Effector Target Research Articles

A Phytophthora capsici RXLR effector targets and inhibits the central immune kinases to suppress plant immunity.

Receptor-like cytoplasmic kinase subfamily VII (RLCK-VII) proteins are the central immune kinases in plant pattern-recognition receptor (PRR) complexes, and they orchestrate a complex array of defense responses against bacterial and fungal pathogens. However, the role of RLCK-VII in plant-oomycete pathogen interactions has not been established. Phytophthora capsici is a notorious oomycete pathogen that infects many agriculturally important vegetables. Here, we report the identification of RXLR25, an RXLR effector that is required for the virulence of P. capsici. In planta expression of RXLR25 significantly enhanced plants' susceptibility to Phytophthora pathogens. Microbial pattern-induced immune activation in Arabidopsis was severely impaired by RXLR25. We further showed that RXLR25 interacts with RLCK-VII proteins. Using nine rlck-vii high-order mutants, we observed that RLCK-VII-6 and RLCK-VII-8 members are required for resistance to P. capsici. The RLCK-VII-6 members are specifically required for Phytophthora culture filtrate (CF)-induced immune responses. RXLR25 directly targets RLCK-VII proteins such as BIK1, PBL8, and PBL17 and inhibits pattern-induced phosphorylation of RLCK-VIIs to suppress downstream immune responses. This study identified a key virulence factor for P. capsici, and the results revealed the importance of RLCK-VII proteins in plant-oomycete interactions.

3D tumor spheroid microarray for high-throughput, high-content natural killer cell-mediated cytotoxicity

Immunotherapy has emerged as a promising approach to treating several forms of cancer. Use of immune cells, such as natural killer (NK) cells, along with small molecule drugs and antibodies through antibody dependent cell-mediated cytotoxicity (ADCC) has been investigated as a potential combination therapy for some difficult to treat solid tumors. Nevertheless, there remains a need to develop tools that support co-culture of target cancer cells and effector immune cells in a contextually relevant three-dimensional (3D) environment to provide a rapid means to screen for and optimize ADCC-drug combinations. To that end, here we have developed a high throughput 330 micropillar-microwell sandwich platform that enables 3D co-culture of NK92-CD16 cells with pancreatic (MiaPaCa-2) and breast cancer cell lines (MCF-7 and MDA-MB-231). The platform successfully mimicked hypoxic conditions found in a tumor microenvironment and was used to demonstrate NK-cell mediated cell cytotoxicity in combination with two monoclonal antibodies; Trastuzumab and Atezolizumab. The platform was also used to show dose response behavior of target cancer cells with reduced EC50 values for paclitaxel (an anti-cancer chemotherapeutic) when treated with both NK cells and antibody. Such a platform may be used to develop more personalized cancer therapies using patient-derived cancer cells.

Abstract 231: Evaluation of pharmacologic mechanisms to overcome IgG1 antibody (Ab) resistance via quantitative systems pharmacology (QSP) modeling of antibody-dependent cell mediated cytotoxicity (ADCC)

