Ibrutinib Responsive Micro-RNAs and Upregulation of Tumor Suppressor Targets in Chronic Lymphocytic Leukemia

Background: MicroRNAs (miRNAs, miRs) are small (~22nt) noncoding RNAs that are involved in post-transcriptional regulation of specific mRNA targets.  Aberrant miRNA expression has been shown to play a role in chronic lymphocytic leukemia (CLL) pathogenesis and to have prognostic implications.  The lymph node (LN) has been demonstrated to be the site of CLL cell proliferation and activation.  In particular, CLL cells in the LN display significantly increased BCR and NF-kB signaling in comparison to CLL cells in the peripheral blood (PB).   Ibrutinib is a clinically approved BTK inhibitor that disrupts BCR and NF-kB signaling; and has been shown to be effective in relapsed or refractory CLL.  We hypothesized that 1) miRNAs would be differentially expressed between activated CLL cells in the LN and CLL cells in the PB; and 2) ibrutinib would alter miRNA expression in CLL, which may affect therapeutic response by regulating the expression of miR target genes. 

Methods: RNA was isolated from CD19 positive selected cells from two separate cohorts of CLL patients. The first group included 17 treatment naïve patients donating paired LN and PB CD19 positive cells (10 IGHV un-mutated [59%], 4 IGHV mutated [23.5%] and 3 IGHV unknown [17.5%]); the second group contained 38 pairs of PB CD19 positive cells (22 IGHV un-mutated [58%] and 16 IGHV mutated [42%]) before and after ibrutinib treatment on day 28. A panel of 22 miRs previously reported to play a role in CLL pathogenesis, prognosis, or BCR signaling was profiled by qPCR. The expression level of a custom panel of genes encoding putative miR targets was quantified using the nanoString nCounter assay and verified by qPCR.  Functional studies were performed in MEC-1 and SP-53 cell lines, which were treated separately with 1 μM ibrutinib  or 1 μM fludarabine for 48 hours.  Live cells were identified by trypan blue staining; miR and mRNA expression was quantified by qPCR.

Results: Nineteen of 22 (86.4%) miRs were significantly increased (p < 0.05) in CLL cells from the LN in comparison to paired CLL cells from the PB, correlating with an activated B cell phenotype. Examination of mRNA transcripts of putative miR targets in 13 paired LN and PB samples showed that multiple tumor suppressor transcripts including CYLD, ARID1B and FOXP1 were significantly decreased in LN-derived CLL cells.   Assay of 22 miRs in PB CLL cells from 38 patients before and after ibrutinib treatment indicated that a subset of miRs (miR-22, miR-34a, miR-146b and miR-181b) were significantly decreased (p< 0.05) in response to ibrutinib. Quantification of mRNA transcripts of putative targets of the ibrutinib responsive miRs by the nanoString nCounter assay showed that several tumor suppressor transcripts (ARID1B, ARID2, ATM, CYLD, FOXP1, HDAC1, IBTK, PTEN and SMAD4) were significantly increased.  Functional studies in MEC-1 and SP-53 cell lines confirmed that miR-22, miR-34a, miR-146b and miR-181b were down-regulated and the tumor suppressor targets of these miRs (ARID1B, ARID2, ATM, CYLD, FOXP1, HDAC1, PTEN and SMAD4) were significantly increased after ibrutinib treatment.  The alteration of miR and gene expression was specific to the effects of ibrutinib as similar changes were not observed post treatment with the chemotherapeutic agent fludarabine.

Conclusions: miRNA expression patterns are significantly altered between CLL cells in the LN and those in the PB. An overall increase in the expression of miRs and down-regulation of mRNAs of tumor suppressor genes (ARID1B, CYLD and FOXP1) was observed in CLL cells in the LN, correlating with an activated B-cell phenotype. Ibrutinib treatment and inhibition of BCR signaling resulted in down-regulation of miR-22, miR-34a, miR-146b and miR-181b and concomitant up-regulation of multiple tumor suppressor targets including ARID1B, ARID2, ATM, CYLD, FOXP1, HDAC1, IBTK, PTEN and SMAD4 in PB CLL cells. Similar findings were also observed in ibrutinib treated MEC-1 and SP-53 cell lines. Additional studies are needed to address how much the down-regulation of miRs and consequent up-regulation of tumor suppressors in response to ibrutinib contributes to the overall disease response and whether this effect could sensitize CLL cells to treatment with cytotoxic agents.