Suppressor Gene Research Articles

Bacteria in the cancer therapy

Human has been fighting with cancer for 130 years since the first tumor radical surgery in 1884. During this 130 years of cancer treatment history, there has developed mainly 4 types of treatment, chemotherapy, radiotherapy, surgery and immunotherapy. .However all of these treatment have their own side-effect, including the harm to normal cells, high cost, long developing time. And traditional cancer treatment are often failed to eliminate all the tumor tissue so few patients can be cure completely. Developing an effective cancer treatment is still a challenge. Luckily, a special approach is developing rapidly which is called bacteriotherapy. It can be combined with traditional treatment like chemotherapy and immunotherapy, use bacteria as a vector to deliver the drug or tumor suppressing gene to the tumor. In this way, the effect of harming normal cells can be reduce and the accuracy of attacking tumor can be improved. There are also tumor-specific bacteria found living in tumor which can be effective in cancer treatment. How to use bacteria well in cancer treatment is remain a challenge. This article reviewed the advantages of using bacteria for cancer treatment and the mechanism of bacteria in cancer treatment, giving example of the use of intestinal bacteria, involving the challenge and future of bacteriotherapy.

Production and characterisation of photorin, a new protein protease inhibitor from the entomopathogenic bacteria <i>photorhabdus laumondii</i>

Entomopathogenic bacteria of the genus Photorhabdus secrete protease S (PrtS), which is considered as a virulence factor. We found that in Photorhabdus genomes, immediately after the prtS genes, there are genes that encode small hypothetical proteins homologous to emfourin, a recently discovered protein inhibitor of metalloproteases. Emfourin-like inhibitor gene from Photorhabdus laumondii subsp. laumondii TT01 was cloned and expressed in Escherichia coli cells. The recombinant protein, named photorin (Phin), was purified by metal chelate affinity and gel permeation chromatography and characterized. It has been established that Phin is a monomer and inhibits the activity of protealysin and thermolysin, which, like PrtS, belong to the M4 peptidase family. The inhibition constants were 1.0 ± 0.3 and 10 ± 2 µM, respectively. It was also demonstrated that Phin is able to suppress the proteolytic activity of P. laumondii culture broth (half-maximal inhibition concentration 3.9 ± 0.3 nM). Polyclonal antibodies to Phin were obtained, and it was shown by immunoblotting that P. laumondii cells produce Phin. Thus, the prtS genes in entomopathogenic bacteria of the genus Photorhabdus are colocalized with the genes of emphorin-like inhibitors, which probably regulate the activity of the enzyme during infection. Strict regulation of the activity of proteolytic enzymes is essential for the functioning of all living systems. At the same time, the principles of regulation of protease activity by protein inhibitors remain poorly understood. Bacterial protease-inhibitor pairs, such as the PrtS and Phin pair, are a promising model for in vivo studies of these principles. Bacteria of the genus Photorhabdus have a complex life cycle with multiple hosts, being both nematode symbionts and powerful insect pathogens. This provides a unique opportunity to use the PrtS and Phin pair as a model for studying the principles of regulation of protease activity by proteinaceous inhibitors in the context of bacterial interactions with different types of hosts.

The deubiquitinating enzyme USP44 suppresses hepatocellular carcinoma progression by inhibiting Hedgehog signaling and PDL1 expression

Hepatocellular carcinoma (HCC) is one of the deadliest malignancies in the world. Research into the key genes that maintain the malignant behavior of cancer cells is crucial for the treatment of HCC. Here, we identified ubiquitin‐specific peptidase 44 (USP44), a member of the deubiquitinase family, as a novel regulator of HCC progression. The tumor suppressive function of USP44 was evaluated in a series of in vitro and in vivo experiments. Through quantitative proteomics examination, we demonstrated that USP44 inhibits HCC PDL1 expression by downregulating the Hedgehog (Hh) signaling pathway. Mechanistically, we found that USP44 directly interacts with Itch, an E3 ligase involved in Hh signaling, and promotes the deubiquitination and stabilization of Itch. These events result in the proteasomal degradation of Gli1 and subsequent inactivation of Hh signaling, which ultimately suppresses PDL1 expression and the progression of HCC. Furthermore, the HCC tissue microarray was analyzed by immunohistochemistry to evaluate the pathological relevance of the USP44/Itch/Gli1/PDL1 axis. Finally, the Gli1 inhibitor GANT61 was found to act in synergy with anti-PDL1 therapy. Overall, USP44 can act as a suppressive gene in HCC by modulating Hh signaling, and co-inhibition of Gli1 and PDL1 might be an effective novel combination strategy for treating HCC patients.

MASLD and aspartame: are new studies in the horizon?

Fatty liver disease has been on the rise in the past few decades, and there is no hope that it will stop. The terminology change that has been recently proposed may not be sufficient to advocate for a reduction of steatogenic foods and a change in lifestyle. A course change may be supported by the recent labeling of aspartame sweetener as a possible carcinogenic compound by the International Association for Research on Cancer (IARC), an agency of the World Health Organization (WHO). Aspartame sweeteners and other edulcorating molecular compounds besides colorings may trigger liver cancer other than fatty liver disease, despite limited data supporting it. An essential bias in human cohort studies is indeed the exclusion of all confounding factors, which may be barely impossible for human studies. In this perspective, we suggest that the activation of the NOD-like receptor-enclosing protein 3 (NLRP3) inflammasome and the stimulation of the tumor suppression gene TP53 may be critical in the progression from fatty liver to liver inflammation and liver cancer. Aspartame reduces a transcriptional coactivator, precisely the peroxisomal proliferator-initiated receptor-γ (gamma) coactivator 1-α (alpha) (or PGC1α). This coactivator upregulates mitochondrial bioformation, oxidative phosphorylation, respiratory capacity, and fatty acid β-oxidation. Aspartame acts in this way, probably through the activation of TP53. These events have been accountable for the variations in the lipid outline in serum and total lipid storage as well as for the impairment of gluconeogenesis in the liver, as supported by the downregulation of the gluconeogenic enzymes in experimental animals, and may be relevant in humans as well.

