Zimmermann, Michael T.

Abstract Background: Peripheral T-cell lymphomas (PTCLs) comprise a heterogeneous group of relatively uncommon and generally aggressive non-Hodgkin lymphomas. Recent studies have begun to reveal their mutational landscape, such as the recurrent mutations affecting RHOA, IDH2, and TET2 in angioimmunoblastic T-cell lymphomas (AITLs) and related PTCLs. Here, we studied the exomes of 63 PTCLs to address several unanswered questions: (1) How frequently do PTCLs have mutations with a strong rationale for being actionable using clinically available targeted therapies? (2) What pathways are enriched in PTCLs that could point to additional targeted therapies? (3) What additional recurrent mutations exist that may provide insight into PTCL pathogenesis and classification? Methods: We performed exome capture and sequencing in 63 frozen PTCL samples (with matched germline controls from 22), including 13 PTCLs, not otherwise specified; 6 AITLs; 37 anaplastic large cell lymphomas (ALCLs: 12 ALK+, 19 ALK-, and 6 cutaneous); 4 extranodal NK/T-cell lymphomas; 2 cutaneous T-cell lymphomas, and 1 enteropathy associated T-cell lymphoma. Following primary variant calling, data were filtered by removing variants that were: outside the capture region, polymorphic (0.1% in ExAC), seen in paired germline or 50 healthy controls, within repetitive elements, or synonymous. Potentially actionable variants required published evidence for altering protein function and demonstrated in vivo or in vitro sensitivity to an available targeted agent (including open clinical trials). Mutated cancer genes (COSMIC) were examined for enrichment of KEGG canonical signaling pathways. A recurrent mutation encoding MSC E116K was validated in tumor DNA and MSC protein was examined by immunohistochemistry. Results: Of 25 unique variants with published evidence suggesting actionability (including ErbB, JAK/STAT, and RAS genes), at least one variant was present in 16/63 patients (25%). Variants in any COSMIC gene were seen in 55/63 samples (87%). Top genes were STAT3, RHOA, ARID1A, MLL2 (KMT2D), PTPRB, TP53, and TET2. The KEGG ErbB pathway was most significantly enriched for mutated genes in our dataset (EGFR, ERBB2, ERBB3, ERBB4, and others; q=3.88x10-3), followed by neurotrophin and mitogen-activated protein kinase signaling pathways. Among 25 recurrent non-synonymous variants, we identified MSC E116K with predicted gain-of-function exclusively in 3 ALK-ALCLs and 1 cutaneous ALCL. All mutated cases strongly expressed MSC protein. Among 96 cases, 13/18 ALK-ALCLs (72%) were positive for MSC, compared to 9/78 (12%) from other PTCL subtypes. Conclusions: Mutations that were potentially actionable based on published variant-specific data were identified in 25% of PTCLs, indicating promise for the role of sequencing in individualizing therapy. Additionally, remaining variants were enriched for several targetable pathways, most significantly ErbB. A novel recurrent mutation, MSC E116K, was seen exclusively in ALK-ALCLs (systemic and cutaneous). MSC protein, a repressor of E2A-mediated transcription of targets such as MYC, was expressed in most ALK- ALCLs and generally was absent in other PTCL subtypes. This finding extends evidence for a genetic relationship between systemic and cutaneous ALCLs and suggests MSC may help distinguish ALK- ALCLs from other PTCLs. Disclosures Link: Genentech: Consultancy, Research Funding; Kite Pharma: Research Funding.