Abstract Novel therapies that enhance ADCC have the potential to overcome Rituximab (RTX) resistance in lymphoma, which can occur due to e.g. CD20 loss, immunogenicity-induced reduction in RTX exposure, or SNPs resulting in lower CD16:Fc affinity. Nonlinear interactions between effector cells (ECs), drug, and target cell (TC) factors in ADCC, however, complicate the prediction of the net effect of potential therapeutic strategies that modulate one or more factors such as CD16 expression ([CD16]) on ECs, CD16:Fc binding affinity, or intrinsic killing propensity (g).We have developed a QSP model of the molecular-level binding reactions and cell-cell interactions governing ADCC using the anti-CD20 IgG1 antibody RTX in lymphoma as a case study. This model takes as inputs [CD16] on ECs, g, Ab Fc:CD16 binding affinity, [RTX], RTX:CD20 affinity, and [CD20] on TCs and predicts the degree of ADCC killing of tumor cells after RTX exposure. Simulations can then be used to estimate how much increase in an input parameter a potential RTX combination partner must provide to compensate for a given mechanism of RTX resistance (MoRR). The model has been successfully calibrated to ex vivo [RTX]-ADCC assays in SUDHL4 and Z138 cell lines (Herter, Mol Cancer Ther, 2013). The model and estimated parameter set well described the observed [RTX]-dependent %ADCC in both cell lines with ECs (PBMCs) from two donors with V158 and F158 CD16 SNP variants. The table below provides the model predictions of the fold increase in model parameters required to offset various MoRRs. For example, a 10-fold loss of [CD20] on TCs is predicted to cause a 100*(1 - 0.16) = 84% drop in ADCC activity, which can be overcome by a 7.8-fold increase in [CD16] on ECs. We have developed a mechanism-based simulation tool which can be used to predict the ability of novel ADCC-enhancing agents to overcome different mechanisms of IgG1 Ab resistance. Mechanism of RTX resistance (MoRR)ADCC fold change due to MoR(90% CI)Fold change in parameter required to restore ADCC (90% CI)CD16 expression on effector cells [CD16]CD16:Fc binding affinity kon16NK killing propensity g10x loss of CD20 on tumor0.16 (0.13,0.20)7.8 (6.3,9.2)10.6 (10.4,10.9)7.4 (6.1,8.7)10x loss of RTX exposure0.17(0.14,0.21)7.0(5.7,8.1)9.36(9.35,9.37)6.9(5.7,8.1)10x decrease in CD16:Fc affinity0.16(0.13,0.20)7.4(6.1,8.7)10(10,10)7.4(6.1,8.6) Citation Format: Kaitlyn E. Johnson, Maria Veronica Ciocanel, Josua Aponte, Nicolas Bajeux, Fanwang Meng, Dean Bottino. Evaluation of pharmacologic mechanisms to overcome IgG1 antibody (Ab) resistance via quantitative systems pharmacology (QSP) modeling of antibody-dependent cell mediated cytotoxicity (ADCC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 231.

Abstract 1539: Development of off-the-shelf B7H3 chimeric antigen receptor NK cell therapeutic with broad applicability across many solid tumors

Abstract Although chimeric antigen receptor (CAR) T cell therapies have demonstrated impressive efficacy in a number of hematological malignancies, successful application of CAR T cells in solid tumor indications has been challenging due to difficulty identifying and targeting suitable tumor associated antigens (TAA) without eliciting significant on-target, off-tumor toxicity. Furthermore, the challenges of tumor heterogeneity and antigen escape need to be addressed when treating bulky solid tumors. Successful development of CAR-targeted cellular therapies for solid tumor indications will consequently require careful selection of target antigen, effector cell, and cell engineering to balance on-tumor efficacy with potential for off-tumor toxicities. In contrast to CAR-T cell therapies, adoptive natural killer (NK) cell immunotherapies have been well tolerated, but conventional, donor-derived strategies have had limited efficacy. B7H3 is an immune checkpoint molecule of the B7 protein superfamily and an ideal TAA; B7H3 has broad expression in cancer and limited expression on healthy tissues. Clinically, upregulation of B7H3 is often associated with metastatic cancer and poor patient prognosis. To target B7H3 with a multifaceted effector cell population, we selected the use of NK cells to leverage their clinical safety profile and versatile innate tumor recognition and cytotoxicity features. Here we describe the optimization and characterization of a novel anti-B7H3 CAR and its application in an induced pluripotent stem cell-derived, off-the-shelf NK (iNK) cell immunotherapy. These anti-B7H3 CAR iNK cells utilize multiplexed genetic engineering to enhance persistence, effector function, and to enable multi-antigen targeting through combinations with monoclonal antibody therapy. Using a novel B7H3 binding domain, CAR constructs were first screened in primary T cells for the optimal spacer length by in vitro and in vivo cytotoxicity assays. Following optimized CAR selection, in vitro proof of concept studies demonstrated broad B7H3 CAR reactivity against an array of tumor cell line targets of both hematologic and solid organ cancers. B7H3 CAR-T cells were highly reactive against cell lines derived from ovarian, prostate, bone, and breast cancers as demonstrated by cytotoxicity and production of IFNγ and TNFα. iNK cells carrying the optimized CAR design were evaluated in vitro for B7H3 specificity using cytotoxicity assays and intracellular cytokine staining. In addition, B7H3 CAR iNK cells synergized with anti-EGFR and anti-HER2 monoclonal antibodies to produce antibody-dependent cellular cytotoxicity, leading to further enhancement of tumor targeting. In summary, these data demonstrate successful targeting of B7H3 using an off-the-shelf, engineered NK cell platform for the effective elimination of multiple tumor types. Citation Format: Ryan Bjordahl, John Goulding, Hui-Yi Chu, Frank Cichocki, Nicholas Zorko, Zachary Davis, Martin Felices, Alex Garcia, Svetlana Gaidarova, Wen-I Yeh, Tom T. Lee, Jeffrey S. Miller, Bahram Valamehr. Development of off-the-shelf B7H3 chimeric antigen receptor NK cell therapeutic with broad applicability across many solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1539.

