Signaling Subunit Research Articles

Signal Transduction of Transient Receptor Potential TRPM8 Channels: Role of PIP5K, Gq-Proteins, and c-Jun.

Transient receptor potential melastatin-8 (TRPM8) is a cation channel that is activated by cold and "cooling agents" such as menthol and icilin, which induce a cold sensation. The stimulation of TRPM8 activates an intracellular signaling cascade that ultimately leads to a change in the gene expression pattern of the cells. Here, we investigate the TRPM8-induced signaling pathway that links TRPM8 channel activation to gene transcription. Using a pharmacological approach, we show that the inhibition of phosphatidylinositol 4-phosphate 5 kinase α (PIP5K), an enzyme essential for the biosynthesis of phosphatidylinositol 4,5-bisphosphate, attenuates TRPM8-induced gene transcription. Analyzing the link between TRPM8 and Gq proteins, we show that the pharmacological inhibition of the βγ subunits impairs TRPM8 signaling. In addition, genetic studies show that TRPM8 requires an activated Gα subunit for signaling. In the nucleus, the TRPM8-induced signaling cascade triggers the activation of the transcription factor AP-1, a complex consisting of a dimer of basic region leucine zipper (bZIP) transcription factors. Here, we identify the bZIP protein c-Jun as an essential component of AP-1 within the TRPM8-induced signaling cascade. In summary, with PIP5K, Gq subunits, and c-Jun, we identified key molecules in TRPM8-induced signaling from the plasma membrane to the nucleus.

Disruption of KLHL6 Fuels Oncogenic Antigen Receptor Signaling in B-Cell Lymphoma.

Pathomechanisms that activate oncogenic B-cell receptor (BCR) signaling in diffuse large B-cell lymphoma (DLBCL) are largely unknown. Kelch-like family member 6 (KLHL6) encoding a substrate-adapter for Cullin-3-RING E3 ubiquitin ligase with poorly established targets is recurrently mutated in DLBCL. By applying high-throughput protein interactome screens and functional characterization, we discovered that KLHL6 regulates BCR by targeting its signaling subunits CD79A and CD79B. Loss of physiologic KLHL6 expression pattern was frequent among the MCD/C5-like activated B-cell DLBCLs and was associated with higher CD79B levels and dismal outcome. Mutations in the bric-a-brac tramtrack broad domain of KLHL6 disrupted its localization and heterodimerization and increased surface BCR levels and signaling, whereas Kelch domain mutants had the opposite effect. Malfunctions of KLHL6 mutants extended beyond proximal BCR signaling with distinct phenotypes from KLHL6 silencing. Collectively, our findings uncover how recurrent mutations in KLHL6 alter BCR signaling and induce actionable phenotypic characteristics in DLBCL. Significance: Oncogenic BCR signaling sustains DLBCL cells. We discovered that Cullin-3-RING E3 ubiquitin ligase substrate-adapter KLHL6 targets BCR heterodimer (CD79A/CD79B) for ubiquitin-mediated degradation. Recurrent somatic mutations in the KLHL6 gene cause corrupt BCR signaling by disrupting surface BCR homeostasis. Loss of KLHL6 expression and mutant-induced phenotypes associate with targetable disease characteristics in B-cell lymphoma. See related commentary by Leveille et al. See related commentary by Corcoran et al.

Abstract 330: EP300 loss drives tumorigenesis in bladder cancer via activation of IL-6/JAK/STAT3 signaling

