Transcriptional Heterogeneity Overcomes Super-Enhancer Disrupting Drug Combinations in Multiple Myeloma.

Multiple myeloma (MM) is a malignancy that is often driven by MYC and that is sustained by IRF4, which are upregulated by super-enhancers. IKZF1 and IKZF3 bind to super-enhancers and can be degraded using immunomodulatory imide drugs (IMiD). Successful IMiD responses downregulate MYC and IRF4; however, this fails in IMiD-resistant cells. MYC and IRF4 downregulation can also be achieved in IMiD-resistant tumors using inhibitors of BET and EP300 transcriptional coactivator proteins; however, in vivo these drugs have a narrow therapeutic window. By combining IMiDs with EP300 inhibition, we demonstrate greater downregulation of MYC and IRF4, synergistic killing of myeloma in vitro and in vivo, and an increased therapeutic window. Interestingly, this potent combination failed where MYC and IRF4 expression was maintained by high levels of the AP-1 factor BATF. Our results identify an effective drug combination and a previously unrecognized mechanism of IMiD resistance. SIGNIFICANCE: These results highlight the dependence of MM on IKZF1-bound super-enhancers, which can be effectively targeted by a potent therapeutic combination pairing IMiD-mediated degradation of IKZF1 and IKZF3 with EP300 inhibition. They also identify AP-1 factors as an unrecognized mechanism of IMiD resistance in MM. See related article by Neri, Barwick, et al., p. 56. See related commentary by Yun and Cleveland, p. 5. This article is featured in Selected Articles from This Issue, p. 4.

Rapid and reversible suppression of ALT by DAXX in osteosarcoma cells.

Many tumors maintain chromosome-ends through a telomerase-independent, DNA-templated mechanism called alternative lengthening of telomeres (ALT). While ALT occurs in only a subset of tumors, it is strongly associated with mutations in the genes ATRX and DAXX, which encode components of an H3.3 histone chaperone complex. The role of ATRX and DAXX mutations in potentiating the mechanism of ALT remains incompletely understood. Here we characterize an osteosarcoma cell line, G292, with wild-type ATRX but a unique chromosome translocation resulting in loss of DAXX function. While ATRX and DAXX form a complex in G292, this complex fails to localize to nuclear PML bodies. We demonstrate that introduction of wild type DAXX suppresses the ALT phenotype and restores the localization of ATRX/DAXX to PML bodies. Using an inducible system, we show that ALT-associated PML bodies are disrupted rapidly following DAXX induction and that ALT is again restored following withdrawal of DAXX.

Novel near-diploid ovarian cancer cell line derived from a highly aneuploid metastatic ovarian tumor.

A new ovarian near-diploid cell line, OVDM1, was derived from a highly aneuploid serous ovarian metastatic adenocarcinoma. A metastatic tumor was obtained from a 47-year-old Ashkenazi Jewish patient three years after the first surgery removed the primary tumor, both ovaries, and the remaining reproductive organs. OVDM1 was characterized by cell morphology, genotyping, tumorigenic assay, mycoplasma testing, spectral karyotyping (SKY), and molecular profiling of the whole genome by aCGH and gene expression microarray. Targeted sequencing of a panel of cancer-related genes was also performed. Hierarchical clustering of gene expression data clearly confirmed the ovarian origin of the cell line. OVDM1 has a near-diploid karyotype with a low-level aneuploidy, but samples of the original metastatic tumor were grossly aneuploid. A number of single nucleotide variations (SNVs)/mutations were detected in OVDM1 and the metastatic tumor samples. Some of them were cancer-related according to COSMIC and HGMD databases (no founder mutations in BRCA1 and BRCA2 have been found). A large number of focal copy number alterations (FCNAs) were detected, including homozygous deletions (HDs) targeting WWOX and GATA4. Progression of OVDM1 from early to late passages was accompanied by preservation of the near-diploid status, acquisition of only few additional large chromosomal rearrangements and more than 100 new small FCNAs. Most of newly acquired FCNAs seem to be related to localized but massive DNA fragmentation (chromothripsis-like rearrangements). Newly developed near-diploid OVDM1 cell line offers an opportunity to evaluate tumorigenesis pathways/events in a minor clone of metastatic ovarian adenocarcinoma as well as mechanisms of chromothripsis.

