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Background: Chimeric antigen receptor (CAR) engineered NK cells (CAR-NK) are a novel approach to the immunotherapy of hematologic malignancies which seeks to overcome some of the challenges faced by CAR-T cells (CAR-T). With few published clinical studies, preclinical studies can identify strategies to accelerate clinical translation. We conducted a systematic review on the preclinical in vivo use of CAR-NK for the treatment of hematologic malignancies to assess these therapies in a holistic and unbiased manner., Methods: Our protocol was registered with PROSPERO (ID: CRD42023438375). We performed a search of OVID MEDLINE, OVID Embase, and Embase for animal studies employing human CAR-NK cells in the treatment of hematologic malignancies. Screening of studies for eligibility criteria was performed in duplicate. Our primary outcomes were survival and reduction in tumor volume. Data extraction from individual experiments was performed by one reviewer using Digitizeit TM software and verified by a second reviewer. Meta-analysis and subgroup analyses were performed using Comprehensive Meta-Analysis TM software. Information for descriptive outcomes was extracted in duplicate by two independent reviewers. Risk of bias was assessed using the SYRCLE Risk of Bias Tool for Animal Studies., Results: A total of 34 papers met eligibility criteria. Overall, CD19 was the most common antigen targeted however there was substantial diversity in antigenic targets, source material for generating CAR-NK cells, and NK cell modifications. Mice treated with CAR-NK therapy survived significantly longer than untreated mice (median survival ratio of 1.18, 95% CI: 1.10-1.27, P < 0.001), and mice treated with nonengineered NK cells (median survival ratio 1.13, 95% CI: 1.03-1.23, P < 0.001). Similarly, treatment with CAR-NK significantly reduced the tumor burden when compared to untreated mice (ratio of mean tumor volume 0.23, 95% CI: 0.17-0.32, P < 0.001) or mice treated with nonengineered NK cells (ratio of mean tumor volume 0.37, 95% CI: 0.28-0.51, P < 0.001). Subgroup analysis showed that cotreatment with IL-15 reduced tumor volume but did not increase survival. In general, CAR-NK cell persistence was short but was increased by IL-15., Conclusions: CAR-NK shows promise for the treatment of hematologic malignancies in preclinical models., Competing Interests: Declaration of Competing Interest MA received research support from Dragonfly Therapeutics and is a SAB member for Aahka Biologics. AV received Honoraria and consulting fees from Janssen, Sanofi, Forus, and Pfizer and research funding from Janssen., (Copyright © 2024 International Society for Cell & Gene Therapy. Published by Elsevier Inc. All rights reserved.)

Inhibition of leucine-rich repeat kinase 2 is a genetically supported mechanism for the treatment of Parkinson's disease. We previously disclosed the discovery of an indazole series lead that demonstrated both safety and translational risks. The safety risks were hypothesized to be of unknown origin, so structural diversity in subsequent chemical matter was prioritized. The translational risks were identified due to a low brain Kp u,u in nonhuman primate studies, which raised concern over the use of an established peripheral biomarker as a surrogate for central target engagement. Given these challenges, the team sought to leverage structure- and property-based drug design and expanded efflux transporter profiling to identify structurally distinct leads with enhanced CNS drug-likeness. Herein, we describe the discovery of a "reinvented" indazole series with improved physicochemical properties and efflux transporter profiles while maintaining excellent potency and off-target kinase selectivity, which resulted in advanced lead, compound 23 .

Natural killer cell-derived extracellular vesicles (NK-EVs) are candidate biotherapeutics against various cancers. However, standardised potency assays are necessary for a reliable assessment of NK-EVs' cytotoxicity. This study aims to thoroughly evaluate a highly sensitive resazurin phenoxazine-based cell viability potency assay (measurement of the cellular redox metabolism) for quantifying the cytotoxicity of NK-EVs against leukaemia K562 cells (suspension model) and breast cancer MDA-MB-231 cells (adherent model) in vitro. The assay was evaluated based on common analytical parameters setforth by regulatory guidelines, including specificity, selectivity,accuracy, precision, linearity, range and stability. Our results revealed that this resazurin-based cell viability potency assay reliably and reproducibly measured a dose-response of NK-EVs' cytotoxic activity against both cancer models. The assay showed precision with 5% and 20% variation for intra-run and inter-run variability. The assay signal showed specificity and selectivity of NK-EVs against cancer target cells, as evidenced by the diminished viability of cancer cells following a 5-hour treatment with NK-EVs, without any detectable interference or background. The linearity analysis of target cancer cells revealed strong linearity for densities of 5000 K562 and 1000 MDA-MB-231 cells per test with a consistent range. Importantly, NK-EVs' dose-response for cytotoxicity showed a strong correlation (| ρ | ∼ 0.8) with the levels of known cytotoxic factors associated with the NK-EVs' corona (FasL, GNLY, GzmB, PFN and IFN-γ), thereby validating the accuracy of the assay. The assay also distinguished cytotoxicity changes in degraded NK-EVs, indicating the ability of the assay to detect the potential loss of sample integrity. Compared to other commonly reported bioassays (i.e., flow cytometry, cell counting, lactate dehydrogenase release assay, DNA-binding reporter assay and confluence assay), our results support this highly sensitive resazurin-based viability potency assay as a high-throughput and quantitative method for assessing NK-EVs' cytotoxicity against both suspension and adherent cancer models for evaluating NK-EVs' biotherapeutics., Competing Interests: Most authors declare no conflict of interest. MA is a Scientific Advisory Board Member for Aakha Therapeutics and is under a contract agreement to perform sponsored research with Dragonfly Therapeutics. Neither consulting nor sponsored research is related to the present article., (© 2024 His Majesty the King in Right of Canada and The Author(s). Journal of Extracellular Biology published by Wiley Periodicals LLC on behalf of International Society for Extracellular Vesicles. Reproduced with the permission of the Minister of Health Canada.)

