A characteristic feature of Burkitt's lymphoma cells is the presence of reciprocal translocations between the c-myc locus on chromosome 8 and one of the immunoglobulin gene loci on chromosome 2, 14, or 22. The most common translocation is the t(8;14). In this translocation, the c-myc gene is covalently linked to the immunoglobulin heavy chain (IgH) 1 gene. As a result of this translocation, the transcription of the translocated c-myc gene is deregulated, whereas the normal c-myc allele is silent. Furthermore, the transcripts initiated from the c-myc P1 promoter, which normally contribute to a minor (10 -20%) population of c-myc mRNA, increase to a level greater than transcripts initiated from the P2 promoter (1-3). These findings support a model in which sequences present in the IgH gene locus deregulate expression from the cis-linked c-myc allele by promoting interactions between c-myc and IgH gene regulatory elements that affect c-myc initiation and elongation. It should be noted, however, that the translocation breakpoint in many sporadic Burkitt's lymphomas separates the c-myc promoter from the coding region (4, 5). In these cases, the regulatory elements of the IgH enhancers apparently activate c-myc transcription without interaction with the c-myc promoter elements. Transcription often initiates in the first intron of c-myc in these sporadic Burkitt's lymphomas.We found that the transcription factors, Nm23H2 and NF-B, activated the c-myc promoter (6, 7). Others have also shown that NF-B is an important regulatory factor for the murine c-myc promoter (8, 9). Because the IgH 3Ј enhancers are linked to the c-myc gene in every Burkitt's lymphoma with the t(8;14) translocation, we sought to identify the transcription factors that bind to sequences in the enhancer region and activate the translocated c-myc gene.Several enhancers have been shown to be important for expression of the IgH gene. Four B cell-specific and cell stagedependent DNase I-hypersensitive sites, MHS1 to MHS4, are located 10 -35 kilobases 3Ј of the C␣ gene (10 -13). The activity of individual enhancer elements varies during B cell differentiation (10,14,15), and these regions have been shown to function as a locus control region in B cells (10). Recently, it has been shown that MHS1-MHS4 increase expression from the c-myc P2 promoter by an increase in histone acetylation. However, this increase in acetylation does not explain the MHS1-4 activation of transcription from the P1 promoter (16). Enhancers have been located downstream of two human C␣ genes (17-19), and these regions share some homology with the murine HS1-4, but only limited functional studies have been performed on the human enhancers.The 3Ј region of the IgH locus is linked to the translocated c-myc gene in all t(8;14) translocations in Burkitt's lymphoma, and it is likely that this region plays a role in the deregulated expression of the translocated c-myc gene. In this study, we show that an NF-B site in the MHS4 enhancer is required for the transcriptional activation of the tra...

SUMMARY Chemotherapy is used to treat most cancer patients, yet our understanding of factors that dictate response and resistance to such drugs remains limited. We report the generation of a quantitative chemical-genetic interaction map in human mammary epithelial cells charting the impact of the knockdown of 625 genes related to cancer and DNA repair on sensitivity to 29 drugs, covering all classes of chemotherapy. This quantitative map is predictive of interactions maintained in other cell lines, identifies DNA-repair factors, predicts cancer cell line responses to therapy, and prioritizes synergistic drug combinations. We identify that ARID1A loss confers resistance to PARP inhibitors in cells and ovarian cancer patients and that loss of GPBP1 causes resistance to cisplatin and PARP inhibitors through the regulation of genes involved in homologous recombination. This map helps navigate patient genomic data and optimize chemotherapeutic regimens by delineating factors involved in the response to specific types of DNA damage.

Ewing's family tumors are characterized by a specific t(11;22) chromosomal translocation that results in the formation of EWS-Fli1 oncogenic fusion protein. To investigate the effects of EWSFli1 on gene expression, we carried out DNA microarray analysis after specific knockdown of EWS-Fli1 through transfection of synthetic siRNAs. EWS-Fli1 knockdown increased expression of genes such as DKK1 and p57 that are known to be repressed by EWS-Fli1 fusion protein.Among other potential EWS-Fli1 targets identified by our microarray analysis, we have focused on the FOXO1 gene since it encodes a potential tumor suppressor and has not been previously reported in Ewing's cells. To better understand how EWS-Fli1 affects FOXO1 expression, we have established a doxycycline-inducible siRNA system to achieve stable and reversible knockdown of EWS-Fli1 in Ewing's sarcoma cells. Here we show that FOXO1 expression in Ewing's cells has an inverse relationship with EWS-Fli1 protein level, and FOXO1 promoter activity is increased after doxycycline-induced EWS-Fli1 knockdown. In addition, we have found that direct binding of EWS-Fli1 to FOXO1 promoter is attenuated after doxycycline-induced siRNA knockdown of the fusion protein. Together, these results suggest that suppression of FOXO1 function by EWS-Fli1 fusion protein may contribute to cellular transformation in Ewing's family tumors.

