TCF4 Expression Research Articles

Abstract 7274: Optimization of intermittent dosing strategies of KRAS G12C inhibitors in preclinical lung cancer model

Abstract Cysteine-binding targeted drugs AMG-510 and MRTX-849 provide a new therapeutic approach for advanced KRASG12C mutant cancers. However, clinical response has been limited. Responders eventually develop adaptive resistance by gaining additional MAPK pathway mutations, resulting in limited therapeutic benefit. High-dose pulsatile treatment with MAPK pathway inhibitors has been proposed as an alternative strategy to alleviate adaptive resistance while maintaining tumor control. The sensitivity of tumor cells to pan-inhibitors of the MAPK pathway, crucial for proliferation, creates a therapeutic opportunity window for immune cells to restore their function during an intermittent regimen. Congruently, we have previously shown that intermittent pan-MAPK pathway inhibitor treatment delays drug resistance, supports T cell activation, and enhances tumor delay when combined with immunotherapy. In this study, we investigated the effects of continuous and pulsatile KRASG12C inhibition on tumor and immune cells in order to design combinatorial strategies with immune-modulating agents that overcome drug resistance. Our data suggest that a weekly single high-dose regimen of AMG-510 is less effective at achieving tumor control in preclinical mouse models of KRAS mutant lung cancer compared to daily treatments. However, supplementing this pulsatile regimen with lower maintenance doses markedly slows the growth of LLC KRASG12C lung cancer. Flow cytometry analysis revealed that high-dose pulse regimen enhances T cell activation, evidenced by increased expression of TCF-1, Ki67, PD-1, and GITR, along with lower expression of co-inhibitory molecules Lag-3 and Tim-3, 3 days after treatment. In vitro, AMG-510 also improves the antigen presentation of OVA-expressing LLC-KML lung cancer cells via MHC class I and, to a lesser extent, MHC class II. Furthermore, we found that while combinatorial therapy with PD-1 blockade alone does not significantly impact tumor control, the combination with CTLA-4 blockade shows efficacy across pulsatile and continuous AMG-510 dosing cohorts. Overall, our findings suggest that the utilization of intermittent dosing regimens for selective KRASG12C inhibitors is a promising approach for improving clinical responses in lung cancers with KRAS mutations. Future experiments will aim to delineate MAPK pathway behavior in vivo and in vitro under pulsatile and continuous treatment, assess immunogenicity, and further characterize immune changes in the different treatment regimens, with the goal of designing more efficient cancer therapies utilizing selective KRAS inhibitors. Citation Format: Valeria Estrada Navarro, Vincent Panneton, Lauren Dong, Hyejin Choi, Divya Venkatesh, Rachana Maniyar, Isabell Schulze, Jedd D. Wolchok, Taha Merghoub. Optimization of intermittent dosing strategies of KRAS G12C inhibitors in preclinical lung cancer model [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 7274.

Abstract 7536: CD4+T cell exhaustion in the tumor microenvironment and antitumor immunity

Abstract Background: Immune checkpoint inhibitors (ICIs) exert clinical efficacy against various types of cancer through reinvigoration of exhausted CD8+ T cells that attack cancer cells directly in the tumor microenvironment (TME). However, compared to CD8+ T cell exhaustion, the role of CD4+ T cell exhaustion in antitumor immunity remains unclear. Methods and Results: From single-cell sequencing for tumor-infiltrating lymphocytes (TILs) from patients with melanoma and mouse models, we found that CXCL13, which is highly expressed in tumor-infiltrating exhausted CD8+ T cells, induces PD-1+CXCR5+CD4+ follicular helper T (TFH)-like cell infiltration, which could contribute to antitumor immunity. Furthermore, a part of the TFH-like cells in the TME exhibits cytotoxicity and directly attacks MHC-II-expressing tumors. RNA velocity and latent time analysis in single-cell sequencing for melanoma TILs showed that the differentiation of TFH-like cells from the naive CD4+ T cells were different from that of other CD4+ T cells. In addition, the TFH-like cytotoxic CD4+ T cells were at the late stage of differentiation and some non-cytotoxic TFH-like cells might differentiate into TFH-like cytotoxic CD4+ T cells. We additionally found considerable overlapped clonotypes between the cytotoxic and non-cytotoxic clusters. TFH-like cytotoxic CD4+ T cells have high LAG-3/BLIMP1 and low TCF1 expression without self-renewal ability, whereas non-cytotoxic TFH-like cells express low LAG-3/BLIMP1 and high TCF1 with self-renewal ability, closely resembling the relationship between terminally differentiated and stem-like progenitor exhaustion in CD8+ T cells, respectively. Conclusion: Our findings provide novel insights into TFH-like CD4+ T cell exhaustion with helper progenitor and cytotoxic differentiated functions, which mediates antitumor immunity orchestrally with CD8+ T cells. Citation Format: Joji Nagasaki, Wenhao Zhou, Shusuke Kawashima, Takamasa Ishino, Katsushige Kawase, Youki Ueda, Kazuo Yamashita, Tomofumi Watanabe, Takashi Inozume, Yosuke Togashi. CD4+T cell exhaustion in the tumor microenvironment and antitumor immunity [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 7536.

