In 2008, the respective laboratories of Dr. Marco Marra and Dr. Randy Gascoyne at Canada’s Michael Smith Genome Sciences Centre (GSC), British Columbia Cancer Agency (BCCA), partnered together to sequence the DNA and RNA of the two most common non-Hodgkin lymphomas (NHLs). Their objective was to diagnostically characterize the NHL tumors by using advanced sequencing techniques. Previous molecular studies had identified a few hallmark abnormalities in these cancers, but a more detailed picture of the genomic landscape was needed. Their comprehensive analysis, supported by the Cancer Genome Characterization Initiative (CGCI), revealed a surprising array of novel alterations1,2,3. Among the most interesting were alterations that pointed to the epigenome as an influential player in lymphomagenesis.
The two NHLs, follicular lymphoma (FL) and diffuse large B cell lymphoma (DLBCL), are cancers of mature B cells. FL is slow-growing and difficult to treat; DLBCL is often aggressive and clinically diverse. In both cancers, processes involved in B cell differentiation are frequently perturbed4. Normal differentiation occurs when an antigen-activated B cell induces formation of a germinal center in the lymph nodes. The B cell localizes to the germinal center and then proliferates, while simultaneously undergoing antibody diversification. The resulting outcome is a diverse collection of B cells with antigen receptors of varying affinities. The mature B cells with the highest affinity receptors are selected to become memory or antibody-secreting plasma cells and exit the germinal center. Those not selected self-destruct through apoptosis.
Lymphomagenesis can occur when acquired alterations block completion of differentiation and cause B cells to divide uncontrollably and evade cell death. Consequently, FL and DLBCL may resemble B cells at a particular stage of differentiation4. Gene expression profiling revealed FL tumors have expression signatures like those of germinal center B cells. Additionally, DLBCL tumors can have signatures like those of germinal center B cells or activated B cells (B cells that have exited the germinal center, but have not fully matured into memory or plasma cells). Accordingly, DLBCL tumors are classified into at least two subgroups: germinal center B cell-like (GCB) and activated B cell-like (ABC). These subgroups show differences in growth behavior and response to treatment, suggesting differences in pathogenesis as well.
Separate genome-wide characterization studies provided additional clues that the underlying pathogenesis differs for each NHL subtype: several genetic abnormalities are specific to lymphomas derived from either germinal center B cells (i.e. FL and GCB DLBCL) or activated B cells (i.e. ABC DLBCL). For example, overexpression of the BCL2 oncoprotein, a result of a translocation between chromosomes 14 and 18, is only found in GCB-derived lymphomas (89% FL and 30-40% GCB DLBCL)5,6. NF-kB signaling dysregulation, on the other hand, is only found in the ABC subtype7,8. These initial molecular profiles were very insightful, but limited in granularity. Dr. Marra and the CGCI-supported investigators at the GSC reasoned that exposing underlying genetic alterations to inform more precise treatments for FL and DLBCL would require higher-resolution, integrative “omic” analyses.
By applying their expertise in sequencing and bioinformatics, the CGCI investigators examined the whole genomes, exomes, and transcriptomes of a large number of FL and DLBCL tissues. They published their results in three papers over the course of several years1,2,3. Integrating the data revealed an interesting trend in these lymphomas: genes encoding histone-modifying proteins and histone proteins themselves were frequently mutated. Of those genes, EZH2 and MLL2 had the most recurrent mutations. Both encode enzymes called methyltransferases that regulate gene expression by adding methyl groups to histones. By methylating histones, these enzymes modify the degree to which the DNA is packed and, thus, the degree to which genes and their promoter regions are accessible to transcriptional machinery. The EZH2 and MLL2 mutations are particularly compelling, because they are both predicted to function in malignancy by turning down gene expression.