Patient Data Supports the Rationale of Low Dose Cyclophosphamide to Potentiate the Anti-Myeloma Activity of Daratumumab through Augmentation of Macrophage-Induced ADCP

Introduction:Daratumumab (Dara) is the first CD38-directed monoclonal antibody approved for the treatment of Multiple Myeloma (MM). Dara is thought to have pleiotropic mechanisms of activity, including antibody-dependent cellular cytotoxicity (ADCC), and antibody-dependent cellular phagocytosis (ADCP). We focused our attention on potential mechanisms of enhancing macrophage (Mφ)-mediated ADCP and have highlighted that exposure of MM cells to low doses of Cyclophosphamide (Cy) led to a secretory response, which greatly augments Mφ-induced ADCP of Dara-coated MM cells1. Here, we sought to confirm this phenomenon in MM and establish the in-vivo relevance of these findings as seen in newly diagnosed MM patients enrolled in CyBorD-DARA phase 1b clinical trial (NCT02951819).Materials and Methods:Bone Marrow (BM) and peripheral blood (PB) samples were collected in EDTA vacutainers from newly diagnosed MM patients recruited to the CyBorD-DARA clinical trial both prior to- and 24hrs after Cy (150-300mg/m2) treatment. Total lymphocyte, monocyte and neutrophil counts were quantified by flow cytometry. Mononuclear cells were prepared and analysed by 8-colour flow cytometry to quantify monocyte, lymphocyte and plasma subsets as well as determine changes in surface expression profiles following treatment. As previously described, MM cells were conditioned with low-dose Cy for 24hrs. Media containing chemotherapy agents were removed and replaced with fresh media for 24hrs (TCM)1. THP-1 Mφ were conditioned with TCM for up to 48hrs and either characterised or incubated at 2:1 effector target ratio with CFSE labelled MM1S cells ± Cytochalasin D for 18hrs in the presence or absence of Dara/Isotype control. The % of Dara-specific tumour cell clearance was calculated to determine if the mechanism of tumour cell clearance was mediated by Mφ-mediated ADCP.Results:In patients receiving Cy for 24hrs, total BM and PB neutrophil numbers were increased (P<0.01, n=11), lymphocyte numbers were decreased (P<0.01, n=11) while monocyte numbers remain unchanged (P>0.05, n=11). More specifically, an increase in CD19+ B cells (P<0.05, n=6) and a decrease in natural killer cells (NK) (P<0.05, n=6) were observed. Interestingly, the number of Mφ precursor monocytes did not change following Cy treatment in both BM and PB of these patients. As innate immune cells are essential in immune surveillance, coupled with our finding that NK cells are reduced, this highlights a potential role for Mφ in Cy induced anti-tumour activity. Additionally, data previously presented highlighted that exposure of Mφ to TCM from Cy treated MM cells significantly enhanced % of Dara-specific clearance of MM cells) p<0.01). We also confirmed ADCP and ADCC as a likely mechanisms of tumour cell clearance in vitro.Furthermore, we observed a marked reduction in expression of the “don't eat me” antigen, CD47, on chemotherapy-treated MM cells in vitro, which we have now confirmed in MM cells isolated from PB and BM of patients (n=2) following Cy treatment, which may enhance phagocytosis. We also observed increased expression of other targetable markers including BCMA (p<0.05), SLAMF7 (p=0.053), PD-L1 (p<0.001) and PD-L2 (p<0.001) in both RPMI8226 or MM1S MM cell lines following low dose Cy treatment in vitro. To date, we can confirm similar findings for both BCMA (p<0.05, n=5) and SLAMF7 (p<0.05, n=3) in patient samples. Furthermore, our in vitro observation of induced CD64 Fc gamma receptor expression on conditioned Mφ following single-agent Cy treatment has been confirmed in Mφ isolated from both BM (P<0.05, n=11) and PB (P<0.01, n=11) of newly diagnosed MM patients following Cy treatment. This finding emphasizes the potential physiological relevance of our in vitro data, implicating Cy in the potentiation of Mφ mediated ADCP in Dara specific anti-tumour immunity.Conclusions:Observations in Mφ precursors isolated from patients' BM and PB following 24hrs Cy treatment, confirm our previous in vitro findings which suggest a role for Cy potentiation of Mφ mediated ADCP in anti-tumour efficacy of Dara. These findings provide a rationale for the efficacy observed to date when combining Cy with Dara in the ongoing CyBorD-DARA clinical trial. Additionally, increases in BCMA and SLAMF7 highlight the potential of other combination immune therapies to further enhance therapeutic efficacy in these patients.1. Rigalou et al, Blood 2016 128:2101 [Display omitted] DisclosuresChiu:Janssen: Employment. Sasser:Janssen Pharmaceuticals: Employment. Ryan:Janssen: Research Funding. O'Dwyer:Onkimmune Ltd: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties; GlycoMimetics Inc: Research Funding; Janssen: Consultancy, Honoraria, Research Funding.

