Structural determinants of mitochondrial STAT3 targeting and function.

Signal transducer and activator of transcription (STAT) 3 has been found within mitochondria in addition to its canonical role of shuttling between cytoplasm and nucleus during cytokine signaling. Mitochondrial STAT3 has been implicated in modulation of cellular metabolism, largely through effects on the respiratory electron transport chain. However, the structural requirements underlying mitochondrial targeting and function have remained unclear. Here, we show that mitochondrial STAT3 partitions between mitochondrial compartments defined by differential detergent solubility, suggesting that mitochondrial STAT3 is membrane associated. The majority of STAT3 was found in an SDS soluble fraction copurifying with respiratory chain proteins, including numerous components of the complex I NADH dehydrogenase, while a minor component was found with proteins of the mitochondrial translation machinery. Mitochondrial targeting of STAT3 required the amino-terminal domain, and an internal linker domain motif also directed mitochondrial translocation. However, neither the phosphorylation of serine 727 nor the presence of mitochondrial DNA was required for the mitochondrial localization of STAT3. Two cysteine residues in the STAT3 SH2 domain, which have been previously suggested to be targets for protein palmitoylation, were also not required for mitochondrial translocation, but were required for its function as an enhancer of complex I activity. These structural determinants of STAT3 mitochondrial targeting and function provide potential therapeutic targets for disrupting the activity of mitochondrial STAT3 in diseases such as cancer.

High-resolution melting assay for rapid, simultaneous detection of JAK2, MPL and CALR variants.

AIMS: Identification of recurrent genetic alterations in JAK2, MPL and CALR remains crucial in the diagnosis of Philadelphia-negative myeloproliferative neoplasms (MPNs). Current laboratory testing algorithms may entail batching and/or sequential testing, involving multiple testing modalities and sometimes send-out testing that increase the technical and economic demands on laboratories while delaying patient diagnoses. To address this gap, an assay based on PCR and high-resolution melting (HRM) analysis was developed for simultaneous evaluation of JAK2 exons 12-14, MPL exon 10 and CALR exon 9, embodied in the HemeScreen® (hereafter 'HemeScreen') MPN assay. METHODS: The HemeScreen MPN assay was validated with blood and bone marrow samples from 982 patients with clinical suspicion for MPN. The HRM assay and Sanger sequencing were performed in independent Clinical Laboratory Improvement Amendments (CLIA)-certified laboratories with Sanger sequencing (supported by droplet digital PCR) serving as the gold standard. RESULTS: HRM and Sanger sequencing had an overall concordance of 99.4% with HRM detecting 133/139 (96%) variants confirmed by sequencing (9/10 MPL, 25/25 CALR, 99/104 JAK2), including 114 single nucleotide variants and 25 indels (3-52 bp). Variants consisted of disease-associated (DA) variants (89%), variants of unclear significance (2%) and non-DA variants (9%) with a positive predictive value of 92.3% and negative predictive value of 99.5%. CONCLUSIONS: These studies demonstrate the exquisite accuracy, sensitivity and specificity of the HRM-based HemeScreen MPN assay, which serves as a powerful, clinically applicable platform for rapid, simultaneous detection of clinically relevant, somatic disease variants.

Proteogenomic Profiling of High-Grade B-Cell Lymphoma With 11q Aberrations and Burkitt Lymphoma Reveals Lymphoid Enhancer Binding Factor 1 as a Novel Biomarker.

High-grade B-cell lymphomas with 11q aberrations (HGBL-11q) represent a World Health Organization-defined group of lymphomas that harbor recurrent chromosome 11q aberrations involving proximal gains and telomeric losses. Although a limited number of HGBL-11q cases evaluated thus far appear to show a similar course and prognosis as Burkitt lymphoma (BL), many molecular differences have been appreciated, most notably the absence of MYC rearrangement. Despite biological differences between BL and HGBL-11q, histomorphologic and immunophenotypic distinction remains challenging. Here, we provide a comparative whole proteomic profile of BL- and HGBL-11q-derived cell lines, identifying numerous shared and differentially expressed proteins. Transcriptome profiling performed on paraffin-embedded tissue samples from primary BL and HGBL-11q lymphomas was additionally performed to provide further molecular characterization. Overlap of proteomic and transcriptomic data sets identified several potential novel biomarkers of HGBL-11q, including diminished lymphoid enhancer-binding factor 1 expression, which was validated by immunohistochemistry staining in a cohort of 23 cases. Altogether, these findings provide a comprehensive multimodal and comparative molecular profiling of BL and HGBL-11q and suggest the use of enhancer-binding factor 1 as an immunohistochemistry target to distinguish between these aggressive lymphomas.

