Influence of PI3K and MAPK Pathway Mutations on Response to Mono and Dual Treatment with Targeted Kinase Inhibitors

Abstract

Genetic heterogeneity and drug resistance has hindered the clinical development of targeted kinase inhibitors for treatment of glioblastoma, the most common and aggressive primary brain tumor. We addressed these issues using immortalized astrocytes harvested from genetically engineered mice with and without phosphoinositide 3‐kinase (PI3K) and/or RAS/mitogen activated protein kinase (MAPK) pathway mutations, specifically Pten deletion and/or oncogenic Kras. We found that the potency of PI3K pathway inhibitors (PI3Ki potency: BEZ235 > BKM120 > LY294002) varied and sensitivity was increased 2 – 19 fold by mutations in Pten, Kras, or both. Sensitivity to MEK inhibitors (MEKi potency: GSK1120212 > PD0325901 > AZD6244) was increased 5 – 53 fold by Pten, but not Kras mutations. To determine how BKM120 alters the kinome, we used multiplexed inhibitor bead‐mass spectrometry (MIB‐MS). In immortalized astrocytes with both Pten and Kras mutations, the PI3Ki BKM120 caused dynamic kinome changes, including sustained inhibition of proximal PI3K, inhibition followed by re‐activation of distal PI3K signaling, and alternative activation of MAPK signaling. These results were confirmed by immunoblot. The more potent dual PI3K/mTOR inhibitor BEZ235 inhibited PI3K and induced alternative activation of MAPK at lower concentrations. Conversely, multiple MEKi caused MAPK inhibition and alternative activation of PI3K signaling. Next we determined whether Pten or Kras mutations alone influenced alternative pathway activation by PI3Ki or MEKi. BKM120 inhibited PI3K signaling and induced alternative activation of MAPK independent of Pten and Kras mutation status. In contrast, treatment with the MEKi AZD6244 induced alternative activation of PI3K in immortalized astrocytes with activated Kras, independent of Pten. These findings suggested that alternative activation of PI3K or MAPK signaling promotes drug resistance in response to monotherapy inhibition of the other arm of receptor tyrosine kinase signaling. Combined treatment of immortalized, Pten‐deleted, Kras‐activated astrocytes with both the PI3Ki BKM120 and the MEKi AZD6244, PD0325901, or GSK1120212 ablated alternative pathway activation and functioned synergistically at physiologically achievable concentrations. Moreover, synergism was achieved at lower PI3Ki doses when a more potent MEKi was used. We then examined how Pten and Kras mutations and MEKi potency interact to influence drug synergism. BKM120 and AZD6244 became synergistic at ≥ 10× lower drug concentrations in immortalized astrocytes with Pten and/or Kras mutations. Combining BKM120 with the more potent MEKi GSK212 was more broadly synergistic and independent of mutation status. Taken together, we conclude that PI3K and MAPK mutation status, as well as drug potency, influences targeted kinase inhibitor response and that dual inhibition may be required to achieve clinical benefits.Support or Funding InformationRSM is a Robert H. Wagner Scholar in the Pathobiology and Translational Sciences Program and a trainee in the HHMI Graduate Training Program in Translational Medicine. CRM was a Damon Runyon‐Genentech Clinical Investigator supported by the Damon Runyon Cancer Research Foundation (CI‐45‐09). This work was supported by grants to CRM from the UNC University Cancer Research Fund (UCRF) and the NIH National Center for Advancing Translational Sciences (NCATS, 550KR21202) and a NIH grant to GLJ (GM101141).