While conventional wisdom initially postulated that PD-L1 serves as the inert ligand for PD-1, an emerging body of literature suggests that PD-L1 has cell-intrinsic functions in immune and cancer cells. In line with these studies, here we show that engagement of PD-L1 via cellular ligands or agonistic antibodies, including those used in the clinic, potently inhibits the type I interferon pathway in cancer cells. Hampered type I interferon responses in PD-L1-expressing cancer cells resulted in enhanced efficacy of oncolytic viruses in vitro and in vivo. Consistently, PD-L1 expression marked tumor explants from cancer patients that were best infected by oncolytic viruses. Mechanistically, PD-L1 promoted a metabolic shift characterized by enhanced glycolysis rate that resulted in increased lactate production. In turn, lactate inhibited type I IFN responses. In addition to adding mechanistic insight into PD-L1 intrinsic function, our results will also help guide the numerous ongoing efforts to combine PD-L1 antibodies with oncolytic virotherapy in clinical trials., (© 2024 Hodgins et al.)

Introduction: Chemotherapy remains the mainstay treatment for triple-negative breast cancer (TNBC) due to the lack of specific targets. Given a modest response of immune checkpoint inhibitors in TNBC patients, improving immunotherapy is an urgent and crucial task in this field. CD73 has emerged as a novel immunotherapeutic target, given its elevated expression on tumor, stromal, and specific immune cells, and its established role in inhibiting anti-cancer immunity. CD73-generated adenosine suppresses immunity by attenuating tumor-infiltrating T- and NK-cell activation, while amplifying regulatory T cell activation. Chemotherapy often leads to increased CD73 expression and activity, further suppressing anti-tumor immunity. While debulking the tumor mass, chemotherapy also enriches heterogenous cancer stem cells (CSC), potentially leading to tumor relapse. Therefore, drugs targeting both CD73, and CSCs hold promise for enhancing chemotherapy efficacy, overcoming treatment resistance, and improving clinical outcomes. However, safe and effective inhibitors of CD73 have not been developed as of now., Methods: We used in silico docking to screen compounds that may be repurposed for inhibiting CD73. The efficacy of these compounds was investigated through flow cytometry, RT-qPCR, CD73 activity, cell viability, tumorsphere formation, and other in vitro functional assays. For assessment of clinical translatability, TNBC patient-derived xenograft organotypic cultures were utilized. We also employed the ovalbumin-expressing AT3 TNBC mouse model to evaluate tumor-specific lymphocyte responses., Results: We identified quercetin and luteolin, currently used as over-the-counter supplements, to have high in silico complementarity with CD73. When quercetin and luteolin were combined with the chemotherapeutic paclitaxel in a triple-drug regimen, we found an effective downregulation in paclitaxel-enhanced CD73 and CSC-promoting pathways YAP and Wnt. We found that CD73 expression was required for the maintenance of CD44 high CD24 low CSCs, and co-targeting CD73, YAP, and Wnt effectively suppressed the growth of human TNBC cell lines and patient-derived xenograft organotypic cultures. Furthermore, triple-drug combination inhibited paclitaxel-enriched CSCs and simultaneously improved lymphocyte infiltration in syngeneic TNBC mouse tumors., Discussion: Conclusively, our findings elucidate the significance of CSCs in impairing anti-tumor immunity. The high efficacy of our triple-drug regimen in clinically relevant platforms not only underscores the importance for further mechanistic investigations but also paves the way for potential development of new, safe, and cost-effective therapeutic strategies for TNBC., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Mediratta, El-Sahli, Marotel, Awan, Kirkby, Salkini, Kurdieh, Abdisalam, Shrestha, Di Censo, Sulaiman, McGarry, Lavoie, Liu, Lee, Li, Sciumè, D’Costa, Ardolino and Wang.)

Cancer stem cells (CSCs), functionally characterized by self-renewal and tumor-initiating activity, contribute to decreased tumor immunogenicity, while fostering tumor growth and metastasis. Targeting G9a histone methyltransferase (HMTase) effectively blocks CSC functions in colorectal tumors by altering pluripotent-like molecular networks; however, existing molecules directly targeting G9a HMTase activity failed to reach clinical stages due to safety concerns. Using a stem cell-based phenotypic drug-screening pipeline, we identified the dopamine transporter (DAT) antagonist vanoxerine, a compound with previously demonstrated clinical safety, as a cancer-specific downregulator of G9a expression. Here we show that gene silencing and chemical antagonism of DAT impede colorectal CSC functions by repressing G9a expression. Antagonizing DAT also enhanced tumor lymphocytic infiltration by activating endogenous transposable elements and type-I interferon response. Our study unveils the direct implication of the DAT-G9a axis in the maintenance of CSC populations and an approach to improve antitumor immune response in colon tumors., (© 2024. The Author(s), under exclusive licence to Springer Nature America, Inc.)

The tumour microenvironment is infiltrated by immunosuppressive cells, such as regulatory T cells (Tregs), which contribute to tumour escape and impede immunotherapy outcomes. Soluble fibrinogen-like protein 2 (sFGL2), a Treg effector protein, inhibits immune cell populations, via receptors FcγRIIB and FcγRIII, leading to downregulation of CD86 in antigen presenting cells and limiting T cell activation. Increased FGL2 expression is associated with tumour progression and poor survival in several different cancers, such as glioblastoma multiforme, lung, renal, liver, colorectal, and prostate cancer. Querying scRNA-seq human cancer data shows FGL2 is produced by cells in the tumour microenvironment (TME), particularly monocytes and macrophages as well as T cells and dendritic cells (DCs), while cancer cells have minimal expression of FGL2. We studied the role of FGL2 exclusively produced by cells in the TME, by leveraging Fgl2 knockout mice. We tested two murine models of cancer in which the role of FGL2 has not been previously studied: epithelial ovarian cancer and melanoma. We show that absence of FGL2 leads to a more activated TME, including activated DCs (CD86+, CD40+) and T cells (CD25+, TIGIT+), as well as demonstrating for the first time that the absence of FGL2 leads to more activated natural killer cells (DNAM-1+, NKG2D+) in the TME. Furthermore, the absence of FGL2 leads to prolonged survival in the B16F10 melanoma model, while the absence of FGL2 synergizes with oncolytic virus to prolong survival in the ID8-p53 -/- Brca2 -/- ovarian cancer model. In conclusion, targeting FGL2 is a promising cancer treatment strategy alone and in combination immunotherapies., (© 2024. The Author(s).)

