Casitas B-lineage Lymphoma Research Articles

BLNK negatively regulates innate antifungal immunity through inhibiting c-Cbl-mediated macrophage migration

B cell linker protein (BLNK) is crucial for orchestrating B cell receptor-associated spleen tyrosine kinase (Syk) signaling. However, the role of BLNK in Syk-coupled C-type lectin receptor (CLR) signaling in macrophages remains unclear. Here, we delineate that CLRs govern the Syk-mediated activation of BLNK, thereby impeding macrophage migration by disrupting podosome ring formation upon stimulation with fungal β-glucans or α-mannans. Mechanistically, BLNK instigates its association with casitas B-lineage lymphoma (c-Cbl), competitively impeding the interaction between c-Cbl and Src-family kinase Fyn. This interference disrupts Fyn-mediated phosphorylation of c-Cbl and subsequent c-Cbl-associated F-actin assembly. Consequently, BLNK deficiency intensifies CLR-mediated recruitment of the c-Cbl/phosphatidylinositol 3-kinase complex to the F-actin cytoskeleton, thereby enhancing macrophage migration. Notably, mice with monocyte-specific BLNK deficiency exhibit heightened resistance to infection with Candida albicans, a prominent human fungal pathogen. This resistance is attributed to the increased infiltration of Ly6C+ macrophages into renal tissue. These findings unveil a previously unrecognized role of BLNK for the negative regulation of macrophage migration through inhibiting CLR-mediated podosome ring formation during fungal infections.

C-CBL/LCK/c-JUN/ETS1/CD28 axis restrains childhood asthma by suppressing Th2 differentiation

BackgroundAsthma is a common immune disease with high morbidity in children. Type 2 inflammation is the center of asthma development, and mainly mediated by a subset of CD4 + T cells, T helper 2 (Th2) cells. Excess Th2 differentiation was generally associated with asthmatic attack. Casitas B-lineage lymphoma (c-CBL) was reported to involved in T cell development and databank showed its decreased expression in CD4 + T cells from peripheral blood of asthmatic children. This study aims to investigate the role of c-CBL in childhood asthma and Th2 differentiation, and explore the underlying mechanism.MethodsWe collected peripheral blood samples from clinical childhood asthma cases and healthy controls, and determined c-CBL expression in CD4 + T cells. Asthma was induced in neonatal mice by ovalbumin (OVA) intraperitoneal injection and aerosol inhalation, and c-CBL expression in CD4 + T cells from peripheral blood and spleen was measured. Gain-of-function experiments was performed to confirm the effects of c-CBL on Th2 differentiation in vitro. Finally, c-CBL was delivered into asthmatic mice via lentivirus infection to verify its effects on experimental asthma.Resultsc-CBL was lowly expressed in CD4 + T cells from asthmatic children than those of healthy controls. Similarly, it was downregulated in CD4 + T cells from peripheral blood and spleen of asthma mice. Overexpression of c-CBL restrained lung pathological injury and type 2 inflammation in experimental asthmatic mice. Gain-of-function experiments demonstrated that c-CBL inhibited Th2 differentiation of CD4 + T cells from healthy children, and mediated the ubiquitination of lymphocyte cell-specific protein-tyrosine kinase (LCK). LCK acted as a kinase to phosphorylate and activate c-JUN, which was predicted to bind promoter sequence of CD28 by bioinformatic analysis. Dual-luciferase reporter assay verified that c-JUN and ETS1 synergically enhanced transcription of CD28, and this transcription activation was aggravated by LCK overexpression.Conclusionc-CBL alleviated asthma and suppressed Th2 differentiation by facilitating LCK ubiquitination, interrupting c-JUN activation and CD28 expression in vivo and in vitro. c-CBL/LCK/c-JUN/ETS1/CD28 axis was partially involved in childhood asthma, and may provide novel insights for clinical treatment for asthma.

YF550-C1, an E3 ligase inhibitor of CBL-B, to demonstrate T cell and NK cell activation and anti-tumor activities in syngeneic and PDX tumor model.