The role of lncRNAKCNQ1OT1/miR-301b/Tcf7 axis in cardiac hypertrophy.

Cardiac hypertrophy, acting as a pathologic process of chronic hypertension and coronary disease, and its underlying mechanisms still need to be explored. Long non-coding RNA (LncRNA) potassium voltage-gated channel subfamily Q member 1 Transcript 1 (KCNQ1OT1) has been implicated in myocardial infarction. However, its role in cardiac hypertrophy remains reported. To explore the regulated effect of lncRNAKCNQ1OT1 and miR-301b in cardiac hypertrophy, gain-and-lose function assays were tested. The expression of lncRNAKCNQ1OT1 and miR-301b were tested by quantitative real time polymerase chain reaction (qRT-PCR). The levels of transcription factor 7 (Tcf7), Proto-oncogene c-myc (c-myc), Brainnatriureticpeptide (BNP)and β-myosin heavy chain (β-MHC) were detected by Western blot. Additionally, luciferase analysis revealed interaction between lncRNAKCNQ1OT1, BNPβ-MHCmiR-301b, and Tcf7. LncRNAKCNQ1OT1 overexpression significantly induced cardiac hypertrophy. Furthermore, lncRNAKCNQ1OT1 acts as a sponge for microRNA-301b, which exhibited lower expression in cardiac hypertrophy model, indicating an anti-hypertrophic role. Furthermore, the BNP and β-MHC expression increased, as well as cardiomyocyte surface area, with Ang II treatment, while the effect was repealed by miR-301b. Moreover, the protein expression of Tcf7 was inversely regulated by miR-301b and Antisense miRNA oligonucleotides (AMO)-301b. Our study has shown that overexpression of lncRNAKCNQ1OT1 could promote the development of cardiac hypertrophy by regulating miR-301b and Tcf7. Therefore, inhibition of lncRNAKCNQ1OT1 might be a potential therapeutic strategy for cardiac hypertrophy.

EP300 regulates the SLC16A1-AS1-AS1/TCF3 axis to promote lung cancer malignancies through the Wnt signaling pathway

ObjectiveTo investigate the regulatory mechanism of EP300 in the interaction between SLC16A1-AS1 and TCF3 to activate the Wnt pathway, thereby promoting malignant progression in lung cancer. MethodsIn lung cancer cell lines, SLC16A1-AS1 was knocked down, and the impact of this knockdown on the malignant progression of lung cancer cells was assessed through clonogenic assays, Transwell assays, and apoptosis experiments. The regulatory relationship between EP300 and SLC16A1-AS1 was investigated through bioinformatic analysis and ChIP experiments. The expression of SLC16A1-AS1 and TCF3 in 56 paired lung cancer tissues was examined using RT-qPCR, and their correlation was analyzed. The interaction between TCF3 and SLC16A1-AS1 was explored through bioinformatic analysis and CoIP experiments. Activation of the Wnt/β-catenin pathway was assessed by detecting the accumulation of β-catenin in the nucleus through Western blotting. The role of EP300 in regulating the effect of SLC16A1-AS1/TCF3-mediated Wnt/β-catenin signaling on lung cancer malignant progression was validated through in vitro and in vivo experiments. ResultsSLC16A1-AS1 is highly expressed in lung cancer and regulates its malignant progression. EP300 mediates histone modifications on the SLC16A1-AS1 promoter, thus controlling its expression. SLC16A1-AS1 exhibits specific interactions with TCF3, and the SLC16A1-AS1/TCF3 complex activates the Wnt/β-catenin pathway. EP300 plays a critical role in regulating the impact of SLC16A1-AS1/TCF3-mediated Wnt/β-catenin signaling on lung cancer malignant progression. ConclusionEP300 regulates the SLC16A1-AS1/TCF3-mediated Wnt/β-catenin signaling pathway, influencing the malignant progression of lung cancer.

OCA-B/Pou2af1 is sufficient to promote CD4+ T cell memory and prospectively identifies memory precursors

The molecular mechanisms leading to the establishment of immunological memory are inadequately understood, limiting the development of effective vaccines and durable antitumor immune therapies. Here, we show that ectopic OCA-B expression is sufficient to improve antiviral memory recall responses, while having minimal effects on primary effector responses. At peak viral response, short-lived effector T cell populations are expanded but show increased Gadd45b and Socs2 expression, while memory precursor effector cells show increased expression of Bcl2, Il7r, and Tcf7 on a per-cell basis. Using an OCA-B mCherry reporter mouse line, we observe high OCA-B expression in CD4+ central memory T cells. We show that early in viral infection, endogenously elevated OCA-B expression prospectively identifies memory precursor cells with increased survival capability and memory recall potential. Cumulatively, the results demonstrate that OCA-B is both necessary and sufficient to promote CD4 T cell memory in vivo and can be used to prospectively identify memory precursor cells.

