Abstract
Simple SummaryOncogenic transformation in response to the activation of MYC requires secondary events that reduce the role of MYC as an inducer of apoptosis and cellular senescence. We made a primary B-cell model in which such secondary processes were reduced and MYC levels could be regulated at the transcriptional level. We studied the changes in transcriptional programs as MYC levels were progressively increased, causing a simultaneous transition to a lymphoma cell phenotype. Two lymphoma-associated MYC mutants (resulting in the substitution of threonine 58 with isoleucine, T58I, or alanine, T58A) were also analyzed and compared with each other and wild type MYC. All three MYC proteins induced cell cycle entry, cell growth and cell proliferation with associated changes in gene expression programs. The MYC-regulated genes overlap with MYC target genes in B-cells during normal activation. A minority of the MYC-regulated genes (enriched in specific aspects of the cell cycle) were regulated significantly differently by the mutant MYC proteins compared to wild type MYC and in most cases between the two mutant MYC proteins. These genes did not overlap significantly with the set of genes regulated during B-cell activation, and may be specific to oncogenesis. Many target genes were more sensitive to regulation by T58A and less sensitive to T58I, compared to wild type MYC, as was also seen for the measured phenotypic processes. This, taken together with the different sets of genes that are differentially regulated by each mutant in relation to wild type MYC, indicates that the mutations augment the oncogenic activity of MYC by different mechanisms. The different effects of the mutations may result from the different effects they are predicted to have on the conformational properties of the intrinsically disordered region of the MYC protein that surrounds the threonine 58 residue.The MYC transcription factor regulates a vast number of genes and is implicated in many human malignancies. In some hematological malignancies, MYC is frequently subject to missense mutations that enhance its transformation activity. Here, we use a novel murine cell system to (i) characterize the transcriptional effects of progressively increasing MYC levels as normal primary B-cells transform to lymphoma cells and (ii) determine how this gene regulation program is modified by lymphoma-associated MYC mutations (T58A and T58I) that enhance its transformation activity. Unlike many previous studies, the cell system exploits primary B-cells that are transduced to allow regulated MYC expression under circumstances where apoptosis and senescence pathways are abrogated by the over-expression of the Bcl-xL and BMI1 proteins. In such cells, transition from a normal to a lymphoma phenotype is directly dependent on the MYC expression level, without a requirement for secondary events that are normally required during MYC-driven oncogenic transformation. A generalized linear model approach allowed an integrated analysis of RNA sequencing data to identify regulated genes in relation to both progressively increasing MYC level and wild type or mutant status. Using this design, a total of 7569 regulated genes were identified, of which the majority (n = 7263) were regulated in response to progressively increased levels of wild type MYC, while a smaller number of genes (n = 917) were differentially regulated, compared to wild type MYC, in T58A MYC- and/or T58I MYC-expressing cells. Unlike most genes that are similarly regulated by both wild type and mutant MYC genes, the set of 917 genes did not significantly overlap with known lipopolysaccharide regulated genes, which represent genes regulated by MYC in normal B cells. The genes that were differently regulated in cells expressing mutant MYC proteins were significantly enriched in DNA replication and G2 phase to mitosis transition genes. Thus, mutants affecting MYC proteins may augment quantitative oncogenic effects on the expression of normal MYC-target genes with qualitative oncogenic effects, by which sets of cell cycle genes are abnormally targeted by MYC as B cells transition into lymphoma cells. The T58A and T58I mutations augment MYC-driven transformation by distinct mechanisms.
Highlights
The transcription factor MYC plays a central role in the onset and progression of several B-cell lymphomas [1]
To understand changes in cellular characteristics and how they differ between wild type (WT) and mutants as increasing MYC levels drive the transduced B-cells towards a lymphoma phenotype, we overexpressed MYC, threonine-58 is substituted with alanine (T58A) and T58I at different levels by progressively increasing the doxycycline concentrations added to the transduced cells for 48, 96 and 144 h (Figure 1A)
We have used a primary B-cell model to determine how progressively increased MYC levels drive cells, which are otherwise predisposed for oncogenic transformation, into a lymphoma phenotype, as well as how this process is augmented by lymphoma-associated mutations affecting the MYC protein
Summary
The transcription factor MYC plays a central role in the onset and progression of several B-cell lymphomas [1]. The defining characteristic of Burkitt’s lymphoma (BL) is a translocation event where MYC is fused to the immunoglobulin heavy chain locus leading to its aberrant expression (t8; 14) (q24; q32) [2]. Less frequently in BL, MYC is translocated to one of two loci encoding for immunoglobulin light chains, but the result is deregulated expression and a BL phenotype. Mutations most commonly lead to a threonine to isoleucine substitution at residue 58 (T58I), while the somewhat less frequently occurring substitution with alanine at the same residue (T58A) mutation causes higher transforming activity than both wild type (WT) and T58I in cellular model systems [10]. Proteins carrying the T58A mutation have been shown to be stabilized while MYC proteins carrying T58I are surprisingly similar to WT in terms of half-life, indicating that additional mechanisms affecting MYC protein levels are affected when the threonine residue at position T58 is converted to other amino acids that cannot be phosphorylated [5,10]
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