Targeted therapy resistance frequently develops in melanoma due to intratumor heterogeneity and epigenetic reprogramming. This also typically induces cross-resistance to immunotherapies. Whether this includes additional modes of therapy has not been fully assessed. We show that co-treatments of MAPKi with VSV-based oncolytics do not function in a synergistic fashion; rather, the MAPKis block infection. Melanoma resistance to vemurafenib further perturbs the cells' ability to be infected by oncolytic viruses. Resistance to vemurafenib can be induced by the loss of SOX10, a common proliferative marker in melanoma. The loss of SOX10 promotes a cross-resistant state by further inhibiting viral infection and replication. Analysis of RNA-seq datasets revealed an upregulation of interferon-stimulated genes (ISGs) in SOX10 knockout populations and targeted therapy-resistant cells. Interestingly, the induction of ISGs appears to be independent of type I IFN production. Overall, our data suggest that the pathway mediating oncolytic resistance is due to the loss of SOX10 during acquired drug resistance in melanoma.

Genetic mutation of the leucine-rich repeat kinase 2 (LRRK2) protein has been associated with Parkinson's disease (PD), a disabling and progressive neurodegenerative disorder that is devoid of efficacious disease-modifying therapies. Herein, we describe the invention of an amidoisoquinoline (IQ)-derived LRRK2 inhibitor lead chemical series. Knowledge-, structure-, and property-based drug design in concert with rigorous application of in silico calculations and presynthesis predictions enabled the prioritization of molecules with favorable CNS "drug-like" physicochemical properties. This resulted in the discovery of compound 8 , which was profiled extensively before human ether-a-go-go (hERG) ion channel inhibition halted its progression. Strategic reduction of lipophilicity and basicity resulted in attenuation of hERG ion channel inhibition while maintaining a favorable CNS efflux transporter profile. Further structure- and property-based optimizations resulted in the discovery of preclinical candidate MK-1468 . This exquisitely selective LRRK2 inhibitor has a projected human dose of 48 mg BID and a preclinical safety profile that supported advancement toward GLP toxicology studies.

The large coding potential of vaccinia virus (VV) vectors is a defining feature. However, limited regulatory switches are available to control viral replication as well as timing and dosing of transgene expression in order to facilitate safe and efficacious payload delivery. Herein, we adapt drug-controlled gene switches to enable control of virally encoded transgene expression, including systems controlled by the FDA-approved rapamycin and doxycycline. Using ribosome profiling to characterize viral promoter strength, we rationally design fusions of the operator element of different drug-inducible systems with VV promoters to produce synthetic promoters yielding robust inducible expression with undetectable baseline levels. We also generate chimeric synthetic promoters facilitating additional regulatory layers for VV-encoded synthetic transgene networks. The switches are applied to enable inducible expression of fusogenic proteins, dose-controlled delivery of toxic cytokines, and chemical regulation of VV replication. This toolbox enables the precise modulation of transgene circuitry in VV-vectored oncolytic virus design., (© 2023. The Author(s).)

Advancements in chimeric antigen receptor engineered T-cell (CAR-T) therapy have revolutionized treatment for several cancer types over the past decade. Despite this success, obstacles including the high price tag, manufacturing complexity, and treatment-associated toxicities have limited the broad application of this therapy. Chimeric antigen receptor engineered natural killer cell (CAR-NK) therapy offers a potential opportunity for a simpler and more affordable "off-the-shelf" treatment, likely with fewer toxicities. Unlike CAR-T, CAR-NK therapies are still in early development, with few clinical trials yet reported. Given the challenges experienced through the development of CAR-T therapies, this review explores what lessons we can apply to build better CAR-NK therapies. In particular, we explore the importance of optimizing the immunochemical properties of the CAR construct, understanding factors leading to cell product persistence, enhancing trafficking of transferred cells to the tumor, ensuring the metabolic fitness of the transferred product, and strategies to avoid tumor escape through antigen loss. We also review trogocytosis, an important emerging challenge that likely equally applies to CAR-T and CAR-NK cells. Finally, we discuss how these limitations are already being addressed in CAR-NK therapies, and what future directions may be possible., Competing Interests: MA received monetary compensation from Alloy Therapeutics for consulting. MA is under a contract agreement to perform sponsored research with Actym Therapeutics and Dragonfly Therapeutics. Neither consulting nor sponsored research is related to the present article. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be constructed as a potential conflict of interest., (Copyright © 2023 Kilgour, Bastin, Lee, Ardolino, McComb and Visram.)

SOX10 is a key regulator of melanoma progression and promotes a melanocytic/differentiated state. Here we identified melanoma cell lines lacking SOX10 expression which retain their in vivo growth capabilities. More importantly, we find that SOX10 can regulate T-cell infiltration in melanoma while also decreasing common cancer stem cell (CSC) properties. We show that SOX10 regulates CEACAM1, a surface protein with immunomodulatory properties. SOX10 directly binds to a distal CEACAM1 promoter region approximately 3-4kbps from the CEACAM1 transcriptional start site. Furthermore, we show that a SOX10-CEACAM1 axis can suppress CD8 + T-cell infiltration as well as reduce CSC pool within tumors, leading to reduced tumor growth. Overall, these results identify SOX10 as a direct regulator of CEACAM1, and uncover both a pro- and anti-tumorigenic roles for SOX10 in melanoma., Competing Interests: The authors declare no competing interests., (Crown Copyright © 2022.)

