Previous studies have shown that human immunodeficiency virus (HIV) protease cleaves procaspase 8 to a fragment, termed Casp8p41, that lacks caspase activity but nonetheless contributes to T cell apoptosis. Herein, we show that Casp8p41 contains a domain that interacts with the BH3-binding groove of pro-apoptotic Bak to cause Bak oligomerization, Bak-mediated membrane permeabilization, and cell death. Levels of active Bak are higher in HIV-infected T cells that express Casp8p41. Conversely, targeted mutations in the Bak-interacting domain diminish Bak binding and Casp8p41-mediated cell death. Similar mutations in procaspase 8 impair the ability of HIV to kill infected T cells. These observations support a novel paradigm in which HIV converts a normal cellular constituent into a direct activator that functions like a BH3-only protein.

To better address the problem of drug resistance during cancer chemotherapy and explore the possibility of manipulating drug response phenotypes, we developed a network-based phenotype mapping approach (P-Map) to identify gene candidates that upon perturbed can alter sensitivity to drugs. We used basal transcriptomics data from a panel of human lymphoblastoid cell lines (LCL) to infer drug response networks (DRNs) that are responsible for conferring response phenotypes for anthracycline and taxane, two common anticancer agents use in clinics. We further tested selected gene candidates that interact with phenotypic differentially expressed genes (PDEGs), which are up-regulated genes in LCL for a given class of drug response phenotype in triple-negative breast cancer (TNBC) cells. Our results indicate that it is possible to manipulate a drug response phenotype, from resistant to sensitive or vice versa, by perturbing gene candidates in DRNs and suggest plausible mechanisms regulating directionality of drug response sensitivity. More important, the current work highlights a new way to formulate systems-based therapeutic design: supplementing therapeutics that aim to target disease culprits with phenotypic modulators capable of altering DRN properties with the goal to re-sensitize resistant phenotypes.

BRCA1 is an important mediator of the DNA damage response, which promotes homologous recombination (HR) and antagonizes 53BP1-dependent non-homologous end joining in S/G2 phase. But how this is achieved remains unclear. Here, we report that the E3 ubiquitin ligase UHRF1 (Ubiquitin-like, with PHD and RING finger domains 1) directly participates in the interplay between BRCA1 and 53BP1. Mechanistically, UHRF1 is recruited to DNA double-strand breaks (DSBs) by BRCA1 in S phase, which requires the BRCT domain of BRCA1 and phosphorylated Ser674 of UHRF1. Subsequently, UHRF1 mediates K63-linked polyubiquitination of RIF1, and results in its dissociation from 53BP1 and DSBs thereby facilitating HR initiation. Thus, UHRF1 is a key regulator of DSB repair choice, which is separate from its role in heterochromatin formation and epigenetic regulator.

Tissue engineering provides unique opportunities for regenerating diseased or damaged tissues using cells obtained from tissue biopsies. Tissue engineered grafts can also be used as high fidelity models to probe cellular and molecular interactions underlying developmental processes. In this study, we co-cultured human umbilical vein endothelial cells (HUVECs) and human mesenchymal stem cells (MSCs) under various environmental conditions to elicit synergistic interactions leading to the colocalized development of capillary-like and bone-like tissues. Cells were encapsulated at the 1:1 ratio in fibrin gel to screen compositions of endothelial growth medium (EGM) and osteogenic medium (OM). It was determined that, to form both tissues, co-cultures should first be supplied with EGM followed by a 1:1 cocktail of the two media types containing bone morphogenetic protein-2. Subsequent studies of HUVECs and MSCs cultured in decellularized, trabecular bone scaffolds for 6 weeks assessed the effects on tissue construct of both temporal variations in growth-factor availability and addition of fresh cells. The resulting grafts were implanted subcutaneously into nude mice to determine the phenotype stability and functionality of engineered vessels. Two important findings resulted from these studies: (i) vascular development needs to be induced prior to osteogenesis, and (ii) the addition of additional hMSCs at the osteogenic induction stage improves both tissue outcomes, as shown by increased bone volume fraction, osteoid deposition, close proximity of bone proteins to vascular networks, and anastomosis of vascular networks with the host vasculature. Interestingly, these observations compare well with what has been described for native development. We propose that our cultivation system can mimic various aspects of endothelial cell-osteogenic precursor interactions in vivo, and could find utility as a model for studies of heterotypic cellular interactions that couple blood vessel formation with osteogenesis.