Abstract Bladder cancer (BLCA) is the sixth most common cancer in the U.S with 83,730 new cases, accounting for 17,200 deaths in the year 2021 and 212,536 deaths worldwide. EP300, a histone acetyltransferase gene, is mutated in ~17% of BLCA and in other cancer types such as endometrial cancer. The mechanisms by which EP300 mutations contribute to tumorigenicity of BLCA is not currently understood. To determine the phenotypic effects of loss-of-function EP300 mutations, we generated isogenic BLCA cells with EP300 knocked out (KO) using CRISPR/CAS9. Conspicuous changes in phenotypes consistent with more aggressive forms of cancer were observed in EP300-null BLCA clones, including luminal-basal histotype plasticity, anchorage-independent growth, enhanced in vitro and in vivo cell proliferation and enhanced invasion in Boyden chamber assays. To identify mechanisms whereby EP300 KO promotes increased oncogenicity, we performed phospho Tandem Tag Mass Spectrometry (TMT MS) using lysates from parental and EP300 KO cells. A notable finding was significantly increased expression of phosphorylated STAT3 (pSTAT3), which we subsequently confirmed by western blot. siRNA knockdown and selective inhibitor experiments indicated JAK1 as the upstream activator of pSTAT3 in BLCA cells. Studies of conditioned media (CM) from the EP300 KO cells using ELISA and neutralizing antibodies demonstrated that secreted IL-6 ligand and soluble IL-6R in the CM drove STAT3 activation through the IL-6 family common signaling subunit, gp130, i.e. trans-signaling. BLCA RT112 cells express an oncogenic FGFR3-TACC3 fusion and are sensitive to the FDA-approved FGFR kinase inhibitor erdafitinib. pSTAT3 expression was not dependent on FGFR3 signaling in EP300 KO RT112 cells, and EP300 KO was sufficient to confer erdafitinib resistance in a BLCA context. In sum, our results uncover that EP300 loss enhances IL-6/JAK/STAT3 signaling which promotes BLCA tumorigenesis and FGFR inhibitor resistance. Our findings elucidate a role for cytokine induced sterile inflammation in EP300-mediated tumorigenesis in bladder cancers and suggest that targeting the IL-6/JAK/STAT3 axis may represent a novel therapy strategy to overcome FGFR3 inhibitor resistance. Citation Format: Sizhi P. Gao, James A. Rodrigues, Amanda R. Sabel, Jiaqian Luo, Chen Ziyu, Xinran Tang, Eduardo A. Mascareno, Naryan Rustgi, Hikmat Al-Ahmadie, Kwanghee Kim, Eugene J. Pietzak, Gopakumar V. Iyer, David B. Solit. EP300 loss drives tumorigenesis in bladder cancer via activation of IL-6/JAK/STAT3 signaling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 330.

Changes in cytokine and cytokine receptor levels during postnatal development of the human dorsolateral prefrontal cortex

In addition to their traditional roles in immune cell communication, cytokines regulate brain development. Cytokines are known to influence neural cell generation, differentiation, maturation, and survival. However, most work on the role of cytokines in brain development investigates rodents or focuses on prenatal events. Here, we investigate how mRNA and protein levels of key cytokines and cytokine receptors change during postnatal development of the human prefrontal cortex. We find that most cytokine transcripts investigated (IL1B, IL18, IL6, TNF, IL13) are lowest at birth and increase between 1.5 and 5 years old. After 5 years old, transcriptional patterns proceeded in one of two directions: decreased expression in teens and young adults (IL1B, p = 0.002; and IL18, p = 0.004) or increased mean expression with maturation, particularly in teenagers (IL6, p = 0.004; TNF, p = 0.002; IL13, p < 0.001). In contrast, cytokine proteins tended to remain elevated after peaking significantly around 3 years of age (IL1B, p = 0.012; IL18, p = 0.026; IL6, p = 0.039; TNF, p < 0.001), with TNF protein being highest in teenagers. An mRNA-only analysis of cytokine receptor transcripts found that early developmental increases in cytokines were paralleled by increases in their ligand-binding receptor subunits, such as IL1R1 (p = 0.033) and IL6R (p < 0.001) transcripts. In contrast, cytokine receptor-associated signaling subunits, IL1RAP and IL6ST, did not change significantly between age groups. Of the two TNF receptors, the ‘pro-death’ TNFRSF1A and ‘pro-survival’ TNFRSF1B, only TNFRSF1B was significantly changed (p = 0.028), increasing first in toddlers and again in young adults. Finally, the cytokine inhibitor, IL13, was elevated first in toddlers (p = 0.006) and again in young adults (p = 0.053). While the mean expression of interleukin-1 receptor antagonist (IL1RN) was highest in toddlers, this increase was not statistically significant. The fluctuations in cytokine expression reported here support a role for increases in specific cytokines at two different stages of human cortical development. The first is during the toddler/preschool period (IL1B, IL18, and IL13), and the other occurs at adolescence/young adult maturation (IL6, TNF and IL13).

The kinetics of signaling through the common FcRγ chain determine cytokine profiles in dendritic cells.

The common Fc receptor γ (FcRγ) chain is a signaling subunit common to several immune receptors, but cellular responses induced by FcRγ-coupled receptors are diverse. We investigated the mechanisms by which FcRγ generates divergent signals when coupled to Dectin-2 and Mincle, structurally similar C-type lectin receptors that induce the release of different cytokines from dendritic cells. Chronological tracing of transcriptomic and epigenetic changes upon stimulation revealed that Dectin-2 induced early and strong signaling, whereas Mincle-mediated signaling was delayed, which reflects their expression patterns. Generation of early and strong FcRγ-Syk signaling by engineered chimeric receptors was sufficient to recapitulate a Dectin-2-like gene expression profile. Early Syk signaling selectively stimulated the activity of the calcium ion-activated transcription factor NFAT, which rapidly altered the chromatin status and transcription of the Il2 gene. In contrast, proinflammatory cytokines, such as TNF, were induced regardless of FcRγ signaling kinetics. These results suggest that the strength and timing of FcRγ-Syk signaling can alter the quality of cellular responses through kinetics-sensing signaling machineries.

