Acute lymphoblastic leukemia (ALL) is a highly heterogeneous disease. B-cell acute lymphoblastic leukemia (B-ALL) is characterized by uncontrolled proliferation of immature lymphoid blasts with suppression of normal hematopoiesis. Phosphoinositide 3-kinases (PI3K) transmit activation signals from diverse transmembrane receptors, leading to generation of phosphatidylinositol- 3,4,5-trisphosphate (PIP3) which promotes proliferation, differentiation, migration, and survival in lymphocytes and various other cell types. A knockout mouse model of the PI3K isoform p110δ demonstrates a unique role of p110δ (PI3Kδ) in B cell receptor (BCR) signaling. This is corroborated by clinical efficacy of the PI3Kδ inhibitor GS-1101 in mature B cell malignancies, especially in chronic lymphocytic leukemia (CLL). In contrast to mature B cell malignancies, expression and function of PI3Kδ in B-ALL has not been well characterized. We therefore analyzed PI3Kδ expression and effects of the PI3Kδ inhibitor GS-1101 in B-ALL.
To screen efficacy of GS-1101 in B-ALL subsets, we performed viability and proliferation assays, using a panel of B-ALL cell lines, derived from different B-cell development stages (Pro-B: REH, RS4;11, Nalm-20, Nalm-21, TOM-1; Pre-B: Nalm-6, Kasumi-2, KOPN-8, SMS-SB, RCH-ACV, 697; Mature: Tanoue, Ball-1 unknown: CCRF-SB). A key downstream effector of PI3K is the serine/threonine kinase Akt, whose phosphorylation is used as a common readout of PI3K activation status. Western Blot analysis of the 15 cell lines showed almost identical levels of phospho-Akt (Ser473) in all tested cell lines, suggesting constitutive PI3K activity. To investigate the ability of GS-1101 to inhibit B-ALL cell proliferation, we performed cell growth experiments. Among the pre-B cell lines 4 of 6 showed a marked decrease in proliferation, 2 other pre-B cell lines showed a minor decrease. In contrast, none of the pro-B or mature B-ALL cell lines were affected by GS-1101. To explore the effects of GS-1101 on cell cycle of B-ALL cells, cell lines were treated with GS-1101 at concentrations ranging from 0.5μM to 5μM. In accordance with the cell growth experiments, G1 phase arrest and reduced numbers of S phase cells were detected in pre-B cell lines after GS-1101 treatment, but not in the pro-B or mature B cell lines. Next, we examined GS-1101 effects on metabolism of B-ALL cells via XTT (sodium 2,3,-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)-carbonyl]-2H-tetrazolium inner salt) staining. Cell lines were treated with GS-1101 concentrations between 0.1μM and 5μM for 3 days prior to XTT measurement. Pre-B cells showed a significant (p-value <0.0001) decrease in normalized absorbance compared to the control (without treatment) indicating a decrease in cellular viability. Finally, preliminary co-culture experiments of primary B-ALL samples and KUSA-H1 bone marrow stromal cells revealed significantly reduced B-ALL cell viability after GS-1101 treatment, signifying that GS-1101 can overcome microenviromental-mediated B-ALL cell protection; this is similar to that in other B cell malignances. In summary, these experiments demonstrate that GS-1101 inhibits growth, cell cycle progression and metabolic activity of pre-B ALL cells. Validation of these data with primary patient samples is ongoing.
Lannutti:Gilead Sciences Inc: Employment.
Asterisk with author names denotes non-ASH members.