Phenotypical and Functional Characteristics of Human Regulatory T Cells during Ex Vivo Maturation from CD4+ T Lymphocytes

Regulatory T cells (Tregs) participate in the negative regulation of inflammatory reactions by suppressing effector cells. In a number of autoimmune disorders, the suppressive function and/or the number of Tregs is compromised. The lack of active functioning Tregs can be restored with adoptive transfer of expanded ex vivo autologous Tregs. In our study, we traced the differentiation and maturation of Tregs CD4+CD25+FoxP3+CD127low over 7 days of cultivation from initial CD4+ T cells under ex vivo conditions. The resulting ex vivo expanded cell population (eTregs) demonstrated the immune profile of Tregs with an increased capacity to suppress the proliferation of target effector cells. The expression of the FoxP3 gene was upregulated within the time of expansion and was associated with gradual demethylation in the promotor region of the T cell-specific demethylation region. Real-time RT-PCR analysis revealed changes in the expression profile of genes involved in cell cycle regulation. In addition to FOXP3, the cells displayed elevated mRNA levels of Ikaros zinc finger transcription factors and the main telomerase catalytic subunit hTERT. Alternative splicing of FoxP3, hTERT and IKZF family members was demonstrated to be involved in eTreg maturation. Our data indicate that expanded ex vivo eTregs develop a Treg-specific phenotype and functional suppressive activity. We suggest that eTregs are not just expanded but transformed cells with enhanced capacities of immune suppression. Our findings may influence further development of cell immunosuppressive therapy based on regulatory T cells.

Exposure to Copper Compromises the Maturational Competency of Porcine Oocytes by Impairing Mitochondrial Function.

Copper (Cu) is an essential trace element for animals, and also an important nutritional component for the normal physiology and metabolism of animal reproductive systems. An excess or lack of Cu will directly or indirectly affect animal reproductive activities. However, the effect of Cu, in particular excessive Cu, on the reproductive performance of sows has not been studied. Here, we report that excessive Cu had negative effects on oocyte maturation and organelle functions. We showed that Cu exposure perturbed porcine oocyte meiotic maturation and impaired spindle/chromosome structure, resulting in a defective spindle assembly, as well as the abnormal distribution of actin dynamics and cortical granules. In addition, single-cell transcriptome analysis identified the target effectors of Cu actions in porcine oocytes, further demonstrating that Cu exposure affects the mitochondrial distribution and function, leading to the high levels of reactive oxygen species, DNA damage, and early apoptosis of porcine oocytes. These findings demonstrate that Cu exposure causes abnormalities in the mitochondrial distribution and function, resulting in the increased oxidative stress and levels of reactive oxygen species, DNA damage, and apoptosis, ultimately leading to a decreased porcine oocyte quality.

