John Wojcik

Targeting the SH2-kinase interface in Bcr-Abl inhibits leukemogenesis.

Summary Chronic myelogenous leukemia (CML) is caused by the constitutively active tyrosine kinase Bcr-Abl and treated with the tyrosine kinase inhibitor (TKI) imatinib. However, emerging TKI resistance prevents complete cure. Therefore, alternative strategies targeting regulatory modules of Bcr-Abl in addition to the kinase active site are strongly desirable. Here, we show that an intramolecular interaction between the SH2 and kinase domains in Bcr-Abl is both necessary and sufficient for high catalytic activity of the enzyme. Disruption of this interface led to inhibition of downstream events critical for CML signaling and, importantly, completely abolished leukemia formation in mice. Furthermore, disruption of the SH2-kinase interface increased sensitivity of imatinib-resistant Bcr-Abl mutants to TKI inhibition. An engineered Abl SH2-binding fibronectin type III monobody inhibited Bcr-Abl kinase activity both in vitro and in primary CML cells, where it induced apoptosis. This work validates the SH2-kinase interface as an allosteric target for therapeutic intervention. PaperFlick

Isoform-specific monobody inhibitors of small ubiquitin-related modifiers engineered using structure-guided library design

Discriminating closely related molecules remains a major challenge in the engineering of binding proteins and inhibitors. Here we report the development of highly selective inhibitors of small ubiquitin-related modifier (SUMO) family proteins. SUMOylation is involved in the regulation of diverse cellular processes. Functional differences between two major SUMO isoforms in humans, SUMO1 and SUMO2/3, are thought to arise from distinct interactions mediated by each isoform with other proteins containing SUMO-interacting motifs (SIMs). However, the roles of such isoform-specific interactions are largely uncharacterized due in part to the difficulty in generating high-affinity, isoform-specific inhibitors of SUMO/SIM interactions. We first determined the crystal structure of a “monobody,” a designed binding protein based on the fibronectin type III scaffold, bound to the yeast homolog of SUMO. This structure illustrated a mechanism by which monobodies bind to the highly conserved SIM-binding site while discriminating individual SUMO isoforms. Based on this structure, we designed a SUMO-targeted library from which we obtained monobodies that bound to the SIM-binding site of human SUMO1 with Kd values of approximately 100 nM but bound to SUMO2 400 times more weakly. The monobodies inhibited SUMO1/SIM interactions and, unexpectedly, also inhibited SUMO1 conjugation. These high-affinity and isoform-specific inhibitors will enhance mechanistic and cellular investigations of SUMO biology.

Broad Ranges of Affinity and Specificity of Anti-Histone Antibodies Revealed by a Quantitative Peptide Immunoprecipitation Assay

Antibodies directed against histone posttranslational modifications (PTMs) are critical tools in epigenetics research, particularly in the widely used chromatin immunoprecipitation (ChIP) experiments. However, a lack of quantitative methods for characterizing such antibodies has been a major bottleneck in accurate and reproducible analysis of histone modifications. Here, we report a simple and sensitive method for quantitatively characterizing polyclonal and monoclonal antibodies for histone PTMs in a ChIP-like format. Importantly, it determines the apparent dissociation constants for the interactions of an antibody with peptides harboring cognate or off-target PTMs. Analyses of commercial antibodies revealed large ranges of affinity, specificity and binding capacity as well as substantial lot-to-lot variations, suggesting the importance of quantitatively characterizing each antibody intended to be used in ChIP experiments and optimizing experimental conditions accordingly. Furthermore, using this method, we identified additional factors potentially affecting the interpretation of ChIP experiments.

Accelerating phage-display library selection by reversible and site-specific biotinylation

Immobilization of a target molecule to a solid support is an indispensable step in phage display library sorting. Here we describe an immobilization method that addresses shortcomings of existing strategies. Our method is based on the use of a polyhistidine-tagged (His-tagged) target molecule and (BT)tris-NTA, a high-affinity capture reagent for His-tags that also contains a biotin moiety. (BT)tris-NTA provides a stable and reversible linkage between a His-tag and a streptavidin-coated solid support. Because His-tags are the de facto standard for recombinant protein purification, this method dramatically simplifies target preparation for phage display library sorting. Here, we demonstrate the utility of this method by selecting high-affinity binding proteins based on the fibronectin type III (FN3) scaffold to two His-tagged protein targets, yeast small ubiquitin-like modifier and maltose-binding protein. Notably, a significant number of FN3 clones binding either targets selected using the new immobilization method exhibited only very weak binding when the same target was immobilized by coating on a polystyrene surface. This suggests that the His-tag-mediated immobilization exposes epitopes that are masked by commonly used passive adsorption methods. Together, these results establish a method with the potential to streamline and enhance many binding-protein engineering experiments.