Generation of Gross Chromosomal Rearrangements by a Single Engineered DNA Double Strand Break.

Gross chromosomal rearrangements (GCRs), including translocations, inversions amplifications, and deletions, can be causal events leading to malignant transformation. GCRs are thought to be triggered by DNA double strand breaks (DSBs), which in turn can be spontaneous or induced by external agents (eg. cytotoxic chemotherapy, ionizing radiation). It has been shown that induction of DNA DSBs at two defined loci can produce stable balanced chromosomal translocations, however, a single engineered DNA DSB could not. Herein, we report that although a single engineered DNA DSB in H2AX "knockdown" cells did not generate GCRs, repair of a single engineered DNA DSB in fibroblasts that had ablated H2ax did produce clonal, stable GCRs, including balanced translocations and megabase-pair inversions. Upon correction of the H2ax deficiency, cells no longer generated GCRs following a single engineered DNA DSB. These findings demonstrate that clonal, stable GCRs can be produced by a single engineered DNA DSB in H2ax knockout cells, and that the production of these GCRs is ameliorated by H2ax expression.

Frequent occurrence of large duplications at reciprocal genomic rearrangement breakpoints in multiple myeloma and other tumors.

Using a combination of array comparative genomic hybridization, mate pair and cloned sequences, and FISH analyses, we have identified in multiple myeloma cell lines and tumors a novel and recurrent type of genomic rearrangement, i.e. interchromosomal rearrangements (translocations or insertions) and intrachromosomal inversions that contain long (1-4000 kb; median ∼100 kb) identical sequences adjacent to both reciprocal breakpoint junctions. These duplicated sequences were generated from sequences immediately adjacent to the breakpoint from at least one-but sometimes both-chromosomal donor site(s). Tandem duplications had a similar size distribution suggesting the possibility of a shared mechanism for generating duplicated sequences at breakpoints. Although about 25% of apparent secondary rearrangements contained these duplications, primary IGH translocations rarely, if ever, had large duplications at breakpoint junctions. Significantly, these duplications often contain super-enhancers and/or oncogenes (e.g. MYC) that are dysregulated by rearrangements during tumor progression. We also found that long identical sequences often were identified at both reciprocal breakpoint junctions in six of eight other tumor types. Finally, we have been unable to find reports of similar kinds of rearrangements in wild-type or mutant prokaryotes or lower eukaryotes such as yeast.

Complex IGH rearrangements in multiple myeloma: Frequent detection discrepancies among three different probe sets.

Primary IGH translocations involving seven recurrent partner loci and oncogenes are present in about 40% of multiple myeloma tumors. Secondary IGH rearrangements, which occur in a smaller fraction of tumors, usually are complex structures, including insertions or translocations that can involve three chromosomes, and often with involvement of MYC. The main approach to detect IGH rearrangements is interphase-but sometimes metaphase-FISH strategies that use a telomeric variable region probe and a centromeric constant region/ Eα enhancer or 3' flanking probe to detect a separation of these two probes, or a fusion of these probes with probes located at nonrandom partner sites in the genome. We analyzed 18 myeloma cell lines for detection discrepancies among Vysis, Cytocell, and in-house IGH probe sets that hybridize with differing sequences in the IGH locus. There were no detection discrepancies for the three telomeric IGH probes, or for unrearranged IGH loci or primary IGH translocations using the centromeric IGH probes. However, the majority of complex IGH rearrangements had detection discrepancies among the three centromeric IGH probes.

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

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.

Multi-layered cancer chromosomal instability phenotype.