Structures suggest an approach for converting weak self-peptide tumor antigens into superagonists for CD8 T cells in cancer

Tumors frequently express unmutated self-tumor-associated antigens (self-TAAs). However, trial results using self-TAAs as vaccine targets against cancer are mixed, often attributed to deletion of T cells with high-affinity receptors (TCRs) for self-TAAs during T cell development. Mutating these weak self-TAAs to produce higher affinity, effective vaccines is challenging, since the mutations may not benefit all members of the broad self-TAA-specific T cell repertoire. We previously identified a common weak murine self-TAA that we converted to a highly effective antitumor vaccine by a single amino acid substitution. In this case the modified and natural self-TAAs still raised very similar sets of CD8 T cells. Our structural studies herein show that the modification of the self-TAA resulted in a subtle change in the major histocompatibility complex I-TAA structure. This amino acid substitution allowed a dramatic conformational change in the peptide during subsequent TCR engagement, creating a large increase in TCR affinity and accounting for the efficacy of the modified self-TAA as a vaccine. These results show that carefully selected, well-characterized modifications to a poorly immunogenic self-TAA can rescue the immune response of the large repertoire of weakly responding natural self-TAA-specific CD8 T cells, driving them to proliferate and differentiate into functional effectors. Subsequently, the unmodified self-TAA on the tumor cells, while unable to drive this response, is nevertheless a sufficient target for the CD8 cytotoxic effectors. Our results suggest a pathway for more efficiently identifying variants of common self-TAAs, which could be useful in vaccine development, complementing other current nonantigen-specific immunotherapies.

Tribbles homolog 2 regulates binding partners expression in bovine granulosa cells

The Tribbles (TRIB) family including TRIB2 are serine/threonine pseudokinases, which function as signaling mediators and scaffolding proteins regulating numerous cellular events such as proliferation, differentiation and cell death through protein stability and activity. However, the signaling pathways affected by TRIB2 in granulosa cells (GC) during the ovarian follicular development is not fully defined. We previously identified and reported TRIB2 as differentially expressed in growing dominant follicles (DF) while downregulated by the luteinizing hormone (LH) surge in ovulatory follicles. In the present study, we used the yeast two-hybrid screening and in vitro co-immunoprecipitation to identify and confirm TRIB2 interactions in GC. We identified CALM1, INHBA, INPPL1, NT5E, SCD, SDHB and RAB14 as TRIB2 binding partners. RT-PCR analyses showed all TRIB2 binding partners are present in GC of DF but differently regulated throughout the different stages of follicular development. TRIB2 inhibition and overexpression using, respectively, CRISPR/Cas9 and pQE systems revealed TRIB2 differently regulates the expression of its binding partners. INPPL1 and INHBA were decreased in TRIB2-inhibited GC and increased in TRIB2-overexpressed GC (P<0.05) while NT5E and SDHB were increased following TRIB2 inhibition (P<0.05). CALM1 was increased following TRIB2 overexpression and both inhibition and overexpression of TRIB2 resulted in increased RAB14 expression (P<0.05). The regulation of these partners by TRIB2 reveal a crucial role of TRIB2 in a number of biological processes in GC, and provide a larger view of potential TRIB2-regulated signal transduction pathways and a strong evidence that TRIB2 may act as a regulator of target effectors during ovarian follicular development.

RhoGTPases and inflammasomes: Guardians of effector-triggered immunity.

Pathogens have evolved smart strategies to invade hosts and hijack their immune responses. One such strategy is the targeting of the host RhoGTPases by toxins or virulence factors to hijack the cytoskeleton dynamic and immune processes. In response to this microbial attack, the host has evolved an elegant strategy to monitor the function of virulence factors and toxins by sensing the abnormal activity of RhoGTPases. This innate immune strategy of sensing bacterial effector targeting RhoGTPase appears to be a bona fide example of effector-triggered immunity (ETI). Here, we review recently discovered mechanisms by which the host can sense the activity of these toxins through NOD and NOD-like receptors (NLRs).

Characterization of Effector-Target Interactions in Necrotrophic Pathosystems Reveals Trends and Variation in Host Manipulation.

