Fernández-Lasquetty presenta la Guía para el manejo integral del paciente con Lesión Medular Crónica

Fernández-Lasquetty presenta la Guía para el manejo integral del paciente con Lesión Medular Crónica

Ataxia, Dementia, and Hypogonadotropism

Background


The combination of ataxia and hypogonadism was first described more than a

century ago, but its genetic basis has remained elusive.

Methods

We performed whole-exome sequencing in a patient with ataxia and hypogonadotropic

hypogonadism, followed by targeted sequencing of candidate genes in similarly

affected patients. Neurologic and reproductive endocrine phenotypes were

characterized in detail. The effects of sequence variants and the presence of an

epistatic interaction were tested in a zebrafish model.   Results

Digenic homozygous mutations in RNF216 and OTUD4, which encode a ubiquitin E3

ligase and a deubiquitinase, respectively, were found in three affected siblings in a

consanguineous family. Additional screening identified compound heterozygous

truncating mutations in RNF216 in an unrelated patient and single heterozygous

deleterious mutations in four other patients. Knockdown of rnf216 or otud4 in zebrafish

embryos induced defects in the eye, optic tectum, and cerebellum; combinatorial

suppression of both genes exacerbated these phenotypes, which were rescued by

nonmutant, but not mutant, human RNF216 or OTUD4 messenger RNA. All patients

had progressive ataxia and dementia. Neuronal loss was observed in cerebellar

pathways and the hippocampus; surviving hippocampal neurons contained ubiquitin-

immunoreactive intranuclear inclusions. Defects were detected   SI QUIERES VER ARTICULO COMPLETO VE AL SIGUIENTE LINK   http://www.nejm.org/doi/pdf/10.1056/NEJMoa1215993   Conclusions

The syndrome of hypogonadotropic hypogonadism, ataxia, and dementia can be

caused by inactivating mutations in RNF216 or by the combination of mutations in

RNF216 and OTUD4. These findings link disordered ubiquitination to neurodegeneration

and reproductive dysfunction and highlight the power of whole-exome sequencing

in combination with functional studies to unveil genetic interactions that

cause disease. (Funded by the National Institutes of Health and others.)

Dengue

Cameron P. Simmons, Ph.D., Jeremy J. Farrar, M.D., Ph.D., Nguyen van Vinh Chau, M.D., Ph.D., and Bridget Wills, M.D., D.M.

N Engl J Med 2012; 366:1423-1432April 12, 2012

Dengue is a self-limited, systemic viral infection transmitted between humans by mosquitoes. The rapidly expanding global footprint of dengue is a public health challenge with an economic burden that is currently unmet by licensed vaccines, specific therapeutic agents, or efficient vector-control strategies.

This review highlights our current understanding of dengue, including its clinical manifestations, pathogenesis, tests that are used to diagnose it, and its management and prevention.

Determinants of the Current Dengue Pandemic

The global burden of dengue is large; an estimated 50 million infections per year occur across approximately 100 countries, with potential for further spread (Figure 1 Figure 1Global Dengue Risk.).1

 Central to the emergence of dengue as a public health problem has been the dispersal of efficient mosquito vectors across much of the tropical and subtropical world. The primary vector, the urban-adapted Aedes aegypti mosquito, has become widely distributed across tropical and subtropical latitudes. It emerged from Africa during the slave trade in the 15th through 19th centuries, spread into Asia through commercial exchanges in the 18th and 19th centuries, and has spread globally with the advent of increased travel and trade in the past 50 years.2 In addition, the geographic range of a secondary vector, A. albopictus, has dramatically expanded in recent years.3 Globalization of trade, in particular the trade of tires from used vehicles, is thought to explain the dispersal of eggs and immature forms of these arboviral vectors into new territories.4 Endemicity has also been facilitated by rapid urbanization in Asia and Latin America, resulting in increased population density with an abundance of vector-breeding sites within crowded urban communities and the areas surrounding them. Dengue infections in Africa remain largely unquantified, but recent outbreaks suggest that substantial parts of the continent may be at risk for increasing dengue transmission. More surveillance is required to assess the true burden of disease (see the Supplementary Appendix, available with the full text of this article at NEJM.org).

Vector control, through chemical or biologic targeting of mosquitoes and removal of their breeding sites, is the mainstay of dengue prevention, but this approach has failed to stop disease transmission in almost all countries where dengue is endemic. Antigenic diversity of the dengue virus is important, since the lack of long-term cross-immunity among the four virus types allows for multiple sequential infections.

Thus, the spread of dengue illustrates how global trade (and the transport of the mosquito vectors), increasing travel within and between countries (and the movement of viremic people), urban crowding (which is conducive to multiple infections from an infected mosquito), and ineffective vector-control strategies have supported a pandemic in the modern era. With the increasingly global spread of dengue, practicing physicians in temperate North America, Europe, Australia, and Japan are more likely than ever to see returning travelers with dengue infection. The diagnosis should be considered in any patient presenting with fever that has developed within 14 days after even a brief trip to the tropics or subtropics, including those regions where dengue has not traditionally been considered an endemic disease.5,6

Virologic Features

Dengue is caused by one of four single-stranded, positive-sense RNA viruses (dengue virus type 1 through dengue virus type 4), also referred to as serotypes) of the genus flavivirus (family Flaviviridae). Infectious virus and the virus-encoded NS1 are present in blood during the acute phase, and high-level early viremia and NS1 antigenemia have been associated with more severe clinical presentations.7-9 The detection of NS1 is also the basis for commercial diagnostic assays.10

Dengue viruses exist in two environments: the urban or endemic setting, where humans and mosquitoes are the only known hosts, and forested areas, where transmission of mosquito-borne viruses occurs between nonhuman primates and, rarely, from these primates to humans.11 Within each dengue virus serotype, multiple genotypes comprise phylogenetically related sequences. Subtle antigenic differences exist between genotypes of the same serotype,12,13 but these may not be clinically relevant, since human infection with one serotype is believed to confer long-lived serotype-specific immunity, but only short-lived cross-immunity between serotypes.

The dynamics of dengue viruses within urban and endemic populations are complex, involving the birth and death of viral lineages.14,16 Although dengue has emerged in multiple new territories over the past 40 years, the viruses themselves are paradoxically “local” in their evolutionary histories, suggesting that the global dispersal of dengue virus has occurred in relatively infrequent “jumps,” most likely by the movement of viremic humans to new geographic settings with a suitable vector and a susceptible population.

Immunopathogenesis

Insights into the pathogenesis of severe dengue are hampered by the lack of an animal model that accurately recreates the transient capillary permeability syndrome accompanied by a decreasing viral burden that is seen in patients (Figure 2Figure 2Immunopathogenesis of Severe Dengue in Secondary Infections.).

 Epidemiologic studies have identified young age, female sex, high body-mass index, virus strain, and genetic variants of the human major-histocompatibility-complex class I–related sequence B and phospholipase C epsilon 1 genes as risk factors for severe dengue.18-21 Secondary infection, in the form of two sequential infections by different serotypes, is also an epidemiologic risk factor for severe disease.17,22,23 Mechanistically, increased risk in secondary infection is thought to be linked to antibody-dependent enhancement of virus infection in Fc receptor–bearing cells and the generation of a large infected cell mass in vivo.24 A consequence of a large virus-infected cell mass is a physiological environment in tissues that promotes capillary permeability; however, this hypothesis is based on temporal associations between immunologic markers and clinical events, without evidence of a direct, mechanistic link to causation (Figure 2).

