Potent Src Family Kinase Inhibitor Research Articles

Abstract 5536: KB-164, a novel and highly potent Src family kinase inhibitor, reduces cancer cell invasion and abrogates downstream oncogenic pathways in triple negative breast cancer

Abstract Src is a non-receptor tyrosine kinase, which acts as an integrator of diverse signaling pathways used by cancer cells for cell proliferation, migration and metastasis. Recent interest in developing target specific therapy for improved benefits with manageable toxic effects has sharply increased the interest in testing new Src family kinase inhibitors for their potential anticancer therapeutic properties. In this study, we sought to investigate the chemical characteristics and the biological effects of the novel Src family kinase inhibitor KB-164, in Src expressing triple negative breast cancer cells. We show that KB-164 acts as a potent Src kinase inhibitor (IC50 1.3 nmol/L) in MDA-MB 231 cells, 100-fold more potent than the most widely used Src family kinase inhibitor dasatinib. At concentrations of 50nmol/L, KB-164 led to complete inhibition of Src activation for 48 hours. Analysis of downstream effectors of Src revealed that treatment with KB-164 inhibits the phosphorylation of several oncogenic pathways i.e. EGFR, ERK1/2, P38MAPK and AKT. A dramatic decrease in cancer cell motility and invasion was observed on treatment with KB-164 in both SUM 149 and MDA-MB 231 triple negative breast cancer cell lines. We also show that triple negative breast cancer cells derived from the patients are equally sensitive to growth inhibition by KB-164 within an IC50 of 1.8-nmol range. No cytotoxicity was observed both in vitro (18 micro mol/L in normal human mammary epithelial cells) and in vivo (70mg/kg in mice). Our findings indicate that KB-164 inhibits signaling pathways involved in controlling tumor cell proliferation and survival, suggesting that KB-164 might prove to be a promising therapeutic agent for breast cancer. Citation Format: Rabia A. Gilani, Kristoffer R. Brandvold, Li Wei Bao, Sameer Phadke, Michael E. Steffey, Matthew B. Soellner, Sofia D. Merajver. KB-164, a novel and highly potent Src family kinase inhibitor, reduces cancer cell invasion and abrogates downstream oncogenic pathways in triple negative breast cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5536. doi:10.1158/1538-7445.AM2015-5536

Src-family tyrosine kinase activities are essential for differentiation of human embryonic stem cells

Embryonic stem (ES) cells are characterized by pluripotency, defined as the developmental potential to generate cell lineages derived from all three primary germ layers. In the past decade, great progress has been made on the cell culture conditions, transcription factor programs and intracellular signaling pathways that control both murine and human ES cell fates. ES cells of mouse vs. human origin have distinct culture conditions, responding to some tyrosine kinase signaling pathways in opposite ways. Previous work has implicated the Src family of non-receptor protein–tyrosine kinases in mouse ES cell self-renewal and differentiation. Seven members of the Src kinase family are expressed in mouse ES cells, and individual family members appear to play distinct roles in regulating their developmental fate. Both Hck and c-Yes are important in self-renewal, while c-Src activity alone is sufficient to induce differentiation. While these findings implicate Src-family kinase signaling in mouse ES cell renewal and differentiation, the role of this kinase family in human ES cells is largely unknown. Here, we explored Src-family kinase expression patterns and signaling in human ES cells during self-renewal and differentiation. Of the eleven Src-related kinases in the human genome, Fyn, c-Yes, c-Src, Lyn, Lck and Hck were expressed in H1, H7 and H9 hES cells, while Fgr, Blk, Srm, Brk, and Frk transcripts were not detected. Of these, c-Yes, Lyn, and Hck transcript levels remained constant in self-renewing human ES cells vs. differentiated EBs, while c-Src and Fyn showed a modest increase in expression as a function of differentiation. In contrast, Lck expression levels dropped dramatically as a function of EB differentiation. To assess the role of overall Src-family kinase activity in human ES cell differentiation, cultures were treated with inhibitors specific for the Src kinase family. Remarkably, human ES cells maintained in the presence of the potent Src-family kinase inhibitor A-419259 retained the morphology of domed, pluripotent colonies and continued to express the self-renewal marker TRA-1-60 despite culture under differentiation conditions. Taken together, these observations support a role for Src-family kinase signaling in the regulation of human ES cell fate, and suggest that the activities of individual Src-family members are required for the initiation of the differentiation program.

Plasmodium induced by SU6656, an Src family kinase inhibitor, is accompanied by a contractile ring defect

We have shown that SU6656, a potent Src family kinase inhibitor, has the ability to induce multinucleation at a high frequency in diverse cells: rat skin fibroblasts, bone marrow adherent cells, 5F9A mesenchymal stem cell-like clones, 2C5 tracheal epithelial cells and MDCK epithelial cells from dog kidney. To gain insight into the mechanism of multinucleation, we observed the process by time-lapse and confocal microscopy. These multinuclei generally seem to exist independently in one cell without any connections with each other. By time-lapse microscopy, multinucleated cells were found to be formed through the mechanism of plasmodium: karyokinesis without cytokinesis. The observation of EGFP-actin transfected cells by time-lapse confocal laser scanning microscopy suggested that plasmodium occurred with deficient contractile ring formation. Although we examined the differentiation of these cells, the multinucleated cells could not be categorized into any type of cell in vivo known to exhibit multinuclei.

