The PI3K Delta Inhibitor Idelalisib Interferes With Pre-B Cell Receptor Signaling In Acute Lymphoblastic Leukemia (ALL): A New Therapeutic Concept

Phosphoinositide-3-kinases (PI3K) play an important role in transmitting signals from surface receptors such as the B-cell receptor (BCR), cytokine receptors and receptor tyrosine kinases, that result in survival and growth of normal and malignant B cells. In mature B-cell malignancies such as CLL and indolent B-NHL, the PI3K pathway is constitutively upregulated and is dependent on PI3Kδ. Idelalisib is a p110δ-isoform-specific PI3K inhibitor that is highly active in patients with CLL and indolent NHL. In contrast to mature B cell malignancies, expression and function of PI3Kδ in B-ALL has not been well characterized.

RNA expression of the PI3K isoforms α, β, γ, and δ was detected in all B-ALL cell lines. Protein expression was analyzed by immunoblotting. We noted that in vitro responsiveness to idelalisib treatment was associated with protein expression of the δ isoform and presence of the Pre-BCR. We found that treatment of B-ALL cell lines with idelalisib at concentrations between 10 nM and 5 µM inhibited metabolism and growth of B-ALL cells expressing a pre-BCR, whereas only minor effects were observed in Pro-B. To monitor the expression and phosphorylation of proteins (CD72, Akt, Plcγ2, S6, Vav) involved in BCR and PI3K signaling, we prepared protein lysates of B-ALL cell lines. Cells were treated with idelalisib with/without stimulation of the µ-heavy chain of the (Pre-) BCR. We were able to show an increase of pAkt^Ser473 and pS6^Ser235/236 after stimulation, as well as a decrease in a dose dependent manner with idelalisib. The decreased amount of Akt phosphorylation was linear with the sensitivity to idelalisib treatment of the cell lines. To investigate intracellular calcium mobilization which occurs in B cells through pre-BCR stimulation, we treated cells with idelalisib and stimulated them with anti-Igµ. A dose dependent decrease in calcium flux was observed in 5 of 6 pre-B cell lines. To examine the effects of idelalisib treatment on the gene expression of pre-B ALL cells, gene expression profiling (GEP) was performed. This revealed down regulation of several genes involved in MAP-Kinase signaling, (Pre-) BCR signaling and Natural Killer cell (NK-cell) mediated cytotoxicity. For the Pre-BCR signaling pathway several genes were differentially expressed, including genes encoding the following proteins, which were found to be down regulated after 3 days of 1 µM idelalisib treatment: BCL-6, CD72, CD79a, Vav, and Plcγ2. To verify the data of the GEP, qPCR analysis was performed. To further investigate the effects of idelalisib on proteins involved in BCR signaling, six Pre-B-ALL cell lines, as well as one mature and one Pro-B cell line were treated with 1 µM idelalisib and the protein expression was quantified after immunoblotting. Most of the proteins that were differentially expressed on genomic levels have also been differentially expressed on proteomic levels and therefore confirmed the effect on Pre-BCR signaling.

In summary, these experiments demonstrate inhibition of Pre-BCR signaling on both gene and protein expression levels via idelalisib treatment of Pre-B-ALL cell lines and support the importance of clinical development of the δ isoform specific PI3K inhibitor idelalisib.