Although 70-80% of newly diagnosed ovarian cancer patients respond to first-line therapy, almost all relapse and five-year survival remains below 50%. One strategy to increase five-year survival is prolonging time to relapse by improving first-line therapy response. However, no biomarker today can accurately predict individual response to therapy. In this study, we present analytical and prospective clinical validation of a new test that utilizes primary patient tissue in 3D cell culture to make patient-specific response predictions prior to initiation of treatment in the clinic. Test results were generated within seven days of tissue receipt from newly diagnosed ovarian cancer patients obtained at standard surgical debulking or laparoscopic biopsy. Patients were followed for clinical response to chemotherapy. In a study population of 44, the 32 test-predicted Responders had a clinical response rate of 100% across both adjuvant and neoadjuvant treated populations with an overall prediction accuracy of 89% (39 of 44, p < 0.0001). The test also functioned as a prognostic readout with test-predicted Responders having a significantly increased progression-free survival compared to test-predicted Non-Responders, p = 0.01. This correlative accuracy establishes the test's potential to benefit ovarian cancer patients through accurate prediction of patient-specific response before treatment.

Interest in preclinical drug development for ovarian cancer has stimulated development of patient-derived xenograft (PDX) or tumorgraft models. However, the unintended formation of human lymphoma in severe combined immunodeficiency (SCID) mice from Epstein-Barr virus (EBV)-infected human lymphocytes can be problematic. In this study, we have characterized ovarian cancer PDXs which developed human lymphomas and explore methods to suppress lymphoproliferative growth. Fresh human ovarian tumors from 568 patients were transplanted intraperitoneally in SCID mice. A subset of PDX models demonstrated atypical patterns of dissemination with mediastinal masses, hepatosplenomegaly, and CD45-positive lymphoblastic atypia without ovarian tumor engraftment. Expression of human CD20 but not CD3 supported a B-cell lineage, and EBV genomes were detected in all lymphoproliferative tumors. Immunophenotyping confirmed monoclonal gene rearrangements consistent with B-cell lymphoma, and global gene expression patterns correlated well with other human lymphomas. The ability of rituximab, an anti-CD20 antibody, to suppress human lymphoproliferation from a patient's ovarian tumor in SCID mice and prevent growth of an established lymphoma led to a practice change with a goal to reduce the incidence of lymphomas. A single dose of rituximab during the primary tumor heterotransplantation process reduced the incidence of CD45-positive cells in subsequent PDX lines from 86.3% (n = 117 without rituximab) to 5.6% (n = 160 with rituximab), and the lymphoma rate declined from 11.1% to 1.88%. Taken together, investigators utilizing PDX models for research should routinely monitor for lymphoproliferative tumors and consider implementing methods to suppress their growth.

Microfluidics, the science of engineering fluid streams at the micrometer scale, offers unique tools for creating and controlling gradients of soluble compounds. Gradient generation can be used to recreate complex physiological microenvironments, but is also useful for screening purposes. For example, in a single experiment, adherent cells can be exposed to a range of concentrations of the compound of interest, enabling high-content analysis of cell behaviour and enhancing throughput. In this study, we present the development of a microfluidic screening platform where, by means of diffusion, gradients of soluble compounds can be generated and sustained. This platform enables the culture of adherent cells under shear stress-free conditions, and their exposure to a soluble compound in a concentration gradient-wise manner. The platform consists of five serial cell culture chambers, all coupled to two lateral fluid supply channels that are used for gradient generation through a source-sink mechanism. Furthermore, an additional inlet and outlet are used for cell seeding inside the chambers. Finite element modeling was used for the optimization of the design of the platform and for validation of the dynamics of gradient generation. Then, as a proof-of-concept, human osteosarcoma MG-63 cells were cultured inside the platform and exposed to a gradient of Cytochalasin D, an actin polymerization inhibitor. This set-up allowed us to analyze cell morphological changes over time, including cell area and eccentricity measurements, as a function of Cytochalasin D concentration by using fluorescence image-based cytometry.

