Small Cell Lung Cancer Exhibits Frequent Inactivating Mutations in the Histone Methyltransferase KMT2D/MLL2: CALGB 151111 (Alliance)

Abstract

Introduction: SCLC 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.

Methods: To identify potential driver mutations in human SCLC we sequenced the whole exomes of 18 primary SCLCs and seven cell lines along with matched normal controls. We extended these data by resequencing a panel of genes across 40 primary SCLCs and 48 cell lines.

Results: We report frequent mutations in the lysine methyltransferase 2D gene (KMT2D) (also known as MLL2), a key regulator of transcriptional enhancer function. KMT2D exhibited truncating nonsense/frameshift/splice site mutations in 8% of SCLC tumors and 17% of SCLC cell lines. We found that KMT2D mutation in human SCLC cell lines was associated with reduced lysine methyltransferase 2D protein levels and reduced monomethylation of histone H3 lysine 4, a mark associated with transcriptional enhancers. We also found mutations in other genes associated with transcriptional enhancer control, including CREB binding protein gene (CREBBP), E1A binding protein p300 gene (EP300), and chromodomain helicase DNA binding protein 7 gene (CHD7), and we report mutations in additional chromatin remodeling genes such as polybromo 1 gene (PBRM1).

Conclusions: These data indicate that KMT2D is one of the major mutated genes in SCLC, and they point to perturbation of transcriptional enhancer control as potentially contributing to SCLC.

Keywords: Genomics; KMT2D; MLL2; SCLC; Small cell lung cancer.

Figure 1

Exome analyses of human SCLC. (A) Number of protein-altering mutations in each sample, separated for missense, nonsense, insertions/deletions (indels), and essential splice site mutations. (B) Patterns of transitions and transversions in primary SCLC and in cell lines. (C) Pie charts showing mutation spectrum in selected SCLC-mutated genes. RB1, retinoblastoma gene; TP53, tumor protein p53 gene; KMT2D, lysine methyltransferase 2D gene; NOTCH1, notch 1 gene; CREBBP, CREB binding protein gene; PBRM1, polybromo 1 gene.

Figure 2

Candidate gene–targeted resequencing in SCLC tumors and cell lines. Oncomap showing mutations in a panel of genes chosen for targeted resequencing analysis in SCLC tumors and cell lines. TP53, tumor protein p53 gene; RB1, retinoblastoma gene; NOTCH1, notch 1 gene; KMT2C, lysine methyltransferase 2C gene; KMT2D, lysine methyltransferase 2D gene; KDM6A, lysine demethylase 6A gene; KMT2A, lysine methyltransferase 2A gene; CREBBP, CREB binding protein gene; EP300, E1A binding protein p300 gene; PBRM1, polybromo 1 gene; ARID1A, AT-rich interaction domain 1 gene; ARID1B, AT-rich interaction domain 1B gene; ARID4A, AT-rich interaction domain 4A gene; CHD7, chromodomain helicase DNA binding protein 20 gene; SETD2, SET domain containing 2 gene; PIK3CA, phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha gene; PTEN, phosphatase and tensin homolog gene; ELAVL2, ELAV like neuron-specific RNA protein binding 2 gene; COBL, cordon-bleu WH2 repeat protein gene; SLIT2, slit guidance ligand 2 gene; EPHA7, EPH receptor A7 gene; PTPRD, protein tyrosine phosphatase, receptor type D gene; RUNX1T1, RUNX1 translocation partner 1 gene.

Figure 3

Mutations in lysine methyltransferase 2D gene (KMT2D) and other chromatin regulators in SCLC. Lollipop plots showing location and type of mutations across a panel of SCLC-mutated chromatin regulating genes. Red circle indicates truncating (nonsense, frameshift, or essential splice site) mutation, and green circle indicates missense mutation. Mutations in KMT2D and polybromo 1 gene (PBRM1) shown also include Sanger sequencing–validated mutations identified through analyses of exome and RNA sequencing data. KDM6A, lysine demethylase 6A gene; ARID1A, AT-rich interaction domain 1 gene; ARID1B, AT-rich interaction domain 1B gene; CHD7, chromodomain helicase DNA binding protein 20 gene.

Figure 4

Mutations in lysine methyltransferase 2D gene (KMT2D) are associated with reduced lysine methyltransferase 2D protein (KMT2D) and reduced histone H3K4 monomethylation. (A) Western blot analysis showing expression of KMT2D across human SCLC cell lines that differ in KMT2D mutation status. Actin is used as a loading control. (B) Western blot analysis of extracted histones showing reduced histone H3K4 monomethylation (H3K4-1me) in the KMT2D-mutant (mut) group. Total histone H3 is used as a loading control. Quantification based on densitometry is shown to the right. A trend toward decreased H3K4 dimethylation (H3K4-2me) is also seen in the KMT2D mutants, although this was not statistically significant. p Values from Student’s t test are shown. WT, wild type; kDa, kilodalton.