EZH2, an enzymatic subunit of the polycomb repressor complex 2, plays an important role in a variety of developmental processes. It tri-methylates lysine 27 on histone 3 (H3K27me3), which generally represses transcription. Expressed in a specific time window during B cell differentiation, EZH2 is required for the formation and functions of the germinal center9. Late-stage differentiating B cells must turn down EZH2 expression, so they can exit the germinal center to become memory or plasma cells. Before the publication of Morin et al. 2010, overexpression of EZH2 was linked to breast, prostate, and other cancers10,11, but mutations in the EZH2 gene itself had not yet been associated with any cancer type.
The CGCI investigators, including Dr. Marra’s then graduate student Ryan Morin, discovered a mutation hot spot in the EZH2 gene in their sequencing of mRNAs of DLBCL and FL cases1. The mutations recurrently affected tyrosine 641 (Y641) in the catalytic domain of EZH2. Interestingly, the Y641 mutations were restricted to GCB-derived lymphomas (21% GCB DLBCL and 7.2% FL), suggesting they are important in GCB-derived B cell malignancy.
To determine the effect of Y641 mutations on EZH2 function, the investigators performed in vitro experiments on several purified mutant proteins. The mutations disrupted the enzyme’s ability to add the first methyl group onto a histone peptide, indicating they were loss of function (inactivating). However, they tested the EZH2 mutant proteins singularly, and not in the presence of the wildtype EZH2. Y641 mutations were heterozygous in all cases identified, meaning each tumor had one copy each of the mutant and wildtype EZH2. Later experiments done by a different group showed that EZH2 mutations were actually gain of function (activating) when paired with the wildtype12,13. Furthermore, EZH2-mutant cell lines have more tri-methylated H3K27 sites as compared to wildtype EZH2 cells, supporting the notion the mutations are indeed gain of function12,14. Reconciling the two in vitro experimental results, researchers theorized that wildtype EZH2 efficiently adds the first methyl group to H3K27, which enables mutated EZH2 to add the second and third methyl groups more readily than the wildtype. The end result is more tri-methylated H3K27 sites and, thus, more repressive chromatin.
The question of precisely how activating mutations in EZH2 contribute to B cell lymphomagenesis has elicited great interest from different research groups and is currently being explored. One recent study in mouse models and human cell lines indicates that mutant EZH2 may continuously silence genes antagonistic to malignancy (e.g. germinal center exit and proliferation checkpoint genes), which are normally transiently repressed in germinal center B cells9. As a result, mature B cells remain locked in a state of incomplete differentiation in the germinal center, where they rapidly grow and divide unhindered. Taking this into consideration, it would make sense that EZH2 mutations would not participate in the pathogenesis of ABC DLBCL, because EZH2 is no longer expressed in activated B cells that have left the germinal center.
MLL2 methylates the fourth lysine of histone 3 (H3K4), which promotes transcription. MLL2 regulates a diverse array of cellular processes and signaling pathways, including the tumor suppressor p53 pathway15, and is frequently mutated in certain cancers. For example, another group of CGCI investigators studying medulloblastoma identified recurrent mutations in MLL2 in 14% of all cases16. The majority of MLL2 mutations identified in this study were predicted to be loss of function, hinting that it may be a tumor suppressor. However, little is known about how MLL2 participates in oncogenesis.
In their analysis of sequence from the genomes, exomes, and transcriptomes of 117 FL and DLBCL tumors, Morin and the CGCI-supported investigators identified MLL2 as one of 109 recurrently mutated genes2. MLL2 showed the greatest mutational frequency, with the majority of identified mutations predicted to disrupt MLL2 function. These largely inactivating MLL2 mutations were found in 89% of FL cases and 32% of DLBCL cases, a prevalence on par with the t(14;18) translocation. Unlike EZH2, MLL2 did not show bias towards any DLBCL subtype. Because the role of MLL2 in B cell differentiation is not well-established, the function of MLL2 mutations in lymphomagenesis remains unclear. However, it is possible that impairment of its H3K4 methylating activity alters gene expression in favor of malignancy. This remains to be determined.
So, What’s Next?
