Exosomes.

Exosomes are small, single-membrane, secreted organelles of ∼30 to ∼200 nm in diameter that have the same topology as the cell and are enriched in selected proteins, lipids, nucleic acids, and glycoconjugates. Exosomes contain an array of membrane-associated, high-order oligomeric protein complexes, display pronounced molecular heterogeneity, and are created by budding at both plasma and endosome membranes. Exosome biogenesis is a mechanism of protein quality control, and once released, exosomes have activities as diverse as remodeling the extracellular matrix and transmitting signals and molecules to other cells. This pathway of intercellular vesicle traffic plays important roles in many aspects of human health and disease, including development, immunity, tissue homeostasis, cancer, and neurodegenerative diseases. In addition, viruses co-opt exosome biogenesis pathways both for assembling infectious particles and for establishing host permissiveness. On the basis of these and other properties, exosomes are being developed as therapeutic agents in multiple disease models.

MAP Kinase Inhibition Promotes T Cell and Anti-tumor Activity in Combination with PD-L1 Checkpoint Blockade.

Targeted inhibition of mitogen-activated protein kinase (MAPK) kinase (MEK) can induce regression of tumors bearing activating mutations in the Ras pathway but rarely leads to tumor eradication. Although combining MEK inhibition with T-cell-directed immunotherapy might lead to more durable efficacy, T cell responses are themselves at least partially dependent on MEK activity. We show here that MEK inhibition did profoundly block naive CD8(+) T cell priming in tumor-bearing mice, but actually increased the number of effector-phenotype antigen-specific CD8(+) T cells within the tumor. MEK inhibition protected tumor-infiltrating CD8(+) T cells from death driven by chronic TCR stimulation while sparing cytotoxic activity. Combining MEK inhibition with anti-programmed death-ligand 1 (PD-L1) resulted in synergistic and durable tumor regression even where either agent alone was only modestly effective. Thus, despite the central importance of the MAP kinase pathway in some aspects of T cell function, MEK-targeted agents can be compatible with T-cell-dependent immunotherapy.

A cell identity switch allows residual BCC to survive Hedgehog pathway inhibition.

Despite the efficacy of Hedgehog pathway inhibitors in the treatment of basal cell carcinoma (BCC)1, residual disease persists in some patients and may contribute to relapse when treatment is discontinued2. Here, to study the effect of the Smoothened inhibitor vismodegib on tumour clearance, we have used a Ptch1-Trp53 mouse model of BCC3 and found that mice treated with vismodegib harbour quiescent residual tumours that regrow upon cessation of treatment. Profiling experiments revealed that residual BCCs initiate a transcriptional program that closely resembles that of stem cells of the interfollicular epidermis and isthmus, whereas untreated BCCs are more similar to the hair follicle bulge. This cell identity switch was enabled by a mostly permissive chromatin state accompanied by rapid Wnt pathway activation and reprogramming of super enhancers to drive activation of key transcription factors involved in cellular identity. Accordingly, treatment of BCC with both vismodegib and a Wnt pathway inhibitor reduced the residual tumour burden and enhanced differentiation. Our study identifies a resistance mechanism in which tumour cells evade treatment by adopting an alternative identity that does not rely on the original oncogenic driver for survival.

Degron tagging of BleoR and other antibiotic-resistance genes selects for higher expression of linked transgenes and improved exosome engineering.

