Cbl Family Research Articles

Discovery, Optimization, and Biological Evaluation of Arylpyridones as Cbl-b Inhibitors.

Casitas B-lymphoma proto-oncogene-b (Cbl-b), a member of the Cbl family of RING finger E3 ubiquitin ligases, has been demonstrated to play a central role in regulating effector T-cell function. Multiple studies using gene-targeting approaches have provided direct evidence that Cbl-b negatively regulates T, B, and NK cell activation via a ubiquitin-mediated protein modulation. Thus, inhibition of Cbl-b ligase activity can lead to immune activation and has therapeutic potential in immuno-oncology. Herein, we describe the discovery and optimization of an arylpyridone series as Cbl-b inhibitors by structure-based drug discovery to afford compound 31. This compound binds to Cbl-b with an IC50 value of 30 nM and induces IL-2 production in T-cells with an EC50 value of 230 nM. Compound 31 also shows robust intracellular target engagement demonstrated through inhibition of Cbl-b autoubiquitination, inhibition of ubiquitin transfer to ZAP70, and the cellular modulation of phosphorylation of a downstream signal within the TCR axis.

Role of the E3 Ubiquitin Ligase CBL Family in Cardiovascular Diseases

Role of the E3 Ubiquitin Ligase CBL Family in Cardiovascular Diseases

B-363 Solid Lipid Nanoparticles Mediated CBLC siRNA Delivery as a Platform to Monitor and Probe Biological Functions

Abstract Background Nanoparticles have been used as a tool for delivering therapeutics since few decades. Researchers has been focusing on advances and modifications to improve the properties of these nanoparticles such as sustained drug delivery and bioavailability. This study focuses on modifying the conventional solid lipid nanoparticles (SLN) with Arginine-Glycine-Aspartic acid tripeptide (RGD). The peptidyl link in these SLN has an ability to bind to the integrin of the target cells, allowing solid adhesion to the cells for sustained drug delivery. The use of small interfering RNA (siRNA) allow better invasion through the cell membranes, silencing the intracellular targets. This study uses Casistas B-lymphoma lineage (CBLC) siRNA as the therapeutics for the treatment of non-small cell lung cancer (NSCLC). CBL family consisting of different proteins such as CBL, CBLB, and CBLC origins from a RING finger class. They are E3 ubiquitin ligases responsible for transferring ubiquitin from E2 to E3 substrate. They function as the negative regulator of Tyrosine Kinase activity in cell cycle signaling. CBLC is abundantly present in epithelial cells and so its expression is noteworthy in lung adenocarcinoma. CBL family also possess Tyrosine Kinase Binding domain (TKB) responsible to decrease the activated Epidermal Growth Factor Receptor (aEGFR). Although, CBLC was initially known to perform the same function as other CBL family members, reports suggest that it is a demethylated target. The expression of CBLC upregulates with the treatment of NSCLC by inhibiting agents for DNA methylation. This process increases the demethylation of the CBLC promoter upregulating CBLC. This causes CBLC to compete with CBL for binding to the aEGFR leading to polyubiquitination of aEGFR. Trafficking of aEGFR in the nucleus makes it more stable progressing lung cancer. CBLC silencing allows tumor cells to be sensitized to the therapy with tyrosine kinase inhibiting agents. Methods SLN were modified with RGDP followed by the loading of CBLC siRNA. To characterize the SLN within the diameter range of 20–100 nm, Scanning Electron Microscopy (SEM) was performed. The Energy-dispersive X-ray Spectroscopy was performed to confirm the presence of Phosphorous and Nitrogen in SLN coming from lipids and RNA. Dynamic Light Scattering (DLS) was done to verify the SEM results. To measure the efficacy of the SLN, the cells were treated with control SLN vs siRNA loaded SLN. The Sybr gold assay and invitro release study was done to confirm the release of siRNA from SLN. The human lung normal cell line NL-20 as control and lung cancer cell line A549 were cultured and treated with synthesized nanoparticles for knocking down CBLC gene. The assays such as MTT, western blot, flow cytometry and mass spectrometry-based proteomics were performed to confirm the knockdown of the CBLC gene. Results The SLN characterization confirmed the diameter in the expected range. The efficacy assays confirmed the encapsulation and release of CBLC siRNA. The results from cell studies showed reduction in tumor growth and viability confirming the knockdown of CBLC gene. Conclusion The data suggests significant effects for the use of synthesized nanoparticles as a therapeutic for the treatment of NSCLC.

