Amplification of the Driving Oncogene, KRAS or BRAF, Underpins Acquired Resistance to MEK1/2 Inhibitors in Colorectal Cancer Cells

Resistance to cancer therapeutics targeting the second kinase in a three-kinase cascade involves amplification of the upstream kinase, not the inhibited kinase. Driving Resistance The promise of using targeted small-molecule kinase inhibitors in treating cancer has been shadowed by the development of resistance to these drugs. Here, Little et al. used colorectal cancer cell lines with oncogenic mutations in either KRAS or BRAF—both of which lead to increased signaling through the ERK (extracellular signal–regulated kinase) signaling pathway—to investigate the mechanisms whereby cells developed resistance to AZD6244, a MEK1/2 (mitogen-activated or extracellular signal–regulated protein kinase kinases 1 and 2) inhibitor now in clinical trials. Rather than developing mutations in MEK1/2, cancer cells became resistant to MEK1/2 through amplification of the driving oncogene (oncogenic KRAS or BRAF) and a consequent increase in signaling through the ERK pathway. These observations have implications for the use of MEK1/2 inhibitors—and possibly other inhibitors that target downstream pathway components rather than the driving oncogene itself—in combination with other antineoplastic therapies. The acquisition of resistance to protein kinase inhibitors is a growing problem in cancer treatment. We modeled acquired resistance to the MEK1/2 (mitogen-activated or extracellular signal–regulated protein kinase kinases 1 and 2) inhibitor selumetinib (AZD6244) in colorectal cancer cell lines harboring mutations in BRAF (COLO205 and HT29 lines) or KRAS (HCT116 and LoVo lines). AZD6244-resistant derivatives were refractory to AZD6244-induced cell cycle arrest and death and exhibited a marked increase in ERK1/2 (extracellular signal–regulated kinases 1 and 2) pathway signaling and cyclin D1 abundance when assessed in the absence of inhibitor. Genomic sequencing revealed no acquired mutations in MEK1 or MEK2, the primary target of AZD6244. Rather, resistant lines showed a marked up-regulation of their respective driving oncogenes, BRAF600E or KRAS13D, due to intrachromosomal amplification. Inhibition of BRAF reversed resistance to AZD6244 in COLO205 cells, which suggested that combined inhibition of MEK1/2 and BRAF may reduce the likelihood of acquired resistance in tumors with BRAF600E. Knockdown of KRAS reversed AZD6244 resistance in HCT116 cells as well as reduced the activation of ERK1/2 and protein kinase B; however, the combined inhibition of ERK1/2 and phosphatidylinositol 3-kinase signaling had little effect on AZD6244 resistance, suggesting that additional KRAS effector pathways contribute to this process. Microarray analysis identified increased expression of an 18-gene signature previously identified as reflecting MEK1/2 pathway output in resistant cells. Thus, amplification of the driving oncogene (BRAF600E or KRAS13D) can drive acquired resistance to MEK1/2 inhibitors by increasing signaling through the ERK1/2 pathway. However, up-regulation of KRAS13D leads to activation of multiple KRAS effector pathways, underlining the therapeutic challenge posed by KRAS mutations. These results may have implications for the use of combination therapies.