e14570 Background: Casitas B-lineage lymphoma b (CBL-B) is one of ligase in E3 ubiquitin family, which mainly expressed in matured hematopoietic cells, including T and NK cells. Study showed that CBL-B deficiency upregulated interleukin-2 and interferon-gamma secretion during anti-CD3 and anti-CD28 stimulated TCR activation, even without co-stimulator anti-CD28[3]. Except T cells, CBL-B plays important role in NK cells as well. CBL-B was upregulated during NK cell activation. While downregulation of CBL-B enhanced the cytotoxicity of primary human NK cells against hematological cancer cell lines. In vivo studies proved that CBL-B deficiency protected mice from tumor growth by inducing antitumor immunity. Thus, CBL-B shows a promising target for cancer therapy. Methods: We developed a small molecule YF550-C1 that targeted CBL-B, and evaluated the activation on T cell and NK cell function, and the therapeutical effect on syngeneic tumor models. The T cell activation was indicated by levels of IL-2 and IFN-γ from CBL-B knockout or WT CD8+ T cells since stimulated with anti-CD3 or anti-CD3/28 for 48 hours. The resistance of PGE2-induced immunosuppressive was evaluated by measuring IL-2 and IFN-γ since human T cells treated with PGE2 and followed by series diluted YF550-C1 or comparable inhibitor cpd23. OVA specific antigen stimulated CD8+ T activation assay was conducted by coculture OT-1 CD8+ T cells and WT DC cells with 50:1 for 72 hours in the present of 5ug/ml OVA peptide and series diluted YF550-C1. The NK cell cytotoxicity was evaluated by co-culture human NK cells and K562-luc cells with 1.25:1 ratio in the present of 1000nM or 333nM YF550-C1, which indicated by luminance. Syngeneic mouse model that inoculated with different types of murine cancer cell lines was used to evaluate tumor growth. Results: CBL-B deficiency suppressed tumor growth in mouse tumor models and enhanced cytokines release in T cells upon TCR engagement. CBL-B inhibitor, YF550-C1, alleviated PGE2-mediated immunosuppression of human T cell activation. CBL-B inhibitor, YF550-C1, enhanced OVA-specific antigen stimulated CD8+ T cell activation. CBL-B inhibitor, YF550-C1, enhanced cytotoxicity of human NK cell against K562-luc cells. CBL-B inhibitor, YF550-C1, or in combinate with anti-PD-L1 suppressed tumor growth in syngeneic tumor models. Single-agent of CBL-B inhibitor YF550-C1 treatment was sufficient to suppress PD1/PD-L1 resistant NSCLC patient-derived tumor growth. Conclusions: These studies provided insight into the activity of the inhibitor of CBL-B, demonstrating that YF550-C1 displays excellently T cell activation, T cell immunosuppression alleviation, NK cell activation and antitumor activity in multiple preclinical tumor models. Our data showed that YF550-C1 is a promising candidate for cancer treatment as monotherapy or in combination with PD-L1 blockade.

Optimization of a Novel DEL Hit That Binds in the Cbl-b SH2 Domain and Blocks Substrate Binding.

We were attracted to the therapeutic potential of inhibiting Casitas B-lineage lymphoma proto-oncogene-b (Cbl-b), a RING E3 ligase that plays a critical role in regulating the activation of T cells. However, given that only protein-protein interactions were involved, it was unclear whether inhibition by a small molecule would be a viable approach. After screening an ∼6 billion member DNA-encoded library (DEL) using activated Cbl-b, we identified compound 1 as a hit for which the cis-isomer (2) was confirmed by biochemical and surface plasmon resonance (SPR) assays. Our hit optimization effort was greatly accelerated when we obtained a cocrystal structure of 2 with Cbl-b, which demonstrated induced binding at the substrate binding site, namely, the Src homology-2 (SH2) domain. This was quite noteworthy given that there are few reports of small molecule inhibitors that bind to SH2 domains and block protein-protein interactions. Structure- and property-guided optimization led to compound 27, which demonstrated measurable cell activity, albeit only at high concentrations.

Deficiency of CBL and CBLB ubiquitin ligases leads to hyper T follicular helper cell responses and lupus by reducing BCL6 degradation

Recent evidence reveals hyper T follicular helper (Tfh) cell responses in systemic lupus erythematosus (SLE); however, molecular mechanisms responsible for hyper Tfh cell responses and whether they cause SLE are unclear. We found that SLE patients downregulated both ubiquitin ligases, casitas B-lineage lymphoma (CBL) and CBLB (CBLs), in CD4+ Tcells. Tcell-specific CBLs-deficient mice developed hyper Tfh cell responses and SLE, whereas blockade of Tfh cell development in the mutant mice was sufficient to prevent SLE. ICOS was upregulated in SLE Tfh cells, whose signaling increased BCL6 by attenuating BCL6 degradation via chaperone-mediated autophagy (CMA). Conversely, CBLs restrained BCL6 expression by ubiquitinating ICOS. Blockade of BCL6 degradation was sufficient to enhance Tfh cell responses. Thus, the compromised expression of CBLs is a prevalent risk trait shared by SLE patients and causative to hyper Tfh cell responses and SLE. The ICOS-CBLs axis may be a target to treat SLE.