CBL Syndrome: At the Intersection of Inborn Error of Immunity and Cancer Predisposition

CBL syndrome is a form of RASopathies due to heterozygous germline mutations of the Casitas B-lineage lymphoma (CBL) gene. CBL encodes the cbl family of proteins which function as E3 ubiquitin ligases that negatively regulate intracellular signaling by various receptor tyrosine kinases including RAS-MAPK pathway. Individuals with CBL syndrome have variable clinical presentation but phenotypically resembles Noonan syndrome with predisposition to JMML. Patients affected by RASopathies are at risk to develop autoimmune disorders given the role of Ras-MAPK signaling pathway in peripheral tolerance. Prior mice studies suggested the role of cbl protein as negative regulator of TCR-mediated signaling. We describe a case of CBL syndrome presenting with multiple autoimmune conditions and reviewed reported cases in literature.A 14-year old male presented with pancytopenia, autoimmune thyroiditis and organomegaly. His past medical history was remarkable for ADHD, speech delay, short stature, idiopathic hepatosplenomegaly and history of acute disseminated encephalomyelitis at age 5. His bone marrow evaluation was normal. A somatic NGS panel identified VUS in CBL (c.1193A>G, p.H398R) with VAF of 53%. Autoimmune work up showed elevated ANA, DNA antibody, RNP antibody titer and mildly positive anti-C3 antibody. Immunophenotyping revealed normal Ig profile, NK cytopenia, T cell lymphopenia, decreased naïve T cells and elevated CD8+ effector memory T cell activation. ALPS genetic panel revealed paternally inherited VUS in CTLA4 which was later determined to be benign. Since his cytopenias did not respond to IVIG, he was treated with corticosteroid with improvement in counts although he remained thrombocytopenic. A skin biopsy was performed and sequencing of patient’s fibroblast DNA demonstrated a germline origin of CBL variant. Parental DNA sequencing confirmed a de novo event. Literature review revealed 28 germline CBL mutations (Figure 1), most of which were missense mutations (78%). At least nine of these mutations (32%) have been associated with immune dysregulation including autoimmune cytopenias, aHUS, RA, autoimmune thyroiditis, vasculitis and HLH. JMML has been reported in more than half of these germline mutations. [Display omitted] In conclusion, immune dysregulation is a common feature of CBL syndrome in addition to JMML. CBL syndrome should be considered in the differential diagnosis of individuals with ALPS-like phenotypes.

The E3 Ubiquitin Ligase TRIM21 Regulates Basal Levels of PDGFRβ.

Activation of platelet-derived growth factor (PDGF) receptors α and β (PDGFRα and PDGFRβ) at the cell surface by binding of PDGF isoforms leads to internalization of receptors, which affects the amplitude and kinetics of signaling. Ubiquitination of PDGF receptors in response to ligand stimulation is mediated by the Casitas b-lineage lymphoma (Cbl) family of ubiquitin ligases, promoting internalization and serving as a sorting signal for vesicular trafficking of receptors. We report here that another E3 ligase, i.e., tripartite motif-containing protein 21 (TRIM21), contributes to the ubiquitination of PDGFRβ in human primary fibroblasts AG1523 and the osteosarcoma cell line U2OS and regulates basal levels of PDGFRβ. We found that siRNA-mediated depletion of TRIM21 led to decreased ubiquitination of PDGFRβ in response to PDGF-BB stimulation, while internalization from the cell surface and the rate of ligand-induced degradation of the receptor were not affected. Moreover, induction of TRIM21 decreased the levels of PDGFRβ in serum-starved cells, and even more in growing cells, in the absence of PDGF stimulation. Consistently, siRNA knockdown of TRIM21 caused accumulation of the total amount of PDGFRβ, both in the cytoplasm and on the cell surface, without affecting mRNA levels of the receptor. We conclude that TRIM21 acts post-translationally and maintains basal levels of PDGFRβ, thus suggesting that ubiquitination of PDGFRβ by TRIM21 may direct a portion of receptor for degradation in growing cells in a ligand-independent manner.

