Faculty Bibliography

RIP1 regulates cell death and inflammation and is believed to play an important role in contributing to a variety of human pathologies, including immune-mediated inflammatory diseases and cancer. While small-molecule inhibitors of RIP1 kinase have been advanced to the clinic for inflammatory diseases and CNS indications, RIP1 inhibitors for oncology indications have yet to be described. Herein we report on the discovery and profile of GSK3145095 (compound 6). Compound 6 potently binds to RIP1 with exquisite kinase specificity and has excellent activity in blocking RIP1 kinase-dependent cellular responses. Highlighting its potential as a novel cancer therapy, the inhibitor was also able to promote a tumor suppressive T cell phenotype in pancreatic adenocarcinoma organ cultures. Compound 6 is currently in phase 1 clinical studies for pancreatic adenocarcinoma and other selected solid tumors.

Imiquimod is a topical toll-like-receptor-7 agonist currently used for treating basal cell carcinoma. Recently, imiquimod has demonstrated tumor regression in melanoma and breast cancer skin metastases. However, the molecular perturbations induced by imiquimod in breast cancer metastases have not been previously characterized. Here, we describe transcriptomic profiles associated with responsiveness to imiquimod in breast cancer skin metastases. Baseline and post-treatment tumor samples from patients treated with imiquimod in a clinical trial were profiled using Nanostring technology. Through an integrative analytic pipeline, we showed that tumors from patients who achieved a durable clinical response displayed a permissive microenvironment, substantiated by the upregulation of transcripts encoding for molecules involved in leukocyte adhesion and migration, cytotoxic functions, and antigen presentation. In responding patients, Imiquimod triggered a strong T-helper-1 (Th-1)/cytotoxic immune response, characterized by the coordinated upregulation of Th-1 chemokines, migration of Th-1 and cytotoxic T cells into the tumor, and activation of immune-effector functions, ultimately mediating tumor destruction. In conclusion, we have shown that topical imiquimod can induce a robust immune response in breast cancer metastases, and this response is more likely to occur in tumors with a pre-activated microenvironment. In this setting, imiquimod could be utilized in combination with other targeted immunotherapies to increase therapeutic efficacy.

The current model of polarized plasma membrane protein sorting in epithelial cells has been largely generated on the basis of experiments characterizing the polarized distribution of a relatively small number of overexpressed model proteins under various experimental conditions. Thus, the possibility exists that alternative roles of various types of sorting machinery may have been underestimated or missed. Here, we utilize domain-selective surface biotinylation combined with stable isotope labeling with amino acids in cell culture (SILAC) and mass spectrometry to quantitatively define large populations of apical and basolateral surface proteins in Madin-Darby canine kidney (MDCK) cells. We identified 313 plasma membrane proteins, of which 38% were apical, 51% were basolateral, and 11% were nonpolar. Silencing of clathrin adaptor proteins (AP) AP-1A, AP-1B, or both caused redistribution of basolateral proteins as expected but also, of a large population of apical proteins. Consistent with their previously reported ability to compensate for one another, the strongest loss of polarity was observed when we silenced AP-1A and AP-1B simultaneously. We found stronger evidence of compensation in the apical pathway compared with the basolateral pathway. Surprisingly, we also found subgroups of proteins that were affected after silencing just one adaptor, indicating previously unrecognized independent roles for AP-1A and AP-1B. While AP-1B silencing mainly affected basolateral polarity, AP-1A silencing seemed to cause comparable loss of apical and basolateral polarity. Our results uncover previously overlooked roles of AP-1 in polarized distribution of apical and basolateral proteins and introduce surface proteomics as a method to examine mechanisms of polarization with a depth not possible until now.

Although bacterial dysbiosis has been previously associated with carcinogenesis and HIV infection, the impact of the virome and these disease states has been less well studied. In this review, we will summarize what is known about the interplay between both the bacterial and the viral components of the microbiome on cancer and HIV pathogenesis. Bacterial dysbiosis has been associated with carcinogenesis such as colorectal cancer (CRC), hepatocellular carcinoma (HCC), lung cancer, breast cancer, and gastric cancer. The dysbiotic pathogenesis may be species-based or community-based and can have varying mechanisms of carcinogenesis. The human virome was also associated with certain cancers. Viruses, such as cytomegalovirus (CMV), Human herpesvirus 8 (HHV-8), human papilloma virus (HPV), hepatitis B virus (HBV), hepatitis C virus (HCV), and Epstein-Barr virus (EBV), all had associations with cancers. It was also reported that an altered bacteriophage community may lead to carcinogenesis by allowing opportunistic, oncogenic bacteria to proliferate in a gastrointestinal biofilm. This mechanism shows the importance of analyzing the bacteriome and the virome concurrently as their interactions can provide insight into new mechanisms in the pathogenesis of not only cancer, but other diseases as well. The enteric bacteriome was shown to be distinctly altered in immunocompromised HIV-infected individuals, and highly active antiretroviral therapy (HAART) was shown to at least partially reverse the alterations that HIV causes in the bacteriome. Studies have shown that the progression to HIV is associated with changes in the plasma concentration of commensal viruses. HIV also acts synergistically with multiple other viruses, such as HPV, EBV, varicella zoster virus (VZV), and HHV-8. Although it has been shown that HIV infection leads to enteric virome expansion in humans, most of the research on HIV's effect on the virome was conducted in non-human primates, and there is a lack of research on the effect of HAART on the virome. Virome-wide analysis is necessary for identifying novel viral etiologies. There is currently a wealth of information on the bacteriome and its associations with cancer and HIV, but more research should be conducted on the virome's associations and reaction to HAART as well as the bacteriome-virome interactions that may play a major role in pathogenesis and recovery.

