The Chlamydia trachomatis Inc Tri1 interacts with TRAF7 to displace native TRAF7 interacting partners.

Chlamydia trachomatis is the leading cause of bacterial sexually transmitted infections in the USA and of preventable blindness worldwide. This obligate intracellular pathogen replicates within a membrane-bound inclusion, but how it acquires nutrients from the host while avoiding detection by the innate immune system is incompletely understood. C. trachomatis accomplishes this in part through the translocation of a unique set of effectors into the inclusion membrane, the inclusion membrane proteins (Incs). Incs are ideally positioned at the host-pathogen interface to reprogram host signaling by redirecting proteins or organelles to the inclusion. Using a combination of co-affinity purification, immunofluorescence confocal imaging, and proteomics, we characterize the interaction between an early-expressed Inc of unknown function, Tri1, and tumor necrosis factor receptor-associated factor 7 (TRAF7). TRAF7 is a multi-domain protein with a RING finger ubiquitin ligase domain and a C-terminal WD40 domain. TRAF7 regulates several innate immune signaling pathways associated with C. trachomatis infection and is mutated in a subset of tumors. We demonstrate that Tri1 and TRAF7 specifically interact during infection and that TRAF7 is recruited to the inclusion. We further show that the predicted coiled-coil domain of Tri1 is necessary to interact with the TRAF7 WD40 domain. Finally, we demonstrate that Tri1 displaces the native TRAF7 binding partners, mitogen-activated protein kinase kinase kinase 2 (MEKK2), and MEKK3. Together, our results suggest that by displacing TRAF7 native binding partners, Tri1 has the capacity to alter TRAF7 signaling during C. trachomatis infection.IMPORTANCEChlamydia trachomatis is the leading cause of bacterial sexually transmitted infections in the USA and preventable blindness worldwide. Although easily treated with antibiotics, the vast majority of infections are asymptomatic and therefore go untreated, leading to infertility and blindness. This obligate intracellular pathogen evades the immune response, which contributes to these outcomes. Here, we characterize the interaction between a C. trachomatis-secreted effector, Tri1, and a host protein involved in innate immune signaling, TRAF7. We identified host proteins that bind to TRAF7 and demonstrated that Tri1 can displace these proteins upon binding to TRAF7. Remarkably, the region of TRAF7 to which these host proteins bind is often mutated in a subset of human tumors. Our work suggests a mechanism by which Tri1 may alter TRAF7 signaling and has implications not only in the pathogenesis of C. trachomatis infections but also in understanding the role of TRAF7 in cancer.

The Chlamydia trachomatis Inc Tri1 interacts with TRAF7 to displace native TRAF7 interacting partners.

Chlamydia trachomatis is the leading cause of bacterial sexually transmitted infections in the US and of preventable blindness worldwide. This obligate intracellular pathogen replicates within a membrane-bound inclusion, but how it acquires nutrients from the host while avoiding detection by the innate immune system is incompletely understood. C. trachomatis accomplishes this in part through the translocation of a unique set of effectors into the inclusion membrane, the inc lusion membrane proteins (Incs). Incs are ideally positioned at the host-pathogen interface to reprogram host signaling by redirecting proteins or organelles to the inclusion. Using a combination of co-affinity purification, immunofluorescence confocal imaging, and proteomics, we characterize the interaction between an early-expressed Inc of unknown function, Tri1, and tumor necrosis factor receptor associated factor 7 (TRAF7). TRAF7 is a multi-domain protein with a RING finger ubiquitin ligase domain and a C-terminal WD40 domain. TRAF7 regulates several innate immune signaling pathways associated with C. trachomatis infection and is mutated in a subset of tumors. We demonstrate that Tri1 and TRAF7 specifically interact during infection and that TRAF7 is recruited to the inclusion. We further show that the predicted coiled-coil domain of Tri1 is necessary to interact with the TRAF7 WD40 domain. Finally, we demonstrate that Tri1 displaces the native TRAF7 binding partners, mitogen activated protein kinase kinase kinase 2 (MEKK2) and MEKK3. Together, our results suggest that by displacing TRAF7 native binding partners, Tri1 has the capacity to alter TRAF7 signaling during C. trachomatis infection. Importance: Chlamydia trachomatis is the leading cause of bacterial sexually transmitted infections in the US and preventable blindness worldwide. Although easily treated with antibiotics, the vast majority of infections are asymptomatic and therefore go untreated, leading to infertility and blindness. This obligate intracellular pathogen evades the immune response, which contributes to these outcomes. Here, we characterize the interaction between a C. trachomatis secreted effector, Tri1, and a host protein involved in innate immune signaling, TRAF7. We identified host proteins that bind to TRAF7 and demonstrate that Tri1 can displace these proteins upon binding to TRAF7. Remarkably, the region of TRAF7 to which these host proteins bind is often mutated in a subset of human tumors. Our work suggests a mechanism by which Tri1 may alter TRAF7 signaling and has implications not only in the pathogenesis of C. trachomatis infections, but also in understanding the role of TRAF7 in cancer.

