Using a biosensor of AKT activity, we showed that initial efficacy of individual PI3K/AKT pathway inhibitors is lost within 24 h, due to previously-described compensatory activation [11]. Vertical combination of 3 or more inhibitors synergistically prevented this compensation, enabling sustained inhibition and toxicity to cancer cells at low concentrations. Clinically-approved inhibitors of multiple PI3K isoforms, and rapamycin as a 4th drug, were also shown to be synergistic in combinations. In vivo, combining two approved inhibitors (targeting PI3Kδ and AKT) with another in clinical trials (targeting PDPK1) showed anti-tumor efficacy without obvious toxicity, suggesting potential for clinical application. Furthermore, many cancers have distinctive upstream pathway activators that are targetable (Fig. 1A), such as kinases activated by the B-cell receptor in lymphoma, or receptor tyrosine kinases in carcinomas (e.g., EGFR or ERBB2).

Dose reduction by synergy may be especially important for PI3K inhibitors, “the ‘poster child’ for a drug class that should have been looking for a minimum-effective dose instead of a maximum-tolerated dose” [12]. Use of PI3K inhibitors has been limited by serious toxicities, likely due to their effect on PI3K (or another target) instead of AKT, since idelalisib used alone does not achieve sustained AKT inhibition. The synergy of vertical combinations may enable use of PI3K inhibitors at lower doses, reducing toxicity and increasing use of these potentially-valuable anticancer agents; however, more studies are needed, given the diverse spectrum of toxicities caused by PI3K inhibitors.

Previous studies of combined PI3K/AKT pathway inhibition in cancer have only targeted mTOR and another pathway member, with two inhibitors or a dual inhibitor [13,14,15]. Our results suggest that vertical combination of 3 or more agents may achieve the long-sought goal of tolerable but effective inhibition of the PI3K/AKT pathway.