Role of Mir-29b in T-Cell Development and in Cutaneous T-Cell Lymphoma Pathogenesis

Abstract

The role of microRNA (miR) is rapidly advancing with numerous functions discovered in cancer pathogenesis. We had previously shown a significant decrease in miR-29b levels in malignant cells isolated from peripheral blood of cutaneous T-cell lymphoma (CTCL) (Kohnken et al. Blood, 2018). Replenishing miR-29b levels in vitro induced apoptosis in leukemic cells suggesting its role as tumor suppressor miR in CTCL patients. In this study, we focused on the role of mir-29b on early T-cell development and homeostasis using the miR-29b-/- mouse model. Using knockout mouse model, we show that the homozygous deletion of miR-29b locus results in an overall decrease in T-cell numbers and density in primary and secondary lymphoid organs. We observed an early thymic involution in miR-29b-/- mice, with a 5-fold decrease in the total number of thymocytes and altered T-cell development. Using the surface expression of CD25 and CD44 on double-negative (DN) cells, we observed significant decrease of the absolute counts in the four early differentiation stages (DN1-4) in miR-29b-/- mice compared to age-matched wild-type (WT) mice: DN1 (miR-29b-/- vs. WT: 4.5 x 105 ± 1.04 vs. 1.12 x 105 ± 0.201, N=4 and 6 respectively, p < 0.05); DN2 (miR-29b-/- vs. WT: 0.83 x 105 ± 0.07 vs. 0.051 x 105 ± 0.021, N=4 and 6 respectively, p < 0.001); DN3 (miR-29b-/- vs. WT: 4.05 x 105 ± 0.38 vs. 0.54 x 105 ± 0.16, N=4 and 6 respectively, p < 0.001) and DN4 (miR-29b-/- vs. WT: 2.871 x 105 ± 0.578 x 105 vs. 0.569 ± 0.14, N=4 and 6 respectively, p < 0.01). Furthermore, miR-29b-/- mice show a significant increase in regulatory T cells in comparison to WT mice in the spleen (miR-29b-/- vs. WT: 18.48 ± 0.89 vs. 10.89 ± 0.41, N=4 and 6 respectively, p < 0.001) thymus (miR-29b-/- vs. WT: 5.80 ± 0.44 vs. 3.65 ± 0.004, N=4 and 6 respectively, p < 0.01) and bone marrow (miR-29b-/- vs. WT: 50.04 ± 3.14 vs. 37.52 ± 3.29, N=4 and 6 respectively, p < 0.05). Using single-cell RNA sequencing (scRNA-seq), we found a framework of putative genes in miR-29b deficient T-cells that overlap with CTCL pathogenesis: Ccr7, Cd69, Cd74, and Lef-1. Among thymic T cells, Ccr7 affects physiologic homing of T-cells to lymph nodes and facilitate nodal metastasis in CTCL patients. As T-cells progenitors originate from the bone marrow, we observed a severe impairment in the progenitor cell population in miR-29b-/- mice and over 4-fold reduction in the absolute number of Lin-Sca1+ckit+ cell (miR-29b-/- vs. WT: 28.42 x 103 ± 4.13 vs. 5.98 x 103 ± 1.01, N=4 and 3 respectively, p < 0.01). Using the surface expression of CD48 and CD150 on Lin-Sca1+ckit+, we observed a significant decrease in hematopoietic stem cells and multipotent progenitor cells in miR-29b-/- mice. Since impairment in T-cells development can be linked to hematopoietic stem cell defects, we interrogated the engraftment capacity of Lin-Sca1+ckit+ cells into committed cell lineages in vivo. To evaluate whether the defect in T-cells development is due to reduced ability of precursor cells, we performed transplantation of Lin-Sca1+ckit+ cells from the bone marrow of miR-29b and WT (CD45.1) mice in lethally irradiated WT (CD45.2) mice. Our results show a significant reduction in T-cells reconstitution in mice transplanted with miR-29b-/- Lin-Sca1+ckit+ cells versus the bone marrow counterpart. These deficits in T-cells populations were observed in peripheral blood, thymus, spleen, and bone marrow in miR-29b-/- transplanted mice. ScRNA-seq profiling of Lin- bone marrow cells show significant changes in miR-29b-/- and WT mice. Among the upregulated genes in the mir-29b-/- mice, several genes showed greater than a 10-fold increase in the thymus (Ifna2, Zmynd10, Plekha4, Etl4, and Gm38004). In conclusion, our results highlight the importance of mir-29b in early defects in T-cells development and help us understand the complex miR-29b regulated cellular transformation machinery in CTCL pathogenesis, thus paving path to develop novel therapeutic approaches targeting miR-29b in CTCL therapy. Disclosures No relevant conflicts of interest to declare.