Targeting Protein Kinase CK2: Evaluating CX-4945 Potential for GL261 Glioblastoma Therapy in Immunocompetent Mice

Abstract

Glioblastoma (GBM) causes poor survival in patients even with aggressive treatment. Temozolomide (TMZ) is the standard chemotherapeutic choice for GBM treatment but resistance always ensues. Protein kinase CK2 (CK2) contributes to tumour development and proliferation in cancer, and it is overexpressed in human GBM. Accordingly, targeting CK2 in GBM may benefit patients. Our goal has been to evaluate whether CK2 inhibitors (iCK2s) could increase survival in an immunocompetent preclinical GBM model. Cultured GL261 cells were treated with different iCK2s including CX-4945, and target effects evaluated in vitro. CX-4945 was found to decrease CK2 activity and Akt(S129) phosphorylation in GL261 cells. Longitudinal in vivo studies with CX-4945 alone or in combination with TMZ were performed in tumour-bearing mice. Increase in survival (p < 0.05) was found with combined CX-4945 and TMZ metronomic treatment (54.7 ± 11.9 days, n = 6) when compared to individual metronomic treatments (CX-4945: 24.5 ± 2.0 and TMZ: 38.7 ± 2.7, n = 6) and controls (22.5 ± 1.2, n = 6). Despite this, CX-4945 did not improve mice outcome when administered on every/alternate days, either alone or in combination with 3-cycle TMZ. The highest survival rate was obtained with the metronomic combined TMZ+CX-4945 every 6 days, pointing to the participation of the immune system or other ancillary mechanism in therapy response.

Anti‐tumour immune response in GL261 glioblastoma generated by Temozolomide Immune‐Enhancing Metronomic Schedule monitored with MRSI‐based nosological images

“…The tumour volume at therapy start (Day 11 p.i.) for this section was 5.4 ± 2.6 mm 3 , which was close to tumour volumes observed in previous work with a similar TMZ schedule (6.0 ± 1.2 mm 3 ) . Tumour volume evolution of vehicle and TMZ‐treated mice is shown in Figure S1b.…”

“…for this section was 5.4 ± 2.6 mm 3 , which was close to tumour volumes observed in previous work with a similar TMZ schedule (6.0 ± 1.2 mm 3 ). 30 Tumour volume evolution of vehicle and TMZ-treated mice is shown in Figure S1b. As described before, 31 TMZ treatment with the IMS administration has a positive impact in the survival of GL261 tumour-bearing mice ( Figure S1d).…”

“…Tissue homogenization and protein extraction. Whole GL261 tumours obtained after euthanization at endpoint were weighted and protein extracted as described in Reference , details can be found in Supporting Information. Protein concentration was determined by Bradford assay (Bio‐Rad) and equal amounts of protein (80 μg) were loaded on 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS‐PAGE), blotted on polyvinylidene fluoride (PVDF) Immobilon‐P membranes (Millipore, Darmstadt, Germany) and immunodetected with the corresponding antibodies using a chemiluminescent detection method (Clarity TM Western ECL Blotting Substrates, Bio‐Rad, California).…”

“…15,34,41,42 However, as relevant as eliciting the immune system is avoiding to impair its amplification, and here the IMS gains importance. In the GL261 mouse model, TMZ produces a significantly better survival rate when administered in a 6-d cycle 30 instead of in daily schedule of five consecutive days. 43 Alkylating agents, including TMZ, are known to induce side effects related to the immune system such as leukopenia and neutropenia, when administered daily.…”

Metabolomics of Therapy Response in Preclinical Glioblastoma: A Multi-Slice MRSI-Based Volumetric Analysis for Noninvasive Assessment of Temozolomide Treatment

“…Metronomic administration, usually referring to administrations of low and equally spaced doses of chemotherapeutics without long rest periods in between [47,48] has been used to improve immune responses to potentiate tumor regression and avoid regrowth [48]. Our group has also used this scheme in work reported in [46], obtaining a survival time slightly higher (38.7 ± 2.7 days, n = 6) than the traditional scheme with three cycles [18] used in the present work (5-2-2 days with a three-day interleave between them (33.9 ± 11.7 days, n = 38)). Still, work reported in [46] highlighted the relevance of the metronomic strategy in comparison with traditional administration schemes in which results suggested an impairment of the immune system related response due to continuous therapy administration, probably interfering with T cell amplification.…”