Due to their crucial role in tumor immunity, NK cells have quickly became a prime target for immunotherapies, with the adoptive transfer of NK cells and the use of NK cell engagers quickly moving to the clinical stage. On the other hand, only a few studies have focused on small molecule drugs capable of unleashing NK cells against cancer. In this context, repurposing small molecules is an attractive strategy to identify new immunotherapies from already approved drugs. Here, we developed a new platform to screen small molecule compounds based on a high-throughput luciferase-release cytotoxicity assay. We tested 1200 FDA approved drugs from the Prestwick Chemical Library, to identify compounds that increase NK cells' cytotoxic potential. We found that the antibiotic colistin sulfate increased the cytotoxicity of human NK cells towards cancer cells. The effect of colistin was short lived and was not observed when NK cells were pretreated with the drug, showing how NK cell activity was potentiated only when the compound was present at the time of recognition of cancer cells. Further studies are needed to uncover the mechanism of action and the pre-clinical efficacy of colistin sulfate in mouse cancer models.

Trogocytosis modulates immune responses, with still unclear underlying molecular mechanisms. Using leukemia mouse models, we found that lymphocytes perform trogocytosis at high rates with tumor cells. While performing trogocytosis, both Natural Killer (NK) and CD8 + T cells acquire the checkpoint receptor PD-1 from leukemia cells. In vitro and in vivo investigation revealed that PD-1 on the surface of NK cells, rather than being endogenously expressed, was derived entirely from leukemia cells in a SLAM receptor-dependent fashion. PD-1 acquired via trogocytosis actively suppressed NK cell antitumor immunity. PD-1 trogocytosis was corroborated in patients with clonal plasma cell disorders, where NK cells that stained for PD-1 also stained for tumor cell markers. Our results, in addition to shedding light on a previously unappreciated mechanism underlying the presence of PD-1 on NK and cytotoxic T cells, reveal the immunoregulatory effect of membrane transfer occurring when immune cells contact tumor cells.

Type 1 innate lymphoid cells (ILC1) are tissue-resident lymphocytes that provide early protection against bacterial and viral infections. Discrete transcriptional states of ILC1 have been identified in homeostatic and pathological contexts. However, whether these states delineate ILC1 with different functional properties is not completely understood. Here, we show that liver ILC1 are heterogeneous for the expression of distinct effector molecules and surface receptors, including granzyme A (GzmA) and CD160, in mice. ILC1 expressing high levels of GzmA are enriched in the liver of adult mice, and represent the main hepatic ILC1 population at birth. However, the heterogeneity of GzmA and CD160 expression in hepatic ILC1 begins perinatally and increases with age. GzmA + ILC1 differ from NK cells for the limited homeostatic requirements of JAK/STAT signals and the transcription factor Nfil3. Moreover, by employing Rorc(γt)-fate map (fm) reporter mice, we established that ILC3-ILC1 plasticity contributes to delineate the heterogeneity of liver ILC1, with RORγt-fm + cells skewed toward a GzmA - CD160 + phenotype. Finally, we showed that ILC1 defined by the expression of GzmA and CD160 are characterized by graded cytotoxic potential and ability to produce IFN-γ. In conclusion, our findings help deconvoluting ILC1 heterogeneity and provide evidence for functional diversification of liver ILC1., (© 2021 The Authors. European Journal of Immunology published by Wiley-VCH GmbH.)

Duchenne's muscular dystrophy (DMD) is a severe muscle wasting disorder characterized by the loss of dystrophin expression, muscle necrosis, inflammation and fibrosis. Ongoing muscle regeneration is impaired by persistent cytokine stress, further decreasing muscle function. Patients with DMD rarely survive beyond their early 20s, with cardiac and respiratory dysfunction being the primary cause of death. Despite an increase in our understanding of disease progression as well as promising preclinical animal models for therapeutic intervention, treatment options for muscular dystrophy remain limited and novel therapeutic targets are required. Many reports suggest that the TGFβ signalling pathway is activated in dystrophic muscle and contributes to the pathology of DMD in part by impairing the differentiation of myoblasts into mature myofibers. Here, we show that in vitro knockdown of the Ste20-like kinase, SLK, can partially restore myoblast differentiation downstream of TGFβ in a Smad2/3 independent manner. In an mdx model, we demonstrate that SLK is expressed at high levels in regenerating myofibers. Muscle-specific deletion of SLK reduced leukocyte infiltration, increased myogenin and utrophin expression and enhanced differentiation. This was accompanied by resistance to eccentric contraction-induced injury in slow fiber type-enriched soleus muscles. Finally, we found that these effects were partially dependent on the upregulation of p38 signalling. Collectively, these results demonstrate that SLK downregulation can restore some aspects of disease progression in DMD., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Sarcomas are a rare malignancy of mesenchymal tissues, comprizing a plethora of unique subtypes, with more than 60 types. The sheer heterogeneity of disease phenotype makes this a particularly difficult cancer to treat. Radiotherapy, chemotherapy and surgery have been employed for over three decades and, although effective in early disease (stages I-II), in later stages, where metastatic tumors are present, these treatments are less effective. Given the spectacular results obtained by cancer immunotherapy in a variety of solid cancers and leukemias, there is now a great interest in appliying this new realm of therapy for sarcomas. The widespread use of immunotherapy for sarcoma relies on immuno-profiling of subtypes, immunomonitoring for prognosis, preclinical studies and insight into the safety profile of these novel therapies. Herein, we discuss preclinical and clinical data highlighting how immunotherapy is being used in soft tissue sarcoma and bone sarcomas., Competing Interests: Competing interests: J-SD is an inventor on patents licensed to Turnstone Biologics, which is commercializing oncolytic Maraba virus. J-SD has patents licensed and also holds equity in Virica Biotech, which is developing oncolytic virus platforms., (© Author(s) (or their employer(s)) 2021. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Natural Killer (NK) cells are innate immune responders critical for viral clearance and immunomodulation. Despite their vital role in viral infection, the contribution of NK cells in fighting SARS-CoV-2 has not yet been directly investigated. Insights into pathophysiology and therapeutic opportunities can therefore be inferred from studies assessing NK cell phenotype and function during SARS, MERS, and COVID-19. These studies suggest a reduction in circulating NK cell numbers and/or an exhausted phenotype following infection and hint toward the dampening of NK cell responses by coronaviruses. Reduced circulating NK cell levels and exhaustion may be directly responsible for the progression and severity of COVID-19. Conversely, in light of data linking inflammation with coronavirus disease severity, it is necessary to examine NK cell potential in mediating immunopathology. A common feature of coronavirus infections is that significant morbidity and mortality is associated with lung injury and acute respiratory distress syndrome resulting from an exaggerated immune response, of which NK cells are an important component. In this review, we summarize the current understanding of how NK cells respond in both early and late coronavirus infections, and the implication for ongoing COVID-19 clinical trials. Using this immunological lens, we outline recommendations for therapeutic strategies against COVID-19 in clearing the virus while preventing the harm of immunopathological responses., (Copyright © 2020 Market, Angka, Martel, Bastin, Olanubi, Tennakoon, Boucher, Ng, Ardolino and Auer.)