Abstract A152: The oncogene MYC as a driver of circadian clock disruption and dedifferentiation in the lung: implications in early lung carcinogenesis

Abstract Circadian rhythms (24-hour cycles) are disrupted in many human cancers, supporting cancer growth. Non-small cell lung cancers (NSCLCs) exhibit a disrupted molecular circadian clock, and associated alterations in circadian gene expression indicate a poor prognosis. However, it remains unclear how clock disruption occurs in NSCLCs and what NSCLCs gain from this disturbance. The oncogene MYC has been functionally implicated in circadian disruption in a variety of cancer cell lines. In NSCLCs, the MYC or one of its related family members is found mutated or amplified in more than 50% of the cases and its expression correlates with worse survival. BMAL1 is a non-redundant component of the circadian clock and is essential for the overall functionality of this machinery and the circadian regulation of gene expression. Using publicly available data we observed an inverse correlation between the expression of MYC and BMAL1 in LUAD. However, it is unknown if MYC drives clock disruption in the lung and whether clock disruption is functionally required for MYC oncogenic effects. Using an organoid model generated from isolated alveolar type II cells (ATII), the cell of origin lung adenocarcinoma (LUAD), the most prevalent type of NSCLC, we showed that MYC suppresses the expression of BMAL1. In parallel, MYC decreased the expression of specific ATII cell-lineage markers, suggesting the activation of a dedifferentiation program. Using an in vivo mouse model of NSCLC with MYC-inducible expression in ATII cells, we further demonstrated that MYC overexpression for 3 days induces an overall suppression of molecular clock genes, including BMAL1. We also observed that MYC increased the expression of proliferative markers such as Ki67 and promoted the loss of cell-lineage markers in ATII cells, which are early events in lung carcinogenesis. However, it is unclear if these effects are applicable to human LUAD. In human LUAD, we found that molecular subtypes that lack epithelial identity markers and are correlated with worse survival, show lower BMAL1 expression. These data corroborate with a potential implication of the molecular clock on the control of differentiation programs and tumor progression. Circadian clock pharmacological restoration in vivo, using the RORα agonist SR1078, attenuated the expression of the proliferation marker Ki67 induced by MYC in the lung, suggesting that clock disruption potentially mediates MYC oncogenic effects to some extent. Additionally, we performed CUT&TAG in a mouse lung epithelial cell line (MLE12) and identified that BMAL1 binds to the promoter of ATII identity genes (Sftpb, Abca3) and cell cycle genes (Cdk4), suggesting that the molecular clock potentially regulates lung epithelial cell identity and proliferation. Overall, our data identify MYC as a driver of circadian clock disruption in lung alveolar cells which is a potential mechanism implicated in MYC-driven proliferation and dedifferentiation in early-lung cancer. Circadian clock restoration might be a valuable tool to target MYC-amplified tumors. Citation Format: Juliana Cazarin de Menezes, Jamison Burchett, Siti Noor Ain Binti Ahmad Shahidan, Rachel E DeRollo, Brian Altman. The oncogene MYC as a driver of circadian clock disruption and dedifferentiation in the lung: implications in early lung carcinogenesis [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr A152.

Abstract A022: CDK2 inhibition demonstrates synthetic lethality in SCLC through apoptotic induction

Abstract Small cell lung cancer (SCLC), a malignant thoracic neoplasm genetically defined by dysregulation of the Retinoblastoma (RB1) gene and tumor suppressor protein 53 (TP53), remains one of the world’s deadliest tumors with a 5-year overall survival rate ranging from 5% to 10%. Despite numerous clinical trials and recent FDA approval of immune checkpoint inhibitors, the prognosis for SCLC has not significantly improved in decades. There remains a strong need for new therapeutic approaches. In this study, we found that the combined effect of dysregulation or deletion of RB1 and TP53, as is found in the majority of SCLC, results in sensitivity to CDK2 inhibition or ablation. This relationship is reflected in the DepMap database, with cell lines harboring deleterious mutations in RB1 demonstrating markedly higher susceptibility to CDK2 inhibition. This relationship is surprising given CDK2’s established role in suppressing the growth inhibition activity of RB and CDK2’s ability to thus facilitate G1/S transition. To validate this discovery, we evaluated the effects of CDK2 inhibition on a panel of SCLC cell lines. ARTS-021 is a potent and highly selective orally administered inhibitor of CDK2 currently in clinical development. In contrast to CCNE1 amplified cells, which undergo G1 arrest upon ARTS-021 treatment, SCLC cells treated with ARTS-021 exhibited minimal G1 accumulation. Instead, ARTS-021 treatment induced apoptosis in SCLC cell lines with loss of functional RB1 and TP53, as demonstrated by a dose-dependent increase in Caspase 3/7 activity and the accumulation of cleaved PARP. This phenotype was further demonstrated to be CDK2 dependent though generation of stable cell lines expressing a dox-inducible shRNA targeting CDK2. Combination studies further demonstrated that this apoptotic phenotype can be enhanced synergistically through inhibition of the anti-apoptotic proteins BCL-2 and BCL-xL with clinical stage inhibitor Navitoclax. In addition, combinations with currently approved standard of care chemotherapies were tested both in vitro and in vivo, and demonstrated potent combinatorial activity. These findings suggest that the clinical development of CDK2 inhibitors for SCLC may represent a novel therapeutic option for this difficult to treat malignancy. Citation Format: Nathan Schomer, Jinhong Chen, Wei Pang, Tingru Zhou, Xipan Liu, Wenhen Liang, Yali Guo, Dai Cheng, Fang Li, Jiaqi Liang, Yaoyu Chen, Qing Sheng. CDK2 inhibition demonstrates synthetic lethality in SCLC through apoptotic induction [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr A022.

Получение рекомбинантного ингибитора рибонуклеаз в E. coli для использования в синтезе мРНК in vitro

Technologies underlying the production of synthetic mRNAs in vitro have significantly expanded the possibilities for research and therapeutic use of this class of molecules. The flagship application area has been the niche of mRNA vaccines, but this class of therapeutic molecules has the potential to be applied in a much broader range of situations. The process of in vitro production of artificial RNA molecules is based on an enzymatic synthesis reaction, one of the components of which is a ribonuclease inhibitor. This protein protects synthesized RNA from attacks by ribonucleases and prevents degradation of the molecules, which is critically important for RNA. Eukaryotic ribonuclease inhibitor synthesized as a recombinant protein in the cells of E. coli bacteria is the most common choice. However, the structure of this protein makes it a difficult product to make in bacteria. This study aimed to test the production of a recombinant ribonuclease inhibitor in various strains of E. coli, and to show the effect that helper polypeptides and cellular chaperones have on this process. Using genetic engineering approaches, we constructed plasmids, from which chimeric ribonuclease inhibitor molecules and helper polypeptides were produced. The influence of various components on solubility of the target recombinant protein was assessed with the help of densitometry, to which we have subjected products of the PAGE electrophoresis. It was determined that combinations of a vector with a strong promoter for the expression of the RNH1 ribonuclease inhibitor gene and helper polypeptides MBP and TIG against the background of increased expression of cellular chaperones dnaK, dnaJ, grpE give the target product yield of 45 mg/l and 60 mg/l, respectively. The selected conditions allow large-scale production of this protein for further use in in vitro RNA synthesis in the context of production of medicines.