Abstract 2548: Substrate binding, not phosphorylation, activates CBL poly-ubiquitination activity

Abstract Casitas B-lineage lymphoma (CBL) is a family of RING-type E3 ligases that target proteins for degradation via the proteasomal pathway. CBL-b specifically functions as a negative effector of T cell activation by down regulating the T cell receptor, whereas c-CBL interacts with various receptor tyrosine kinases involved in cell signaling and activation, targeting them for degradation. Therefore, the CBL family regulates the biology of immune cells and targeting CBL-b or c-CBL is a potential therapeutic strategy for the treatment of cancer, infection, or autoimmune diseases. In this study, we used TR-FRET (Time-Resolved Fluorescence Resonance Energy Transfer) to uncover new mechanistic aspects of CBL activation. Our assays were designed and optimized using purified components (E1/E2/c-CBL or CBL-b E3 ligase and kinase substrate Tyro3 or Axl) to recreate the polyubiquitination cascade in vitro. The assays use a Europium cryptate-labeled Ubiquitin (donor) and a Cy5-labeled Ubiquitin (acceptor) to complete the TR-FRET pairing. Since both the TR-FRET donor and acceptor are incorporated into poly-ubiquitin chains formed on the E3 ligase, the technique measures only poly-ubiquitination and not mono-ubiquitination. This FRET-based assay is homogeneous, making it especially suitable for screening applications and real-time kinetics. Validation of the assay indicated that the presence of a kinase substrate (Tyro3 or Axl) activates CBL-b and c-CBL, which are known to display a similar mechanism of activation. Importantly, our assay design allowed us to show that known CBL-B inhibitor CBL-B-IN-1 is not strictly selective toward CBL-B but also affects c-CBL E3 ligase activity to similar extend since the IC50 of the compound for c-CBL and CBL-B differ by only 2-fold. Finally, it has been proposed that CBL-b phosphorylation at Y106, Y133, and Y363 by Tyro3 kinase is required for its activation. Indeed, in our assay, we observed that CBL-b mutation at Y363F decreases TYRO3 ubiquitination drastically. Surprisingly, phospho-mimetic mutation Y363E did not restore CBL-b-mediated ubiquitination of Tyro3, indicating that substrate binding close to Y363 is the predominant requirement for CBL activation. The weak E3 ligase activity of CBL-b Y363E mutant was insensitive to CBL-B-IN-1 treatment.In conclusion, our data indicates that no significant difference in the parameters of substrate ubiquitination exists between CBL-B and c-CBL, and that ubiquitination of receptor tyrosine kinase Tyro3 by either c-CBL or CBL-B depends on the kinase binding to CBL but not on the phosphorylation of CBL. Citation Format: Kasia Zientara-Rytter, Veronique Baron, Pavel Shashkin, Henry Zhu. Substrate binding, not phosphorylation, activates CBL poly-ubiquitination activity [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 2548.

Abstract 3474: Identification of the selective CBL-b inhibitors which effectively prevent T cells from exhaustion and demonstrate synergistic anti-tumor activity in combination with an anti-PD1 antibody