The oncogenic TLS-ERG fusion protein is found in human myeloid leukemia and Ewing's sarcoma as a result of specific chromosomal translocation. To unveil the potential mechanism(s) underlying cellular transformation, we have investigated the effects of TLS-ERG on both gene transcription and RNA splicing. Here we show that the TLS protein forms complexes with RNA polymerase II (Pol II) and the serine-arginine family of splicing factors in vivo. Deletion analysis of TLS-ERG in both mouse L-G myeloid progenitor cells and NIH 3T3 fibroblasts revealed that the RNA Pol II-interacting domain of TLS-ERG resides within the first 173 amino acids. While TLS-ERG repressed expression of the luciferase reporter gene driven by glycoprotein IX promoter in L-G cells but not in NIH 3T3 cells, the fusion protein was able to affect splicing of the E1A reporter in NIH 3T3 cells but not in L-G cells. To identify potential target genes of TLS-ERG, the fusion protein and its mutants were stably expressed in both L-G and NIH 3T3 cells through retroviral transduction. Microarray analysis of RNA samples from these cells showed that TLS-ERG activates two different sets of genes sharing little similarity in the two cell lines. Taken together, these results suggest that the oncogenic TLS-ERG fusion protein transforms hematopoietic cells and fibroblasts via different pathways.In acute myelogenous leukemia, chronic myelogenous leukemia in BLAST crisis, and certain myelodysplastic syndromes, the TLS (translocation liposarcoma) gene is fused to the ERG (ets-related gene) through a recurrent t(16;21) chromosomal translocation (18). Interestingly, the same t(16;21) rearrangement and the resultant TLS-ERG chimeric fusion protein were also reported in Ewing's sarcoma (36). The TLS-ERG fusion protein retains the N-terminal domain of TLS, but the Cterminal domain of TLS is replaced by the DNA-binding domain of ERG. Previous studies have demonstrated that TLS-ERG fusion protein is capable of transforming mouse cell lines (19) as well as normal human hematopoietic cells (28).The TLS gene was originally cloned as a fusion partner with the CHOP gene in human myxoid liposarcoma (9, 33). TLS belongs to a family of closely related proteins that include the Ewing's sarcoma protein EWS (11) and the TATA-binding protein-associated factor TAF II 68 (3). EWS is known to interact with the transcription coactivator CBP/p300 (35). TLS has been reported to be a target of the BCR/ABL oncoprotein and binds to DNA in a phosphorylation-dependent manner (29,30). In addition, transient-expression experiments revealed that TLS binds to RNA polymerase II (Pol II) through the N-terminal domain of TLS and interacts with splicing factors through the C-terminal domain of TLS (8,42,43).TLS-ERG was originally speculated to act as a chimeric transcription factor leading to transformation through deregulation of gene transcription (31), but accumulating evidence suggests that TLS-ERG and the related EWS-FLI-1 fusion proteins may lead to cellular abnormalities by deregulating both gen...

Synthetic lethal screens have the potential to identify new vulnerabilities incurred by specific cancer mutations but have been hindered by lack of agreement between studies. In the case of KRAS, we identify that published synthetic lethal screen hits significantly overlap at the pathway rather than gene level. Analysis of pathways encoded as protein networks could identify synthetic lethal candidates that are more reproducible than those previously reported. Lack of overlap likely stems from biological rather than technical limitations as most synthetic lethal phenotypes are strongly modulated by changes in cellular conditions or genetic context, the latter determined using a pairwise genetic interaction map that identifies numerous interactions that suppress synthetic lethal effects. Accounting for pathway, cellular and genetic context nominates a DNA repair dependency in KRAS-mutant cells, mediated by a network containing BRCA1. We provide evidence for why most reported synthetic lethals are not reproducible which is addressable using a multi-faceted testing framework.