Cardiolipin (CL) is a mitochondrial phospholipid with a very specific and functionally important fatty acid composition, generated by tafazzin. However, in vitro tafazzin catalyzes a promiscuous acyl exchange that acquires specificity only in response to perturbations of the physical state of lipids. To identify the process that imposes acyl specificity onto CL remodeling in vivo, we analyzed a series of deletions and knockdowns in Saccharomyces cerevisiae and Drosophila melanogaster, including carriers, membrane homeostasis proteins, fission-fusion proteins, cristae-shape controlling and MICOS proteins, and the complexes I-V. Among those, only the complexes of oxidative phosphorylation (OXPHOS) affected the CL composition. Rather than any specific complex, it was the global impairment of the OXPHOS system that altered CL and at the same time shortened its half-life. The knockdown of OXPHOS expression had the same effect on CL as the knockdown of tafazzin in Drosophila flight muscles, including a change in CL composition and the accumulation of monolyso-CL. Thus, the assembly of OXPHOS complexes induces CL remodeling, which, in turn, leads to CL stabilization. We hypothesize that protein crowding in the OXPHOS system imposes packing stress on the lipid bilayer, which is relieved by CL remodeling to form tightly packed lipid-protein complexes.

The many steps of gene expression, from the transcription of a gene to the production of its protein product, are well understood. Yet, transcriptional regulation has been the focal point for the study of gene expression during development. However, quantitative studies reveal that messenger RNA (mRNA) levels are not necessarily good predictors of the respective proteins' levels in a cell. This discrepancy is, at least in part, the result of developmentally regulated, translational mechanisms that control the spatiotemporal regulation of gene expression. In this review, we focus on translational regulatory mechanisms mediating global transitions in gene expression: the shift from the maternal to the embryonic developmental program in the early embryo and the switch from the self-renewal of stem cells to differentiation in the adult.

Background/UNASSIGNED:The purpose of this study is to evaluate the ability of preoperative and postoperative radiographic union scores for tibia fractures (RUST) to predict treatment success of tibia fracture nonunion. Materials and methods/UNASSIGNED:Patients presenting for operative treatment of tibia fracture nonunion were enrolled in a prospective data registry. Enrolled patients were followed at regular intervals for 12 months. Preoperative and 12 week postoperative radiographs were reviewed and scored using the RUST criteria. Postoperative time to union was determined by clinical and radiographic measures. Multivariate regressions were conducted to predict time to union using preoperative and postoperative RUST while controlling for treatment method. Receiver operating characteristic (ROC) curve was conducted to determine the accuracy of preoperative RUST in predicting failure of treatment. Results/UNASSIGNED:Sixty-eight patients with aseptic tibia fracture nonunion treated operatively were identified. Sixty-one patients achieved union. Mean preoperative RUST was 7.5 (SD 1.4). Mean postoperative RUST was 9.2 (SD 1.4). Multivariate linear regressions demonstrated that preoperative (p = 0.043) and postoperative (p = 0.007) RUST are significant predictors of time to union after tibia fracture nonunion surgery. ROC curve demonstrated preoperative RUST below 7 was a good predictor of developing persistent tibia fracture nonunion (AUC = 0.83, Sensitivity = 1.000, Specificity = 0.745). Conclusions/UNASSIGNED:RUST preoperatively and postoperatively predicts outcome after nonunion surgery. RUST can be used as part of the complete clinical picture to shape patient expectations and guide treatment.

Mitochondria contain their own genomes that, unlike nuclear genomes, are inherited only in the maternal line. Owing to a high mutation rate and low levels of recombination of mitrochondrial DNA (mtDNA), special selection mechanisms exist in the female germline to prevent the accumulation of deleterious mutations^1-5. However, the molecular mechanisms that underpin selection are poorly understood⁶. Here we visualize germline selection in Drosophila using an allele-specific fluorescent in situ-hybridization approach to distinguish wild-type from mutant mtDNA. Selection first manifests in the early stages of Drosophila oogenesis, triggered by reduction of the pro-fusion protein Mitofusin. This leads to the physical separation of mitochondrial genomes into different mitochondrial fragments, which prevents the mixing of genomes and their products and thereby reduces complementation. Once fragmented, mitochondria that contain mutant genomes are less able to produce ATP, which marks them for selection through a process that requires the mitophagy proteins Atg1 and BNIP3. A reduction in Atg1 or BNIP3 decreases the amount of wild-type mtDNA, which suggests a link between mitochondrial turnover and mtDNA replication. Fragmentation is not only necessary for selection in germline tissues, but is also sufficient to induce selection in somatic tissues in which selection is normally absent. We postulate that there is a generalizable mechanism for selection against deleterious mtDNA mutations, which may enable the development of strategies for the treatment of mtDNA disorders.

Developmental programs that generate the astonishing neuronal diversity of the nervous system are not completely understood and thus present a major challenge for clinical applications of guided cell differentiation strategies. Using direct neuronal programming of embryonic stem cells, we found that two main vertebrate proneural factors, Ascl1 and neurogenin 2 (Neurog2), induce different neuronal fates by binding to largely different sets of genomic sites. Their divergent binding patterns are not determined by the previous chromatin state, but are distinguished by enrichment of specific E-box sequences that reflect the binding preferences of the DNA-binding domains. The divergent Ascl1 and Neurog2 binding patterns result in distinct chromatin accessibility and enhancer activity profiles that differentially shape the binding of downstream transcription factors during neuronal differentiation. This study provides a mechanistic understanding of how transcription factors constrain terminal cell fates, and it delineates the importance of choosing the right proneural factor in neuronal reprogramming strategies.