The PI3Kdelta Inhibitor, CAL-101, Inhibits Migration of Primary Pre-B ALL to SDF1alpha: Treatment Implications for Overcoming Cell-Adhesion-Mediated Drug Resistance

BACKGROUND: 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 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. In addition, we have shown that treatment with CAL-101, a commercially available PI3Kδ inhibitor, de-adheres pre-B ALL cells from the α4-counter-receptor VCAM1, downregulates p-Akt and induces apoptosis in primary pre-B ALL cells. However, the microenvironmental stimuli that activate PI3K/AKT in ALL remain unknown. PI3Kδ can be activated by extracellular signals upon engagement with extracellular matrix, however specific ligands remain elusive, the identification of which would be critical to understand the mechanism of PI3Kδ inhibition. These studies were designed to determine which extracellular ligands activate PI3K/AKT in patient-derived (primary) pre-B ALL cells and whether CAL-101 affects adhesion and migration of ALL cells.

METHODS: Four samples ofpatient-derived (primary) pre-B ALL cells, co-cultured with murine calvaria-derived mesenchymal stromal (OP9) cells were treated with a commercially available PI3Kδ inhibitor, CAL-101 (Selleckchem), or its vehicle DMSO control. Culture plates with and without transwells were used for in vitro assays. Annexin V/7-AAD staining was used for viability determination by flow cytometry and Western Blot was used for determination of total Akt and p-Akt expression.

RESULTS: To determine which ligands activate p-Akt, ALL cells were adhered to fibronectin (FN), VCAM-1 (both counter-receptors of integrin α4), laminin-1 (hLam-1; counter-receptor for integrin α6) and albumin (BSA). OP9 cells were used in direct contact with ALL cells or separated through a transwell insert. α-MEM or RPMI were used as media controls. hVCAM-1 and hLam-1 activated p-Akt in one out of three samples. In all 3 samples, OP9 cells activated p-Akt compared to media control, but only in direct contact, not when separated through a transwell. This suggests that direct adhesion of ALL cells to stromal cells rather than diffusible factors are associated with p-Akt activation. Next, we determined if CAL-101 de-adheres ALL cells independently from the ligands ALL cells are bound to. Primary pre-B ALL cells were plated in triplicates onto FN, VCAM-1, hLam1-coated 96-well plates and incubated with CAL-101 (10µM) ± HUTS-21 antibody (20µg/mL). HUTS-21 is an antibody that binds only to the active form of integrin β1, CD29, and can activate outside-in signaling via integrin β1 stimulation. CAL-101 inhibited cell-adhesion from FN (15±4.2% vs. 51±9.9%, p=0.04) and VCAM-1 (54.6 x±8.6%vs. 95.8±3.8%, p=0.03), but not from hLam1 indicating that it acts specifically on the VCAM-1/FN-mediated adhesion of ALL cells. HUTS-21 restored cell-adhesion of ALL cells to FN (67.0±1.4% vs. 81±7.1%, p=0.19) and VCAM-1 (42.5±16.2% vs. 50.6±8.6%, p=0.59) indicating adhesion recovering upon HUTS-21 addition to CAL-101-treated cells. Next, we determined if CAL-101 has an impact on migration of ALL cells. ALL cells were treated with CAL-101 (10μM) or DMSO control for 24 hours and added to a migration assay. We observed that migration of ALL cells to stromal cell-derived factor 1alpha (SDF-1alpha; CXCL12) was significantly decreased (1.7 x10³ ±0.3x10³ vs. 3.1x10³ ±2.4x10³ p=0.004), while migration to OP9 cells was partially inhibited compared to DMSO control (2.1 x10³ ±0.5x10³ vs. 4.2 x10³ ±0.5x10³ p=0.005).

CONCLUSION: Taken together, our data demonstrate that the PI3Kδ/AKT pathway is stimulated by extracellular matrices and that PI3Kδ inhibition with CAL-101 inhibits adhesion of ALL cells to FN/VCAM-1 but not Lam1, which can be restored using HUTS-21. Data derived from further studies will determine the potential of the FDA-approved PI3Kδinhibitor Idelalisib as a novel therapy for pre-B ALL.