Allosteric Inhibition of Bcr-Abl Kinase by High Affinity Monobody Inhibitors Directed to the Src Homology 2 (SH2)-Kinase Interface.

Bcr-Abl is a constitutively active kinase that causes chronic myelogenous leukemia. We have shown that a tandem fusion of two designed binding proteins, termed monobodies, directed to the interaction interface between the Src homology 2 (SH2) and kinase domains and to the phosphotyrosine-binding site of the SH2 domain, respectively, inhibits the Bcr-Abl kinase activity. Because the latter monobody inhibits processive phosphorylation by Bcr-Abl and the SH2-kinase interface is occluded in the active kinase, it remained undetermined whether targeting the SH2-kinase interface alone was sufficient for Bcr-Abl inhibition. To address this question, we generated new, higher affinity monobodies with single nanomolar KD values targeting the kinase-binding surface of SH2. Structural and mutagenesis studies revealed the molecular underpinnings of the monobody-SH2 interactions. Importantly, the new monobodies inhibited Bcr-Abl kinase activity in vitro and in cells, and they potently induced cell death in chronic myelogenous leukemia cell lines. This work provides strong evidence for the SH2-kinase interface as a pharmacologically tractable site for allosteric inhibition of Bcr-Abl.

Actionable mutations in canine hemangiosarcoma

Background Angiosarcomas (AS) are rare in humans, but they are a deadly subtype of soft tissue sarcoma. Discovery sequencing in AS, especially the visceral form, is hampered by the rarity of cases. Most diagnostic material exists as archival formalin fixed, paraffin embedded tissue which serves as a poor source of high quality DNA for genome-wide sequencing. We approached this problem through comparative genomics. We hypothesized that exome sequencing a histologically similar tumor, hemangiosarcoma (HSA), that occurs in approximately 50,000 dogs per year, may lead to the identification of potential oncogenic drivers and druggable targets that could also occur in angiosarcoma. Methods Splenic hemangiosarcomas are common in dogs, which allowed us to collect a cohort of archived matched tumor and normal tissue samples suitable for whole exome sequencing. Mapping of the reads to the latest canine reference genome (Canfam3) demonstrated that >99% of the targeted exomal regions were covered, with >80% at 20X coverage and >90% at 10X coverage. Results and conclusions Sequence analysis of 20 samples identified somatic mutations in PIK3CA, TP53, PTEN, and PLCG1, all of which correspond to well-known tumor drivers in human cancer, in more than half of the cases. In one case, we identified a mutation in PLCG1 identical to a mutation observed previously in this gene in human visceral AS. Activating PIK3CA mutations present novel therapeutic targets, and clinical trials of targeted inhibitors are underway in human cancers. Our results lay a foundation for similar clinical trials in canine HSA, enabling a precision medicine approach to this disease.

Imaging and histological appearance of pleomorphic hyalinizing angiectatic tumors: A case series and literature review

Pleomorphic hyalinizing angiectatic tumors (PHATs) are rare mesenchymal soft tissue tumors of uncertain lineage and intermediate malignancy. The present study assesses two cases of PHAT and discusses the histological and immunophenotypical features, as well as the imaging appearance of these tumors on ultrasound, computed tomography (CT), magnetic resonance imaging and positron emission tomography/CT scans. The current study also reviews the literature and discusses the clinical management of these tumors. Wide local excision with tumor free margins is the current recommended treatment for PHAT. Surgical excision may be combined with low-dose radiation to reduce the risk of local recurrence. Patients should be followed up with serial imaging, as PHAT lesions tend to recur locally.

Abstract A10: Integrated proteome and epigenetic analysis reveals crosstalk between chromatin and cancer pathways in malignant peripheral nerve sheath tumors