Mesothelin-targeting T cells bearing a novel T cell receptor fusion construct (TRuC) exhibit potent antitumor efficacy against solid tumors

ABSTRACT T cell Receptor (TCR) Fusion Construct (TRuC®) T cells harness all signaling subunits of the TCR to activate T cells and eliminate tumor cells, with minimal release of cytokines. While adoptive cell therapy with chimeric antigen receptor (CAR)-T cells has shown unprecedented clinical efficacy against B-cell malignancies, monotherapy with CAR-T cells has suboptimal clinical efficacy against solid tumors, probably because of the artificial signaling properties of the CAR. TRuC-T cells may address the suboptimal efficacy of existing CAR-T therapies for solid tumors. Here, we report that mesothelin (MSLN)-specific TRuC-T cells (referred to as TC-210 T cells) potently kill MSLN+ tumor cells in vitro and efficiently eradicate MSLN+ mesothelioma, lung, and ovarian cancers in xenograft mouse tumor models. When benchmarked against MSLN-targeted BBζ CAR-T cells (MSLN-BBζ CAR-T cells), TC-210 T cells show an overall comparable level of efficacy; however, TC-210 T cells consistently show faster tumor rejection kinetics that are associated with earlier intratumoral accumulation and earlier signs of activation. Furthermore, in vitro and ex vivo metabolic profiling suggests TC-210 T cells have lower glycolytic activity and higher mitochondrial metabolism than MSLN-BBζ CAR-T cells. These data highlight TC-210 T cells as a promising cell therapy for treating MSLN-expressing cancers. The differentiated profile from CAR-T cells may translate into better efficacy and safety of TRuC-T cells for solid tumors.

Abstract 557: Engineering off-the-shelf anti mesothelin t-cell receptor fusion construct (TRuC™) t-cells

Abstract We have previously described the generation of autologous T Cell Receptor Fusion Construct (TRuC™) T cells which are engineered to express a fusion protein comprised of an antibody-derived binder tethered to the extracellular domain of the CD3ε signaling subunit. Upon integration of the TRuC into the T cell receptor (TCR), it targets specific tumor surface antigens independent of HLA and uses the complete receptor complex to trigger a comprehensive T cell response. Here, we report about engineering of off-the-shelf TRuC-T cells directed against mesothelin (MSLN). To eliminate the alloreactivity of α/β T cells and reduce the risk of graft-versus-host-disease (GvHD), the TRAC locus is knocked-out. To enable the re-assembly of the TCR, the endogenous TCRα and β subunits are replaced with fusion proteins comprised of antibody-derived binders fused to the TCRγ and δ constant domains. Allogeneic anti-MSLN TRuC-T cells upregulate activation markers, secrete robust cytokines, and lyse tumor cells in an antigen-specific manner without allo-reactivity. In comparison with control autologous TRuC-T cells targeting mesothelin (TC-210), allogeneic TRuC-Ts demonstrate extended tumor clearance in NSG xenograft models due to their enhanced expansion and persistence. To reduce host rejection and further boost the persistence of the allogeneic TRuC-T cells, we eliminated MHC class I surface expression by B2M gene knockout and over-expressed membrane-bound IL15 tethered to its receptor alpha. In summary we have engineered persistence enhanced, allogeneic TRuC-T cells that maintain the signaling properties of the TCR complex with improved efficacy compared to donor-matched autologous TRuC-T cells. Citation Format: Julie Donaghey, Cecilia Kwong, Allison Powell, Troy Patterson, Ella Liberzon, Richard Decker, Julio Gomez Rodriguez, Darby Kreienberg, Jennifer Bian, Urmi Patankar, Holly Horton, Jian Ding, Robert Hofmeister, Dario Gutierrez, Robert Tighe. Engineering off-the-shelf anti mesothelin t-cell receptor fusion construct (TRuC™) t-cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 557.

Abstract CT250: A first-in-human phase 1 study of NL-201 in patients with relapsed or refractory cancer

