Activation of the phosphoinositide-3 kinase (PI3K) pathway contributes to mantle cell lymphoma (MCL) pathogenesis, but early phase studies of the p110δ selective inhibitor GS-1101 demonstrate inferior responses in MCL compared to CLL and indolent NHL. The relative importance of the class Ia PI3K isoforms - p110α, p110β and p110δ in MCL is not clear. While p110δ is enriched in leucocytes, p110α gene amplification has been reported in 68% of MCL. Loss of PTEN expression occurs in approximately 15% of MCL and evidence in solid tumors suggests an essential role for p110β in PI3K signaling associated with PTEN loss. We previously reported significant up-regulation of p110α expression in relapsed disease as well as significantly greater in vitro cytotoxicity with GDC0941, a predominantly p110α/δ inhibitor, compared to the p110δ selective inhibitor GS-1101 in MCL. To investigate this further, we used isoform selective inhibitors to study the relative contribution of class Ia isoforms to PI3K signaling in the context of B-cell receptor (BCR) activation, p110α over-expression and PTEN deletion in this disease. We also screened tumor cells for activating mutations in PIK3CA and PIK3R1, the genes encoding p110α and p85α respectively.
Peripheral blood mononuclear cells (PBMCs) and lymph nodes from MCL patients were used in addition to two MCL cell lines Jeko1 and Granta519. Goat anti-human IgM f(a'b) fragments were used for BCR stimulation. Immunohistochemistry (IHC) and western blotting were used to assess PTEN expression. In addition to GDC0941 and GS-1101, A66 was used for p110α-selective inhibition and TGX-221 for p110β inhibition. GS-1101 was purchased from Active Biochem and all other inhibitors from Selleck Chem. Changes in downstream targets of PI3K (p-Akt t308, p-Akt s473 and p-S6 s235/236) were evaluated using western blotting. DNA was extracted from 10 primary samples and 2 cell lines. Exons 9, 10, 11, 13, 15 and 16 of PIK3R1 and exons 9 and 20 of PIK3CA, were sequenced and screened for activating mutations.
p110δ is expressed at high levels in MCL and p110δ selective inhibition with GS-1101 was sufficient to block BCR-stimulated activation of the PI3K pathway in MCL cells. In order to investigate the relevance of increased p110α in relapsed MCL, that we previously reported, we treated the Granta519 MCL cell line, which exhibits p110α gene amplification and constitutive Akt phosphorylation, with GS-1101 and GDC0941. In GS-1101 treated cells, phosphorylation of Akt was still detectable at low levels after 2 hours of treatment while p-Akt was undetectable in cells treated with equimolar concentrations (1μM) of GDC0941. At 24 hours, GS-1101 treated cells showed an increase in Akt phosphorylation compared to 2 hour levels suggesting ongoing p110α mediated signaling despite p110δ inhibition, whereas p-Akt remained undetectable in GDC-0941 treated cells. These changes were reflected in downstream phosphorylation of ribosomal S6. The addition of the highly selective p110β inhibitor TGX221 to GS-1101 did not have any additional effect on p-Akt whereas adding a p110α selective inhibitor (A66) mimicked the activity of GDC0941, further supporting the role of p110α in p110δ-independent PI3K signaling. We found loss of PTEN expression in 17% (25/147) of MCL biopsies by IHC. There was no change in the pattern of class Ia isoform expression in tumors with loss of PTEN and adding a p110β inhibitor to GS-1101 did not increase cytotoxicity in MCL primaries that had loss of PTEN expression. No activating PIK3CA or PIK3R1 mutations were detected in 10 primary MCL samples and 2 cell lines.
In healthy B-cells, p110δ is essential for BCR mediated PI3K signaling but both p110α and p110δ contribute to tonic PI3K signaling. Our studies confirm a similar essential role for p110δ in MCL cells both by its expression and its role in BCR signaling. However, over-expression of p110α in MCL, especially in relapsed disease, increases the contribution of this isoform to tonic PI3K signaling thereby limiting the efficacy of p110δ-selective inhibition. The increased expression of p110α with relapse may reflect reduced dependence of tumor cells on survival signals from their microenvironment. We conclude that whereas PI3K p110β does not appear to play a significant role in MCL, inhibition of both p110α and δ isoforms appears essential for effective PI3K inhibition in this disease.
Auer:Pharmacyclics: Research Funding. Gribben:Celgene: Honoraria; Roche: Honoraria; Pharmacyclics: Honoraria; GSK: Honoraria; Mundipharma: Honoraria; Gilead: Honoraria.
Asterisk with author names denotes non-ASH members.