Erica Salerno

Correcting miR-15a/16 genetic defect in New Zealand Black mouse model of CLL enhances drug sensitivity

Alterations in the human 13q14 genomic region containing microRNAs mir-15a and mir-16-1 are present in most human chronic lymphocytic leukemia (CLL). We have previously found the development of CLL in the New Zealand Black murine model to be associated with a point mutation in the primary mir-15a/16-1 region, which correlated with a decrease in mature miR-16 and miR-15a levels. In this study, addition of exogenous miR-15a and miR-16 led to an accumulation of cells in G1 in non–New Zealand Black B cell and New Zealand Black–derived malignant B-1 cell lines. However, the New Zealand Black line had significantly greater G1 accumulation, suggesting a restoration of cell cycle control upon exogenous miR-15a/16 addition. Our experiments showed a reduction in protein levels of cyclin D1, a miR-15a/16 target and cell cycle regulator of G1/S transition, in the New Zealand Black cell line following miR-15a/16 addition. These microRNAs were shown to directly target the cyclin D1 3′ untranslated region using a green fluorescent protein lentiviral expression system. miR-16 was also shown to augment apoptosis induction by nutlin, a mouse double minute 2 (MDM2) antagonist, and genistein, a tyrosine kinase inhibitor, when added to a B-1 cell line derived from multiple in vivo passages of malignant B-1 cells from New Zealand Black mice with CLL. miR-16 synergized with nutlin and genistein to induce apoptosis. Our data support a role for the mir-15a/16-1 cluster in cell cycle regulation and suggest that these mature microRNAs in both the New Zealand Black model and human CLL may be targets for therapeutic efficacy in this disease. [Mol Cancer Ther 2009;8(9):2684–92]

Role of microRNA-15a in autoantibody production in interferon-augmented murine model of lupus ☆

MicroRNAs (miRNAs) are involved in the regulation of immunity via targeting of mRNA encoding immune response elements. In this report, alterations in the expression of microRNAs as autoantibody levels increase was investigated. The (NZB×NZW)F1 or B/W mouse model of systemic lupus erythematosus (SLE) naturally has increased autoantibodies with aging. IFNα (type I IFN) accelerates B/W disease, however, the effects of a related IFN, IFNλ, which is a type III IFN, is largely unknown. The purpose of the study was to investigate the relationship between IFN-accelerated disease, microRNAs, immunoregulatory B cell subsets and autoantibody production in the autoimmune-prone environment in vivo. B/W mice received osmotic pumps to chronically deliver IFNα and IFNλ for up to 16 weeks. Urine protein level was monitored weekly by urine strips and proteinuria was used as the disease marker. Splenic cells were taken for flow analysis of B cell subsets and levels of microRNAs determined. Plasma were analyzed for autoantibodies and microRNA levels. As a result of treatment, IFNα accelerated proteinuria in a dose dependent manner, while IFNλ single treatment did not show a significant effect, but greatly enhanced low dose IFNα effects in the combination treatment. Both the splenic cellular and plasma miR-15a were elevated in diseased compared to pre-diseased mice as well as autoantibody levels. Autoantibodies and miR-15a levels were significantly correlated. The immunosuppressive B subpopulation, B-10, was reduced following IFNα treatment. In addition in diseased mice, B-10 versus B-2 ratios were reduced in IFN-treated B/W compared to the control PBS treated group. In all B/W the miR-15a was highly expressed in the B-10 subset and this increased with disease development, suggesting that miR-15a has a specific negative effect on the B-10 subpopulation. In conclusion, our data support the involvement of elevated miR-15a in autoimmune disease development in B/W mice and suggest that decreasing this microRNA might be beneficial in B/W mice.

The New Zealand black mouse as a model for the development and progression of chronic lymphocytic leukemia

Similar to a subset of human patients who progress from monoclonal B lymphocytosis (MBL) to chronic lymphocytic leukemia (CLL), New Zealand Black (NZB) mice have an age‐associated progression to CLL. The murine disease is linked to a genetic abnormality in microRNA mir‐15a/16‐1 locus, resulting in decreased mature miR‐15a/16.

Regulatory T cells as central regulators of both autoimmunity and B cell malignancy in New Zealand Black mice.

Regulatory T cells (Tregs) play an important role in protection against autoimmune disease and are also known to be potent inhibitors of anti-tumor immune responses. The New Zealand Black (NZB) mouse is a murine model for both autoimmune diseases, since high levels of autoantibodies are present, and human CLL, due to the expansion of malignant B-1 cells. In this study, we examined the functional role of CD4(+)CD25(+) Foxp3(+) Tregs in these different manifestations. Flow cytometric analysis showed increased levels of Tregs in NZB mice compared to healthy C57Bl/6 controls. Aged NZB mice that have developed a B-1 cell malignancy identified as IgM(+)CD5(+), have the most pronounced increase in Tregs. Ex vivo treatment of splenocytes from NZB mice with IFN-alpha resulted in a decrease in the frequency of Tregs and malignant B-1 cells. In vivo treatment of both NZB and C57Bl/6 mice with poly (I:C), a potent inducer of IFN-alpha, also led to a decrease in the levels of Tregs and malignant B-1 cells (NZB only) while amplifying autoimmune manifestations. These results indicate that while high levels of Tregs found in NZB mice might suppress a more severe autoimmune disease, they may also contribute to the development of the B cell malignancy.