Great strides have been made in defining the details of the plant defense response involving biotrophic fungal and bacterial pathogens. The groundwork for the current model was laid by H.H. Flor and others who defined the gene-for-gene hypothesis, which is now known to involve effector-triggered immunity (ETI). PAMP-triggered immunity (PTI) is also a highly effective response to most pathogens because of the recognition of common pathogen molecules by pattern recognition receptors. In this article, we consider the three pathogens that make up the foliar disease complex of wheat, Zymoseptoria tritici, Pyrenophora tritici-repentis, and Parastagonospora nodorum, to review the means by which necrotrophic pathogens circumvent, or outright hijack, the ETI and PTI pathways to cause disease.

Nuclear Localization of HopA1Pss61 Is Required for Effector-Triggered Immunity.

Plant resistance proteins recognize cognate pathogen avirulence proteins (also named effectors) to implement the innate immune responses called effector-triggered immunity. Previously, we reported that hopA1 from Pseudomonas syringae pv. syringae strain 61 was identified as an avr gene for Arabidopsis thaliana. Using a forward genetic screen approach, we cloned a hopA1-specific TIR-NBS-LRR class disease resistance gene, RESISTANCE TO PSEUDOMONAS SYRINGAE6 (RPS6). Many resistance proteins indirectly recognize effectors, and RPS6 is thought to interact with HopA1Pss61 indirectly by surveillance of an effector target. However, the involved target protein is currently unknown. Here, we show RPS6 is the only R protein that recognizes HopA1Pss61 in Arabidopsis wild-type Col-0 accession. Both RPS6 and HopA1Pss61 are co-localized to the nucleus and cytoplasm. HopA1Pss61 is also distributed in plasma membrane and plasmodesmata. Interestingly, nuclear localization of HopA1Pss61 is required to induce cell death as NES-HopA1Pss61 suppresses the level of cell death in Nicotiana benthamiana. In addition, in planta expression of hopA1Pss61 led to defense responses, such as a dwarf morphology, a cell death response, inhibition of bacterial growth, and increased accumulation of defense marker proteins in transgenic Arabidopsis. Functional characterization of HopA1Pss61 and RPS6 will provide an important piece of the ETI puzzle.

547 Regulatory T cell production of IFN-γ in vitiligo

Vitiligo is a common autoimmune skin disease that affects 1-2% of the population. In vitiligo, CD8+ effector T cells (Teffs) target melanocytes, the pigment producing cells. Over time, this targeted destruction leads to patchy skin depigmentation, which can result in social stigma and emotional distress to affected individuals. Regulatory T cells (Tregs) help maintain tolerance in peripheral tissues by suppressing Teffs that target self-antigens and thus limit vitiligo disease severity. We performed single cell RNA sequencing on cells isolated from vitiligo patients and determined that Tregs adopt a type 1 cytokine expression profile in lesions, but not nonlesional skin. Consistent with this profile, human lesional Tregs produce IFN-γ, which we confirmed in our mouse model of vitiligo. IFN-γ production by Tregs could indicate reduced function with conversion to effector status, or in contrast, improved function by adopting a similar profile to their effector targets. Additional studies will determine what role IFN-γ plays in Treg function during the progression, maintenance, and stabilization of vitiligo.

Anti-Angiogenic Therapy: Current Challenges and Future Perspectives.

Anti-angiogenic therapy is an old method to fight cancer that aims to abolish the nutrient and oxygen supply to the tumor cells through the decrease of the vascular network and the avoidance of new blood vessels formation. Most of the anti-angiogenic agents approved for cancer treatment rely on targeting vascular endothelial growth factor (VEGF) actions, as VEGF signaling is considered the main angiogenesis promotor. In addition to the control of angiogenesis, these drugs can potentiate immune therapy as VEGF also exhibits immunosuppressive functions. Despite the mechanistic rational that strongly supports the benefit of drugs to stop cancer progression, they revealed to be insufficient in most cases. We hypothesize that the rehabilitation of old drugs that interfere with mechanisms of angiogenesis related to tumor microenvironment might represent a promising strategy. In this review, we deepened research on the molecular mechanisms underlying anti-angiogenic strategies and their failure and went further into the alternative mechanisms that impact angiogenesis. We concluded that the combinatory targeting of alternative effectors of angiogenic pathways might be a putative solution for anti-angiogenic therapies.