Rapid and Automated Semiconductor-Based Next-Generation Sequencing for Simultaneous Detection of Somatic DNA and RNA Aberrations in Myeloid Neoplasms.

Evaluation of suspected myeloid neoplasms involves testing for recurrent, diagnostically and therapeutically relevant genetic alterations. Current molecular testing requires multiple technologies, different domains of expertise, and unconnected workflows, resulting in variable, lengthy turnaround times that can delay treatment. To address this unmet clinical need, we evaluated the Oncomine Myeloid Assay GX panel on the Ion Torrent Genexus platform, a rapid, integrated nucleic acid to report next-generation sequencing platform for detecting clinically relevant genetic aberrations in myeloid disorders. Specimens included synthetic DNA (101 targets) and RNA (9 targets) controls and real-world nucleic acid material derived from bone marrow or peripheral blood samples (40 patients). Ion Torrent Genexus results and performance indices were compared with those obtained from clinically validated genomic testing workflows in 2 separate clinical laboratories. The Ion Torrent Genexus identified 100% of DNA and RNA control variants. For primary patient specimens, the Ion Torrent Genexus reported 82 of 107 DNA variants and 19 of 19 RNA gene fusions identified on clinically validated assays, yielding an 80% overall detection rate. Reanalysis of exported, unfiltered Ion Torrent Genexus data revealed 15 DNA variants not called by the filtered on-board bioinformatics pipeline, yielding a 92% potential detection rate. These results hold promise for the implementation of an integrated next-generation sequencing system to rapidly detect genetic aberrations, facilitating accurate, genomics-based diagnoses and accelerated time to precision therapies in myeloid neoplasms.

FBXW7ß isoform drives transcriptional activation of the proinflammatory TNF cluster in human pro-B cells.

Noncanonical exon usage plays many important roles in cellular phenotypes, but its contribution to human B-cell development remains sketchily understood. To fill this gap, we collected various B-cell fractions from bone marrow (BM) and tonsil donors, performed RNA sequencing, and examined transcript variants. We identified 150 genes that harbor local splicing variations in all pairwise comparisons. One of them encodes FBXW7, an E3 ubiquitin ligase implicated as a driver in several blood cancers. Surprisingly, we discovered that in normal human pro-B cells, the predominant transcript used an alternative first exon to produce the poorly characterized FBXW7ß isoform, previously thought to be restricted to neural tissues. The FBXW7ß transcript was also abundant in cell lines and primary samples of pediatric B-cell acute lymphoblastic leukemia (B-ALL), which originates in the BM. When overexpressed in a heterologous cell system, this transcript yielded the expected protein product, as judged by anti-FLAG immunoblotting and mass spectrometry. Furthermore, in REH B-ALL cells, FBXW7ß mRNA was the only FBXW7 isoform enriched in the polyribosome fraction. To shed light on possible functions of FBXW7ß, we used gain- and loss-of-function approaches and identified an FBXW7-dependent inflammatory gene signature, apparent in a subset of B-ALL with high FBXW7ß expression. This signature contained several members of the tumor necrosis factor superfamily, including those comprising the HLA Class III cluster (LTB, LST1, NCR3, LTA, and NFKBIL1). Our findings suggest that FBXW7ß expression drives proinflammatory responses, which could contribute to normal B-cell development, leukemogenesis, and responses to anticancer therapies.

BCL10 Mutations Define Distinct Dependencies Guiding Precision Therapy for DLBCL.

