Chronic lymphocytic leukemia (CLL) is the most common leukemia in the Western World and remains incurable with standard therapies. Despite significant advances, novel treatments are essential to improve outcomes. A number of therapeutic agents have recently been developed including some that have shown significant activity and tolerability in clinical trials. Among these drugs, are small molecule kinase inhibitors that inhibit B-cell receptor (BCR)-mediated signaling pathways and disrupt essential CLL cell-microenvironment interactions. Specifically, when primary CLL cells are treated with GS-1101, a PI3 kinase delta-specific isoform (PI3Kδ) inhibitor, Bruton's tyrosine kinase (Btk) inhibitors or spleen tyrosine kinase (Syk) inhibitors, inhibition of signaling pathways downstream of the BCR, cell killing, and disruption of chemokine-mediated CLL cell migration are observed. Although significant clinical activity has been observed in patients treated with single agents that target these critical pathways, very little is known about the effects of inhibiting multiple nodes in the BCR pathway. Simultaneous inhibition of multiple pathways downstream of the BCR has the potential to result in a synergistic response that may overcome the resistance observed with single compound use. These considerations prompted us to assess the effects of PI3Kd and Syk inhibition and to determine if dual inhibition might enhance antitumor effects in CLL.
CLL patient samples were assessed for growth inhibition, chemokine release, and pathway activation status using a tetrazolium-based MTS assay, ELISA, and flow cytometry, respectively. PBMCs were isolated from primary patient samples using a Ficoll-Paque gradient. GS-1101, a PI3Kδ inhibitor, and GS-9973, a novel Syk inhibitor, were plated either alone in concentrations known to span their IC50 (.6nM to 10uM), or in combination using equimolar concentrations of each drug (.6nM to 10uM) in 96-well plates. 5×105 primary cells were cultured in triplicate in HS-5 stromal cell conditioned media and growth inhibition was determined after 72 hours.
To explore potential additive, synergistic, or antagonistic interactions between GS-1101 and GS-9973, dose response and interaction indices were calculated using R (Lee et al. 2007). Given the importance of microenvironmental signals in the ability of malignant cells to survive, proliferate, and resist standard therapies, we investigated the effect of combining PI3Kd and Syk inhibitors for use on CLL cells in the presence of stroma-conditioned media. Immunoblotting and flow cytometry were used to measure the inhibitory effects of each drug alone or in combination on downstream targets known to be associated with BCR activation, including AKT, ERK, and S6 in Ramos cells, a Burkitt's lymphoma cell line, and in primary CLL cells. Of the 14 primary CLL samples treated with the single agent GS-9973, the median cell viability IC50 for all samples was 3.7 μM and an IC50 of ≤ 2μM was obtained in 7 samples (50%). Interaction indices were calculated from combination studies using GS-1101 and GS-9973 in 7 samples. At least one significantly synergistic concentration of the combination of GS-1101 and GS-9973 was found in five of the seven samples. The two remaining CLL samples showed additive responses when treated with the combination. Patient samples cultured in the presence of conditioned media resulted in increased CCL2, CCL3, and CCL4 levels. Production of these chemokines by CLL cells was reduced by both GS-1101 and GS-9973, alone and in combination. Furthermore, treatment with individual inhibitors decreased S6 and ERK, phosphorylation, an effect further enhanced by the combination of PI3Kd and Syk inhibition.
Our findings indicate that both PI3Kd and Syk inhibition reduces CLL survival. Dual targeting can also induce synergistic growth inhibition and further disrupt chemokine signaling. Given the complexity of BCR signaling pathways, simultaneous targeting of multiple kinases has the potential to significantly increase clinical activity. Since inhibition of BCR mediated kinases has demonstrated good patient tolerability, combination therapy targeting both PI3Kd and Syk may provide a novel treatment approach, especially in patients with poor risk disease that may not respond optimally to single agents.
Clarke:Gilead Sciences Inc: Employment. Meadows:Gilead sciences: Employment. Loriaux:Gilead Sciences Inc: Research Funding. Maciejewski:Gilead Sciences Inc: Employment. Di Paolo:Gilead Sciences Inc: Employment. Lannutti:Gilead Sciences Inc: Employment. Druker:Gilead: Consultancy, Membership on an entity's Board of Directors or advisory committees; Cylene: Consultancy, Membership on an entity's Board of Directors or advisory committees. Spurgeon:Gilead: Research Funding.
Asterisk with author names denotes non-ASH members.