Abstract Background: NL-201 is a selective and long-acting computationally designed alpha-independent agonist of the IL-2 and IL-15 receptors, which share beta and gamma signaling subunits. NL-201 is being developed as a potent activator of CD8+ T cells and NK cells for cancer immunotherapy. Binding to the beta and gamma subunits stimulates dose-dependent expansion and tumor infiltration of cytotoxic CD8+ T cells and NK cells, thereby enhancing the immune response in the tumor. The absence of binding to the IL-2 alpha subunit reduces the undesirable effects of traditional IL-2 therapies, such as vascular leak syndrome and expansion of immunosuppressive regulatory T cells. As such, NL-201 is designed to promote the desired immunomodulatory anti-tumor effects of IL-2 with an improved safety profile. Methods: NL201-101 is a Phase 1 first-in-human, open-label, dose-escalation and cohort expansion study consisting of two parts. Part 1 is a monotherapy dose-escalation study in up to 60 adult patients with advanced and/or refractory solid tumors to define the safety profile and the recommended Phase 2 dose (RP2D) and schedule of NL-201. During dose escalation, two different schedules will be evaluated: dosing every 21 days (Schedule A), or on days 1 and 8 of each 21-day cycle (Schedule B). Tumor response will be assessed by Response Evaluation Criteria in Solid Tumours (RECIST) 1.1 and/or RECIST for use in cancer immunotherapy trials (iRECIST). In Part 2, patients with pathologically proven diagnosis of melanoma and renal cell carcinoma (up to N=30/cohort), who have advanced and/or refractory measurable disease and have failed at least one line of treatment, which may include checkpoint inhibitors, will be enrolled. Key exclusion criteria include history of brain cancer or active brain metastases, carcinomatous meningitis, neurologic autoimmune disease; patients previously receiving CAR-T or IL-2-based therapies are not eligible. Recruitment of Part 1 began in April 2021, and the trial is actively enrolling. Clinicaltrials.gov identifier: NCT04659629. Citation Format: Aung Naing, Margaret Callahan, Brian A. Costello, Brendan Curti, Evan Hall, Aaron Hansen, Georgina V. Long, Anthony M. Joshua, Cynthia Wetmore, Andrew Weickhardt. A first-in-human phase 1 study of NL-201 in patients with relapsed or refractory cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr CT250.

Pediatric and young adult leukemia adoptive therapy (PLAT)-08: A phase 1 study of SC-DARIC33 in pediatric and young adults with relapsed or refractory CD33+ AML.

TPS7078 Background: The acute and late effects of acute myeloid leukemia (AML) therapy in children can be devastating, and relapse rates remain high. Targeted therapies are needed to decrease toxicity and improve cure rates. Most AML patients express CD33 on their leukemic blasts. CD33 is a viable target with CD33 targeted therapies approved for AML. CD33 targeted CAR T cell therapies are being developed, but safety and efficacy remain a challenge. Safety concerns include cytokine release syndrome, neurotoxicity and aplasia. Efficacy is limited, with antigen escape and T cell exhaustion proposed as mechanisms of treatment failure. Dimerizing Agent Regulated ImmunoReceptor Complex (DARIC) is a next-generation technology that utilizes a split receptor design separating the antigen binding and intracellular signaling subunits (Leung, 2019). These subunits remain inactive until the administration of rapamycin which allows for dimerization of the subunits, enabling T cell activation when antigen is present. Modulating CAR T cell activation may aid hematopoietic recovery, enhance CAR T cell function and persistence, and mitigate toxicity. We developed a DARIC T cell product, SC-DARIC33, that couples the DARIC technology with a lentiviral construct that incorporates the use of a novel humanized camelid nanobody for targeting the C2 splice isoform within the membrane proximal domain of CD33. Methods: Up to 18 patients will be enrolled in this phase 1 study (NCT05105152). Patients must have early first relapse of AML (≤ 6 months from diagnosis), first relapse refractory to at least one reinduction attempt, second or greater relapse, or refractory de novo AML. The first 3 patients must be ≥18 years, all patients who meet eligibility criteria must be ≤28 years. The primary objectives of this Phase I study (NCT05105152) are assessment of safety/toxicity and feasibility of manufacturing of SC-DARIC33. Secondary objectives include assessment of efficacy, on/off rate of DARIC expression, engraftment, expansion and persistence of SC-DARIC33. Also, the effect of cessation of rapamycin on activation state of SC-DARIC 33 and on hematopoietic recovery will be evaluated. Subjects receive lymphodepletion prior to a single dose of SC-DARIC33 T cells on Day 0 followed by initiation of rapamycin on Day +2 with the purpose of activating the T cells. Rapamycin continues until Day 21 and bone marrow evaluation occurs on Day 28. Subsequent cycles of rapamycin are allowed for patients who respond. The trial will implement a BOIN design with three possible dose levels (1 x 106, 5 x 106, and 10 x 106 DARIC+ cells/kg) to determine the maximum tolerated dose (MTD). The trial is open for enrollment at Seattle Children’s Hospital. Clinical trial information: NCT05105152.

Betulinic acid accelerates diabetic wound healing by modulating hyperglycemia-induced oxidative stress, inflammation and glucose intolerance.