HER2 is overexpressed in 20-30% of all breast cancers and is associated with an invasive disease and poor clinical outcome. The Ste20-like kinase (SLK) is activated downstream of HER2/Neu and is required for efficient epithelial-to-mesenchymal transition, cell cycle progression, and migration in the mammary epithelium. Here we show that loss of SLK in a murine model of HER2/Neu-positive breast cancers significantly accelerates tumor onset and decreases overall survival. Transcriptional profiling of SLK knockout HER2/Neu-derived tumor cells revealed a strong induction in the triple-negative breast cancer marker, Sox10, accompanied by an increase in mammary stem/progenitor activity. Similarly, we demonstrate that SLK and Sox10 expression are inversely correlated in patient samples, with the loss of SLK and acquisition of Sox10 marking the triple-negative subtype. Furthermore, pharmacological inhibition of AKT reduces SLK-null tumor growth in vivo and is rescued by ectopic Sox10 expression, suggesting that Sox10 is a critical regulator of tumor growth downstream of SLK/AKT. These findings highlight a role for SLK in negatively regulating HER2-induced mammary tumorigenesis and provide mechanistic insight into the regulation of Sox10 expression in breast cancer.

Natural killer (NK) cells are innate cytotoxic lymphocytes involved in the surveillance and elimination of cancer. As we have learned more and more about the mechanisms NK cells employ to recognize and eliminate tumor cells, and how, in turn, cancer evades NK cell responses, we have gained a clear appreciation that NK cells can be harnessed in cancer immunotherapy. Here, we review the evidence for NK cells' critical role in combating transformed and malignant cells, and how cancer immunotherapies potentiate NK cell responses for therapeutic purposes. We highlight cutting-edge immunotherapeutic strategies in preclinical and clinical development such as adoptive NK cell transfer, chimeric antigen receptor-expressing NK cells (CAR-NKs), bispecific and trispecific killer cell engagers (BiKEs and TriKEs), checkpoint blockade, and oncolytic virotherapy. Further, we describe the challenges that NK cells face (e.g., postsurgical dysfunction) that must be overcome by these therapeutic modalities to achieve cancer clearance.

Checkpoint blockade immunotherapy targeting the PD-1/PD-L1 inhibitory axis has produced remarkable results in the treatment of several types of cancer. Whereas cytotoxic T cells are known to provide important antitumor effects during checkpoint blockade, certain cancers with low MHC expression are responsive to therapy, suggesting that other immune cell types may also play a role. Here, we employed several mouse models of cancer to investigate the effect of PD-1/PD-L1 blockade on NK cells, a population of cytotoxic innate lymphocytes that also mediate antitumor immunity. We discovered that PD-1 and PD-L1 blockade elicited a strong NK cell response that was indispensable for the full therapeutic effect of immunotherapy. PD-1 was expressed on NK cells within transplantable, spontaneous, and genetically induced mouse tumor models, and PD-L1 expression in cancer cells resulted in reduced NK cell responses and generation of more aggressive tumors in vivo. PD-1 expression was more abundant on NK cells with an activated and more responsive phenotype and did not mark NK cells with an exhausted phenotype. These results demonstrate the importance of the PD-1/PD-L1 axis in inhibiting NK cell responses in vivo and reveal that NK cells, in addition to T cells, mediate the effect of PD-1/PD-L1 blockade immunotherapy.

Many NK cells express inhibitory receptors that bind self-MHC class I (MHC I) molecules and prevent killing of self-cells, while enabling killing of MHC I-deficient cells. But tolerance also occurs for NK cells that lack inhibitory receptors for self-MHC I, and for all NK cells in MHC I-deficient animals. In both cases, NK cells are unresponsive to MHC I-deficient cells and hyporesponsive when stimulated through activating receptors, suggesting that hyporesponsiveness is responsible for self-tolerance. We generated irradiation chimeras, or carried out adoptive transfers, with wild-type (WT) and/or MHC I-deficient hematopoietic cells in WT or MHC I-deficient C57BL/6 host mice. Unexpectedly, in WT hosts, donor MHC I-deficient hematopoietic cells failed to induce hyporesponsiveness to activating receptor stimulation, but did induce tolerance to MHC I-deficient grafts. Therefore, these two properties of NK cells are separable. Both tolerance and hyporesponsiveness occurred when the host was MHC I deficient. Interestingly, infections of mice or exposure to inflammatory cytokines reversed the tolerance of NK cells that was induced by MHC I-deficient hematopoietic cells, but not the tolerance induced by MHC I-deficient nonhematopoietic cells. These data have implications for successful bone marrow transplantation, and suggest that tolerance induced by hematopoietic cells versus nonhematopoietic cells may be imposed by distinct mechanisms., (Copyright © 2016 by The American Association of Immunologists, Inc.)