Abstract 2116: Telisotuzumab vedotin (Teliso-V)-mediated c-Met internalization, endolysosomal trafficking, and potent cytotoxic activity are unaffected by MET exon-14 skipping mutation

Abstract Background: Telisotuzumab vedotin (Teliso-V) is an antibody-drug conjugate (ADC) composed of a c-Met (MET protein) antibody coupled to the microtubule inhibitor monomethyl auristatin E (MMAE). Teliso-V was granted FDA Breakthrough Therapy Designation for advanced/metastatic EGFR WT, NSQ NSCLC with high c-MET overexpression. For MMAE drug delivery, Teliso-V must first undergo receptor internalization and endolysosomal (EL) trafficking. Hepatocyte growth factor (HGF)-dependent MET EL trafficking and degradation depend on the E3 ubiquitin ligase Casitas B-lineage lymphoma (CBL). METex14 skipping and CBL-binding site mutations, which delete the binding site for CBL, occur in ~ 3% of NSCLC. The role of CBL in Teliso-V-MMAE delivery and activity was assessed in this work. Methods: WT-human c-Met, c-Met-Y1003F/N (CBL binding site mutations in MET), and c-Met-ex14∆ (47 aa deletion to mimic ex14 skipping) or CBL-L620f, G807*, and W802* (CBL inactivating mutants) were expressed in NIH3T3, Ba/F3 cells, or A549 tumor cells using retroviral transduction. Genetically modified cells were then used to define the role of CBL in c-Met surface expression, intracellular signaling, EL trafficking (using pHrodo Red-labeled Teliso-V), and Teliso-V sensitivity in 2D/3D cell cultures. Results: c-Met expression was required for Teliso-V cytotoxicity and intracellular payload delivery in all cell lines. While minimal changes in cell surface c-Met levels were observed, CBL binding mutants had increased phosphorylated c-Met in the absence of HGF, and ex14∆-MET and CBL binding site mutants showed increased sensitivity to small molecule c-Met inhibitors. Compared to WT c-Met, ex14∆ and CBL-binding mutants showed similar EL trafficking and no change in sensitivity to Teliso-V. Co-treatment with a pan-CBL inhibitor also failed to alter Teliso-V EL trafficking and anti-tumor activity. Like most ADCs, EL trafficking was dependent on a dynamin-mediated process with reduced EL trafficking by co-treatment with Dyngo4a. To further understand the role of CBL in Teliso-V sensitivity, we expressed CBL inactivating mutations in A549 tumor cells. While the L602F and W802* mutants increased spheroid size, as well as activated p-MET, there was no change in sensitivity to Teliso-V. Conclusions: Loss of CBL-binding sites on c-Met via ex14 deletion or Y1003F/N leads to increased MET-targeted TKI sensitivity and increased phospho-c-Met levels but does not impact Teliso-V sensitivity or EL trafficking. Our results indicate that Teliso-V traffics to the EL via a dynamin-mediated process. Therefore, Teliso-V delivers MMAE through a robust ADC-mediated EL-dependent mechanism that is independent of CBL, suggesting that high c-Met expression in Ex14skipping or CBL binding site mutant NSCLC patients should be further investigated for response to Teliso-V. Citation Format: Izhar Batth, Ronald Pandoy, Shamim Khaja, Dolonchampa Maji, Tamar Uziel, Athan Vasilopoulos, Peter Ansell, PK Epling-Burnette. Telisotuzumab vedotin (Teliso-V)-mediated c-Met internalization, endolysosomal trafficking, and potent cytotoxic activity are unaffected by MET exon-14 skipping mutation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2116.

Abstract 661: An oral Cbl-b inhibitor with sustained T cell activation demonstrated robust anti-tumor efficacy along with enhanced infiltration and activation of functional T cells

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, which associates with multi-immune response regulations including Teff and NK activation, Treg differentiation. Cbl-b KO mice showed enhanced immune responses and resistance to tumor transplantation. Previously, we reported a novel Cbl-b inhibitor, ZM-8026, which robustly activated T cells and efficiently inhibited tumor growth as monotherapy or in combination with anti-PD-1. Here, we further demonstrated that Cbl-b inhibition restores the Teff cells' function in immune suppression conditions, such as in the presence of PGE2, A2AR agonist, and TGF-β. CD8+T proliferation and cytokines induction were maintained in the presence of MDSC. Furthermore, CD8+T function was reversed from an exhausting status induced by a continuous anti-CD3/CD28 activation for a long period. To investigate whether continuous target occupancy was necessary for sustained TCR activation, the dynamic impacts of Cbl-b inhibition on CD8+T cell activation were explored using a washout assay. The results showed that the high-level activated T cells lasted for 48 h after more than 4hs preincubation with 1 µM Cbl-b inhibitor followed by a washout, however, short and weak activation states were found with less than 4hs preincubation or low inhibitor concentration treatment (< 0.1 uM). This finding revealed sufficient inhibitor concentration and treatment time ensure high T-cell activation. Further, in vivo efficacy studies demonstrated less anti-tumor growth efficacy for intermittent dosing (Q2D, Q3D) compared with QD dosing. In vitro and in vivo pharmacokinetics studies exhibited favorable ADME profiles and bioavailability of our Cbl-b inhibitor for oral administration in mice, rats, and dogs. At least in two syngeneic models, our Cbl-b inhibitor exhibited efficient anti-tumor growth with more than 70% TGI (tumor growth inhibition). In a CT26 syngeneic model, complete tumor growth inhibition in 6 of 8 mice was found in the Cbl-b inhibitor combined with an anti-PD1 antibody. The complete regression mice were re-challenged with the same tumor cells, and no measurable tumors were found for up to 30 days, suggesting the treatment-induced immune memory. Tumor samples in mice were collected, RNAseq showed that activated functions of tumor-infiltrated T, NK, DC, and macrophage were dramatically increased in the Cbl-b inhibitor treatment group, and the T and NK activation signature genes such as Gzmb, Ifng were significantly upregulated by RT-PCR. In conclusion, our Cbl-b inhibitor exhibited a favorable PK profile in multi-preclinical animals. The sustained T cell activation in vitro was identified and robust anti-tumor growth in vivo was demonstrated with optimized dosing frequency. Citation Format: Feng Zhou, Guimei Yang, Yajing Liu, Liting Xue, Weijie Chen, Zhengtao Li, Xiaowu Liu, Jian Li, Renhong Tang. An oral Cbl-b inhibitor with sustained T cell activation demonstrated robust anti-tumor efficacy along with enhanced infiltration and activation of functional T cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 661.

