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Physiologically, MYC levels must be precisely set to faithfully amplify the transcriptome, but in cancer MYC is quantitatively misregulated. Here, we study the variation of MYC amongst single primary cells (B-cells and murine embryonic fibroblasts, MEFs) for the repercussions of variable cellular MYC-levels and setpoints. Because FUBPs have been proposed to be molecular "cruise controls" that constrain MYC expression, their role in determining basal or activated MYC-levels was also examined. Growing cells remember low and high-MYC setpoints through multiple cell divisions and are limited by the same expression ceiling even after modest MYC-activation. High MYC MEFs are enriched for mRNAs regulating inflammation and immunity. After strong stimulation, many cells break through the ceiling and intensify MYC expression. Lacking FUBPs, unstimulated MEFs express levels otherwise attained only with stimulation and sponsor MYC chromatin changes, revealed by chromatin marks. Thus, the FUBPs enforce epigenetic setpoints that restrict MYC expression.

Cancer treatments often require combinations of molecularly targeted agents to be effective. mTORi (rapamycin) and HDACi (MS-275/entinostat) inhibitors have been shown to be effective in limiting tumor growth, and here we define part of the cooperative action of this drug combination. More than 60 human cancer cell lines responded synergistically (CI<1) when treated with this drug combination compared with single agents. In addition, a breast cancer patient-derived xenograft, and a BCL-XL plasmacytoma mouse model both showed enhanced responses to the combination compared with single agents. Mice bearing plasma cell tumors lived an average of 70 days longer on combination treatment compared with single agents. A set of 37 genes cooperatively affected (34 downregulated; 3 upregulated) by the combination responded pharmacodynamically in human myeloma cell lines, xenografts, and a P493 model, and were both enriched in tumors, and correlated with prognostic markers in myeloma patient datasets. Genes downregulated by the combination were overexpressed in several untreated cancers (breast, lung, colon, sarcoma, head and neck, myeloma) compared with normal tissues. The MYC/E2F axis, identified by upstream regulator analyses and validated by immunoblots, was significantly inhibited by the drug combination in several myeloma cell lines. Furthermore, 88% of the 34 genes downregulated have MYC-binding sites in their promoters, and the drug combination cooperatively reduced MYC half-life by 55% and increased degradation. Cells with MYC mutations were refractory to the combination. Thus, integrative approaches to understand drug synergy identified a clinically actionable strategy to inhibit MYC/E2F activity and tumor cell growth in vivo Mol Cancer Ther; 16(9); 2008-21. ©2017 AACR ., (©2017 American Association for Cancer Research.)

50 years ago, Vincent Allfrey and colleagues discovered that lymphocyte activation triggers massive acetylation of chromatin. However, the molecular mechanisms driving epigenetic accessibility are still unknown. We here show that stimulated lymphocytes decondense chromatin by three differentially regulated steps. First, chromatin is repositioned away from the nuclear periphery in response to global acetylation. Second, histone nanodomain clusters decompact into mononucleosome fibers through a mechanism that requires Myc and continual energy input. Single-molecule imaging shows that this step lowers transcription factor residence time and non-specific collisions during sampling for DNA targets. Third, chromatin interactions shift from long range to predominantly short range, and CTCF-mediated loops and contact domains double in numbers. This architectural change facilitates cognate promoter-enhancer contacts and also requires Myc and continual ATP production. Our results thus define the nature and transcriptional impact of chromatin decondensation and reveal an unexpected role for Myc in the establishment of nuclear topology in mammalian cells., (Published by Elsevier Inc.)

