PI3Kdelta Inhibitor, CAL-101, De-Adheres Primary Pre-B ALL from VCAM-1 and Induces Apoptosis in Primary Pre-B ALL
Authors: Khazal Sajad , Jiang Enzi , Shishido Stephanie , Kosoyan Osanna , Hye-Na Kim , Velasquez Alexa , Hsieh Yao-Te , Abdel-Azim Hisham , Mohrbacher Ann M , Merchant Akil , Wayne Alan S. , Heisterkamp Nora , Kim Yong-Mi 
Published: October 2019

Abstract
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 (α4fl/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 α4fl/fl cells were cultured under lymphoid-skewing conditions and oncogenically transformed using BCR-ABL1 (p210). Subsequent transduction with CreERT2 or EmptyERT2 generated leukemia cells in which α4 ablation could be induced (CreERT2) or not (EmptyERT2) 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.

Disclosures
Wayne: MedImmune: Honoraria, Research Funding, Travel Support Other; NIH: Co-inventor on investigational products with patents, Co-inventor on investigational products with patents Other.