MYC Partner Research Articles

Clinicopathological features of lymphoma/leukemia patients carrying both BCL2 and MYC translocations

Lymphoid neoplasm with 18q21.3/BCL2 and 8q24/MYC translocation to immunoglobulin (IG) genes as dual-hit lymphoma/leukemia is very rare and known to have a poor clinical outcome. To clarify the clinicopathological characteristics of this malignancy, we analyzed 27 cases of cytogenetically proven dual-hit lymphoma/leukemia. Dual-hit lymphoma/leukemia was diagnosed at presentation in 22 cases and at relapse or disease progression in 5 cases. At the time of diagnosis of dual-hit lymphoma/leukemia, extranodal involvement was found in 25 cases (93%) and central nervous system involvement occurred in 15 cases (56%). The median survival and 1-year survival rate of the 27 cases were only 6 months and 22%, respectively, after diagnosis of the dual-hit lymphoma/leukemia. Seven cases of triple-hit lymphoma/leukemia (dual-hit lymphoma/leukemia with 3q27/BCL6 translocation) were included; the median survival of these patients was only 4 months from the diagnosis of the dual-hit lymphoma/leukemia. The duration of survival of the patients with a triple-hit malignancy was shorter than that of the other 20 cases of dual-hit lymphoma/leukemia (p=0.02). The translocation partner of MYC subdivided the dual-hit cases into two groups; 14 cases of IGH and 13 cases of IGK/L. The MIB-1 index was investigated in 14 cases with aggressive B-cell lymphoma, and was higher in the group with MYC-IGH translocation (n=7) than in the MYC-IGK/L group (n=7) (p=0.02). Overall survival was not different between the MYC-IGH translocation group (n=14) and the MYC-IGK or MYC-IGL translocation group (n=13). Dual-hit lymphoma/leukemia is a rare but distinct mature B-cell neoplasm with an extremely poor prognosis characterized by frequent extranodal involvement and central nervous system progression with either of the translocation partners of MYC.

Stabilizers of the Max Homodimer Identified in Virtual Ligand Screening Inhibit Myc Function

Many human cancers show constitutive or amplified expression of the transcriptional regulator and oncoprotein Myc, making Myc a potential target for therapeutic intervention. Here we report the down-regulation of Myc activity by reducing the availability of Max, the essential dimerization partner of Myc. Max is expressed constitutively and can form unstable homodimers. We have isolated stabilizers of the Max homodimer by applying virtual ligand screening (VLS) to identify specific binding pockets for small molecule interactors. Candidate compounds found by VLS were screened by fluorescence resonance energy transfer, and from these screens emerged a potent, specific stabilizer of the Max homodimer. In vitro binding assays demonstrated that the stabilizer enhances the formation of the Max-Max homodimer and interferes with the heterodimerization of Myc and Max in a dose-dependent manner. Furthermore, this compound interferes with Myc-induced oncogenic transformation, Myc-dependent cell growth, and Myc-mediated transcriptional activation. The Max-Max stabilizer can be considered a lead compound for the development of inhibitors of the Myc network.

Lymphomas with Concurrent T(14;18) and 8q24 Translocations Are Under- Reported and Clinical Outcome Depends on the MYC Partner

