNA-106a targets autophagy and enhances sensitivity of lung cancer cells to Src inhibitors

ObjectivesSrc tyrosine kinase inhibitors (TKIs) significantly inhibit cell migration and invasion in lung cancer cell lines with minor cytotoxic effects. In clinical trials, however, they show modest activity in combination with chemotherapeutic agents. Possible resistance mechanisms include the induction of cytoprotective autophagy upon Src inhibition. Autophagy is a cellular recycling process that allows cell survival in response to a variety of stress stimuli including responses to various treatments. Material and methodsWe screened autophagic activity in A549, H460, and H1299 NSCLC cell lines treated with two different Src-TKIs (saracatinib, dasatinib) or shRNA targeting SRC. The autophagy response was determined by LC3B-I to −II conversion, increased ULK1 epxression and increased GFP-LC3B dot formation. Autophagy was inhibited by pharmacological (bafilomycin A, chloroquine) or genetic (ULK1 shRNA) means. Expression of miR-106a and miR-20b was analyzed by qPCR, and we used different lentivral vectors for ectopic expression of either miR-106a mimetics, anti-sense miR-106a or different miR-106a-363 cluster constructs. ResultsIn the current study we found that Src-TKIs induce autophagy in lung adenocarcinoma cell lines and that a combination of autophagy and Src tyrosine kinase inhibition results in cell death. Moreover, Src-TKI induced autophagy depends on the induction of the key autophagy kinase ULK1. This ULK1 upregulation is caused by downregulation of the ULK1-targeting microRNA-106a. An inverse correlation of miR-106a and ULK1 expression was seen in lung adenocarcinoma. Accordingly, ectopic expression of miR-106a in combination with Src-TKI treatment resulted in significant cell death as compared to control transduced cells. ConclusionsAutophagy protects lung adenocarcinoma cells from Src-TKIs via a newly identified miR-106a-ULK1 signaling pathway. The combined inhibition of Src and ULK1/autophagy might represent a promising treatment option for future clinical trials. Lastly, our data might challenge the term “oncogenic” miR-106a as it can promote sensitivity to Src-TKIs thereby underlining the context-dependent function of miRNAs.

Abstract 950: Src tyrosine kinase contributes to lapatinib resistance in human breast cancer models.

Abstract Background. The dual EGFR/HER2 tyrosine kinase inhibitor (TKI) lapatinib is approved in metastatic breast cancer patients after trastuzumab failure. Unfortunately, several patients do not respond to treatment due to constitutive resistance, and even in responders the occurrence of disease progression is often observed. Recently, the activation of Src TK has been associated with resistance to anti-HER2 drugs, both trastuzumab and lapatinib, in HER2 overexpressing breast cancer cells. However, the molecular mechanisms by which Src activation may affect response to HER2 antagonists are still partially unclear. Methods. We used a panel of HER2 expressing human breast cancer cell lines with different degree of response to lapatinib: SKBR3, BT474 and MDA-MB-361 (high sensitivity), KPL-4 (moderate sensitivity), JIMT-1 (low sensitivity). Moreover, we generated a cell line with acquired resistance to lapatinib, MDA-MB-361-LR (Lapatinib Resistant). On these models, we investigated the effect of Src silencing via small interference RNA (siRNA) or pharmacological inhibition by saracatinib on lapatinib response, both in vitro and in vivo. Results. The combination of Src inhibition with lapatinib synergistically inhibited survival of human breast cancer cells with both intrinsic or acquired resistance to lapatinib, by interfering with signal transducers involved in cell proliferation and survival. Moreover, both saracatinib and Src specific siRNAs were able to reduce migration and invasion capabilities of breast cancer cells, by affecting Focal Adhesion Kinase (FAK) and paxillin activation. In nude mice xenografted with JIMT-1 cells, the combined treatment with saracatinib and lapatinib produced a cooperative antitumor effect with inhibition of tumor growth, interference with signal transduction and prolongation of mice survival. The combination strongly reduced the incidence of lung metastasis, as revealed through experimental metastasis assays. Interestingly, a significant reduction of EGFR/HER2 co-immunoprecipitation was found in resistant cells, thus suggesting that growth signals could be transduced by other type of TKRs dimers in these cells. Moreover, in breast cancer cell lines with both intrinsic or acquired resistance to lapatinib, Src interacted with EGFR rather than with HER2, in opposition to what was observed in sensitive cells. Consistently, EGFR inhibition through cetuximab or EGFR specific siRNAs was able to partially restore lapatinib sensitivity in resistant cells, by interfering with the reciprocal activation of EGFR and Src. Conclusions. Src plays an important role in the onset of resistance to lapatinib in breast cancer cell lines, and its interaction with EGFR may be involved in this event. Therefore, the combined inhibition of Src and EGFR/HER2 axis may represent a potentially effective therapeutic strategy in order to prevent or overcome resistance to lapatinib. Citation Format: Roberta Rosa, Lucia Nappi, Luigi Formisano, Claudia D'Amato, Valentina D'Amato, Vincenzo Damiano, Roberta Marciano, Ana Paula De Maio, Lucia Raimondo, Umberto Malapelle, Giancarlo Troncone, Sabino De Placido, Roberto Bianco. Src tyrosine kinase contributes to lapatinib resistance in human breast cancer models. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 950. doi:10.1158/1538-7445.AM2013-950 Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.