Background Identifying the biologic functions of groups of genes identified in high-throughput studies currently requires considerable time and/or bioinformatics experience. This is due in part to each resource housed within separate databases, requiring users to know about them, and integrate across them. Time consuming and often repeated for each study, integrating across resources and merging with data under study is an increasingly common bioinformatics task. Methods We developed an open-source R software package for assisting researchers in annotating their genesets with functions, pathways, and their interconnectivity across a diversity of network resources. Results We present rapid integration of term annotation and network resources (RITAN) for the rapid and comprehensive annotation of a list of genes using functional term and pathway resources and their relationships among each other using multiple network biology resources. Currently, and to comply with data redistribution policies, RITAN allows rapid access to 16 term annotations spanning gene ontology, biologic pathways, and immunologic modules, and nine network biology resources, with support for user-supplied resources; we provide recommendations for additional resources and scripts to facilitate their addition to RITAN. Having the resources together in the same system allows users to derive novel combinations. RITAN has a growing set of tools to explore the relationships within resources themselves. These tools allow users to merge resources together such that the merged annotations have a minimal overlap with one another. Because we index both function annotation and network interactions, the combination allows users to expand small groups of genes using links from biologic networks—either by adding all neighboring genes or by identifying genes that efficiently connect among input genes—followed by term enrichment to identify functions. That is, users can start from a core set of genes, identify interacting genes from biologic networks, and then identify the functions to which the expanded list of genes contribute. Conclusion We believe RITAN fills the important niche of bridging the results of high-throughput experiments with the ever-growing corpus of functional annotations and network biology resources. Availability Rapid integration of term annotation and network resources is available as an R package at github.com/MTZimmer/RITAN and BioConductor.org .

AbstractDiffuse idiopathic skeletal hyperostosis (DISH) is a disorder principally characterized by calcification and ossification of spinal ligaments and entheses. Fibrodysplasia ossificans progressiva (FOP) is a rare autosomal dominant disabling disorder characterized by progressive ossification of skeletal muscle, fascia, tendons, and ligaments. These conditions manifest phenotypic overlap in the ossification of tendons and ligaments. We describe herein a patient with DISH, exhibiting heterotopic ossification of the posterior longitudinal ligament where clinical whole exome sequencing identified a variant within ACVR1, a gene implicated in FOP. This variant, p.K400E, is a novel variant, not identified previously, and occurs in a highly conserved region across orthologs. We used sequence‐based predicative algorithms, molecular modeling, and molecular dynamics simulations, to test the potential for p.K400E to alter the structure and dynamics of ACVR1. We applied the same modeling and simulation methods to established FOP variants, to identify the detailed effects that they have on the ACVR1 protein, as well as to act as positive controls against which the effects of p.K400E could be evaluated. Our in silico molecular analyses support p.K400E as altering the behavior of ACVR1. In addition, functional testing to measure the effect of this variant on BMP‐pSMAD 1/5/8 target genes was carried out which revealed this variant to cause increased ID1 and Msx2 expression compared with the wild‐type receptor. This analysis supports the potential for the variant of uncertain significance to contribute to the patient's phenotype.

AbstractHeterochromatin protein 1α (HP1α) is a protein that mediates cancer‐associated processes in the cell nucleus. Proteomic experiments, reported here, demonstrate that HP1α complexes with importin α (IMPα), a protein necessary for its nuclear transport. This data is congruent with Simple Linear Motif (SLiM) analyses that identify an IMPα‐binding motif within the linker that joins the two globular domains of this protein. Using molecular modeling and dynamics simulations, we develop a model of the IMPα‐HP1α complex and investigate the impact of phosphorylation and genomic variants on their interaction. We demonstrate that phosphorylation of the HP1α linker likely regulates its association with IMPα, which has implications for HP1α access to the nucleus, where it functions. Cancer‐associated genomic variants do not abolish the interaction of HP1α but instead lead to rearrangements where the variant proteins maintain interaction with IMPα, but with less specificity. Combined, this new mechanistic insight bears biochemical, cell biological, and biomedical relevance.

AbstractAnaplastic large cell lymphomas (ALCLs) represent a relatively common group of T-cell non-Hodgkin lymphomas (T-NHLs) that are unified by similar pathologic features but demonstrate marked genetic heterogeneity. ALCLs are broadly classified as being anaplastic lymphoma kinase (ALK)+ or ALK−, based on the presence or absence of ALK rearrangements. Exome sequencing of 62 T-NHLs identified a previously unreported recurrent mutation in the musculin gene, MSCE116K, exclusively in ALK− ALCLs. Additional sequencing for a total of 238 T-NHLs confirmed the specificity of MSCE116K for ALK− ALCL and further demonstrated that 14 of 15 mutated cases (93%) had coexisting DUSP22 rearrangements. Musculin is a basic helix-loop-helix (bHLH) transcription factor that heterodimerizes with other bHLH proteins to regulate lymphocyte development. The E116K mutation localized to the DNA binding domain of musculin and permitted formation of musculin–bHLH heterodimers but prevented their binding to authentic target sequence. Functional analysis showed MSCE116K acted in a dominant-negative fashion, reversing wild-type musculin-induced repression of MYC and cell cycle inhibition. Chromatin immunoprecipitation–sequencing and transcriptome analysis identified the cell cycle regulatory gene E2F2 as a direct transcriptional target of musculin. MSCE116K reversed E2F2-induced cell cycle arrest and promoted expression of the CD30–IRF4–MYC axis, whereas its expression was reciprocally induced by binding of IRF4 to the MSC promoter. Finally, ALCL cells expressing MSCE116K were preferentially targeted by the BET inhibitor JQ1. These findings identify a novel recurrent MSC mutation as a key driver of the CD30–IRF4–MYC axis and cell cycle progression in a unique subset of ALCLs.