Whole-chromosomal instability (W-CIN) - unequal chromosome distribution during cell division - is a characteristic feature of a majority of cancer cells distinguishing them from their normal counterparts. The precise molecular mechanisms that may cause mis-segregation of chromosomes in tumor cells just recently became more evident. The consequences of W-CIN are numerous and play a critical role in carcinogenesis. W-CIN mediates evolution of cancer cell population under selective pressure and can facilitate the accumulation of genetic changes that promote malignancy. It has both tumor-promoting and tumor-suppressive effects, and their balance could be beneficial or detrimental for carcinogenesis. The characterization of W-CIN as a complex multi-layered adaptive phenotype highlights the intra- and extracellular adaptations to the consequences of genome reshuffling. It also provides a framework for targeting aggressive chromosomally unstable cancers.

Integrated high-resolution array CGH and SKY analysis of homozygous deletions and other genomic alterations present in malignant mesothelioma cell lines.

High-resolution oligonucleotide array comparative genomic hybridization (aCGH) and spectral karyotyping (SKY) were applied to a panel of malignant mesothelioma (MMt) cell lines. SKY has not been applied to MMt before, and complete karyotypes are reported based on the integration of SKY and aCGH results. A whole genome search for homozygous deletions (HDs) produced the largest set of recurrent and non-recurrent HDs for MMt (52 recurrent HDs in 10 genomic regions; 36 non-recurrent HDs). For the first time, LINGO2, RBFOX1/A2BP1, RPL29, DUSP7, and CCSER1/FAM190A were found to be homozygously deleted in MMt, and some of these genes could be new tumor suppressor genes for MMt. Integration of SKY and aCGH data allowed reconstruction of chromosomal rearrangements that led to the formation of HDs. Our data imply that only with acquisition of structural and/or numerical karyotypic instability can MMt cells attain a complete loss of tumor suppressor genes located in 9p21.3, which is the most frequently homozygously deleted region. Tetraploidization is a late event in the karyotypic progression of MMt cells, after HDs in the 9p21.3 region have already been acquired.

Targeting karyotypic complexity and chromosomal instability of cancer cells.

Multiple karyotypic abnormalities and chromosomal instability are characteristic features of many cancers that are relatively resistant to chemotherapeutic agents currently used in the clinic. These same features represent potentially targetable "states" that are essentially tumor specific. The assessment of the chromosomal state of a cancer cell population may provide a guide for the selection or development of drugs active against aggressive and intractable cancers.

Efficient repair of DNA double-strand breaks in malignant cells with structural instability.

Aberrant repair of DNA double-strand breaks (DSBs) is thought to be important in the generation of gross chromosomal rearrangements (GCRs). To examine how DNA DSBs might lead to GCRs, we investigated the repair of a single DNA DSB in a structurally unstable cell line. An I-SceI recognition site was introduced into OVCAR-8 cells between a constitutive promoter (EF1alpha) and the Herpes simplex virus thymidine kinase (TK) gene, which confers sensitivity to gancyclovir (GCV). Expression of I-SceI in these cells caused a single DSB. Clones with aberrant repair could acquire resistance to GCV by separation of the EF1alpha promoter from the TK gene, or deletion of either the EF1alpha promoter or the TK gene. All mutations that we identified were interstitial deletions. Treatment of cells with etoposide or bleomycin, agents known to produce DNA DSBs following expression of I-SceI also did not generate GCRs. Because we identified solely interstitial deletions using the aforementioned negative selection system, we developed a positive selection system to produce GCR. A construct containing an I-SceI restriction site immediately followed by a hygromycin phosphotransferase cDNA, with no promoter, was stably integrated into OVCAR-8 cells. DNA DSBs were produced by an I-SceI expression vector. None of the hygromycin resistant clones recovered had linked the hygromycin phosphotransferase cDNA to an endogenous promoter, but had instead captured a portion of the I-SceI expression vector. These results indicate that even in a structurally unstable malignant cell line, the majority of DNA DSBs are repaired by religation of the two broken chromosome ends, without the introduction of a GCR.

Chromosomal instability is associated with higher expression of genes implicated in epithelial-mesenchymal transition, cancer invasiveness, and metastasis and with lower expression of genes involved in cell cycle checkpoints, DNA repair, and chromatin maintenance.