Malignant peripheral nerve sheath tumors (MPNST) are rare, aggressive sarcomas. Recent work has identified loss-of-function mutations in PRC2 components in a subset of these tumors. PRC2 mutation leads to a loss of trimethylation at lysine 27 of histone H3 (H3K27me3), and is linked to transcriptional upregulation of the Ras pathway in cell culture models of MPNST. We hypothesized that PRC2 loss would result in global alterations in histone post-translational modifications (PTMs), and such changes would be associated with altered protein expression in oncogenic pathways. We thus implemented protein and histone extraction methods directly on formalin-fixed, paraffin-embedded human tumor samples and performed parallel histone PTM and proteome analysis. Data were merged using our in-house developed chromatin and proteomics bioinformatics tools. Methods: Human tumors were obtained from pathology archives. Total protein was isolated from 3-mm tissue cores, following deparaffinization, antigen retrieval, lysis, and homogenization. Histone bands were excised following SDS-PAGE of total lysate. For cell lines, total protein was obtained following lysis in urea and histones were obtained via nuclear isolation and acid precipitation. Cell proteome and histone PTMs were analyzed using nano-liquid chromatography and tandem mass spectrometry. The proteome was characterized using MaxQuant. Histone PTMs were identified and quantified using lab-developed software. Statistical analysis was performed using Student9s t-test, Gene Ontology enrichment was assessed using hypergeometric distribution annotations, and GSEA analysis was used to identify signatures of pathway activation. Results: PRC2 loss was associated with decreased H3K27me3 and increased H3K27 acetylation (p=0.02 and p=0.01). This was accompanied by increased hyperacetylation of H4, a marker of open chromatin. We also observed an increase in overall H3K36me2 (p=0.0001). By quantifying combinatorial PTMs, we observed that tandem H3K27me2K36me2 and H3K27me3K36me2 marks were markedly decreased in MPNST with PRC2 loss (p=0.04 and p=0.004). These alterations result in chromatin in which the active H3K36me2 mark is not flanked by repressive PTMs. At the level of the proteome, PRC2 loss was associated with increased expression of chromatin remodelers, particularly members of the SWI/SNF complex. We confirmed increased Ras pathway expression and noted increases in other tumor pathways, and decreases in proteins associated with nerve sheath differentiation and immune surveillance. To assess whether PRC2 function was directly linked to the proteome changes seen in the human tumors, we restored PRC2 function in PRC2-mutant MPNST cell lines and measured the proteome response. PRC2 reconstitution downregulated pathways that were upregulated with PRC2 loss in human tumors and restored expression of markers of nerve sheath differentiation and immune surveillance. The same effects were observed in PRC2 mutant cell lines upon shRNA silencing of NSD2, the methyltransferase responsible for H3K36me2, suggesting that, for a subset of genes, H3K27me3 and H3K36me2 function in tandem to control expression. These results link PRC2 loss to increased active chromatin signatures that promote pathways associated with aggressive tumor behavior. Significantly, the effects of PRC2 loss were ameliorated by restoration of PRC2 function, or inhibition of NSD2 function, revealing crosstalk between H3K27me and H3K36me marks, and suggesting that multiple agents targeting components of the active chromatin state may be beneficial in this rare and deadly disease. Citation Format: John B. Wojcik, Simone Sidoli, Kumarasen Cooper, Benjamin A. Garcia. Integrated proteome and epigenetic analysis reveals crosstalk between chromatin and cancer pathways in malignant peripheral nerve sheath tumors [abstract]. In: Proceedings of the AACR Conference on Advances in Sarcomas: From Basic Science to Clinical Translation; May 16-19, 2017; Philadelphia, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(2_Suppl):Abstract nr A10.

Clear Cell Chondrosarcoma With Chondroblastoma-Like Features: A Case for Team Diagnosis

Clear cell chondrosarcoma (CCCS) is a rare variant of conventional chondrosarcoma with low-grade malignant features that may be confused radiographically and histologically with chondroblastoma. We report a case of a 50-year-old female who presented with 6 months of left hip pain. Initial radiographs demonstrated an osteolytic lesion with adjacent area of sclerosis in the proximal left femur. Magnetic resonance imaging demonstrated a marrow-infiltrative lesion with periosteal reaction and thickened enhancing periosteum. Biopsy of the sclerotic area demonstrated chondroblastoma-like findings, whereas biopsy of the lytic area showed features suggestive of CCCS. The patient eventually underwent en bloc resection and reconstruction with a proximal femoral megaprosthesis. The final diagnosis was CCCS. We present this unusual case with review of the radiographic and histologic features of CCCS with attention to its ability to mimic chondroblastomas. This case highlights the importance of sampling radiographically heterogeneous areas within a bone lesion to facilitate accurate diagnosis and appropriate management.

Epigenetic Alterations in Bone and Soft Tissue Tumors

Human malignancies are driven by heritable alterations that lead to unchecked cellular proliferation, invasive growth and distant spread. Heritable changes can arise from changes in DNA sequence, or, alternatively, through altered gene expression rooted in epigenetic mechanisms. In recent years, high-throughput sequencing of tumor genomes has revealed a central role for mutations in epigenetic regulatory complexes in oncogenic processes. Through interactions with or direct modifications of chromatin, these proteins help control the accessibility of genes, and thus the transcriptional profile of a cell. Dysfunction in these proteins can lead to activation of oncogenic pathways or silencing of tumor suppressors. Although epigenetic regulators are altered across a broad spectrum of human malignancies, they play a particularly central role in tumors of mesenchymal and neuroectodermal origin. This review will focus on recent advances in the understanding of the molecular pathogenesis of a subset of tumors in which alterations in the polycomb family of chromatin modifying complexes, the SWI/SNF family of nucleosome remodelers, and histones play a central role in disease pathogenesis. Although this review will focus predominantly on the molecular mechanisms underlying these tumors, each section will also highlight areas in which an understanding of the molecular pathogenesis of these diseases has led to the adoption of novel immunohistochemical and molecular markers.