Unlabelled: Immune cells promote the initial metastatic dissemination of carcinoma cells from primary tumors. In contrast to their well-studied functions in the initial stages of metastasis, the specific roles of immunocytes in facilitating progression through the critical later steps of the invasion-metastasis cascade remain poorly understood. Here, we define novel functions of neutrophils in promoting intraluminal survival and extravasation at sites of metastatic dissemination. We show that CD11b(+)/Ly6G(+) neutrophils enhance metastasis formation via two distinct mechanisms. First, neutrophils inhibit natural killer cell function, which leads to a significant increase in the intraluminal survival time of tumor cells. Thereafter, neutrophils operate to facilitate extravasation of tumor cells through the secretion of IL1β and matrix metalloproteinases. These results identify neutrophils as key regulators of intraluminal survival and extravasation through their cross-talk with host cells and disseminating carcinoma cells., Significance: This study provides important insights into the systemic contributions of neutrophils to cancer metastasis by identifying how neutrophils facilitate intermediate steps of the invasion-metastasis cascade. We demonstrate that neutrophils suppress natural killer cell activity and increase extravasation of tumor cells. Cancer Discov; 6(6); 630-49. ©2016 AACR.This article is highlighted in the In This Issue feature, p. 561., (©2016 American Association for Cancer Research.)

Increasing evidence supports a role for innate immune effector cells in tumor surveillance. Natural killer (NK) cells and myeloid cells represent the two main subsets of innate immune cells possessing efficient but quite different tumor suppressive abilities. Here, we describe the germline-encoded NK cell receptors that play a role in suppressing tumor development and describe briefly the cellular pathways leading to the upregulation of their ligands in tumor cells. We also describe mechanisms underlying the elimination of tumor cells by macrophages and a recently characterized mechanism dedicated to sensing cytosolic DNA that is implicated in antitumor immune responses., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Recent extraordinary advances in cancer immunotherapy rely primarily on marshaling T cell responses. Here we discuss how NK cell responses can be amplified. We find that MHC I-deficient tumors induce anergy of NK cells but that cytokine therapy restores NK cell activity and increases the survival of mice bearing MHC I-deficient tumors.

Various cytokines have been evaluated as potential anticancer drugs; however, most cytokine trials have shown relatively low efficacy. Here, we found that treatments with IL-12 and IL-18 or with a mutant form of IL-2 (the "superkine" called H9) provided substantial therapeutic benefit for mice specifically bearing MHC class I-deficient tumors, but these treatments were ineffective for mice with matched MHC class I+ tumors. Cytokine efficacy was linked to the reversal of the anergic state of NK cells that specifically occurred in MHC class I-deficient tumors, but not MHC class I+ tumors. NK cell anergy was accompanied by impaired early signal transduction and was locally imparted by the presence of MHC class I-deficient tumor cells, even when such cells were a minor population in a tumor mixture. These results demonstrate that MHC class I-deficient tumor cells can escape from the immune response by functionally inactivating NK cells, and suggest cytokine-based immunotherapy as a potential strategy for MHC class I-deficient tumors. These results suggest that such cytokine therapies would be optimized by stratification of patients. Moreover, our results suggest that such treatments may be highly beneficial in the context of therapies to enhance NK cell functions in cancer patients.

Natural killer (NK) cells represent a first line of defense against pathogens and tumor cells. The activation of NK cells is regulated by the integration of signals deriving from activating and inhibitory receptors expressed on their surface. However, different NK cells respond differently to the same stimulus, be it target cells or agents that crosslink activating receptors. The processes that determine the level of NK cell responsiveness have been referred to collectively as NK cell education. NK cell education plays an important role in steady state conditions, where potentially auto-reactive NK cells are rendered tolerant to the surrounding environment. According to the "tuning" concept, the responsiveness of each NK cell is quantitatively adjusted to ensure self tolerance while at the same time ensuring useful reactivity against potential threats. MHC-specific inhibitory receptors displayed by NK cells play a major role in tuning NK cell responsiveness, but recent studies indicate that signaling from activating receptors is also important, suggesting that the critical determinant is an integrated signal from both types of receptors. An important and still unresolved question is whether NK cell education involves interactions with a specific cell population in the environment. Whether hematopoietic and/or non-hematopoietic cells play a role is still under debate. Recent results demonstrated that NK cell tuning exhibits plasticity in steady state conditions, meaning that it can be re-set if the MHC environment changes. Other evidence suggests, however, that inflammatory conditions accompanying infections may favor high responsiveness, indicating that inflammatory agents can over-ride the natural tendency of NK cells to adjust to the steady state environment. These findings raise many questions such as whether viruses and tumor cells manipulate NK cell responsiveness to evade immune-recognition. As knowledge of the underlying processes grows, the possibility of modulating NK cell responsiveness for therapeutic purposes is becoming increasingly attractive, and is now under serious investigation in clinical studies., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

In recent years, roles of the immune system in immune surveillance of cancer have been explored using a variety of approaches. The roles of the adaptive immune system have been a major emphasis, but increasing evidence supports a role for innate immune effector cells such as natural killer (NK) cells in tumor surveillance. Here, we discuss some of the evidence for roles in tumor surveillance of innate immune cells. In particular, we focus on NK cells and other immune cells that express germline-encoded receptors, often labeled NK receptors. The impact of these receptors and the cells that express them on tumor suppression is summarized. We discuss in detail some of the pathways and events in tumor cells that induce or upregulate cell-surface expression of the ligands for these receptors, and the logic of how those pathways serve to identify malignant, or potentially malignant cells. How tumors often evade tumor suppression mediated by innate killer cells is another major subject of the review. We end with a discussion on some of the implications of the various findings with respect to possible therapeutic approaches., (© 2014 Elsevier Inc. All rights reserved.)