Influence of immunosuppressive drugs on natural killer cells in therapeutic drug exposure in liver transplantation.

Natural killer (NK) cells are enriched in the liver and are the main regulators in liver transplantation regarding rejection or tolerance, viral infection, or tumor recurrence. Immunosuppression consists of a triple drug standard regimen comprising tacrolimus (TAC) and corticosteroids (CS) with either mycophenolate mofetil (MMF) or sirolimus (SIR)/everolimus (EVE). The aim of this study was to evaluate the impact of trough levels of these regimens under clinical conditions and exposure on human NK-cell activity and function in order to better understand the antiviral and anti-tumor effects of mammalian target of rapamycin inhibitor (mTORI). Peripheral blood mononuclear cells (PBMCs) were collected from liver transplant recipients and healthy controls. Number and phenotypes of NK cells in vivo were analyzed by flow cytometry. In this study we simulated the immunosuppressive microenvironment in vitro. PBMCs were cultured at the clinically effective plasma concentration of drugs for 3 d to detect the effect of immunosuppressants on NK cells. Drug type and concentration: single drug [EVE, 5 ng/mL; SIR, 5 ng/mL; TAC, 5 ng/mL; cyclosporine A (CSA), 125 ng/mL; MMF, 15 µg/mL; CS, 0.5 µg/mL] and combined immunosuppressants (Group 1: TAC, 5 ng/mL + MMF, 15 µg/mL + CS, 0.5 µg/mL; Group 2: TAC, 5 ng/mL + SIR, 5 ng/mL + CS, 0.5 µg/mL; Group 3: TAC, 5 ng/mL + EVE, 5 ng/mL + CS, 0.5 µg/mL). In addition, NK cells were sorted from PBMCs and treated under the above conditions to detect NK cell killing function and RNA transcription characteristics. CS significantly impaired the cytolytic activity of NK cells, followed by MMF and SIR/EVE. CS and TAC/CSA significantly decreased the secretion of IFN-γ and CD107a. NK cell function in liver transplant recipients was most pronouncedly inhibited by a triple immunosuppressive regimen, with CS playing the most prominent role compared with the other drugs. The MMF-containing regimen demonstrated a significant increase in the expression of suppressive genes, especially of the Siglec7/9 family. The SIR group had stronger NK cell activity compared with that of the MMF group, although liver transplantation patients have lower NK cell activity and function. Despite an overall comparable immunosuppressive efficiency in terms of prevention of acute rejection, a mTORIs-including regimen might be considered as having less impact on NK cell function.

A T cell receptor β chain-directed antibody fusion molecule activates and expands subsets of T cells to promote antitumor activity.

Despite the success of programmed cell death-1 (PD-1) and PD-1 ligand (PD-L1) inhibitors in treating solid tumors, only a proportion of patients respond. Here, we describe a first-in-class bifunctional therapeutic molecule, STAR0602, that comprises an antibody targeting germline Vβ6 and Vβ10 T cell receptors (TCRs) fused to human interleukin-2 (IL-2) and simultaneously engages a nonclonal mode of TCR activation with costimulation to promote activation and expansion of αβ T cell subsets expressing distinct variable β (Vβ) TCR chains. In solution, STAR0602 binds IL-2 receptors in cis with Vβ6/Vβ10 TCRs on the same T cell, promoting expansion of human Vβ6 and Vβ10 CD4+ and CD8+ T cells that acquire an atypical central memory phenotype. Monotherapy with a mouse surrogate molecule induced durable tumor regression across six murine solid tumor models, including several refractory to anti-PD-1. Analysis of murine tumor-infiltrating lymphocyte (TIL) transcriptomes revealed that expanded Vβ T cells acquired a distinct effector memory phenotype with suppression of genes associated with T cell exhaustion and TCR signaling repression. Sequencing of TIL TCRs also revealed an increased T cell repertoire diversity within targeted Vβ T cell subsets, suggesting clonal revival of tumor T cell responses. These immunological and antitumor effects in mice were recapitulated in studies of STAR0602 in nonhuman primates and human ex vivo models, wherein STAR0602 boosted human antigen-specific T cell responses and killing of tumor organoids. Thus, STAR0602 represents a distinct class of T cell-activating molecules with the potential to deliver enhanced antitumor activity in checkpoint inhibitor-refractory settings.

Ectoine Globally Hypomethylates DNA in Skin Cells and Suppresses Cancer Proliferation.

Epigenetic modifications, mainly aberrant DNA methylation, have been shown to silence the expression of genes involved in epigenetic diseases, including cancer suppression genes. Almost all conventional cancer therapeutic agents, such as the DNA hypomethylation drug 5-aza-2-deoxycytidine, have insurmountable side effects. To investigate the role of the well-known DNA protectant (ectoine) in skin cell DNA methylation and cancer cell proliferation, comprehensive methylome sequence analysis, 5-methyl cytosine (5mC) analysis, proliferation and tumorigenicity assays, and DNA epigenetic modifications-related gene analysis were performed. The results showed that extended ectoine treatment globally hypomethylated DNA in skin cells, especially in the CpG island (CGIs) element, and 5mC percentage was significantly reduced. Moreover, ectoine mildly inhibited skin cell proliferation and did not induce tumorigenicity in HaCaT cells injected into athymic nude mice. HaCaT cells treated with ectoine for 24 weeks modulated the mRNA expression levels of Dnmt1, Dnmt3a, Dnmt3b, Dnmt3l, Hdac1, Hdac2, Kdm3a, Mettl3, Mettl14, Snrpn, and Mest. Overall, ectoine mildly demethylates DNA in skin cells, modulates the expression of epigenetic modification-related genes, and reduces cell proliferation. This evidence suggests that ectoine is a potential anti-aging agent that prevents DNA hypermethylation and subsequently activates cancer-suppressing genes.