BackgroundDiabetes significantly delays wound healing through oxidative stress, inflammation and impaired re-epithelialization that lead to defective regulation of the healing process, although the related mechanism remains unclear. Here, we aim to investigate the potential role and mechanism for the beneficial effect of betulinic acid (BA) on diabetic wound healing.MethodsThe molecular effect of BA on hyperglycemia-mediated gene expression, oxidative stress, inflammation and glucose uptake was evaluated in endothelial, fibroblast and muscle cells. Burn injury was introduced to streptozotocin-induced diabetic rats and BA administration through either an intraperitoneal (IP) or topical (TOP) technique was used for wound treatment. Glucose tolerance was evaluated in both muscle tissue and fibroblasts, while oxidative stress and inflammation were determined in both the circulatory system and in wound tissues. The effect of BA on the wound healing process was also evaluated.ResultsBA treatment reversed hyperglycemia-induced glucose transporter type 4 (GLUT4) suppression in both muscle and fibroblast cells. This treatment also partly reversed hyperglycemia-mediated suppression of endothelial nitric oxide synthase (eNOS), nuclear factor erythroid 2-related factor 2 (Nrf2) signaling and nuclear factor NFκB p65 subunit (NFκB p65) activation in endothelial cells. An in vivo rat study showed that BA administration ameliorated diabetes-mediated glucose intolerance and partly attenuated diabetes-mediated oxidative stress and inflammation in both the circulatory system and wound tissues. BA administration by both IP and TOP techniques significantly accelerated diabetic wound healing, while BA administration by either IP or TOP methods alone had a significantly lower effect.ConclusionsBA treatment ameliorates hyperglycemia-mediated glucose intolerance, endothelial dysfunction, oxidative stress and inflammation. Administration of BA by both IP and TOP techniques was found to significantly accelerate diabetic wound healing, indicating that BA could be a potential therapeutic candidate for diabetic wound healing.

Abstract P032: Expression of an IL-15 receptor fusion protein enhances the persistence of TRuC-T cells

Abstract Adoptive cell therapies have shown great promise in hematological malignancies. To realize the potential of T cell therapies in solid tumors, we have developed T cell receptor fusion construct (TRuC®) T cells, which are equipped with an engineered T cell receptor that utilizes all TCR signaling subunits and recognizes tumor-associated antigens independent of HLA. In clinical trials, mesothelin-targeting TRuC-T cells (aka TC-210 or gavo-cel) have shown unprecedented results in patients suffering from advanced mesothelioma and ovarian cancer. To potentially increase the effector function and persistence of TRuC-T cells in the hostile tumor microenvironment, we generated TC-210 T cells that express a membrane-tethered IL15Rα-IL15 fusion protein. IL-15 is a common γ chain cytokine that promotes the differentiation, maintenance, and effector function of memory CD8+ T cell subsets and confers resistance to IL-2-mediated activation induced cell death (AICD). In vitro, the co-expression of the IL-15 fusion protein enhances T cell proliferation and persistence upon repeated stimulation with MSLN+ cancer cell lines, while exhaustion marker expression is decreased. Furthermore, IL-15 enhanced TC-210 T cells sustain a significantly higher TCF-1+ population. When tested in a mesothelioma xenograft mouse model, the presence of the IL-15 fusion protein increased tumor infiltration and persistence of TC-210 T cells. Altogether, the presented data support clinical studies that explore the impact of IL-15 enhancement on the persistence of TC-210 T cells and depth of response in patients with MSLN+ malignancies. Citation Format: Michelle Fleury, Courtney Anderson, Amy Watt, Holly Horton, Adam Zieba, Jian Ding, Robert Tighe, Robert Hofmeister, Derrick McCarthy, Dario Gutierrez. Expression of an IL-15 receptor fusion protein enhances the persistence of TRuC-T cells [abstract]. In: Abstracts: AACR Virtual Special Conference: Tumor Immunology and Immunotherapy; 2021 Oct 5-6. Philadelphia (PA): AACR; Cancer Immunol Res 2022;10(1 Suppl):Abstract nr P032.

Discovery of a novel potentially transforming somatic mutation in CSF2RB gene in breast cancer.

The colony stimulating factor 2 receptor subunit beta (CSF2RB) is the common signaling subunit of the cytokine receptors for IL‐3, IL‐5, and GM‐CSF. Several studies have shown that spontaneous and random mutants of CSF2RB can lead to ligand independence in vitro. To date, no report(s) have been shown for the presence of potentially transforming and oncogenic CSF2RB mutation(s) clinically in cancer patients until the first reported case of a leukemia patient in 2016 harboring a germline‐activating mutation (R461C). We combined exome sequencing, pathway analyses, and functional assays to identify novel somatic mutations in KAIMRC1 cells and breast tumor specimen. The patient’s peripheral blood mononuclear cell (PBMC) exome served as a germline control in the identification of somatic mutations. Here, we report the discovery of a novel potentially transforming and oncogenic somatic mutation (S230I) in the CSF2RB gene of a breast cancer patient and the cell line, KAIMRC1 established from her breast tumor tissue. KAIMRC1 cells are immortalized and shown to survive and proliferate in ligand starvation condition. Immunoblot analysis showed that mutant CSF2RB signals through JAK2/STAT and PI3K/mTOR pathways in ligand starvation conditions. Screening a small molecule kinase inhibitor library revealed potent JAK2 inhibitors against KAIMRC1 cells. We, for the first time, identified a somatic, potentially transforming, and oncogenic CSF2RB mutation (S230I) in breast cancer patients that seem to be an actionable mutation leading to the development of new therapeutics for breast cancer.