Abstract 2485: Combined Src and aurora kinase inhibition in medulloblastoma

Abstract Src kinase is overexpressed in many human cancers, including medulloblastoma (MB), the most common malignant pediatric brain tumor. Src plays a central role in many oncogenic signaling pathways, and has been implicated in cell migration. Recently, we observed that blockade of aurora kinases A and B, which regulate cell cycle progression and are expressed by MB cells, also inhibits MB proliferation and migration. Because metastatic disease is such a powerful predictor of survival in MB patients, we hypothesized that combined Src and aurora kinase inhibition may act in a synergistic manner to more effectively inhibit MB growth and progression. To test this hypothesis, we investigated the effects of Dasatinib, a potent Src inhibitor, and AT9283, an inhibitor of aurora kinases A and B, alone and in combination, against a panel of MB cells in vitro and in a mouse flank tumor model in vivo. In two human MB cell lines (DAOY, D556), we show a dose-dependent decrease in phosphorylation of Src and histone-H3 when treated with Dasatinib and AT9283 respectively. This correlated with decreased cell viability measured by MTT assay after treatment with each agent (Dasatinib 1nM, AT9283 10nM). When treated at hour 0 and measured at hour 96, without drug replenishment, Dasatinib inhibits cell viability by 57% (DAOY) and 86% (D556), and AT9283 inhibits viability by 47% (DAOY) and 76% (D556), compared to control. Viability was more potently inhibited in DAOY cells when used in combination at the above concentrations, decreasing by 81% compared to control. Similarly, in a mouse MB cell line (PS125), viability decreased by 47% and 48% after treatment with Dasatinib at 10nM and AT9283 at 10nM respectively. Interestingly, in PS125 cells, Dasatinib at 3nM did not impact viability, but when combined with AT9283 at 10nM, a marked decrease was observed compared to AT9283 at 10nM alone (70% vs 48%). Each agent also inhibits migration when measured by scratch assay. When treated with Dasatinib at 1nM, cell movement decreased by 94% (DAOY) and 60% (D556) at 24 hours, and 29% (DAOY) and 50% (D556) at 48 hours, without drug replenishment. Similarly, movement decreased by 42% (DAOY) and 23% (D556) at 24 hours, and 30% (DAOY) and 3% (D556) at 48 hours, when treated with AT9283 at 10nM. Movement was more potently inhibited when drugs were combined at the above concentrations, decreasing by 100% (DAOY) and 85% (D556) at 24 hours, and 82% (DAOY) and 90% (D556) at 48 hours, compared to control. Additionally, our preliminary in vivo data suggest that Dasatinib inhibits tumor growth in mice with implanted syngeneic flank tumors (PS125 cells). By day 20 of Dasatinib treatment (15 mg/kg po daily M-F), tumors were 36% smaller in treated mice compared to controls, without obvious differences in toxicity. We conclude that combined inhibition of Src and aurora kinases appears to synergistically inhibit proliferation and migration of MB cells in vitro, and Src inhibition appears to inhibit MB tumor growth in vivo. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2485. doi:1538-7445.AM2012-2485

Combined Blockade of Src Kinase and Epidermal Growth Factor Receptor with Gemcitabine Overcomes STAT3-Mediated Resistance of Inhibition of Pancreatic Tumor Growth

