Supplementary Figure 1. Knockout of Mcl-1 from H1299 by CRISPR/Cas9 system results in decreased cell proliferation and increased caspase 3/7 activity. Supplementary Figure 2. Expression of constitutive active form of Akt in H1299 Mcl-1 knockout cells restores cell growth. Supplementary Figure 3. Depletion of Mcl-1 by shRNA retards the growth of H460 lung xenografts. Supplementary Figure 4. Expression of WT Mcl-1 but not the PEST deletion mutant Mcl-1 in H1299 Mcl-1 knockout cells restores Akt/PDK1 and Akt/mTORC2 interactions. Supplementary Figure 5. Treatment of H1299 cells with PH-687 results in increased intramolecular PH/KD interactions in Akt and decreased interaction of Akt with PDK1 or mTORC2. Supplementary Figure 6. Mcl-1/Akt signaling pathway is essential for PH-687 inhibition of cancer cell growth. Supplementary Figure 7. Knockout of Mcl-1 or treatment of cells with PH-687 inhibits growth factor (s)-stimulated Akt activation. Supplementary Figure 8. In vivo toxicity analysis. Supplementary Methods
ARTICLE ABSTRACTMcl-1 is a unique antiapoptotic Bcl2 family protein that functions as a gatekeeper in manipulating apoptosis and survival in cancer cells. Akt is an oncogenic kinase that regulates multiple cellular functions and its activity is significantly elevated in human cancers. Here we discovered a cross-talk between Mcl-1 and Akt in promoting lung cancer cell growth. Depletion of endogenous Mcl-1 from human lung cancer cells using CRISPR/Cas9 or Mcl-1 shRNA significantly decreased Akt activity, leading to suppression of lung cancer cell growth in vitro and in xenografts. Mechanistically, Mcl-1 directly interacted via its PEST domain with Akt at the pleckstrin homology (PH) domain. It is known that the interactions between the PH domain and kinase domain (KD) are important for maintaining Akt in an inactive state. The binding of Mcl-1/PH domain disrupted intramolecular PH/KD interactions to activate Akt. Intriguingly, Mcl-1 expression correlated with Akt activity in tumor tissues from patients with non–small cell lung cancer. Using the Mcl-1–binding PH domain of Akt as a docking site, we identified a novel small molecule, PH-687, that directly targets the PH domain and disrupts Mcl-1/Akt binding, leading to suppression of Akt activity and growth inhibition of lung cancer in vitro and in vivo. By targeting the Mcl-1/Akt interaction, this mechanism-driven agent provides a highly attractive strategy for the treatment of lung cancer.
These findings indicate that targeting Mcl-1/Akt interaction by employing small molecules such as PH-687 represents a potentially new and effective strategy for cancer treatment.