Activated B cell-like diffuse large B-cell lymphomas (ABC-DLBCL) have unfavorable outcomes and chronic activation of CARD11-BCL10-MALT1 (CBM) signal amplification complexes that form due to polymerization of BCL10 subunits, which is affected by recurrent somatic mutations in ABC-DLBCLs. Herein, we show that BCL10 mutants fall into at least two functionally distinct classes: missense mutations of the BCL10 CARD domain and truncation of its C-terminal tail. Truncating mutations abrogated a motif through which MALT1 inhibits BCL10 polymerization, trapping MALT1 in its activated filament-bound state. CARD missense mutations enhanced BCL10 filament formation, forming glutamine network structures that stabilize BCL10 filaments. Mutant forms of BCL10 were less dependent on upstream CARD11 activation and thus manifested resistance to BTK inhibitors, whereas BCL10 truncating but not CARD mutants were hypersensitive to MALT1 inhibitors. Therefore, BCL10 mutations are potential biomarkers for BTK inhibitor resistance in ABC-DLBCL, and further precision can be achieved by selecting therapy based on specific biochemical effects of distinct mutation classes. SIGNIFICANCE: ABC-DLBCLs feature frequent mutations of signaling mediators that converge on the CBM complex. We use structure-function approaches to reveal that BCL10 mutations fall into two distinct biochemical classes. Both classes confer resistance to BTK inhibitors, whereas BCL10 truncations confer hyperresponsiveness to MALT1 inhibitors, providing a road map for precision therapies in ABC-DLBCLs. See related commentary by Phelan and Oellerich, p. 1844. This article is highlighted in the In This Issue feature, p. 1825.

Functional characterization of NPM1-TYK2 fusion oncogene.

Gene fusions are known to drive many human cancers. Therefore, the functional characterization of newly discovered fusions is critical to understanding the oncobiology of these tumors and to enable therapeutic development. NPM1-TYK2 is a novel fusion identified in CD30 + lymphoproliferative disorders, and here we present the functional evaluation of this fusion gene as an oncogene. The chimeric protein consists of the amino-terminus of nucleophosmin 1 (NPM1) and the carboxyl-terminus of tyrosine kinase 2 (TYK2), including the kinase domain. Using in vitro lymphoid cell transformation assays and in vivo tumorigenic xenograft models we present direct evidence that the fusion gene is an oncogene. NPM1 fusion partner provides the critical homodimerization needed for the fusion kinase constitutive activation and downstream signaling that are responsible for cell transformation. As a result, our studies identify NPM1-TYK2 as a novel fusion oncogene and suggest that inhibition of fusion homodimerization could be a precision therapeutic approach in cutaneous T-cell lymphoma patients expressing this chimera.

Optimization of Sources of Circulating Cell-Free DNA Variability for Downstream Molecular Analysis.

Circulating cell-free DNA (ccfDNA) is used increasingly as a cancer biomarker for prognostication, as a correlate for tumor volume, or as input for downstream molecular analysis. Determining optimal blood processing and ccfDNA quantification are crucial for ccfDNA to serve as an accurate biomarker as it moves into the clinical realm. Whole blood was collected from 50 subjects, processed to plasma, and used immediately or frozen at -80°C. Plasma ccfDNA was extracted and concentration was assessed by real-time quantitative PCR (qPCR), fluorimetry, and droplet digital PCR (ddPCR). For the 24 plasma samples from metastatic pancreatic cancer patients, the variant allele fractions (VAF) of KRAS G12/13 pathogenic variants in circulating tumor DNA (ctDNA) were measured by ddPCR. Using a high-speed (16,000 × g) or slower-speed (4100 × g) second centrifugation step showed no difference in ccfDNA yield or ctDNA VAF. A two- versus three-spin centrifugation protocol also showed no difference in ccfDNA yield or ctDNA VAF. A higher yield was observed from fresh versus frozen plasma by qPCR and fluorimetry, whereas a higher yield was observed for frozen versus fresh plasma by ddPCR, however, no difference was observed in ctDNA VAF. Overall, our findings suggest factors to consider when implementing a ccfDNA extraction and quantification workflow in a research or clinical setting.

Mastermind: A Comprehensive Genomic Association Search Engine for Empirical Evidence Curation and Genetic Variant Interpretation.