We previously established a mechanistic rationale for Src inhibition as a novel therapeutic target in pancreatic cancer and have identified activated STAT3 as a potential biomarker of resistance to Src inhibition. The purpose of this study was to translate the current understanding of complementary activated tyrosine kinase signaling pathways by targeting Src kinase and epidermal growth factor receptor (EGFR). IC(50) values for dasatinib, a Src kinase inhibitor, erlotinib, an EGFR tyrosine kinase inhibitor and gemcitabine were determined and sensitive and resistant pancreatic cancer cell lines were identified. The in vitro and in vivo effects of these agents on multiple signaling pathways and tumorigenicity in pancreatic cancer were investigated. The combination of dasatinib, erlotinib, and gemcitabine resulted in cooperative inhibition of cell migration and invasion of both sensitive and resistant pancreatic cancer cells as well as cooperative inhibition of multiple signaling pathways including FAK, AKT, ERK, JNK, MAPK, and STAT3 at concentrations that were ineffective as individual agents or as double combinations of agents. The triple combination of agents was also most effective at inhibiting the growth of xenografts of both sensitive and resistant pancreatic cancer cells in vivo without increasing toxicity. Furthermore, combined inhibition of Src and EGFR with gemcitabine inhibited constitutively activated STAT3 in vitro and in vivo. These results provide evidence that combined targeted biological therapy in addition to cytotoxic chemotherapy can overcome treatment resistance. Such treatment strategies may be used to tailor therapy based on identified biomarkers of resistance to targeted monotherapy.

Integrative Genomic and Proteomic Analyses Identify Targets for Lkb1-Deficient Metastatic Lung Tumors

In mice, Lkb1 deletion and activation of Kras(G12D) results in lung tumors with a high penetrance of lymph node and distant metastases. We analyzed these primary and metastatic de novo lung cancers with integrated genomic and proteomic profiles, and have identified gene and phosphoprotein signatures associated with Lkb1 loss and progression to invasive and metastatic lung tumors. These studies revealed that SRC is activated in Lkb1-deficient primary and metastatic lung tumors, and that the combined inhibition of SRC, PI3K, and MEK1/2 resulted in synergistic tumor regression. These studies demonstrate that integrated genomic and proteomic analyses can be used to identify signaling pathways that may be targeted for treatment.

Abstract A258: Combined blockade of Src kinase and epidermal growth factor receptor synergistically inhibits growth of pancreas cancer

Abstract The failure of conventional chemotherapeutic regimes to produce any meaningful impact on survival in patients with pancreatic cancer highlights a desperate need for novel treatment strategies. The purpose of this study was to translate the current understanding of activated tyrosine kinase signaling pathways in pancreas cancer into improved patient therapies by targeting two tyrosine kinases, the epidermal growth factor receptor (EGFR) and the non-receptor Src kinase, which act in complementary pathways. The biochemical effects of individual and combined drug treatment with dasatinib (Sprycel, BMS-354825), a potent Src kinase inhibitor, erlotinib (Tarceva, OSI-774), a tyrosine kinase inhibitor to EGFR and the nucleoside analog gemcitabine were examined in pancreas cancer cell lines in vitro and in vivo. IC50 for dasatinib (DTB), erlotinib and gemcitabine was determined by the MTT proliferation assay against 9 human pancreas cancer cell lines, and sensitive and resistant cell lines were identified. In vitro regulation of cell motility, proliferation, survival, migration, invasion and in vivo tumor growth inhibition was determined. Expression of Ki67, cleaved caspase 3, phosphorylated Src and AKT in tumor xenografts was determined following exposure to combination of drugs. The combined inhibition of Src with DTB and EGFR with erlotinib results in cooperative attenuation of cell migration, invasion, proliferation and increases in apoptosis of pancreatic cancer cells. Combined inhibition of Src and EGFR also results in cooperative inhibition of multiple signaling pathways including FAK, AKT, Stat3, ERK, JNK, MAPK and decreases cyclin D1 at concentrations that are ineffective as individual agents. The addition of gemcitabine enhances the effects of combined inhibition of Src and EGFR compared to that seen with individual agents alone or in double combination. Mice with tumor xenografts of BxPC3 (sensitive cell line) and Panc1 (resistant cell line) cells treated with DTB, erlotinib or gemcitabine either individually or as two drug or three drug combinations showed that the most effective in vivo anti-tumor effects were seen with the triple combination of DTB, erlotinib and gemcitabine even in xenografts of resistant pancreatic cells. Pharmacological inhibition of EGFR and Src, in addition to gemcitabine treatment, results in cooperative inhibition of pancreatic tumor cell growth and appears to overcome resistance of single and double agent therapy. These results show that activation of Src and EGFR signaling pathways is evident in pancreas cancer and may be a driver of poor prognosis and chemotherapy resistance and provide a rationale for combination therapy with erlotinib, DTB and gemcitabine in clinical trials of pancreas cancer. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A258.