Chromosomal instability-a hallmark of epithelial cancers-is an ongoing process that results in aneuploidy and karyotypic heterogeneity of a cancer cell population. Previously, we stratified cancer cell lines in the NCI-60 drug discovery panel based on their karyotypic complexity and heterogeneity. Using this stratification in conjunction with drug response data for the cell lines allowed us to identify classes of chemical compounds whose growth-inhibitory activity correlates with karyotypic complexity and chromosomal instability. In this article, we asked the question: What are the biological processes, pathways, or genes associated with chromosomal instability of cancer cells? We found that increased instability of the chromosomal content in a cancer cell population, particularly, persistent gains and losses of chromosomes, is associated with elevated expression of genes involved with aggressive cellular behavior, including invasion- and metastasis-associated changes in cell communication, adhesion, motility, and migration. These same karyotypic features are negatively correlated with the expression of genes involved in cell cycle checkpoints, DNA repair, and chromatin maintenance.

Secondary genomic rearrangements involving immunoglobulin or MYC loci show similar prevalences in hyperdiploid and nonhyperdiploid myeloma tumors.

The pathogenesis of multiple myeloma (MM) is thought to involve at least two pathways, which generate hyperdiploid (HRD) or nonhyperdiploid (NHRD) tumors, respectively. Apart from chromosome content, the two pathways are distinguished by five primary immunoglobulin heavy chain (IGH) rearrangements (4p16, FGFR3, and MMSET; 6p21, CCND3; 11q13, CCND1; 16q23, MAF; 20q12, MAFB) that are present mainly in NHRD tumors. To determine the prevalence and structures of IGH, immunoglobulin (IG) light chain, and MYC genomic rearrangements in MM, we have done comprehensive metaphase fluorescent in situ hybridization analyses on 48 advanced MM tumors and 47 MM cell lines. As expected, the prevalence of the five primary IGH rearrangements was nearly 70% in NHRD tumors, but only 12% in HRD tumors. However, IGH rearrangements not involving one of the five primary partners, and IG light chain rearrangements, have a similar prevalence in HRD and NHRD tumors. In addition, MYC rearrangements, which are thought to be late progression events that sometimes do not involve an IG heavy or light chain locus, also have a similar prevalence in HRD and NHRD tumors. In contrast to the primary IGH rearrangements, which usually are simple balanced translocations, these other IG rearrangements usually have complex structures, as previously described for MYC rearrangements in MM. We conclude that IG light chain and MYC rearrangements, as well as secondary IGH rearrangements, make similar contributions to the progression of both HRD and NHRD MM tumors.

Aberrant immunoglobulin class switch recombination and switch translocations in activated B cell-like diffuse large B cell lymphoma.

To elucidate the mechanisms underlying chromosomal translocations in diffuse large B cell lymphoma (DLBCL), we investigated the nature and extent of immunoglobulin class switch recombination (CSR) in these tumors. We used Southern blotting to detect legitimate and illegitimate CSR events in tumor samples of the activated B cell-like (ABC), germinal center B cell-like (GCB), and primary mediastinal B cell lymphoma (PMBL) subgroups of DLBCL. The frequency of legitimate CSR was lower in ABC DLBCL than in GCB DLBCL and PMBL. In contrast, ABC DLBCL had a higher frequency of internal deletions within the switch mu (Smu) region compared with GCB DLBCL and PMBL. ABC DLBCLs also had frequent deletions within Sgamma and other illegitimate switch recombinations. Sequence analysis revealed ongoing Smu deletions within ABC DLBCL tumor clones, which were accompanied by ongoing duplications and activation-induced cytidine deaminase-dependent somatic mutations. Unexpectedly, short fragments derived from multiple chromosomes were interspersed within Smu in one case. These findings suggest that ABC DLBCLs have abnormalities in the regulation of CSR that could predispose to chromosomal translocations. Accordingly, aberrant switch recombination was responsible for translocations in ABC DLBCLs involving BCL6, MYC, and a novel translocation partner, SPIB.

