Tumor Antigen ROR1 Targeted Delivery of MiR-29b in Chronic Lymphocytic Leukemia Induces Epigenetic Reprograming Resulting in Cell Cycle Arrest

Aggressive Chronic lymphocytic leukemia (CLL) exhibits reduced miR-29b with poor outcome. Strategically increasing miR-29b to reprogram CLL cells provides therapeutic potential. However, miRs are sensitive to degradation in circulation and encapsulation by cationic lipocomplex leads non-specific delivery, thus causing limited uptake in CLL cells. To overcome these limitations, we designed miR29b-encapsuled immunoliposome (2A2-miR-29b-ILP) targeting ROR1, a receptor tyrosine kinase expressed in leukemic but not normal B cells. The 2A2-ILP enhanced miR-29b uptake by >20-fold compared to free miR29b and 3-fold to IgG-ILP control. Interestingly, 2A2-miR-29b-ILP treatment did not directly affect cellular viability nor BCL2 pathways, but significantly decreased the global DNA methylation through downregulation of DNMTs and SP1 in primary CLL cells and a human CLL cell line OSU-CLL. (n=3 CLL, DNMT1: p= 0.0115; DNMT3a: p=0.0231, SP1; p=0.0031). Consistent with the downregulation of DNMTs, decreased global DNA methylation was observed in OSU-CLL cell line one week after treatment with 2A2-ILP-miR-29b (n=3, p=0.0003). Furthermore, using a double transgenic Eμ-TCL-1-ROR1 mouse model expressing human ROR1 (hROR1) on leukemic B cells, we further demonstrated the therapeutic benefit of enhanced survival with 2A2-miR-29b-ILP via cellular reprograming by downregulation of DNMT1 and DNMT3a in-vivo. Using the hROR1+CD19+CD5+ leukemic cell adoptive transfer model, we observed significant in-vivo efficacy of 2A2-miR-29b-ILP (also referred as 2A2-29b) formulation, compared to scramble control (referred as 2A2-SC), associated with a) decreased number of circulating leukemic B220+CD5+ cells (58±3% decrease in 2A2-29b treatment, p=0.0184; 2A2-29b: n=9; 2A2-SC: n=7) b) reduced splenomegaly (53±2% decrease in 2A2-29b treatment, p=0.0002, n=4) c) with extended survival (p=0.0075, 2A2-29b: n=9; 2A2-SC: n=7). Moreover, our RNAseq profiling of hROR1xTCL1 splenocyte implied reprogramming of cell cycle by miR-29b treatment relieving the leukemic progression. Gene expression of miR-29b treated leukemic splenocytes revealed 128 of 233 differentially expressed genes to be associated with cell growth and proliferation. The in-vivo miR29b-treated splenocytes exhibited compromised cell cycle, associated with decreased SP1 and increased p21 protein expression in our immunoblotting analysis. Importantly, SP1-knockdown resulted in significant decrease in miR29b mediated cell cycle arrest implicating SP1 is involved in miR-29b cell cycle arrest/suppression. In conclusion, 2A2-ILP effectively delivered functional miR-29b, resulting in DNMTs downregulation, reduction of DNA methylation and anti-leukemic activity. These studies form a basis of leukemic cell targeted delivery of miR29b as a promising therapeutic approach for CLL without compromising the ROR1-ve normal B cell counterparts.

[Chi-Ling Chiang is a recipient of Pelotonia Graduate Student Fellowship. This work was supported by NIH Grants]