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SUMMARY RNAi screens have implicated hundreds of host proteins as HIV-1 dependency factors (HDFs). While informative, these early studies overlap poorly due to false positives and false negatives. To ameliorate these issues, we combined information from the existing HDF screens together with new screens performed with multiple orthologous RNAi reagents (MORR). In addition to being traditionally validated, the MORR screens and the historical HDF screens were quantitatively integrated by the adaptation of an established analysis program, RIGER, for the collective interpretation of each gene’s phenotypic significance. False positives were addressed by the removal of poorly expressed candidates through gene expression filtering, as well as with GESS, which identifies off-target effects. This workflow produced a quantitatively integrated network of genes that modulate HIV-1 replication. We further investigated the roles of GOLGI49, SEC13, and COG in HIV-1 replication. Collectively, the MORR-RIGER method minimized the caveats of RNAi screening and improved our understanding of HIV-1–host cell interactions., Graphical Abstract

The systematic identification of effective drug combinations has been hindered by the unavailability of methods that can explore the large combinatorial search space of drug interactions. Here we present a multiplex screening method named MuSIC (Multiplex Screening for Interacting Compounds), which expedites the comprehensive assessment of pair-wise compound interactions. We examined ~500,000 drug pairs from 1000 FDA-approved or clinically tested drugs and identified drugs that synergize to inhibit HIV replication. Our analysis reveals an enrichment of anti-inflammatory drugs in drug combinations that synergize against HIV, indicating HIV benefits from inflammation that accompanies its infection. Multiple drug pairs identified in this study, including glucocorticoid and nitazoxanide, synergize by targeting different steps of the HIV life cycle. As inflammation accompanies HIV infection, our findings indicate that inhibiting inflammation could curb HIV propagation. MuSIC can be applied to a wide variety of disease-relevant screens to facilitate efficient identification of compound combinations.

shRNAs can trigger effective silencing of gene expression in mammalian cells, thereby providing powerful tools for genetic studies, as well as potential therapeutic strategies. Specific shRNAs can interfere with the replication of pathogenic viruses and are currently being tested as antiviral therapies in clinical trials. However, this effort is hindered by our inability to systematically and accurately identify potent shRNAs for viral genomes. Here we apply a recently developed highly parallel sensor assay to identify potent shRNAs for HIV, hepatitis C virus (HCV), and influenza. We observe known and previously unknown sequence features that dictate shRNAs efficiency. Validation using HIV and HCV cell culture models demonstrates very high potency of the top-scoring shRNAs. Comparing our data with the secondary structure of HIV shows that shRNA efficacy is strongly affected by the secondary structure at the target RNA site. Artificially introducing secondary structure to the target site markedly reduces shRNA silencing. In addition, we observe that HCV has distinct sequence features that bias HCV-targeting shRNAs toward lower efficacy. Our results facilitate further development of shRNA based antiviral therapies and improve our understanding and ability to predict efficient shRNAs.

TGFβ may act as a tumor suppressor early in tumorigenesis; however, TGFβ is also known to support tumor growth via multiple mechanisms, including promotion of epithelial-mesenchymal transition, fibroblast recruitment, ECM deposition, neovascularization, and immunosuppression of both adaptive and innate immune cells. SAR439459 is pan-neutralizing TGFβ antibody that is being developed for the treatment of solid tumors. In this study, we investigated the effect of SAR439459 on the immune system in primary human immune cells in vitro and in murine syngeneic tumor models in vivo. Exogenous TGFβ inhibited proliferation of CD8pos primary human T cells and suppressed the production of inflammatory cytokines such as IFNγ. The addition of SAR439459 blocked these effects. Additionally, TGFβ enhanced the development of inducible Tregs by approximately 4-fold, while the inclusion of SAR439459 abrogated this effect. The effects of exogenous TGFβ and SAR439459 were next examined in the context of T Cell Receptor signaling in a co-culture system of Jurkat cells expressing luciferase reporter under the control of NFAT-responsive promoter and PD-L1pos APCs. The addition of anti-PD1 stimulated TCR signaling in this system, while the addition of TGFβ inhibited anti-PD1-mediated response. SAR439459 restored the ability of anti-PD1 to stimulate the TCR signaling. These data support the notion that TGFβ neutralization could positively impact T effector cells and inhibit Tregs development. Therefore, combining a TGFβ-neutralizing antibody with anti-PD1 could enhance T cell responsiveness. Compared to checkpoint inhibitors, which are suggested to cause the reactivation and de-repression of exhausted T cells, the effects of TGFβ on the immune system are thought to be distinct. To test this, the combination of anti-PD1 and SAR439459 was examined in syngeneic mouse models. Established MC38 tumors were treated with anti-PD1, SAR439459 or the combination of both antibodies. In tumor-bearing mice treated with either anti-PD1 or SAR439459 alone, neither Ab resulted in significant tumor growth inhibition. However, the combination of anti-PD-1 and SAR439459 led to tumor regression in the majority of tumor-bearing mice. These results provide the rationale for combining TGF-β neutralization with checkpoint inhibitors, specifically anti-PD1, for the treatment of human cancers. Citation Format: Richard C. Gregory, Rita Greco, Hongjing Qu, Natalia Malkova, Mikhail Levit, Keli Perron, Waldemar Racki, Fangxian Sun, Gary Shapiro, Christopher Winter, Dmitri Wiederschain, Tun Tun Lin, Jack Pollard. The anti-TGFβ neutralizing antibody, SAR439459, blocks the immunosuppressive effects of TGFβ and inhibits the growth of syngeneic tumors in combination with anti-PD1 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2790.