U2 snRNA structure is influenced by SF3A and SF3B proteins but not by SF3B inhibitors.

U2 snRNP is an essential component of the spliceosome. It is responsible for branch point recognition in the spliceosome A-complex via base-pairing of U2 snRNA with an intron to form the branch helix. Small molecule inhibitors target the SF3B component of the U2 snRNP and interfere with A-complex formation during spliceosome assembly. We previously found that the first SF3B inhibited-complex is less stable than A-complex and hypothesized that SF3B inhibitors interfere with U2 snRNA secondary structure changes required to form the branch helix. Using RNA chemical modifiers, we probed U2 snRNA structure in A-complex and SF3B inhibited splicing complexes. The reactivity pattern for U2 snRNA in the SF3B inhibited-complex is indistinguishable from that of A-complex, suggesting that they have the same secondary structure conformation, including the branch helix. This observation suggests SF3B inhibited-complex instability does not stem from an alternate RNA conformation and instead points to the inhibitors interfering with protein component interactions that normally stabilize U2 snRNP's association with an intron. In addition, we probed U2 snRNA in the free U2 snRNP in the presence of SF3B inhibitor and again saw no differences. However, increased protection of nucleotides upstream of Stem I in the absence of SF3A and SF3B proteins suggests a change of secondary structure at the very 5' end of U2 snRNA. Chemical probing of synthetic U2 snRNA in the absence of proteins results in similar protections and predicts a previously uncharacterized extension of Stem I. Because this stem must be disrupted for SF3A and SF3B proteins to stably join the snRNP, the structure has the potential to influence snRNP assembly and recycling after spliceosome disassembly.

Next-Generation Sequencing of Retinoblastoma Identifies Pathogenic Alterations beyond RB1 Inactivation That Correlate with Aggressive Histopathologic Features.

PURPOSE: To determine the usefulness of a comprehensive, targeted-capture next-generation sequencing (NGS) assay for the clinical management of children undergoing enucleation for retinoblastoma. DESIGN: Cohort study. PARTICIPANTS: Thirty-two children with retinoblastoma. METHODS: We performed targeted NGS using the UCSF500 Cancer Panel (University of California, San Francisco, San Francisco, CA) on formalin-fixed, paraffin-embedded tumor tissue along with constitutional DNA isolated from peripheral blood, buccal swab, or uninvolved optic nerve. Peripheral blood samples were also sent to a commercial laboratory for germline RB1 mutation testing. MAIN OUTCOME MEASURES: Presence or absence of germline RB1 mutation or deletion, tumor genetic profile, and association of genetic alterations with clinicopathologic features. RESULTS: Germline mutation or deletion of the RB1 gene was identified in all children with bilateral retinoblastoma (n = 12), and these NGS results were 100% concordant with commercial germline RB1 mutation analysis. In tumor tissue tested with NGS, biallelic inactivation of RB1 was identified in 28 tumors and focal MYCN amplification was identified in 4 tumors (2 with wild-type RB1 and 2 with biallelic RB1 inactivation). Additional likely pathogenic alterations beyond RB1 were identified in 13 tumors (41%), several of which have not been reported previously in retinoblastoma. These included focal amplifications of MDM4 and RAF1, as well as damaging mutations involving BCOR, ARID1A, MGA, FAT1, and ATRX. The presence of additional likely pathogenetic mutations beyond RB1 inactivation was associated with aggressive histopathologic features, including higher histologic grade and anaplasia, and also with both unilateral and sporadic disease. CONCLUSIONS: Comprehensive NGS analysis reliably detects relevant mutations, amplifications, and chromosomal copy number changes in retinoblastoma. The presence of genetic alterations beyond RB1 inactivation correlates with aggressive histopathologic features.

A requirement for STAG2 in replication fork progression creates a targetable synthetic lethality in cohesin-mutant cancers.

Cohesin is a multiprotein ring that is responsible for cohesion of sister chromatids and formation of DNA loops to regulate gene expression. Genomic analyses have identified that the cohesin subunit STAG2 is frequently inactivated by mutations in cancer. However, the reason STAG2 mutations are selected during tumorigenesis and strategies for therapeutically targeting mutant cancer cells are largely unknown. Here we show that STAG2 is essential for DNA replication fork progression, whereby STAG2 inactivation in non-transformed cells leads to replication fork stalling and collapse with disruption of interaction between the cohesin ring and the replication machinery as well as failure to establish SMC3 acetylation. As a consequence, STAG2 mutation confers synthetic lethality with DNA double-strand break repair genes and increased sensitivity to select cytotoxic chemotherapeutic agents and PARP or ATR inhibitors. These studies identify a critical role for STAG2 in replication fork procession and elucidate a potential therapeutic strategy for cohesin-mutant cancers.