Abstract The E3 ubiquitin ligase Casitas B-lineage lymphoma b (Cbl-b) is a member of the highly conserved family of Cbl (casitas b-lineage lymphoma) proteins. CBL-b is widely expressed in immune cells and plays as a “gate keeper” to negatively regulate the activation of immune cells. Engagement of TCR/BCR triggers the Cbl-b-mediated ubiquitination and degradation of the key components in TCR axis, such as Vav1 and PLCγ, and consequently attenuates TCR/BCR signal axis which finally results in T/B cells exhaustion. Besides, Cbl-b was reported to negatively regulate the activation of dendritic cells, NK, NKT, and mast cells. Cbl-b KO mice show resistance to tumor transplantation, and the anti-tumor activities depend on the T and NK cells. According to the above observation, Cbl-b emerges as a novel intracellular immune checkpoint target to prevent immune cells from exhaustion and improve the antitumor immunotherapy. Here we identified a series of highly potent Cbl-b inhibitors. The inhibition of Cbl-b protein was evaluated by an in vitro CBL-b/E2-ubiquitin assay. Results showed that our candidates strongly inhibited Cbl-b with IC50s at single-digital nanomolar level, and meanwhile, these compounds displayed over than 10-fold selectivity against c-CBL/E2-ubiquitin, a member of the Cbl family, which was reported to synergistically enhance toxicity caused by Cbl-b. Consistent with the Cbl-b inhibition, these compounds further enhanced activation of Jurkat T cells as well as human PBMCs with dramatical increase of IL-2 or IFNɣ secretion in presence of anti-CD3/CD28, and without any significant cytotoxicity even up to 10 µM concertation. Furthermore, these compounds exhibited the restoring function in exhausted T cells in presence with PEG2 or adenosine treatment. Favorable ADME and bioavailability supported the oral administration. In naïve mice, our inhibitors dose-dependently promoted the cytokine IL-2 and IFNɣ production in serum and spleens following the anti-CD3 challenge. In a syngeneic model, CT26, oral administration of our Cbl-b inhibitor effectively inhibited tumor growth, while completely inhibited tumor growth in combination with an anti-PD1 antibody. Further study demonstrated that the increased lymphocytes infiltration and cytokines release in tumor tissues were observed either in Cbl-b mono or in anti-PD1 combo groups, which correlated well with anti-tumor efficacy. In conclusion, our selective Cbl-b inhibitors demonstrated robust immune activation and anti-tumor activity in vitro and in vivo and showed synergistic activities with immune checkpoint inhibitors, such as anti-PD(L)1. Currently, one of our leading molecules is in IND enabling studies. Citation Format: Feng Zhou, Zhengtao Li, Yajing Liu, Guimei Yang, Wansun Mai, Wenqing Yang, Liting Xue, Ping Chen, Renhong Tang. Identification of the selective CBL-b inhibitors which effectively prevent T cells from exhaustion and demonstrate synergistic anti-tumor activity in combination with an anti-PD1 antibody [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 3474.

Genome-wide identification of CBL family genes in Nicotiana tabacum and the functional analysis of NtCBL4A-1 under salt stress

The CALCINEURIN B-LIKE PROTEIN family genes (CBLs) encode a group of plant-specific calcium sensor proteins that play critical roles in plant development and stress response. Although genome-wide analysis of CBL family genes has been conducted in several diploid plant species, less is known about their functions in allopolyploid plant species. This study conducted a detailed analysis of tobacco CBL family genes. Of the 24 NtCBL genes identified, 14 were derived from the maternal genome donor N. sylvestris, and 10 were from the paternal genome donor N. tomentosiformis. Phylogenetic analysis revealed that NtCBLs could be clustered into three different groups with distinct N-terminal characteristics. All NtCBLs contain EF-hand motifs, but amino acid substitutions and configuration changes may be responsible for variations in their Ca2+-binding abilities. Alternative splicing is common for NtCBL gene transcripts, which may contribute to variations in translation efficiency or protein interaction preferences. Several NtCBL genes exhibited shoot- or root-predominant expression patterns, and some were responsive to salt and/or drought stress. NtCBL4A-1, an ortholog of Arabidopsis thaliana AtCBL4 (SOS3), was predominantly expressed in roots and exhibited a lower gene expression level under salt stress. Further functional analysis showed that overexpression of NtCBL4A-1 increased the sensitivity of transgenic tobacco to Na+-induced salt stress, which is inconsistent with the role of AtCBL4 in contributing to salt tolerance. This study provides data for identifying CBL gene functions and selecting candidate stress-tolerant genes in N. tabacum. Additionally, the results may aid gene function studies in other allopolyploid plant species.