The deregulation of expression of the c-myc gene in Burkitt's lymphoma results from the translocation that links one c-myc allele to one of the immunoglobulin genes. This physical linkage promotes interactions between c-myc and immunoglobulin gene regulatory elements that affect c-myc transcription initiation and elongation. We have located a region in the c-myc promoter that is required for the complete activation by the immunoglobulin heavy chain gene enhancer. This regulatory element contains a core sequence, GGGAGG, similar to the GA box recognized by the transcription factor Myc-associated zinc finger protein (MAZ). UV cross-link analysis indicated that the mass of this protein did not correspond to that of MAZ, suggesting that a protein related to but distinct from MAZ bound to this site. Mutation of this regulatory element resulted in a loss of promoter activity induced by the immunoglobulin heavy chain gene enhancer. This site was also required for the c-myc promoter shift from P2 to P1. In vivo footprinting revealed that this site was occupied on the translocated c-myc allele but not on the untranslocated allele. Taken together, these findings suggest that this regulatory element is required for the full activation of c-myc promoter activity by the immunoglobulin heavy chain gene enhancer.Burkitt's lymphoma is characterized by specific chromosomal translocations that juxtapose the proto-oncogene c-myc on chromosome 8 to one of the Ig loci on chromosome 2, 14, or 22. The most common form of the translocation is t(8;14) where the c-myc gene is covalently linked to the immunoglobulin heavy chain gene (IgH)1 . The translocated c-myc gene is highly expressed, whereas the normal allele is silent. Furthermore, the transcripts initiated from the c-myc P1 promoter, which normally contribute to a minor (10 -20%) population of c-myc mRNA, increase to a level greater than the transcripts initiated from the P2 promoter, a phenomenon known as promoter shift (1-3). It is assumed that the physical linkage of the c-myc gene to one of the immunoglobulin loci promotes the interactions between c-myc and Ig regulatory elements that affect c-myc transcriptional initiation and elongation. In support of this view, it has been demonstrated that linkage of the murine IgH 3Ј enhancer region, MHS1234, to a 2.3-kb region of the human c-myc promoter in an episomal vector is sufficient to reproduce the activation of c-myc transcription and the promoter shift in stably transfected Raji cells (4). Similar results have been obtained with the Ig (5) and Ig (6) light chain enhancers. Despite these findings, most of the cis-acting enhancer and promoter elements that contribute to the deregulation of expression of the c-myc gene remain unidentified.We have shown previously that an NF-B site in the MHS4 region of the IgH enhancer is required for the transcriptional activation of the translocated c-myc gene and is involved in inducing the c-myc promoter shift from P2 to P1 (7). Others have found that NF-B and PU.1 sites are critical for the d...

C/EBPα, -β, and -δ are members of the CCAAT/enhancer binding protein family of transcriptional regulators. All three of these factors are expressed by bone marrow-derived macrophages, with the DNA binding activity of C/EBPβ and -δ increased by treatment with LPS while that of C/EBPα is decreased. We have ectopically expressed each C/EBP protein in P388 lymphoblasts. The expression of any of these transcription factors is sufficient to confer the LPS-inducible expression of IL-6 and monocyte chemoattractant protein-1 to lymphoblasts, which normally lack C/EBP factors and do not display LPS induction of proinflammatory cytokines. Thus, the activities of C/EBPα, -β, and -δ are redundant in regard to the expression of IL-6 and monocyte chemoattractant protein-1. Since C/EBPβ-deficient mice have been reported to be largely normal in their expression of proinflammatory cytokines, it is likely that the lack of C/EBPβ is compensated for by the induction of C/EBPδ upon LPS treatment.

SUMMARYSignaling networks balance the activities of many physically interacting proteins and perturbations to this network influence downstream signaling, potentially leading to oncogenic states. Using affinity purification-mass spectrometry we defined this network for all 90 human tyrosine kinases revealing 1,463 mostly novel interactions between these key cancer proteins and diverse molecular complexes. Modulation of interactor levels altered growth phenotypes associated with corresponding tyrosine kinase partners suggesting that tumors may alter the stoichiometries of interactors to maximize oncogenic signaling. We show that the levels of EGFR interactors delineates this form of network oncogenesis in 19% of EGFR wild-type lung cancer patients which were mostly otherwise oncogene negative, predicting sensitivity to EGFR inhibitors in vitro and in vivo. EGFR network oncogenesis occurs through mechanistically distinct network alleles often in cooperation with weak oncogenes in the MAPK pathway. Network oncogenesis may be a common and targetable convergent mechanism of oncogenic pathway activation in cancer.HIGHLIGHTSA human tyrosine kinome protein interaction map reveals novel physical and functional associations.Dependence on oncogenic tyrosine kinases is modulated through perturbation of their interactors.EGFR network oncogenesis in up to 19% of EGFR wild-type lung cancers is targetable.EGFR network oncogenesis cooperates with weak oncogenes in the MAPK pathway.