Pathophysiology of Endothelial Dysfunction

There is no evidence that the virus infects endothelial cells, and only minor nonspecific changes have been detected in histopathological studies of the microvasculature.25,26 Although no specific pathway has been identified linking known immunopathogenic events with definitive effects on microvascular permeability, thromboregulatory mechanisms, or both, preliminary data suggest that transient disruption in the function of the endothelial glycocalyx layer occurs.27,28 This layer functions as a molecular sieve, selectively restricting molecules within plasma according to their size, charge, and shape. Hypoalbuminemia and proteinuria are observed during dengue infection; proteins up to and including the size of albumin are preferentially lost; this is consistent with a small but crucial change in the filtration characteristics of the glycocalyx.29 Both the virus itself and dengue NS1 are known to adhere to heparan sulfate, a key structural element of the glycocalyx, and increased urinary heparan sulfate excretion has been detected in children with severe infection.30,31

Differential Diagnosis and Disease Classification

Although most dengue virus infections are asymptomatic, a wide variety of clinical manifestations may occur, ranging from mild febrile illness to severe and fatal disease.1 The differential diagnosis is broad and varies as the disease evolves. During the febrile phase, it includes other arboviral infections as well as measles, rubella, enterovirus infections, adenovirus infections, and influenza. Other diseases that should be considered as part of the differential diagnosis, depending on the clinical picture and local disease prevalence, include typhoid, malaria, leptospirosis, viral hepatitis, rickettsial diseases, and bacterial sepsis.

Patients were previously classified as having either dengue fever or dengue hemorrhagic fever, with the latter classified as grade 1, 2, 3, or 4. Over a number of years, there was increasing concern regarding the complexity and usefulness of this classification system. In particular, there was concern regarding the requirement that all four specific criteria (fever lasting 2 to 7 days, tendency to hemorrhage evidenced by a positive tourniquet test or spontaneous bleeding, a platelet count of less than 100×10⁹ per liter, and evidence of a plasma leak based on changes in the hematocrit and pleural effusions) be met to support a diagnosis of dengue hemorrhagic fever — such that some patients with clinically severe disease were categorized inappropriately.32-34 With the recent revision of the World Health Organization (WHO) dengue classification scheme, patients are now classified as having either dengue or severe dengue.1,33,35 Patients who recover without major complications are classified as having dengue, whereas those who have any of the following conditions are designated as having severe dengue: plasma leakage resulting in shock, accumulation of serosal fluid sufficient to cause respiratory distress, or both; severe bleeding; and severe organ impairment. It is hoped that this system will prove more effective for triage and clinical management and will improve the quality of surveillance and epidemiologic data collected globally. Continued efforts through prospective multicenter studies are warranted to define the most appropriate classification scheme.

Clinical Manifestations

After an incubation period of 3 to 7 days, symptoms start suddenly and follow three phases — an initial febrile phase, a critical phase around the time of defervescence, and a spontaneous recovery phase.

Febrile Phase

The initial phase is typically characterized by high temperature (≥38.5°C) accompanied by headache, vomiting, myalgia, and joint pain, sometimes with a transient macular rash. Children have high fever but are generally less symptomatic than adults during this phase of the illness. Mild hemorrhagic manifestations such as petechiae (Figure 3AFigure 3Hemorrhagic Manifestations of Dengue Infection.) and bruising, particularly at venipuncture sites (Figure 3B), and a palpable liver are commonly noted.

Laboratory findings include mild-to-moderate thrombocytopenia and leukopenia, often with a moderate elevation of hepatic aminotransferase levels. This phase lasts for 3 to 7 days, after which most patients recover without complications.

Critical Phase

In a small proportion of patients, typically in children and young adults, a systemic vascular leak syndrome becomes apparent around the time of defervescence, evidenced by increasing hemoconcentration, hypoproteinemia, pleural effusions, and ascites. Initially, physiological compensatory mechanisms are up-regulated in an attempt to maintain adequate circulation to critical organs, resulting in narrowing of the pulse pressure when loss of plasma volume becomes critical. If the pulse pressure narrows to 20 mm Hg or less, accompanied by signs of peripheral vascular collapse, dengue shock syndrome is diagnosed and urgent, although careful, resuscitation is required. Systolic pressure may remain normal or even elevated at this time, and the patient may appear deceptively well, but once hypotension develops, systolic pressure decreases rapidly and irreversible shock and death may follow despite aggressive attempts at resuscitation. During the transition from the febrile to the critical phase, between days 4 and 7 of the illness, it is crucial for the clinician to be aware of warning signs that clinically significant vascular leakage may be developing in the patient. These signs of impending deterioration include persistent vomiting, increasingly severe abdominal pain, tender hepatomegaly, a high or increasing hematocrit level that is concurrent with a rapid decrease in the platelet count, serosal effusions, mucosal bleeding, and lethargy or restlessness.

Hemorrhagic manifestations are most common during this critical period. In children, clinically significant bleeding occurs only rarely, usually in association with profound and prolonged shock. However, major skin bleeding, mucosal bleeding (gastrointestinal or vaginal), or both may occur in adults with no obvious precipitating factors and only minor plasma leakage (Figure 3C).36 Moderate-to-severe thrombocytopenia is common, with nadir platelet counts below 20×10⁹ per liter often observed during the critical phase, followed by rapid improvement during the recovery phase. A transient increase in the activated partial-thromboplastin time and a decrease in fibrinogen levels are also frequently noted. However, the coagulation profile is not typical of disseminated intravascular coagulation, and the underlying mechanisms remain unclear.37-39 Infrequently, other severe manifestations, including liver failure, myocarditis, and encephalopathy, occur, often with minimal associated plasma leakage.

Recovery Phase

The altered vascular permeability is short-lived, reverting spontaneously to a normal level after approximately 48 to 72 hours, and is concurrent with rapid improvement in the patient's symptoms. A second rash may appear during the recovery phase, ranging from a mild maculopapular rash to a severe, itchy lesion suggesting leukocytoclastic vasculitis that resolves with desquamation over a period of 1 to 2 weeks (Figure 3D). Adults may have profound fatigue for several weeks after recovery.

Diagnostic Tests

Laboratory diagnosis of dengue is established directly by detection of viral components in serum or indirectly by serologic means. The sensitivity of each approach is influenced by the duration of the patient's illness (Figure 4Figure 4Laboratory Diagnostic Options in a Patient with Suspected Dengue Infection.).10

During the febrile phase, detection of viral nucleic acid in serum by means of reverse-transcriptase–polymerase-chain-reaction (RT-PCR) assay or detection of the virus-expressed soluble nonstructural protein 1 (NS1) by means of enzyme-linked immunosorbent assay (ELISA) or the lateral-flow rapid test (not currently available in the United States) is sufficient for a confirmatory diagnosis.

