Histone Code Aberrancies in Small Cell Lung Cancer
Authors: Eric E. Gardner, John T. Poirier, Charles M. Rudin
Journal: Journal of Thoracic Oncology (2017)

SCLC is characterized by inactivation of the tumor suppressor genes tumor protein p53 gene (TP53) and retinoblastoma gene (RB1), neuroendocrine histologic features, and high proliferative capacity, leading to early metastasis and an overall aggressive clinical course. Recent evidence in both human and murine models of SCLC have shown tumor suppressor roles for phosphatase and tensin homolog1Cui M. Augert A. Rongione M. et al.PTEN is a potent suppressor of small cell lung cancer.Mol Cancer Res. 2014; 12: 654-659Crossref PubMed Scopus (79) Google Scholar, 2McFadden D.G. Papagiannakopoulos T. Taylor-Weiner A. et al.Genetic and clonal dissection of murine small cell lung carcinoma progression by genome sequencing.Cell. 2014; 156: 1298-1311Abstract Full Text Full Text PDF PubMed Scopus (197) Google Scholar and NOTCH,3George J. Lim J.S. Jang S.J. et al.Comprehensive genomic profiles of small cell lung cancer.Nature. 2015; 524: 47-53Crossref PubMed Scopus (1215) Google Scholar as well as oncogenic roles for MYC family members4Huijbers I.J. Bin Ali R. Pritchard C. et al.Rapid target gene validation in complex cancer mouse models using re-derived embryonic stem cells.EMBO Mol Med. 2014; 6: 212-225Crossref PubMed Scopus (69) Google Scholar, 5Mollaoglu G. Guthrie M.R. Bohm S. et al.MYC drives progression of small cell lung cancer to a variant neuroendocrine subtype with vulnerability to aurora kinase inhibition.Cancer Cell. 2017; 31: 270-285Abstract Full Text Full Text PDF PubMed Scopus (275) Google Scholar and nuclear factor I/B.6Denny S.K. Yang D. Chuang C.H. et al.Nfib promotes metastasis through a widespread increase in chromatin accessibility.Cell. 2016; 166: 328-342Abstract Full Text Full Text PDF PubMed Scopus (219) Google Scholar, 7Dooley A.L. Winslow M.M. Chiang D.Y. et al.Nuclear factor I/B is an oncogene in small cell lung cancer.Genes Dev. 2011; 25: 1470-1475Crossref PubMed Scopus (125) Google Scholar, 8Semenova E.A. Kwon M.C. Monkhors K. et al.Transcription factor NFIB is a driver of small cell lung cancer progression in mice and marks metastatic disease in patients.Cell Rep. 2016; 16: 631-643Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar, 9Wu N. Jia D. Ibrahim A.H. Bachurski C.J. Gronostajski R.M. MacPherson D. NFIB overexpression cooperates with Rb/p53 deletion to promote small cell lung cancer.Oncotarget. 2016; 7: 57514-57524Crossref PubMed Scopus (48) Google Scholar Now, new sequencing data on primary SCLC and a reanalysis of existing data sets highlight frequent inactivation of the lysine methyltransferase 2D gene (KMT2D/MLL2). In this issue of the Journal of Thoracic Oncology, an article from Augert et al.10Augert A. Zhang Q. Bates B. et al.Small cell lung cancer exhibits frequent inactivating mutations in the histone methyltransferase KMT2D/MLL2: CALGB 151111 (Alliance).J Thorac Oncol. 2017; 12: 704-713Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar adds to the growing evidence that SCLC is an epigenetically perturbed disease. This study demonstrates that nonsense/truncating mutations of KMT2D/MLL2 occur in primary tumors and cell lines of SCLC origin. Lysine methyltransferase 2D (KMT2D) is one of at least five members of the KMT2 family of lysine methyltransferase enzymes involved in methylating histone H3 at lysine residue 4 (K4). KMT2D functions principally in monomethylating H3K4 and designating distal enhancer elements, which are often bound by factors known to direct cell fate.11Lee J.E. Wang C. Xu S. et al.H3K4 mono- and di-methyltransferase MLL4 is required for enhancer activation during cell differentiation.Elife. 2013; 2: e01503Crossref Scopus (300) Google Scholar Using 18 primary, mostly limited-stage SCLC samples with matched normal DNA, the authors performed whole exome sequencing and identified two of 18 cases with nonsense mutations in KMT2D. They supplemented these findings by sequencing seven SCLC cell lines with matched normal Epstein-Barr virus–transformed lymphoblast cells and targeted sequencing of a set of 40 primary and 48 SCLC cell lines. Reanalysis of two larger data sets on primary SCLC also showed a frequency of inactivating mutations in KMT2D in more than 10% of cases. When cell lines were analyzed, heterozygous and homozygous truncating mutations in KMT2D were shown to express lower levels of the KMT2D protein by Western blot, and purified histones from these cell lines displayed a lower level of H3K4me1 in the KMT2D-mutant cells lines, but not H3K4me2, when normalized against total levels of histone H3. Importantly, this work demonstrates that in addition to the previously described inactivating mutations in the histone acetyltransferase enzymes E1A binding protein p300, CREB binding protein, and (to some extent) lysine acetyltransferase 6B,3George J. Lim J.S. Jang S.J. et al.Comprehensive genomic profiles of small cell lung cancer.Nature. 