Casein kinase 2 (CK2) is a highly conserved and constitutively active serine-threonine protein kinase that plays a fundamental role in maintaining cell survival through pro-proliferative, anti-apoptotic and pro-angiogenic signaling. CK2 promotes malignant cell viability by regulating critical downstream signaling pathways including PI3K/AKT, and overexpression and hyperactivation of this kinase has been well described in B-cell malignancies including chronic lymphocytic leukemia (CLL) and multiple myeloma (MM).
In B-cell malignancies, the B-cell receptor (BCR) represents another key survival pathway. BCR activation results in downstream signaling of multiple pro-survival kinases including PI3K and AKT. Drugs that inhibit the transmission of signals through the BCR have been shown to reduce cell survival. Given that BCR-mediated kinase signaling relies on the convergence of many regulatory inputs, we hypothesize that inhibiting this pathway at more than one site would be most effective at killing cells.
CX-4945 is the first orally bioavailable small molecule inhibitor of CK2 to progress to human clinical trials. CX-4945 has been found to be highly selective for CK2 and has been shown to inhibit AKT phosphorylation. We have previously reported pre-clinical single agent activity of CX-4945 across a broad range of primary hematologic malignancy samples including CLL. Many of the samples sensitive to CX-4945 were also sensitive to other inhibitors of the BCR pathway. Here we present our findings combining CX-4945 with GS-1101, the isoform selective inhibitor of PI3KΔ, or fludarabine in CLL.
Fresh peripheral blood mononuclear cells from CLL patients were purified using a Ficoll-Paque gradient. Purified cells were then added to wells (5 × 104 per well) containing HS-5 stromal cell conditioned media and serial dilutions (ranging from 4.5 nM to 10 μM) of CX-4945 alone and in combination with serial dilutions of GS-1101 and fludarabine (both ranging from 4.5 nM to 10 μM). Cells were cultured for 72 hours and a tetrazolium-based MTS assay was performed to measure cell viability. Synergy analysis was performed using R or CalcuSyn.
Ramos, a Burkitt's lymphoma cell line, and primary patient samples were treated with increasing concentrations of CX-4945 alone and in combination with GS-1101, and phosphorylation of the BCR pathway including AKT and ERK were evaluated by immunoblotting.
Synergy was observed between CX-4945 and GS-1101 in all samples tested (9 primary CLL patient samples). The median combination index (CI) for all samples was < 1, which indicates overall synergy between CX-4945 and GS-1101. The mean IC50 of CX-4945 alone was 4.3 μM, and the mean IC50 for GS-1101 alone was 4.2 μM suggesting limited single agent activity of each individual drug in the samples tested. However, in combination, the mean IC50 was reduced to 321 nM. Synergy was also observed in 3 of 4 CLL samples treated with a combination of CX-4945 and fludarabine.
Downstream targets of CK2 and the BCR were examined. Decreased levels of phospho-AKT and phospho-ERK were seen in response to treatment with CX-4945 alone and in combination with GS-1101.
Our data confirms that CX-4945 demonstrates significant pre-clinical activity in CLL when combined with GS-1101 or fludarabine. As such, CX-4945 may be an attractive compound for clinical development in lymphoid malignancies in combination with other agents, especially those that target other kinases. Additional studies focused on evaluating the efficacy of CX-4945 in combination with other inhibitors of the BCR signaling pathway and in combination with other cytotoxic chemotherapies are underway and will be presented.
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.