Design and interpretation of genome sequencing assays in clinical diagnostics and research labs is complicated by an inability to identify information from the medical literature and related databases quickly, comprehensively and reproducibly. This challenge is compounded by the complexity and heterogeneity of nomenclatures used to describe diseases, genes and genetic variants. Mastermind is a widely-used bioinformatic platform of genomic associations that has indexed more than 7.5 M full-text articles and 2.5 M supplemental datasets. It has automatically identified, disambiguated and annotated >6.1 M genetic variants and identified >50 K disease-gene associations. Here, we describe how Mastermind improves the sensitivity and reproducibility of clinical variant interpretation and produces comprehensive genomic landscapes of genetic variants driving pharmaceutical research. We demonstrate an alarmingly high degree of heterogeneity across commercially available panels for hereditary cancer that is resolved by evidence from Mastermind. We further examined the sensitivity of Mastermind for variant interpretation by examining 108 clinically-encountered variants and comparing the results to alternate methods. Mastermind demonstrated a sensitivity of 98.4% compared to 4.4, 45.6, and 37.4% for alternatives PubMed, Google Scholar, and ClinVar, respectively, and a specificity of 98.5% compared to 45.1, 57.6, and 68.8% as well as an increase in content yield of 22.6-, 2.2-, and 2.6-fold. When curated for clinical significance, Mastermind identified more than 4.9-fold more pathogenic variants than ClinVar for representative genes. For structural variants, we compared Mastermind's ability to sensitively identify evidence for 10 representative disease-causing CNVs versus results identified in PubMed, as well as its ability to identify evidence for fusion events compared to COSMIC. Mastermind demonstrated a 4.0- to 43.9-fold increase in references for specific CNVs compared to PubMed, as well as 5.4-fold more fusion genes when compared with COSMIC's curated database. Additionally, Mastermind produced an 8.0-fold increase in reference citations for fusion events common to Mastermind and outside databases. Taken together, these results demonstrate the utility and superiority of Mastermind in terms of both sensitivity and specificity of automated results for clinical diagnostic variant interpretation for multiple genetic variant types and highlight the potential benefit in informing pharmaceutical research.

FBXW7 Triggers Degradation of KMT2D to Favor Growth of Diffuse Large B-cell Lymphoma Cells.

Mature B-cell neoplasms are the fifth most common neoplasm. Due to significant heterogeneity at the clinical and genetic levels, current therapies for these cancers fail to provide long-term cures. The clinical success of proteasome inhibition for the treatment of multiple myeloma and B-cell lymphomas has made the ubiquitin pathway an important emerging therapeutic target. In this study, we assessed the role of the E3 ligase FBXW7 in mature B-cell neoplasms. FBXW7 targeted the frequently inactivated tumor suppressor KMT2D for protein degradation, subsequently regulating gene expression signatures related to oxidative phosphorylation (OxPhos). Loss of FBXW7 inhibited diffuse large B-cell lymphoma cell growth and further sensitized cells to OxPhos inhibition. These data elucidate a novel mechanism of regulation of KMT2D levels by the ubiquitin pathway and uncover a role of FBXW7 in regulating oxidative phosphorylation in B-cell malignancies. SIGNIFICANCE: These findings characterize FBXW7 as a prosurvival factor in B-cell lymphoma via degradation of the chromatin modifier KMT2D.

Enabling Precision Oncology Through Precision Diagnostics.

Genomic testing enables clinical management to be tailored to individual cancer patients based on the molecular alterations present within cancer cells. Genomic sequencing results can be applied to detect and classify cancer, predict prognosis, and target therapies. Next-generation sequencing has revolutionized the field of cancer genomics by enabling rapid and cost-effective sequencing of large portions of the genome. With this technology, precision oncology is quickly becoming a realized paradigm for managing the treatment of cancer patients. However, many challenges must be overcome to efficiently implement the transition of next-generation sequencing from research applications to routine clinical practice, including using specimens commonly available in the clinical setting; determining how to process, store, and manage large amounts of sequencing data; determining how to interpret and prioritize molecular findings; and coordinating health professionals from multiple disciplines.

Impact of Genetics on Mature Lymphoid Leukemias and Lymphomas.