2015; 524: 47-53Crossref PubMed Scopus (1215) Google Scholar, 12Simo-Riudalbas L. Perez-Salvia M. Setien F. et al.KAT6B is a tumor suppressor histone H3 lysine 23 acetyltransferase undergoing genomic loss in small cell lung cancer.Cancer Res. 2015; 75: 3936-3945Crossref PubMed Scopus (53) Google Scholar SCLC can also be characterized by inactivation of histone methyltransferase enzymes in the mixed lineage leukemia (MLL) family. Although these genes are relatively large, the authors were keen to note that mutations in other KMT2 family members were not detected at such frequency, despite being of similar size. Previous consortia-based sequencing efforts have identified the KMT family members MLL2 and lysine methyltransferase 2C gene (KMT2C/MML3) to be among the most frequently mutated or rearranged genes in cancer.13Kandoth C. McLellan M.D. Vandin F. et al.Mutational landscape and significance across 12 major cancer types.Nature. 2013; 502: 333-339Crossref PubMed Scopus (2935) Google Scholar Mutations in KMT2D/MLL2 have been previously described to concentrate in either the SET or PHD domains of the protein, affecting methyltransferase activity or KMT complex assembly and targeting, respectively.14Rao R.C. Dou Y. Hijacked in cancer: the KMT2 (MLL) family of methyltransferases.Nat Rev Cancer. 2015; 15: 334-346Crossref PubMed Scopus (353) Google Scholar The MLL family members were originally identified as defining alterations in subgroups of aggressive leukemias; however, unlike the inactivating mutations identified by Augert et al.,10Augert A. Zhang Q. Bates B. et al.Small cell lung cancer exhibits frequent inactivating mutations in the histone methyltransferase KMT2D/MLL2: CALGB 151111 (Alliance).J Thorac Oncol. 2017; 12: 704-713Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar the MLL family is typically rearranged in the form of translocations, genomic amplifications, or tandem duplications, yielding MLL fusions that create "chimeric" versions possessing chromatin-targeting function in addition to recruitment of components of the transcriptional elongation complex.15Mohan M. Lin C. Guest E. Shilatifard A. Licensed to elongate: a molecular mechanism for MLL-based leukaemogenesis.Nat Rev Cancer. 2010; 10: 721-728Crossref PubMed Scopus (142) Google Scholar Previous strategies targeting MLL and MLL fusions have relied on interrupting protein-protein interactions that are critical to the downstream function of fusions,16Borkin D. He S. Miao H. et al.Pharmacologic inhibition of the menin-MLL interaction blocks progression of MLL leukemia in vivo.Cancer Cell. 2015; 27: 589-602Abstract Full Text Full Text PDF PubMed Scopus (232) Google Scholar or more recently, inhibiting degradation pathways intrinsic to the wild-type MLL protein, effectively competing with the fusion for chromatin occupancy.17Liang K. Volk A.G. Haug J.S. et al.Therapeutic targeting of MLL degradation pathways in MLL-rearranged leukemia.Cell. 2017; 168 (e13): 59-72Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar It is not yet clear whether KMT2D-mutant SCLC will benefit from therapeutic inhibition of the H3K4 demethylase lysine demethylase 1A (LSD1). A previous study examining the single-agent activity of GSK2879552 in SCLC demonstrated an association between global DNA hypomethylation and sensitivity to LSD1 chemical inhibition.18Mohammad H.P. Smitheman K.N. Kamat C.D. et al.A DNA hypomethylation signature predicts antitumor activity of LSD1 inhibitors in SCLC.Cancer Cell. 2015; 28: 57-69Abstract Full Text Full Text PDF PubMed Scopus (350) Google Scholar Cells lines found by the authors to be heterozygous and homozygous KMT2D mutant include examples previously reported as either sensitive or resistant to LSD1 chemical inhibition.18Mohammad H.P. Smitheman K.N. Kamat C.D. et al.A DNA hypomethylation signature predicts antitumor activity of LSD1 inhibitors in SCLC.Cancer Cell. 2015; 28: 57-69Abstract Full Text Full Text PDF PubMed Scopus (350) Google Scholar This observational association suggests that KMT2D status alone is not a predictor of sensitivity to this class of LSD1 chemical inhibitor; however, we note that many of these SCLC cell lines have additional