AbstractBackgroundBeta‐galactosidase‐1 (GLB1) is a lysosomal hydrolase that is responsible for breaking down specific glycoconjugates, particularly GM1 (monosialotetrahexosylganglioside). Pathogenic variants in GLB1 cause two different lysosomal storage disorders: GM1 gangliosidosis and mucopolysaccharidosis type IVB. In GM1 gangliosidosis, decreased β‐galactosidase‐1 enzymatic activity leads to the accumulation of GM1 gangliosides, predominantly within the CNS. We present a 22‐month‐old proband with GM1 gangliosidosis type II (late‐infantile form) in whom a novel homozygous in‐frame deletion (c.1468_1470delAAC, p.Asn490del) in GLB1 was detected.MethodsWe used an experimental protein structure of β‐galactosidase‐1 to generate a model of the p.Asn490del mutant and performed molecular dynamic simulations to determine whether this mutation leads to altered ligand positioning compared to the wild‐type protein. In addition, residual mutant enzyme activity in patient leukocytes was evaluated using a fluorometric assay.ResultsMolecular dynamics simulations showed the deletion to alter the catalytic site leading to misalignment of the catalytic residues and loss of collective motion within the model. We predict this misalignment will lead to impaired catalysis of β‐galactosidase‐1 substrates. Enzyme assays confirmed diminished GLB1 enzymatic activity (~3% of normal activity) in the proband.ConclusionsWe have described a novel, pathogenic in‐frame deletion of GLB1 in a patient with GM1 gangliosidosis type II.

Objective: Bringing together genomics, transcriptomics, proteomics, and other –omics technologies is an important step towards developing highly personalized medicine. However, instrumentation has advances far beyond expectations and now we are able to generate data faster than it can be interpreted. Materials and Methods: We have developed PANDA ( P athway AND A nnotation) Explorer, a visualization tool that integrates gene-level annotation in the context of biological pathways to help interpret complex data from disparate sources. PANDA is a web-based application that displays data in the context of well-studied pathways like KEGG, BioCarta, and PharmGKB. PANDA represents data/annotations as icons in the graph while maintaining the other data elements (i.e. other columns for the table of annotations). Custom pathways from underrepresented diseases can be imported when existing data sources are inadequate. PANDA also allows sharing annotations among collaborators. Results : In our first use case, we show how easy it is to view supplemental data from a manuscript in the context of a user’s own data. Another use-case is provided describing how PANDA was leveraged to design a treatment strategy from the somatic variants found in the tumor of a patient with metastatic sarcomatoid renal cell carcinoma. Conclusion : PANDA facilitates the interpretation of gene-centric annotations by visually integrating this information with context of biological pathways. The application can be downloaded or used directly from our website http://bioinformaticstools.mayo.edu/research/panda-viewer/.

Loeys-Dietz syndrome (LDS) is a connective tissue disorder characterized by vascular findings of aneurysm and/or dissection of cerebral, thoracic, or abdominal arteries and skeletal findings. We report a case of a novel pathogenic variant in TGFBR2 and phenotype consistent with classic LDS. The proband was a 10-year-old presenting to the genetics clinic with an enlarged aortic root (Z-scores 5-6), pectus excavatum, and congenital contractures of the right 2nd and 3rd digit. Molecular testing of TGFBR2 was sent to a commercial laboratory and demonstrated a novel, likely pathogenic, variant in exon 4, c.1061T>C, p.(L354P). Molecular modeling reveals alteration of local protein structure as a result of this pathogenic variant. This pathogenic variant has not been previously reported in LDS and thus expands the pathogenic variant spectrum of this condition.