D-Ala2, D-Leu5]-enkephalin (DADLE) provides protection against myocardial ischemia reperfusion injury by inhibiting Wnt/β-Catenin pathway

BackgroundAcute myocardial infarction is one of the leading causes of death worldwide. Myocardial ischemia reperfusion (MI/R) injury occurs immediately after the coronary reperfusion and aggravates myocardial ischemia. Whether the Wnt/β-Catenin pathway is involved in the protection against MI/R injury by DADLE has not been evaluated. Therefore, the present study aimed to investigate the protective effect of DADLE against MI/R injury in a mouse model and to further explore the association between DADLE and the Wnt/β-Catenin pathway.MethodsForty-four mice were randomly allocated to four groups: Group Control (PBS Control), Group D 0.25 (DADLE 0.25 mg/kg), Group D 0.5 (DADLE 0.5 mg/kg), and Group Sham. In the control and DADLE groups, myocardial ischemia injury was induced by occluding the left anterior descending coronary artery (LAD) for 45 min. PBS and DADLE were administrated, respectively, 5 min before reperfusion. The sham group did not go through LAD occlusion. 24 h after reperfusion, functions of the left ventricle were assessed through echocardiography. Myocardial injury was evaluated using TTC double-staining and HE staining. Levels of myocardial enzymes, including CK-MB and LDH, in the serum were determined using ELISA kits. Expression of caspase-3, TCF4, Wnt3a, and β-Catenin was evaluated using the Western blot assay.ResultsThe infarct area was significantly smaller in the DADLE groups than in the control group (P < 0.01). The histopathology score and serum levels of myocardial enzymes were significantly lower in the DADLE groups than in the control group (P < 0.01). DADLE significantly improved functions of the left ventricle (P < 0.01), decreased expression of caspase-3 (P < 0.01), TCF4 (P < 0.01), Wnt3a (P < 0.05), and β-Catenin (P < 0.01) compared with PBS.ConclusionsThe present study showed that DADLE protected the myocardium from MI/R through suppressing the expression of caspase-3, TCF4, Wnt3a, and β-Catenin and consequently improving functions of the left ventricle in I/R model mice. The TCF4/Wnt/β-Catenin signaling pathway might become a therapeutic target for MI/R treatment.

ECSIT facilitates memory CD8+ T cell development by mediating fumarate synthesis during viral infection and tumorigenesis.

Memory CD8+ T cells play a crucial role in infection and cancer and mount rapid responses to repeat antigen exposure. Although memory cell transcriptional programmes have been previously identified, the regulatory mechanisms that control the formation of CD8+ T cells have not been resolved. Here we report ECSIT as an essential mediator of memory CD8+ T cell differentiation. Ablation of ECSIT in T cells resulted in loss of fumarate synthesis and abrogated TCF-1 expression via demethylation of the TCF-1 promoter by the histone demethylase KDM5, thereby impairing memory CD8+ T cell development in a cell-intrinsic manner. In addition, ECSIT expression correlated positively with stem-like memory progenitor exhausted CD8+ T cells and the survival of patients with cancer. Our study demonstrates that ECSIT-mediated fumarate synthesis stimulates TCF-1 activity and memory CD8+ T cell development during viral infection and tumorigenesis and highlights the utility of therapeutic fumarate analogues and PD-L1 inhibition for tumour immunotherapy.

Clinical and immunological relevance of SLAMF6 expression in the tumor microenvironment of breast cancer and melanoma

Compelling evidence shows that the frequency of T cells in the tumor microenvironment correlates with prognosis as well as response to immunotherapy. However, considerable heterogeneity exists within tumor-infiltrating T cells, and significance of their genomic and transcriptomic landscape on clinical outcomes remains to be elucidated. Signaling lymphocyte activation molecule 6 (SLAMF6) is expressed on intra-tumoral progenitor-exhausted T cells, which exhibit the capacity to proliferate, self-renew and produce terminally-exhausted T cells in pre-clinical models and patients. Here, we investigated the impact of SLAMF6 expression on prognosis in two immunologically different tumor types using publicly available databases. Our findings demonstrate that high SLAMF6 expression is associated with better prognosis, expression of TCF7 (encoding T-cell factor 1), and increased gene signatures associated with conventional type 1 dendritic cells and effector function of T cells in melanoma and breast cancer. Single-cell profiling of breast cancer tumor microenvironment reveals SLAMF6 expression overlaps CD8 T cells with a T-effector signature, which includes subsets expressing TCF7, memory and effector-related genes, analogous to progenitor-exhausted T cells. These findings illustrate the significance of SLAMF6 in the tumor as a marker for better effector responses, and provide insights into the predictive and prognostic determinants for cancer patients.