Severe acute respiratory syndrome coronavirus 2 targeted antibodies cocktail and B cell receptor interplay: interventions to trigger vaccine development

Coronavirus disease-2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus (SARS-CoV)-2 spread globally and creates an alarming situation. Following the SARS-CoV-2 paradigm, therapeutic efficacy is achieved via repurposing several antiviral, antibacterial, and antimalarial drugs. Innate and adaptive immune cells work close to combat infection through the intricate production of antibodies (Abs) and inflammatory cytokines. As an essential component of the immune system, Abs play an important role in eliminating viruses and maintaining homeostasis. B lymphocytes (B cells) are effector cells, stringent to produce neutralizing Abs to combat infection. After recognizing SARS-CoV-2 antigens by a surface receptor called B cell receptors (BCRs) on the plasma membrane, the BCRs transmembrane signal transduction and immune activation results in Ab production and development of immune memory. Thus, it ensures that plasma B cells can quickly start an intricate immune response to generate efficient protective Abs to clear the pathogen. Nevertheless, considering therapeutic challenges in the context of the new coronavirus pandemic, this review addresses the molecular mechanism of the immune activation and function of novel SARS-CoV-2 specific B cells in the production of SARS-CoV-2 specific Abs. Additionally, these studies highlighted the Ab-mediated pathogenesis, the intriguing role of nano-scale signaling subunits, non-structural proteins during COVID-19 infection, and structural insights of SARS-CoV-2 specific Abs.

Novel Insights into the Molecular Regulation of Ribonucleotide Reductase in Adrenocortical Carcinoma Treatment.

Simple SummaryThe current clinical gold standard etoposide, doxorubicin, cisplatin, and mitotane (EDP-M) is not satisfying for the treatment of adrenocortical carcinoma (ACC). However, clinical translation of novel, preclinically promising therapies were unfortunately disappointing in recent years, indicating that utilized tumor models inadequately predicted clinical applicability of novel pharmacological approaches. In an attempt to optimize the current preclinical armamentarium, our workgroup initiated a comparative drug screen of clinically relevant chemotherapies and therapies targeting IGF, EGF, and Wnt signaling pathways in the classical NCI-H295R cell line and, for the first time, in the recently developed highly drug-resistant MUC-1 cell line. These testings revealed gemcitabine and cisplatin as a promising combination, but further investigations also indicated developing drug resistance mechanisms on the molecular level. We aimed to decipher underlying resistance mechanisms, identified ribonucleotide reductase as an important player, and successfully targeted the involved DNA damage/repair mechanism.Current systemic treatment options for patients with adrenocortical carcinomas (ACCs) are far from being satisfactory. DNA damage/repair mechanisms, which involve, e.g., ataxia-telangiectasia-mutated (ATM) and ataxia-telangiectasia/Rad3-related (ATR) protein signaling or ribonucleotide reductase subunits M1/M2 (RRM1/RRM2)-encoded ribonucleotide reductase (RNR) activation, commonly contribute to drug resistance. Moreover, the regulation of RRM2b, the p53-induced alternative to RRM2, is of unclear importance for ACC. Upon extensive drug screening, including a large panel of chemotherapies and molecular targeted inhibitors, we provide strong evidence for the anti-tumoral efficacy of combined gemcitabine (G) and cisplatin (C) treatment against the adrenocortical cell lines NCI-H295R and MUC-1. However, accompanying induction of RRM1, RRM2, and RRM2b expression also indicated developing G resistance, a frequent side effect in clinical patient care. Interestingly, this effect was partially reversed upon addition of C. We confirmed our findings for RRM2 protein, RNR-dependent dATP levels, and modulations of related ATM/ATR signaling. Finally, we screened for complementing inhibitors of the DNA damage/repair system targeting RNR, Wee1, CHK1/2, ATR, and ATM. Notably, the combination of G, C, and the dual RRM1/RRM2 inhibitor COH29 resulted in previously unreached total cell killing. In summary, we provide evidence that RNR-modulating therapies might represent a new therapeutic option for ACC.