For primary infections in persons who have not been infected previously (which is typical in the case of most travelers), the diagnostic sensitivity of NS1 detection in the febrile phase can exceed 90%, and antigenemia may persist for several days after the resolution of fever. 40-42 The sensitivity of NS1 detection in the febrile phase is lower in secondary infections (60 to 80%), reflecting an anamnestic serologic response due to a previous dengue virus or related flavivirus infection.43

Serologic diagnosis of dengue relies on the detection of high levels of serum IgM that bind dengue virus antigens in an ELISA or a lateral-flow rapid test; IgM can be detected as early as 4 days after the onset of fever. IgM seroconversion between paired samples is considered a confirmatory finding, whereas detection of IgM in a single specimen obtained from a patient with a clinical syndrome that is consistent with dengue is widely used to establish a presumptive diagnosis. Commercially available IgM tests with acceptable performance characteristics have recently been identified.44

Serologic diagnosis of dengue can be confounded if the patient has very recently been infected or vaccinated with an antigenically related flavivirus (e.g., a virus associated with yellow fever or Japanese encephalitis). In addition, patients with secondary infections mount rapid anamnestic antibody responses in which dengue virus–reactive IgG may predominate over IgM. In clinical settings where methods of molecular detection (e.g., RT-PCR) are not available, investigation for elevated levels of dengue virus–reactive IgM or soluble NS1 in serum is a pragmatic diagnostic approach in a patient in whom dengue is suspected.43,45

Management

Currently, no effective antiviral agents to treat dengue infection are available, and treatment remains supportive, with particular emphasis on careful fluid management. 1 Patients who have no complications and are able to tolerate oral fluids may remain at home with instructions to return to the hospital immediately if bleeding or warning signs suggestive of vascular leakage develop. However, our practice is to evaluate these patients daily in a medical clinic with a complete blood count to monitor hematocrit and platelet values.

Development of any warning sign indicates the need for hospitalization and close observation, with judicious use of parenteral fluids in patients with inadequate oral intake or a rapidly increasing hematocrit. If the condition progresses to the dengue shock syndrome, prompt fluid resuscitation to restore plasma volume is imperative, followed by ongoing fluid therapy to support the circulation at a level just sufficient to maintain critical organ perfusion. Isotonic crystalloid solutions should be used, and isotonic colloid solutions should be reserved for patients presenting with profound shock or those who do not have a response to initial crystalloid therapy.46 To limit the risk of the development of fluid overload, parenteral fluid therapy should be kept to the minimum required to maintain cardiovascular stability until permeability reverts to a normal level.

Blood transfusion can be lifesaving for patients with severe bleeding that compromises cardiovascular function, but it should be undertaken with care because of the risk of fluid overload. Platelet concentrates, fresh-frozen plasma, and cryoprecipitate may also be needed depending on the coagulation profile. However, at present, there is no evidence that prophylactic platelet transfusions are of any value in patients who do not have clinically significant bleeding, even when thrombocytopenia is profound.47,48 The use of prophylactic platelet transfusions is increasing in countries where dengue is endemic, but given the associated clinical risks and the financial costs, controlled trials need to be performed before this becomes established as the standard of care. In patients with severe dengue infection, adjuvant therapy, including vasopressor and inotropic therapies, renal-replacement therapy, and further treatment of organ impairment, may be necessary.

The establishment of a therapeutic pipeline and the design of randomized, controlled trials of drugs targeting the virus or the immune response are recent developments. Recent trials have assessed chloroquine,49 oral prednisolone (A Randomized, Placebo-Controlled, Partially Blinded [Drug versus Placebo] Trial of Early Corticosteroid Therapy in Vietnamese Children and Young Adults with suspected Dengue Infection; Current Controlled Trials number, ISRCTN39575233), and balapiravir (A Randomized, Double-Blind, Placebo-Controlled Study to Evaluate the Safety and Efficacy of the Dengue Virus Polymerase Inhibitor [Balapiravir] in Male Patients with Confirmed Dengue Virus Infection; ClinicalTrials.gov number, NCT01096576), and further trials of statins and other antiviral drugs are planned. Currently, there is no evidence in favor of the use of any specific therapeutic agent for dengue.

Effects on Health Care Systems

Dengue imposes major demands on health care systems. Although severe dengue occurs in only a small proportion of dengue infections, early identification of high-risk patients is difficult and patients with uncomplicated infections are frequently hospitalized for observation. Rapid and effective triage by experienced personnel at the primary health care level, efficient and affordable transportation systems to facilitate daily clinical assessment, and public education campaigns to increase awareness of the disease all help to reduce unnecessary admissions. Among hospitalized patients, meticulous attention to detail is necessary to limit iatrogenic complications, including fluid overload.

Ideally, patients with severe dengue infection should be treated in dedicated high-dependency units where frequent clinical observations by experienced staff with immediate access to repeated hematocrit measurements can ensure that fluid therapy is carefully titrated as needed. In such circumstances, mortality of less than 1% is achievable among patients with shock, and the need for ventilatory support and intensive care is minimized. Improvements in the early diagnosis and risk prediction of severe disease are urgently needed, especially in areas with a high case burden, where appropriate allocation of limited resources is crucial to the outcome. Ongoing research aims to refine the WHO 2009 classification scheme, particularly with regard to warning signs for the development of severe disease.

New Approaches to Targeting the Vector

New vector-control approaches include the release of genetically modified male mosquitoes that sterilize the wild-type female population, thereby reducing egg output and the population size of the next generation that would be available for potential transmission of the dengue virus.50 An alternative strategy involves embryonic introduction of strains of the obligate intracellular bacterium wolbachia into A. aegypti. Strikingly, wolbachia-infected A. aegypti are partially resistant to dengue virus infection51,52 and can invade natural A. aegypti populations,51,53 suggesting the possibility of induction of widespread biologic resistance to dengue viruses in A. aegypti populations.

Vaccines

The leading dengue vaccine candidate, ChimeriVax (Sanofi Pasteur), is a tetravalent formulation of attenuated yellow fever 17D vaccine strains expressing the dengue virus prM and E proteins.54 It has been difficult to develop a vaccine for dengue that is safe and elicits balanced neutralizing antibody responses to all four serotypes. However, in the past 5 years, remarkable progress has been made, and multicenter phase 2–3 clinical trials that are designed to determine the efficacy of this three-dose vaccine are under way. Data on immunologic correlates of immunity are lacking. Long-term follow-up of vaccinees will be essential to understand whether waning vaccine-elicited immunity predisposes recipients to more severe outcomes on subsequent natural infection. Other candidates in early phases of clinical development include vaccines containing live attenuated dengue viruses and recombinant subunit vaccines.55

Future Directions

The field of dengue research has been invigorated over the past decade, fueled by the growing recognition of the burden of disease coupled with the prospect of a dengue vaccine. However, no vaccine can be an immediate global panacea, and efforts to improve treatment through application of existing best practices in triage and fluid management, along with efforts to develop new antiviral or other therapeutic drugs, must continue. Similarly, innovative approaches to preventing transmission of the virus, such as through modification of mosquito populations, should be fostered. An improved understanding of the current epidemiology of the disease and the potential for its future spread would also assist policymakers in allocating resources to combat this global public health challenge.

Dr. Simmons reports that his institution receives consulting fees on his behalf from Unither Virology and Tibotec and grant support on his behalf from Hoffmann–La Roche. No other potential conflict of interest relevant to this article was reported.

Disclosure forms provided by the authors are available with the full text of this article at NEJM.org.

Source Information

From the Oxford University Clinical Research Unit and Wellcome Trust Major Overseas Programme (C.P.S., J.J.F., B.W.), Hospital for Tropical Diseases (N.V.C.), Ho Chi Minh City, Vietnam; and the Centre for Tropical Medicine, University of Oxford, Churchill Hospital, Oxford, United Kingdom (C.P.S., J.J.F., B.W.).
Address reprint requests to Dr. Farrar at the Hospital for Tropical Diseases, Oxford University Clinical Research Unit, 190 Ben Ham Tu, Quan 5, Ho Chi Minh City, Vietnam, or at jfarrar@oucru.org.