Background: Lymphomas with concurrent t(14;18) and 8q24 translocations (BCL2+/MYC+) are associated with a poor overall survival (OS). Recently, Savage et al. (Ann Oncol 19: 4, 2008, a186) reported that 4% (6/163) of patients with newly diagnosed diffuse large B cell lymphoma (DLBCL) have BCL2+/MYC+ rearrangements and have a variable clinical outcome. We identified the clinical and cytogenetic characteristics of a larger cohort of patients with BCL2+/MYC+ rearrangements to determine correlations with outcome.Methods: Cases of non-Hodgkin lymphoma (NHL) with concurrent 18q21 and 8q24 breakpoints were identified by karyotype and/or fluorescence in-situ hybridization (FISH) analysis. Histological diagnoses were determined according to the World Health Organization. All MYC rearrangements were confirmed by FISH using a commercial MYC break-apart probe. OS was calculated from the MYC+ biopsy date to last follow up date or death.Results: 55 of 1480 NHL samples (4%) were identified and confirmed to have BCL2+/MYC+ rearrangements. The histological diagnoses were follicular lymphoma (FL)(1), DLBCL(17) and high-grade B cell lymphoma unclassifiable (HGBCL) (37). The HGBCL included cases that were considered intermediate between DLBCL and Burkitt lymphoma (BL), Tdt+ lymphoblastic lymphoma (LBL) and surface immunoglobulin (IG)+ acute lymphoblastic leukemia (sIG+ ALL). All HGBCL cases with available Bcl-2 protein staining were positive in contrast to 5/14 DLBCL cases that were Bcl-2 protein negative despite having t(14;18). By karyotype, 31/55 had MYC translocations involving the IG loci (11 t(8;14), 11 t(8;22), 3 t(2;8) and 6 complex t(8,14,18)). The remaining MYC rearrangements involved different chromosome partners, the most common being t(8;9) (q24;p13) (12/31). The other MYC partners included 1p36, 3p25, 3q27, 4p13, 5q13, 12p11 and 13q31. There was a high correlation between the MYC partner and the resulting histology. 28/31 of the IG/MYC translocations were associated with HGBCL. 14/17 of DLBCL cases had non-IG/MYC translocations and those with IG/MYC translocations were either Bcl-2 protein-negative (2) or had a complex t(8;14;18)(1). HGBCL histology, Bcl-2 protein expression and the presence of an IG/MYC partner all correlated with a poor outcome. HGBCL was associated with a median OS of 3 months compared to 3 y for DLBCL (p<0.0001). In contrast to the DLBCL patients, the HGBCL patients were older (median age 62 y vs 54 y), more often developed transformation from antecedent indolent lymphoma (44% vs 30%) and were less likely to complete anthracycline-based chemotherapy (76% vs 94%). Bcl-2 protein-negative biopsies were associated with median OS of 5.7 years vs 5 months for Bcl-2 protein+ cases (p = 0.05). An IG/MYC partner correlated with a median OS of 3 months compared to 7 months for non-IG/MYC partner (p=0.02). Thus, a favorable outcome was associated with DLBCL morphology, a non-IG/MYC rearrangement and Bcl-2 protein-negative biopsy. IG/MYC+ and Bcl-2 protein+ biopsies were associated with HGBCL and a median OS of < 4 months (p = 0.02).Conclusion: NHL with BCL2+/MYC+ rearrangements are under-recognized due to lack of routine karyotyping or FISH analysis of all high-grade lymphomas. Comprehensive analysis of BCL2 and MYC status should be performed on all HGBCL in the new WHO (2008) classification. The presence of an IG-related MYC partner correlates with a poor prognosis. The favorable survival associated with the few Bcl-2 protein-negative cases suggests that the anti-apoptotic effect of Bcl-2 protein explains the negative impact on prognosis when NHL have concurrent t(14;18) and MYC translocations. Thus, Bcl-2 targeted therapy should be ideally suited for the more frequent Bcl-2 protein+ cases.

Hypoxia-inducible factor-2α regulates the expression of TRAIL receptor DR5 in renal cancer cells

To understand the role of hypoxia-inducible factor (HIF)-2α in regulating sensitivity of renal cancer cells to tumor necrosis factor-related apoptosis inducing ligand (TRAIL)-induced apoptosis, we transfected wild-type and mutant von Hippel Lindau (VHL) proteins into TRAIL-sensitive, VHL-negative A498 cells. We find that wild-type VHL, but not the VHL mutants S65W and C162F that do not degrade HIF proteins, cause TRAIL resistance. Knock down of the HIF-2α protein by RNA interference (short hairpin RNA) blocked TRAIL-induced apoptosis, decreased the level of TRAIL receptor (DR5) protein and inhibited the transcription of DR5 messenger RNA. By using luciferase constructs containing the upstream region of the DR5 promoter, we demonstrate that HIF-2α stimulates the transcription of the DR5 gene by activating the upstream region between −448 and −1188. Because HIF-2α is thought to exert its effect on gene transcription by interacting with the Max protein partner of Myc in the Myc/Max dimer, small interfering RNAs to Myc were used to lower the levels of this protein. In multiple renal cancer cell lines decreasing the levels of Myc blocked the ability of HIF-2α to stimulate DR5 transcription. PS-341 (VELCADE, bortezomib), a proteasome inhibitor used to treat human cancer, increases the levels of both HIF-2α and c-Myc and elevates the level of DR5 in renal cancer, sensitizing renal cancer cells to TRAIL therapy. Similarly, increasing HIF-2α in prostate and lung cancer cell lines increased the levels of DR5. Thus, in renal cancer cell lines expressing HIF-2α, this protein plays a role in regulating the levels of the TRAIL receptor DR5.