Antigen-presenting B cells promote TCF-1+ PD1- stem-like CD8+ T-cell proliferation in glioblastoma.

Understanding the spatial relationship and functional interaction of immune cells in glioblastoma (GBM) is critical for developing new therapeutics that overcome the highly immunosuppressive tumor microenvironment. Our study showed that B and T cells form clusters within the GBM microenvironment within a 15-μm radius, suggesting that B and T cells could form immune synapses within the GBM. However, GBM-infiltrating B cells suppress the activation of CD8+ T cells. To overcome this immunosuppression, we leveraged B-cell functions by activating them with CD40 agonism, IFNγ, and BAFF to generate a potent antigen-presenting B cells named BVax. BVax had improved antigen cross-presentation potential compared to naïve B cells and were primed to use the IL15-IL15Ra mechanism to enhance T cell activation. Compared to naïve B cells, BVax could improve CD8 T cell activation and proliferation. Compared to dendritic cells (DCs), which are the current gold standard professional antigen-presenting cell, BVax promoted highly proliferative T cells in-vitro that had a stem-like memory T cell phenotype characterized by CD62L+CD44- expression, high TCF-1 expression, and low PD-1 and granzyme B expression. Adoptive transfer of BVax-activated CD8+ T cells into tumor-bearing brains led to T cell reactivation with higher TCF-1 expression and elevated granzyme B production compared to DC-activated CD8+ T cells. Adoptive transfer of BVax into an irradiated immunocompetent tumor-bearing host promoted more CD8+ T cell proliferation than adoptive transfer of DCs. Moreover, highly proliferative CD8+ T cells in the BVax group had less PD-1 expression than those highly proliferative CD8+ T cells in the DC group. The findings of this study suggest that BVax and DC could generate distinctive CD8+ T cells, which potentially serve multiple purposes in cellular vaccine development.

Deficiency of Cbfβ in articular cartilage leads to osteoarthritis-like phenotype through Hippo/Yap, TGFβ, and Wnt/β-catenin signaling pathways.

Osteoarthritis (OA) is the most prevalent degenerative joint disorder, causing physical impairments among the elderly. Core binding factor subunit β (Cbfβ) has a critical role in bone homeostasis and cartilage development. However, the function and mechanism of Cbfβ in articular cartilage and OA remains unclear. We found that Cbfβf/fAggrecan-CreERT mice with Cbfβ-deficiency in articular cartilage developed a spontaneous osteoarthritis-like phenotype with articular cartilage degradation. Immunofluorescence staining showed that Cbfβf/fAggrecan-CreERT mice exhibited a significant increase in the expression of articular cartilage degradation markers and inflammatory markers in the knee joints. RNA-sequencing analysis demonstrated that Cbfβ orchestrated Hippo/Yap, TGFβ/Smad, and Wnt/β-catenin signaling pathways in articular cartilage, and Cbfβ deficiency resulted in the abnormal expression of downstream genes involved in maintaining articular cartilage homeostasis. Immunofluorescence staining results showed Cbfβ deficiency significantly increased active β-catenin and TCF4 expression while reducing Yap, TGFβ1, and p-Smad 2/3 expression. Western blot and qPCR validated gene expression changes in hip articular cartilage of Cbfβ-deficient mice. Our results demonstrate that deficiency of Cbfβ in articular cartilage leads to an OA-like phenotype via affecting Hippo/Yap, TGFβ, and Wnt/β-catenin signaling pathways, disrupting articular cartilage homeostasis and leading to the pathological process of OA in mice. Our results indicate that targeting Cbfβ may be a potential therapeutic target for the design of novel and effective treatments for OA.

ABCA8 Elevation Predicts the Prognosis and Exerts the Anti-oncogenic Effects on the Malignancy of Non-small Cell Lung Cancer via TCF21-Mediated Inactivation of PI3K/AKT.