CD38-Directed, Single-Chain T Cell-Engager Targets Leukemia Stem Cells through IFNγ-Induced CD38 Expression

Leukemia stem cells (LSCs), enriched in the CD34+CD38- AML population, are considered the main drivers of relapse in acute myeloid leukemia (AML). While more differentiated progenitors and bulk AML blasts reside in the CD34+CD38+ fraction, targeting CD38 in AML is less pursued, as LSCs are mainly CD38-. We show IFNγ (10 ng/ml) induced an increase in CD38 mRNA (mean fold change IFNγ over vehicle = 7.7, p=0.03, n=3) and surface CD38 (mean CD38 High% population IFNγ 56%±17 vs vehicle 16%±10, p<0.0001) in both CD34+ and CD34- AML primary blasts, and decreased the clonogenic activities of primary AML cells (colony forming unit IFNγ 50±36 vs vehicle 216±117, p=0.002, n=4) regardless of cytogenetics. scRNA and total RNA sequencing data in CD34+ AML primary blasts showed that upon IFNγ treatment, CD38 and Interferon Regulatory Factor 1 (IRF-1) were consistently the most upregulated genes compared to vehicle (>2.5 Log 2 Fold Change; p=0; FDR=0). Furthermore, gene set enrichment analysis (FDR=0) indicated IFNγ induced an upregulation of genes involved in apoptosis and DNA repair, and a suppression of genes in the pro-oncogenic Wnt beta catenin pathways. CD38 luciferase-based reporter assays, chromatin immunoprecipitation, and IRF-1 knockdown experiments in AML cells showed that, upon IFNγ treatment, IRF1 is pivotal in CD38 transcriptional regulation. Because CD4+T helper type 1 and CD8+ cytotoxic T cells release IFNγ once engaged on their target, we hypothesized a CD38-directed T cell engager will convert CD34+CD38- LSC into CD34+CD38+ blasts via the secreted IFNγ to kill LSCs and blasts. We created a single chain construct (BN-CD38), inserting a CD38 nanobody between the light and heavy chains of an anti-CD3 Fab using two short peptide linkers. Based on modeling, BN-CD38 reproduces the distance between T cell receptor and the major histocompatibility complex, mimicking the natural immunological synapse. Surface plasmon resonance and cytometry studies showed BN-CD38 had strong binding affinity to both CD38 (K D 4.77xE -10 M) and CD3 (K D 4.27xE -8 M). The IC 50 for BN-CD38 at 1:1 E:T at 24hrs in different CD38+ AML cell lines (n=6) was within the 0.01 to 0.1 ng/ml range, whereas the IC 50 of a non-binding CD38 construct (BN-CD38Mut) was 100-fold higher. By 24hrs of treatment, we noted >60% CD4+ and CD8+ T cell activation (CD69+, CD25+, IFNγ+) with BN-CD38 (e.g., CD69: 60%±27) in contrast to controls (e.g., CD69: IgG 7%±0.8 and BN-CD38Mut 11%±10, p<0.0001). In primary AML samples (n=10) BN-CD38 activated >70% T cells against autologous leukemia cells (CD69: BN-CD38 73%±16, BN-CD38Mut 10%±9, IgG 11%±10, p<0.0001). CyTOF analysis showed BN-CD38 but not controls significantly reduced CD34+CD38+ AML blasts (%change to IgG range: BN-CD38 -27 to -82% vs BN-CD38Mut 1% to 7%, p = 0.01) and CD34+CD38- LSCs (%change to IgG range: BN-CD38 -78 to -99% vs BN-CD38Mut -7 to 11%, p= 0.0003) and expanded CD8+ effector memory (p<0.01), CD8+ terminally differentiated effector memory CD45RA+ (p<0.05), NKT cells (p<0.01), and central memory Tregs (p<0.0001). Anti-human-IFNγ neutralizing antibody reverted BN-CD38 induced expression of CD38 and BN-CD38 killing activity on CD34+CD38- AML cells (p<0.05). BN-CD38 (2.5 mg/kg, i.v, n=6/group) co-injected weekly with healthy 3x10 6 T cells induced near-complete disease eradication in mice xenografted with luciferase-CD38+ THP1 (p=0.003) and U937 (p=0.004) cells. Long-term survival analysis showed 38% of BN-CD38-treated THP-1 engrafted mice achieved complete cure compared to IgG (OS median: BN-CD38 61.5d, control IgG 29d, p=0.0005). Consistently, BN-CD38 suppressed AML cell engraftment, splenomegaly, and prolonged survival in three patient-derived xenografts (PDX) with complex karyotypes including TP53 mutation (PDX1: OS median BN-CD38 undefined, BN-CD38Mut 28d (p=0.002), and IgG 27d (p=0.002); PDX2: OS median BN-CD38 39d, BN-CD38Mut and control IgG 32d (p<0.01); PDX3: OS median BN-CD38 undefined, BN-CD38Mut 92d (p=0.001), and IgG 81d (p=0.0007)). BN-CD38 did not affect normal hematopoietic stem cell clonogenicity and the development of multi-lineage human immune cells in CD34+ humanized mice. In sum, BN-CD38 induces an effective T cell synapse and IFNγ release, while concomitantly blocking the clonogenicity and inducing CD38 expression on LSCs. These studies suggest a new mechanism to unmask and target LSCs, providing the rationale for using BN-CD38 to effectively treat AML.

