Olfactomedin 4 Mediates All- Trans Retinoic Acid Induced Growth Inhibition, Differentiation and Apoptosis in Myeloid Leukemia Cells and Inhibits 4E-BP1 Phosphorylation.

Abstract 2395 Poster Board II-372 Introduction: All- trans retinoic acid (ATRA) has been shown to induce cellular differentiation and growth inhibition of acute promyelocytic leukemia cells. Clinical application of ATRA has proved extremely successful in inducing clinical remission in most acute promyelocytic leukemia patients. Although the mechanisms of retinoid-dependent gene transcription regulation are well understood, the target genes that mediate retinoid-induced biological responses still remain to be defined. Olfactomedin 4 (OLFM4, also called hGC-1 and GW112) is a member of olfactomedin-related glycoprotein family. It is constitutively expressed in myeloid cells and gastrointestinal tract. It has been recently reported that OLFM4 expression is up-regulated in stomach and colon cancer patients. The purpose of this study is to examine its expression in myeloid leukemia patients, its regulation by ATRA and potential biological functions in myeloid leukemia. Results: 1) In this study, we found that OLFM4 expression was up-regulated in the peripheral leukocytes of chronic myeloid leukemia patients (91%, 22 cases) and acute myeloid leukemia patients (30%, 10 cases). OLFM4 expression in accelerated phase of chronic myeloid leukemia patients was significantly higher than that in chronic phase. 2) We identified that OLFM4 is a novel target gene of retinoic acids in myeloid leukemia cells. Treatment of HL-60 cells with ATRA and 9- cis -RA induced OLFM4 expression. The expression level of OLFM4 is correlated with the myeloid cell differentiation stage. Deletion analysis led to the identification of a positive retinoic acid response element (DR5) and a negative response element (DR1) within OLFM4 promoter. Furthermore, electrophoretic mobility-shift assays and transfection study in COS-7 cells demonstrated that RARα/RXRα binds to the DR5 site and mediates ATRA induced transactivation of OLFM4 promoter. 3) We showed that OLFM4 over-expression in HL-60 cells lead to growth inhibition, differentiation and apoptosis and potentates ATRA mediated these effects. Conversely, silencing of endogenous OLFM4 by lentiviral shRNA against OLFM4 in AML-193 cells reduces ATRA induced growth inhibition, differentiation and apoptosis. 4) We further investigated the molecular mechanism that OLFM4 is involved in leukemia cell growth and differentiation. We found that over-expression of OLFM4 in HL60 cells inhibited ATRA induced phosphorylation of translation repressor 4E-BP1. This inhibitory effect was further confirmed in 293T cells, in which over-expression of OLFM4 inhibits 4E-BP1 phosphorylation at all four phosphorylation sites (Thr37/46, Thr70 and Ser65). Application of lentiviral shRNA against OLFM4 in AML-193 increased phosphorylation of 4E-BP1 compared with control shRNA. Over-expression of OLFM4 in HL60 cells does not affect phosphorylation of Akt, p70S6 kinase, GSK3β and ERK1/2 . Conclusion: 1) OLFM4 expression is up-regulated in chronic and acute myeloid leukemia patients and is correlated with the stage of chronic myeloid leukemia. 2) OLFM4 is a novel target gene of retinoic acids. RARα/RXRα binds to the DR5 site of OLFM4 promoter and mediates the ATRA induced transactivation of OLFM4. 3) OLFM4 mediates ATRA induced growth inhibition, differentiation and apoptosis of myeloid leukemia cells. 4) OLFM4 acts an inhibitor of 4E-BP1 phosphorylation down stream of Akt and mTOR, suggesting OLFM4 may inhibit protein synthesis that is observed with the differentiation of myeloid leukemia cells. Our results suggest that OLFM4 up-regulation in the leukocytes of leukemia patients might have a feedback effect to restrain cell growth, and induce differentiation and apoptosis rather than act as an initiator of leukemiagenesis. As OLFM4 is a secreted glycoprotein, it could represent a promising therapeutic agent in the treatment of myeloid leukemia patients. The effect of purified OLFM4 on leukemia cells is currently under investigation. Disclosures: No relevant conflicts of interest to declare.