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Promiscuous MYC locus rearrangements hijack enhancers but mostly super-enhancers to dysregulate MYC expression in multiple myeloma.

Affer M, Chesi M, Chen WD, Keats JJ, Demchenko YN, Tamizhmani K, Garbitt VM, Riggs DL, Brents LA, Roschke AV, Van Wier S, Fonseca R, Bergsagel PL, Kuehl WM - Leukemia (2014)

Bottom Line: MYC locus rearrangements-often complex combinations of translocations, insertions, deletions and inversions-in multiple myeloma (MM) were thought to be a late progression event, which often did not involve immunoglobulin genes.Rearrangements reposition MYC near a limited number of genes associated with conventional enhancers, but mostly with super-enhancers (e.g., IGH, IGL, IGK, NSMCE2, TXNDC5, FAM46C, FOXO3, IGJ, PRDM1).Our data suggest that MYC rearrangements, regardless of when they occur during MM pathogenesis, provide one event that contributes to tumor autonomy.

View Article: PubMed Central - PubMed

Affiliation: Department of Hematology Oncology, Mayo Clinic Arizona, Comprehensive Cancer Center, Scottsdale, AZ, USA.

ABSTRACT
MYC locus rearrangements-often complex combinations of translocations, insertions, deletions and inversions-in multiple myeloma (MM) were thought to be a late progression event, which often did not involve immunoglobulin genes. Yet, germinal center activation of MYC expression has been reported to cause progression to MM in an MGUS (monoclonal gammopathy of undetermined significance)-prone mouse strain. Although previously detected in 16% of MM, we find MYC rearrangements in nearly 50% of MM, including smoldering MM, and they are heterogeneous in some cases. Rearrangements reposition MYC near a limited number of genes associated with conventional enhancers, but mostly with super-enhancers (e.g., IGH, IGL, IGK, NSMCE2, TXNDC5, FAM46C, FOXO3, IGJ, PRDM1). MYC rearrangements are associated with a significant increase of MYC expression that is monoallelic, but MM tumors lacking a rearrangement have biallelic MYC expression at significantly higher levels than in MGUS. We also have shown that germinal center activation of MYC does not cause MM in a mouse strain that rarely develops spontaneous MGUS. It appears that increased MYC expression at the MGUS/MM transition usually is biallelic, but sometimes can be monoallelic if there is an MYC rearrangement. Our data suggest that MYC rearrangements, regardless of when they occur during MM pathogenesis, provide one event that contributes to tumor autonomy.

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Rearrangements cis-dysregulate MYC expressionA. Affymetrix Hu133Plus2 arrays were used to estimate the normalized expression of MYC mRNA in CD138-selected plasma cells from normal bone marrow (BMPC), MGUS, MM without MYC rearrangements (MYC NR), MM with MYC rearrangements (MM R), MM with MYC rearrangements involving IgH or IgL (IgH or IgL) and MM cell lines (MMCL).B. The arrays were also used to calculate the proliferation index in the same cell populations.C. MM RNA from tumors heterozygous for SNPs rs4645958 or rs2070582 was analyzed by RT-PCR for allele specific expression. For both SNPs the polymorphism alters an Ici1 restriction site allowing the two alleles to be distinguished after restriction digest of the PCR product. Shown is the 4% agarose gel electrophoresis of the PCR digest for three patients with each polymorphism, providing examples of MM tumors that express each allele alone, and both together. Also provided is a table summarizing the allele specific expression of MYC in MM tumors and cell lines.D. Regions adjacent to MYC breakpoints in XG6 and L363 are shown, with the location of fragments cloned for enhancer assays indicated. The fragments were located in predicted MM.1S super-enhancers35. The relative ability of the various fragments to enhance the transcription of a MYC promoter driven luciferase construct is shown as relative luciferase activity (RLA) normalized to the activity of the MYC promoter without enhancer.E. Expression of genes adjacent to putative super-enhancers involved in MYC locus rearrangements during in vitro B cell differentiation to long lived plasma cells. The log2-normalized data from Cocco et al is shown for the progressive stages of in vitro plasma cell differentiation23. In addition to IgH, IgK, and IgL (not shown), also IGJ, TXNDC5, FAM46C, FOXO3, PRDM1 (but not FAM188A or ANKRD55) are markedly up regulated with plasma cell differentiation.
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Figure 2: Rearrangements cis-dysregulate MYC expressionA. Affymetrix Hu133Plus2 arrays were used to estimate the normalized expression of MYC mRNA in CD138-selected plasma cells from normal bone marrow (BMPC), MGUS, MM without MYC rearrangements (MYC NR), MM with MYC rearrangements (MM R), MM with MYC rearrangements involving IgH or IgL (IgH or IgL) and MM cell lines (MMCL).B. The arrays were also used to calculate the proliferation index in the same cell populations.C. MM RNA from tumors heterozygous for SNPs rs4645958 or rs2070582 was analyzed by RT-PCR for allele specific expression. For both SNPs the polymorphism alters an Ici1 restriction site allowing the two alleles to be distinguished after restriction digest of the PCR product. Shown is the 4% agarose gel electrophoresis of the PCR digest for three patients with each polymorphism, providing examples of MM tumors that express each allele alone, and both together. Also provided is a table summarizing the allele specific expression of MYC in MM tumors and cell lines.D. Regions adjacent to MYC breakpoints in XG6 and L363 are shown, with the location of fragments cloned for enhancer assays indicated. The fragments were located in predicted MM.1S super-enhancers35. The relative ability of the various fragments to enhance the transcription of a MYC promoter driven luciferase construct is shown as relative luciferase activity (RLA) normalized to the activity of the MYC promoter without enhancer.E. Expression of genes adjacent to putative super-enhancers involved in MYC locus rearrangements during in vitro B cell differentiation to long lived plasma cells. The log2-normalized data from Cocco et al is shown for the progressive stages of in vitro plasma cell differentiation23. In addition to IgH, IgK, and IgL (not shown), also IGJ, TXNDC5, FAM46C, FOXO3, PRDM1 (but not FAM188A or ANKRD55) are markedly up regulated with plasma cell differentiation.

