The phosphatidylinositol 3‑kinase/protein kinase B (PI3K/AKT) pathway promotes uncontrolled growth and survival in many cancers.(1) Small-molecule inhibitors of the PI3K/AKT pathway are approved for certain lymphoid neoplasms, but have several limitations.(2) Because PI3K inhibitors are ATP-competitive, they have low specificity; furthermore, because the PI3K/AKT pathway is important in many normal cells, pathway inhibitors are dose-limited by side effects, leading to low efficacy and/or resistance development.(3)
PI3K/AKT pathway inhibitors are more potent at reducing AKT activity than viable cell number.
We hypothesized that PI3K/AKT pathway inhibition might be improved by combining inhibitors acting at different levels (Fig. 1A), which we term “vertical” combination. We first assessed PI3K/AKT pathway inhibitors, used individually, for their ability to inhibit AKT activity measured with a FRET-based biosensor (4, 5) in two cell lines representing diffuse large B-cell lymphoma. “Kinetic” monitoring showed that idelalisib (targeting the PI3Kδ isoform), GSK2334470 (PDPK1 inhibitor), and ipatasertib (pan-AKT inhibitor) decreased AKT activity substantially within minutes (Fig. S1). However, for idelalisib and GSK2334470, the IC50 concentration for inhibition of cell growth after 4 days (Fig. S2) was much higher than their AKT-inhibitory IC50 concentrations determined after 1-hr incubations (Fig. S3 and 1B).
Single PI3K/AKT inhibitors cause only transient decrease of AKT activity.
To investigate discrepancies in potency of individual PI3K/AKT inhibitors at AKT inhibition vs. growth reduction, we evaluated AKT activity over multiple timepoints. Maximal inhibition was reached at 0.5-1 hours, and sustained for ~6 hours (Fig. S4). AKT activity rebounded thereafter, however, and reached essentially normal levels at 24 hours (Fig. 1C). This increase was not due to inhibitor degradation: medium from treated cultures was equally able to inhibit AKT when cultured with untreated cells (Fig. S5).
Instead, inhibitor release experiments confirmed previous reports that compensatory responses to PI3K/AKT inhibitors normalize AKT activity.(6) Cells treated with individual inhibitors for 24 hours, when washed and allowed to recover in inhibitor-free medium, showed a rapid increase in AKT activity to supranormal levels (Fig. 1D). Counter-regulation was also implied by the PI3K/AKT pathway inhibitor rapamycin, whose target (mTOR) is downstream of AKT; although rapamycin was effective at reducing cell growth (Fig. S2), it caused an increase in AKT activity (Fig. S3), as previously reported.(7)
Combined PI3K/AKT inhibitors provide lasting AKT activity inhibition and synergistic reduction in cell growth
GSK2334470 and ipatasertib produced lasting inhibition of AKT activity when combined with idelalisib (Fig. 1E), but not without idelalisib (Fig. 1F). Maximal AKT inhibition with this triple combination was reached between 3 and 12 hours after treatment initiation (Fig. 1E), later than with single inhibitors (Fig. 1C). When cross-titrations of pairs of PI3K/AKT inhibitors acting at different levels were evaluated for their effect on cell viability, all tested combinations showed strong synergy (Fig. 2A and Fig. S6). In contrast, paired AKT inhibitors (ipatasertib and MK2206) showed little or no synergy (Fig. S7).
Vertical combination lowers the concentrations of PI3K/AKT pathway inhibitors needed for growth inhibition, and provides safe and effective antitumor activity in vivo.
We hypothesized that combination would lower the growth-inhibitory IC50 concentrations of PI3K/AKT inhibitors. From cross-titrations, we determined a highly-synergistic fixed molar ratio of inhibitors for further testing: idelalisib/GSK2334470/ipatasertib/rapamycin = 1/10/5/0.00005. When 2 to 4 inhibitors were used together in these ratios, IC50 concentrations were substantially decreased by each additional inhibitor (Fig. 2B and Fig. S8).
We tested vertical PI3K/AKT inhibition of syngeneic tumor growth in vivo (Fig. 2C), using the lymphoma cell line A20 in immune-competent BALB/c mice.(8) PI3K/AKT inhibitors were first tested for A20 growth inhibition in vitro; combination in the fixed molar ratio above reduced their IC50 concentrations, as compared to individual use (Fig. S9), from which in vivo dosing was determined (see Supplement). A20 was resistant to rapamycin, which was not used for in vivo experiments.
Tumor growth was significantly reduced by combination treatment (Fig. 2D, S10, and S11), resulting in longer survival (Fig. 2E). Vertical combination showed no side effects on body weight (Fig. S12), liver or renal function (Fig. S13), or blood counts (Fig. S14), including at the time of sacrifice (Fig. S15). This suggests that this triple combination is not toxic, and that higher doses might have been tolerated, perhaps improving tumor control.