Five antibiotic resistance (AR) genes have been used to select for transgenic eukaryotic cell lines, with the BleoR, PuroR, HygR, NeoR, and BsdR cassettes conferring resistance to zeocin, puromycin, hygromycin, geneticin/G418, and blasticidin, respectively. We recently demonstrated that each AR gene establishes a distinct threshold of transgene expression below which no cell can survive, with BleoR selecting for the highest level of transgene expression, nearly ∼10-fold higher than in cells selected using the NeoR or BsdR markers. Here, we tested the hypothesis that there may be an inverse proportionality between AR protein function and the expression of linked, transgene-encoded, recombinant proteins. Specifically, we fused each AR protein to proteasome-targeting degron tags, used these to select for antibiotic-resistant cell lines, and then measured the expression of the linked, recombinant protein, mCherry, as a proxy marker of transgene expression. In each case, degron-tagged AR proteins selected for higher mCherry expression than their cognate WT AR proteins. ER50BleoR selected for the highest level of mCherry expression, greater than twofold higher than BleoR or any other AR gene. Interestingly, use of ER50BleoR as the selectable marker translated to an even higher, 3.5-fold increase in the exosomal loading of the exosomal cargo protein, CD63/Y235A. Although a putative CD63-binding peptide, CP05, has been used to decorate exosome membranes in a technology known as "exosome painting," we show here that CP05 binds equally well to CD63-/- cells, WT 293F cells, and CD63-overexpressing cells, indicating that CP05 may bind membranes nonspecifically. These results are of high significance for cell engineering and especially for exosome engineering.

A shared, stochastic pathway mediates exosome protein budding along plasma and endosome membranes.

Exosomes are small extracellular vesicles of ∼30 to 150 nm that are secreted by all cells, abundant in all biofluids, and play important roles in health and disease. However, details about the mechanism of exosome biogenesis are unclear. Here, we carried out a cargo-based analysis of exosome cargo protein biogenesis in which we identified the most highly enriched exosomal cargo proteins and then followed their biogenesis, trafficking, and exosomal secretion to test different hypotheses for how cells make exosomes. We show that exosome cargo proteins bud from cells (i) in exosome-sized vesicles regardless of whether they are localized to plasma or endosome membranes, (ii) ∼5-fold more efficiently when localized to the plasma membrane, (iii) ∼5-fold less efficiently when targeted to the endosome membrane, (iv) by a stochastic process that leads to ∼100-fold differences in their abundance from one exosome to another, and (v) independently of small GTPase Rab27a, the ESCRT complex-associated protein Alix, or the cargo protein CD63. Taken together, our results demonstrate that cells use a shared, stochastic mechanism to bud exosome cargoes along the spectrum of plasma and endosome membranes and far more efficiently from the plasma membrane than the endosome. Our observations also indicate that the pronounced variation in content between different exosome-sized vesicles is an inevitable consequence of a stochastic mechanism of small vesicle biogenesis, that the origin membrane of exosome-sized extracellular vesicles simply cannot be determined, and that most of what we currently know about exosomes has likely come from studies of plasma membrane-derived vesicles.

Choice of selectable marker affects recombinant protein expression in cells and exosomes.

Transgenic mammalian cells are used for numerous research, pharmaceutical, industrial, and clinical purposes, and dominant selectable markers are often used to enable the selection of transgenic cell lines. Using HEK293 cells, we show here that the choice of selectable marker gene has a significant impact on both the level of recombinant protein expression and the cell-to-cell variability in recombinant protein expression. Specifically, we observed that cell lines generated with the NeoR or BsdR selectable markers and selected in the antibiotics G418 or blasticidin, respectively, displayed the lowest level of recombinant protein expression as well as the greatest cell-to-cell variability in transgene expression. In contrast, cell lines generated with the BleoR marker and selected in zeocin yielded cell lines that expressed the highest levels of linked recombinant protein, approximately 10-fold higher than those selected using the NeoR or BsdR markers, as well as the lowest cell-to-cell variability in recombinant protein expression. Intermediate yet still-high levels of expression were observed in cells generated with the PuroR- or HygR-based vectors and that were selected in puromycin or hygromycin, respectively. Similar results were observed in the African green monkey cell line COS7. These data indicate that each combination of selectable marker and antibiotic establishes a threshold below which no cell can survive and that these thresholds vary significantly between different selectable markers. Moreover, we show that choice of selectable marker also affects recombinant protein expression in cell-derived exosomes, consistent with the hypothesis that exosome protein budding is a stochastic rather than determinative process.

Exosome-mediated mRNA delivery in vivo is safe and can be used to induce SARS-CoV-2 immunity.