Eradication of HIV-1 by the "kick and kill" strategy requires reactivation of latent virus to cause death of infected cells by either HIV-induced or immune-mediated apoptosis. To date this strategy has been unsuccessful, possibly due to insufficient cell death in reactivated cells to effectively reduce HIV-1 reservoir size. As a possible cause for this cell death resistance, we examined whether leading latency reversal agents (LRAs) affected apoptosis sensitivity of CD4 T cells. Multiple LRAs of different classes inhibited apoptosis in CD4 T cells. Protein kinase C (PKC) agonists bryostatin-1 and prostratin induced phosphorylation and enhanced neutralizing capability of the anti-apoptotic protein BCL2 in a PKC-dependent manner, leading to resistance to apoptosis induced by both intrinsic and extrinsic death stimuli. Furthermore, HIV-1 producing CD4 T cells expressed more BCL2 than uninfected cells, both in vivo and after ex vivo reactivation. Therefore, activation of BCL2 likely contributes to HIV-1 persistence after latency reversal with PKC agonists. The effects of LRAs on apoptosis sensitivity should be considered in designing HIV cure strategies predicated upon the "kick and kill" paradigm.

With the emergence of genome editing technologies and synthetic biology, it is now possible to engineer genetic circuits driving a cell's phenotypic response to a stressor. However, capturing a continuous response, rather than simply a binary 'on' or 'off' response, remains a bioengineering challenge. No tools currently exist to identify gene candidates responsible for predetermining and fine-tuning cell response phenotypes. To address this gap, we devised a novel Regulostat Inferelator (RSI) algorithm to decipher intrinsic molecular devices or networks that predetermine cellular phenotypic responses. The RSI algorithm is designed to extract gene expression patterns from basal transcriptomic data in order to identify 'regulostat' constituent gene pairs, which exhibit rheostat-like mode-of-cooperation capable of fine-tuning cellular response. Our proof-of-concept study provides computational evidence for the existence of regulostats and that these networks predetermine cellular response prior to exposure to a stressor or drug. In addition, our work, for the first time, provides evidence of context-specific, drug-regulostat interactions in predetermining drug response phenotypes in cancer cells. Given RSI-inferred regulostat networks offer insights for prioritizing gene candidates capable of rendering a resistant phenotype sensitive to a given drug, we envision that this tool will be of great value in bioengineering and medicine.

The fluoropyrimidines 5-fluorouracil (5-FU) and FdUrd (5-fluorodeoxyuridine; floxuridine) are the backbone of chemotherapy regimens for colon cancer and other tumors. Despite their widespread use, it remains unclear how these agents kill tumor cells. Here, we have analyzed the checkpoint and DNA repair pathways that affect colon tumor responses to 5-FU and FdUrd. These studies demonstrate that both FdUrd and 5-FU activate the ATR and ATM checkpoint signaling pathways, indicating that they cause genotoxic damage. Notably, however, depletion of ATM or ATR does not sensitize colon cancer cells to 5-FU, whereas these checkpoint pathways promote the survival of cells treated with FdUrd, suggesting that FdUrd exerts cytotoxicity by disrupting DNA replication and/or inducing DNA damage, whereas 5-FU does not. We also found that disabling the base excision (BER) repair pathway by depleting XRCC1 or APE1 sensitized colon cancer cells to FdUrd but not 5-FU. Consistent with a role for the BER pathway, we show that small molecule poly(ADP-ribose) polymerase 1/2 (PARP) inhibitors, AZD2281 and ABT-888, remarkably sensitized both mismatch repair (MMR)-proficient and -deficient colon cancer cell lines to FdUrd but not to 5-FU. Taken together, these studies demonstrate that the roles of genotoxin-induced checkpoint signaling and DNA repair differ significantly for these agents and also suggest a novel approach to colon cancer therapy in which FdUrd is combined with a small molecule PARP inhibitor.

We describe a phase II clinical trial of the combination of ribociclib and letrozole for treatment of relapsed oestrogen receptor (ER)-positive ovarian cancer (OC) and endometrial cancer (EC). The primary endpoint was the proportion of patients alive, progression-free survival (PFS), and still on treatment at 12 weeks (PFS12), with 45% or greater considered positive.