Correlation of dasatinib (DAS) peak levels with interleukin-8 (IL-8) and monocyte chemotactic protein-1 (MCP-1) levels in patients with castration-resistant progressive prostate cancer (CRPC).

4665 Background: IL-8 has been reported to promote CRPC growth [Am J Path1999;1503]. Further, MCP-1 mediates tumor- induced osteoclastogenesis and bone resorption [Cancer Res 2007;3646]. Dasatinib (DAS), a potent SRC-family kinase inhibitor, affects tumor growth and bone turnover induced by CRPC, the latter, by potentially blocking osteoclast activation Thus, the effects of DAS in CRPC might in part be mediated through reduction of either IL-8 and/or MCP-1 levels. Here, we analyzed the correlation between serum peak DAS levels with IL-8 and MCP-1 levels in the phase II portion of a phase I/II study of DAS and docetaxel (D) for treatment of metastatic CRPC. Data was analyzed for patients enrolled at M. D. Anderson Cancer Center (MDACC). Methods: Male pts with progressive CRPC and testosterone levels ≤50 ng/dL requiring chemotherapy were enrolled. Escalating doses of DAS and D were evaluated. The doses selected for the included pts was DAS 100mg QD and D 75mg/m2 Q21D. DAS, IL-8 and MCP-1 levels were measure...

Phase II study of dasatinib in the treatment of head and neck squamous cell carcinoma (HNSCC)

6022 Background: Dasatinib is a potent inhibitor of src-family kinases, ephA2, PDGFR, Abl, and c-kit. A single-center, open-label, phase II trial was conducted to evaluate the safety, tolerability, pharmacokinetics (PK), and efficacy of dasatinib in recurrent or metastatic HNSCC. Methods: Pts with measurable disease by RECIST, who received 0 or 1 prior regimen for recurrent or metastatic HNSCC with an ECOG performance status 0–1 and tumor tissue appropriate for IHC and FISH were eligible. Dasatinib 100 mg bid was given for 28-day cycles. Primary endpoints were 12-wk progression-free survival (PFS) and objective response rate (ORR). Pts who took at least 1 dose of dasatinib and who died or left study before 12 wks were counted as progressive disease (PD). A 2 stage design, closure after accrual of 15 pts was required if PFS was 45% or less and ORR was 0. Otherwise, planned accrual was 35. Response was assessed at 4 and 12 wks. PK was studied in pts receiving dasatinib per PEG. Biomarkers relevant to Src pathway were planned in tissue and blood. Results: Fifteen pts were accrued. To date, 13 pts are evaluable for response, and 15 pts for toxicity. No grade 3/4 hematologic toxicities were noted. Grade 2–4 nonhematologic toxicities(n): pleural effusion(2), nausea/vomiting(2), dehydration(1), diarrhea(1), dyspnea(1). Toxicity led to hospitalization of 4 pts and drug discontinuation in 5 pts. ORR was 0. One pt was stable at 12 wks (PFS: 7.6%). This pt stopped drug at 15 wks due to toxicity, but also had PD. One pt died on study and cause was deemed unlikely related. Conclusions: Dosed at 100mg bid, dasatinib led to a characteristic toxicity profile in this pt population. Rates of hospitalization and discontinuation for toxicity were fairly high. Final efficacy parameters are pending evaluation of 2 pts. Evaluation of PK and tissue/blood biomarkers is ongoing. No significant financial relationships to disclose.

AP23846, a novel and Highly potent Src family kinase inhibitor, reduces vascular endothelial growth factor and interleukin-8 expression in human solid tumor cell lines and abrogates downstream angiogenic processes

AP23846, a novel and Highly potent Src family kinase inhibitor, reduces vascular endothelial growth factor and interleukin-8 expression in human solid tumor cell lines and abrogates downstream angiogenic processes

AP23846, a novel and highly potent Src family kinase inhibitor, reduces vascular endothelial growth factor and interleukin-8 expression in human solid tumor cell lines and abrogates downstream angiogenic processes

