Purpose: Patients with anaplastic lymphoma kinase (ALK) gene rearrangements often manifest dramatic responses to crizotinib, a small-molecule ALK inhibitor. Unfortunately, not every patient responds and acquired drug resistance inevitably develops in those who do respond. This study aimed to define molecular mechanisms of resistance to crizotinib in patients with ALK⁺ non–small cell lung cancer (NSCLC). Experimental Design: We analyzed tissue obtained from 14 patients with ALK⁺ NSCLC showing evidence of radiologic progression while on crizotinib to define mechanisms of intrinsic and acquired resistance to crizotinib. Results: Eleven patients had material evaluable for molecular analysis. Four patients (36%) developed secondary mutations in the tyrosine kinase domain of ALK. A novel mutation in the ALK domain, encoding a G1269A amino acid substitution that confers resistance to crizotinib in vitro, was identified in two of these cases. Two patients, one with a resistance mutation, exhibited new onset ALK copy number gain (CNG). One patient showed outgrowth of epidermal growth factor receptor (EGFR) mutant NSCLC without evidence of a persistent ALK gene rearrangement. Two patients exhibited a KRAS mutation, one of which occurred without evidence of a persisting ALK gene rearrangement. One patient showed the emergence of an ALK gene fusion–negative tumor compared with the baseline sample but with no identifiable alternate driver. Two patients retained ALK positivity with no identifiable resistance mechanism. Conclusions: Crizotinib resistance in ALK⁺ NSCLC occurs through somatic kinase domain mutations, ALK gene fusion CNG, and emergence of separate oncogenic drivers. Clin Cancer Res; 18(5); 1472–82. ©2012 AACR.

Four papers in this issue tackle the hot topic of chromatin remodelling, specifically, how methyl marks on chromatin are 'read' by the proteins that interact with them. Two report on BPTF (bromodomain and PHD domain transcription factor), a subunit of NURF, the nucleosome remodelling factor. It contains a domain known as a PHD finger, which is shown to bind to histone H3 trimethylated at lysine 4 (H3K4) and to maintain proper activity at developmentally critical HOX genes. The accompanying structural study of the complex explains how the site specificity for H3K4 is achieved. The two other papers reveal that the PHD domain of tumour suppressor ING2 also recognizes trimethylated H3K4, and link the histone mark to repression of transcription. The four papers together establish certain PHD finger domains as previously unrecognized chromatin-binding modules. In a News and Views piece, Peter B. Becker discusses what these papers tell us about the function of the chemical modifications of histone tails. One of four papers establishing certain PHD domains as effectors of trimethylated histone H3K4, a chromatin mark generally associated with active transcription. The interaction between H3K4me3 and the ING2 PHD domain is enhanced after DNA damage, recruiting the repressive complex to the promoters of proliferation genes. Dynamic regulation of diverse nuclear processes is intimately linked to covalent modifications of chromatin1,2. Much attention has focused on methylation at lysine 4 of histone H3 (H3K4), owing to its association with euchromatic genomic regions3,4. H3K4 can be mono-, di- or tri-methylated. Trimethylated H3K4 (H3K4me3) is preferentially detected at active genes, and is proposed to promote gene expression through recognition by transcription-activating effector molecules5. Here we identify a novel class of methylated H3K4 effector domains—the PHD domains of the ING (for inhibitor of growth) family of tumour suppressor proteins. The ING PHD domains are specific and highly robust binding modules for H3K4me3 and H3K4me2. ING2, a native subunit of a repressive mSin3a–HDAC1 histone deacetylase complex6, binds with high affinity to the trimethylated species. In response to DNA damage, recognition of H3K4me3 by the ING2 PHD domain stabilizes the mSin3a–HDAC1 complex at the promoters of proliferation genes. This pathway constitutes a new mechanism by which H3K4me3 functions in active gene repression. Furthermore, ING2 modulates cellular responses to genotoxic insults, and these functions are critically dependent on ING2 interaction with H3K4me3. Together, our findings establish a pivotal role for trimethylation of H3K4 in gene repression and, potentially, tumour suppressor mechanisms.

Four papers in this issue tackle the hot topic of chromatin remodelling, specifically, how methyl marks on chromatin are 'read' by the proteins that interact with them. Two report on BPTF (bromodomain and PHD domain transcription factor), a subunit of NURF, the nucleosome remodelling factor. It contains a domain known as a PHD finger, which is shown to bind to histone H3 trimethylated at lysine 4 (H3K4) and to maintain proper activity at developmentally critical HOX genes. The accompanying structural study of the complex explains how the site specificity for H3K4 is achieved. The two other papers reveal that the PHD domain of tumour suppressor ING2 also recognizes trimethylated H3K4, and link the histone mark to repression of transcription. The four papers together establish certain PHD finger domains as previously unrecognized chromatin-binding modules. In a News and Views piece, Peter B. Becker discusses what these papers tell us about the function of the chemical modifications of histone tails. One of four papers establishing certain PHD domains as effectors of trimethylated histone H3K4, a chromatin mark generally associated with active transcription. This paper describes the structure of the PHD finger from the tumour suppressor ING2 complexed with H3K4me3. Covalent modifications of histone tails have a key role in regulating chromatin structure and controlling transcriptional activity. In eukaryotes, histone H3 trimethylated at lysine 4 (H3K4me3) is associated with active chromatin and gene expression1,2,3,4. We recently found that plant homeodomain (PHD) finger of tumour suppressor ING2 (inhibitor of growth 2) binds H3K4me3 and represents a new family of modules that target this epigenetic mark5. The molecular mechanism of H3K4me3 recognition, however, remains unknown. Here we report a 2.0 Å resolution structure of the mouse ING2 PHD finger in complex with a histone H3 peptide trimethylated at lysine 4. The H3K4me3 tail is bound in an extended conformation in a deep and extensive binding site consisting of elements that are conserved among the ING family of proteins. The trimethylammonium group of Lys 4 is recognized by the aromatic side chains of Y215 and W238 residues, whereas the intermolecular hydrogen-bonding and complementary surface interactions, involving Ala 1, Arg 2, Thr 3 and Thr 6 of the peptide, account for the PHD finger's high specificity and affinity. Substitution of the binding site residues disrupts H3K4me3 interaction in vitro and impairs the ability of ING2 to induce apoptosis in vivo. Strong binding of other ING and YNG PHD fingers suggests that the recognition of H3K4me3 histone code is a general feature of the ING/YNG proteins. Elucidation of the mechanisms underlying this novel function of PHD fingers provides a basis for deciphering the role of the ING family of tumour suppressors in chromatin regulation and signalling.