PI3-Kinase Inhibition Decreases the Proliferation of ALL Cells, Potentiates mTOR Inhibition, and Abrogates Growth Factor-Mediated Reversal of mTOR Inhibition.

Biologically targeted cancer agents, including signal transduction inhibitors, have shown great promise in treating hematologic malignancies. However, used as single agents, these drugs may not be curative secondary to innate or acquired cellular resistance. Thus, acute lymphoblastic leukemia (ALL) and other cancer cells may become resistant to rapamycin, an mTOR inhibitor (MTI), following extended exposure to the drug. A strategy to overcome such resistance is to combine targeted agents, and thereby inhibit multiple targets simultaneously. Previously, we have shown activity of MTI in models of both human and murine ALL. In mouse models, treatment of ALL with MTI prolongs survival but may not cure disease. IL-7, a lymphoid growth factor important in the regulation of progenitor B cell development and proliferation, can reverse the inhibitory effects of MTI on human and murine pre-B ALL cells. We wished to further explore the mechanisms by which IL-7-mediated signaling protects ALL cells from the inhibitory effects of MTI, through the investigation of modulators of growth factor signaling in ALL. Thus, we have evaluated the impact of LY294002, an inhibitor of phosphatidyl inositol-3 kinase (PI3K). PI3K is a critical signaling molecule in cell survival and proliferation, with one of its central roles being signal transduction from growth factor receptors to the activation of AKT (an upstream regulator of mTOR). PI3K/AKT pathway over-activation has been implicated in many different cancers. Treatment of ALL cell lines with the PI3K inhibitor LY294002 markedly decreased cell proliferation in a dose-dependent manner. More importantly, the inhibitory effects of LY294002 were additive or synergistic with the inhibitory effects of MTI, and prevented the ability of IL-7 to reverse the inhibitory effects of rapamycin. Treatment of pre-B ALL cell lines with 2.5 μM LY294002 resulted in decreased proliferation to 20–45% of baseline as compared to untreated cells, whereas treatment with a higher dose (5 μM) reduced cell proliferation to 10–20%. Combinations of LY294002 and rapamycin, even at low doses, inhibited cell proliferation to a greater degree than each drug individually. Co-treatment with 2.5 μM LY294002 and low dose rapamycin (1 ng/ml) resulted in profound inhibition of proliferation to <=5%, compared to 20–30% with rapamycin alone. Furthermore, co-treatment with low-dose LY294002 and low-dose rapamycin resulted in greater inhibition than even higher doses of each of these agents individually. While the addition of IL-7 (1 U/ml) to rapamycin-treated cells resulted in the reversal of rapamycin-mediated cell inhibition, the further addition of 2.5 μM LY294002 significantly antagonized this growth factor rescue of MTI-treated ALL cells. The blockade by LY294002 of the IL-7 effect was most apparent in ALL cell lines that were IL-7 dependent, with cell proliferation reduced to <20%. However, the effects were still significant in IL-7 independent cell lines, with proliferation reduced to 20–60%. Similar results were seen using human ALL cell lines. These data suggest that

1. the PI3K signaling pathway serves as a potential rescue pathway from mTOR inhibition, mediating the ability of growth factors to rescue cells from rapamycin;

2. PI3K itself is a therapeutic target for ALL; and

3. combination therapy with MTI and PI3K inhibitors may be more active than either agent alone.