Preclinical study on the role of human endogenous retroviruses as potential markers of disease and prognosis of chronic lymphocytic leukemia

Background. The dysregulated expression of human endogenous retrovirus K (HERV-K) at transcriptional and protein levels, as well as viral particle production, have been found in tissues, sera, and cell lines isolated from different types of tumors, such as ovarian, breast, prostate, teratocarcinoma, lymphomas, leukemias, sarcomas, and melanoma, and has been associated with the onset, progression and acquisition of aggressive features of many cancers. The mechanisms underlying HERV-K oncogenic activity could depend on the expression of oncogenic viral proteins, on the induction of immune escape mechanisms, on the regulation of gene expression mediated by the long terminal repeat sequences, or by the ability of retro-transposition to determine genomic instability and alteration of the expression of neighboring genes. Cancer stem cells (CSCs) are essential for tumour growth and spread and have been characterized by the presence of different markers, among which the surface marker CD133 and the embryonic factors OCT4, NANOG, and KLF4. In the field of oncohematology, some studies have identified alterations of HERV-K and others HERV families’ expression in human lymphoid leukemic cells and the presence of circulating anti HERV-K antibodies. Moreover, very few studies describe the potential involvement of embryonic genes in chronic lymphocytic leukemia (CLL). On these bases, aim of this thesis is the evaluation of distinct HERVs families and embryonic genes in CLL to understand the potential implication in the etiopathogenesis of the disease and their use as biomarkers and prognostic markers of CLL. Materials and methods: Peripheral blood mononuclear cells (PBMCs) were isolated from peripheral venous blood of 75 patients with CLL diagnosis and 49 healthy donors (HDs). CLL patients were divided according to the treatment regimen received. In PBMCs, the transcriptional activity of the ENV gene of HERV-K (HML-2), HERV-H, HERV-W, and pathogenic HERV-W, as well as of the embryonic genes OCT4 and KLF4, and stemness marker CD133 were analyzed by RT-Real Time PCR. Also, the percentage of HERV-K ENV positive cells in B lymphocyte subpopulations (CD19+CD5+) were characterized by flow cytometry analysis. In order to identify associations between biomarkers in a multivariate manner, factor analysis was performed on all CLL patients and patients divided based on therapy. Multivariate general linear models were used to evaluate the association of HERVs and embryonic genes with prognostic markers of CLL. Results: Expression profiles of HERVs and embryonic genes have been defined in CLL: the molecular analysis showed a significantly higher expression of HERV-K, HERV-H, HERV-W, OCT4, and KLF4 in the patients compared to HDs, and, although less modulated, also of pHERV-W and CD133. Significant differences in all genes analyzed between untreated patients and those treated with chemotherapeutic or biological drugs patients were found. Moreover, a higher level of ENV HERV-K protein in CD19+CD5+ cells of CLL patients and its modulation in the presence of therapy were found. The PCA analysis revealed specific expression profiles of HERVs and embryonic genes in CLL patients. The identified expression profiles were found associated to the different therapeutic regiments and to clinical features and unfavorable prognostic factors. Discussion and conclusion: These results could help to clarify the complexity of CLL, proposing HERVs and embryonic genes as potential new diagnostic and prognostic markers, suggesting their involvement in the etiopathogenesis of the disease. This scenario opens new avenues for investigating the use of HERVs and embryonic genes as potential therapeutics in combination with standard therapies.