Sequencing the DNA and RNA of FL and DLBCL tumors revealed many frequent alterations in histone-modifying genes, most notably in genes affecting H3K27 (EZH2) and H3K4 (MLL2) methylation. The investigators at the GSC, along with several other groups, are following up by studying how the EZH2 and MLL2 alterations specifically contribute to lymphoma biology and how they may be exploited to improve treatment strategies for these cancers. Because both genes might regulate chromatin marks in the same promoter regions and H3K4 methylation opposes H3K27 methylation17, researchers speculate that both alterations produce the same outcome in differentiating B cells: increased H3K27-methylated chromatin and repressed transcription of genes antagonistic to malignancy18. However, additional and/or alternative theories for their roles in lymphomagenesis are entirely plausible. Both EZH2 and MLL2 have widespread functions that are not fully characterized and their exact mechanisms in B cell differentiation are not completely or clearly defined.
For EZH2, the path to drug development is more straightforward, because the recurrent mutations result in increased activity, which can be counteracted by chemical inhibition. The biopharmaceutical company, Epizyme, recently reported tumor growth inhibition in NHL mouse models bearing the cancer-causing EZH2 mutations when treated with a small molecule inhibitor of EZH (EPZ-6438)19. They have since initiated a phase I/II clinical trial to test the safety and efficacy of using EPZ-6438 to treat lymphomas harboring those mutations20. For MLL2, therapeutic development is trickier, because the recurrent mutations result in decreased activity, rendering chemical inhibition a non-starter. To circumvent this problem, Dr. Marra’s lab is working on identifying synthetic lethal interactions in MLL2 mutant tumors as potential alternative drug targets21.
The Emerging Role of the Epigenome
In addition to EZH2 and MLL2 mutations, the CGCI-supported investigators and other groups detected frequent mutations in other genes encoding previously identified epigenetic modifiers (e.g. CREBBP/EP300)2,3, 22. They also found novel mutations in genes encoding histone proteins (e.g. HIST1H1C) and transcription factors that recruit epigenetic modifiers (e.g. MEF2B), confirming the importance of epigenetic dysregulation in B cell lymphoma biology. Clearly, more research must be done to elucidate the epigenome’s specific mechanistic contributions.
Dr. Marra and colleagues are currently applying their savvy sequencing and bioinformatics approaches to examine the epigenome of NHL to gain deeper insight into its role in lymphomagenesis. Considering the mysteries that have been revealed in their previous studies of the genome and transcriptome, the NHL epigenome could prove equally illuminating.
- Morin RD, Johnson NA, Severson TM, Mungall AJ, An J, Goya R, et al. (2010). Somatic mutations altering EZH2 (Tyr641) in follicular and diffuse large B-cell lymphomas of germinal-center origin. Nature Genetics 42(2):181–5 (PMID: 20081860)
- Morin RD, Mendez-Lago M, Mungall AJ, Goya R, Mungall KL, Corbett RD, et al. (2011). Frequent mutation of histone-modifying genes in non-Hodgkin lymphoma. Nature 476(7360):298–303 (PMID: 21796119)
- Morin RD, Mungall K, Pleasance E, Mungall AJ, Goya R, Huff R, et al. (2013). Mutational and structural analysis of diffuse large B-cell lymphoma using whole genome sequencing. Blood 122(7):1256-65 (PMID: 23699601)
- Shaffer AL, Young RM, Staudt LM (2012). Pathogenesis of human B cell lymphomas. Annual Review of Immunology 30:565–610. (PMID: 22224767)