Functional delivery of mRNA has high clinical potential. Previous studies established that mRNAs can be delivered to cells in vitro and in vivo via RNA-loaded lipid nanoparticles (LNPs). Here we describe an alternative approach using exosomes, the only biologically normal nanovesicle. In contrast to LNPs, which elicited pronounced cellular toxicity, exosomes had no adverse effects in vitro or in vivo at any dose tested. Moreover, mRNA-loaded exosomes were characterized by efficient mRNA encapsulation (∼90%), high mRNA content, consistent size, and a polydispersity index under 0.2. Using an mRNA encoding the red light-emitting luciferase Antares2, we observed that mRNA-loaded exosomes were superior to mRNA-loaded LNPs at delivering functional mRNA into human cells in vitro. Injection of Antares2 mRNA-loaded exosomes also led to strong light emission following injection into the vitreous fluid of the eye or into the tissue of skeletal muscle in mice. Furthermore, we show that repeated injection of Antares2 mRNA-loaded exosomes drove sustained luciferase expression across six injections spanning at least 10 weeks, without evidence of signal attenuation or adverse injection site responses. Consistent with these findings, we observed that exosomes loaded with mRNAs encoding immunogenic forms of the SARS-CoV-2 Spike and Nucleocapsid proteins induced long-lasting cellular and humoral responses to both. Taken together, these results demonstrate that exosomes can be used to deliver functional mRNA to and into cells in vivo.

MAP4K4 regulates integrin-FERM binding to control endothelial cell motility.

Cell migration is a stepwise process that coordinates multiple molecular machineries. Using in vitro angiogenesis screens with short interfering RNA and chemical inhibitors, we define here a MAP4K4-moesin-talin-ß1-integrin molecular pathway that promotes efficient plasma membrane retraction during endothelial cell migration. Loss of MAP4K4 decreased membrane dynamics, slowed endothelial cell migration, and impaired angiogenesis in vitro and in vivo. In migrating endothelial cells, MAP4K4 phosphorylates moesin in retracting membranes at sites of focal adhesion disassembly. Epistasis analyses indicated that moesin functions downstream of MAP4K4 to inactivate integrin by competing with talin for binding to ß1-integrin intracellular domain. Consequently, loss of moesin (encoded by the MSN gene) or MAP4K4 reduced adhesion disassembly rate in endothelial cells. Additionally, α5ß1-integrin blockade reversed the membrane retraction defects associated with loss of Map4k4 in vitro and in vivo. Our study uncovers a novel aspect of endothelial cell migration. Finally, loss of MAP4K4 function suppressed pathological angiogenesis in disease models, identifying MAP4K4 as a potential therapeutic target.

Chemotherapy Combines Effectively with Anti-PD-L1 Treatment and Can Augment Antitumor Responses.

Immunotherapy with checkpoint inhibitors has proved to be highly effective, with durable responses in a subset of patients. Given their encouraging clinical activity, checkpoint inhibitors are increasingly being tested in clinical trials in combination with chemotherapy. In many instances, there is little understanding of how chemotherapy might influence the quality of the immune response generated by checkpoint inhibitors. In this study, we evaluated the impact of chemotherapy alone or in combination with anti-PD-L1 in a responsive syngeneic tumor model. Although multiple classes of chemotherapy treatment reduced immune cell numbers and activity in peripheral tissues, chemotherapy did not antagonize but in many cases augmented the antitumor activity mediated by anti-PD-L1. This dichotomy between the detrimental effects in peripheral tissues and enhanced antitumor activity was largely explained by the reduced dependence on incoming cells for antitumor efficacy in already established tumors. The effects of the various chemotherapies were also agent specific, and synergy with anti-PD-L1 was achieved by different mechanisms that ultimately helped establish a new threshold for response. These results rationalize the combination of chemotherapy with immunotherapy and suggest that, despite the negative systemic effects of chemotherapy, effective combinations can be obtained through distinct mechanisms acting within the tumor.

PRC2-mediated repression of SMARCA2 predicts EZH2 inhibitor activity in SWI/SNF mutant tumors.