The induction of cellular senescence is an important mechanism by which p53 suppresses tumorigenesis. Using a mouse model of liver carcinoma, where cellular senescence is triggered in vivo by inducible p53 expression, we demonstrated that NK cells participate in the elimination of senescent tumors. The elimination of senescent tumor cells is dependent on NKG2D. Interestingly, p53 restoration neither increases ligand expression nor increases the sensitivity to lysis by NK cells. Instead, p53 restoration caused tumor cells to secrete various chemokines with the potential to recruit NK cells. Antibody-mediated neutralization of CCL2, but not CCL3, CCL4 or CCL5, prevented NK cell recruitment to the senescent tumors and reduced their elimination. Our findings suggest that elimination of senescent tumors by NK cells occurs as a result of the cooperation of signals associated with p53 expression or senescence, which regulate NK cell recruitment, and other signals that induce NKG2D ligand expression on tumor cells.

The immunoreceptors NKG2D and NKp46 are known for their capacity to activate natural killer (NK) cell cytotoxicity and secretory responses in the contexts of tumors and infections, yet their roles in NK cell education remain unclear. Here, we provide the first characterization of mice deficient for both NKG2D and NKp46 receptors to address the relevance of their concomitant absence during NK cell development and function. Our findings reveal that NK cells develop normally in double-mutant (DKO) mice. Mice lacking NKG2D but not NKp46 showed subtle differences in the percentages of NK cells expressing inhibitory Ly49 receptors and the adhesion molecule DNAM-1. A slightly increased percentage of terminally differentiated NK cells and functional response to in vitro stimuli was observed in some experiments. These alterations were modest and did not affect NK cell function in vivo in response to mouse cytomegalovirus infection. NKp46 deficiency alone, or in combination with NKG2D deficiency, had no effect on frequency or function of NK cells.

Importance: Inpatient care providers often order laboratory tests without any appreciation for the costs of the tests., Objective: To determine whether we could decrease the number of laboratory tests ordered by presenting providers with test fees at the time of order entry in a tertiary care hospital, without adding extra steps to the ordering process., Design: Controlled clinical trial., Setting: Tertiary care hospital., Participants: All providers, including physicians and nonphysicians, who ordered laboratory tests through the computerized provider order entry system at The Johns Hopkins Hospital., Intervention: We randomly assigned 61 diagnostic laboratory tests to an "active" arm (fee displayed) or to a control arm (fee not displayed). During a 6-month baseline period (November 10, 2008, through May 9, 2009), we did not display any fee data. During a 6-month intervention period 1 year later (November 10, 2009, through May 9, 2010), we displayed fees, based on the Medicare allowable fee, for active tests only., Main Outcome Measures: We examined changes in the total number of orders placed, the frequency of ordered tests (per patient-day), and total charges associated with the orders according to the time period (baseline vs intervention period) and by study group (active test vs control)., Results: For the active arm tests, rates of test ordering were reduced from 3.72 tests per patient-day in the baseline period to 3.40 tests per patient-day in the intervention period (8.59% decrease; 95% CI, -8.99% to -8.19%). For control arm tests, ordering increased from 1.15 to 1.22 tests per patient-day from the baseline period to the intervention period (5.64% increase; 95% CI, 4.90% to 6.39%) (P < .001 for difference over time between active and control tests)., Conclusions and Relevance: Presenting fee data to providers at the time of order entry resulted in a modest decrease in test ordering. Adoption of this intervention may reduce the number of inappropriately ordered diagnostic tests.

Importance: Poor health care provider communication across health care settings may lead to adverse outcomes., Objective: To determine the frequency with which inpatient providers report communicating directly with outpatient providers and whether direct communication was associated with 30-day readmissions., Design: We conducted a single-center prospective study of self-reported communication patterns by discharging health care providers on inpatient medical services from September 2010 to December 2011 at The Johns Hopkins Hospital., Setting: A 1000-bed urban, academic center., Participants: There were 13 954 hospitalizations in this time period. Of those, 9719 were for initial visits. After additional exclusions, including patients whose outpatient health care provider was the inpatient attending physician, those who had planned or routine admissions, those without outpatient health care providers, those who died in the hospital, and those discharged to other healthcare facilities, we were left with 6635 hospitalizations for analysis., Interventions: Self-reported communication was captured from a mandatory electronic discharge worksheet field. Thirty-day readmissions, length of stay (LOS), and demographics were obtained from administrative databases., Data Extraction: We used multivariable logistic regression models to examine, first, the association between direct communication and patient age, sex, LOS, race, payer, expected 30-day readmission rate based on diagnosis and illness severity, and physician type and, second, the association between 30-day readmission and direct communication, adjusting for patient and physician-level factors., Results: Of 6635 included hospitalizations, successful direct communication occurred in 2438 (36.7%). The most frequently reported reason for lack of direct communication was the health care provider's perception that the discharge summary was adequate. Predictors of direct communication, adjusting for all other variables, included patients cared for by hospitalists without house staff (odds ratio [OR], 1.81 [95% CI, 1.59-2.08]), high expected 30-day readmission rate (OR, 1.18 [95% CI, 1.10-1.28] per 10%), and insurance by Medicare (OR, 1.35 [95% CI, 1.16-1.56]) and private insurance companies (OR, 1.35 [95% CI, 1.18-1.56]) compared with Medicaid. Direct communication with the outpatient health care provider was not associated with readmissions (OR, 1.08 [95% CI, 0.92-1.26]) in adjusted analysis., Conclusions and Relevance: Self-reported direct communication between inpatient and outpatient providers occurred at a low rate but was not associated with readmissions. This suggests that enhancing interprovider communication at hospital discharge may not, in isolation, prevent readmissions.