Thermodynamics of b-HLH-LZ Protein Binding to DNA: The Energetic Importance of Protein−DNA Contacts in Site-Specific E-Box Recognition by the Complete Gene Product of the Max p21 Transcription Factor

The Myc/Mad/Max network of dimeric basic region-helix-loop-helix-leucine zipper (b-HLH-LZ) transcription factors bind to enhancer box sequences (E-box) in the promotors of a large set of genes that control cell metabolism, proliferation, and differentiation. Max (Myc-associated factor X) is the obligate heterodimerization partner of Myc and Mad proteins. On the other hand, Max is the only member of the family capable of forming a stable homodimer. As part of the transcriptional regulation mechanism, Myc/Max and Mad/Max heterodimers and Max homodimers are thought to compete for binding to the E-box target sequences. E-box recognition is structurally supported by the b-HLH-LZ structural motif, which also promotes dimerization. However, the actual dimerization and heterodimerization constants of the complete gene products and their affinities for E-box sequences are not known. Also, the detailed thermodynamic characterization of DNA binding by these transcription factors has not been done yet. Such knowledge is necessary for complete understanding of the transcriptional regulation carried out by the Myc/Mad/Max network. Here, we report the first in-depth thermodynamic characterization of the stability and specific DNA binding of a full length gene product of the Myc/Mad/Max family, namely, Max protein isoform p21 (Max p21). Using calorimetric methods (DSC and ITC) we have determined the dimerization constant of Max p21 in the low micromolar range, and the Max p21/E-box complex dissociation constant in the low nanomolar range at 37 degrees C. The association is driven by a large exothermic effect, which is partly compensated by entropic factors. The energetic contribution to binding affinity of seven highly conserved residues that contact the DNA was probed by X-to-Ala mutagenesis. The results demonstrate that high binding affinity critically relies on the side chain of Arg 26. Furthermore, the mutational analysis points to the important role of the persistent helical turn that comprises this residue at the junction of the basic region and helix H1. Altogether, the study supports the idea that Max p21 can bind E-box sequences in vivo and likely participates directly in the regulation of transcription as homodimer.

Proteomic Discovery of Max as a Novel Interacting Partner of C/EBPa: A Myc/Max/Mad Link.

In the present study, we sought to identify novel C/EBPa interacting proteins in vivo through immunoprecipitation using mass spectrometry-based proteomic techniques. We identified Max, a heterodimeric partner of Myc, as one of the interacting proteins of C/EBPa in our screen. We confirmed the in vivo interaction of C/EBPa with Max and showed that this interaction involves the basic region of C/EBPa. Endogenous C/EBPa and Max, but not Myc and Max co-localize in intranuclear structures during granulocytic differentiation of myeloid U937 cells. Max enhanced the transactivation capacity of C/EBPa on a minimal promoter. A chromatin-immunoprecipitation assay revealed occupancy of the human C/EBPa promoter in vivo by Max under cellular settings and by C/EBPa and Max under retinoic acid induced granulocytic differentiation. Interestingly, enforced expression of Max and C/EBPa, results in granulocytic differentiation of the human hematopoietic CD34+ cells as evidenced by CD11b and CD15 expression and by real-time PCR for various genes. Silencing of Max by short hairpin RNA in CD34+ and U937 cells strongly reduced the differentiation-inducing potential of C/EBPa, indicating the importance of C/EBPa-Max in myeloid progenitor differentiation. Taken together, our data reveal Max as a novel co-activator of C/EBPa functions thereby suggesting a possible link between CEBPa and Myc-Max-Mad network.

Proteomic discovery of Max as a novel interacting partner of C/EBPα: a Myc/Max/Mad link

The transcription factor CCAAT/enhancer binding protein a (C/EBPalpha) is important in the regulation of granulopoiesis and is disrupted in human acute myeloid leukemia. In the present study, we sought to identify novel C/EBPalpha interacting proteins in vivo through immunoprecipitation using mass spectrometry-based proteomic techniques. We identified Max, a heterodimeric partner of Myc, as one of the interacting proteins of C/EBPalpha in our screen. We confirmed the in vivo interaction of C/EBPalpha with Max and showed that this interaction involves the basic region of C/EBPalpha. Endogenous C/EBPalpha and Max, but not Myc and Max, colocalize in intranuclear structures during granulocytic differentiation of myeloid U937 cells. Max enhanced the transactivation capacity of C/EBPalpha on a minimal promoter. A chromatin immunoprecipitation assay revealed occupancy of the human C/EBPalpha promoter in vivo by Max and Myc under cellular settings and by C/EBPalpha and Max under retinoic acid induced granulocytic differentiation. Interestingly, enforced expression of Max and C/EBPalpha results in granulocytic differentiation of the human hematopoietic CD34(+) cells, as evidenced by CD11b, CD15 and granulocyte colony-stimulating factor receptor expression. Silencing of Max by short hairpin RNA in CD34(+) and U937 cells strongly reduced the differentiation-inducing potential of C/EBPalpha, indicating the importance of C/EBPalpha-Max in myeloid progenitor differentiation. Taken together, our data reveal Max as a novel co-activator of C/EBPalpha functions, thereby suggesting a possible link between C/EBPalpha and Myc-Max-Mad network.

