Growth-Inhibitory Effects of a Class I PI3K Inhibitor GDC-0941 Are Modulated by Hypoxia-Induced mTOR Pathway Activation in Mantle Cell Lymphoma

Abstract 1424

Hypoxia confers pro-survival signals to tumor cells via multiple mechanisms including activation of phosphatidylinositol 3-kinase (PI3K)/Akt/mTOR pathway. Constitutive activation of PI3K/Akt signaling pathway is documented in aggressive blastoid variants of mantle cell lymphoma (MCL) and is implicated in the pathogenesis of MCL. mTOR signaling, one of the important downstream targets of Akt, positively regulates protein translation by phosphorylating p70 ribosomal S6 kinase (S6K) or eukaryotic initiation factor 4E-binding protein-1 (4E-BP1). We have reported that the Class IA PI3K isoforms, mainly p110α and partially p110δ, are responsible for PI3K/Akt/mTOR signaling activation in blastoid MCL cells (Zhou et al., ASH abstract 2010). In this study, the activity and molecular mechanisms of action of a selective pan-isoform class I PI3K inhibitor GDC-0941 (Genentech) were investigated in the context of hypoxic microenvironment in MCL cells. For hypoxia experiments, MCL cells were cultured under 1% O₂ for at least 14 days to assure their sustained proliferation and survival. Under hypoxia, more cells accumulated in G₀/G₁ phase, indicating that hypoxic conditions promote cell cycle quiescence in MCL cells. GDC-0941 treatment in normoxia resulted in a reduction of cell proliferation in a dose-dependent manner (IC₅₀ at 48 hrs; 0.7 μM for Granta519, 0.5 μM for JVM2, 1.0 μM for MINO, 0.7 μM for Jeko-1, MTT test). In hypoxia, higher doses of GDC-0941 were required to cause cell growth inhibition compared to normoxic conditions in Granta519 (IC₅₀; 1.7 μM), JVM2 (3.5 μM) and MINO (1.2 μM). Further, GDC-0941 failed to inhibit growth of Jeko-1 cells under hypoxia. GDC-0941 (0.5 μM) induced G_0/1 cell cycle arrest under both normoxic and hypoxic conditions in Granta519, JVM2 and MINO cells. No significant induction of apoptosis by GDC-0941 (0.5 μM) was observed in any of the tested MCL cells. GDC-0941 completely inhibited phosphorylated (p-Ser473) Akt in all cell lines analyzed under both normoxic and hypoxic conditions, which resulted in decreased expression of p-GSK-3β and its downstream target cyclin D1. GDC-0941 further downregulated the mTOR downstream targets p-S6K and p-4EBP1 in Granta519, JVM2 and MINO cells under both, normoxia and hypoxia. On the contrary, GDC-0941 failed to affect hypoxia-induced p-S6K and p-4EBP1 expression levels in Jeko-1 cells, whereas potently suppressed these proteins at ambient oxygen level. These results suggest that in Jeko-1 cells hypoxia activates mTOR through PI3K-independent pathways. To test this, we examined consequences of mTOR blockade in Jeko-1 cells. mTOR inhibitor rapamycin (100 nM) moderately diminished cell growth under normoxic conditions causing accumulation of cells in G_0/1 cell cycle phase but no apoptosis. In turn, under hypoxia rapamycin profoundly inhibited cell growth, inducing >80% of MCL cells to undergo cell death.

These findings indicate that in certain MCL cells hypoxia activates mTOR through alternative, PI3K-independent pathways, which facilitate cell survival under hypoxia. Hence, blockade of class I PI3K may eliminate circulating MCL cells but not tissue-resident cells in hypoxic areas of the bone marrow or lymph nodes. In turn, simultaneous therapeutic ablation of PI3K/Akt and mTOR pathway may represent a promising strategy to target the aggressive blastoid variants of MCL cells in the hypoxic microenvironments.