The malignant growth and metastatic potential of non-small-cell lung cancer (NSCLC) are the major causes for its poor prognosis. ATP-binding cassette (ABC) subfamily A member 8 (ABCA8) exerts contradictive roles in the development of several cancers. Nevertheless, its role in NSCLC remains unclear. In this study, three GEO datasets and bioinformatics databases (GEPIA2 and UALCAN) revealed the obvious down-regulation of ABCA8 in NSCLC tissues and cells, and this expression was associated with cancer stages and lymph node metastasis. Low expression of ABCA8 predicted poor survival in NSCLC. ABCA8 elevation inhibited cell proliferation and induced cell apoptosis. Moreover, ABCA8 overexpression suppressed cancer cell invasion. Mechanistically, ABCA8 was associated with TCF21 in NSCLC specimens and its overexpression enhanced TCF21 expression. ABCA8 elevation inactivated the PI3K/AKT signaling, which was reversed after TCF21 knockdown. Additionally, targeting TCF21 overturned the anti-oncogenic effects of ABCA8 elevation on cell proliferation, apoptosis and invasion. Thus, the current findings highlight that ABCA8 may be a promising prognostic marker and may act as a suppressor gene to regulate the malignancy of NSCLC cells via TCF21-mediated inactivation of PI3K/AKT signaling, supporting a new promising target for the treatment of NSCLC.

Mass cytometry and transcriptomic profiling reveal PD1 blockade induced alterations in oral carcinogenesis.

Oral squamous cell carcinoma is the predominant subtype of head and neck squamous cell carcinoma, characterized by a challenging prognosis. In this study, we established a murine model of oral carcinogenesis using 4-nitroquinoline-1-oxide (4-NQO) induction to investigate the impact of immunotherapy on microenvironmental alterations. Mice in the precancerous condition were randomly divided into two groups:one receiving programmed death-1 (PD1) monoclonal antibody treatment and the other, control immunoglobulin G. Our observations showed that while PD1 blockade effectively delayed the progression of carcinogenesis, it did not completely impede or reverse it. To unravel the underlying reasons for the limited effectiveness of PD1 blockade, we collected tongue lesions and applied mass cytometry (CyTOF) and RNA sequencing (RNA-seq) to characterize the microenvironment. CyTOF analysis revealed an increased macrophage subset (expressing high levels of IFNγ and iNOS) alongside a diminished Th1-like subset (exhibiting low expression of TCF7) and three myeloid-derived suppressor cell subsets (displaying low expression of MHC Class II or IFNγ) following anti-PD1 treatment. Notably, we observed an increased presence of cancer-associated fibroblasts (CAFs) expressing collagen-related genes after PD1 blockade. Furthermore, we found a negative correlation between the infiltration levels of CAFs and CD8+ T cells. These findings were validated in murine tongue tissue slides, and publicly available multi-omics datasets. Our results suggest that CAFs may impair the therapeutic efficacy of PD1 blockade in oral carcinogenesis by the remodeling of the extracellular matrix.

The PES1/FOXM1 heterodimer suppresses TCF21 and ERβ expression in ovarian endometriosis

AbstractTranscription factor 21 (TCF21) and estrogen receptor beta (ERβ, encoded by ESR2) are highly expressed in endometriotic stromal cells (ESCs) and contribute to the pathogenesis of endometriosis. However, the exploration of TCF21 and ERβ expression regulation at the molecular level remains limited. Here, by using bioinformatics analysis and experimental verification, we identified PES1, also known as Pescadillo, as a negative regulator in the development of endometriosis that downregulates TCF21 and ERβ expression in ESCs. PES1 overexpression regulated critical biological processes involved in endometriosis development, such as invasion and apoptosis. A coimmunoprecipitation assay showed that PES1 could form a complex with Forkhead box M1 (FOXM1). Further analyses elucidated that siPES1 in ectopic lesions decreased the stability of FOXM1 protein and reduced the binding activities of FOXM1 to TCF21 and ESR2 promoters, thus weakening the transcriptional inhibition of TCF21 and ERβ by FOXM1. Moreover, in an endometriosis mouse model, overexpressing PES1 effectively reduced the growth of ectopic lesions and suppressed TCF21 and ERβ expression, which suggests a promising therapeutic strategy for endometriosis. Collectively, our results indicate that the loss of PES1 in ectopic lesions contributes to endometriosis progression by upregulating ERβ and TCF21 expression through heterodimer formation with FOXM1. Moreover, targeting PES1 could serve as a treatment method for endometriosis.

Resistomycin Inhibits Wnt/β-Catenin Signaling to Induce the Apoptotic Death of Human Colorectal Cancer Cells.

Resistomycin is a natural antibiotic related to quinone that has been shown to exhibit robust antitumor activity. To further characterize the mechanistic basis for such activity, human colorectal cancer (CRC) cells were selected as a model to explore the role of Wnt/β-catenin signaling in the ability of resistomycin to induce apoptotic cell death. These analyses revealed that resistomycin was able to suppress β-catenin, TCF4, and GSK-3β expression, together with that of the downstream targets c-Myc and survivin. This coincided with elevated cleaved caspase-3 and Bax protein levels and a decline in Bcl-2 content. When β-catenin was silenced, this further enhanced the ability of resistomycin to induce apoptotic CRC cell death, whereas this apoptotic process was partially ablated when cells were treated using lithium chloride to activate Wnt/β-catenin signaling. Overall, these results support a model wherein resistomycin inhibits Wnt/β-catenin signaling within CRC cells, thereby inducing apoptotic death. Further research may be warranted to better clarify the potential utility of this compound as a candidate drug for use in the treatment of patients suffering from this form of cancer.