Genes differentially expressed in breast cancer (BC) samples as compared to normal breast tissue from women without cancer

9593 Background: We have determined the gene profile correlated to doxorubicin resistance in 38 BC patients (pt) treated with primary chemotherapy and 37 genes were differentially expressed in tumors according to response. Among the genes more expressed in sensitive tumors as compared to resistant tumors were ephrin receptor and c-src; JunD; and CDKN2B. Genes more expressed in resistant tumors were fibroblast growth factor receptor 2 and lymphocyte protein TK; Max (Myc partner); and MMP12 (metalloproteinase). We have now analyzed gene expression of these 38 tumors as compared to normal breast tissue (perilesional) obtained from 5 women without cancer submitted to resection of benign lesions. Methods: A tissue specific cDNA microarray glass slide was assembled based on genes expressed in mammary tissue and BC. Genes hyperexpressed in BC as determined in SAGE libraries were also selected. Sequences representing 496 genes and 195 ESTs were chosen in an ORESTES (open reading frame expressed sequence tags) bank (Instituto Ludwig de Pesquisa sobre o Câncer/FAPESP) or synthesized in PCR reactions and spotted 3 or 6 times on glass slides. Tissue was hand dissected, RNA was extracted, amplified, and co-hybridized with a HB4A (normal mammary epithelial cell line) probe against cDNA microarray slides. Results: Median age of BC pt was 51 y; pt were clinically staged as IIA (3), IIB (2), IIIA (17), IIIB (15) and IV (1 pt). Sixty three molecules were differentially expressed between tumors and normal breast tissue. Genes more expressed in tumors included KRT8 and KRT19 (cytoskeleton proteins); MMP11 and MMP12; transmembrane 4 superfamily member tetraspan NET-7; NME2 (tumor suppressor gene); REL (oncogene); MSH2 (DNA repair); XIAP (inhibitor of apoptosis) associated factor 1. Genes more expressed in normal tissue were CCND3 (cyclin D3); KRT14 and KRT15; LAMA3 and LAMB3 (laminin); maspin (proteinase inhibitor); GSTP1 (detoxifying enzyme); ITGB4 (adhesion molecule); JUN, FOS, MYC (transcription factors) and MAX; and ABCG2 (ATP binding cassette). Conclusions: This gene profile seems to characterize breast malignancy. Supported by FAPESP 01/00146–8 No significant financial relationships to disclose.

C-myc is required for osteoclast differentiation.

The role of the receptor activator of nuclear factor kappaB (NF-kappaB) ligand (RANKL)-a tumor necrosis factor (TNF)-related cytokine-in osteoclast formation has been established clearly. However, the downstream signaling pathways activated by this cytokine remain largely unknown. To identify genes that play a role in osteoclastogenesis, we used RAW 264.7 mouse monocytes as a model system for the differentiation of multinucleated osteoclasts from mononucleated precursors. RAW 264.7 cells were induced with RANKL to form multinucleated giant osteoclast-like cells (OCLs) that expressed a number of osteoclast-specific markers and were able to form resorption pits on both calcium phosphate films and bone slices. This system was used to identify genes that are regulated by RANKL and may play a role in osteoclast differentiation. The proto-oncogene c-myc was strongly up-regulated in RANKL-induced OCLs but was absent in undifferentiated cells. Expression of Myc partners Max and Mad, on the other hand, was constant during OCL differentiation. We expressed a dominant negative Myc in RAW 264.7 cells and were able to block RANKL-induced OCL formation. Northern Blot analysis revealed a delay and a significant reduction in the level of messenger RNA (mRNA) for tartrate-resistant acid phosphatase (TRAP) and cathepsin K. We conclude that c-myc is a downstream target of RANKL and its expression is required for RANKL-induced osteoclastogenesis.