Many tumors are characterized by recurrent translocations between a tissue-specific gene and a proto-oncogene. The juxtaposition of the Ig heavy chain gene and Myc in Burkitt's lymphoma and in murine plasmacytoma is a classic example. Regulatory elements within the heavy chain constant region locus are required for Myc translocation and/or deregulation. However, many genes are regulated by cis-acting elements at distances up to 1,000 kb outside the locus. Such putative distal elements have not been examined for the heavy chain locus, particularly in the context of Myc translocations. We demonstrate that a transgene containing the Ig heavy chain constant region locus, inserted into five different chromosomal locations, can undergo translocations involving Myc. Furthermore, these translocations are able to generate plasmacytomas in each transgenic line. We conclude that the heavy chain constant region locus itself includes all of the elements necessary for both the translocation and the deregulation of the proto-oncogene.

Immunoglobulin (Ig) isotype switching is a recombination event that changes the constant domain of antibody genes and is catalyzed by activation-induced cytidine deaminase (AID). Upon recruitment to Ig genes, AID deaminates cytidines at switch (S) recombination sites, leading to the formation of DNA breaks. In addition to their role in isotype switching, AID-induced lesions promote Igh-cMyc chromosomal translocations and tumor development. However, cMyc translocations are also present in lymphocytes from healthy humans and mice, and thus, it remains unclear whether AID directly contributes to the dynamics of B cell transformation. Using a plasmacytoma mouse model, we show that AID(+/-) mice have reduced AID expression levels and display haploinsufficiency both in the context of isotype switching and plasmacytomagenesis. At the Ig loci, AID(+/-) lymphocytes show impaired intra- and inter-switch recombination, and a substantial decrease in the frequency of S mutations and chromosomal breaks. In AID(+/-) mice, these defects correlate with a marked decrease in the accumulation of B cell clones carrying Igh-cMyc translocations during tumor latency. These results thus provide a causality link between the extent of AID enzymatic activity, the number of emerging Igh-cMyc-translocated cells, and the incidence of B cell transformation.

Background: Gene-targeted iMycEmu mice that carry a His6-tagged mouse Myc(c-myc)cDNA, MycHis, just 5' of the immunoglobulin heavy-chain enhancer, Emu, are prone to B cell and plasma cell neoplasms, such as lymphoblastic B-cell lymphoma (LBL) and plasmacytoma (PCT). Cell lines derived from Myc-induced neoplasms of this sort may provide a good model system for the design and testing of new approaches to prevent and treat MYC-driven B cell and plasma cell neoplasms in human beings. To test this hypothesis, we used the LBL-derived cell line, iMycEmu-1, and the newly established PCT-derived cell line, iMycEmu-2, to evaluate the growth inhibitory and death inducing potency of the cancer drug candidate, CDDO-imidazolide (CDDO-Im)., Methods: Morphological features and surface marker expression of iMycEmu-2 cells were evaluated using cytological methods and FACS, respectively. mRNA expression levels of the inserted MycHis and normal Myc genes were determined by allele-specific RT-PCR and qPCR. Myc protein was detected by immunoblotting. Cell cycle progression and apoptosis were analyzed by FACS. The expression of 384 "pathway" genes was assessed with the help of Superarray cDNA macroarrays and verified, in part, by RT-PCR., Results: Sub-micromolar concentrations of CDDO-Im caused growth arrest and apoptosis in iMycEmu-1 and iMycEmu-2 cells. CDDO-Im-dependent growth inhibition and apoptosis were associated in both cell lines with the up-regulation of 30 genes involved in apoptosis, cell cycling, NFkappaB signaling, and stress and toxicity responses. Strongly induced (> or = 10 fold) were genes encoding caspase 14, heme oxygenase 1 (Hmox1), flavin-containing monooxygenase 4 (Fmo4), and three members of the cytochrome P450 subfamily 2 of mixed-function oxygenases (Cyp2a4, Cyp2b9, Cyp2c29). CDDO-Im-dependent gene induction coincided with a decrease in Myc protein., Conclusion: Growth arrest and killing of neoplastic mouse B cells and plasma cells by CDDO-Im, a closely related derivative of the synthetic triterpenoid 2-cyano-3,12-dioxooleana-1,9-dien-28-oic acid, appears to be caused, in part, by drug-induced stress responses and reduction of Myc.