Epigenetics cancer

The principal tenet in oncology is that cancer is a disease that is initiated and driven by somatic aberrations of our genome. The challenge is to decipher how these genomic alterations culminate in malignant transformation and how they can be targeted for therapeutic gain. Molecular insights provided by the study of hematopoietic cancers have elucidated many fundamental principles in cancer biology.

Recurrent chromosomal translocations involving transcriptional regulators in several hematopoietic cancers illustrate the importance of transcriptional dysregulation in cancer.1 The earliest detected translocations provided examples of abnormalities in both transcriptional activation and transcriptional repression.1,2 The introduction of more refined techniques in molecular biology has led to the identification of further recurrent translocations and somatic mutations that result in pathognomonic transcriptional alterations.3 These gene-expression signatures have diagnostic utility as well as prognostic significance, but as yet few new treatments have emerged. The task of cataloguing all the genomic aberrations in cancer has now begun in earnest.4 Using next-generation sequencing platforms, the International Cancer Genome Consortium has already provided an unparalleled annotation of recurrent somatic mutations in protein-coding genes for a large variety of cancers.5 These efforts have brought into focus a new central theme: recurrent mutations in epigenetic regulators, which are especially prevalent in the hematopoietic cancers (Table 1Table 1Epigenetic Regulators with Reader Domains Recurrently Mutated in Cancer.).

Epigenetics and Chromatin Biology

The term “epigenetics” (see the Glossary) remains the object of contention and ambiguity.6,7 It was originally coined by Waddington to describe heritable changes in gene expression and cellular phenotype that were independent of alterations in the DNA sequence.7 Epigenetics is most frequently used to describe the study of chromatin biology, and this will be the definition used in this review. Chromatin is the macromolecular complex of DNA and histone proteins. It provides the scaffold for the packaging of our entire genome and contains the heritable material of eukaryotic cells.
The basic functional unit of chromatin is the nucleosome, which consists of an octamer containing two each of the histones H2A, H2B, H3, and H4, around which 147 bp of DNA are wrapped6 (Figure 1AFigure 1The Nucleosome.). Nucleosomes compact and package DNA in a dynamic and highly controlled fashion that caters to the multitude of DNA-based processes. Consecutive nucleosomes are separated by unwrapped linker DNA, typically between 20 and 50 bp in length.8 Wrapped nucleosomal DNA is inherently less accessible than linker DNA, thus the genomic positioning and compaction of nucleosomes strongly influences the ability of proteins to bind target sequences within DNA and to carry out their function. Broadly speaking, chromatin can be divided into two disparate states: heterochromatin, which is tightly packaged and contains primarily inactive genes, and euchromatin, which has a more relaxed conformation that provides a more permissive environment for active transcription. Several factors influence both local and global chromatin architecture, but perhaps the most influential elements that coordinate this process are the covalent modifications of either DNA or histones (Figure 1B).8 These chromatin modifications play an instructive role in regulating DNA-templated processes, including transcription, repair, and replication.6,9
Chromatin modifications serve two main functions: first, they physically enhance or weaken the noncovalent interactions between histones or between histones and DNA, determining accessibility to specific DNA loci, and second, they provide an informative platform for the recruitment of epigenetic regulators. At least 4 different DNA modifications10,11 and at least 16 distinct classes of histone modification have been described (Figure 1B).9,12 All of these chemical modifications are dynamic; they are laid down by “chromatin writers” and removed by “chromatin erasers” in a highly regulated manner (Figure 2AFigure 2Epigenetic Regulation.). Chromatin immunoprecipitation coupled with next-generation sequencing has made it possible to annotate comprehensive global maps in specific cellular contexts for many of these modifications.13,14 The vast array of modifications are not random and often adopt a predictable genomic distribution that can be used to define specific cellular processes, such as active transcription.14 A fundamental advance in our understanding of chromatin regulation was the realization that many chromatin regulators possess specialized domains that allow these proteins to survey the epigenetic landscape and dock at specific regions within the genome. The binding modules within these “chromatin readers” recognize different covalent modifications of the nucleosome and assemble functional complexes onto specific loci to facilitate DNA-templated processes (Figure 2B).15,16
In contrast with the static somatic alterations in DNA, the dynamic plasticity of the epigenome lends itself well to therapeutic manipulation. Epigenetic therapies targeting the catalytic activity of chromatin regulators have been developed and approved by the Food and Drug Administration for use in a small number of hematopoietic cancers.17-19 These therapies include small molecules that target epigenetic writers (DNA methyltransferase inhibitors)17,18 and epigenetic erasers (histone deacetylase inhibitors).19 Despite these clinical successes, the pleiotropic effects of these compounds have made it difficult to decipher their exact molecular mechanism of action and have hampered their broader application in oncology. More recently, it has been possible to develop and deploy small molecules that specifically target the protein–protein interaction modules of certain epigenetic readers. This new therapeutic approach will be the primary subject of this review.

Epigenetic Readers

Many chromatin regulators, including several catalytic enzymes, act as “chromatin readers,” possessing specialized domains that bind to distinct covalent modifications of the nucleosome and respond to information conveyed by upstream signaling cascades.15 Critical residues within the binding pocket of the reader domain confer a preference for specific modification states, whereas residues outside the binding pocket contribute to determining the histone-sequence specificity. This modular combination allows proteins with similar binding domains to dock at different modified residues, or at the same amino acid displaying a different modification state.
For example, lysine residues have been shown to contain at least eight different covalent modifications, including acetylation, methylation, ubiquitylation, and sumoylation. Further complexity exists by virtue of the fact that each lysine residue may be unmethylated, monomethylated, dimethylated, or trimethylated. For instance, the plant homeodomain (PHD) finger is a region within a protein that is capable of detecting methylated histones. The PHD fingers of the proteins BHC80 and DNMT3L preferentially bind unmethylated lysine residues,20,21 whereas the PHD finger of ING2 binds most avidly to dimethylated and trimethylated lysines.22,23 Other, similar methyl–lysine-recognition motifs exist, including chromodomains, tudor domains, and the PWWP domain, named for its characteristic proline–tryptophan–tryptophan–proline motif (Figure 2B).24-26 Notably, when the same lysine residue undergoes another modification, such as acetylation, it may then provide docking sites for other proteins containing acetyl–lysine binding domains, such as bromodomains.27,28 Finally, to add to the complexity, many chromatin regulators have more than one type of reader domain, and their chromatin binding can be further influenced by neighboring histone modifications, so-called multivalent engagement-of-histone modifications.29 These examples highlight the multifaceted mechanisms that chromatin readers use to decipher the intricate epigenetic landscape. (The best-characterized protein-binding pockets contained within chromatin-associated proteins are summarized in Figure 2B.)
The importance of chromatin reader domains in maintaining homeostasis was initially revealed in the seminal observation that mutations in the PHD finger of RAG2 abrogate the protein's ability to bind trimethylated-H3K4, reduce V(D)J recombination (in which variable, diverse, and joining gene segments are randomly combined), and result in immunodeficiency syndromes.30 Mutations that abrogate the chromatin-reading capacity of many epigenetic regulators play an influential role in a variety of diseases, including cancer.31 Using these chromatin reader modules as therapeutic targets may offer a unique opportunity to tailor therapies to specific diseases. An exemplar of this process has recently been provided with small molecules that specifically and avidly inhibit the tandem bromodomains of the bromodomain and extraterminal (BET) family of proteins.32-34