Well-differentiated papillary mesothelioma of the peritoneum is genetically defined by mutually exclusive mutations in TRAF7 and CDC42.

Well-differentiated papillary mesothelioma is an uncommon mesothelial neoplasm that most frequently arises in the peritoneal cavity of women of reproductive age. Whereas malignant mesothelioma is an aggressive tumor associated with poor outcome, well-differentiated papillary mesothelioma typically exhibits indolent behavior. However, histologically differentiating between these two entities can be challenging, necessitating the development of distinguishing biomarkers. While the genetic alterations that drive malignant mesothelioma have recently been determined, the molecular pathogenesis of well-differentiated papillary mesothelioma is unknown. Here we performed genomic profiling on a cohort of ten well-differentiated papillary mesothelioma of the peritoneum. We identified that all tumors harbored somatic missense mutations in either the TRAF7 or CDC42 genes, and lacked alterations involving BAP1, NF2, CDKN2A, DDX3X, SETD2, and ALK that are frequent in malignant mesothelioma. We recently identified that another mesothelial neoplasm, adenomatoid tumor of the genital tract, is genetically defined by somatic missense mutations in the TRAF7 gene, indicating a shared molecular pathogenesis between well-differentiated papillary mesothelioma and adenomatoid tumors. To the best of our knowledge, well-differentiated papillary mesothelioma is the first human tumor type found to harbor recurrent mutations in the CDC42 gene, which encodes a Rho family GTPase. Immunohistochemistry demonstrated intact BAP1 expression in all cases of well-differentiated papillary mesothelioma, indicating that this is a reliable marker for distinguishing well-differentiated papillary mesothelioma from malignant mesotheliomas that frequently display loss of expression. Additionally, all well-differentiated papillary mesothelioma demonstrated robust expression of L1 cell adhesion molecule (L1CAM), a marker of NF-kB pathway activation, similar to that observed in adenomatoid tumors. In contrast, we have previously shown that L1CAM staining is not observed in normal mesothelial cells and malignant mesotheliomas of the peritoneum. Together, these studies demonstrate that well-differentiated papillary mesothelioma is genetically defined by mutually exclusive mutations in TRAF7 and CDC42 that molecularly distinguish this entity from malignant mesothelioma.

Cell of origin and mutation pattern define three clinically distinct classes of sebaceous carcinoma.

Sebaceous carcinomas (SeC) are cutaneous malignancies that, in rare cases, metastasize and prove fatal. Here we report whole-exome sequencing on 32 SeC, revealing distinct mutational classes that explain both cancer ontogeny and clinical course. A UV-damage signature predominates in 10/32 samples, while nine show microsatellite instability (MSI) profiles. UV-damage SeC exhibited poorly differentiated, infiltrative histopathology compared to MSI signature SeC (p = 0.003), features previously associated with dissemination. Moreover, UV-damage SeC transcriptomes and anatomic distribution closely resemble those of cutaneous squamous cell carcinomas (SCC), implicating sun-exposed keratinocytes as a cell of origin. Like SCC, this UV-damage subclass harbors a high somatic mutation burden with >50 mutations per Mb, predicting immunotherapeutic response. In contrast, ocular SeC acquires far fewer mutations without a dominant signature, but show frequent truncations in the ZNF750 epidermal differentiation regulator. Our data exemplify how different mutational processes convergently drive histopathologically related but clinically distinct cancers.

Adenomatoid tumors of the male and female genital tract are defined by TRAF7 mutations that drive aberrant NF-kB pathway activation.

Adenomatoid tumors are the most common neoplasm of the epididymis, and histologically similar adenomatoid tumors also commonly arise in the uterus and fallopian tube. To investigate the molecular pathogenesis of these tumors, we performed genomic profiling on a cohort of 31 adenomatoid tumors of the male and female genital tracts. We identified that all tumors harbored somatic missense mutations in the TRAF7 gene, which encodes an E3 ubiquitin ligase belonging to the family of tumor necrosis factor receptor-associated factors (TRAFs). These mutations all clustered into one of five recurrent hotspots within the WD40 repeat domains at the C-terminus of the protein. Functional studies in vitro revealed that expression of mutant but not wild-type TRAF7 led to increased phosphorylation of nuclear factor-kappa B (NF-kB) and increased expression of L1 cell adhesion molecule (L1CAM), a marker of NF-kB pathway activation. Immunohistochemistry demonstrated robust L1CAM expression in adenomatoid tumors that was absent in normal mesothelial cells, malignant peritoneal mesotheliomas and multilocular peritoneal inclusion cysts. Together, these studies demonstrate that adenomatoid tumors of the male and female genital tract are genetically defined by TRAF7 mutation that drives aberrant NF-kB pathway activation.