A potential therapeutic strategy for chronic lymphocytic leukemia by combining Idelalisib and GS-9973, a novel spleen tyrosine kinase (Syk) inhibitor

Abstract

Agents that target B-cell receptor (BCR) signaling in lymphoid malignancies including idelalisib (GS-1101) and fostamatinib which inhibit the delta isoform of PI3 kinase (PI3Kd) and spleen tyrosine kinase (Syk) respectively have shown significant clinical activity. By disrupting B-cell signaling pathways, idelalisib treatment has been associated with a dramatic lymph node response, but eradication of disease and relapse in high risk disease remain challenges. Targeting the BCR signaling pathway with simultaneous inhibition of PI3Kd and Syk has not yet been reported. We evaluated the pre-clinical activity of idelalisib combined with the novel and selective Syk inhibitor GS-9973 in primary peripheral blood and bone marrow Chronic Lymphocytic Leukemia (CLL) samples. Both PI3Kd and Syk inhibition reduced CLL survival and in combination induced synergistic growth inhibition and further disrupted chemokine signaling at nanomolar concentrations including in bone marrow derived and poor risk samples. Simultaneous targeting of these kinases may significantly increase clinical activity.

Figure 1

(A) A heat map displaying interaction indices at individual concentration points for each CLL sample. R values >1 (antagonistic), 1 (additive) and <1 (synergistic) are color coded as green, black and red, respectively. Specifically, the heat map was created using TreeView by transforming the interaction index for each concentration point of the primary samples by log₂. Each row represents an individual sample. Peripheral blood samples are labeled “PB” and bone marrow samples are labeled “BM.” The columns represent (from left to right) increasing equimolar concentrations of each drug drug ranging from .977 nM to 10 μM. (B) A representative viability curve for CLL BM1 is shown with viability on the y axis and increasing equimolar concentrations of drug (μM) on the x axis. Resistance to each agent as monotherapy is seen (top 2 curves), an effect that is overcome by the drug combination (bottom curve)when the combination is used. (C)) Viability curve for a sample representative of having synergy. Although all concentrations of the drug combination were synergistic, (CI < 1), some concentrations had values that lost significance when accounting for the 95% confidence interval. Therefore, asterisks are provided which denote points that are significantly synergistic within the 95% confidence interval as defined by R. Idelalisib (GS-1101) treated cells are shown in red, GS-9973 in blue, and the combination in green.

Figure 2. Treatment of primary CLL cells (n =14) co-cultured with HS5 stromal cells with idelalisib (100 nM) or GS-9973 (100 nM), alone or in combination (100 nM each), results in decreased AKT phosphorylation

(A) while treatment with the combination significantly decreases S6 phosphorylation as compared to treatment with each drug alone (p = 0.011 for the combination compared to GS-1101 treatment alone and p= 0.019 compared to GS-9973 alone). Each condition was compared using a two tailed t-test. (B)The effect of idelalisib (GS-1101) and/or GS-9973 on CCL2, CCL3, CCL4, and CCL22 expression after CLL-HS-5 co-culture. The bar diagrams represent the mean supernatant concentrations (+/− SEM) of CCL2 (C), CCL3 (D), CCL4 (E), CCL22 (F) from CLL cells co-cultured with or without (controls) HS-5 cells and various concentrations of idelalisib, GS-9973, or a combination of idelalisib and/GS-9973 (50 nM plus 50 nM, 50 nM plus 500 nM for each drug, and 500 nM plus 500 nM for each drug) in 6 different patient samples assessed after 24 hours. Chemokine expression after treatment with individual drug or the combination was compared using a two tailed t-test. The combination of idelalisib and GS-9973 showed significant effect on decreasing CCL2, CCL3, CCL4, CCL22. A single asterisk denotes significant (p < .05) and a double asterisk (**) represents a significance of p < .01. Differences between single agent treatment and the combinations are shown. Panels G-J show increased chemokine expression when CLL cells are cultured in the presence of primary bone marrow derived stromal cells (n=3). Similar to the HS5 co-culture system, co-culture with primary bone marrow leads to increased chemokine expression (mean +/− SEM supernatant concentrations) which is abrogated by treatment with idelalisib (100 nM) and GS-9973( 100 nM) for 24 hours. As shown in the last column in each figure, this effect that is significantly enhanced by combining these two agents (p < .05, two tailed t-test).