BET Bromodomain Inhibitors

Bromodomains are highly conserved motifs present in a number of proteins throughout phylogeny.27 More than 40 different human proteins contain a bromodomain, and some have multiple bromodomains. They can be clustered into nine major families according to sequence identity. Although the acetyl–lysine binding pocket for all bromodomains is hydrophobic, there can be considerable variation in the electrostatic interactions at the opening of the pocket among bromodomain families. This variation determines the specificity of individual bromodomains and provides the opportunity to develop specific small molecules that are targeted against certain families of bromodomains, including the BET proteins.35,36
The BET family has four members, including bromodomain-containing proteins 2, 3, and 4 (BRD2, BRD3, and BRD4), whose expression is ubiquitous, and BRDT, whose expression is confined to the germ cells. BET proteins share a common structural design, featuring a tandem bromodomain at the N-terminal end of the protein, and play an integral role in transcription and cell growth.37 Early studies characterizing the BET proteins showed that BRD4 is associated with the mediator complex,38 an important multicomponent protein complex facilitating the initiation of transcription.39 BRD4 also associates with the active form of positive transcription elongation factor b (P-TEFb), a complex that regulates RNA polymerase II activity at the onset of transcriptional elongation.40,41 BET proteins also maintain an association with chromatin throughout mitosis, and this feature facilitates “gene bookmarking,” a process of rapid transcriptional reactivation of critical genes after mitosis.42 Underscoring the physiological importance of the BET proteins in cellular homeostasis is the observation that the experimental knockout of either Brd2 43 or Brd4 44 in mice results in early embryonic lethality.
The essential nature of the BET proteins and their fundamental importance in the regulation of transcription has increased interest in elucidating the molecular mechanisms of their action. These efforts will be aided considerably by the recent identification of the complete nuclear BET-protein interactome.32 In this study, three complementary global proteomic strategies were used to isolate the nuclear complexes containing the BET proteins. These data show that BET proteins are integral components of a large number of nuclear protein complexes that play a role in DNA replication, chromatin remodeling, DNA damage, and transcriptional regulation. Recurrent translocations of both BRD3 and BRD4 underpin the pathogenesis of NUT (or nuclear protein in testis) midline carcinoma,33 and more recently, RNA interference (RNAi) screening strategies have been used to identify a central role for BRD4 in acute myeloid leukemia.45 These findings have provided the impetus for investigating the potential of BET bromodomain inhibitors as novel anticancer agents.

BET Inhibitors in Cancer

Three different BET inhibitors (I-BET762,34 JQ1,33 and I-BET15132) show highly specific binding to both of the tandem domains of BRD2, BRD3, and BRD4 and inhibit their ability to engage with acetyl–lysine residues (Figure 3AFigure 3The BET Inhibitors.). These agents inhibit growth in a range of hematopoietic malignant cell lines32,45-47 and in human NUT midline carcinoma cell lines.33 Although the therapeutic efficacy of BET inhibition has not yet been tested in patients, its efficacy has been shown in vivo in murine models.

NUT Midline Carcinoma

NUT midline carcinoma is a rare but aggressive epithelial malignant disease that is invariably fatal, with a mean survival of less than 1 year.48 It is most often manifested in the midline of the upper aerodigestive tract and mediastinum. Histologic diagnosis of NUT midline carcinoma is difficult, and confirmation relies primarily on the demonstration of rearrangement of the NUT gene with the use of fluorescence in situ hybridization.48 BRD4 and BRD3 represent the NUT translocation partners in the majority of cases, and these fusions result in the aberrant localization of NUT to chromatin. Knockdown of the NUT fusions resulted in dramatic differentiation and growth arrest of the malignant cells, providing a therapeutic rationale for the targeting of BET proteins. Remarkably, the BET bromodomain inhibitor JQ1 was able to displace the BRD4–NUT fusion protein from chromatin and induce a rapid differentiation and arrest of proliferation in NUT midline carcinoma cell lines.33 In addition, JQ1 showed excellent efficacy in murine xenograft models of NUT midline carcinoma, resulting in tumor differentiation and regression and increased survival.33

Acute Myeloid Leukemia with MLL Translocations

Recurrent translocations of MLL, the mixed-lineage leukemia gene, result in aggressive leukemias with a poor prognosis that are often refractory to conventional therapies.49 More than 70 different MLL-translocation partners have been identified in leukemia.49 Despite this variation, a central abnormality in transcriptional elongation appears to underpin the molecular pathogenesis of this disease.50 Many MLL fusion partners are members of the superelongation complex (SEC), an important regulator of transcriptional elongation.51-53 Moreover, the N-terminal portion of MLL, a region conserved in all MLL fusion proteins, associates with another critical transcriptional complex called the polymerase-associated factor complex (PAFc).54,55 The functional integrity of these complexes is critical for malignant transformation by MLL fusion proteins.52-55 Two recent studies using complementary approaches have identified an essential role for the BET proteins in a broad range of acute myeloid leukemias, including MLL-translocated leukemias.32,45
Dawson and colleagues used a global proteomic approach that identified BRD3 and BRD4 as key components of both PAFc and SEC.32 In contrast, Zuber and colleagues used an RNAi screen to show that the depletion of BRD4 dramatically reduced the viability of MLL-AF9 leukemia in vitro and in vivo.45 Both JQ1 and the novel BET inhibitor I-BET151 showed remarkable efficacy in vitro and in vivo against MLL fusion leukemia, resulting in the rapid induction of cell-cycle arrest and apoptosis. Both studies highlighted a reduction in expression of critical regulators of transformation, including MYC, BCL2, and CDK6 (a cyclin-dependent protein kinase), after treatment with a BET inhibitor. Dawson and colleagues also found that the therapeutic efficacy of I-BET151 could be attributed at least in part to the inhibition of BRD3–BRD4-mediated recruitment of PAFc and SEC to chromatin (Figure 3). These molecular events resulted in reduced recruitment of RNA polymerase II to the promoters of crucial oncogenes such as MYC, BCL2, and CDK6.32 BET inhibition was also effective against primary human acute myeloid leukemia cells in vitro.32,45 Notably, in primary human MLL-translocated leukemia cells, BET inhibition dramatically reduced the clonogenic capacity of the leukemia stem-cell compartment, suggesting that disease eradication may be possible.
Although Zuber and colleagues focused primarily on MLL-translocated leukemias, they also showed the efficacy of BET inhibition in a range of acute myeloid leukemia subtypes in which MLL rearrangement was absent. These findings raise the intriguing possibility that BET inhibition may be a more broadly applicable therapy in this genetically heterogeneous disease.

