CDK4/6 inhibition can also overcome resistance to vemurafenib in mutant melanoma cell lines (44)

CDK4/6 inhibition can also overcome resistance to vemurafenib in mutant melanoma cell lines (44). AKT pathway. Analysis of these CEP-1347 cells revealed that CDK4/6 inhibition failed to induce cell cycle arrest or senescence. Mechanistic investigations showed that resistant cells coordinately upregulated expression of cyclins A, E and D1, activated phospho-CDK2 and phospho-S477/T479 AKT. Treatment with GSK2334470 or the CDK2 inhibitor dinaciclib was sufficient to reverse these events and restore the sensitivity of ribociclib-resistant cells to CDK4/6 inhibitors. Ribociclib in combination with GSK2334470 or the PI3K inhibitor alpelisib decreased xenograft tumor growth more potently than each drug alone. Taken together, our results highlight a role for the PI3K-PDK1 signaling pathway in mediating acquired resistance to CDK4/6 inhibitors. and using two independent siRNAs, each in combination with 0.25 M ribociclib, in MCF-7, T47D, HCC1428, and HCC1500 ER+ breast cancer cells. Individually, ribociclib treatment and PDK1 siRNA transfection inhibited proliferation of all four cell lines (Fig. 1C). However, combined inhibition of CDK4/6 (with ribociclib) and of PDK1 (with siRNA) led to a statistically significant reduction in cell proliferation in MCF-7, T47D, and HCC1500 cell lines, consistent with the results of the kinome screen. This effect was CEP-1347 greater in wildCtype cell lines (HCC1428 and HCC1500). Knockdown of resulted in decreased phosphorylation of S6, a downstream effector of the PDK1 target p70S6K (Fig. 1D and Supplementary Fig. S1B). To subscribe CDK4-specificity to the effects of ribociclib, we treated MCF-7 cells with CDK4 and PDK1 siRNA oligonucleotides, individually and in combination. Treatment with both siRNAs inhibited cell viability more potently than each alone while simultaneously reducing levels of PDK1, CDK4, and P-Rb (Supplementary Fig. 1C), suggesting the effects of ribociclib may extend to other CDK 4/6 inhibitors. Pharmacological blockade of PDK1 and CDK4/6 synergistically inhibits ER+ breast cancer cell proliferation We next examined the effect of pharmacological inhibition of PDK1 in combination with CDK4/6 inhibitors. GSK2334470 is a highly specific small molecule inhibitor of PDK1 with a published inhibitory activity in the nanomolar range (16, Mouse monoclonal to INHA 25). GSK2334470 suppresses T-loop phosphorylation and subsequent activation of the PDK1 substrates AKT, S6K, RSK2, and SGK architecture and growth rates of cancers (28C30). Thus, we next extended our findings to cells growing in Matrigel in three-dimensional (3D) culture. Under these conditions, GSK2334470 enhanced the anti-proliferative effect of both ribociclib and palbociclib against MCF-7, T47D, and HCC1500 cells (Fig. 2C). Of note, the combined effect of CDK4/6 and PDK1 inhibitors in wild-type HCC1428 and HCC1500 cells was less pronounced than in < 0.01; ****, CEP-1347 < 0.0001 by ANOVA). Combination therapies with CDK4/6 inhibitors are also being evaluated in other advanced solid tumors (REF 31). To test whether these findings in ER+ breast cancer cells can be translated to other tumor types, we treated triple negative breast cancer, ovarian/endometrial, melanoma, and glioblastoma cell lines with ribociclib, GSK23334470, or the combination. Results showed that the combination induced greater inhibition of cell viability compared to each drug alone (Supplementary Fig. S3B,C). These observations suggest that PDK1 plays a role in mediating resistance to CDK4/6 inhibition in a variety of tumor types where CDK4/6 inhibitors are being investigated clinically (31). In addition to cell cycle arrest, CDK4/6 inhibitors can induce senescence through regulation of FoxM1-mediated transcription (32). Consistent with this, we observed a decrease in FoxM1 levels and an increase in senescence-associated (SA) -galactosidase positive cells upon treatment with ribociclib, which was unaffected by the PDK1 inhibitor (Fig. 2E,F). Treatment with GSK2334470 alone or in combination with CEP-1347 ribociclib induced apoptosis as measured by increased annexin V staining (Fig. 2G) and poly (ADP) ribose polymerase (PARP) cleavage (Fig. 2H), compared to DMSO or ribociclib treated MCF-7 cells. These findings suggest that inhibition of PDK1 with GSK2334470 induces apoptosis without counteracting the effect of ribociclib on tumor cell senescence resulting in the synergistic growth inhibition of ER+ breast cancer cells. Inhibition of PI3K/PDK1 enhances the anti-tumor effect of ribociclib < 0.05 vs. single-agent ribociclib or GSK2334470). Numbers in parenthesis represent the.