Emergence and fate of stem cell–like Tcf7 + CD8 + T cells during a primary immune response to viral infection

In response to infection, naïve CD8 + T (T N ) cells yield a large pool of short-lived terminal effector (T TE ) cells that eliminate infected host cells. In parallel, a minor population of stem cell–like central memory (T CM ) cells forms, which has the capacity to maintain immunity after pathogen clearance. It has remained uncertain whether stem-like T CM cells arise by dedifferentiation from a subset of cytolytic T TE cells or whether priming generates stem-like cells capable of seeding the T CM compartment and, if so, when cytolytic T TE cells branch off. Here, we show that CD8 + T cells with stem-like properties, which are identified by the expression of TCF1 (encoded by Tcf7 ), are present across the primary response to infection. Priming programs T N cells to undergo multiple cell divisions, over the course of which TCF1 expression is maintained. These TCF1 + cells further expand relatively independently of systemic inflammation, antigen dose, or affinity, and they quantitatively yield TCF1 + T CM cells after pathogen clearance. Inflammatory signals suppress TCF1 expression in early divided TCF1 + cells. TCF1 down-regulation is associated with the irreversible loss of self-renewal capacity and the silencing of stem/memory genes, which precedes the stable acquisition of a T TE state. TCF1 expression restrains cell cycling, explaining in part the limited expansion of TCF1 + relative to TCF1 − cells during the primary response. Thus, our data are consistent with terminal differentiation of effector cells being a step-wise process that is initiated by inflammation in primed stem-like cells, which would otherwise become central memory cells by default.

TCF21 gene and cardiovascular events in a coronary population

Abstract Background Recent research showed that TCF21 expression in precursor cells that migrate into the disease lesions contributes to the fibrous cap, stabilizing the lesions and preventing heart attacks. Targeted deletion of the transcription factor encoded by the TCF21 was associated with vascular smooth muscle cell disruption impairing the fibrous cap structure, increasing cardiovascular(CV) disease risk. Its association with CV events is unknown. Purpose Analyze the TCF21 rs12190287 variant G&amp;gt;C evaluating its association with atherosclerosis severity and the appearance of cardiovascular (CV) events. Methods We performed a prospective study with 1,716 coronary artery disease (CAD) patients (mean age 53.3±7.8 years). TCF21 rs12190287 G&amp;gt;C was genotyped by TaqMan genotyping assay (Applied Biosystems) in all patients. The severity of CAD was graded according to the number of obstructed coronary arteries with at least 70% narrowed lumen. Chi-squared tests were used to determine differences in CAD severity by genotype. We performed a first multivariate logistic regression analysis to assess the independent risk variables associated with CAD severity. After that, we presented a second multivariate Cox regression to evaluate independent variables related to CV events. Kaplan Meier estimated the survival curves. Results 48.0% of the CC genotype carriers were associated with more than two obstructed coronary arteries compared to the GG wild genotype, which was associated with only 9.2% (p=0.002). Multivariate analysis (logistic regression) showed that the CC genotype had a high risk of CAD severity (OR=2.95; p=0.001) than GG. After Cox regression analysis, which takes into account the time to the first event, the CC genotype remained in the equation with an HR of 1.38; 95%CI 1.02-1.88; p=0.040. The survival events free at ten years is 49.1% in the CAD patients with the GG genotype, then drops to 39.6 % with the CC risk genotype. Conclusion This work shows that the GG wild genotype protects against CAD severity. In contrast, the CC genotype is associated with an increased risk of CAD severity. The patients with this allelic variant had a worse survival event free. Future research with the implementation of epigenetic and genetic therapeutics targeted to deleterious genes will allow the eradication of coronary heart disease worldwide.

Proenkephalin-A secreted by renal proximal tubules functions as a brake in kidney regeneration

Organ regeneration necessitates precise coordination of accelerators and brakes to restore organ function. However, the mechanisms underlying this intricate molecular crosstalk remain elusive. In this study, the level of proenkephalin-A (PENK-A), expressed by renal proximal tubular epithelial cells, decreases significantly with the loss of renal proximal tubules and increased at the termination phase of zebrafish kidney regeneration. Notably, this change contrasts with the role of hydrogen peroxide (H2O2), which acts as an accelerator in kidney regeneration. Through experiments with penka mutants and pharmaceutical treatments, we demonstrate that PENK-A inhibits H2O2 production in a dose-dependent manner, suggesting its involvement in regulating the rate and termination of regeneration. Furthermore, H2O2 influences the expression of tcf21, a vital factor in the formation of renal progenitor cell aggregates, by remodeling H3K4me3 in renal cells. Overall, our findings highlight the regulatory role of PENK-A as a brake in kidney regeneration.

