Casein Kinase II Inhibition By CX-4945 and Epigenetic Modulation By Decitabine Demonstrate Significant Antiproliferative Activity As Single Agents As Well As in Combination in Acute B-Lymphoblastic Leukemia Cells

Introduction: Aberrant PI3K/AKT signaling is frequently observed in B cell precursor acute lymphoblastic leukemia (B-ALL) resulting in increased cell proliferation and survival. Casein kinase II (CK2) is a regulator of both, PI3K signaling and epigenetic mechanisms in hematological neoplasms. CK2 induces downregulation of de novo methylation and DNA damage response control. However, it is unclear if maintenance methylation is also influenced by CK2. We performed extended in vitro and in vivo analyses with CK2 inhibitor CX-4945 (CX) alone and in combination with hypomethylating agent Decitabine (DEC) or PI3K pathway inhibitors to enhance anti-leukemic efficiency. We hypothesized that demethylation and CK2 blockade act synergistically and have anti-leukemic activity.

Methods: B-ALL cell lines SEM, RS4;11, REH, NALM-6 and de novo B-ALL cells were exposed to CX alone or combined with DEC, Idelalisib, Ibrutinib, Sorafenib, BKM120, Perifosine or RAD001 for up to 72 h. B-ALL cells had a broad variety of genetic aberrations. PI3K downstream protein, metabolism, apoptosis and proliferation analysis were performed by western blot, WST-1 assay, Annexin V/propidium iodide and trypan blue staining. Synergistic effects were calculated using the Bliss formula. Whole genome methylation and B cell receptor (BCR) signaling expression were analyzed using Illumina Infinium MethylationEPIC BeadChip and AmpliSeq targeted sequencing after 24 h and 48 h, respectively. For in vivo studies NOD scid gamma (NSG) mice were i.v. injected (d0) with dual firefly luciferase (ffluc) and GFP expression plasmid transduced ALL cell lines or de novo patient-derived (PDX) B-ALL cells. Mice were treated with saline, CX (50 mg/kg per dose BID d7-12), DEC (0.4 mg/kg d7-10) or both. Leukemic engraftment and drug response were monitored twice weekly by peripheral blood (PB) flow cytometry and in vivo bioluminescence imaging (BLI) for up to 30 days.

Results: CX significantly inhibited proliferation and metabolism in all cell lines (p < 0.05). In addition, apoptosis induction and decreased phosphorylation of PTEN, AKT and ERK were observed. Combination of CX with any PI3K pathway inhibitor resulted in significantly stronger reduced metabolic activity (p < 0.05 vs single CX or inhibitor). Synergistic effects were generally stronger with substances targeting upstream signaling molecules of the pathway than with those affecting downstream targets. DEC alone and in combination with CX significantly reduced proliferation and metabolism while protein phosphorylation remained unchanged.

In order to identify potential CX-mediated regulation of gene expression, targeted sequencing of 179 genes related to BCR signaling was performed, identifying expression changes in interleukin, mTOR and cyclin related genes. Combination with DEC barely modulated CX-induced expression patterns. Methylome profiling revealed 54 demethylated and 82 hypermethylated genes after CX incubation. Addition of DEC increased demethylated genes to 1,323, mainly being involved in vesicle transport, NFκB and WNT signaling. Interestingly, DEC alone demethylated 1,974 genes and showed stronger demethylating effects than the combination.

In vivo , BLI revealed slower increase of the tumor burden during and shortly after CX therapy in cell line xenograft models (SEM-ffluc d14: 3.6E+08 ± 3.0E+08 (n=6)) compared to saline treated xenograft mice (5.8E+08 ± 6.3E+08 (n=9), p < 0.05). CX also reduced PB blast frequency in SEM-ffluc (d30, 9.5 ± 6.1 % vs 43.7 ± 26.2 %, p < 0.01), RS4;11-ffluc (d30, 8.3 ± 4.3 % vs 13.8 ± 7.0 %, p = 0.21) and PDX xenograft models (d32, 16.5 ± 2.5 % vs 32.3 ± 1.2 %, p = 0.06). Spleen and bone marrow (BM) infiltration remained similar to the controls. Combined therapy of CX and DEC (n=5) significantly decreased leukemic infiltration in spleen (d30, 18.3 ± 13.5 % vs 54.4 ± 14.8 % in CX, p < 0.01) and reduced PB (p < 0.01) and BM (p = 0.09) blast frequency. However, effects were not stronger than achieved by DEC alone.

Conclusion: Herein we demonstrate that CK2 inhibitor CX-4945 and hypomethylating agent DEC have significant in vitro and in vivo antiproliferative acitivity in precursor B-ALL cells of different genetic background. While the combination of CX and DEC acted synergistically in vitro , this effect could not be demonstrated in vivo . We conclude that both CK2 and hypermethylation targeting strategies should be explored in B-ALL in a clinical setting.