Integrating data on DNA copy number with gene expression levels and drug sensitivities in the NCI-60 cell line panel.

Chromosome rearrangement, a hallmark of cancer, has profound effects on carcinogenesis and tumor phenotype. We used a panel of 60 human cancer cell lines (the NCI-60) as a model system to identify relationships among DNA copy number, mRNA expression level, and drug sensitivity. For each of 64 cancer-relevant genes, we calculated all 4,096 possible Pearson's correlation coefficients relating DNA copy number (assessed by comparative genomic hybridization using bacterial artificial chromosome microarrays) and mRNA expression level (determined using both cDNA and Affymetrix oligonucleotide microarrays). The analysis identified an association of ERBB2 overexpression with 3p copy number, a finding supported by data from human tumors and a mouse model of ERBB2-induced carcinogenesis. When we examined the correlation between DNA copy number for all 353 unique loci on the bacterial artificial chromosome microarray and drug sensitivity for 118 drugs with putatively known mechanisms of action, we found a striking negative correlation (-0.983; 95% bootstrap confidence interval, -0.999 to -0.899) between activity of the enzyme drug L-asparaginase and DNA copy number of genes near asparagine synthetase in the ovarian cancer cells. Previous analysis of drug sensitivity and mRNA expression had suggested an inverse relationship between mRNA levels of asparagine synthetase and L-asparaginase sensitivity in the NCI-60. The concordance of pharmacogenomic findings at the DNA and mRNA levels strongly suggests further study of L-asparaginase for possible treatment of a low-synthetase subset of clinical ovarian cancers. The DNA copy number database presented here will enable other investigators to explore DNA transcript-drug relationships in their own domains of research focus.

Drugs aimed at targeting characteristic karyotypic phenotypes of cancer cells.

The karyotypic features of cancer cells have not been a particular focus of anticancer drug targeting either as guidance for treatment or as specific drug targets themselves. Cancer cell lines typically have considerable, characteristic, and variable chromosomal aberrations. Here, we consider small-molecule screening data across the National Cancer Institute's 60 tumor cell line drug screening panel (NCI-60) analyzed for specific association with karyotypic variables (numerical and structural complexity and heterogeneity) determined for these same cell lines. This analysis is carried out with the aid of a self-organizing map allowing for a simultaneous assessment of all screened compounds, revealing an association between karyotypic variables and a unique part of the cytotoxic response space. Thirteen groups of compounds based on related specific chemical structural motifs are identified as possible leads for anticancer drug discovery. These compounds form distinct groups of molecules associated with relatively unexplored regions of the NCI-60 self-organizing map where anticancer agents currently standard in the clinic are not present. We suggest that compounds identified in this study may represent new classes of potential anticancer agents.

Targeting cancer cells by exploiting karyotypic complexity and chromosomal instability.

Multiple karyotypic abnormalities and chromosomal instability are particular hallmarks of many cancers that are relatively resistant to long term control by current chemotherapeutic agents. We have asked whether these same hallmarks, karyotypic complexity and instability, can be used as determinants for the screening of potential anticancer compounds. Using a panel of well characterized cancer cell lines we have been able to identify specific groups of chemical compounds that are more cytotoxic toward the relatively more karyotypically complex and unstable panel members. Thus, we delineate an approach for the identification of "lead compounds" for anticancer drug discovery complementary to approaches that are focused at the outset on a given gene or pathway.

Karyotypic "state" as a potential determinant for anticancer drug discovery.

Cancer is a genetic disease caused by genomic instability. In many cancers, this instability is manifested by chromosomal reconfigurations and karyotypic complexity. These features are particular hallmarks of the epithelial cancers that are some of the malignancies most resistant to long term control by current chemotherapeutic agents. We have asked whether we could use karyotypic complexity and instability as determinants for the screening of potential anticancer compounds. Using a panel of well characterized cancer cell lines, we have been able to identify specific groups of chemical compounds that are more cytotoxic toward the relatively more karyotypically complex and unstable panel members. Thus, we delineate an approach for the identification of "lead compounds" for anticancer drug discovery complementary to those that are focused at the outset on a given gene or pathway.