E3 ubiquitin ligase CBLB regulates innate immune responses and bacterial dissemination during nontuberculous mycobacteria infection.

Nontuberculous mycobacteria (NTM) are emerging opportunistic pathogens causing pulmonary infection to fatal disseminated disease. NTM infections are steadily increasing in children and adults, and immune-compromised individuals are at a greater risk of fatal infections. The NTM disease's adverse pathology and resistance to antibiotics have further worsened the therapeutic measures. Innate immune regulators are potential targets for therapeutics to NTM, especially in a T cell-suppressed population, and many ubiquitin ligases modulate pathogenesis and innate immunity during infections, including mycobacterial infections. Here, we investigated the role of an E3 ubiquitin ligase, Casitas B-lineage lymphoma proto-oncogene B (CBLB), in immunocompromised mouse models of NTM infection. We found that CBLB is essential to prevent bacterial growth and dissemination. Cblb deficiency debilitated natural killer cells, inflammatory monocytes, and macrophages in vivo. However, Cblb deficiency in macrophages did not wane its ability to inhibit bacterial growth or production of reactive oxygen species or interferon γ production by natural killer cells in vitro. CBLB restricted NTM growth and dissemination by promoting early granuloma formation in vivo. Our study shows that CBLB bolsters innate immune responses and helps prevent the dissemination of NTM during compromised T cell immunity.

Long non-coding RNA small nucleolar RNA host gene 29 drives chronic myeloid leukemia progression via microRNA-483-3p/Casitas B-lineage Lymphoma axis-mediated activation of the phosphoinositide 3-kinase/Akt pathway.

The aberrant expression of the long non-coding RNA (lncRNA) Small Nucleolar RNA Host Gene 29 (SNHG29) has been associated with various human cancers. However, the role of SNHG29 in chronic myeloid leukemia (CML) remains elusive. Therefore, this study aimed to investigate the function of SNHG29 in CML and unveil its potential underlying mechanisms. Herein, peripheral blood samples from 44 CML patients and 17 healthy subjects were collected. The expressions of SNHG29, microRNA-483-3p (miR-483-3p), and Casitas B-lineage Lymphoma (CBL) were measured using quantitative polymerase chain reaction (qPCR) or Western Blot. Cell viability, apoptosis, and cell cycle progression were evaluated using the Cell Counting Kit-8 assay, 5-ethynyl-2'-deoxyuridine incorporation, and flow cytometry, respectively. Western Blot analysis was employed to assess protein expressions related to cellular proliferation, apoptosis, and oncogenesis. RNA immunoprecipitation and dual-luciferase reporter assays were utilized to verify the interactions among SNHG29, miR-483-3p, and CBL. SNHG29 was significantly overexpressed in both blood samples of CML patients and CML cell lines. In CML, increased expression of SNHG29 was positively correlated with clinical staging, and patients with high SNHG29 expression had poorer survival outcomes. Functionally, knocking down SNHG29 effectively inhibited CML cell proliferation and promoted apoptosis. Mechanistically, SNHG29 acted as a competing endogenous RNA for miR-483-3p to modulate CBL expression, thereby activating the Phosphoinositide 3-Kinase/Akt signaling pathway and mediating CML progression. In summary, these findings reveal that SNHG29 promotes tumorigenesis in CML, offering a potential therapeutic strategy for CML treatment.

The role of CBL family ubiquitin ligases in cancer progression and therapeutic strategies.