Bclxl Prevents Progressive Exhaustion in BCMA CAR T Cells with Maintenance of High TCF1 Expressing T Cells

Although BCMA targeted CAR T cell therapies are highly effective in the treatment of multiple myeloma (MM), patients with high disease burden have not fully benefited from this therapy with shorter disease remissions despite initial responses. It is postulated that high disease burden results in chronic antigenic stimulation of CAR T cells with the induction of exhaustion-associated gene signatures and CAR T cells contraction, limiting their in vivo potency. We have previously reported that BCL2L1 (gene encoding for BCLxL) armoring of BCMA CAR T cells enhanced CAR T cells functional fitness and improved proliferation and cytotoxicity after chronic antigenic stimulation in vitro, as well as superior tumor clearance in xenograft models of MM. In the current study we aimed to define the molecular mechanisms that mediates BCL2L1-armored CAR T cell and examine the effect of BCLxL variants on CAR T cells fitness. In in vivo studies, NSG mice were systemically injected with OPM2 cells (stably transduced with firefly luciferase) and led to develop large MM disease burden, treatment with BCLxL armored (BCMA_BCL2L1) vs unarmored CARs (BCMA_CAR) or control T cells completely eliminated the MM disease and significantly improved animal survival. In animals treated with armored CARs (BCMA_BCL2L1) no disease recurrence was noted 180 days post treatment (in contrast to unarmored CARs where disease recurrence was noted within 60 days). BCLxL also improved CARs persistence, with armored CAR T cells being detectable in the marrow of these mice 7 and 14 days post treatment. Of note, BCLxL armored CAR T cells did not demonstrate uncontrolled proliferation as they were no longer detectable on day 120 post-infusion. Furthermore, CITEseq profiling on harvested human T cells 7 days post CAR T cells infusion to diseased mice revealed a differential T cell repertoires and phenotypes between BCMA_CAR and BCMA_BCL2L1 CARs. While unarmored CARs were significantly enriched for exhausted T cells (CD8_Tex), BCMA_BCL2L1 CAR T were enriched with early activated non-exhausted CD8 T cells, precursor exhausted T cells (CD8_Tpex) and naïve CD4 and CD8 T cells. Importantly, single cell genes set enrichment analysis (ssGSEA) revealed a differential metabolic signature with higher oxidative phosphorylation in BCMA_BCL2L1 CAR T cells compared to unarmored CARs. These findings suggested that armoring CAR T cells with BCL2L1 maintain CAR T cells in an activated and precursor exhausted T cell states with improved mitochondrial and metabolic fitness, preventing terminal T cells exhaustion. To further evaluate the impact of BCLxL in suppressing BCMA CAR T cells' exhaustion, we engineered BCMA CAR T cells with armored expression of two BCLxL mutants (BCLxL Δ26-83 or BCLxL Δ46-83) harboring large or small deletions involving a BCLxL regulatory loop. These mutants were previously reported to enhance BCLxL function (Chang et al The EMBO Journal 1997,16:968-977). Under chronic antigenic stimulation (CAS) or hypoxic conditions, wild type or mutants BClxL overexpression in BCMA CAR T cells resulted in higher proliferation rates compared to unarmored CARs. Compared to wild type BClxL, BCLxL Δ26-83 and BCLxL Δ46-83 mutants demonstrated higher proliferation capacity post CARs or under hypoxic conditions. Intriguingly, majority of BCLxL-overexpressing BCMA CAR-T cells, particularly BCMA CAR-T BCLxL_Δ26-83 cells, maintained a central memory (T CM) phenotype following a long co-culture with MM tumor cells with lower levels of exhaustion markers. Importantly BCLxL-overexpressing BCMA CAR-T cells had substantially higher level of TCF1 with lower TOX/TOX2 expression compared to unarmored BCMA CAR T cells. TCF1 high population was also enriched in unstimulated BCLxL-overexpressing BCMA CAR T cells suggesting a positive regulatory impact of BCL2L1 on TCF1 expression, either through direct or indirect pathways. Expectedly, TCF1 high BCMA CAR-T cells overexpressing a BCLxL were mainly associated with the naive/T CM phenotype. In summary, we have identified a role for BCLxL and two mutant variants lacking a loop regulatory domain (BCLxL Δ26-83 or BCLxL Δ46-83), in maintenance of BCMA CAR T cells in central memory and precursor exhausted states with retained high TCF1 expression, even under hypoxic and chronic antigenic stimulation conditions. These studies further support the clinical development of a BCL2L1 armored CAR T cells for the treatment of MM.