The negative regulation of adaptive immunity is relevant to maintain lymphocyte homeostasis. Several studies on natural killer (NK) cells have shown a previously unappreciated immunomodulatory role, as they can negatively regulate T cell-mediated immune responses by direct killing and by secretion of inhibitory cytokines. The molecular mechanisms of T cell suppression by NK cells, however, remained elusive. Only in the last few years has it become evident that, upon activation, human T cells express MICA-B, ULBP1-3, and PVR, ligands of the activating receptors NKG2D and DNAM-1, respectively. Their expression renders T cells targets of NK cell lysis, representing a new mechanism taking part to the negative regulation of T cell responses. Studies on the expression of NKG2D and DNAM-1 ligands on T cells have also contributed in understanding that the activation of ATM (ataxia-telangiectasia, mutated)/ATR (ATM/Rad3-related) kinases and the DNA damage response is a common pathway regulating the expression of activating ligands in different types of cells and under different conditions. The functional consequences of NKG2D and DNAM-1 ligand expression on activated T cells are discussed in the context of physiologic and pathologic processes such as infections, autoimmunity, and graft versus host disease.

Activation-induced cytidine deaminase (AID) is specifically induced in germinal center B cells to carry out somatic hypermutation and class-switch recombination, two processes responsible for antibody diversification. Because of its mutagenic potential, AID expression and activity are tightly regulated to minimize unwanted DNA damage. Surprisingly, AID expression has been observed ectopically during pathogenic infections. However, the function of AID outside of the germinal centers remains largely uncharacterized. In this study, we demonstrate that infection of human primary naïve B cells with Kaposi's sarcoma-associated herpesvirus (KSHV) rapidly induces AID expression in a cell intrinsic manner. We find that infected cells are marked for elimination by Natural Killer cells through upregulation of NKG2D ligands via the DNA damage pathway, a pathway triggered by AID. Moreover, without having a measurable effect on KSHV latency, AID impinges directly on the viral fitness by inhibiting lytic reactivation and reducing infectivity of KSHV virions. Importantly, we uncover two KSHV-encoded microRNAs that directly regulate AID abundance, further reinforcing the role for AID in the antiviral response. Together our findings reveal additional functions for AID in innate immune defense against KSHV with implications for a broader involvement in innate immunity to other pathogens.

An important role for natural killer (NK) cells in the regulation of T-cell responses is emerging, although the receptor pairs regulating the NK-T-cell interaction have still not been identified. We found that superantigen-stimulated T cells express Nectin-2 (CD112) and poliovirus receptor (PVR; CD155), the ligands of the activating NK receptor DNAX accessory molecule-1 (DNAM-1; CD226). Interestingly, only PVR was present at the T cell surface, particularly on cells in the S and G(2)/M phases of the cell cycle. The up-regulation of PVR expression involves DNA-damage response (DDR)-dependent pathways, because we found that pharmacologic inhibition of ATM and ATR kinases reduced PVR expression and that PVR was almost exclusively induced on cells expressing the DDR marker γH2AX. Oxidative stress contributed to DDR activation, and our results showed impaired PVR levels in the presence of the reactive oxygen species (ROS) scavenger N-acetyl-cysteine (NAC), being monocytes the main ROS source needed for optimal PVR expression on activated T cells. Interestingly, in accordance with ligand expression, NK cells lysed allogeneic proliferating more efficiently than nonproliferating T lymphocytes, with a mechanism requiring the cooperation between DNAM-1 and NKG2D. These results could contribute to unraveling the role of NK cells in the down-regulation of T-cell responses in physiologic and pathologic processes such as autoimmunity or GVHD.

NKG2D is an activating receptor expressed on CD8(+)alphabeta(+) T cells, gammadelta(+) T cells, natural killer (NK) cells, and some CD4(+) T cells. For a long time, the interaction of NKG2D with its ligands (NKG2DLs) MICA, MICB, and ULBP1-3 has been considered a mechanism for recognition and elimination of tumor, infected, or otherwise "stressed" cells. However, a new role for NKG2D as an immunoregulatory receptor is emerging. Here, we show that NKG2D is strongly down-modulated on antigen-activated CD8(+) T cells but only if CD4(+) T cells are present. Down-modulation was caused by soluble factors produced by CD4(+) T cells, and in particular soluble NKG2DLs were found in the supernatants of antigen-activated T-cell cultures. MICB was the ligand released at higher levels when CD4(+) T cells were present in the cell cultures, suggesting that it could be the major player of NKG2D down-modulation. CD8(+) T cells expressing low levels of NKG2D had impaired effector functions, as evaluated by proliferation, cytokine production, and cytotoxicity assays after combined triggering of NKG2D and TCR-CD3 complex. These findings show that activated CD4(+) T cells expressing NKG2DLs can efficiently prevent NKG2D-mediated CD8(+) T-cell functions, and suggest that the NKG2D/NKG2DL interaction can regulate immune responses.

New computer tools for physicians, nurses, and the medical care team will become common in the 1990s. This paper describes a clinical workstation (CWS) development project that uses new technology that moves the technical support for medical decision making from the computer room to the nursing station. Collection, processing, and display of clinical information including patient identification, laboratory, and radiology results and current medications are carried out in the environment of a multi-windowed computer workstation. Easy access to automated medical literature databases from the workstation is also provided. This pilot project has successfully demonstrated a CWS operating on an acute general neurology and neurosurgical inpatient nursing unit and a critical care unit at The Johns Hopkins Hospital.