Subunits of the SWI/SNF chromatin remodeling complex are frequently mutated in human cancers leading to epigenetic dependencies that are therapeutically targetable. The dependency on the polycomb repressive complex (PRC2) and EZH2 represents one such vulnerability in tumors with mutations in the SWI/SNF complex subunit, SNF5; however, whether this vulnerability extends to other SWI/SNF subunit mutations is not well understood. Here we show that a subset of cancers harboring mutations in the SWI/SNF ATPase, SMARCA4, is sensitive to EZH2 inhibition. EZH2 inhibition results in a heterogenous phenotypic response characterized by senescence and/or apoptosis in different models, and also leads to tumor growth inhibition in vivo. Lower expression of the SMARCA2 paralog was associated with cellular sensitivity to EZH2 inhibition in SMARCA4 mutant cancer models, independent of tissue derivation. SMARCA2 is suppressed by PRC2 in sensitive models, and induced SMARCA2 expression can compensate for SMARCA4 and antagonize PRC2 targets. The induction of SMARCA2 in response to EZH2 inhibition is required for apoptosis, but not for growth arrest, through a mechanism involving the derepression of the lysomal protease cathepsin B. Expression of SMARCA2 also delineates EZH2 inhibitor sensitivity for other SWI/SNF complex subunit mutant tumors, including SNF5 and ARID1A mutant cancers. Our data support monitoring SMARCA2 expression as a predictive biomarker for EZH2-targeted therapies in the context of SWI/SNF mutant cancers.

Patient Perception of Orthopedic Surgeon Reimbursement.

Significant attention has been directed at evaluating reimbursement rates to orthopedic surgeons for various surgical procedures. To evaluate patients' understanding of the surgeon reimbursement process, studies using patient surveys have been conducted to determine patients' perceptions of orthopedic surgeon compensation. To date, there has been no systematic review to consolidate the data of these studies. This study aimed to synthesize the findings of these individual studies across multiple subspecialties of orthopedic surgery to evaluate the potential discrepancy between how much patients believe orthopedic surgeons are reimbursed and the actual reimbursement rate. We performed a systematic review following the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines to identify studies that report findings of patient perceptions of orthopedic surgeon reimbursement for various procedures. Searches were conducted using MEDLINE through PubMed, Embase, and Scopus. Summary estimates of reimbursement discrepancies across subspecialties and overall were reported as unweighted averages of the individual study results within each group. Twelve studies were identified that met inclusion criteria, constituting 4309 surveys. These survey studies measured patients' perceptions of how much orthopedic surgeons are reimbursed for common procedures, including anterior cruciate ligament reconstruction, arthroscopic meniscectomy, carpal tunnel release, rotator cuff repair, multiple spine procedures and total shoulder, hip, and knee arthroplasty. It was found that patients reported reasonable surgeon's fees to be 11.2 times more than actual Medicare reimbursement. Among individual studies, the largest discrepancies were seen in total hip arthroplasty (26 times), whereas the smallest difference was in anterior cruciate ligament reconstruction (1.6 times). On average, patients estimated Medicare reimbursement rates to be 5.9 times higher than the actual surgeon reimbursement. Patients consistently overestimate how much orthopedic surgeons are reimbursed for common orthopedic procedures. The results of this systematic review suggest that patients may value these procedures more than what Medicare reimburses. Such information may help educate the public, direct policy, and increase transparency between orthopedic surgeons and patients.

Design of a brain-penetrant CDK4/6 inhibitor for glioblastoma.