Single-Cell RNA-Seq Analysis Reveals Distinct Tumor and Immunosuppressive T Cell Phenotypes in CLL Patients Treated with Ibrutinib

Introduction: The development of Bruton tyrosine kinase inhibitors (BTKIs) and their introduction into clinical practice represents a major advance in the treatment of chronic lymphocytic leukemia (CLL). Monotherapy with ibrutinib or other BTKIs generally do not induce complete remissions or undetectable minimal residual disease (uMRD) even with extended therapy. The reason why some patients relapse, and some have minimal residual disease is unknown. Therefore, there is a need to understand the differences between ibrutinib sensitive and resistant CLL cells along with the immune microenvironment to identify novel therapeutic targets. Methods: Here, we investigate the cellular heterogeneity of peripheral blood mononuclear cells from patients with CLL treated with ibrutinib using single-cell RNA sequencing. To understand ibrutinib resistance mechanisms, samples were collected from 8 patients at baseline (before treatment) (1), where BTK C481S is present with low variant allele frequency (VAF) (2) and at time of relapse (3). Data from these were compared to ibrutinib sensitive patient samples at baseline (1) (before treatment), 3yr (2) and 5yr (3) on treatment timepoints. Single cells were isolated using the 10X Genomics 5' immune profiling kit. Data were processed with CellRanger v3.1.0. Poor quality cells were filtered out based on a high proportion of mitochondrial reads and low overall feature count. Sample variation was reduced using Batchelor to aid clustering. Clustering was performed using the partitioning and Leiden methods. Cluster top markers were calculated using functions from monocle3. Identity mapping was performed using Seurat and a published PBMC reference dataset. Results: Single-cell RNA sequencing revealed transcriptional heterogeneity within the B cell cluster (Fig 1A). Three subpopulations of B cells were defined based on differential representation in the ibrutinib-sensitive and resistant patients. Ibrutinib sensitive cells showed enrichment of B cell populations with upregulation of MHC I molecules and TNF family members. We also identified that inflammatory response and metabolic-related pathways were decreased, whereas cellular response to stress and DNA repair programs were increased in the ibrutinib resistance samples. Gene module analysis showed that ibrutinib sensitive samples had an increase in T cell activation, RNA splicing, cell adhesion and migration programs during therapy. We hypothesized that the emergence of BTK C481S mutation in ibrutinib-resistant cells would lead to a distinct transcriptional profile. Long-read sequencing was performed using Oxford Nanopore technology. BTK-mutant cells were identified based on the presence of two or more unique RNA molecules detected with the C481S mutation and these were predominantly identified in the low-VAF and relapse samples. However, there was no significant difference between BTK-mutant cells and the remaining B cells. To see if T cell phenotypes reflected the differences observed in B cell transcriptional state, we characterized and quantified T cell populations according to ibrutinib response and investigated the changes in the T cell population. Ibrutinib markedly increased CD4+ and CD8+ T cell numbers in both resistant and sensitive CLL patients. However, there was a significant (p<0.05) enrichment of Tregs in the ibrutinib-resistant samples. We found a connection between the immunosuppressive phenotype in the microenvironment demonstrating tumor's intrinsic immunological reactive program with exhausted T cells. Also, we found significant difference in the expression of checkpoint molecules and suppressive genes in T cells (Fig 1B) and NK cells. Conclusion: Our findings demonstrate that the B cell population in ibrutinib-resistant and ibrutinib sensitive CLL patients exhibit distinct transcriptional states. At the single-cell level, our findings demonstrate a systematic picture of diverse malignancy and microenvironmental changes in CLL. Overall, these findings provide an insight into the complex molecular and cellular interactions of CLL that may have a crucial role in BTKI resistance.

Integrating Proteomics and Functional Genomics to Identify Targets for Improving Glucocorticoid-Based Treatment of B-Lymphoblastic Leukemia

Glucocorticoids are a critical component of successful combination chemotherapy regimens for B-lymphoblastic leukemia (B-ALL). Despite advances in immunotherapies, glucocorticoid-based chemotherapy continues to be the essential frontline treatment of patients with B-ALL. This approach has led to high cure rates but at the risk of both acute, potentially life-threatening complications and chronic toxicities. Additionally, glucocorticoids remain ineffective for some patients with B-ALL, which predicts poor outcomes. To achieve better outcomes and reduce the toxicity of treatment, we take a functional genomics approach to understand how glucocorticoids induce cell death and identify strategies to make them more effective. Glucocorticoids work through the glucocorticoid receptor (GR), a ligand-activated transcription factor, to induce cell death in B-ALL. By integrating gene expression and RNA interference screening, we have identified both effector genes (genes whose regulation by glucocorticoids contributes to B-ALL cell death) and sources of resistance that can be targeted to improve treatment (Kruth et al Blood 2017). This identified the network of BCL-2 family proteins (e.g., BCL2, BCL2L11, BMF), whose regulation contributes to apoptosis in B-ALL. Surprisingly, it also showed that suppression of B-cell development genes (e.g., EBF1, PAX5, IL7R) contributes to B-ALL cell death. However, only a fraction of the genes that affect the glucocorticoid sensitivity of B-ALL are effector genes. We therefore sought to identify functional interaction partners of GR, defined as proteins that physically interact with GR and influence glucocorticoid sensitivity. To identify proteins that physically interact with GR, we used proximity labeling. We fused an ascorbate peroxidase domain (APEX2) to the N-terminus of GR and expressed it REH cells, a B-ALL cell line that does not express GR protein. After verifying that APEX2-GR restored glucocorticoid sensitivity in the REH cells, we performed proximity labeling in the presence and absence of dexamethasone. After optimizing these reactions, cells were lysed and labeled proteins were isolated using magnetic streptavidin beads and identified by mass spectrometry. We were able to detect more than 4,000 proteins that interact with GR, including hundreds that are enriched upon addition of dexamethasone. To identify functional interaction, we intersected the proteins enriched upon addition of dexamethasone with proteins shown by us to modulate glucocorticoid sensitivity in B-ALL (Kruth et al Blood 2017). Supporting our working model, we found functional interaction partners that are involved in B-cell development and apoptosis. We found that GR interacts with EBF1 and MEF2C, B-cell development transcription factors that restrain glucocorticoid-induced cell death. This indicates that GR activity and glucocorticoid sensitivity is directly blunted by B-cell development, and further supports the model that glucocorticoids restrain B-cell development. The mechanism of how these interactions influence glucocorticoid sensitivity will guide rational targeting to improve treatment of B-ALL both in general and in different B-ALL subtypes. Additionally, the mitochondrial voltage dependent anion channel 2 (VDAC2) also interacts with GR in a dexamethasone-dependent manner and is integral to glucocorticoid-induced cell death. VDAC2 is important for nucleating and perhaps triggering pore formation through BAK and BAX. How GR influences this activity is not known but suggests a non-genomic role of GR in glucocorticoid-induced cell death. In summary, integration of functional genomics and proteomics provides rich data sets that will help understand the mechanism of glucocorticoid-induced B-ALL cell death. Targeting of non-canonical (B-cell development) and non-genomic (VDAC2) functions of GR will allow for optimization of glucocorticoid-based chemotherapy regimens.