The Peach (Prunus persica) CBL and CIPK Family Genes: Protein Interaction Profiling and Expression Analysis in Response to Various Abiotic Stresses.

The plant calcineurin B-like protein-CBL interacting protein kinase (CBL-CIPK) signaling pathway is a Ca2+-related signaling pathway that responds strongly to both biological and abiotic environmental stimuli. This study identified eight CBL and eighteen CIPK genes from peach for the first time. Their basic properties and gene structure were analyzed, and the CBL and CIPK members from Arabidopsis and apple were combined to study their evolutionary relationships. Using RT-qPCR and RNA-seq data, we detected the expression patterns of PprCBLs and PprCIPKs in different tissues and fruit development stages of peach. Among them, the expression levels of PprCBL1 and PprCIPK18 were stable in various tissues and stages. The expression patterns of other members showed specificity between cultivars and developmental stages. By treating shoots with drought and salt stress simulated using PEG6000 and NaCl, it was found that PprCIPK3, PprCIPK6, PprCIPK15 and PprCIPK16 were strongly responsive to salt stress, and PprCIPK3, PprCIPK4, PprCIPK10, PprCIPK14, PprCIPK15, PprCIPK16 and PprCIPK18 were sensitive to drought stress. Three genes, PprCIPK3, PprCIPK15 and PprCIPK16, were sensitive to both salt and drought stress. We cloned four PprCBL and several PprCIPK genes and detected their interaction by yeast two-hybrid assay (Y2H). The results of Y2H show not only the evolutionary conservation of the interaction network of CBL-CIPK but also the specificity among different species. In conclusion, CBL and CIPK genes are important in peach and play an important role in the response to various abiotic stresses.

Evolution, gene expression, and protein‒protein interaction analyses identify candidate CBL-CIPK signalling networks implicated in stress responses to cold and bacterial infection in citrus

BackgroundCold is a major abiotic stress and Huanglongbing and citrus canker disease are two devastating bacterial diseases for citrus. The Ca2+-CBL-CIPK network is known to regulate different types of stress signalling in plants. How do CBL–CIPK signalling networks function in response to cold and infection by CLas or Xcc in citrus?ResultsEight calcineurin B-like proteins (CBLs) and seventeen CBL-interacting protein kinases (CIPKs) were identified from the cold-tolerant satsuma mandarin ‘Guijing2501’ (Citrus. unshiu) and CLas/Xcc-sensitive sweet orange (C. sinensis). Phylogenetic analysis revealed that both CBL and CIPK family members in citrus were classified into an ancient and a recent clade according to their conserved domain characteristics and/or intron/exon structures. Genome duplication analysis suggested that both tandem and segmental duplications contributed to the amplification of the CBL and CIPK gene families in citrus under intense purifying selection, and the duplication events only existed in the recent clades. Expression comparison of the duplicated gene pairs indicated that the duplicated CBL and CIPK genes underwent functional differentiation. Further expression analysis identified that CBL1, 5, 6, and 8 and CIPK2, 8, 12, 15, 16, and 17 were significantly regulated by multiple stresses, including cold, Xcc infection and/or CLas infection, in citrus, whereas CBL2/7 and CIPK1/4/5/11/13/14 were independently highly regulated by cold and CIPK3 was uniquely responsive to Xcc infection. The combination analyses of targeted Y2H assay and expression analysis revealed that CBL6-CIPK8 was the common signalling network in response to cold and Xcc infection, while CBL6/CBL8-CIPK14 was uniquely responsive to cold in citrus. Further stable transformation and cold tolerance assay indicated that overexpression of CuCIPK16 enhanced the cold tolerance of transgenic Arabidopsis with higher POD activity and lower MDA content.ConclusionsIn this study, evolution, gene expression and protein‒protein interaction analyses of citrus CBLs and CIPKs were comprehensively conducted over a genome-wide range. The results will facilitate future functional characterization of individual citrus CBLs and CIPKs under specific stresses and provide clues for the clarification of cold tolerance and disease susceptibility mechanisms in corresponding citrus cultivars.

Negative regulation of receptor tyrosine kinases by ubiquitination: Key roles of the Cbl family of E3 ubiquitin ligases.