Endometriosis is a highly prevalent, chronic, heterogeneous, fibro-inflammatory disease that remains recalcitrant to conventional therapy. We previously showed that loss of KLF11, a transcription factor implicated in uterine disease, results in progression of endometriosis. Despite extensive homology, co-expression, and human disease association, loss of the paralog Klf10 causes a unique inflammatory, cystic endometriosis phenotype in contrast to fibrotic progression seen with loss of Klf11. We identify here for the first time a novel role for KLF10 in endometriosis. In an animal endometriosis model, unlike wild-type controls, Klf10(-/-) animals developed cystic lesions with massive immune infiltrate and minimal peri-lesional fibrosis. The Klf10(-/-) disease progression phenotype also contrasted with prolific fibrosis and minimal immune cell infiltration seen in Klf11(-/-) animals. We further found that lesion genotype rather than that of the host determined each unique disease progression phenotype. Mechanistically, KLF10 regulated CD40/CD154-mediated immune pathways. Both inflammatory as well as fibrotic phenotypes are the commonest clinical manifestations in chronic fibro-inflammatory diseases such as endometriosis. The complementary, paralogous Klf10 and Klf11 models therefore offer novel insights into the mechanisms of inflammation and fibrosis in a disease-relevant context. Our data suggests that divergence in underlying gene dysregulation critically determines disease-phenotype predominance rather than the conventional paradigm of inflammation being precedent to fibrotic scarring. Heterogeneity in clinical progression and treatment response are thus likely from disparate gene regulation profiles. Characterization of disease phenotype-associated gene dysregulation offers novel approaches for developing targeted, individualized therapy for recurrent and recalcitrant chronic disease.

Current understandings of individual disease etiology and therapeutics are limited despite great need. To fill the gap, we propose a novel computational pipeline that collects potent disease gene cooperative pathways to envision individualized disease etiology and therapies. Our algorithm constructs individualized disease modules de novo, which enables us to elucidate the importance of mutated genes in specific patients and to understand the synthetic penetrance of these genes across patients. We reveal that importance of the notorious cancer drivers TP53 and PIK3CA fluctuate widely across breast cancers and peak in tumors with distinct numbers of mutations and that rarely mutated genes such as XPO1 and PLEKHA1 have high disease module importance in specific individuals. Furthermore, individualized module disruption enables us to devise customized singular and combinatorial target therapies that were highly varied across patients, showing the need for precision therapeutics pipelines. As the first analysis of de novo individualized disease modules, we illustrate the power of individualized disease modules for precision medicine by providing deep novel insights on the activity of diseased genes in individuals.

Endoxifen, a secondary tamoxifen metabolite, is a potent antiestrogen exhibiting estrogen receptor alpha (ERα) binding at nanomolar concentrations. Phase I/II clinical trials identified clinical activity of Z-endoxifen (ENDX), in endocrine-refractory metastatic breast cancer as well as ERα+ solid tumors, raising the possibility that ENDX may have a second, ERα-independent, mechanism of action. An unbiased mass spectrometry approach revealed that ENDX concentrations achieved clinically with direct ENDX administration (5 µM), but not low concentrations observed during tamoxifen treatment (<0.1 µM), profoundly altered the phosphoproteome of the aromatase expressing MCF7AC1 cells with limited impact on the total proteome. Computational analysis revealed protein kinase C beta (PKCβ) and protein kinase B alpha or AKT1 as potential kinases responsible for mediating ENDX effects on protein phosphorylation. ENDX more potently inhibited PKCβ1 kinase activity compared to other PKC isoforms, and ENDX binding to PKCβ1 was confirmed using Surface Plasma Resonance. Under conditions that activated PKC/AKT signaling, ENDX induced PKCβ1 degradation, attenuated PKCβ1-activated AKTSer473 phosphorylation, diminished AKT substrate phosphorylation, and induced apoptosis. ENDX's effects on AKT were phenocopied by siRNA-mediated PKCβ1 knockdown or treatment with the pan-AKT inhibitor, MK-2206, while overexpression of constitutively active AKT diminished ENDX-induced apoptosis. These findings, which identify PKCβ1 as an ENDX target, indicate that PKCβ1/ENDX interactions suppress AKT signaling and induce apoptosis in breast cancer.