The Resistance Mechanism to BET-Protac in Multiple Myeloma

Objective ：Explore the resistance mechanism of BET protein degradation targeting chimera (BET-PROTAC) in multiple myeloma (MM) cells, and find strategies to enhance the efficacy of BET-PROTAC. So as to provide new theoretical basis for the treatment of multiple myeloma. Methods ：The changes of TCF7 and c-Myc mRNA level were detected by RT-qPCR after BET-PROTAC (ARV-771) treatment of RPMI-8226 cells, the transcriptional activity of β-catenin in ARV-771 tolerant cells was detected by double luciferase reporter assay. Constructing lentiCRISPR-v2 vector to knockout β-catenin in RPMI-8226 cells, and detecting the sensitivity changes to ARV-771 and the formation of drug-resistant clones; MM cell lines resistant to ARV-771 were induced by dose escalation method, and detect the sensitivity of drug-resistant cells to other BET inhibitors and anti-tumor drugs; RNA-seq was used to detect the significantly changed genes and signal modules in drug-resistant cells, and verify this in drug-resistant cells; The drug resistant cells were treated with inhibitors or knockout the resistance gene to observe whether the drug resistance was reversible; The CRISPR knockout gene library was used to screen the candidate genes related to the up-regulation of drug resistance genes in drug-resistant cells, the expression of candidate genes was interfered, and the expression of drug resistance proteins was detected. The correlation between these candidate genes and the prognosis of MM patients was analyzed through public databases. Results ： In ARV-771 tolerant RPMI-8226 cells, the mRNA expression of TCF7 was significantly elevated, but the expression of c-Myc was still at a low level. TOPFlash assay showed activation of β-catenin signal in tolerant cells; Knockout of β-catenin in RPMI-8226 cells by CRISPR/cas9 increased apoptosis induced by ARV-771. Colony formation assay showed that there was no significant difference in the formation rate and number of drug-resistant clones after β-catenin knockout. ARV-771 resistant RPMI-8226 cells were successfully induced by dose increasing method, and three drug-resistant clones (AR1, AR2 and AR3) were obtained, the IC50 of these cells were 80, 120 and 8 times higher than the parental cells, respectively. The drug-resistant cells were cultured in drug-free medium for another 6 months, but the sensitivity did not recover. AR1 and AR2 cells showed decreased sensitivity to ARV-825, MZ-1, OTX-015, I-BET151, Daunorubicin and Epirubicin. RNA-seq showed that ABCB1 was significantly up-regulated in drug-resistant cells, and we verified the up-regulation of ABCB1 at mRNA and protein levels in all drug-resistant cells. Combined use of ABCB1 inhibitors (verapamil, cyclosporin A, Elacridar) or knockout of ABCB1 could significantly reduce the IC50 of drug-resistant cells, increase the apoptosis rate after ARV-771 treatment, and increase the degradation of BRD4 and the down-regulation of c-Myc. Using CRISPR/Cas9 library screening technology, we preliminarily selected 39 genes that may be involved in the regulation of ABCB1 expression, among which the downregulation of 14 genes could reduce the protein expression of ABCB1 in drug-resistant cells. Further through public databases, we found that C1orf112, CCDC167 and CRIP2 are significantly correlated with the prognosis of myeloma patients and are highly expressed in a variety of tumor tissues. These three genes will be the focus of subsequent studies. Conclusions ：Our results showed that BET-PROTAC resistance in MM cells wasindependent of β-catenin activation. The up-regulation of ABCB1 expression was the key mechanism mediating the resistance of myeloma cells to BET-PROTAC. C1orf112, CCDC167 and CRIP2 might be associated with drug resistance in myeloma and could affect prognosis, and their mechanisms in myeloma need to be further investigated. Keywords ：Multiple myeloma; BET; Protein degradation targeting chimera; Drug resistance; CRISPR library; ABCB1

TCF-1 limits intraepithelial lymphocyte antitumor immunity in colorectal carcinoma.

Intraepithelial lymphocytes (IELs), including αβ and γδ T cells (T-IELs), constantly survey and play a critical role in maintaining the gastrointestinal epithelium. We show that cytotoxic molecules important for defense against cancer were highly expressed by T-IELs in the small intestine. In contrast, abundance of colonic T-IELs was dependent on the microbiome and displayed higher expression of TCF-1/TCF7 and a reduced effector and cytotoxic profile, including low expression of granzymes. Targeted deletion of TCF-1 in γδ T-IELs induced a distinct effector profile and reduced colon tumor formation in mice. In addition, TCF-1 expression was significantly reduced in γδ T-IELs present in human colorectal cancers (CRCs) compared with normal healthy colon, which strongly correlated with an enhanced γδ T-IEL effector phenotype and improved patient survival. Our work identifies TCF-1 as a colon-specific T-IEL transcriptional regulator that could inform new immunotherapy strategies to treat CRC.