The CBL (Casitas B-lineage lymphoma) family, as a class of ubiquitin ligases, can regulate signal transduction and activate receptor tyrosine kinases through various tyrosine kinase-dependent pathways. There are three members of the family: c-CBL, CBL-b, and CBL-c. Numerous studies have demonstrated the important role of CBL in various cellular pathways, particularly those involved in the occurrence and progression of cancer, hematopoietic development, and regulation of T cell receptors. Therefore, the purpose of this review is to comprehensively summarize the function and regulatory role of CBL family proteins in different human tumors, as well as the progress of drug research targeting CBL family, so as to provide a broader clinical measurement strategy for the treatment of tumors.

Discovery of a Novel Benzodiazepine Series of Cbl-b Inhibitors for the Enhancement of Antitumor Immunity.

Casitas B-lineage lymphoma proto-oncogene-b (Cbl-b) is a RING finger E3 ligase that is responsible for repressing T-cell, natural killer (NK) cell, and B-cell activation. The robust antitumor activity observed in Cbl-b deficient mice arising from elevated T-cell and NK-cell activity justified our discovery effort toward Cbl-b inhibitors that might show therapeutic promise in immuno-oncology, where activation of the immune system can drive the recognition and killing of cancer cells. We undertook a high-throughput screening campaign followed by structure-enabled optimization to develop a novel benzodiazepine series of potent Cbl-b inhibitors. This series displayed nanomolar levels of biochemical potency, as well as potent T-cell activation. The functional activity of this class of Cbl-b inhibitors was further corroborated with ubiquitin-based cellular assays.

Investigation of CBL-b, c-CBL expressions in colorectal cancer patients and their relationship with clinicopathological characteristics

Introduction: Colorectal cancer (CRC) is the most common gastrointestinal cancer and a major cause of cancer-related mortality worldwide. Molecular indicators of pathogenic factors that regulate CRC, which are known as oncogenes, are desirable for response to treatment and improvement of CRC patient management. Several risk factors play a role in the development of this disease, which can occur genetically, familial, or sporadically. CRC tumorigenesis is stimulated by the proto-oncogene β-catenin (wnt/β-catenin). Casitas B-lineage lymphoma (c-Cbl) inhibits CRC tumor growth through an unknown mechanism that affects nuclear β-catenin. The current objective of this study is to evaluate the expression levels of Cbl-b and c-Cbl genes to determine if their transcripts can be used as suitable diagnostic indicators. Additionally, we aim to investigate the correlation between clinicopathological information of CRC patients and the levels of Cbl-b and c-Cbl. Materials and Methods: Quantitative Real-Time PCR (qRT-PCR) method was used to evaluate the expression levels of Cbl-b and c-Cbl in 45 colorectal tumor tissues and 45 adjacent control tissues. Furthermore, we analyzed the diagnostic power of Cbl-b and c-Cbl by plotting receiver operating characteristic (ROC) curves. Results: Our results showed that the expression levels of Cbl-b and c-Cbl were significantly increased in CRC patients compared to the adjacent control group (P<0.003, P<0.004). Analysis of clinicopathological features of CRC patients revealed that the expression of Cbl-b and c-Cbl was associated differently with TMN stage, LVI+ (P<0.0003, P<0.0001) (P<0.003, P<0.07). Conclusion: These results indicate that the levels of Cbl-b and c-Cbl may serve as potential diagnostic indicators for CRC.

Results of Phase I/II Study of Azacitidine in Combination with Quizartinib for Patients with Myelodysplastic Syndromes and Myelodysplastic/Myeloproliferative Neoplasms with FLT3 or CBL Mutations