Follicular lymphoma (FL), an indolent neoplasm caused by a t(14;18) chromosomal translocation that juxtaposes the BCL2 gene and immunoglobulin locus, has a variable clinical course and frequently undergoes transformation to an aggressive lymphoma. Although BCL2 mutations have been previously described, their relationship to FL progression remains unclear. In this study, we evaluated the frequency and nature of BCL2 mutations in 2 independent cohorts of grade 1 and 2 FLs, along with the correlation between BCL2 mutations, transformation risk, and survival. The prevalence of BCL2 coding sequence mutations was 12% in FL at diagnosis and 53% at transformation (P < .0001). The presence of these BCL2 mutations at diagnosis correlated with an increased risk of transformation (hazard ratio 3.6; 95% CI, 2.0-6.2; P < .0001) and increased risk of death due to lymphoma (median survival of 9.5 years with BCL2 mutations vs 20.4 years without; P = .012). In a multivariate analysis, BCL2 mutations and high FL international prognostic index were independent risk factors for transformation and death due to lymphoma. Some mutant Bcl-2 proteins exhibited enhanced antiapoptotic capacity in vitro. Accordingly, BCL2 mutations can affect antiapoptotic Bcl-2 function, are associated with increased activation-induced cytidine deaminase expression, and correlate with increased risk of transformation and death due to lymphoma.

Protein-based hydrogels with distinct conformations which enable encapsulation or differentiation of cells are of great interest in 3D cancer research models. Conformational changes may cause macroscopic shifts in the hydrogels, allowing for its use as biosensors and drug carriers. In depth knowledge on how 3D conformational changes in proteins may affect cell fate and tumor formation is required. Thus, this study reports an enzymatically crosslinked silk fibroin (SF) hydrogel system that can undergo intrinsic conformation changes from random coil to β-sheet conformation. In random coil status, the SF hydrogels are transparent, elastic, and present ionic strength and pH stimuli-responses. The random coil hydrogels become β-sheet conformation after 10 days in vitro incubation and 14 days in vivo subcutaneous implantation in rat. When encapsulated with ATDC-5 cells, the random coil SF hydrogel promotes cell survival up to 7 days, whereas the subsequent β-sheet transition induces cell apoptosis in vitro. HeLa cells are further incorporated in SF hydrogels and the constructs are investigated in vitro and in an in vivo chick chorioallantoic membrane model for tumor formation. In vivo, Angiogenesis and tumor formation are suppressed in SF hydrogels. Therefore, these hydrogels provide new insights for cancer research and uses of biomaterials.

The sequential chain of interactions altering the binary state of a biomolecule represents the 'information flow' within a cellular network that determines phenotypic properties. Given the lack of computational tools to dissect context-dependent networks and gene activities, we developed NetDecoder, a network biology platform that models context-dependent information flows using pairwise phenotypic comparative analyses of protein-protein interactions. Using breast cancer, dyslipidemia and Alzheimer's disease as case studies, we demonstrate NetDecoder dissects subnetworks to identify key players significantly impacting cell behaviour specific to a given disease context. We further show genes residing in disease-specific subnetworks are enriched in disease-related signalling pathways and information flow profiles, which drive the resulting disease phenotypes. We also devise a novel scoring scheme to quantify key genes-network routers, which influence many genes, key targets, which are influenced by many genes, and high impact genes, which experience a significant change in regulation. We show the robustness of our results against parameter changes. Our network biology platform includes freely available source code (http://www.NetDecoder.org) for researchers to explore genome-wide context-dependent information flow profiles and key genes, given a set of genes of particular interest and transcriptome data. More importantly, NetDecoder will enable researchers to uncover context-dependent drug targets.