Receptor tyrosine kinases (RTKs) serve as transmembrane receptors that participate in a broad spectrum of cellular processes including cellular growth, motility, differentiation, proliferation, and metabolism. Hence, elucidating the regulatory mechanisms of RTKs involved in an assortment of diseases such as cancers attracts increasing interest from researchers. Members of the Cbl family ubiquitin ligases (c-Cbl, Cbl-b and Cbl-c in mammals) have emerged as negative regulators of activated RTKs. Upon activation of RTKs by growth factors, Cbl binds to RTKs via its tyrosine kinase binding (TKB) domain and targets them for ubiquitination, thus facilitating their degradation and negative regulation of RTK signaling. RTKs such as epidermal growth factor receptor (EGFR), platelet-derived growth factor receptor (PDGF), fibroblast growth factor receptor (FGFR) and hepatocyte growth factor receptor (HGFR) undergo ubiquitination upon interaction with Cbl family members. In this review, we summarize the current knowledge related to the negative regulation of RTKs by Cbl family proteins.

Ablation of Cbl-b and c-Cbl in dendritic cells causes spontaneous liver cirrhosis via altering multiple properties of CD103+ cDC1s

The Casitas B-lineage lymphoma (Cbl) family proteins are E3 ubiquitin ligases implicated in the regulation of various immune cells. However, their function in dendritic cells (DCs) remains unclear. To investigate the role of Cbl family members in DCs, we created dendritic cell double-deficient Casitas B lymphoma-b (Cbl-b) and Casitas B lineage lymphoma (c-Cbl) mice by crossing Cbl-b−/− mice with c-Cblflox/flox CD11c-Cre+ mice. We found that specific deletion of Cbl-b and c-Cbl in CD11c+ cells, predominantly in DCs, led to liver fibrosis, cirrhosis, and accumulation of systemic conventional Type I DCs (cDC1s) due to enhanced cell proliferation and decreased cell apoptosis. In addition to a change in DC number, double knockout (dKO) cDC1s exhibited a partially activated status as indicated by high basal expression levels of certain cytokines and possessed an enhanced capacity to prime T cells. After adoptive transfer, dKO cDC1s could drive liver fibrosis too. In further experiments, we demonstrated that Cbl-b and c-Cbl could target signal transducer and activator of transcription 5 (STAT5), a transcriptional repressor for the pro-apoptotic protein Bim, to promote ubiquitination-mediated degradation and cell apoptosis in cDC1s. Further extensive experiments revealed that Cbl-b mediated K27-linked ubiquitination of lysine 164 of STAT5a while c-Cbl induced K29-linked ubiquitination of lysine 696 of STAT5a and K27-linked ubiquitination of lysine 140 and 694 of STAT5b. Thus, our findings indicate a functional redundancy of Cbl-b and c-Cbl in cDC homeostasis and maturation.

EP289: Non-cirrhotic portal hypertension: Expanding the phenotype of CBL syndrome-related vasculopathy

CBL syndrome is a form of “RASopathies”, a class of developmental disorders caused by germline variants in genes that encode components of the RAS signaling pathway. CBL is a tumor suppressor gene that encodes the cbl family of proteins which function as E3 ubiquitin ligases that negatively regulate intracellular signaling by various receptor tyrosine kinases. RAS/MAPK pathway is central to the cellular function of cell survival, proliferation, and differentiation as well as senescence in response to extracellular stimuli and modulates developmental processes.

Stabilization of AURKA by the E3 ubiquitin ligase CBLC in lung adenocarcinoma.

