Hsa-Mir-199b-5p Targets PODXL and DDR1: Decreased Levels of Mir-199b-5p Correlate with Elevated Expressions of PODXL and DDR1 in Acute Myeloid Leukemia

Abstract 1463

Discovery of new prognostic factors in AML is driving our understanding of disease biology and development of new therapeutic targets. Aberrant expression of Podocalyxin (PODXL), a CD34 orthologue, has recently been associated with a subset of aggressive cancers including acute myeloid and lymphoid leukemia, myeloid sarcomas, and certain breast, liver, pancreatic and kidney tumors. Herein, via tissue microarray (TMA) approach, we discovered 50% of M1 and 100% of M2 and M4 FAB-subtype patients showed significantly elevated PODXL levels in bone marrow compared to normal subjects. Importantly, several recent investigations have linked aberrant miRNA expression with AML. A recent study evaluated miRNA expression in 122 untreated adult AML cases using a microarray platform. Interestingly, one of the significantly down regulated miRNA in Trisomy 8 associated AML was miR-199b. Via applying TargetScan prediction algorithm, we discovered that PODXL is a highly conserved target of miR-199b-5p. To test the silencing potential of miR-199b-5p on PODXL, miRIDIAN Mimic hsa-miR-199b-5p (and negative control) were transfected in THP-1 and HeLa cells. Analysis of PODXL transcript levels post transfection confirmed that PODXL is indeed a strong target of miR-199b-5p. This was further confirmed via generating stable cell lines expressing miR-199b-5p. Further prediction analyses of miR-199b- 5p targets identified Discoidin domain receptor 1 (DDR1) as another highly conserved target (4x-3'UTR sites). DDR1 is a class of collagen receptor, expressed on human leukocytes, including neutrophils, monocytes, lymphocytes and podocytes. In addition, DDR1 is significantly overexpressed in several human tumors of breast, ovarian, esophageal, and brain origin. Experimental validation via a similar approach outlined for PODXL confirmed that DDR1 like PODXL is also a specific target of miR-199b-5p. In addition, this was further confirmed via employing 3'UTR-luciferase assays for both PODXL and DDR1 where seed regions in the respective 3'UTRs were mutated for miR-199b-5p binding. Experimental results showed that mutation of seed regions significantly inhibited the ability of miR-199b-5p to target PODXL and DDR1. Interestingly, previous studies have demonstrated both PODXL and DDR1 play very similar functional roles in kidney by regulating slit diaphragms and foot processes. Therefore, we next evaluated the expression of DDR1 in the same AML patient set via TMA approach. For the first time, IHC analyses showed DDR1 levels were highly up regulated in AML. Interestingly, DDR1 expression was significantly up regulated in 100% of M1 AML cases and in 25% of M2 and M4 AML patients. Significantly, DDR1 expression was highly elevated in the same AML cases where PODXL levels were increased. Most importantly, via extracting miRNA from paraffin embedded bone marrow tissues of these AML patients and quantitative RT-PCR based assay, we found that miR-199b-5p levels were significantly decreased in most of the AML patients with elevated PODXL and DDR1 expressions. The miRNA qRT-PCR data was normalized using RNU6 and SNORD48 as internal controls. Furthermore, 55% of these AML cases were patients with persistent AML. A recent investigation revealed that overexpression of miR-199b-5p in Medulloblastoma (MB) cancer stem cells results in down regulation of CD133+ tumor initiating cells and causes depletion of the side population compartment of MB tumor stem cell population via negatively regulating Hes1 mediated Notch signaling pathway. Therefore, it is tempting to hypothesize that down regulation of hsa-miR-199b-5p in AML may provide proliferative advantage for leukemic stem cells in addition to accelerating migration and invasion functions via up regulation of PODXL and DDR1. Current investigations are focused on employing retroviral transduction of miR-199b-5p antagomir into human bone marrow derived CD34 cells to test the leukemogenic potential of miR-199b-5p silencing via colony forming assays. Taken together, our studies have identified that miR-199b-5p targets PODXL and DDR1 and demonstrate that concurrent increased expressions of PODXL and DDR1 in AML correlates with decreased expression of miR-199b-5p in AML.