mutations in chromatin-modifying enzymes and differential expression of other H3K4 demethylases, complicating mechanistic interpretation. Indeed, a more straightforward path in addressing the mechanistic contribution of KMT2D to SCLC pathogenesis and therapeutic vulnerabilities lies in modeling loss of Kmt2d in the mouse. As the MacPherson group has expertise in mouse modeling of SCLC, it will be important to understand the contribution that heterozygous and homozygous inactivating KMT2D (Kmt2d/Mll4 in mice) alleles have in the development, metastatic potential, and chemosensitivity of murine SCLC. The ability to demonstrate whether KMT2D can function as a haploinsufficient tumor suppressor gene in SCLC will strengthen what we know about SCLC biology. Combinations of inactivating alleles in other chromatin remodeling enzymes such as p300 and CREB binding protein, which are both frequently inactivated in almost mutual exclusivity in primary SCLC data sets, will additionally teach the SCLC community about putative tumor suppressors functions in maintenance of the SCLC epigenome. The utility of modeling these changes in mouse models of SCLC will aid the interrogation of epigenome-specific changes that occur during the development and progression of KMT2D-mutated SCLC. Sequencing approaches interrogating changes in histone modifications (e.g., chromatin immunoprecipitation sequencing) and chromatin accessibility (e.g., assay for transposase-accessible chromatin sequencing) will be critical in understanding how these chromatin remodeling enzymes change the path of disease in an epigenome-wide manner. It is intriguing to consider the consequences of losing KMT2D function in SCLC in the context of what we currently know about the epigenetic landscape. Studies from other groups have shown that enhancer regions within the epigenome harbor high levels of both H3K4me1 and one of several potential H3K27 marks, termed bivalent enhancers.19Jiang Y. Dominguez P.M. Melnick A.M. The many layers of epigenetic dysfunction in B-cell lymphomas.Curr Opin Hematol. 2016; 23: 377-384Crossref PubMed Scopus (30) Google Scholar Depending on whether that H3K27 mark is active (e.g., H3K27Ac) or repressive (H3K27me3), the enhancer element is considered to be in an "active" or "poised" state, respectively. Considering that SCLC is characterized by loss of RB1, E2F family members are disinhibited, driving high expression levels of enhancer of zeste 2 polycomb repressive complex 2 subunit, which is responsible for H3K27me3 deposition. This observation is further underscored by reported lysine demethylase 6A gene (KDM6A)/UTX demethylase mutations in a small number of patients with SCLC in this report and others. Taken together, individual or combined losses of certain histone lysine-, methyl-, and acetyltransferases with high level expression of enhancer of zeste 2 polycomb repressive complex 2 subunit and LSD1 paint an image of a largely repressed chromatin landscape that is consistent with the exceptionally high level of global DNA methylation also seen in SCLC and reported to be associated with poorer prognosis.20Poirier J.T. Gardner E.E. Connis N. et al.DNA methylation in small cell lung cancer defines distinct disease subtypes and correlates with high expression of EZH2.Oncogene. 2015; 34: 5869-5878Crossref PubMed Scopus (152) Google Scholar, 21Saito Y. Nagae G. Motoi N. et al.Prognostic significance of CpG island methylator phenotype in surgically resected small cell lung carcinoma.Cancer Sci. 2016; 107: 320-325Crossref PubMed Scopus (18) Google Scholar With additional evidence for frequent mutations of switch/sucrose nonfermentable components hSWI/SNF across a spectrum of solid tumors,22Kadoch C. Hargreaves D.C. Hodges C. et al.Proteomic and bioinformatic analysis of mammalian SWI/SNF complexes identifies extensive roles in human malignancy.Nat Genet. 2013; 45: 592-601Crossref PubMed Scopus (823) Google Scholar future work will need to determine which subsets of SCLC are likely to benefit from current approaches to target chromatin dynamic states. Small Cell Lung Cancer Exhibits Frequent Inactivating Mutations in the Histone Methyltransferase KMT2D/MLL2: CALGB 151111 (Alliance)Journal of Thoracic OncologyVol. 12Issue 4PreviewSCLC is a lethal neuroendocrine tumor type that is highly prone to metastasis. There is an urgency to understand the mutated genes that promote SCLC, as there are no approved targeted therapies yet available. SCLC is rarely resected, limiting the number of samples available for genomic analyses of somatic mutations. Full-Text PDF Open Archive