CDK4 and CDK6 are kinases with similar sequences that regulate cell cycle progression and are validated targets in the treatment of cancer. Glioblastoma is characterized by a high frequency of CDKN2A/CCND2/CDK4/CDK6 pathway dysregulation, making dual inhibition of CDK4 and CDK6 an attractive therapeutic approach for this disease. Abemaciclib, ribociclib, and palbociclib are approved CDK4/6 inhibitors for the treatment of HR+/HER2- breast cancer, but these drugs are not expected to show strong activity in brain tumors due to poor blood brain barrier penetration. Herein, we report the identification of a brain-penetrant CDK4/6 inhibitor derived from a literature molecule with low molecular weight and topological polar surface area (MW = 285 and TPSA = 66 Å2), but lacking the CDK2/1 selectivity profile due to the absence of a basic amine. Removal of a hydrogen bond donor via cyclization of the pyrazole allowed for the introduction of basic and semi-basic amines, while maintaining in many cases efflux ratios reasonable for a CNS program. Ultimately, a basic spiroazetidine (cpKa = 8.8) was identified that afforded acceptable selectivity over anti-target CDK1 while maintaining brain-penetration in vivo (mouse Kp,uu = 0.20-0.59). To probe the potency and selectivity, our lead compound was evaluated in a panel of glioblastoma cell lines. Potency comparable to abemaciclib was observed in Rb-wild type lines U87MG, DBTRG-05MG, A172, and T98G, while Rb-deficient cell lines SF539 and M059J exhibited a lack of sensitivity.

Extracellular vesicles carry microRNA-195 to intrahepatic cholangiocarcinoma and improve survival in a rat model.

The cancer microenvironment plays a central role in cancer development, growth, and homeostasis. This paradigm suggests that cancer fibroblasts support cancers, probably in response to stimuli received from the cancer cells. We aimed at investigating whether extracellular vesicles (EVs) can shuttle microRNA (miR) species between cancer-associated fibroblasts (CAFs) and cancer cells. To this end, we extracted EVs according to published protocols. EVs were studied for their miR content by quantitative reverse-transcription polymerase chain reaction. EVs were transfected with select miR species and utilized in vitro as well as in vivo in a rat model of cholangiocarcinoma (CCA). We found that miR-195 is down-regulated in CCA cells, as well as in adjoining fibroblasts. Furthermore, we report that EVs shuttle miR-195 from fibroblasts to cancer cells. Last, we show that fibroblast-derived EVs, loaded with miR-195, can be administered in a rat model of CCA, concentrate within the tumor, decrease the size of cancers, and improve survival of treated rats. CONCLUSION: EVs play a salient role in trafficking miR species between cancer cells and CAFs in human CCA. Understanding of these mechanisms may allow devising of novel therapeutics. (Hepatology 2017;65:501-514).

Carcinogenicity assessment of the Hedgehog pathway inhibitor, vismodegib in Tg.rasH2 mice and Sprague-Dawley rats.

Vismodegib (also known as GDC-0449) is a novel small molecule inhibitor of the Hedgehog (Hh) signaling pathway currently approved for the treatment of metastatic or locally advanced basal cell carcinoma (BCC) in humans. Its tumorigenic potential was assessed in dedicated carcinogenicity studies in rasH2 transgenic (Tg.rasH2) mice and Sprague Dawley (SD) rats. Tumorigenicity potential of vismodegib was identified in rats only and was limited to benign hair follicle tumors, including pilomatricomas and keratoacanthomas at exposures of ≥0.1-fold and ≥0.6-fold, respectively, of the steady-state exposure (AUC0-24h) of the recommended human dose. No malignant tumors were identified in either species. Overall, the totality of pharmacology and nonclinical safety data (lack of genotoxicity, in vitro secondary pharmacological binding, and immunoregulatory effects, and limited effects on the endocrine system) suggests that the development of the benign hair follicle tumors may be related to pharmacologically-mediated disruption of hair follicle morphogenesis, although the exact mechanism of tumorigenesis is unclear. Hair follicle tumors have not been reported in vismodegib-treated patients. The relevance of this finding in rats to patients is uncertain.

Brain Distribution and Efficacy of the Brain Penetrant PI3K Inhibitor GDC-0084 in Orthotopic Mouse Models of Human Glioblastoma.