Recurrent genetic aberrations have long been recognized in mature lymphoid leukemias and lymphomas. As conventional karyotypic and molecular cloning techniques evolved in the 1970s and 1980s, multiple cytogenetic aberrations were identified in lymphomas, often balanced translocations that juxtaposed oncogenes to the immunoglobulin (IG) or T-cell receptor (TR) loci, leading to dysregulation. However, genetic characterization and classification of lymphoma by conventional cytogenetic methods is limited by the infrequent occurrence of recurrent karyotypic abnormalities in many lymphoma subtypes and by the frequent difficulty in growing clinical lymphoma specimens in culture to obtain informative karyotypes. As higher-resolution genomic techniques developed, such as array comparative genomic hybridization and fluorescence in situ hybridization, many recurrent copy number changes were identified in lymphomas, and copy number assessment of interphase cells became part of routine clinical practice for a subset of diseases. Platforms to globally examine mRNA expression led to major insights into the biology of several lymphomas, although these techniques have not gained widespread application in routine clinical settings. With the advent of next-generation sequencing (NGS) techniques in the early 2000s, numerous insights into the genetic landscape of lymphomas were obtained. In contrast to the myeloid malignancies, most common lymphomas exhibit an at least somewhat mutationally complex genome, with few single driver mutations in the majority of patients. However, many recurrently mutated pathways have been identified across lymphoma subtypes, informing targeted therapeutic approaches that are beginning to make meaningful changes in the treatment of lymphoma. In addition to the ability to identify possible therapeutic targets, NGS techniques are highly amenable to the tracking of residual lymphoma following therapy, because of the presence of unique genetic "fingerprints" in lymphoma cells due to V(D)-J recombination at the antigen receptor loci. This review will provide an overview of the impact of novel genetic technologies on lymphoma classification, biology, and therapy.

What is new in mature T-cell leukemias?

Mature T-cell and NK-cell leukemias represent a clinically heterogeneous group of diseases, ranging from indolent expansions of large granular lymphocytes, to aggressive diseases that are associated with a fulminant clinical course. Recent advances in genomic methodologies have massively increased the understanding of the pathogenesis of this group of diseases. While the entities are genetically heterogeneous, JAK-STAT pathway activation appears to be important across these disorders. The identification of constitutively activated pathways and the emergence of novel targeted pharmaceutical agents raise the expectation that more effective therapies will be identified for these disorders in the coming years.

The mechanism of cancer drug addiction in ALK-positive T-Cell lymphoma.

Rational new strategies are needed to treat tumors resistant to kinase inhibitors. Mechanistic studies of resistance provide fertile ground for development of new approaches. Cancer drug addiction is a paradoxical resistance phenomenon, well-described in MEK-ERK-driven solid tumors, in which drug-target overexpression promotes resistance but a toxic overdose of signaling if the inhibitor is withdrawn. This can permit prolonged control of tumors through intermittent dosing. We and others showed previously that cancer drug addiction arises also in the hematologic malignancy ALK-positive anaplastic large-cell lymphoma (ALCL) resistant to ALK-specific tyrosine kinase inhibitors (TKIs). This is driven by the overexpression of the fusion kinase NPM1-ALK, but the mechanism by which ALK overactivity drives toxicity upon TKI withdrawal remained obscure. Here we reveal the mechanism of ALK-TKI addiction in ALCL. We interrogated the well-described mechanism of MEK/ERK pathway inhibitor addiction in solid tumors and found it does not apply to ALCL. Instead, phosphoproteomics and confirmatory functional studies revealed that the STAT1 overactivation is the key mechanism of ALK-TKI addiction in ALCL. The withdrawal of TKI from addicted tumors in vitro and in vivo leads to overwhelming phospho-STAT1 activation, turning on its tumor-suppressive gene-expression program and turning off STAT3's oncogenic program. Moreover, a novel NPM1-ALK-positive ALCL PDX model showed a significant survival benefit from intermittent compared with continuous TKI dosing. In sum, we reveal for the first time the mechanism of cancer drug addiction in ALK-positive ALCL and the benefit of scheduled intermittent dosing in high-risk patient-derived tumors in vivo.

Colony-Stimulating Factor 1 Receptor (CSF1R) Activates AKT/mTOR Signaling and Promotes T-Cell Lymphoma Viability.

