The Addition of the Estrogen Receptor to the Carboxy Terminus of c-Myc Inhibits the Fas/CD-95 Apoptotic Pathway Associated with the Myc-Mediated Block in Myeloid Differentiation.
Authors: D’Angelo Santo A. , Liebermann Dan A. , Hoffman Barbara 
Published: October 2019

Tightly regulated c-Myc expression is crucial for normal hematopoiesis, and alterations in the level of c-Myc expression or protein structure are associated with many hematological malignancies. Deregulated Myc expression has been shown to block myeloid terminal differentiation as well as concomitantly induce p53 independent apoptosis through the Fas/CD-95 pathway. This observed apoptosis was not completely penetrant, however it impacted on the neoplastic potential of the blocked myeloid cells. It has been previously shown that deregulated c-Myc, in combination with the deregulation of an apoptotic suppressor such as Bcl-2, can effectively transform hematopoietic cells. Since acute myeloid leukemia (AML) results from a clonal expansion of myeloid cells that are blocked from differentiating, there is significance to the mechanisms behind the apoptosis seen during the Myc mediated block in myeloid differentiation. To facilitate the study of Myc, conditional alleles were used that employed fusing the estrogen homone receptor binding (ER) domain onto the carboxy terminus of the Myc protein. This MycER mutant was originally determined to have no impact on the various proliferative or apoptotic functions of Myc. Consequently, this conditional ER chimeric Myc has been widely used in a variety of studies as a means of titrating Myc activity. Here, we show that the presence of the ER binding domain on the c-terminus of Myc alters its ability to induce apoptosis during the Myc mediated block in myeloid differentiation by affecting the Fas/CD-95 ligand receptor pathway. Our data showed that M1 myeloid leukemic cells stably transfected with MycER (M1MycER), and stimulated to differentiate with interleukin-6 (IL-6) were blocked for terminal differentiation but lacked the apoptotic phenotype normally seen with M1 cells expressing the wild type Myc transgene (M1Myc). Further study of M1MycER cells showed low expression of the death receptor signaling protein RIP when compared to M1Myc cells. RIP has been shown to be an important component of the Fas/CD-95 death receptor pathway. Furthermore, during IL-6 treatment, M1MycER cells showed down-regulation of GADD45 alpha, as well as increased levels of activated Akt, both indicative of NFkB activation. Others have shown that NFkB can be activated through the Fas/CD-95 pathway during the inhibition of Fas/CD-95 mediated apoptosis. The presence of activated Akt in the stimulated M1MycER cells coincides with an increase in the transcriptional level of MCL-1. MCL-1 is an anti-apoptotic member of the Bcl-2 family induced by Akt. In M1MycER cells, increased transcriptional levels of DAD-1 and Bax, both binding partners of MCL-1, suggest possible mechanisms for affecting cytochrome-c release in the mitochondria and cell survival. Taken as a whole, these results offer new insights into how mutations in cMyc as well as the various components of the NF-kB branch of the Fas/CD-95 pathway can impact on the aggressiveness of leukemias.