Phospho-kinase profile of colorectal tumors guides in the selection of multi-kinase inhibitors

Abstract

Protein kinases play a central role in the oncogenesis of colorectal tumors and are attractive druggable targets. Detection of activated kinases within a tumor could open avenues for drug selection and optimization of new kinase inhibitors. By using a phosphokinase arrays with human colorectal tumors we identified activated kinases, including the Epidermal Growth Factor Receptor (EGFR), components of the PI3K/mTOR pathway (AKT and S6), and STAT, among others. A pharmacological screening with kinase inhibitors against these proteins helped us to identify a new kinase inhibitor, termed EC-70124 that showed the highest anti-proliferative activity in cell lines. EC-70124 also inhibited cell migration and biochemical experiments demonstrated its effect targeting the PI3K/mTOR pathway. This drug also arrested cells at G2/M and induced apoptosis. Experiments in combination with standard chemotherapy used in the clinical setting indicated a synergistic effect. EC-70124 also reduced tumor growth in vivo and inhibited pS6 in the implanted tumors. In conclusion, by studying the kinase profile of colorectal tumors, we identified relevant activated pathways, and a new multi-kinase compound with significant antitumor properties.

The multikinase inhibitor EC‐70124 synergistically increased the antitumor activity of doxorubicin in sarcomas

“…The efficient inhibition of the phosphorilation of S6 correlates with the high affinity (low Kd) of EC‐70124 for S6K1 (Supporting Information Table S3). This data are in line with previously reported activity of EC‐70124 in breast and colon cancer . By combining EC‐70124 or midostaurin with the mTOR inhibitor torin, we studied the role of mTOR signaling in the cytotoxic effect of indolocarbazole analogs.…”

“…This data are in line with previously reported activity of EC-70124 in breast and colon cancer. 27,30 By combining EC-70124 or midostaurin with the mTOR inhibitor torin, we studied the role of mTOR signaling in the cytotoxic effect of indolocarbazole analogs. Torin induced certain level of toxicity and did not synergize with EC-70124, which was an efficient mTOR inhibitor by itself and therefore did not allow for a mechanistic synergy with torin.…”

“…In line with this aim, EC‐70124 is a hybrid indolocarbazole analog with a potent multikinase inhibitor spectrum affecting key signaling kinases implicated in pro‐survival and proliferative pathways. Thus, EC‐70124 have shown antitumor activity in breast, glioblastoma, colorectal and prostate tumors as well as in acute myeloid leukemia (AML) associated to the inhibition of a wide variety of targets including components of the PI3K/AKT/mTOR, JAK/STAT, NFκB or FLT3 pathways . Interestingly, midostaurin (PKC‐412), an indolocarbazole clinically approved for AML treatment, has shown antitumor activity in certain sarcoma subtypes …”

“…Thus, EC-70124 have shown antitumor activity in breast, glioblastoma, colorectal and prostate tumors as well as in acute myeloid leukemia (AML) associated to the inhibition of a wide variety of targets including components of the PI3K/AKT/mTOR, JAK/STAT, NFκB or FLT3 pathways. [26][27][28][29][30] Interestingly, midostaurin (PKC-412), an indolocarbazole clinically approved for AML treatment, has shown antitumor activity in certain sarcoma subtypes. [31][32][33] Here, we evaluated the antitumor effect properties of EC-70124 and midostaurin in relevant sarcoma models and patient-derived primary cell lines.…”

IMiDs mobilize acute myeloid leukemia blasts to peripheral blood through downregulation of CXCR4 but fail to potentiate AraC/Idarubicin activity in preclinical models of non del5q/5q- AML

“…The cytotoxic effect of IMiDs on BM-MSCs was assessed using MTT assays as previously described. 21,22 Briefly, 2 × 10 4 cells were cultured with increasing drug concentrations in 96-well plates for 48 h. To determine the chemoprotective effect of BM-MSCs a total of 2 × 10 4 BM-MSCs were plated in 96-well plates 18 h before addition of AML cells (2 × 10 4 for cell lines and 2 × 10 5 for primary cells). BM-MSC:AML co-cultures were treated with IC 25 concentrations of the AraC (77nM) and Idarubicin (7nM) and 10 µM of lenalidomide/pomalidomide for 48–72 h. Apoptosis of CD33 + AML cells was measured using the annexin-V/7-AAD apoptosis detection kit (BD Biosciences) on a FACSCanto-II cytometer using FACSDiva software (BD Biosciences) as previously described.…”