Introduction: FMS-like tyrosine kinase 3 ( FLT3) mutations occur in approximately 1% of patients (pts) with newly diagnosed myelodysplastic syndrome (MDS) and up to 19% of pts at time of failure to hypomethylating agents (HMA). In addition, mutations in the Casitas B-lineage Lymphoma gene ( CBL) are observed in 10% of pts with MDS/MPN and up to 15% of pts with CMML. Preclinical studies suggest that CBL-mutant cells are dependent on FLT3 signaling. Here we present results of a phase I-II study of the FLT3 inhibitor quizartinib in combination with azacitidine for pts with FLT3 and CBL mutant MDS and MDS/MPN. Methods: We conducted a phase I/II clinical trial of azacitidine in combination with quizartinib for pts with MDS and MDS/MPN with detectable FLT3 and/or CBL mutations and with/without prior failure to HMA (HMA-F). The study included an initial phase I dose-escalation portion, following a 3+3 design, followed by a phase II dose expansion portion. Dose escalation included 3 dose levels of quizartinib: 30, 40 and 60 mg administered p.o daily on days 1-28 of each 28 day cycle. Therapy consisted on quizartinib (at corresponding dose level) in combination with azacitidine 75 mg/m 2/day i.v os s.c on days 1-5. The primary endpoint was to evaluate safety, tolerability and maximum tolerated dose of quizartinib. Responses were evaluated following 2006 IWG criteria. The Kaplan-Meier product-limit method was used to estimate median survival. Results: Between July 2020 and June 2023 a total of 16 pts have been treated: 12 in the phase I portion and 4 in the phase II. A total of 4 pts had MDS with excess blasts, 2 had MDS/MPN with neutrophilia and 10 had CMML. Eight (50%) pts had FLT3-ITD mutations and 8 (50%) had CBL mutations. Four (25%) pts had HMA-F one of which had received 4 lines of therapy including hematopoietic stem-cell transplant (HSCT) prior to enrollment. Based on the Molecular IPSS, 8 (50%), 3 (19%) and 5 (31%) pts had very high, high and moderate high risk disease. Among CMML pts, 3 (30%) pts had high and 7 (70%) had intermediate-2 risk by CPSS-Molecular score. In the phase I portion, 3 pts received quizartinib at dose level 1, 3 at dose level 2 and 6 at dose level 3. No dose limiting toxicities were detected during the 28-day DLT evaluation window. Dose level 2 was selected as the P2RD. Most common adverse events (AEs) were constipation (56%), fatigue (50%), insomnia (44%), anorexia (38%), cough (38%), diarrhea (38%) and arthralgia (31%). Most common grade 3-4 AEs were anemia (31%), thrombocytopenia (31%), lung infection (13%), skin infection (13%), hyponatremia (13%) and sepsis (13%). Arrythmias were observed 5 pts: atrial fibrillation (grade 2, n=2; grade 3, n=1), Mobitz type II atrioventricular block (grade 3, n=1), atrial flutter (grade 2, n=1), QTC prolongation (grade 2, n=1). The 4-week and 8-week cumulative incidences of mortality were 0%. Median number of days to cycle 2 was 30 (range 27-51). Median number of days to cycle 2 of therapy by dose level were: 30, 30 and 37 days for dose levels 1, 2 and 3, respectively (p=0.301). Dose reductions due to myelosuppression were required in 5 (31%) pts including dose reductions of azacitidine in 3 pts and of quizartinib in 3 pts. Median number of cycles was 4 (range 1-17). Median cycles to to best response was 1 (range 1-6). The overall response rate was 69% (n=11): CR in 1 (6%), mCR with HI in 2 (13%), mCR in 8 (50%) pts. FLT3 mutant pts were more likely to respond to therapy (100% vs 50%, p=0.038). Clearance (n=6, 75%) or allelic burden reductions (n=2, 25%) of FLT3-ITD mutations were observed in all FLT3 mutant pts. No clearance of CBL mutations were observed. Dynamics of absolute neutrophil count, platelets and hemoglobin during cycle 1 are shown in Figure 1A. Median response duration was 3.5 months (range 0-22 moths). With a median follow up of 19.1 months (95% CI 2.6-35.6), the median event-free survival has not been reached and the median overall survival is 17.5 months (NR vs 10.1 months in FLT3 vs CBL mutant pts, p=0.084, Figure 1B) Five (31%) pts discontinued study to proceed with HSCT at time of best response, 3 (19%) due to transformation to AML, 2 (12%) due to pt choice, 1 (6%) due to treating physician choice and 1 (6%) due to relapse. Four (25%) pts remain on study. Conclusion: Therapy with azacitidine in combination with quizartinib for pts with higher-risk MDS and MDS/MPN with FLT3 or CBL mutations has acceptable toxicity profile and is associated with promising responses mainly among FLT3-mutant pts.

A First-in-Human Phase 1 Trial of NX-1607, a First-in-Class Oral CBL-B Inhibitor, in Patients with Advanced Malignancies Including DLBCL