Mentions: Exonic and intronic polymorphisms were determined in the MMCL and some tumor samples by Sanger sequencing of PCR products generated from different regions of the MYC gene. Expression of MYC polymorphisms that crossed an intron for exonic polymorphisms or total RNA treated with DNase for intronic polymorphisms (Table S2, Table S4). Restriction enzyme polymorphisms of genomic variants rs4645958 and rs2070582 were used to analyze allele specific expression in some MMRC MM tumors (Figure 2C, Table S4). Additional details in Supplemental Methods.


Promiscuous MYC locus rearrangements hijack enhancers but mostly super-enhancers to dysregulate MYC expression in multiple myeloma.

Affer M, Chesi M, Chen WD, Keats JJ, Demchenko YN, Tamizhmani K, Garbitt VM, Riggs DL, Brents LA, Roschke AV, Van Wier S, Fonseca R, Bergsagel PL, Kuehl WM - Leukemia (2014)

Rearrangements cis-dysregulate MYC expressionA. Affymetrix Hu133Plus2 arrays were used to estimate the normalized expression of MYC mRNA in CD138-selected plasma cells from normal bone marrow (BMPC), MGUS, MM without MYC rearrangements (MYC NR), MM with MYC rearrangements (MM R), MM with MYC rearrangements involving IgH or IgL (IgH or IgL) and MM cell lines (MMCL).B. The arrays were also used to calculate the proliferation index in the same cell populations.C. MM RNA from tumors heterozygous for SNPs rs4645958 or rs2070582 was analyzed by RT-PCR for allele specific expression. For both SNPs the polymorphism alters an Ici1 restriction site allowing the two alleles to be distinguished after restriction digest of the PCR product. Shown is the 4% agarose gel electrophoresis of the PCR digest for three patients with each polymorphism, providing examples of MM tumors that express each allele alone, and both together. Also provided is a table summarizing the allele specific expression of MYC in MM tumors and cell lines.D. Regions adjacent to MYC breakpoints in XG6 and L363 are shown, with the location of fragments cloned for enhancer assays indicated. The fragments were located in predicted MM.1S super-enhancers35. The relative ability of the various fragments to enhance the transcription of a MYC promoter driven luciferase construct is shown as relative luciferase activity (RLA) normalized to the activity of the MYC promoter without enhancer.E. Expression of genes adjacent to putative super-enhancers involved in MYC locus rearrangements during in vitro B cell differentiation to long lived plasma cells. The log2-normalized data from Cocco et al is shown for the progressive stages of in vitro plasma cell differentiation23. In addition to IgH, IgK, and IgL (not shown), also IGJ, TXNDC5, FAM46C, FOXO3, PRDM1 (but not FAM188A or ANKRD55) are markedly up regulated with plasma cell differentiation.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4126852&req=5