BAFF signaling in health and disease.

BAFF is a critical cytokine supporting the survival of mature naïve B cells, acting through the BAFFR receptor. Recent studies show that BAFF and BAFFR are also required for the survival of memory B cells, autoimmune B cells as well as malignant chronic lymphocytic leukaemia (CLL) cells. BAFFR cooperates with other receptors, notably the B cell antigen receptor (BCR), a process which is critical for the expansion of autoimmune and CLL cells. This crosstalk may be mediated by TRAF3 which interacts with BAFFR and with CD79A, a signalling subunit of the BCR and the downstream SYK kinase, inhibiting its activity. BAFF binding to BAFFR leads to degradation of TRAF3 which may relieve inhibition of SYK activity transducing signals to pathways required for B cell survival. BAFFR activates both canonical and non-canonical NF-κB signalling and both pathways play important roles in the survival of B cells and CLL cells.

Abstract 1514: Engineering off-the-shelf T Cell Receptor Fusion Construct (TRuC) T cells

Abstract The natural T cell receptor (TCR) recognizes its cognate peptide antigen only when presented on human leukocyte antigen (HLA) molecules and requires HLA matching of α/β TCR-engineered T cells for cancer therapy. To bypass the need for HLA matching, we have previously described the generation of autologous T Cell Receptor Fusion Construct (TRuC™) T cells which are engineered to express a fusion protein that comprises an antibody-derived binder tethered to the CD3ϵ signaling subunit. Upon integration of the TRuC into the TCR, it recognizes tumor surface antigens independent of HLA and uses the complete receptor complex to trigger a comprehensive T cell response. Here, we report the engineering of off-the-shelf TRuC-T cells directed against CD19 and mesothelin, respectively. To eliminate the alloreactivity of α/β T cells and reduce the risk of graft-versus-host-disease (GvHD), the TRAC locus is knocked-out. To enable the re-assembly of the TCR, the endogenous TCRα and β subunits are replaced with fusion proteins comprising antibody binders fused to the murine TCRα or β constant domains, or alternatively, to the TCRγ and δ constant domains. Similar to their autologous counterparts, allogeneic CD19 and MSLN-targeting TRuC-T cells upregulate activation markers, secrete cytokines, and lyse tumor cells in an antigen-specific manner. Importantly, TRuC-T cells lack alloreactivity as demonstrated in mixed lymphocyte reactions and clear tumors in NSG xenograft model comparable to autologous anti-CD19 TRuC-T cells, but without signs of GvHD. Likewise, the functional activity and efficacy of allogeneic TRuC-T cells directed against mesothelin is similar to that of autologous anti-MSLN TRuC-T cells. To reduce host rejection and enhance the persistence of the allogeneic TRuC-T cells, MHC class I expression on the surface of the TRuC-T cells was down-regulated by means of RNAi. In summary we have engineered allogeneic TRuC-T cells that maintain the signaling properties of the TCR complex with comparable efficacy as donor-matched autologous TRuC-T cells; moreover, these T cells have the potential to persist in an allogeneic host by diminishing the risk of rejection by the host. Citation Format: Julie Donaghey, Philippe Kieffer-Kwon, Julio Gomez-Rodriguez, Troy Patterson, Jessica Gierut, Tiffany Chan, Ryan Milione, Joshua Daniel, Holly Horton, Robert Tighe, Robert Hofmeister, Dario A. Gutierrez. Engineering off-the-shelf T Cell Receptor Fusion Construct (TRuC) T cells [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 1514.

Allosteric activation of Tcell antigen receptor signaling by quaternary structure relaxation.

SummaryThe mechanism of T cell antigen receptor (TCR-CD3) signaling remains elusive. Here, we identify mutations in the transmembrane region of TCRβ or CD3ζ that augment peptide T cell antigen receptor (pMHC)-induced signaling not explicable by enhanced ligand binding, lateral diffusion, clustering, or co-receptor function. Using a biochemical assay and molecular dynamics simulation, we demonstrate that the gain-of-function mutations loosen the interaction between TCRαβ and CD3ζ. Similar to the activating mutations, pMHC binding reduces TCRαβ cohesion with CD3ζ. This event occurs prior to CD3ζ phosphorylation and at 0°C. Moreover, we demonstrate that soluble monovalent pMHC alone induces signaling and reduces TCRαβ cohesion with CD3ζ in membrane-bound or solubilised TCR-CD3. Our data provide compelling evidence that pMHC binding suffices to activate allosteric changes propagating from TCRαβ to the CD3 subunits, reconfiguring interchain transmembrane region interactions. These dynamic modifications could change the arrangement of TCR-CD3 boundary lipids to license CD3ζ phosphorylation and initiate signal propagation.

