Marta Chesi

BET Bromodomain Inhibition as a Therapeutic Strategy to Target c-Myc

MYC contributes to the pathogenesis of a majority of human cancers, yet strategies to modulate the function of the c-Myc oncoprotein do not exist. Toward this objective, we have targeted MYC transcription by interfering with chromatin-dependent signal transduction to RNA polymerase, specifically by inhibiting the acetyl-lysine recognition domains (bromodomains) of putative coactivator proteins implicated in transcriptional initiation and elongation. Using a selective small-molecule bromodomain inhibitor, JQ1, we identify BET bromodomain proteins as regulatory factors for c-Myc. BET inhibition by JQ1 downregulates MYC transcription, followed by genome-wide downregulation of Myc-dependent target genes. In experimental models of multiple myeloma, a Myc-dependent hematologic malignancy, JQ1 produces a potent antiproliferative effect associated with cell-cycle arrest and cellular senescence. Efficacy of JQ1 in three murine models of multiple myeloma establishes the therapeutic rationale for BET bromodomain inhibition in this disease and other malignancies characterized by pathologic activation of c-Myc.

Genetics and Cytogenetics of Multiple Myeloma A Workshop Report

Much has been learned regarding the biology and clinical implications of genetic abnormalities in multiple myeloma. Because of recent advances in the field, an International Workshop was held in Paris in February of 2003. This summary describes the consensus recommendations arising from that meeting with special emphasis on novel genetic observations. For instance, it is increasingly clear that translocations involving the immunoglobulin heavy-chain locus are important for the pathogenesis of one-half of patients. As a corollary, it also clear that the remaining patients, lacking IgH translocations, have hyperdiploidy as the hallmark of their disease. Several important genetic markers are associated with a shortened survival such as chromosome 13 monosomy, hypodiploidy, and others. The events leading the transformation of the monoclonal gammopathy of undetermined significance (MGUS) to myeloma are still unclear. One of the few differential genetic lesions between myeloma and MGUS is the presence of ras mutations in the latter. Gene expression platforms are capable of detecting many of the genetic aberrations found in the clonal cells of myeloma. Areas in need of further study were identified. The study of the genetic aberrations will likely form the platform for targeted therapy for the disease.

International Myeloma Working Group molecular classification of multiple myeloma: spotlight review

Myeloma is a malignant proliferation of monoclonal plasma cells. Although morphologically similar, several subtypes of the disease have been identified at the genetic and molecular level. These genetic subtypes are associated with unique clinicopathological features and dissimilar outcome. At the top hierarchical level, myeloma can be divided into hyperdiploid and non-hyperdiploid subtypes. The latter is mainly composed of cases harboring IgH translocations, generally associated with more aggressive clinical features and shorter survival. The three main IgH translocations in myeloma are the t(11;14)(q13;q32), t(4;14)(p16;q32) and t(14;16)(q32;q23). Trisomies and a more indolent form of the disease characterize hyperdiploid myeloma. A number of genetic progression factors have been identified including deletions of chromosomes 13 and 17 and abnormalities of chromosome 1 (1p deletion and 1q amplification). Other key drivers of cell survival and proliferation have also been identified such as nuclear factor- B-activating mutations and other deregulation factors for the cyclin-dependent pathways regulators. Further understanding of the biological subtypes of the disease has come from the application of novel techniques such as gene expression profiling and array-based comparative genomic hybridization. The combination of data arising from these studies and that previously elucidated through other mechanisms allows for most myeloma cases to be classified under one of several genetic subtypes. This paper proposes a framework for the classification of myeloma subtypes and provides recommendations for genetic testing. This group proposes that genetic testing needs to be incorporated into daily clinical practice and also as an essential component of all ongoing and future clinical trials.

