Abstract
Abstract 2897
Specific inhibition of signaling elements essential for chronic lymphocytic leukemia (CLL) cell survival offers great promise for the design of improved therapies against this still incurable malignancy. The serine/threonine protein kinase CK2 is frequently upregulated in cancer, and mounting evidence implicates CK2 in tumorigenesis. Here, we evaluated whether CK2 is a valid target for therapeutic intervention in CLL, by testing the efficacy of CX-4945, a potent and highly specific orally available ATP-competitive inhibitor of CK2 that is undergoing phase I clinical trials for solid tumors and multiple myeloma.
We previously showed that CK2 phosphorylates and thereby inactivates PTEN in primary T-ALL and CLL cells, leading to the hyperactivation of PI3K signaling pathway, and consequently promoting leukemia cell survival (Silva et al, JCI 2008; Martins et al, Blood 2010). Therefore, we first analyzed the impact of CX-4945 on PTEN phosphorylation and PI3K pathway activation. Incubation of CLL cells with 20 μM CX-4945 for 2h resulted in striking downregulation of PTEN phosphorylation, indicative of increased PTEN activity, and a concomitant decrease in the activity of PI3K downstream targets Akt and PKC, as determined by Akt (S473), PKCβ (S660) and PKCδ (T550) phosphorylation in both MO1043 and primary CLL cells collected and isolated to >90% purity from the peripheral blood of untreated patients. Importantly, we confirmed that Akt phosphorylation on the CK2 direct target site (S129) was also inhibited by CX-4945. Next, we evaluated the functional impact of the CK2 inhibitor on CLL cell viability. Primary CLL cells (n=11) were cultured with 10 and 20 μM CX-4945. Both drug concentrations exerted clear pro-apoptotic effects in all cases (P<0.0001 for each dose, 2-tailed paired Student's t test), as determined by Annexin V-APC/7-AAD staining. Moreover, the effect of CX-4945 was time- and dose-dependent in 4 out of 4 cases that were more thoroughly analyzed. Similar results were obtained using MEC1, MEC2, WaC3CD5, JVM3 and MO1043 cell lines whose IC50 ranged between 3.1 and 5.8μM. Notably, although co-culture with OP9 stromal cells promoted primary leukemia cell survival, it did not prevent CX-4945-mediated apoptosis of CLL cells. Most importantly, CX-4945 induced a stronger decrease in the viability of CLL cells from patients with higher percentage of malignant cells in the blood (R2=0.4176, P=0.0317, n=11, Pearson correlation), Binet stage B/C (P=0.0424, n=10, 2-tailed unpaired Student's t test) or higher plasma β2 microglobulin levels (P=0.0239, n=9). Furthermore, CLL cells with a higher proliferation rate (LDT < 12 months) were also more sensitive to CX-4945 (P=0.0007, n=11). In accordance, the need for treatment positively correlated with the sensitivity to CX-4945 (R2=0.4504, P = 0.0238). These observations suggest that treatment with CK2 inhibitors may be especially beneficial to patients with more advanced or aggressive disease.
The promising results obtained in vitro prompted us to assess the impact of CX-4945 on CLL tumor development in vivo. We implanted MO1043 CLL cells subcutaneously into Swiss nude mice. At day 3, all animals presented palpable tumor masses of approximately 150 mm3, and were randomly assigned into 4 groups (n=6 per group) to receive either CX-4945 alone (75mg/kg, bid, p.o.), fludarabine alone (34mg/kg, i.p., 5 days + 2 days rest, every week), the combination of both drugs, or vehicle control. A significant delay in tumor growth was observed in all of the treatment groups when compared to the control group (P<0.0001, 2-way ANOVA). Notably, CX-4945 was as effective as fludarabine when used as a single agent, and the combination of the two drugs was significantly more effective than fludarabine alone (P=0.0375). All treatments were well tolerated as evidenced by the maintenance of body weight and the inexistence of signs of overt toxicity.
Overall, our data indicate that pharmacological inhibition of CK2 is a promising therapeutic strategy in CLL that may be of special benefit to patients with aggressive and advanced stage disease. Moreover, our studies pave the way to the development of clinical trials using CX-4945 or other CK2 antagonists to manage CLL.
Disclosures:
Stansfield: Cylene Pharmaceuticals Inc.: Employment. Drygin:Cylene Pharmaceuticals Inc.: Employment.
Complement alternative pathway activation by chronic lymphocytic leukemia cells: its role in their hepatosplenic localization