c-Src is frequently activated in human malignancies, including colon, breast, and pancreatic carcinomas. Several recent studies have shown that activation of Src family kinases leads to tumor progression and metastasis by increasing cellular migration and invasion, promoting cell growth and survival, and deregulating expression of proangiogenic molecules. Therefore, selective inhibitors of Src are being developed for cancer therapy. In this study, we characterize the biological effects of the novel ATP-based Src family kinase inhibitor, AP23846, in tumor cells with high Src activity. As a lead compound, AP23846 is a potent c-Src kinase inhibitor (IC50 approximately 0.5 nmol/L in vitro, approximately 10-fold more potent than PP2, the most widely used commercially available Src family kinase inhibitor). At concentrations of 1 micromol/L, AP23846 led to complete Src inhibition for 48 hours in cells. No cytotoxicity was observed under these conditions, although proliferation rates were slower. Therefore, this was an excellent inhibitor to examine Src-regulated signaling pathways in tumor cells. AP23846 reduced cellular migration, vascular endothelial growth factor, and interleukin-8 in a dose-dependent fashion in pancreatic adenocarcinoma cells grown in vitro. Correspondingly, cell culture supernatants from L3.6pl pancreatic adenocarcinoma cells pretreated with AP23846 failed to promote migration of hepatic endothelial cells in vitro and failed to support angiogenesis into gel foams implanted s.c. in mice in vivo. These results suggest that Src inhibitors affect biological properties of tumor progression and may be useful as cancer therapeutic agents in more advanced disease.

363 Targeting of PDGF receptors; combination effects in various solid tumors and identification of a PDGF dependent brain tumor subset

Embryonic stem (ES) cells are characterized by pluripotency, defined as the developmental potential to generate cell lineages derived from all three primary germ layers. In the past decade, great progress has been made on the cell culture conditions, transcription factor programs and intracellular signaling pathways that control both murine and human ES cell fates. ES cells of mouse vs. human origin have distinct culture conditions, responding to some tyrosine kinase signaling pathways in opposite ways. Previous work has implicated the Src family of non-receptor protein–tyrosine kinases in mouse ES cell self-renewal and differentiation. Seven members of the Src kinase family are expressed in mouse ES cells, and individual family members appear to play distinct roles in regulating their developmental fate. Both Hck and c-Yes are important in self-renewal, while c-Src activity alone is sufficient to induce differentiation. While these findings implicate Src-family kinase signaling in mouse ES cell renewal and differentiation, the role of this kinase family in human ES cells is largely unknown. Here, we explored Src-family kinase expression patterns and signaling in human ES cells during self-renewal and differentiation. Of the eleven Src-related kinases in the human genome, Fyn, c-Yes, c-Src, Lyn, Lck and Hck were expressed in H1, H7 and H9 hES cells, while Fgr, Blk, Srm, Brk, and Frk transcripts were not detected. Of these, c-Yes, Lyn, and Hck transcript levels remained constant in self-renewing human ES cells vs. differentiated EBs, while c-Src and Fyn showed a modest increase in expression as a function of differentiation. In contrast, Lck expression levels dropped dramatically as a function of EB differentiation. To assess the role of overall Src-family kinase activity in human ES cell differentiation, cultures were treated with inhibitors specific for the Src kinase family. Remarkably, human ES cells maintained in the presence of the potent Src-family kinase inhibitor A-419259 retained the morphology of domed, pluripotent colonies and continued to express the self-renewal marker TRA-1-60 despite culture under differentiation conditions. Taken together, these observations support a role for Src-family kinase signaling in the regulation of human ES cell fate, and suggest that the activities of individual Src-family members are required for the initiation of the differentiation program.

Ornithine Decarboxylase- and ras-Induced Cell Transformations: Reversal by Protein Tyrosine Kinase Inhibitors and Role of pp130CAS

We have found that overexpression of human ornithine decarboxylase (ODC) induces cell transformation in NIH 3T3 and Rat-1 cells (M. Auvinen, A. Paasinen, L. C. Andersson, and E. Hölttä, Nature (London) 360:355-358, 1992). The ODC-transformed cells display increased levels of tyrosine phosphorylation, in particular of a cluster of 130-kDa proteins. Here we show that one of the proteins with enhanced levels of tyrosine phosphorylation in ODC-overexpressing cells is the previously described p130 substrate of pp60v-src, known to associate also with v-Crk and designated p130CAS. We also studied the role of protein tyrosine phosphorylation in the ODC-induced cell transformation by exposing the cells to herbimycin A, a potent inhibitor of Src-family kinases, and to other inhibitors of protein tyrosine kinases. Treatment with the inhibitors reversed the phenotype of ODC-transformed cells to normal, with an organized, filamentous actin cytoskeleton. Coincidentally, the tyrosine hyperphosphorylation of p130 was markedly reduced, while the level of activity of ODC remained highly elevated. A similar reduction in pp130 phosphorylation and reversion of morphology by herbimycin A were observed in v-src- and c-Ha-ras-transformed cells. In addition, we show that expression of antisense mRNA for p130CAS resulted in reversion of the transformed phenotype of all these cell lines. An increased level of tyrosine kinase activity, not caused by c-Src or c-Abl, was further detected in the cytoplasmic fraction of ODC-transformed cells. Preliminary characteristics of this kinase are shown. These data indicate that p130CAS is involved in cell transformation by ODC, c-ras, and v-src oncogenes, raise the intriguing possibility that p130CAS may be generally required for transformation, and imply that there is at least one protein tyrosine kinase downstream of ODC that is instrumental for cell transformation.