Aberrant Stabilization of c-Myc Protein in Lymphoblastic and Myelogenous Leukemia Cell Lines.

Abstract

The c-Myc oncoprotein is a key regulator of cell fate decisions including proliferation, differentiation, and apoptosis. Orderly control of c-Myc protein levels is important in maintaining regulated cell proliferation in normal cells. While c-Myc overexpression is seen in many hematopoietic malignancies, the reason for high protein levels in most cases is unknown and, in general, is not the result of translocations or gene amplification. C-Myc levels vary with cell cycle and are kept very low in quiescent cells. Protein half-life is controlled by phosphorylation at two specific N-terminal sites, Serine 62 and Threonine 58, which regulate c-Myc degradation by the ubiquitin proteasome pathway. Two Ras-dependent signaling pathways (Raf/MEK/ERK and PI(3)K/Akt) modulate phosphorylation at Serine 62, which stabilizes the protein, and Threonine 58, which targets Myc for ubiquitination and subsequent degradation. We recently reported that a stabilized form of c-Myc (c-Myc T58A) contributes to oncogenic transformation of human cells in culture (Yeh et al, Nat. Cell Bio. 6:308–318, 2004). Here we describe the role of c-Myc protein stabilization in 2 pediatric ALL cell lines (REH and Sup-B15), 1 AML cell line (HL-60), and 1 CML cell line (K562). Markedly higher expression of c-Myc protein was seen in all 4 cell lines as compared to normal peripheral blood mononuclear cells (PBMCs). FISH analysis demonstrated amplification of the c-myc gene in HL-60 cells as has been previously reported, but not in REH, Sup-B15, or K562 cells. Using [35S]methionine pulse-chase analysis we demonstrate that the half-life of c-Myc in REH (55 minutes), Sup-B15 (47 minutes), and K562 (40 minutes) cells is longer than in normal PBMCs (9 to 15 minutes), but is not significantly prolonged in HL60 cells. We provide additional functional evidence for aberrant protein stabilization based on greater elevation of c-Myc protein after proteasome inhibition in PBMCs and HL-60 cells than in REH, Sup-B15, or K562 cells. These results suggest that that abnormalities in c-Myc degradation exist upstream of ubiquitination in the ALL and CML cell lines. Consistent with this hypothesis, experimental inhibition of the PI(3)K pathway knocked down c-Myc levels in REH and Sup-B15 cells, an effect that was abrogated by concomitant proteasome inhibition. This result suggests that abnormal activation of the PI(3)K pathway could participate in c-Myc stabilization in these cells. In addition, destabilization of c-Myc by PI(3)K inhibition correlated with a significant decrease in cell proliferation. In conclusion, we demonstrate that aberrant stabilization of c-Myc protein occurs in human leukemia cell lines. Affecting the c-Myc degradation pathway in hematopoietic malignancies that have stabilized c-Myc may constitute a novel therapeutic target. Additional experiments are ongoing to assess c-Myc stability in primary cells from leukemic bone marrow samples.