- Horsman DE, Okamoto I, Ludkovski O, Le N, Harder L, Gesk S, Siebert R, et al. (2003). Follicular lymphoma lacking the t(14;18)(q32;q21): identification of two disease subtypes. Br. J. Haematol. 120(3):424–433 (PMID: 12580956)
- Iqbal J, Sanger WG, Horsman DE, Rosenwald A, Pickering DL, Dave B, et al. (2004). BCL2 translocation defines a unique tumor subset within the germinal center B-cell-like diffuse large B-cell lymphoma. Am. J. Pathol. 165(1):159–166 (PMID: 16418494)
- Davis RE, Brown KD, Siebenlist U, Staudt LM (2001) Constitutive nuclear factor κB activity is required for survival of activated B cell-like diffuse large B cell lymphoma cells. J. Exp. Med. 194(12):1861–1874 (PMID: 11748286)
- Lenz G, Davis RE, Ngo VN, Lam L, George TC, Wright GW, Dave SS, et al. (2008). Oncogenic CARD11 mutations in human diffuse large B cell lymphoma. Science 319(5870):1676–1679 (PMID: 18323416)
- Béguelin W, Popovic R, Teater M, Jiang Y, Bunting KL, Rosen M, et al. (2013). EZH2 is required for germinal center formation and somatic EZH2 mutations promote lymphoid transformation. Cancer Cell 23(5):677–92 (PMID: 23680150)
- Varambally S, Dhanasekaran SM, Zhou M, Barrette TR, Kumar-Sinha C, et al. (2002). The polycomb group protein EZH2 is involved in progression of prostate cancer. Nature 419(6907):624–629 (PMID: 12374981)
- Kleer CG, Cao Q, Varambally S, Shen R, Ota I, Tomlins SA, Ghosh D, et al. (2003). EZH2 is a marker of aggressive breast cancer and promotes neoplastic transformation of breast epithelial cells. Proc. Natl. Acad. Sci. USA 100(20):11606–11611 (PMID: 14500907)
- Sneeringer CJ, Scott MP, Kuntz KW, Knutson SK, Pollock RM, Richon VM, Copeland RA (2010). Coordinated activities of wild-type plus mutant EZH2 drive tumor-associated hypertrimethylation of lysine 27 on histone H3 (H3K27) in human B-cell lymphomas. Proc. Natl Acad. Sci. USA 107(49):20980–20985 (PMID: 21078963)
- Yap DB, Chu J, Berg T, Schapira M, Cheng SW, Moradian A, Morin RD, et al. (2011). Somatic mutations at EZH2 Y641 act dominantly through a mechanism of selectively altered PRC2 catalytic activity, to increase H3K27 trimethylation. Blood 117:2451–2459 (PMID: 21190999)
- McCabe MT, Graves AP, Ganji G, Diaz E, Halsey WS, Jiang Y, et al. (2012). Mutation of A677 in histone methyltransferase EZH2 in human B-cell lymphoma promotes hypertrimethylation of histone H3 on lysine 27 (H3K27). Proc. Natl Acad. Sci. USA 109(8):2989–2994 (PMID: 22323599)
- Guo C, Chang CC, Wortham M, Chen LH, Kernagis DN, Qin X, Cho YW, et al. (2012). Global identification of MLL2-targeted loci reveals MLL2’s role in diverse signaling pathways Proc. Natl Acad. Sci. USA 109(43):17603-17608 (PMID: 23045699)
- Parsons DW, Li M, Zhang X, Jones S, Leary RJ, Lin JC, Boca SM, Carter H, et al. (2011).The genetic landscape of the childhood cancer medulloblastoma. Science 331(6016):435–439 (PMID: 21163964)
- Shaknovich R, Melnick A (2011). Epigenetics and B-cell lymphoma. Current Opinion in Hematology 18(4):293–9 (PMID: 21577103)
- Mills AA (2010). Throwing the cancer switch: reciprocal roles of polycomb and trithorax proteins. Nature Reviews Cancer 10(10):669–82 (PMID: 20865010)
- Knutson SK, Kawano S, Minoshima Y, Warholic N, Huang KC, et al. (2014). Selective inhibition of EZH2 by EPZ-6438 leads to potent antitumor activity in EZH2 mutant non-Hodgkin lymphoma. Mol. Cancer Therapeutics. Epub ahead of print, Feb 21, 2014 (PMID: 24563539)
- Pasqualucci L1, Dominguez-Sola D, Chiarenza A, Fabbri G, Grunn A, et al. (2011). Inactivating mutations of acetyltransferase genes in B-cell lymphoma. Nature 471(7337):189-95 (PMID: 21390126)