Iadademstat and Gilteritinib for the Treatment of FLT3-Mutated Relapsed/Refractory Acute Myeloid Leukemia: The Frida Study

Background: Frontline and relapsed /refractory (R/R) acute myeloid leukemia (AML) patients carrying fms-related tyrosine kinase 3 [FLT3]-mutations (FLT3 mut+) benefit from FLT3 inhibitors (FLT3i) that have emerged as active therapeutic agents for this subset of AML encompassing up to 30-40% of all AML cases. The FLT3i gilteritinib, as monotherapy for R/R AML pts, has improved outcomes but the duration of remission achieved is transient and often brief. The Phase 3 randomized ADMIRAL trial of gilteritinib demonstrated a 20% CR rate and 2.8 months event-free-survival (EFS) (Perl, et al., NEJM 2019). The development of new targeted agents which synergize with FLT3 inhibitors, and ideally also inhibit the emergence of resistance, is therefore a major medical need in AML. Iadademstat (iada/ORY-1001) is a specific, oral, potent, covalent inhibitor of the epigenetic Lysine-Specific Demethylase 1 (LSD1/KDMA1) enzyme. Epigenetic dysregulation is a hallmark of AML. Up to 70% of recurring mutations in AML patients target epigenetic regulators of gene expression, resulting in myeloid differentiation blockade and enhanced leukemic stem cell renewal, underscoring the potential of epigenetic therapies to change the natural history of the disease. Preclinically, iada produces striking synergy with FLT3is, particularly gilteritinib in FLT3 wild-type and FLT3 mut+ AML cells and in derived cell lines resistant to venetoclax, azacitidine and other FLT3is (Sacilotto et al., 2022 Eur J. of Cancer 174S1). The novel MOAs generating this synergy in combinations include activation of a pro-differentiating epigenetic transcriptional program with simultaneous suppression of MYC-driven target genes (Yashar, et al. 2023, Mol. Cancer Res). The recently completed Phase 2 study of iada in combination with azacitidine (ALICE) produced a high rate of composite remission, including complete remission (CR) and CR with incomplete count recovery (CRi) (mostly MRD negative), as well as durable responses, in treatment naïve, unfit AML patients (pts), without exacerbating the toxicity profile of azacitidine (Salamero et al., 2022, Blood S1). The FRIDA study (NCT05546580) aims to establish the safety, tolerability, and the recommended phase 2 dose (RP2D) of the combination of iada plus gilteritinib in FLT3 mut+ R/R AML. Methods: Adult pts with body weight ≥50 Kg and ECOG 0-2, with FLT3 mut+ R/R AML, after no more than 2 prior lines of therapy, are enrolling. Certain patients with prior exposure to FLT3is including gilteritinib may be eligible. Up to 18 pts, in a 3+3 escalation phase, will receive iada at doses of 75 to 150 ug, orally, in 5 days ON - 2 days OFF schedule, with continuous gilteritinib, at 120 mg/day orally. In the expansion phase, up to 14 pts at the selected safe and pharmacologically active dose/s (determined based on all available data from escalation pts including PK, target engagement (TE), safety, tolerability, and emerging activity) will be enrolled. Primary endpoints of the study are safety and RP2D determination, based on the same 5 criteria outlined above. Bayesian posterior probability efficacy monitoring will be performed periodically for each dose cohort in the expansion phase. Bayesian efficacy futility and early stopping boundary will be applied during the monitoring. Posterior probability criterion (Prob (CR>0.3) ≥ 0.60) at the end of the study will warrant additional development. The safety of the combination treatment will also be continuously evaluated during expansion following a Bayesian design stopping rule.Secondary endpoints include overall survival, EFS, CR, CR/CR with partial hematologic recovery (CRh), overall response rates, time to response, duration of response, and transfusion rate. Exploratory endpoints include measurable residual disease and gene mutational analysis. At the time of the submission, FRIDA is enrolling a second dose level cohort in escalation phase and plans to have the 15 sites open to accrual in the US by the end of 2023. Additional sites will be added for a subsequent randomized controlled double-blinded FRIDA 2 study to assess the efficacy of the iada and gilteritinib combination in R/R FLT3 mut+ AML.

Leukemia Blasts Induce NK-like Dysfunction in AML-Specific T Cells

Introduction Efforts to enhance T cell-mediated acute myeloid leukemia (AML) eradication face challenges possibly due to unique mechanisms of leukemia-induced T-cell dysfunction compared to exhaustion observed in solid tumors(Penter et al., Blood 2023). We previously showed the efficacy of Wilms' Tumor (WT) Antigen 1-specific T-cell receptor (TCR C4) inserted into CD8 + T cells (T TCR-C4) in preventing AML recurrence after hematopoietic cell transplantation (HCT) (Chapuis et al., Nat Med 2019). This led us to investigate the safety and efficacy of T TCR-C4 in post-HCT relapsed patients. Methods In this Phase I/II study conducted at Fred Hutchinson Cancer Center, 15 patients were enrolled and prospectively monitored for disease response. We manufactured T TCR-C4 using a lentiviral transduction system (Chapuis et al, Nat Med 2019) and performed comprehensive analyses of bone marrow (BM) and peripheral blood (PB) samples using single-cell RNA sequencing (scRNAseq) and TCRseq. Results T TCR-C4 treatment showed no survival advantage compared to matched controls (n =17). We hypothesized that active AML exacerbates T-cell dysfunction compared to patients who received prophylactic T TCR-C4 treatment. Identifying tumor-specific T cells poses a challenge in scRNAseq. By utilizing the WT1-transgene (WT1tg) sequence, we built a custom reference genome to dissect the transcriptional state of leukemia-specific cells. In PB scRNAseq profiles of 4 patients (24,472 CD8 + T cells) with post-infusion persistent blasts and T TCR-C4, we identified 5 main CD8 + T-cell signatures: Naïve, Stem-like, Activated-Exhausted, WT1tg positive, and NK-like (Figure 1). The NK-like signature has been associated with dysfunction in CAR-T cells (Good et al., Cell 2021). T TCR-C4 cells exhibited an NK-like skewing, although the expression of these markers was not as high as in endogenous NK-like CD8 + T cells. Trajectory inference revealed that NK-like endogenous CD8 + T cells represented the terminal state of differentiation, while T TCR-C4, despite expressing some NK-like markers, were in an intermediate state of differentiation along the trajectory (Figure 2). Combining scTCRseq data with proliferation signatures (Zheng et al., Science 2021) we found that WT1tg exhibited clonal expansion and proliferation, suggesting a functional intermediate state. We validated the 5 CD8 + T-cell differentiation states in BM samples (7,719 CD8 + T cells) from our cohort and in a publicly available large-scale BM CD8 + T-cell scRNAseq dataset (68,545 CD8+ T cells), with NK-like as the last differentiation state along the trajectory. Differential gene expression (DGE) of CD8 + T cells between active AML (AML+) and blasts-not-detectable (AML-) timepoints revealed reduced expression of stem-like and increased expression of NK-like markers in AML+ vs. AML- suggesting that leukemia drives NK-like terminal differentiation in T TCR-C4. To investigate if the NK-like derangement is AML-specific or a general dysfunction mechanism, we performed DGE between T TCR-C4 vs. TCR-T cells mesothelin-specific (T MSLN). The analysis revealed overexpression of exhaustion markers (CTLA4, PDCD1, TOX, CXCR6, NR4A2) in T MSLN and NK-like markers (NKG7, KLRD1, KLRG1, S1PR5) in T TCR-C4. To explore leukemia immune-escape mechanisms, we studied blasts gene expression. One patient who initially responded to T TCR-C4, exhibited monocytic blast differentiation upon relapse. We found overexpression of immune suppression genes (IL10, TNF, reactive oxygen species) in monocytic blasts at relapse vs. blasts before T TCR-C4 infusion. Published AML scRNAseq confirmed NK-like skewing in monocytic leukemia patients. Ongoing in-vitro assays will help to dissect the mechanisms of T TCR-C4 dysfunction. Discussion Our study reveals a novel AML-specific NK-like signature of T-cell dysfunction, exacerbated when AML blasts are present, potentially contributing to the restricted efficacy observed in TCR-T cell therapies. We observed that monocytic blast differentiation and the subsequent formation of an immune-suppressive environment imply the existence of intricate immune escape mechanisms. This study provides a critical foundation for the development of innovative interventions aimed at circumventing the dysfunction observed in cell-based therapies and fostering long-term remission in AML patients.

