The Role of MXD3 in Human Precursor B Cell Acute Lymphoblastic Leukemia

Abstract 3523 Disregulation of the Hedgehog (Hh) signaling pathway is known to drive proliferation in several cancers, including hematopoietic malignancies. The role of the Hh pathway in precursor B cell (preB) acute lymphoblastic leukemia (ALL), the most common leukemia in children, is unclear; however, recent reports suggest that Hh signaling is involved in the development of B cell precursors as well as the self-renewal and survival of B cell malignancies. MXD3 is a transcription factor previously shown in our lab to be a novel member of the Hh pathway (Yun, Rust, Ishimaru, Diaz, Mol Bio Cell, 2007). We have previously shown that MXD3 is expressed in medulloblastoma (the most common brain tumor in children) and that its knockdown reduces proliferation of human medulloblastoma cell lines (Barisone et al., PLoS, 2012). In the current study, we investigated a possible role for MXD3 in preB ALL cell proliferation. Using quantitative real-time reverse-transcription PCR (qRT-PCR) we observed high levels of MXD3 mRNA expression in 8 primary preB ALL samples as well as in the preB ALL cell lines Reh and JM1. However, MXD3 levels were very low in mobilized peripheral blood mononuclear cells from healthy donors, mouse bone marrow and spleen. Indeed, MXD3 levels in the primary ALL cells and cell lines ranged between 13 and 35 fold increase when compared to the normal cells. Immunoblot analysis with anti-MXD3 monoclonal antibodies confirmed that the protein was present in the ALL samples but not in normal cells. We investigated the role of MXD3 in cell proliferation and survival, by silencing MXD3 in the Reh cell line. We used lentiviral delivery to knockdown MXD3 using an RNA interference approach. Briefly, lentiviruses were produced carrying a short hairpin RNA sequence specific for MXD3 (shMXD3) or a negative control sequence (shCTRL). Upon transduction by the viral vector, MXD3 knockdown was confirmed at both the RNA and protein level. Within 48 hours after transduction, MXD3 protein levels were reduced >90% in cells infected with the shMXD3 virus but not with the control virus. Interestingly, MXD3 knockdown resulted in decreased proliferation in Reh cells, supporting our hypothesis that it may be involved in the maintenance of this malignancy. As a first step toward understanding the mechanisms by which MXD3 exerts its function in leukemia cells, we analyzed cell cycle progression and apoptosis levels after knockdown using flow cytometry. We observed no significant differences in the G0/G1, S or G2/M populations between experimental and control samples. However, samples where MXD3 had been knocked down showed higher levels of apoptosis when compared to controls. Taken together, our results suggest that MXD3 is important for preB ALL cell proliferation, possibly by acting as an anti-apoptotic factor. Therefore, MXD3 may represent a suitable candidate for future efforts in developing targeted therapies against preB ALL. Disclosures: No relevant conflicts of interest to declare.