Karyotypic complexity of the NCI-60 drug-screening panel.

We used spectral karyotyping to provide a detailed analysis of karyotypic aberrations in the diverse group of cancer cell lines established by the National Cancer Institute for the purpose of anticancer drug discovery. Along with the karyotypic description of these cell lines we defined and studied karyotypic complexity and heterogeneity (metaphase-to-metaphase variations) based on three separate components of genomic anatomy: (a) ploidy; (b) numerical changes; and (c) structural rearrangements. A wide variation in these parameters was evident in these cell lines, and different association patterns between them were revealed. Analysis of the breakpoints and other specific features of chromosomal changes across the entire set of cell lines or within particular lineages pointed to a striking lability of centromeric regions that distinguishes the epithelial tumor cell lines. We have also found that balanced translocations are as frequent in absolute number within the cell lines derived from solid as from hematopoietic tumors. Important similarities were noticed between karyotypic changes in cancer cell lines and that seen in primary tumors. This dataset offers insights into the causes and consequences of the destabilizing events and chromosomal instability that may occur during tumor development and progression. It also provides a foundation for investigating associations between structural genome anatomy and cancer molecular markers and targets, gene expression, gene dosage, and resistance or sensitivity to tens of thousands of molecular compounds.

Apoptotic susceptibility of cancer cells selected for camptothecin resistance: gene expression profiling, functional analysis, and molecular interaction mapping.

To study the molecular mechanisms by which drug resistance develops, we compared DU145 humanprostate cancer cells with a subline selected for resistance to camptothecin. Differences in gene expression level were assessed by hybridizing the two cell types against each other using quadruplicate "Oncochip" cDNA microarrays that included 1648 cancer-related genes. Expression levels differing by a factor of >1.5 were detected for 181 of the genes. These differences were judged statistically reliable on the basis of a stratum-adjusted Kruskal-Wallis test, after taking into account a dye-dependent variable. The 181 expression-altered genes included a larger than expected number of the "apoptosis-related" genes (P = 0.04). To assess whether this observation reflected a generalized resistance of RCO.1 to apoptosis, we exposed the cells to a range of stresses (cisplatin, staurosporine, UV, ionizing radiation, and serum starvation) and found greatly reduced apoptotic responses for RC0.1 (relative to DU145) using flow cytometric Annexin V and terminal deoxynucleotidyl transferase-mediated nick end labeling assays. We next examined the apoptosis-related genes in the context of a molecular interaction map and found expression differences in the direction "expected" on the basis of the apoptosis-resistance of RC0.1 for BAD, caspase-6, and genes that signal via the Akt pathway. Exposure of the cells to wortmannin, an inhibitor of the Akt effector phosphatidylinositol 3-kinase, provided functional support for involvement of the Akt pathway. However, closer examination of the molecular interaction map revealed a paradox: many of the expression differences observed for apoptosis-related genes were in the direction "contrary" to that expected given the resistance of RC0.1. The map indicated that most of these unexpected expression differences were associated with genes involved in the nuclear factor kappa B and transforming growth factor beta pathways. Overall, the patterns that emerged suggested a two-step model for the selection process that led to resistance in RC0.1 cells. The first hypothesized step would involve a decrease in apoptotic susceptibility through changes in the apoptosis-control machinery associated with the Bcl-2 and caspase gene families, and also in antiapoptotic pathways operating through Akt/PKB. The second step would involve changes in multifunctional upstream genes (including some genes in the nuclear factor kappa B and transforming growth factor beta pathways) that can facilitate apoptosis but that would also tend to contribute to cell proliferation in the presence of drug. Thus, we propose that a downstream blockade of apoptosis was "permissive" for the selection of upstream pathway changes that would otherwise have induced apoptosis. This model is analogous to one suggested previously for the relationship between oncogene function and apoptosis in carcinogenesis.