LincRNA Expression Discriminates Cytogenetic Subtypes In B-Lymphoblastic Leukemia and Plays a Functional Role In Leukemia Cell Survival

It has now become clear that a recently described class of non-coding RNA, so-called long intergenic non-coding RNA (lincRNA), has a vital role in gene regulation. Dysregulation of their expression has been shown to be associated with the development of cancers. However, the role of lincRNAs in B-lymphoblastic leukemia (B-Acute Lymphoblastic Leukemia; B-ALL) has not been investigated thus far. Here, we analyzed lincRNA expression in B-ALL samples to investigate their role in pathogenesis and disease severity.

Using microarray technology, we analyzed 44 cases of B-ALL of three different cytogenetic subtypes, namely t(12;21), TEL-AML1 translocated; t(1;19) E2A-PBX translocated; and 11q23 (MLL)-rearranged cases and found that lincRNAs were expressed in distinctive patterns across the 3 different cytogenetic subtypes. We named the top 10 differentially expressed lincRNAs as B-ALL associated long intergenic RNAs, or BALIRs. Using RT-qPCR, differential lincRNA expression was confirmed in both the original samples and in an independent cohort. In both original and independent samples, four lincRNAs were consistently differentially expressed in cases with MLL rearrangement, which constitute a poor prognostic subgroup in B-ALL.

We also found that the expression of BALIR-2 and BALIR-6, two of the four lincRNAs that we queried by RT-qPCR, was significantly different in prepreB immunophenotype. Looking at clinicopathologic correlates for the patients in these studies, we found that BALIR-2 was significantly higher in patients who had poor survival and diminished response to prednisone treatment.

We further characterized BALIR-2 using B-ALL cell lines. The chromosomal location of BALIR-2 was conserved in mice and humans. Using RACE, we confirmed the 5’ and the 3’ end of the transcript. In order to further characterize the prednisone response in the patients, we treated RS411and NALM6 cell lines with prednisolone. We found a 30-50% decrease in expression of BALIR-2 following prednisolone treatment. Next, we knocked down BALIR-2 expression using a lentiviral miRNA-formatted siRNA system. BALIR-2 knockdown resulted in reduced cell proliferation and a modest increase in apoptosis of B-ALL cell lines both at steady state and when treated with prednisolone.

To examine the mechanism by which BALIR-2 affects proliferation and apoptosis in B-ALL cell lines, we examined gene expression by microarray in RS411 cell lines with BALIR-2 knockdown with and without prednisolone treatment. Clustering analyses revealed several differentially expressed genes; of these, one of the top clusters included genes involved in the prednisolone response / glucocorticoid receptor signaling pathway (HSPA6, SGK1, IL8, JUN, SERPINE1, CDKN1A and ICAM1). We are currently confirming the expression of several of these genes in multiple cell lines by RT-qPCR and Western Blotting.

In conclusion, this data suggests that BALIR-2 may play a functional role in the survival of B-ALL cells and thereby contribute to the inferior survival observed in patients with high levels of BALIR-2 expression. Based on the microarray experiments, BALIR-2 may be regulating the expression of glucocorticoid receptor signaling pathway genes and thereby apoptosis. These results suggest that knockdown of BALIR-2 could be utilized as a therapeutic approach in treating B-ALL, particularly in poor-prognostic group cases with MLL rearrangement.