Hypomethylating Agents and Casein Kinase 2 Inhibitor Act Synergistic and Reveal Significant Anti-Leukemic Effects in Acute Lymphoblastic Leukemia Cells
Authors: Roolf Catrin , Richter Anna , Konkolefski Christoph , Khodamoradi Yascha , Knuebel Gudrun , Sekora Anett , Stenzel Jan , Krause Bernd Joachim , Vollmar Brigitte , Jeremias Irmela , Panse Jens P. , Murua Escobar Hugo , Junghanss Christian 
Published: October 2019

Abstract
Introduction: Aberrant methylation of tumor suppressor gene promoters is frequently observed in acute lymphoblastic leukemia (ALL). Decitabine (DAC) and Azacitidine (AZA) are methyltransferase inhibitors (hypomethylating agents, HMA) which partially reverse aberrant DNA methylation. Recently, it was reported that DNA methylation patterns are regulated by casein kinase 2 (CK2) mediated phosphorylation of DNMT3a. CK2 is a key regulator of cell proliferation and survival and modulates hematopoiesis associated signaling cascades by phosphorylation of PTEN and AKT. Elevated CK2 expression has been demonstrated in hematological malignancies and plays a significant role in cell survival. However, it is not yet clear if CK2 inhibition is effective in ALL cells. Therefore, we examined the impact of conventional cytostatics, demethylation and CK2 inhibition on B- and T-ALL cells in single application and in combination. We hypothesized that demethylation and inhibition of CK2 act synergistically inducing greater impact on DNA hypomethylation and ALL cell proliferation.
Methods: Several B- and T-ALL cell lines (SEM, RS4;11, Jurkat, CEM) as well as de novo ALL cells were treated with DAC and AZA in mono application. Further, DAC was combined with AraC, Doxorubicin (Doxo) or a CK2 inhibitor for up to 72 h. Cell proliferation and metabolism were determined (trypan blue staining & WST-1 assay). Methylation patterns of bisulfite converted DNA samples were examined using methylation specific qPCR on LINE-1 and the CDH13 gene. For in vivo studies, the SEM cell line was stably transfected with a dual firefly luciferase (ffluc) and GFP expression plasmid. NOD scid gamma (NSG) mice were intravenously injected with 2.5x106 SEM-ffluc-GFP cells. Starting on day (d) 7, mice were treated intraperitoneal. with a vehicle (saline: d7-d10), daily 0.5 mg/kg DAC (d7-d10), daily with 150 mg/kg AraC (d7, d8), or both. Leukemic engraftment and drug response were investigated weekly by flow cytometry (GFP) and bioluminescence imaging (ffluc) for up to 31 days, respectively. Additionally, for therapy monitoring 18F-FDG metabolism of spleen was evaluated using PET/CT on d21 and d28.
Results : Mono application of HMA reduced metabolic activity and proliferation significantly (p<0.05) in B- and T-ALL cell lines and de novo cells. Notably, strongest effects were obtained with DAC in B- and T-ALL cells. Hereby, methylation status of the LINE-1 element significantly decreased in cells with the most pronounced effects at 24 h and 48 h after treatment with DAC. Methylation status of CDH13 was not affected. Furthermore, enhanced anti-proliferative effects on ALL cells were detected when DAC was combined with AraC or Doxo. Here, metabolic activity decreased significantly when DAC and Doxo or Ara-C were given simultaneously.
Moreover, we evaluated the in vivo efficacy of DAC and DAC+AraC. Transplantation of SEM-ffluc-GFP into NSG mice resulted in stable engraftment of ALL cells. Using bioluminescence imaging, leukemic organ infiltration in bone marrow, spleen, lung, liver and brain increased in saline treated mice from d7: 3.0x107 ± 2.3x107 to d31: 6.2x109 ± 5.7x108 ph/s (n=9) continuously. In contrast, treatment with DAC alone or in combination with AraC significantly inhibited the engraftment of ALL cells in vivo. Notably, strongest anti-leukemic effects were induced with DAC alone and not in combination with AraC (d31: DAC: 3.1 x109 ± 1.7 x109 ph/s; n=11 vs. DAC+ AraC: 4.8 x109 ± 1.7 x109 ph/s, n=11). In addition, metabolic spleen volume determined by 18F-FDG-PET/CT on d28 was also markedly reduced in DAC-treated (DAC: 22.8 ± 15.5 mm3, DAC+AraC: 48.7 ± 15.4 mm3) compared to saline treated mice (81.1 ± 21.8 mm3).
In vitro combination of DAC with an CK2 inhibitor (CX-4945) induced strong synergistic effects. Here, metabolic activity decreased significantly after 72 h (DAC+CX-4945: 32.2 ± 2.4 % compared to DAC: 72.6 ± 6.4 % and CX-4945: 68.6 ± 8.4 %; DMSO-control: 100 %). The efficacy of DAC and CK2 inhibition in an ALL-xenograft model is currently evaluated.
In conclusion, we have shown that HMA significantly inhibit ALL cell proliferation (DAC more than AZA). Further, in vivo combination of DAC with AraC was less effective than DAC alone pointing at antagonistic effects. Of note, our in vitro data indicate that treatment with DAC and CK2 inhibition is synergistic and reveals significant anti-leukemic effects which have to be further elucidated.

Disclosures
No relevant conflicts of interest to declare.