Conservation of Aging and Cancer Epigenetic Signatures across Human and Mouse.

Aging and cancer are two interrelated processes, with aging being a major risk factor for the development of cancer. Parallel epigenetic alterations have been described for both, although differences, especially within the DNA hypomethylation scenario, have also been recently reported. Although many of these observations arise from the use of mouse models, there is a lack of systematic comparisons of human and mouse epigenetic patterns in the context of disease. However, such comparisons are significant as they allow to establish the extent to which some of the observed similarities or differences arise from pre-existing species-specific epigenetic traits. Here, we have used reduced representation bisulfite sequencing to profile the brain methylomes of young and old, tumoral and nontumoral brain samples from human and mouse. We first characterized the baseline epigenomic patterns of the species and subsequently focused on the DNA methylation alterations associated with cancer and aging. Next, we described the functional genomic and epigenomic context associated with the alterations, and finally, we integrated our data to study interspecies DNA methylation levels at orthologous CpG sites. Globally, we found considerable differences between the characteristics of DNA methylation alterations in cancer and aging in both species. Moreover, we describe robust evidence for the conservation of the specific cancer and aging epigenomic signatures in human and mouse. Our observations point toward the preservation of the functional consequences of these alterations at multiple levels of genomic regulation. Finally, our analyses reveal a role for the genomic context in explaining disease- and species-specific epigenetic traits.

Successful Partnerships: Exploring the Potential of Immunogenic Signals Triggered by TMZ, CX-4945, and Combined Treatment in GL261 Glioblastoma Cells.

BACKGROUND: The relevance of the cancer immune cycle in therapy response implies that successful treatment may trigger the exposure or the release of immunogenic signals. Previous results with the preclinical GL261 glioblastoma (GB) showed that combination treatment of temozolomide (TMZ) + CX-4945 (protein kinase CK2 inhibitor) outperformed single treatments, provided an immune-friendly schedule was followed. Our purpose was to study possible immunogenic signals released in vitro by GB cells. METHODS: GL261 GB cells were treated with TMZ and CX-4945 at different concentrations (25 µM-4 mM) and time frames (12-72 h). Cell viability was measured with Trypan Blue and propidium iodide. Calreticulin exposure was assessed with immunofluorescence, and ATP release was measured with bioluminescence. RESULTS: TMZ showed cytostatic rather than cytotoxic effects, while CX-4945 showed remarkable cytotoxic effects already at low concentrations. Calreticulin exposure after 24 h was detected with TMZ treatment, as well as TMZ/CX-4945 low concentration combined treatment. ATP release was significantly higher with CX-4945, especially at high concentrations, as well as with TMZ/CX-4945. CONCLUSIONS: combined treatment may produce the simultaneous release of two potent immunogenic signals, which can explain the outperformance over single treatments in vivo. A word of caution may be raised since in vitro conditions are not able to mimic pharmacokinetics observed in vivo fully.

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

Glioblastomas (GB) are brain tumours with poor prognosis even after aggressive therapy. Improvements in both therapeutic and follow-up strategies are urgently needed. In previous work we described an oscillatory pattern of response to Temozolomide (TMZ) using a standard administration protocol, detected through MRSI-based machine learning approaches. In the present work, we have introduced the Immune-Enhancing Metronomic Schedule (IMS) with an every 6-d TMZ administration at 60 mg/kg and investigated the consistence of such oscillatory behaviour. A total of n = 17 GL261 GB tumour-bearing C57BL/6j mice were studied with MRI/MRSI every 2 d, and the oscillatory behaviour (6.2 ± 1.5 d period from the TMZ administration day) was confirmed during response. Furthermore, IMS-TMZ produced significant improvement in mice survival (22.5 ± 3.0 d for controls vs 135.8 ± 78.2 for TMZ-treated), outperforming standard TMZ treatment. Histopathological correlation was investigated in selected tumour samples (n = 6) analyzing control and responding fields. Significant differences were found for CD3+ cells (lymphocytes, 3.3 ± 2.5 vs 4.8 ± 2.9, respectively) and Iba-1 immunostained area (microglia/macrophages, 16.8% ± 9.7% and 21.9% ± 11.4%, respectively). Unexpectedly, during IMS-TMZ treatment, tumours from some mice (n = 6) fully regressed and remained undetectable without further treatment for 1 mo. These animals were considered "cured" and a GL261 re-challenge experiment performed, with no tumour reappearance in five out of six cases. Heterogeneous therapy response outcomes were detected in tumour-bearing mice, and a selected group was investigated (n = 3 non-responders, n = 6 relapsing tumours, n = 3 controls). PD-L1 content was found ca. 3-fold increased in the relapsing group when comparing with control and non-responding groups, suggesting that increased lymphocyte inhibition could be associated to IMS-TMZ failure. Overall, data suggest that host immune response has a relevant role in therapy response/escape in GL261 tumours under IMS-TMZ therapy. This is associated to changes in the metabolomics pattern, oscillating every 6 d, in agreement with immune cycle length, which is being sampled by MRSI-derived nosological images.

Up-Regulation of the Alpha Prime Subunit of Protein Kinase CK2 as a Marker of Fast Proliferation in GL261 Cultured Cells.

Glioblastoma (GB) is the most prevalent malignant primary brain tumor in adults. The preclinical glioblastoma model GL261 is widely used for investigating new therapeutic strategies. GL261 cultured cells are used for assessing preliminary in vitro data for this model although very little is known about the molecular characteristics of this cell line. Protein Kinase CK2 is a pleiotropic serine-threonine kinase and its inhibition may be a promising therapeutic strategy for GB treatment. In our group we follow treatment response with CK2 inhibitors in vivo using the GL261 murine model. For that, it is of our interest to assess the differential expression of α, α', ß CK2 subunits as well as CK2 activity in the GL261 GB model. CK2α' expression changed along the growth curve of GL261 cells, undergoing downregulation in postconfluent phase cells, whereas CK2α and CK2ß expression remained essentially unchanged. Furthermore, a marked decrease in CK2 activity in slowly proliferating postconfluent phase GL261 cells was observed. Finally, CK2α' expression in orthotopic GL261 tumors was intermediate between CK2α' expression found in cultured cells in exponentially growing or postconfluent phase, reflecting the heterogeneous nature of GL261 tumours growing in vivo. The results obtained suggest that, in the GL261 cell line, CK2α' could play a specific role in highly proliferative cells. Also, the decrease in CK2 activity in slowly proliferating GL261 cells could imply a differential susceptibility to subunit-specific CK2 inhibitors in this cell line, although further studies are needed to confirm this hypothesis.

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

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.