Clonal competition with alternating dominance in multiple myeloma

Emerging evidence indicates that tumors can follow several evolutionary paths over a patients disease course. With the use of serial genomic analysis of samples collected at different points during the disease course of 28 patients with multiple myeloma, we found that the genomes of standard-risk patients show few changes over time, whereas those of cytogenetically high-risk patients show significantly more changes over time. The results indicate the existence of 3 temporal tumor types, which can either be genetically stable, linearly evolving, or heterogeneous clonal mixtures with shifting predominant clones. A detailed analysis of one high-risk patient sampled at 7 time points over the entire disease course identified 2 competing subclones that alternate in a back and forth manner for dominance with therapy until one clone underwent a dramatic linear evolution. With the use of the Vk*MYC genetically engineered mouse model of myeloma we modeled this competition between subclones for predominance occurring spontaneously and with therapeutic selection.

MMSET regulates histone H4K20 methylation and 53BP1 accumulation at DNA damage sites

p53-binding protein 1 (53BP1) is known to be an important mediator of the DNA damage response, with dimethylation of histone H4 lysine 20 (H4K20me2) critical to the recruitment of 53BP1 to double-strand breaks (DSBs). However, it is not clear how 53BP1 is specifically targeted to the sites of DNA damage, as the overall level of H4K20me2 does not seem to increase following DNA damage. It has been proposed that DNA breaks may cause exposure of methylated H4K20 previously buried within the chromosome; however, experimental evidence for such a model is lacking. Here we found that H4K20 methylation actually increases locally upon the induction of DSBs and that methylation of H4K20 at DSBs is mediated by the histone methyltransferase MMSET (also known as NSD2 or WHSC1) in mammals. Downregulation of MMSET significantly decreases H4K20 methylation at DSBs and the subsequent accumulation of 53BP1. Furthermore, we found that the recruitment of MMSET to DSBs requires the γH2AX–MDC1 pathway; specifically, the interaction between the MDC1 BRCT domain and phosphorylated Ser 102 of MMSET. Thus, we propose that a pathway involving γH2AX–MDC1–MMSET regulates the induction of H4K20 methylation on histones around DSBs, which, in turn, facilitates 53BP1 recruitment.

AID-Dependent Activation of a MYC Transgene Induces Multiple Myeloma in a Conditional Mouse Model of Post-Germinal Center Malignancies

By misdirecting the activity of Activation-Induced Deaminase (AID) to a conditional MYC transgene, we have achieved sporadic, AID-dependent MYC activation in germinal center B cells of Vk*MYC mice. Whereas control C57BL/6 mice develop benign monoclonal gammopathy with age, all Vk*MYC mice progress to an indolent multiple myeloma associated with the biological and clinical features highly characteristic of the human disease. Furthermore, antigen-dependent myeloma could be induced by immunization with a T-dependent antigen. Consistent with these findings in mice, more frequent MYC rearrangements, elevated levels of MYC mRNA, and MYC target genes distinguish human patients with multiple myeloma from individuals with monoclonal gammopathy, implicating a causal role for MYC in the progression of monoclonal gammopathy to multiple myeloma.

Clinical and Biological Implications of MYC Activation: A common difference between MGUS and newly diagnosed multiple myeloma

Events mediating transformation from the pre-malignant monoclonal gammopathy of undetermined significance (MGUS) to multiple myeloma (MM) are unknown. We analyzed gene expression data sets generated on the Affymetrix U133 platform from 22 MGUS and 101 MM patients using gene-set enrichment analysis. Genes overexpressed in MM were enriched for cell cycle, proliferation and MYC activation gene sets. Upon dissecting the relationship between MYC and cell-cycle gene sets, we identified and validated an MYC activation signature dissociated from proliferation. Applying this signature, MYC is activated in 67% of myeloma, but not in MGUS. This was further confirmed by immunohistochemistry (IHC) using membrane CD138 and nuclear MYC double staining. We also showed that almost all tumors with RAS mutations expressed the MYC activation signature, and multiple mechanisms may be involved in activating MYC. MYC activation, whether assessed by gene-expression signature or IHC, is associated with hyperdiploid MM and shorter survival even in tumors that are not proliferative. Bortezomib treatment is able to overcome the survival disadvantage in patients with MYC activation.