Epigenetic silencing of microRNA-199a-5p promotes the proliferation of non-small cell lung cancer cells by increasing AKAP1 expression.

MicroRNA (miR)-199a-5p expression is downregulated in a variety of malignancies, including non-small cell lung cancer (NSCLC), and its low expression is associated with a poor prognosis. However, to the best of our knowledge, the mechanism underlying miR-199a-5p downregulation in NSCLC and its target effectors remain to be elucidated. The present study revealed the downregulation of miR-199a-5p expression in NSCLC tissues and cell lines compared with in para-carcinoma tissues and a lung epithelial cell line. Further experiments indicated that the methylation levels of the miR-199a promoter were markedly higher in NSCLC tissues compared with in para-carcinoma tissues. The DNA methyltransferase inhibitor 5-Aza-2′-deoxycytidine markedly increased the expression levels of miR-199a-5p in NSCLC cells. Furthermore, it was identified that miR-199a-5p mimics transfection decreased the expression levels of A-kinase anchoring protein 1 (AKAP1) at both the mRNA and protein levels by targeting the 3′ untranslated region of AKAP1 mRNA. The in vitro experiments demonstrated that miR-199a-5p overexpression inhibited the proliferation and tumorigenicity of NSCLC cells, whereas overexpression of AKAP1 partially recovered the malignant phenotypes, suggesting that AKAP1 may be a downstream effector targeted by miR-199a-5p. Collectively, the present findings indicated that miR-199a-5p may be a novel regulator of AKAP1, and that miR-199a-5p may be a potential tumor suppressor in NSCLC.

Potato StMPK7 is a downstream component of StMKK1 and promotes resistance to the oomycete pathogen Phytophthora infestans

A cascade formed by phosphorylation events of mitogen‐activated protein kinases (MAPKs) takes part in plant stress responses. However, the roles of these MAPKs in resistance of potato (Solanum tuberosum) against Phytophthora pathogens is not well studied. Our previous work showed that a Phytophthora infestans RXLR effector targets and stabilizes the negative regulator of MAPK kinase 1 of potato (StMKK1). Because in Arabidopsis thaliana the AtMPK4 is the downstream phosphorylation target of AtMKK1, we performed a phylogenetic analysis and found that potato StMPK4/6/7 are closely related and are orthologs of AtMPK4/5/11/12. Overexpression of StMPK4/7 enhances plant resistance to P. infestans and P. parasitica. Yeast two‐hybrid analysis revealed that StMPK7 interacts with StMKK1, and StMPK7 is phosphorylated on flg22 treatment and by expressing constitutively active StMKK1 (CA‐StMKK1), indicating that StMPK7 is a direct downstream signalling partner of StMKK1. Overexpression of StMPK7 in potato enhances potato resistance to P. infestans. Constitutively active StMPK7 (CA‐StMPK7; StMPK7D198G, E202A) was found to promote immunity to Phytophthora pathogens and to trigger host cell death when overexpressed in Nicotiana benthamiana leaves. Cell death triggered by CA‐StMPK7 is SGT1/RAR1‐dependent. Furthermore, cell death triggered by CA‐StMPK7 is suppressed on coexpression with the salicylate hydroxylase NahG, and StMPK7 activation promotes salicylic acid (SA)‐responsive gene expression. We conclude that potato StMPK7 is a downstream signalling component of the phosphorelay cascade involving StMKK1 and StMPK7 plays a role in immunity to Phytophthora pathogens via an SA‐dependent signalling pathway.

Thinking Small: Small Molecules as Potential Synergistic Adjuncts to Checkpoint Inhibition in Melanoma.

Metastatic melanoma remains the deadliest form of skin cancer. Immune checkpoint inhibition (ICI) immunotherapy has defined a new age in melanoma treatment, but responses remain inconsistent and some patients develop treatment resistance. The myriad of newly developed small molecular (SM) inhibitors of specific effector targets now affords a plethora of opportunities to increase therapeutic responses, even in resistant melanoma. In this review, we will discuss the multitude of SM classes currently under investigation, current and prospective clinical combinations of ICI and SM therapies, and their potential for synergism in melanoma eradication based on established mechanisms of immunotherapy resistance.