Introduction: Recurrent diffuse large B cell lymphoma (DLBCL) remains a high unmet medical need despite advances in treatment such as cell- and immune-mediated therapies. T cell dysfunction and emerging resistance to T cell-mediated therapies such as CAR-T and T cell engagers underscores the need for novel therapies that can enhance T cell function, counteract a suppressive tumor microenvironment (TME) and prevent tumor escape associated with low tumor antigen expression. Casitas B-lineage lymphoma proto-oncogene B (CBL-B) is an E3 ubiquitin ligase expressed in immune cells and is a master regulator of T, NK, and dendritic cell activation, collectively curtailing their anti-tumor functionality. Notably, it has been observed that, in the absence of CBL-B, T cell activation can occur even when antigen expression on tumor cells is low, suggesting a potential strategy for circumventing tumor resistance mechanisms [Stromnes et al. 2010]. NX-1607 is an oral, small molecule inhibitor of CBL-B that has been shown to enhance antigen recall, reduce T cell exhaustion and increase cytokine production upon T cell receptor stimulation, overcoming suppressive signals from the TME [Gosling et al. 2019; Rountree et al. 2021; Gallotta et al. 2022]. Moreover, preclinical studies in mouse lymphoma models have demonstrated that NX-1607 can induce robust, T cell-dependent tumor regression. By bolstering the effectiveness of inherent T- and NK-mediated anti-tumor responses and enhancing antigen recall, NX-1607 offers potential as a supportive and rejuvenating agent for CAR-T or NK cell therapies in patients with hematologic malignancies who have developed resistance. Methods: NX-1607-101 is a first-in-human, multicenter, open-label, Phase 1a/1b dose-escalation/expansion trial evaluating safety, tolerability, pharmacokinetics/pharmacodynamics (PK/PD) and preliminary anti-tumor activity of NX-1607, an oral investigational new drug, in several advanced malignancies, including DLBCL (see Figure). Key eligibility criteria: adults with selected measurable metastatic/unresectable malignancies for which standard therapy with proven clinical benefit does not exist or is no longer effective or appropriate. Eligible tumor types - see Figure. Prior treatment with checkpoint inhibitors and CAR-T therapy is allowed with protocol-defined washout. In the ongoing Phase 1a portion (3+3 dose escalation), patients receive NX-1607 monotherapy at doses ranging from 5 up to 100 mg each day of a 21-day cycle. The primary objective of Phase 1a is to evaluate safety/tolerability and establish the maximum tolerated dose and recommended Phase 1b dose (RP1bD). In the Phase 1b portion (dose expansion), patients with select advanced malignancies, including DLBCL are being enrolled and receive NX-1607 monotherapy at the RP1bD determined during Phase 1a. The primary objective of Phase 1b is to evaluate the anti-tumor activity of NX-1607 at the RP1bD in expansion cohorts. Tumor response will be assessed based on RECIST v1.1, modified RECIST, or Revised Response Criteria for Malignant Lymphoma for DLBCL. All patients are evaluable for safety based on standard safety criteria. Up to 304 patients will be enrolled at approximately 20 sites in the UK and US and treated until disease progression or unacceptable toxicity. Dose escalation is ongoing. Clinical trial information: NCT05107674. Study contact: nx1607101@nurixtx.com

Cbl-b negatively regulates TLR/MyD88-mediated anti-Toxoplasma gondii immunity.

Toxoplasma gondii is an important intracellular protozoan, which needs to exploit host nutrition for successful parasitism. We previously reported that Casitas B-lineage lymphoma-b (Cbl-b) was screened as a host dependency factor of T. gondii by using lentiviral CRISPR-Cas9-single guide RNA (sgRNA) libraries. Furthermore, the detailed mechanism of Cbl-b being required by T. gondii infection was explored in this study. The proliferation of T. gondii was found to be significantly inhibited in Cbl-b knockdown cell lines, and the Cbl-b expression level increased with prolonged T. gondii infection, while the MyD88 level was significantly decreased in the T. gondii infection group. Toll-like receptor (TLR)/MyD88 is a conserved innate cellular immune signaling pathway against pathogens infection. Cbl-b was found to interact with MyD88 and mediate MyD88 ubiquitination in the fluorescence resonance energy transfer and co-immunoprecipitation experiments. A Cbl-b knockout (KO) C57BL/6J lineage was then constructed and, together with the wild-type (WT) mice, was infected with the same amount of T. gondii tachyzoites of ME49 strain. At 13 days post infection, all the mice in the WT group died, while 80% of the mice in the Cbl-b KO group still survived, even to the end of the experiment. The parasitic burden in the liver, lung, and brain of the Cbl-b KO mice was significantly lower than that of the WT mice (P < 0.05). In Cbl-b KO infected mice, the percentage of B cells was higher, whereas that of macrophages was lower, and the interferon-γ and interleukin-6 levels in the serum were higher than that in the WT infected mice; the difference was significant (P < 0.05). Furthermore, when host cells were infected by T. gondii, host's Cbl-b was found to interact with MyD88 to ubiquitinate MyD88 for ribosome-dependent degradation. Therefore, the host's innate immunity against T. gondii through the TLR/MyD88 pathway was negatively regulated by Cbl-b.IMPORTANCEThis is the first report that a human E3 ubiquitin ligase, Casitas B-lineage lymphoma proto-oncogene B (Cbl-b), functions as a host dependency factor for the intracellular protozoan Toxoplasma gondii and the mechanism for how T. gondii infection inhibits the TLR/MyD88 innate immunity pathway through MyD88 degradation mediated by Cbl-b. This finding is an impactful contribution for understanding the host cell immunity against T. gondii infection.