Phenotype Molding of Monocytes in the Multiple Myeloma Microenvironment

Human monocytes are mononuclear phagocytes that comprise a major population of innate immune cells. Various monocyte-derived cells, including osteoclasts, dendritic cells, and macrophages, have been implicated in immunosuppression within the multiple myeloma (MM) tumor microenvironment (TME). Despite recent developments in understanding the link between monocyte-derived cells and immunosuppression in myeloma, there are still critical knowledge gaps in the association between monocytes themselves and immunosuppression within the context of MM. Specifically, the functional and developmental alterations of monocytes in the MM TME have not been explored based on the single-cell studies. In the present study, we generated a comprehensive single-cell map of peripheral blood (PB) and bone marrow (BM) monocytes from both healthy donors (HDs) and MM patients. Specifically, Heparin-anticoagulated PB samples or BM aspirates were collected from seven newly diagnosed MM (NDMM) patients and healthy donors (HDs). Paired BM aspirates were obtained from 10 MM patients at the time of diagnosis and after induction therapy. Differential expression analysis showed that type I interferon (IFN) response was upregulated in MM monocytes. And BM monocytes derived from MM patients exhibited impaired T cell attraction abilities and demonstrated an elevated capacity for suppressing T cell function. Moreover, immune checkpoint molecules, including CD274 (encoding PD-L1) and PDCD1LG2 (encoding PD-L2), were significantly upregulated in MM monocytes. Additionally, a positive correlation was observed between type I IFN response and T cell suppression capacity of MM monocytes. Trajectory analyses revealed an IFN-focused disruption of developmental pathways of both BM and PB monocytes in MM. Lastly, we included 10 MM patients at the time of diagnosis and after induction therapy to monitor transcriptional changes over time. Our results revealed that anti-tumor therapy successfully mitigated the excessive type-I interferon response of BM monocytes in MM. We document that MM monocytes exhibited an augmented type-I IFN response compared to monocytes from HDs, while their overall heterogeneity remained unchanged. Furthermore, we extended the knowledge on immunosuppression of the MM TME that the excessive type-I IFN response in MM had a significant impact on the differentiation of both BM and PB monocytes, leading to T-cell immunosuppression, specifically in BM monocytes. In the cohort that we longitudinally followed, we observed a decrease in the type-I IFN response across nearly all monocyte subsets in MM BM monocytes after induction therapy, accompanied by a reduction in the expression of the T-cell suppression gene set. In sum, our findings indicates that monocytes in the BM promotes the immunosuppression of the MM TME and represents potential therapeutic targets for modulating the MM TME.

Dynamic Interplay of Metabolic and Transcriptional Responses in Shrimp during Early and Late Infection Stages of Enterocytozoon hepatopenaei (EHP)

Enterocytozoon hepatopenaei (EHP) is a microsporidian parasite that infects Litopenaeus vannamei, causing severe hepatopancreatic microsporidiosis (HPM) and resulting in significant economic losses. This study utilizes a combined analysis of transcriptomics and metabolomics to unveil the dynamic molecular interactions between EHP and its host, the Pacific white shrimp, during the early and late stages of infection. The results indicate distinct immunological, detoxification, and antioxidant responses in the early and late infection phases. During early EHP infection in shrimp, immune activation coincides with suppression of genes like Ftz-F1 and SEPs, potentially aiding parasitic evasion. In contrast, late infection shows a refined immune response with phagocytosis-enhancing down-regulation of Ftz-F1 and a resurgence in SEP expression. This phase is characterized by an up-regulated detoxification and antioxidant response, likely a defense against the accumulated effects of EHP, facilitating a stable host–pathogen relationship. In the later stages of infection, most immune responses return to baseline levels, while some immune genes remain active. The glutathione antioxidant system is suppressed early on but becomes activated in the later stages. This phenomenon could facilitate the early invasion of EHP while assisting the host in mitigating oxidative damage caused by late-stage infection. Notably, there are distinctive events in polyamine metabolism. Sustained up-regulation of spermidine synthase and concurrent reduction in spermine levels suggest a potential role of polyamines in EHP development. Throughout the infection process, significant differences in genes such as ATP synthase and hexokinase highlight the continuous influence on energy metabolism pathways. Additionally, growth-related pathways involving amino acids such as tryptophan, histidine, and taurine are disrupted early on, potentially contributing to the growth inhibition observed during the initial stages of infection. In summary, these findings elucidate the dynamic interplay between the host, Litopenaeus vannamei, and the parasite, EHP, during infection. Specific phase differences in immune responses, energy metabolism, and antioxidant processes underscore the intricate relationship between the host and the parasite. The disruption of polyamine metabolism offers a novel perspective in understanding the proliferation mechanisms of EHP. These discoveries significantly advance our comprehension of the pathogenic mechanisms of EHP and its interactions with the host.