Toxoplasma gondii association with host mitochondria requires key mitochondrial protein import machinery

Host mitochondrial association (HMA) is a well-known phenomenon during Toxoplasma gondii infection of the host cell. The T. gondii locus mitochondrial association factor 1 (MAF1) is required for HMA and MAF1 encodes distinct paralogs of secreted dense granule effector proteins, some of which mediate the HMA phenotype (MAF1b paralogs drive HMA; MAF1a paralogs do not). To identify host proteins required for MAF1b-mediated HMA, we performed unbiased, label-free quantitative proteomics on host cells infected with type II parasites expressing MAF1b, MAF1a, and an HMA-incompetent MAF1b mutant. Across these samples, we identified ∼1,360 MAF1-interacting proteins, but only 13 that were significantly and uniquely enriched in MAF1b pull-downs. The gene products include multiple mitochondria-associated proteins, including those that traffic to the mitochondrial outer membrane. Based on follow-up endoribonuclease-prepared short interfering RNA (esiRNA) experiments targeting these candidate MAF1b-targeted host factors, we determined that the mitochondrial receptor protein TOM70 and mitochondria-specific chaperone HSPA9 were essential mediators of HMA. Additionally, the enrichment of TOM70 at the parasitophorous vacuole membrane interface suggests parasite-driven sequestration of TOM70 by the parasite. These results show that the interface between the T. gondii vacuole and the host mitochondria is characterized by interactions between a single parasite effector and multiple target host proteins, some of which are critical for the HMA phenotype itself. The elucidation of the functional members of this complex will permit us to explain the link between HMA and changes in the biology of the host cell.

Bacterial effector targeting of a plant iron sensor facilitates iron acquisition and pathogen colonization.

Acquisition of nutrients from different species is necessary for pathogen colonization. Iron is an essential mineral nutrient for nearly all organisms, but little is known about how pathogens manipulate plant hosts to acquire iron. Here, we report that AvrRps4, an effector protein delivered by Pseudomonas syringae bacteria to plants, interacts with and targets the plant iron sensor protein BRUTUS (BTS) to facilitate iron uptake and pathogen proliferation in Arabidopsis thaliana. Infection of rps4 and eds1 by P. syringae pv. tomato (Pst) DC3000 expressing AvrRps4 resulted in iron accumulation, especially in the plant apoplast. AvrRps4 alleviates BTS-mediated degradation of bHLH115 and ILR3(IAA-Leucine resistant 3), two iron regulatory proteins. In addition, BTS is important for accumulating immune proteins Enhanced Disease Susceptibility1 (EDS1) at both the transcriptional and protein levels upon Pst (avrRps4) infections. Our findings suggest that AvrRps4 targets BTS to facilitate iron accumulation and BTS contributes to RPS4/EDS1-mediated immune responses.

A Conserved Oomycete CRN Effector Targets Tomato TCP14-2 to Enhance Virulence.

Phytophthora spp. secrete vast arrays of effector molecules during infection to aid in host colonization. The crinkling and necrosis (CRN) protein family forms an extensive repertoire of candidate effectors that accumulate in the host nucleus to perturb processes required for immunity. Here, we show that CRN12_997 from Phytophthora capsici binds a TCP transcription factor, SlTCP14-2, to inhibit its immunity-associated activity against Phytophthora spp. Coimmunoprecipitation and bimolecular fluorescence complementation studies confirm a specific CRN12_997-SlTCP14-2 interaction in vivo. Coexpression of CRN12_997 specifically counteracts the TCP14-enhanced immunity phenotype, suggesting that CRN mediated perturbation of SlTCP14-2 function. We show that SlTCP14-2 associates with nuclear chromatin and that CRN12_997 diminishes SlTCP14-2 DNA binding. Collectively, our data support a model in which SlTCP14-2 associates with chromatin to enhance immunity. The interaction between CRN12_997 and SlTCP14-2 reduces DNA binding of the immune regulator. We propose that the modulation of SlTCP14-2 chromatin affinity, caused by CRN12-997, enhances susceptibility to P. capsici.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.