Diosgenin prevents dexamethasone-induced myotube atrophy in C2C12 cells

Several pathophysiological abnormalities, including a sedentary lifestyle, chronic diseases, and oxidative stress, can contribute to muscle atrophy triggered by an imbalance in muscle protein synthesis and degradation. Resolving muscle atrophy is a critical issue as it can reduce the quality of life. Here, one of the promising functional food factors, diosgenin (a steroidal sapogenin) showed strong preventive activities against dexamethasone (Dex)-induced muscle atrophy, as determined by the expression levels and morphology of the myosin heavy chain in C2C12 myotubes. Diosgenin inhibited protein expressions of Dex-induced skeletal muscle-specific ubiquitin ligase, including muscle RING finger 1 (MuRF1) and casitas B-lineage lymphoma protooncogene b (Cbl-b) but not atrogin-1. Diosgenin ameliorated Dex-induced declines of Akt phosphorylation at Ser473 and FoxO3a phosphorylation at Ser253, which probably at least partially contributed to the suppression of MuRF1, Cbl-b, and atrogin-1 gene expression. Additionally, diosgenin inhibited Dex-induced nuclear translocation of the glucocorticoid receptor (GR), diosgenin therefore may competitively inhibit the interaction between Dex and GR. These findings suggest that diosgenin is an effective functional food for preventing glucocorticoid-induced skeletal muscle atrophy.

Clinical significance of genetic profiling based on different anatomic sites in patients with mucosal melanoma who received or did not receive immune checkpoint inhibitors

BackgroundTo date, data on the efficacy of targeted therapies for mucosal melanoma (MM) are limited. In this study, we analyzed genetic alterations according to the primary site of origin, which could provide clues for targeted therapy for MM.MethodsWe conducted a retrospective cohort study of 112 patients with MM. Targeted sequencing was performed to analyze genetic aberrations. Kaplan–Meier analysis was conducted with the log-rank test to compare the significance among subgroups.ResultsIn total, 112 patients with MM were included according to the anatomic sites: 38 (33.9%) in the head and neck, 22 (19.6%) in the genitourinary tract, 21 (18.8%) in the anorectum, 19 (17.0%) in the esophagus, 10 (8.9%) in the uvea, and 2 (1.8%) in the small bowel. The most significantly mutated genes included BRAF (17%), KIT (15%), RAS (15%), TP53 (13%), NF1 (12%), SF3B1 (11%), GNA11 (7%), GNAQ (5%), and FBXW7 (4%). A large number of chromosomal structural variants was found. The anatomic sites of esophagus and small bowel were independent risk factors for progression-free survival (PFS, hazard ratio [HR] 4.78, 95% confidence interval [CI] 2.42–9.45, P < 0.0001) and overall survival (OS, HR 5.26, 95% CI 2.51–11.03, P < 0.0001). Casitas B-lineage lymphoma (CBL) mutants showed significantly poorer PFS and OS. In contrast, MM patients who received immune checkpoint inhibitors (ICIs) had a significantly more favorable OS (HR 0.39, 95% CI 0.20–0.75, P = 0.008).ConclusionsOur findings reveal the genetic features of patients with MM, mainly across six anatomic sites, offering a potential avenue for targeted therapies.

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 462: A novel and potent Cblb inhibitor demonstrates robust immunological profile and anti-tumor efficacy

Abstract Background: Casitas B-lineage lymphoma b (Cbl-b), a RING finger E3 ligase, is a negative regulator of immune cell activation1. Genetic deletion or pharmacological inhibition of Cbl-b resulted in hyper-reactive and co-stimulation independent T cell activation and cytokine production1. In syngeneic tumor models, CD8 T-cell and NK-cell mediated rejection of tumours were observed1. These findings point to Cbl-b as a therapeutic target in cancer immunotherapy. Inhibition of Cbl-b also demonstrated the potential to enhance the efficacy of check-point blockers like anti-PD-1 antibody, an unmet need in the clinic. Methods: Using intuitive medicinal chemistry design supported by computational approaches, we identified a lead Cbl-b inhibitor. SAR was developed using a battery of biochemical assays, functional read-outs and primary human in vitro T-cell activation and exhaustion assays. In vivo efficacy was demonstrated in syngeneic mouse colon tumor model. Results: Our lead Cbl-b inhibitor demonstrated potent binding to Cbl-b, robust anti-tumor cytokine secretion in human and mouse T cells, whole blood and potent reversal of T cell exhaustion. A strong tumor growth inhibition was demonstrated by the lead compound in a mouse colon tumor model. Compared to single agent, a combination of the lead compound with anti-PD-1 antibody induced enhanced complete tumor rejections. Conclusions: We have identified a novel, potent and orally bioavailable Cbl-b inhibitor that demonstrated robust in vitro and in vivo anti-tumor profiles. Acknowledgements: We thank Sanjib Das, Ajit Patil, Gauri Gawas, Savita Pandita, Priya Yadav, Sneha Pusadkar, Mayura Behere, Subhadip Das, Shravankumar Kolli and Radheshyam Yadav for their contributions to the project