Novel Function Of Chromosome 7 Open Reading Frame 41 Gene To Promote Leukemic Megakaryocyte Differentiation By Modulating TPA-Induced MAPK/ERK, SAPK/JNK, and NF-κB Signaling

Phorbol 12-myristate 13-acetate (TPA) primarily activates PKC and subsequently leads to activation of downstream signaling pathways including MAPK/ERK, SAPK/JNK, and NF-κB, which causes gene expression alteration and leukemic cell differentiation. How these TPA-induced genes may contribute to leukemic cell differentiation remains to be addressed. We noticed that a novel gene chromosome 7 open reading frame 41 (C7ORF41) was one of TPA-induced genes without any known functions. Differential expression of C7ORF41 has been identified in human embryo development and predicted to function in hematopoiesis based on hierarchical clustering analysis. To support this, we found high expression level of C7ORF41 in bone marrow. By using K562 cell as a model, we discovered that ectopic expression of C7ORF41 significantly promoted TPA-induced megakaryocyte differentiation evidenced by an increase of CD61 expression. Consistently, two types of transcription factors critical for megakaryopoiesis, RUNX1 and ETS proteins, were simultaneously upregulated by C7ORF41. Furthermore, cytoplasmic distribution of C7ORF41 suggests that it may act as a signaling molecule. As expected, C7ORF41 overexpression enhanced ERK and JNK phosphorylation. In contrast, C7ORF41 knockdown led to an opposite phenotype: impaired megakaryocyte differentiation, attenuated signaling, and reduced transcription factor expression. These observations suggest that C7ORF41 may promote megakaryocyte differentiation by enhancing ERK and JNK signaling that subsequently leads to upregulation of RUNX1 and ETS proteins. Indeed, C7ORF41 overexpression rescued megakaryocyte differentiation blocked by PD98059, a potent ERK inhibitor, while JNK inhibition abrogated the effect of C7ORF41 on upregulation of ETS proteins. In addition, C7ORF41 was highly conserved in evolution and several tyrosine residues including Y34 were strictly preserved, suggesting the importance of tyrosine phosphorylation in C7ORF41 function. In fact, mutant C7ORF41 with Y34 substitution by phenylalanine functioned to inhibit megakaryocyte differentiation. Finally, NF-κB appeared to be the major activator of C7ORF41 that in turn repressed NF-κB activity by inhibiting its phosphorylation at serine 536. Taken together, we have identified novel function of a new gene C7ORF41 that may promote leukemic megakaryocyte differentiation through a novel mechanism in which C7ORF41 forms a well-balanced regulatory network in TPA-induced signaling. In this network, initial TPA treatment primes downstream signaling including MAPK/ERK, SAPK/JNK, and NF-kB. TPA-induced NF-κB activation further upregulates C7ORF41 that may serve to amplify TPA-induced ERK and JNK signaling to ensure megakaryocyte differentiation. On the other hand, C7ORF41 upregulation also serves as a negative regulator of NF-κB activity that may quench TPA-indcued NF-κB signaling. In addition, enhanced ERK signaling feeds back to damp C7ORF41 upregulation that may tune TPA-induced signaling under controllable level. Our findings shed light on understanding forced differentiation in leukemic cells and may provide useful information for rational differentiation therapy.