Multiple Myeloma and Burkitt's Lymphoma

Multiple myeloma is an incurable plasma-cell dyscrasia. Although the disease is genetically heterogeneous,56 disordered expression of the myelocytomatosis viral oncogene homolog MYC is a prominent feature, providing the rationale to test for BET inhibition.57 Similarly, Burkitt's lymphoma, an aggressive lymphoproliferative disorder, is characterized by recurrent translocations of MYC, most frequently involving the IGH locus that results in constitutive high-level expression of MYC. Several multiple myeloma cell lines show a dramatic response to BET inhibition with JQ1.46,47 Treatment with JQ1 leads to a profound cell-cycle arrest and apoptosis of the cell lines; this cellular phenotype is associated with a reduction in MYC transcription and protein expression. This transcriptional repression of MYC is linked to a reduction in the binding of chromatin by BRD4, upstream of the MYC promoter. Delmore and colleagues further translated these findings into work with primary human multiple myeloma cells and murine models of multiple myeloma, further indicating the therapeutic potential of BET inhibition in this disease.46 In addition, Mertz and colleagues extended their findings to reveal a survival benefit in murine xenograft models of Burkitt's lymphoma and acute myeloid leukemia.47
Taken together, the studies involving BET bromodomain inhibitors further validate the use of epigenetic targets in cancer therapy. They show that it is possible to target epigenetic readers as well as catalytic writers and erasers. Moreover, the studies establish that the specific targeting of protein–protein interactions can have antitumor effects in vitro and in animal models. Several questions warrant further investigation. It is unclear why these drugs have effects in hematopoietic cancers but not in the majority of common solid tumors, including malignant tumors of the breast and cervix.47 It is also unclear why a compound that inhibits the localization of proteins germane to transcription in general alters the expression of only a few hundred specific and reproducible genes. Although we have some insights into the molecular mechanism determining efficacy in the MLL fusion leukemias32 and in NUT midline carcinoma,33 it is largely unclear what molecular events dictate efficacy in the other sensitive cancers. A central theme in all the studies cited is the down-regulation of MYC. However, even though MYC has a prominent role in cancer,58 it is unlikely that the profound effects observed with the introduction of BET inhibition are mediated solely by MYC inhibition. There are many malignant cell lines that overexpress MYC yet fail to respond to BET inhibition47; MYC expression is not always affected by BET inhibition,47 and MYC down-regulation is not predictive of a response to BET inhibition.32,45 Furthermore, MYC overexpression fails to prevent the apoptosis induced by BET inhibition.45 Identification of the complete BET protein interactome has shown that the BET proteins are integral components of a large number of nuclear protein complexes.32 This finding suggests that BET proteins are involved with a variety of molecular mechanisms.

Conclusions

Cancer epigenetics is an area of ongoing research that continues to inform our understanding of the molecular pathogenesis of cancer and to identify novel therapeutic targets. Advances in medicinal chemistry have now made it possible to specifically target not just the catalytic activity of epigenetic regulators but also the protein–protein interaction modules that localize many of these proteins to chromatin. However, as is the case for the majority of tumors sensitive to BET inhibition, epigenetic therapeutic targets are not necessarily mutated in sensitive tumor types. Therefore, simple mutational screening may not provide a predictor of response. Potential means of identifying sensitive tumor types include combining drug sensitivity with mutational screening, as has recently been reported in two large screening studies,59,60 and using transcriptional or epigenetic biomarkers to predict response. The successful use of the bromodomain inhibitors in cancer therapy is likely to further energize basic scientists, clinicians, and the pharmaceutical industry to search for compounds that may similarly target and disrupt other chromatin reader motifs, including chromodomains and PHD fingers, which are already known to play key roles in certain cancers.31

Disclosure forms provided by the authors are available with the full text of this article at NEJM.org.

We thank Andy Bannister for reviewing an earlier version of this article and for helpful comments; and Rab Prinjha and Chun Wa Chung for providing the chemical structures and surface structure used in Figure 3A.

Source Information

From the Department of Haematology, Cambridge Institute for Medical Research (M.A.D., B.J.P.H.), Cambridge University Hospitals National Health Service Foundation Trust (M.A.D., B.J.P.H.), the Gurdon Institute and Department of Pathology (M.A.D., T.K.), and the Wellcome Trust and Medical Research Council Cambridge Stem Cell Institute (B.J.P.H.), University of Cambridge — all in Cambridge, United Kingdom.
Address reprint requests to Dr. Huntly at the Department of Haematology, Cambridge Institute for Medical Research, University of Cambridge, Cambridge CB2 0XY, United Kingdom, or at bjph2@cam.ac.uk.

Targeting Epigenetic Readers in Cancer

Mark A. Dawson, M.D., Ph.D., Tony Kouzarides, Ph.D., and Brian J.P. Huntly, M.D., Ph.D.

N Engl J Med 2012; 367:647-657August 16, 2012

The principal tenet in oncology is that cancer is a disease that is initiated and driven by somatic aberrations of our genome. The challenge is to decipher how these genomic alterations culminate in malignant transformation and how they can be targeted for therapeutic gain. Molecular insights provided by the study of hematopoietic cancers have elucidated many fundamental principles in cancer biology.

Recurrent chromosomal translocations involving transcriptional regulators in several hematopoietic cancers illustrate the importance of transcriptional dysregulation in cancer.1 The earliest detected translocations provided examples of abnormalities in both transcriptional activation and transcriptional repression.1,2 The introduction of more refined techniques in molecular biology has led to the identification of further recurrent translocations and somatic mutations that result in pathognomonic transcriptional alterations.3 These gene-expression signatures have diagnostic utility as well as prognostic significance, but as yet few new treatments have emerged. The task of cataloguing all the genomic aberrations in cancer has now begun in earnest.4 Using next-generation sequencing platforms, the International Cancer Genome Consortium has already provided an unparalleled annotation of recurrent somatic mutations in protein-coding genes for a large variety of cancers.5 These efforts have brought into focus a new central theme: recurrent mutations in epigenetic regulators, which are especially prevalent in the hematopoietic cancers (Table 1Table 1

Epigenetic Regulators with Reader Domains Recurrently Mutated in Cancer.).

 

 

Epigenetics and Chromatin Biology

The term “epigenetics” (see the Glossary) remains the object of contention and ambiguity.6,7 It was originally coined by Waddington to describe heritable changes in gene expression and cellular phenotype that were independent of alterations in the DNA sequence.7 Epigenetics is most frequently used to describe the study of chromatin biology, and this will be the definition used in this review. Chromatin is the macromolecular complex of DNA and histone proteins. It provides the scaffold for the packaging of our entire genome and contains the heritable material of eukaryotic cells.

The basic functional unit of chromatin is the nucleosome, which consists of an octamer containing two each of the histones H2A, H2B, H3, and H4, around which 147 bp of DNA are wrapped6 (Figure 1AFigure 1The Nucleosome.).

 Nucleosomes compact and package DNA in a dynamic and highly controlled fashion that caters to the multitude of DNA-based processes.

Consecutive nucleosomes are separated by unwrapped linker DNA, typically between 20 and 50 bp in length.8 Wrapped nucleosomal DNA is inherently less accessible than linker DNA, thus the genomic positioning and compaction of nucleosomes strongly influences the ability of proteins to bind target sequences within DNA and to carry out their function. Broadly speaking, chromatin can be divided into two disparate states: heterochromatin, which is tightly packaged and contains primarily inactive genes, and euchromatin, which has a more relaxed conformation that provides a more permissive environment for active transcription. Several factors influence both local and global chromatin architecture, but perhaps the most influential elements that coordinate this process are the covalent modifications of either DNA or histones (Figure 1B).8 These chromatin modifications play an instructive role in regulating DNA-templated processes, including transcription, repair, and replication.6,9

Chromatin modifications serve two main functions: first, they physically enhance or weaken the noncovalent interactions between histones or between histones and DNA, determining accessibility to specific DNA loci, and second, they provide an informative platform for the recruitment of epigenetic regulators. At least 4 different DNA modifications10,11 and at least 16 distinct classes of histone modification have been described (Figure 1B).9,12 All of these chemical modifications are dynamic; they are laid down by “chromatin writers” and removed by “chromatin erasers” in a highly regulated manner (Figure 2AFigure 2Epigenetic Regulation.).