The healthspan of mice is enhanced by killing senescent cells using a transgenic suicide gene. Achieving the same using small molecules would have a tremendous impact on quality of life and the burden of age-related chronic diseases. Here, we describe the rationale for identification and validation of a new class of drugs termed senolytics, which selectively kill senescent cells. By transcript analysis, we discovered increased expression of pro-survival networks in senescent cells, consistent with their established resistance to apoptosis. Using siRNA to silence expression of key nodes of this network, including ephrins (EFNB1 or 3), PI3Kδ, p21, BCL-xL, or plasminogen-activated inhibitor-2, killed senescent cells, but not proliferating or quiescent, differentiated cells. Drugs targeting these same factors selectively killed senescent cells. Dasatinib eliminated senescent human fat cell progenitors, while quercetin was more effective against senescent human endothelial cells and mouse BM-MSCs. The combination of dasatinib and quercetin was effective in eliminating senescent MEFs. In vivo, this combination reduced senescent cell burden in chronologically aged, radiation-exposed, and progeroid Ercc1(-/Δ) mice. In old mice, cardiac function and carotid vascular reactivity were improved 5 days after a single dose. Following irradiation of one limb in mice, a single dose led to improved exercise capacity for at least 7 months following drug treatment. Periodic drug administration extended healthspan in Ercc1(-/∆) mice, delaying age-related symptoms and pathology, osteoporosis, and loss of intervertebral disk proteoglycans. These results demonstrate the feasibility of selectively ablating senescent cells and the efficacy of senolytics for alleviating symptoms of frailty and extending healthspan.

PARP inhibitors (PARPi) have activity in homologous recombination (HR) repair-deficient, high-grade serous ovarian cancers (HGSOC). However, even responsive tumors develop PARPi resistance, highlighting the need to delay or prevent the appearance of PARPi resistance. Here, we showed that the ALK kinase inhibitor ceritinib synergizes with PARPis by inhibiting complex I of the mitochondrial electron transport chain, which increases production of reactive oxygen species (ROS) and subsequent induction of oxidative DNA damage that is repaired in a PARP-dependent manner. In addition, combined treatment with ceritinib and PARPi synergized in HGSOC cell lines irrespective of HR status, and a combination of ceritinib with the PARPi olaparib induced tumor regression more effectively than olaparib alone in HGSOC patient-derived xenograft (PDX) models. Notably, the ceritinib and olaparib combination was most effective in PDX models with preexisting PARPi sensitivity and was well tolerated. These findings unveil suppression of mitochondrial respiration, accumulation of ROS, and subsequent induction of DNA damage as novel effects of ceritinib. They also suggest that the ceritinib and PARPi combination warrants further investigation as a means to enhance PARPi activity in HGSOC, particularly in tumors with preexisting HR defects. SIGNIFICANCE: The kinase inhibitor ceritinib synergizes with PARPi to induce tumor regression in ovarian cancer models, suggesting that ceritinib combined with PARPi may be an effective strategy for treating ovarian cancer.

Cytotoxic CD8+ T cells (CTL) are a crucial component of the immune system notable for their ability to eliminate rapidly proliferating malignant cells. However, the T-cell intrinsic factors required for human CTLs to accomplish highly efficient antitumor cytotoxicity are not well defined. By evaluating human CD8+ T cells from responders versus nonresponders to treatment with immune checkpoint inhibitors, we sought to identify key factors associated with effective CTL function. Single-cell RNA-sequencing analysis of peripheral CD8+ T cells from patients treated with anti-PD-1 therapy showed that cells from nonresponders exhibited decreased expression of the cytolytic granule-associated molecule natural killer cell granule protein-7 (NKG7). Functional assays revealed that reduced NKG7 expression altered cytolytic granule number, trafficking, and calcium release, resulting in decreased CD8+ T-cell-mediated killing of tumor cells. Transfection of T cells with NKG7 mRNA was sufficient to improve the tumor-cell killing ability of human T cells isolated from nonresponders and increase their response to anti-PD-1 or anti-PD-L1 therapy in vitro. NKG7 mRNA therapy also improved the antitumor activity of murine tumor antigen-specific CD8+ T cells in an in vivo model of adoptive cell therapy. Finally, we showed that the transcription factor ETS1 played a role in regulating NKG7 expression. Together, our results identify NKG7 as a necessary component for the cytotoxic function of CD8+ T cells and establish NKG7 as a T-cell-intrinsic therapeutic target for enhancing cancer immunotherapy.See related article by Li et al., p. 154.