Figure 2: Rearrangements cis-dysregulate MYC expressionA. Affymetrix Hu133Plus2 arrays were used to estimate the normalized expression of MYC mRNA in CD138-selected plasma cells from normal bone marrow (BMPC), MGUS, MM without MYC rearrangements (MYC NR), MM with MYC rearrangements (MM R), MM with MYC rearrangements involving IgH or IgL (IgH or IgL) and MM cell lines (MMCL).B. The arrays were also used to calculate the proliferation index in the same cell populations.C. MM RNA from tumors heterozygous for SNPs rs4645958 or rs2070582 was analyzed by RT-PCR for allele specific expression. For both SNPs the polymorphism alters an Ici1 restriction site allowing the two alleles to be distinguished after restriction digest of the PCR product. Shown is the 4% agarose gel electrophoresis of the PCR digest for three patients with each polymorphism, providing examples of MM tumors that express each allele alone, and both together. Also provided is a table summarizing the allele specific expression of MYC in MM tumors and cell lines.D. Regions adjacent to MYC breakpoints in XG6 and L363 are shown, with the location of fragments cloned for enhancer assays indicated. The fragments were located in predicted MM.1S super-enhancers35. The relative ability of the various fragments to enhance the transcription of a MYC promoter driven luciferase construct is shown as relative luciferase activity (RLA) normalized to the activity of the MYC promoter without enhancer.E. Expression of genes adjacent to putative super-enhancers involved in MYC locus rearrangements during in vitro B cell differentiation to long lived plasma cells. The log2-normalized data from Cocco et al is shown for the progressive stages of in vitro plasma cell differentiation23. In addition to IgH, IgK, and IgL (not shown), also IGJ, TXNDC5, FAM46C, FOXO3, PRDM1 (but not FAM188A or ANKRD55) are markedly up regulated with plasma cell differentiation.
Mentions: Exonic and intronic polymorphisms were determined in the MMCL and some tumor samples by Sanger sequencing of PCR products generated from different regions of the MYC gene. Expression of MYC polymorphisms that crossed an intron for exonic polymorphisms or total RNA treated with DNase for intronic polymorphisms (Table S2, Table S4). Restriction enzyme polymorphisms of genomic variants rs4645958 and rs2070582 were used to analyze allele specific expression in some MMRC MM tumors (Figure 2C, Table S4). Additional details in Supplemental Methods.

Bottom Line: MYC locus rearrangements-often complex combinations of translocations, insertions, deletions and inversions-in multiple myeloma (MM) were thought to be a late progression event, which often did not involve immunoglobulin genes.Rearrangements reposition MYC near a limited number of genes associated with conventional enhancers, but mostly with super-enhancers (e.g., IGH, IGL, IGK, NSMCE2, TXNDC5, FAM46C, FOXO3, IGJ, PRDM1).Our data suggest that MYC rearrangements, regardless of when they occur during MM pathogenesis, provide one event that contributes to tumor autonomy.

View Article: PubMed Central - PubMed

Affiliation: Department of Hematology Oncology, Mayo Clinic Arizona, Comprehensive Cancer Center, Scottsdale, AZ, USA.

ABSTRACT
MYC locus rearrangements-often complex combinations of translocations, insertions, deletions and inversions-in multiple myeloma (MM) were thought to be a late progression event, which often did not involve immunoglobulin genes. Yet, germinal center activation of MYC expression has been reported to cause progression to MM in an MGUS (monoclonal gammopathy of undetermined significance)-prone mouse strain. Although previously detected in 16% of MM, we find MYC rearrangements in nearly 50% of MM, including smoldering MM, and they are heterogeneous in some cases. Rearrangements reposition MYC near a limited number of genes associated with conventional enhancers, but mostly with super-enhancers (e.g., IGH, IGL, IGK, NSMCE2, TXNDC5, FAM46C, FOXO3, IGJ, PRDM1). MYC rearrangements are associated with a significant increase of MYC expression that is monoallelic, but MM tumors lacking a rearrangement have biallelic MYC expression at significantly higher levels than in MGUS. We also have shown that germinal center activation of MYC does not cause MM in a mouse strain that rarely develops spontaneous MGUS. It appears that increased MYC expression at the MGUS/MM transition usually is biallelic, but sometimes can be monoallelic if there is an MYC rearrangement. Our data suggest that MYC rearrangements, regardless of when they occur during MM pathogenesis, provide one event that contributes to tumor autonomy.

Show MeSH
Related in: MedlinePlus