TGFβ is a potent immunosuppressive cytokine, acting on multiple cell types of both the innate and adaptive immune systems. Emerging preclinical and clinical data implicate TGFβ in tumor immune evasion, poor prognosis and resistance to PD1/PDL1 directed checkpoint therapy. Thus, neutralization of TGFβ is suggested to relieve immunosuppression via mechanisms that are distinct yet complementary to checkpoint inhibitors such as PD1. SAR439459 is a potent human pan-neutralizing anti-TGFβ antibody that has entered first-in-human studies in advanced solid tumors (NCT03192345*). I. Biomarkers for indication selection: A gene expression signature of TGFβ pathway activation was derived from analysis of TGFβ-stimulated versus naïve cancer cell lines. Queries of the signature against The Cancer Genome Atlas (TCGA) revealed that primary head and neck, ovarian, and colorectal cancers are enriched for activation. II. Biomarkers for patient selection: An analysis of the patients enriched for activation revealed that mesenchymal tumors predominate. Machine learning methods were applied to panels of gene expression data for colorectal (CRC), ovarian serous and head and neck squamous cell carcinoma to derive a two-gene biomarker for selection of patients with mesenchymal tumors. Implementation in a logistic regression model achieves good for prediction in CRC, and similar performance in other indications. Patient cohorts are enriched over three-fold for the mesenchymal subtype and false-negative rates are acceptable. Implementation of the two-gene biomarker on a low complexity platform and its validation on an independent panel of samples is described. III. Biomarkers for assessment of the tumor microenvironment: In partnership with NeoGenomics, the immune contexture of patient tumors was evaluated using MultiOmyx, a multiplex IHC assay, on CRC and melanoma. Multiplexing was conducted with 12 biomarkers (jointly describing 22 immune cell types) on single FFPE section from each tumor sample. Pilot studies included a range of inflammation to evaluate how well the analytics assess each tumor type and correlate to possible treatment effects. Statistical methods were developed to assess differences at the cell population level, including replicate concordance, volcano plots for analyses of variance, and correlation matrices. The MultiOmyx assay demonstrated excellent technical reproducibility and precision, a favorable dynamic range, inflammation status differences in select immune cells and regions of interest, and included both positive and negative correlations between cell populations. * https://clinicaltrials.gov/ct2/show/NCT03192345 Citation Format: Joachim Theilhaber, Jean Cavallo, Stephen L. Madden, Charlene Manning, Sherry Cao, Parminder Mankoo, Robert Pomponio, Hongjing Qu, Natalia Malkova, Gary Shapiro, Christopher Winter, Dmitri Wiederschain, Michele Sanicola-Nadel, Fangxian Sun, Tun Tun Lin, Richard C. Gregory, Jack Pollard. Translational biomarkers for SAR439459, an anti-TGFβ antibody for cancer immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5550.