The basic region/helix-loop-helix/leucine repeat transcription factor USF interferes with Ras transformation.

Upstream stimulatory factor (USF) is a transcription factor of the basic region/helix-loop-helix/leucine repeat family. It shares the same DNA-binding sequence as the myc oncogene. Based on the three-dimensional structures, its DNA-binding domain is structurally related to that of Max, the partner of Myc. In addition, USF can form heterodimers with a related factor, Fos-interacting protein/upstream stimulatory factor 2 (FIP/USF2), which has been shown to directly interact with Fos. In view of the provocative relationship of USF with other factors involved in cell proliferation, we investigated whether USF could also play a role in cellular growth control. In this study, we report that USF is not an oncogene, but interferes with Ras-driven transformation. This inhibitory effect is independent of USF transactivating domains, but requires its DNA-binding activity. However, the minimal USF DNA-binding domain does not display this inhibitory effect, and even slightly enhances Ras transformation. On the basis of these data, we propose that USF may play an important role in the control of cell growth and proliferation, through both binding to promoter sequences and specific protein/protein interactions.

Basal levels of max are sufficient for the cotransformation of C3H10T1/2 cells by ras and myc

The ras and myc oncoproteins cooperate to transform the established murine fibroblast cell line C3H10T1/2. To determine the impact of overexpression of the myc oncoprotein on the phenotype of C3H10T1/2 cells, two C3H10Tl/2- myc clonal cell lines, SVc- myc 11A and myc neo 13A, were isolated and characterized. Although both C3H10Tl/2- myc cell lines are morphologically indistinguishable from wild-type C3H10T1/2 cells and possess growth properties similar to those of C3H10T1/2 cells, each displays a predisposition to transformation following transfection with the activated form of the human H- ras gene. In C3H10T1/2 cells overexpressing the v- myc or H- ras oncogenes, the levels of mRNA encoding max, the recently identified oligomerization partner of myc, remain unchanged, suggesting that the endogenous level of max in C3H10T1/2 cells is sufficient for a high frequency of transformation by ras and myc. Based on these studies, the C3H10Tl/2- myc clonal cell lines we describe are suitable model systems for examining the molecular role of the myc protein in transformation and for characterizing additional factors that synergize with myc in multistep transformation.

Activation domains of L-Myc and c-Myc determine their transforming potencies in rat embryo cells.

Members of the Myc family of proteins share a number of protein motifs that are found in regulators of gene transcription. Conserved stretches of amino acids found in the N-terminal transcriptional activation domain of c-Myc are required for cotransforming activity. Most of the Myc proteins contain the basic helix-loop-helix zipper (bHLH-Zip) DNA-binding motif which is also required for the cotransforming activity of c-Myc. L-Myc, the product of a myc family gene that is highly amplified in many human lung carcinomas, was found to cotransform primary rat embryo cells with an activated ras gene. However, L-Myc cotransforming activity was only 1 to 10% of that of c-Myc (M. J. Birrer, S. Segal, J. S. DeGreve, F. Kaye, E. A. Sausville, and J. D. Minna, Mol. Cell. Biol. 8:2668-2673, 1988). We sought to determine whether functional differences between c-Myc and L-Myc in either the N-terminal or the C-terminal domain could account for the relatively diminished L-Myc cotransforming activity. Although the N-terminal domain of L-Myc could activate transcription when fused to the yeast GAL4 DNA-binding domain, the activity was only 5% of that of a comparable c-Myc domain. We next determined that the interaction of the C-terminal bHLH-Zip region of L-Myc or c-Myc with that of a Myc partner protein, Max, was equivalent in transfected cells. A Max expression vector was found to augment the cotransforming activity of L-Myc as well as that of c-Myc. In addition, a bacterially synthesized DNA-binding domain of L-Myc, like that o c-Myc, heterodimerizes with purified Max protein to bind the core DNA sequence CACGTG. To determine the region of L-Myc responsible for its relatively diminished cotransforming activity, we constructed chimeras containing exons 2 (constituting activation domains) and 3 (constituting DNA-binding domains) of c-Myc fused to those of L-Myc. The cotransforming potencies of these chimeras were compared with those of full-length L-Myc of c-Myc in rat embryo cells. The relative cotransforming activities suggest that the potencies of the activation domains determine the cotransforming efficiencies for c-Myc and L-Myc. This correlation supports the hypothesis that the Myc proteins function in neoplastic cotransformation as transcription factors.