BackgroundPromotor hypermethylation of CpG islands is common in B cell precursor acute lymphoblastic leukemia (BCP-ALL) with mixed lineage leukemia (MLL) gene rearrangements. Hypomethylating agents (HMA) such as azacitidine (AZA) and decitabine (DEC) reduce DNA hypermethylation by incorporation into DNA and were successfully introduced into the clinic for the treatment of myeloid neoplasias.MethodsHere, we investigated whether HMA induce comparable biological effects in MLL-positive BCP-ALL. Further, efficacy of HMA and concomitant application of cytostatic drugs (cytarabine and doxorubicin) were evaluated on established SEM and RS4;11 cell lines. In addition, promising approaches were studied on BCP-ALL cell line- and patient-derived xenograft models.ResultsIn general, DEC effects were stronger compared to AZA on MLL-positive BCP-ALL cells. DEC significantly reduced proliferation by induction of cell cycle arrest in G0/G1 phase and apoptosis. Most sensitive to HMA were SEM cells which are characterized by a fast cell doubling time. The combination of low-dose HMA and conventional cytostatic agents revealed a heterogeneous response pattern. The strongest antiproliferative effects were observed when ALL cells were simultaneously exposed to HMA and cytostatic drugs. Most potent synergistic effects of HMA were induced with cytarabine. Finally, the therapeutic potential of DEC was evaluated on BCP-ALL xenograft models. DEC significantly delayed leukemic proliferation in xenograft models as demonstrated longitudinally by non-invasive bioluminescence as well as 18F-FDG-PET/CT imaging. Unexpectedly, in vivo concomitant application of DEC and cytarabine did not enhance the antiproliferative effect compared to DEC monotherapy.ConclusionsOur data reveal that DEC is active in MLL-positive BCP-ALL and warrant clinical evaluation.Electronic supplementary materialThe online version of this article (10.1186/s13045-018-0607-3) contains supplementary material, which is available to authorized users.

Background The tumor suppressor protein phosphatase and tensin homolog (PTEN) is a key regulator of the PI3K/AKT pathway which is frequently altered in a variety of tumors including a subset of acute B-lymphoblastic leukemias (B-ALL). While PTEN mutations and deletions are rare in B-ALL, promoter hypermethylation and posttranslational modifications are the main pathways of PTEN inactivation. Casein Kinase II (CK2) is often upregulated in B-ALL and phosphorylates both PTEN and DNA methyltransferase 3A, resulting in increased PI3K/AKT signaling and offering a potential mechanism for further regulation of tumor-related pathways. Methods Here, we evaluated the effects of CK2 inhibitor CX-4945 alone and in combination with hypomethylating agent decitabine on B-ALL proliferation and PI3K/AKT pathway activation. We further investigated if CX-4945 intensified decitabine-induced hypomethylation and identified aberrantly methylated biological processes after CK2 inhibition. In vivo tumor cell proliferation in cell line and patient derived xenografts was assessed by longitudinal full body bioluminescence imaging and peripheral blood flow cytometry of NSG mice. Results CX-4945 incubation resulted in CK2 inhibition and PI3K pathway downregulation thereby inducing apoptosis and anti-proliferative effects. CX-4945 further affected methylation patterns of tumor-related transcription factors and regulators of cellular metabolism. No overlap with decitabine-affected genes or processes was detected. Decitabine alone revealed only modest anti-proliferative effects on B-ALL cell lines, however, if combined with CX-4945 a synergistic inhibition was observed. In vivo assessment of CX-4945 in B-ALL cell line xenografts resulted in delayed proliferation of B-ALL cells. Combination with DEC further decelerated B-ALL expansion significantly and decreased infiltration in bone marrow and spleen. Effects in patient-derived xenografts all harboring a t(4;11) translocation were heterogeneous. Conclusions We herein demonstrate the anti-leukemic potential of CX-4945 in synergy with decitabine in vitro as well as in vivo identifying CK2 as a potentially targetable kinase in B-ALL. Electronic supplementary material The online version of this article (10.1186/s12885-019-5411-0) contains supplementary material, which is available to authorized users.

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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.