CBL family proteins (CBL, CBLB and CBLC in mammals) are E3 ubiquitin ligases of protein tyrosine kinases. CBL mediates the lysosomal degradation of activated EGFR through K63-linked ubiquitination, while CBLC has an oncogenic function by positively regulating EGFR activation through K6 and K11-linked ubiquitination in EGFR mutant lung adenocarcinoma (LAD). Here, we used immunoprecipitation and mass spectrometry to study the CBLC interactome, and found that CBLC is also involved in cell cycle regulation by stabilizing Aurora kinase A (AURKA). CBLC interacted with the kinase domain of AURKA and positively regulated the stability of AURKA by conjugating monoubiquitination and K11/K63-linked polyubiquitination, which are protective from degrading K11/K48 polyubiquitination. CBLC depletion markedly decreased the half-life of AURKA in cycloheximide-treated LAD cells. When LAD cells were synchronized with double thymidine block at the G1/S boundary and then released into mitotic arrest, CBLC depletion delayed the accumulation and activation of AURKA and prevented cancer cells from entering mitosis. CBLC deficiency significantly delayed cell cycle progression, reduced the mitotic population, and increased apoptosis of LAD cells. Targeting CBLC inhibited tumor growth of LAD cells and enhanced their sensitivity to paclitaxel in xenograft models. Immunohistochemical staining of the tissue microarray also revealed a positive correlation between the expression of CBLC and AURKA in normal and LAD tissues, further supporting the positive regulation of AURKA expression by CBLC. In summary, these findings indicate that the oncogenic E3 ligase CBLC plays a role in mitotic entry by stabilizing AURKA via ubiquitination in LAD. This work demonstrates that targeting CBLC combined with paclitaxel might be a potential option for the treatment of LAD patients who have no available targeted therapies.

The deficiency of Cbl family in human elicits heightened T-dependent immune activation with augmentation of TFH responses

Abstract T follicular helper (TFH) cells are functional subset CD4+ T cells in providing help to B cells and aberrant expansion of TFH cells is observed in systemic lupus erythematosus (SLE), which suggests a global defect in the maintenance of T and B cell tolerance. The mechanisms underlying uncontrolled TFH cells development and differentiation in SLE pathogenesis is fragmentary understand. Casitas B-lineage lymphoma (Cbl) family is an E3 ubiquitin ligase and has been implicated in SLE pathogenesis as a potent regulator in activating the immune cells and maintaining peripheral tolerance. The deficiency of cbl-b mice spontaneously develops autoimmunity and is highly susceptible to develop pathogenic auto-antibodies of autoimmune diseases. Through CRISPR KO system, we identified the deficiency of cbl/cbl-b in human naïve CD4+ T cells induces the TFH expansion in T cell stimulation with Activin A & IL12. Indeed, higher levels of chemokine (C-X-C motif) ligand 13 (CXCL13) which is constitutively produced in germinal center (GC) TFH cells were detected. Functionally, cbl/cbl-b KO TFH cells are more potent B cell activators compared to cbl/cbl-b competent TFH cells. There was increased plasmablast differentiation which secrete IgG. These data suggest that the deficiency of Cbl family in human elicits heightened T-dependent immune activation with augmentation of TFH responses, thus regulating humoral immune responses and the susceptibility to SLE in human. This project will advance our understanding of the mechanism of activation and maintenance of TFH cells in lupus and dissect possible avenues of therapeutic intervention in disease.

Genome-wide analysis of CBL and CIPK family genes in cotton: conserved structures with divergent interactions and expression.

The online version contains supplementary material available at (doi:10.1007/s12298-021-00943-1).

Targeting USP13-mediated drug tolerance increases the efficacy of EGFR inhibition of mutant EGFR in non-small cell lung cancer.

In non‐small cell lung cancer (NSCLC), activating mutations in the epidermal growth factor receptor (EGFR) induce sensitivity to EGFR tyrosine kinase inhibitors. Despite impressive clinical responses, patients ultimately relapse as a reservoir of drug‐tolerant cells persist, which ultimately leads to acquired resistance mechanisms. We performed an unbiased high‐throughput siRNA screen to identify proteins that abrogate the response of EGFR‐mutant NSCLC to EGFR‐targeted therapy. The deubiquitinase USP13 was a top hit resulting from this screen. Targeting USP13 increases the sensitivity to EGFR inhibition with small molecules in vitro and in vivo. USP13 selectively stabilizes mutant EGFR in a peptidase‐independent manner by counteracting the action of members of the Cbl family of E3 ubiquitin ligases. We conclude that USP13 is a strong mutant EGFR‐specific cotarget that could improve the treatment efficacy of EGFR‐targeted therapies.