 Chromatin immunoprecipitation coupled with next-generation sequencing has made it possible to annotate comprehensive global maps in specific cellular contexts for many of these modifications.13,14 The vast array of modifications are not random and often adopt a predictable genomic distribution that can be used to define specific cellular processes, such as active transcription.14 A fundamental advance in our understanding of chromatin regulation was the realization that many chromatin regulators possess specialized domains that allow these proteins to survey the epigenetic landscape and dock at specific regions within the genome. The binding modules within these “chromatin readers” recognize different covalent modifications of the nucleosome and assemble functional complexes onto specific loci to facilitate DNA-templated processes (Figure 2B).15,16

In contrast with the static somatic alterations in DNA, the dynamic plasticity of the epigenome lends itself well to therapeutic manipulation. Epigenetic therapies targeting the catalytic activity of chromatin regulators have been developed and approved by the Food and Drug Administration for use in a small number of hematopoietic cancers.17-19 These therapies include small molecules that target epigenetic writers (DNA methyltransferase inhibitors)17,18 and epigenetic erasers (histone deacetylase inhibitors).19 Despite these clinical successes, the pleiotropic effects of these compounds have made it difficult to decipher their exact molecular mechanism of action and have hampered their broader application in oncology. More recently, it has been possible to develop and deploy small molecules that specifically target the protein–protein interaction modules of certain epigenetic readers. This new therapeutic approach will be the primary subject of this review.

Epigenetic Readers

Many chromatin regulators, including several catalytic enzymes, act as “chromatin readers,” possessing specialized domains that bind to distinct covalent modifications of the nucleosome and respond to information conveyed by upstream signaling cascades.15 Critical residues within the binding pocket of the reader domain confer a preference for specific modification states, whereas residues outside the binding pocket contribute to determining the histone-sequence specificity. This modular combination allows proteins with similar binding domains to dock at different modified residues, or at the same amino acid displaying a different modification state.

For example, lysine residues have been shown to contain at least eight different covalent modifications, including acetylation, methylation, ubiquitylation, and sumoylation. Further complexity exists by virtue of the fact that each lysine residue may be unmethylated, monomethylated, dimethylated, or trimethylated. For instance, the plant homeodomain (PHD) finger is a region within a protein that is capable of detecting methylated histones. The PHD fingers of the proteins BHC80 and DNMT3L preferentially bind unmethylated lysine residues,20,21 whereas the PHD finger of ING2 binds most avidly to dimethylated and trimethylated lysines.22,23 Other, similar methyl–lysine-recognition motifs exist, including chromodomains, tudor domains, and the PWWP domain, named for its characteristic proline–tryptophan–tryptophan–proline motif (Figure 2B).24-26 Notably, when the same lysine residue undergoes another modification, such as acetylation, it may then provide docking sites for other proteins containing acetyl–lysine binding domains, such as bromodomains.27,28 Finally, to add to the complexity, many chromatin regulators have more than one type of reader domain, and their chromatin binding can be further influenced by neighboring histone modifications, so-called multivalent engagement-of-histone modifications.29 These examples highlight the multifaceted mechanisms that chromatin readers use to decipher the intricate epigenetic landscape. (The best-characterized protein-binding pockets contained within chromatin-associated proteins are summarized in Figure 2B.)

The importance of chromatin reader domains in maintaining homeostasis was initially revealed in the seminal observation that mutations in the PHD finger of RAG2 abrogate the protein's ability to bind trimethylated-H3K4, reduce V(D)J recombination (in which variable, diverse, and joining gene segments are randomly combined), and result in immunodeficiency syndromes.30 Mutations that abrogate the chromatin-reading capacity of many epigenetic regulators play an influential role in a variety of diseases, including cancer.31 Using these chromatin reader modules as therapeutic targets may offer a unique opportunity to tailor therapies to specific diseases. An exemplar of this process has recently been provided with small molecules that specifically and avidly inhibit the tandem bromodomains of the bromodomain and extraterminal (BET) family of proteins.32-34

BET Bromodomain Inhibitors

Bromodomains are highly conserved motifs present in a number of proteins throughout phylogeny.27 More than 40 different human proteins contain a bromodomain, and some have multiple bromodomains. They can be clustered into nine major families according to sequence identity. Although the acetyl–lysine binding pocket for all bromodomains is hydrophobic, there can be considerable variation in the electrostatic interactions at the opening of the pocket among bromodomain families. This variation determines the specificity of individual bromodomains and provides the opportunity to develop specific small molecules that are targeted against certain families of bromodomains, including the BET proteins.35,36

The BET family has four members, including bromodomain-containing proteins 2, 3, and 4 (BRD2, BRD3, and BRD4), whose expression is ubiquitous, and BRDT, whose expression is confined to the germ cells. BET proteins share a common structural design, featuring a tandem bromodomain at the N-terminal end of the protein, and play an integral role in transcription and cell growth.37 Early studies characterizing the BET proteins showed that BRD4 is associated with the mediator complex,38 an important multicomponent protein complex facilitating the initiation of transcription.39 BRD4 also associates with the active form of positive transcription elongation factor b (P-TEFb), a complex that regulates RNA polymerase II activity at the onset of transcriptional elongation.40,41 BET proteins also maintain an association with chromatin throughout mitosis, and this feature facilitates “gene bookmarking,” a process of rapid transcriptional reactivation of critical genes after mitosis.42 Underscoring the physiological importance of the BET proteins in cellular homeostasis is the observation that the experimental knockout of either Brd2 43 or Brd4 44 in mice results in early embryonic lethality.

The essential nature of the BET proteins and their fundamental importance in the regulation of transcription has increased interest in elucidating the molecular mechanisms of their action. These efforts will be aided considerably by the recent identification of the complete nuclear BET-protein interactome.32 In this study, three complementary global proteomic strategies were used to isolate the nuclear complexes containing the BET proteins. These data show that BET proteins are integral components of a large number of nuclear protein complexes that play a role in DNA replication, chromatin remodeling, DNA damage, and transcriptional regulation. Recurrent translocations of both BRD3 and BRD4 underpin the pathogenesis of NUT (or nuclear protein in testis) midline carcinoma,33 and more recently, RNA interference (RNAi) screening strategies have been used to identify a central role for BRD4 in acute myeloid leukemia.45 These findings have provided the impetus for investigating the potential of BET bromodomain inhibitors as novel anticancer agents.

BET Inhibitors in Cancer

Three different BET inhibitors (I-BET762,34 JQ1,33 and I-BET15132) show highly specific binding to both of the tandem domains of BRD2, BRD3, and BRD4 and inhibit their ability to engage with acetyl–lysine residues (Figure 3AFigure 3The BET Inhibitors.). These agents inhibit growth in a range of hematopoietic malignant cell lines32,45-47 and in human NUT midline carcinoma cell lines.33 Although the therapeutic efficacy of BET inhibition has not yet been tested in patients, its efficacy has been shown in vivo in murine models.