Nanoscience and nanotechnology require development of nanomaterials that are amiable for molecular design from bottom up. Molecular designer self-assembling peptides are one of such nanomaterials that will become increasingly important for the endeavor. Peptides have not only been used in all aspects of biomedical and pharmaceutical research and medical products, but also have had enormous impact in nascent field of designed biological materials. We here report the dynamic structures of lipid-like designer peptide A6D (AAAAAAD) and A6K (AAAAAAK) that undergo self-assembly into nanotubes in water and salt solution. We not only analyzed their self-assemblies using dynamic light scattering to determine the critical aggregation concentration (CAC), but also use atomic force microscope to observe their nanostructures. We also propose a simple scheme by which these lipid-like peptides self-assemble into dynamic nanostructures. Since the knowledge of CAC is important for uses of these peptides for a variety of applications, these findings may have significant implications in the study of molecular self-assembly and for a wide range of utilities of designer self-assembling peptide materials.

An epistasis map (E-MAP) was constructed in the fission yeast, Schizosaccharomyces pombe , by systematically measuring the phenotypes associated with pairs of mutations. This high-density, quantitative genetic interaction map focused on various aspects of chromosome function, including transcription regulation and DNA repair/replication. The E-MAP uncovered a previously unidentified component of the RNA interference (RNAi) machinery ( rsh1 ) and linked the RNAi pathway to several other biological processes. Comparison of the S. pombe E-MAP to an analogous genetic map from the budding yeast revealed that, whereas negative interactions were conserved between genes involved in similar biological processes, positive interactions and overall genetic profiles between pairs of genes coding for physically associated proteins were even more conserved. Hence, conservation occurs at the level of the functional module (protein complex), but the genetic cross talk between modules can differ substantially.

Nanoscience and nanotechnology require development of nanomaterials that are amiable for molecular design from bottom up. Molecular designer self-assembling peptides are one of such nanomaterials that will become increasingly important for the endeavor. Peptides have not only been used in all aspects of biomedical and pharmaceutical research and medical products, but also have had enormous impact in nascent field of designed biological materials. We here report the dynamic structures of lipid-like designer peptide A6D (AAAAAAD) and A6K (AAAAAAK) that undergo self-assembly into nanotubes in water and salt solution. We not only analyzed their self-assemblies using dynamic light scattering to determine the critical aggregation concentration (CAC), but also use atomic force microscope to observe their nanostructures. We also propose a simple scheme by which these lipid-like peptides self-assemble into dynamic nanostructures. Since the knowledge of CAC is important for uses of these peptides for a variety of applications, these findings may have significant implications in the study of molecular self-assembly and for a wide range of utilities of designer self-assembling peptide materials.

HIV-1 depends on many host factors for propagation. Other host factors, however, antagonize HIV-1 and may have profound effects on viral activation. Curing HIV-1 requires the reduction of latent viral reservoirs that remain in the face of antiretroviral therapy (ART). Using orthologous genetic screens, we identified bromodomain containing 4 (BRD4) as a negative regulator of HIV-1 replication. Antagonism of BRD4, via RNA interference or with a small molecule inhibitor, JQ1, both increased proviral transcriptional elongation and alleviated HIV-1 latency in cell line models. In multiple instances, JQ1 when used in combination with the NF-κB activators, Prostratin or PHA, enhanced the in vitro reactivation of latent HIV-1 in primary human T cells. These data are consistent with a model wherein BRD4 competes with the virus for HIV-1 dependency factors (HDFs) and suggests that combinatorial therapies that activate HDFs and antagonize HIV-1 competitive factors may be useful for curing HIV-1 infection.

SummaryIntroduction of disulfide bonds into proteins entering the secretory pathway is catalyzed by Ero1p, which generates disulfide bonds de novo, and Pdi1p, which transfers disulfides to substrate proteins. A sufficiently oxidizing environment must be maintained in the endoplasmic reticulum (ER) to allow for disulfide formation, but a pool of reduced thiols is needed for isomerization of incorrectly paired disulfides. We have found that hyperoxidation of the ER is prevented by attenuation of Ero1p activity through noncatalytic cysteine pairs. Deregulated Ero1p mutants lacking certain cysteines show increased enzyme activity, a decreased lag phase in kinetic assays, and growth defects in vivo. We hypothesize that noncatalytic cysteine pairs in Ero1p sense the level of potential substrates in the ER and correspondingly modulate Ero1p activity as part of a homeostatic regulatory system governing the thiol-disulfide balance in the ER.