Elucidation of the molecular interactions that enable stable assembly and structural diversity in multicomponent immune receptors

Multicomponent immune receptors are essential complexes in which distinct ligand-recognition and signaling subunits are held together by interactions between acidic and basic residues of their transmembrane helices. A 2:1 acidic-to-basic motif in the transmembrane domains of the subunits is necessary and sufficient to assemble these receptor complexes. Here, we study a prototype for these receptors, a DAP12-NKG2C 2:1 heterotrimeric complex, in which the two DAP12 subunits each contribute a single transmembrane Asp residue, and the NKG2C subunit contributes a Lys to form the complex. DAP12 can also associate with 20 other subunits using a similar motif. Here, we use molecular-dynamics simulations to understand the basis for the high affinity and diversity of interactions in this group of receptors. Simulations of the transmembrane helices with differing protonation states of the Asp-Asp-Lys triad identified a structurally stable interaction in which a singly-protonated Asp-Asp pair forms a hydrogen-bonded carboxyl-carboxylate clamp that clasps onto a charged Lys side chain. This polar motif was also supported by density functional theory and a Protein Data Bank-wide search. In contrast, the helices are dynamic at sites distal to the stable carboxyl-carboxylate clamp motif. Such a locally stable but globally dynamic structure is well suited to accommodate the sequence and structural variations in the transmembrane helices of multicomponent receptors, which mix and match subunits to create combinatorial functional diversity from a limited number of subunits. It also supports a signaling mechanism based on multisubunit clustering rather than propagation of rigid conformational changes through the membrane.

Cooperative ectodomain interaction among TCRαβ, CD3δɛ and CD3γɛ

Abstract The T-cell receptor (TCR) complex comprises of the ligand-binding subunit TCRαβ, and the signaling subunits CD3δɛ, CD3γɛ and CD3ζζ, with the Cα/Cβ in proximity to both CD3δɛ and CD3γɛ extracellularly. Direct measurements of the ectodomain interactions had not been successful, although they are believed to be important for TCR stability and functionality. Mechanical force has been shown to modulate TCR–ligand interactions. The TCR mechanosensor hypothesis predicts that force-encoded information may transmit from pMHC to CD3 via TCR-CD3 interaction. Evaluating ectodomain interactions among TCRαβ, CD3δɛ and CD3γɛ can help elucidate the TCR triggering mechanism and further guide the design of TCR-based immunotherapy. Using two mechanical based assays, we were able to measure the weak two-dimensional (2D) affinities among ectodomains of human TCRαβ (2B4-LC13), and human CD3δɛ or CD3γɛ and showed catch bond formation where lifetimes of TCRαβ–CD3δɛ and TCRαβ–CD3γɛ bonds are prolonged by forces &amp;lt;15 pN. Remarkably, CD3δɛ and CD3γɛ bind TCRαβ cooperatively, forming more bonds that last longer when both CD3s interact with TCRαβ as a whole than the sum of either CD3 interacting with TCRαβ individually. Interestingly, these measurements are comparable to 2D affinity and force-dependent bond lifetime of the 2B4 TCR interaction with its cognate ligand K5:I-Ek, supporting their relevance to TCR function. Using molecular dynamics simulations based on a published Cryo-EM structure, we identified the formation of long-lasting CD3δɛ–TCRαβ–CD3γɛ trimolecular bonds as the structural mechanisms of the cooperativity. Our work helps explain TCR function under force and suggests strategies for engineering of TCR for immunotherapy applications.

Semaphorin3F Drives Dendritic Spine Pruning Through Rho-GTPase Signaling.

Dendritic spines of cortical pyramidal neurons are initially overproduced then remodeled substantially in the adolescent brain to achieve appropriate excitatory balance in mature circuits. Here we investigated the molecular mechanism of developmental spine pruning by Semaphorin 3F (Sema3F) and its holoreceptor complex, which consists of immunoglobulin-class adhesion molecule NrCAM, Neuropilin-2 (Npn2), and PlexinA3 (PlexA3) signaling subunits. Structure-function studies of the NrCAM-Npn2 interface showed that NrCAM stabilizes binding between Npn2 and PlexA3 necessary for Sema3F-induced spine pruning. Using a mouse neuronal culture system, we identified a dual signaling pathway for Sema3F-induced pruning, which involves activation of Tiam1-Rac1-PAK1-3 -LIMK1/2-Cofilin1 and RhoA-ROCK1/2-Myosin II in dendritic spines. Inhibitors of actin remodeling impaired spine collapse in the cortical neurons. Elucidation of these pathways expands our understanding of critical events that sculpt neuronal networks and may provide insight into how interruptions to these pathways could lead to spine dysgenesis in diseases such as autism, bipolar disorder, and schizophrenia.