NUT Midline Carcinoma

NUT midline carcinoma is a rare but aggressive epithelial malignant disease that is invariably fatal, with a mean survival of less than 1 year.48 It is most often manifested in the midline of the upper aerodigestive tract and mediastinum. Histologic diagnosis of NUT midline carcinoma is difficult, and confirmation relies primarily on the demonstration of rearrangement of the NUT gene with the use of fluorescence in situ hybridization.48 BRD4 and BRD3 represent the NUT translocation partners in the majority of cases, and these fusions result in the aberrant localization of NUT to chromatin. Knockdown of the NUT fusions resulted in dramatic differentiation and growth arrest of the malignant cells, providing a therapeutic rationale for the targeting of BET proteins. Remarkably, the BET bromodomain inhibitor JQ1 was able to displace the BRD4–NUT fusion protein from chromatin and induce a rapid differentiation and arrest of proliferation in NUT midline carcinoma cell lines.33 In addition, JQ1 showed excellent efficacy in murine xenograft models of NUT midline carcinoma, resulting in tumor differentiation and regression and increased survival.33

Acute Myeloid Leukemia with MLL Translocations

Recurrent translocations of MLL, the mixed-lineage leukemia gene, result in aggressive leukemias with a poor prognosis that are often refractory to conventional therapies.49 More than 70 different MLL-translocation partners have been identified in leukemia.49 Despite this variation, a central abnormality in transcriptional elongation appears to underpin the molecular pathogenesis of this disease.50 Many MLL fusion partners are members of the superelongation complex (SEC), an important regulator of transcriptional elongation.51-53 Moreover, the N-terminal portion of MLL, a region conserved in all MLL fusion proteins, associates with another critical transcriptional complex called the polymerase-associated factor complex (PAFc).54,55 The functional integrity of these complexes is critical for malignant transformation by MLL fusion proteins.52-55 Two recent studies using complementary approaches have identified an essential role for the BET proteins in a broad range of acute myeloid leukemias, including MLL-translocated leukemias.32,45

Dawson and colleagues used a global proteomic approach that identified BRD3 and BRD4 as key components of both PAFc and SEC.32 In contrast, Zuber and colleagues used an RNAi screen to show that the depletion of BRD4 dramatically reduced the viability of MLL-AF9 leukemia in vitro and in vivo.45 Both JQ1 and the novel BET inhibitor I-BET151 showed remarkable efficacy in vitro and in vivo against MLL fusion leukemia, resulting in the rapid induction of cell-cycle arrest and apoptosis. Both studies highlighted a reduction in expression of critical regulators of transformation, including MYC, BCL2, and CDK6 (a cyclin-dependent protein kinase), after treatment with a BET inhibitor. Dawson and colleagues also found that the therapeutic efficacy of I-BET151 could be attributed at least in part to the inhibition of BRD3–BRD4-mediated recruitment of PAFc and SEC to chromatin (Figure 3). These molecular events resulted in reduced recruitment of RNA polymerase II to the promoters of crucial oncogenes such as MYC, BCL2, and CDK6.32 BET inhibition was also effective against primary human acute myeloid leukemia cells in vitro.32,45 Notably, in primary human MLL-translocated leukemia cells, BET inhibition dramatically reduced the clonogenic capacity of the leukemia stem-cell compartment, suggesting that disease eradication may be possible.

Although Zuber and colleagues focused primarily on MLL-translocated leukemias, they also showed the efficacy of BET inhibition in a range of acute myeloid leukemia subtypes in which MLL rearrangement was absent. These findings raise the intriguing possibility that BET inhibition may be a more broadly applicable therapy in this genetically heterogeneous disease.

Multiple Myeloma and Burkitt's Lymphoma

Multiple myeloma is an incurable plasma-cell dyscrasia. Although the disease is genetically heterogeneous,56 disordered expression of the myelocytomatosis viral oncogene homolog MYC is a prominent feature, providing the rationale to test for BET inhibition.57 Similarly, Burkitt's lymphoma, an aggressive lymphoproliferative disorder, is characterized by recurrent translocations of MYC, most frequently involving the IGH locus that results in constitutive high-level expression of MYC. Several multiple myeloma cell lines show a dramatic response to BET inhibition with JQ1.46,47 Treatment with JQ1 leads to a profound cell-cycle arrest and apoptosis of the cell lines; this cellular phenotype is associated with a reduction in MYC transcription and protein expression. This transcriptional repression of MYC is linked to a reduction in the binding of chromatin by BRD4, upstream of the MYC promoter. Delmore and colleagues further translated these findings into work with primary human multiple myeloma cells and murine models of multiple myeloma, further indicating the therapeutic potential of BET inhibition in this disease.46 In addition, Mertz and colleagues extended their findings to reveal a survival benefit in murine xenograft models of Burkitt's lymphoma and acute myeloid leukemia.47

Taken together, the studies involving BET bromodomain inhibitors further validate the use of epigenetic targets in cancer therapy. They show that it is possible to target epigenetic readers as well as catalytic writers and erasers. Moreover, the studies establish that the specific targeting of protein–protein interactions can have antitumor effects in vitro and in animal models. Several questions warrant further investigation. It is unclear why these drugs have effects in hematopoietic cancers but not in the majority of common solid tumors, including malignant tumors of the breast and cervix.47 It is also unclear why a compound that inhibits the localization of proteins germane to transcription in general alters the expression of only a few hundred specific and reproducible genes. Although we have some insights into the molecular mechanism determining efficacy in the MLL fusion leukemias32 and in NUT midline carcinoma,33 it is largely unclear what molecular events dictate efficacy in the other sensitive cancers. A central theme in all the studies cited is the down-regulation of MYC. However, even though MYC has a prominent role in cancer,58 it is unlikely that the profound effects observed with the introduction of BET inhibition are mediated solely by MYC inhibition. There are many malignant cell lines that overexpress MYC yet fail to respond to BET inhibition47; MYC expression is not always affected by BET inhibition,47 and MYC down-regulation is not predictive of a response to BET inhibition.32,45 Furthermore, MYC overexpression fails to prevent the apoptosis induced by BET inhibition.45 Identification of the complete BET protein interactome has shown that the BET proteins are integral components of a large number of nuclear protein complexes.32 This finding suggests that BET proteins are involved with a variety of molecular mechanisms.

Conclusions

Cancer epigenetics is an area of ongoing research that continues to inform our understanding of the molecular pathogenesis of cancer and to identify novel therapeutic targets. Advances in medicinal chemistry have now made it possible to specifically target not just the catalytic activity of epigenetic regulators but also the protein–protein interaction modules that localize many of these proteins to chromatin. However, as is the case for the majority of tumors sensitive to BET inhibition, epigenetic therapeutic targets are not necessarily mutated in sensitive tumor types. Therefore, simple mutational screening may not provide a predictor of response. Potential means of identifying sensitive tumor types include combining drug sensitivity with mutational screening, as has recently been reported in two large screening studies,59,60 and using transcriptional or epigenetic biomarkers to predict response. The successful use of the bromodomain inhibitors in cancer therapy is likely to further energize basic scientists, clinicians, and the pharmaceutical industry to search for compounds that may similarly target and disrupt other chromatin reader motifs, including chromodomains and PHD fingers, which are already known to play key roles in certain cancers.31

Disclosure forms provided by the authors are available with the full text of this article at NEJM.org.

We thank Andy Bannister for reviewing an earlier version of this article and for helpful comments; and Rab Prinjha and Chun Wa Chung for providing the chemical structures and surface structure used in Figure 3A.

 

Source Information

From the Department of Haematology, Cambridge Institute for Medical Research (M.A.D., B.J.P.H.), Cambridge University Hospitals National Health Service Foundation Trust (M.A.D., B.J.P.H.), the Gurdon Institute and Department of Pathology (M.A.D., T.K.), and the Wellcome Trust and Medical Research Council Cambridge Stem Cell Institute (B.J.P.H.), University of Cambridge — all in Cambridge, United Kingdom.
Address reprint requests to Dr. Huntly at the Department of Haematology, Cambridge Institute for Medical Research, University of Cambridge, Cambridge CB2 0XY, United Kingdom, or at bjph2@cam.ac.uk.