PI3Kdelta Inhibitor, CAL-101, De-Adheres Primary Pre-B ALL from VCAM-1 and Induces Apoptosis in Primary Pre-B ALL

BACKGROUND: Relapse remains a major problem in the treatment pre-B acute lymphoblastic leukemia (ALL). ALL cells are physically anchored in the bone marrow (BM) microenvironment through a network of adhesion molecules. Adhesion also creates intracellular signals that regulate proliferation and cell death. We have previously identified integrin α4 (α4) as a critical molecule for such cell-adhesion-mediated drug resistance (CAM-DR) in pre-B ALL. In chronic lymphocytic leukemia (CLL), α4-mediated activation of the PI3K/AKT pathway was reported. In ALL, stromal cell protection of B-lineage ALL cells has also been shown to require active Akt. However, it remains elusive how this target can be therapeutically harnessed. Therefore, we investigated if directly targeting the PI3K/AKT pathway downstream of α4 can overcome CAM-DR in pre-B ALL using a novel, FDA-approved PI3Kδ inhibitor, CAL-101

METHODS: For in vitro studies, murine integrin α4 conditional knockout mouse model (α4^fl/fl) oncogenically transformed with BCR-ABL1+ and patient-derived (primary) pre-B ALL cells (Philadelphia chromosome negative) co-cultured with murine calvaria-derived mesenchymal stromal (OP9) cells were used. Annexin V/7-AAD staining was used for viability determination by flow cytometry and Western Blot was used for determination of PI3Kδ and p-Akt expression. For in vivo experiments, we used a NOD/SCID IL2Rγ^-/- xenograft model of primary pre-B ALL.

RESULTS: Integrin α4^fl/fl cells were cultured under lymphoid-skewing conditions and oncogenically transformed using BCR-ABL1 (p210). Subsequent transduction with CreER^T2 or EmptyER^T2 generated leukemia cells in which α4 ablation could be induced (CreER^T2) or not (EmptyER^T2) by addition of Tamoxifen while plated on murine VCAM-1. In the absence of a4, mouse pre-B ALL cells contain markedly reduced levels of pAKT (ser⁴⁷³). In a parallel experiment, we observed in primary pre-B ALL cells co-cultured with OP9 cells an increase in p-AKT compared to media-only cultured ALL cells indicating activation of the PI3K/AKT pathway upon adhesion to stroma. Under starvation conditions, primary pre-B ALL cells (LAX7R) were incubated for 4 hours without serum or stroma, and subsequently treated for 48 hours with an integrin α4 blocking antibody or with control IgG4. By Western Blot, we detected decreased levels of activated p-AKT in the cells with α4 blockade, but not with control, indicating that the AKT pathway is affected by modulation of α4.To directly target the PI3K/AKT pathway, we used CAL-101, a small molecule inhibitor highly selective for the δ isoform, which was recently FDA-approved for treatment of CLL and Non-Hodgkin Lymphoma (NHL). Previously, in primary samples from CLL patients and ALL cell lines, CAL-101 blocked PI3Kδ-AKT signaling and promoted apoptosis. However, a preclinical evaluation in primary pre-B ALL patients in vitro and in vivo are missing. For these reasons, we used CAL-101 in the following experiments as a specific PI3Kδ inhibitor in primary pre-B ALL. Expression of PI3Kδ was demonstrated by Western Blot in 8 out of 8 cases of primary pre-B ALL. After incubation of primary pre-B ALL cells with different concentrations of CAL-101 (0.1-10 µM), dose-dependent downregulation of pAkt was observed. ALL cells were plated on immobilized VCAM1 (the counter-receptor for α4) or OP9 cells and treated with vehicle control (DMSO) or CAL-101 (10 µM) for 2 days. CAL-101 de-adhered ALL cells 69.5%±0.7% from VCAM1, and only 13%±0.5% from OP-9 cells, indicating an inhibition of the of the VCAM-1 and α4 interaction. ALL cells treated with CAL-101 (10 µM) for 5 days showed a marked decrease in viability as demonstrated by AnnexinV negative and 7AAD negative staining compared to DMSO-treated controls (66.2%±1.5% vs 87.9%±1.0%, p=0.0003) indicating that mono-treatment with CAL-101-decreases viability of ALL cells. Further studies addressing whether PI3Kδ inhibition is beneficial in the setting of combination chemotherapy for ALL in vitro and in vivo are ongoing.

CONCLUSION: Taken together, our data demonstrate that the AKT pathway is affected by modulation of α4. PI3Kδ inhibition with CAL-101 de-adheres pre-B ALL cells from the α4-counter-receptor VCAM1, downregulates pAkt and induces apoptosis in ALL. Data derived from further studies will determine the potential of the FDA-approved PI3Kδinhibitor CAL-101 as a novel therapy for pre-B ALL.