Glioblastoma multiforme (GBM) is the most common primary brain tumor in adults. Limited treatment options have only marginally impacted patient survival over the past decades. The phophatidylinositol 3-kinase (PI3K) pathway, frequently altered in GBM, represents a potential target for the treatment of this glioma. 5-(6,6-Dimethyl-4-morpholino-8,9-dihydro-6H-[1,4]oxazino[4,3-e]purin-2-yl)pyrimidin-2-amine (GDC-0084) is a PI3K inhibitor that was specifically optimized to cross the blood-brain barrier. The goals of our studies were to characterize the brain distribution, pharmacodynamic (PD) effect, and efficacy of GDC-0084 in orthotopic xenograft models of GBM. GDC-0084 was tested in vitro to assess its sensitivity to the efflux transporters P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP) and in vivo in mice to evaluate its effects on the PI3K pathway in intact brain. Mice bearing U87 or GS2 intracranial tumors were treated with GDC-0084 to assess its brain distribution by matrix-assisted laser desorption ionization (MALDI) imaging and measure its PD effects and efficacy in GBM orthotopic models. Studies in transfected cells indicated that GDC-0084 was not a substrate of P-gp or BCRP. GDC-0084 markedly inhibited the PI3K pathway in mouse brain, causing up to 90% suppression of the pAkt signal. MALDI imaging showed GDC-0084 distributed evenly in brain and intracranial U87 and GS2 tumors. GDC-0084 achieved significant tumor growth inhibition of 70% and 40% against the U87 and GS2 orthotopic models, respectively. GDC-0084 distribution throughout the brain and intracranial tumors led to potent inhibition of the PI3K pathway. Its efficacy in orthotopic models of GBM suggests that it could be effective in the treatment of GBM. GDC-0084 is currently in phase I clinical trials.

Translational pharmacokinetic/pharmacodynamic analysis in cancer pharmacology: a tool to maximize the value of antitumor efficacy from tumor bearing mice.

Translational pharmacokinetic/pharmacodynamic (PK/PD) analysis is becoming an increasingly important tool for the identification and selection of new anticancer agents. There are two important elements of effectively using PK/PD analysis to translate preclinical antitumor efficacy from tumor bearing mice (xenografts and allografts) to cancer patients. These two sometimes overlapping elements are termed translation 'WITHIN' and 'ACROSS' species. Translating 'WITHIN' species refers to the quantitative characterization of drug action and disease behavior within tumor bearing mice using PK/PD modeling in order to use this information to make predictions of drug response in humans. Translating 'ACROSS' species refers to use of PK/PD modeling to quantify species similarities and differences in drug response in order to understand the clinical relevance of preclinical efficacy data.

Fragment-based identification and optimization of a class of potent pyrrolo[2,1-f][1,2,4]triazine MAP4K4 inhibitors.

MAP4K4 has been shown to regulate key cellular processes that are tied to disease pathogenesis. In an effort to generate small molecule MAP4K4 inhibitors, a fragment-based screen was carried out and a pyrrolotriazine fragment with excellent ligand efficiency was identified. Further modification of this fragment guided by X-ray crystal structures and molecular modeling led to the discovery of a series of promising compounds with good structural diversity and physicochemical properties. These compounds exhibited single digit nanomolar potency and compounds 35 and 44 achieved good in vivo exposure.

Discovery of 5,6,7,8-tetrahydropyrido[3,4-d]pyrimidine inhibitors of Erk2.

The discovery and optimization of a series of tetrahydropyridopyrimidine based extracellular signal-regulated kinase (Erks) inhibitors discovered via HTS and structure based drug design is reported. The compounds demonstrate potent and selective inhibition of Erk2 and knockdown of phospho-RSK levels in HepG2 cells and tumor xenografts.

A paracrine requirement for hedgehog signalling in cancer.

Ligand-dependent activation of the hedgehog (Hh) signalling pathway has been associated with tumorigenesis in a number of human tissues. Here we show that, although previous reports have described a cell-autonomous role for Hh signalling in these tumours, Hh ligands fail to activate signalling in tumour epithelial cells. In contrast, our data support ligand-dependent activation of the Hh pathway in the stromal microenvironment. Specific inhibition of Hh signalling using small molecule inhibitors, a neutralizing anti-Hh antibody or genetic deletion of smoothened (Smo) in the mouse stroma results in growth inhibition in xenograft tumour models. Taken together, these studies demonstrate a paracrine requirement for Hh ligand signalling in the tumorigenesis of Hh-expressing cancers and have important implications for the development of Hh pathway antagonists in cancer.