PURPOSE: Peripheral T-cell lymphomas are clinically aggressive and usually fatal, as few complete or durable remissions are achieved with currently available therapies. Recent evidence supports a critical role for lymphoma-associated macrophages during T-cell lymphoma progression, but the specific signals involved in the cross-talk between malignant T cells and their microenvironment are poorly understood. Colony-stimulator factor 1 receptor (CSF1R, CD115) is required for the homeostatic survival of tissue-resident macrophages. Interestingly, its aberrant expression has been reported in a subset of tumors. In this article, we evaluated its expression and oncogenic role in T-cell lymphomas. EXPERIMENTAL DESIGN: Loss-of-function studies, including pharmacologic inhibition with a clinically available tyrosine kinase inhibitor, pexidartinib, were performed in multiple in vitro and in vivo models. In addition, proteomic and genomic screenings were performed to discover signaling pathways that are activated downstream of CSF1R signaling. RESULTS: We observed that CSF1R is aberrantly expressed in many T-cell lymphomas, including a significant number of peripheral and cutaneous T-cell lymphomas. Colony-stimulating factor 1 (CSF1), in an autocrine or paracrine-dependent manner, leads to CSF1R autophosphorylation and activation in malignant T cells. Furthermore, CSF1R signaling was associated with significant changes in gene expression and in the phosphoproteome, implicating PI3K/AKT/mTOR in CSF1R-mediated T-cell lymphoma growth. We also demonstrated that inhibition of CSF1R in vivo and in vitro models is associated with decreased T-cell lymphoma growth. CONCLUSIONS: Collectively, these findings implicate CSF1R in T-cell lymphomagenesis and have significant therapeutic implications.

Improving Cancer Diagnosis and Care: Patient Access to High-Quality Oncologic Pathology.

Drawing on discussions at a workshop hosted by the National Cancer Policy Forum, current challenges in pathology are reviewed and practical steps to facilitate high­quality cancer diagnosis and care through improved patient access to expertise in oncologic pathology are highlighted.

Clinical Implications of Plasma-Based Genotyping With the Delivery of Personalized Therapy in Metastatic Non-Small Cell Lung Cancer.

Importance: The clinical implications of adding plasma-based circulating tumor DNA next-generation sequencing (NGS) to tissue NGS for targetable mutation detection in non-small cell lung cancer (NSCLC) have not been formally assessed. Objective: To determine whether plasma NGS testing was associated with improved mutation detection and enhanced delivery of personalized therapy in a real-world clinical setting. Design, Setting, and Participants: This prospective cohort study enrolled 323 patients with metastatic NSCLC who had plasma testing ordered as part of routine clinical management. Plasma NGS was performed using a 73-gene commercial platform. Patients were enrolled at the Hospital of the University of Pennsylvania from April 1, 2016, through January 2, 2018. The database was locked for follow-up and analyses on January 2, 2018, with a median follow-up of 7 months (range, 1-21 months). Main Outcomes and Measures: The number of patients with targetable alterations detected with plasma and tissue NGS; the association between the allele fractions (AFs) of mutations detected in tissue and plasma; and the association of response rate with the plasma AF of the targeted mutations. Results: Among the 323 patients with NSCLC (60.1% female; median age, 65 years [range, 33-93 years]), therapeutically targetable mutations were detected in EGFR, ALK, MET, BRCA1, ROS1, RET, ERBB2, or BRAF for 113 (35.0%) overall. Ninety-four patients (29.1%) had plasma testing only at the discretion of the treating physician or patient preference. Among the 94 patients with plasma testing alone, 31 (33.0%) had a therapeutically targetable mutation detected, thus obviating the need for an invasive biopsy. Among the remaining 229 patients who had concurrent plasma and tissue NGS or were unable to have tissue NGS, a therapeutically targetable mutation was detected in tissue alone for 47 patients (20.5%), whereas the addition of plasma testing increased this number to 82 (35.8%). Thirty-six of 42 patients (85.7%) who received a targeted therapy based on the plasma result achieved a complete or a partial response or stable disease. The plasma-based targeted mutation AF had no correlation with depth of Response Evaluation Criteria in Solid Tumors response (r = -0.121; P = .45). Conclusions and Relevance: Integration of plasma NGS testing into the routine management of stage IV NSCLC demonstrates a marked increase of the detection of therapeutically targetable mutations and improved delivery of molecularly guided therapy.