Prognostic factors for hyperdiploid-myeloma: effects of chromosome 13 deletions and IgH translocations

Chromosomal hyperdiploidy is the defining genetic signature in 40–50% of myeloma (MM) patients. We characterize hyperdiploid-MM (H-MM) in terms of its clinical and prognostic features in a cohort of 220 H-MM patients entered into clinical trials. Hyperdiploid-myeloma is associated with male sex, kappa immunoglobulin subtype, symptomatic bone disease and better survival compared to nonhyperdiploid-MM (median overall survival 48 vs 35 months, log-rank P=0.023), despite similar response to treatment. Among 108 H-MM cases with FISH studies for common genetic abnormalities, survival is negatively affected by the existence of immunoglobulin heavy chain (IgH) translocations, especially those involving unknown partners, while the presence of chromosome 13 deletion by FISH did not significantly affect survival (median overall survival 50 vs 47 months, log-rank P=0.47). Hyperdiploid-myeloma is therefore a unique genetic subtype of MM associated with improved outcome with distinct clinical features. The existence of IgH translocations but not chromosome 13 deletion by FISH negatively impacts survival and may allow further risk stratification of this population of MM patients.

Identification of Copy Number Abnormalities and Inactivating Mutations in Two Negative Regulators of Nuclear Factor-κB Signaling Pathways in Waldenström's Macroglobulinemia

Waldenströms macroglobulinemia (WM) is a distinct clinicobiological entity defined as a B-cell neoplasm characterized by a lymphoplasmacytic infiltrate in bone marrow (BM) and IgM paraprotein production. Cytogenetic analyses were historically limited by difficulty in obtaining tumor metaphases, and the genetic basis of the disease remains poorly defined. Here, we performed a comprehensive analysis in 42 WM patients by using a high-resolution, array-based comparative genomic hybridization approach to unravel the genetic mechanisms associated with WM pathogenesis. Overall, 83% of cases have chromosomal abnormalities, with a median of three abnormalities per patient. Gain of 6p was the second most common abnormality (17%), and its presence was always concomitant with 6q loss. A minimal deleted region, including MIRN15A and MIRN16-1, was delineated on 13q14 in 10% of patients. Of interest, we reported biallelic deletions and/or inactivating mutations with uniparental disomy in tumor necrosis factor (TNF) receptor-associated factor 3 and TNFalpha-induced protein 3, two negative regulators of the nuclear factor-kappaB (NF-kappaB) signaling pathway. Furthermore, we confirmed the association between TRAF3 inactivation and increased transcriptional activity of NF-kappaB target genes. Mutational activation of the NF-kappaB pathway, which is normally activated by ligand receptor interactions within the BM microenvironment, highlights its biological importance, and suggests a therapeutic role for inhibitors of NF-kappaB pathway activation in the treatment of WM.

Drug response in a genetically engineered mouse model of multiple myeloma is predictive of clinical efficacy

The attrition rate for anticancer drugs entering clinical trials is unacceptably high. For multiple myeloma (MM), we postulate that this is because of preclinical models that overemphasize the antiproliferative activity of drugs, and clinical trials performed in refractory end-stage patients. We validate the Vk*MYC transgenic mouse as a faithful model to predict single-agent drug activity in MM with a positive predictive value of 67% (4 of 6) for clinical activity, and a negative predictive value of 86% (6 of 7) for clinical inactivity. We identify 4 novel agents that should be prioritized for evaluation in clinical trials. Transplantation of Vk*MYC tumor cells into congenic mice selected for a more aggressive disease that models end-stage drug-resistant MM and responds only to combinations of drugs with single-agent activity in untreated Vk*MYC MM. We predict that combinations of standard agents, histone deacetylase inhibitors, bromodomain inhibitors, and hypoxia-activated prodrugs will demonstrate efficacy in the treatment of relapsed MM.