A novel role of the calcium sensor CBL1 in response to phosphate deficiency in Arabidopsis thaliana

Phosphorus acts as an essential macroelement in plant growth and development. A lack of phosphate (Pi) in arable soil and phosphate fertilizer resources is a vital limiting factor in crop yields. Calcineurin B-like proteins (CBLs) act as one of the most important calcium sensors in plants; however, whether CBLs are involved in Pi deficiency signaling pathway remains largely elusive. In this study, we utilized a reverse genetic strategy to screen Arabidopsis thaliana T-DNA insertion mutants belonging to the CBL family under Pi deficiency conditions. The cbl1 mutant exhibited a relatively tolerant phenotype, with longer roots, lower anthocyanin content, and elevated Pi content under Pi deficiency, and a more sensitive phenotype to arsenate treatment compared with wild-type plants. Moreover, CBL1 was upregulated, and the mutation of CBL1 caused phosphate starvation-induced (PSIs) genes to be significantly induced under Pi deficiency. Histochemical staining demonstrated that the cbl1 mutant has decreased acid phosphatase activity and hydrogen peroxide concentrations under Pi deficiency. Collectively, our results have revealed a novel role of CBL1 in maintaining Pi homeostasis.

Cbls boost B cells.

T cell regulation of antibody-mediated immunity is critical for health. In this issue of JEM, Li et al. (https://doi.org/10.1084/jem.20191537) identify the Cbl family of E3 ubiquitin ligases as B cell-intrinsic gatekeepers of T cell-dependent humoral immunity.

Abstract 4852: CBLC functions as a novel therapeutic target to enhance the efficacy of paclitaxel on EGFR wild-type lung adenocarcinoma by downregulating AURKA

Abstract Patients with lung adenocarcinoma harboring driver genes can be treated with target therapies, such as tyrosine kinase inhibitors for epithelial growth factor receptor (EGFR) mutations, anaplastic lymphoma kinase (ALK) inhibitors for ALK gene rearrangements, and so on. However, a large portion of lung adenocarcinoma patients, especially for those with wild-type EGFR, still do not have valid target therapies in clinics. The CBL family proteins (CBL, CBLB, and CBLC) are E3 ubiquitin ligases of protein tyrosine kinases (PTKs), including EGFR, hepatocyte growth factor receptor (MET), and SRC proto-oncogene (SRC). It has been reported that the K63-linkaged ubiquitination mediated by CBL can negatively regulate activated EGFR (aEGFR) signaling through lysosomal degradation. Previously, we discovered that CBLC, unlike CBL, possesses an oncogenic function and positively regulates aEGFR stability through polyubiquitination via K6 and K11 linkages. The CBLC-mediated conjugation of polyubiquitin promotes aEGFR preferentially recycled back to the plasma membrane, or trafficked to the cell nucleus, leading to the sustained activation of EGFR signaling in lung adenocarcinoma. Aside from aEGFR dysregulation, we recently found that CBLC assumes a novel role in positively regulating the expression of Aurora kinase A (AURKA), which contributes to tumor development by promoting cell proliferation, metastasis, epithelial-mesenchymal transition, and drug resistance. In lung adenocarcinoma cells with wild-type EGFR, CBLC knockdown significantly reduced cell viability, delayed cell cycle progression and increased apoptotic death. Our study also showed that CBLC knockdown in EGFR wild type cells was associated with increased sensitivity to paclitaxel, probably through downregulating AURKA. Taken together, these findings indicated that CBLC may play a role in promoting cell cycle progression and may be a novel therapeutic target to enhance the efficacy of paclitaxel on EGFR wild-type lung adenocarcinoma. Citation Format: Shiao-Ya Hong, Yu-Rung Kao, Yi-Ping Lin, Meng-Hsuan Lee, Jyun-Yi Wu, Cheng-Wen Wu. CBLC functions as a novel therapeutic target to enhance the efficacy of paclitaxel on EGFR wild-type lung adenocarcinoma by downregulating AURKA [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4852.