Taste receptor type 1 member 3 regulates Western diet-induced male infertility

Western diet (WD), characterized by a high intake of fats and sugary drinks, is a risk factor for male reproductive impairment. However, the molecular mechanisms underlying this remain unclear. Taste receptor type 1 member 3 (TAS1R3), activated by ligands of WD, is highly expressed in extra-oral tissues, particularly in the testes. Here, we investigated to determine the effects of WD intake on male reproduction and whether TAS1R3 mediates WD-induced impairment in male reproduction. Male C57BL/6 J wild-type (WT) and Tas1r3 knockout (KO) mice were fed either a normal diet and plain water (ND) or a 60 % high-fat-diet and 30 % (w/v) sucrose water (WD) for 18 weeks (n = 7–9/group). Long-term WD consumption significantly impaired sperm count, motility and testicular morphology in WT mice with marked Tas1r3 overexpression, whereas Tas1r3 KO mice were protected from WD-induced reproductive impairment. Testicular transcriptome analysis revealed downregulated AMP-activated protein kinase (AMPK) signaling and significantly elevated AMPK-targeted nuclear receptor 4A1 (Nr4a1) expression in WD-fed Tas1r3 KO mice. In vitro studies further validated that Tas1r3 knockdown in Leydig cells prevented the suppression of Nr4a1 and downstream steroidogenic genes (Star, Cyp11a1, Cyp17a1, and Hsd3b1) caused by high glucose, fructose, and palmitic acid levels, and maintained the levels of testosterone. Additionally, we analyzed the public human dataset to assess the clinical implications of our findings and confirmed a significant association between TAS1R3 and male-infertility-related diseases. Our findings suggest that TAS1R3 regulates WD-induced male reproductive impairment via the AMPK/NR4A1 signaling and can be a novel therapeutic target for male infertility.

Enhancing productivity of Chinese hamster ovary (CHO) cells: synergistic strategies combining low-temperature culture and mTORC1 signaling engineering

Introduction: The growing demand for recombinant proteins in medicine has prompted biopharmaceutical companies to seek ways to maximize the manufacturing process. Despite its known negative impact on cell growth, temperature shift (TS) has emerged as a cost-effective strategy to enhance protein quantity and quality in Chinese Hamster Ovary cells (CHO). As cells adapt their growth and protein synthesis rate to the environment through influencing mTOR complex 1 (mTORC1), here we evaluated the potential of mTORC1 signaling engineering to improve the production of granulocyte-macrophage colony-stimulating factor (GM-CSF) protein in stable CHO cells at low temperature.Methods: First, the expression of genes that negatively control mTORC1 functions in response to environmental fluctuations, including TSC1, AMPK, MAPKAPK5, and MARK4 genes, was assessed via real-time qPCR in CHO-K1 after a temperature shift from 37°C to 30°C. Then, plasmids harboring the shRNAs targeting these genes were constructed into the PB513B-1 plasmid with expression driven by either the constitutive CMV promoter or the cold-inducible HSP90 promoter. Finally, the impact of transient gene downregulation was evaluated on GM-CSF and mTOR proteins productivity in GM-CSF-producing CHO-K1 cells using ELISA and Western-blot assays, respectively. The growth rate of the transfected cells at the two temperatures was evaluated using flow cytometry.Results: Hypothermic conditions promote the upregulation of mTORC1 inhibitor genes, especially TSC1 and MAPKAPK5, while downregulating S6K, a key effector of the mTORC1 signaling pathway, in CHO-K1 cells. Transcription and protein levels of mTOR increased upon transfection, “pB513-b CMV-P/4shRNAs/GFP” plasmid, “pB513-bHSP90-P/4sh-RNAs/GFP” and pB513B-1 plasmid as mock group in GM-CSF-producing CHO-K1 cells (approximately 60%), along with a high transcript level of S6K. Cell growth-related characteristics were improved, albeit with distinct effects at different temperatures. Notably, these changes were more efficient at 30°C when utilizing the HSP90 promoter, resulting in a three-fold increase in GM-CSF production after 3 days.Conclusion: This study highlights the importance of temperature regulation and mTORC1 modulation in CHO cellular processes, particularly in recombinant protein production. Understanding these mechanisms paves the way for developing innovative strategies to enhance cell growth, protein synthesis, and overall bioprocess performance, particularly in manufacturing human therapeutic proteins.

CsMYB67 participates in the flavonoid biosynthesis of summer tea leaves.

Flavonoids are important compounds in tea leaves imparting bitter and astringent taste, which also play key roles in tea plants responding to environmental stress. Our previous study showed that the expression level of CsMYB67 was positively correlated with the accumulation of flavonoids in tea leaves as exposed to sunlight. Here, we newly reported the function of CsMYB67 in regulating flavonoid biosynthesis in tea leaves. CsMYB67 was localized in the nucleus and responded to temperature. The results of transient expression assays showed the co-transformation of CsMYB67 and CsTTG1 promoted the transcription of CsANS promoter in the tobacco system. CsTTG1 was bound to the promoter of CsANS based on the results of yeast one-hybrid (Y1H) and transient expression assays, while CsMYB67 enhanced the transcription of CsANS through protein interaction with CsTTG1 according to the results of yeast two-hybrid (Y2H) and bimolecular fluorescence complementation (BiFC). Thus, CsMYB67-CsTTG1 module enhanced the anthocyanin biosynthesis through up-regulating the transcription of CsANS. Besides, CsMYB67 also enhanced the transcription of CsFLS and CsUFGT through forming transcription factor complexes. The function of CsMYB67 on flavonoid biosynthesis in tea leaves was validated by gene suppression assay. As CsMYB67 was suppressed, the transcriptional level of CsFLS was greatly reduced, leading to a significant increase in the contents of total catechins and total anthocyanidins. Hence, CsMYB67 plays an important role in regulating the downstream pathway of flavonoid biosynthesis in summer tea leaves.