Single-Cell Sequencing Technology Reveals the Heterogeneity of the Immune Microenvironment and Key Molecules of Drug Resistance in Angioimmunoblastic T-Cell Lymphoma

Objective: Angioimmunoblastic T-cell lymphoma (AITL) is a common subtype of peripheral T-cell lymphoma (PTCL). AITL is an aggressive malignancy with a poor prognosis, and its clinical manifestations vary greatly among individuals. The current chemotherapy regimens based on anthracycline show limited efficacy, and there is no best rescue treatment for patients with relapsed and refractory (RR) AITL. In addition, the lack of optimal AITL models in vitro greatly limits the basic research on the mechanism of disease occurrence and progression, and also hinders the development of new drugs and preclinical trials. Our study aims to deeply analyze the tumor heterogeneity and clonal evolution of AITL, discovering key molecules of drug resistance and potential theraputic targets. Methods : We detected fresh lymph node samples from newly diagnosed and relapsed/ refractory AITL patients using single-cell RNA sequencing, combined with imaging mass cytometry (IMC) and whole exome sequencing. IMC was performed to analyze the spatial position relationship and protein expression characteristics of different subgroups in the tumor microenvironment of AITL. In addition, AITL patient-derived organoid model was established to study the regulatory role of YY1 and its inhibitors in relapsed and refractory AITL. Results : ScRNA-seq revealed the significant differences in the tumor microenvironment of newly diagnosed and RR-AITL patients (Fig A,B). B cells and myeloid subgroups may play important roles in the development of AITL (Fig C). Transcription factor YY1, highly expressed in follicular helper T cell (Tfh) of RR-AITL patients, promoted the proliferation and drug resistance of AITL cells (Fig E,F). The proportion of CD8+ T cells in the RR-AITL sample was reduced, while the proportion of Treg was increased, as well as the depletion of T cells (Fig G,H). Furthermore, the stemness of B cells in RR-AITL was enhanced and exhibits significant malignant characteristics (Fig C,I-K). We also found decreased interaction in RR-AITL samples (Fig L,M). Moreover, for the first time, we established AITL patient-derived organoid models that can be stablely cultured in vitro (Fig N). On this basis, we could further clarify the important roles of transcription factor YY1 in the drug resistance of AITL, evaluate the cytotoxic effect of YY1 inhibitor NP-001 on AITL tumor cells. Conclusion : In conclusion, our study revealed the differences between newly diagnosed and relapsed /refractory AITL in terms of immune microenvironment, single-cell transcriptomes, and signal pathway activation. YY1 may serve as an novel target for drug resistance for RR-AITL patients. These findings may provide a theoretical foundation for improving the clinical treatment of AITL. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Kynurenine derivative 3-HAA is an agonist ligand for transcription factor YY1

The 3-hydroxyanthranilic acid (3-HAA), a derivative of kynurenine, was reported to suppress tumor growth. However, the function of 3-HAA largely remains unclear. Here, we report that 3-hydroxyanthranilic acid (3-HAA) is lower in tumor cells, while adding exogenous 3-HAA induces apoptosis in hepatocellular carcinoma by binding YY1. This 3-HAA binding of YY1 leads to phosphorylation of YY1 at the Thr 398 by PKCζ, concomitantly enhances YY1 chromatin binding activity to increase expression of target genes. These findings demonstrate that 3-HAA is a ligand of YY1, suggesting it is a promising therapeutic candidate for HCC.

Inhibition of pyrimidine biosynthesis targets protein translation in AML

The mitochondrial enzyme dihydroorotate dehydrogenase (DHODH) catalyzes one of the rate-limiting steps in de novo pyrimidine biosynthesis, a pathway that provides essential metabolic precursors for nucleic acids, glycoproteins and phospholipids. DHODH inhibitors (DHODHi) are clinically used for autoimmune diseases and are emerging as a novel class of anti-cancer agents, especially in acute myeloid leukemia (AML) where pyrimidine starvation was recently shown to reverse the characteristic differentiation block in AML cells. Herein we show that DHODH blockade rapidly shuts down protein translation in leukemic stem cells (LSCs) by down-regulation of the multi-functional transcription factor YY1, has potent activity against AML in vivo and is well tolerated with minimal impact on normal blood development. Moreover, we find that ablation of CDK5, a gene that is recurrently deleted in AML and related disorders, increases the sensitivity of AML cells to DHODHi. Our studies provide important molecular insights and identify a potential biomarker for an emerging strategy to target AML.

LINC01224 promotes colorectal cancer progression through targeting miR-485-5p/MYO6 axis

Background Long noncoding RNAs (lncRNAs) are related to colorectal cancer (CRC) development. However, the role and mechanism of lncRNA LINC01224 in CRC development are largely unknown. Methods LINC01224, Yin Yang 1 (YY1), microRNA (miR)-485-5p, and myosins of class VI (MYO6) levels were examined using quantitative reverse transcription polymerase chain reaction and western blotting. Functional analyses were processed through CCK-8, colony formation, flow cytometry, transwell, and xenograft analyses. Dual-luciferase reporter, chromatin immunoprecipitation (ChIP), RNA immunoprecipitation, and pull-down assays were conducted to analyze the binding interaction. Results LINC01224 abundance was elevated in CRC tissue samples and cell lines. Elevated LINC01224 might indicate the lower 5-year overall survival in 52 CRC patients. LINC01224 was upregulated via the transcription factor YY1. LINC01224 knockdown restrained CRC cell proliferation, migration, and invasion and increased apoptosis. MiR-485-5p was sponged by LINC01224, and miR-485-5p downregulation relieved the influence of LINC01224 interference on CRC progression. MYO6 was targeted via miR-485-5p and regulated via LINC01224/miR-485-5p axis. MiR-485-5p overexpression suppressed CRC cell proliferation, migration, and invasion and facilitated apoptosis. MYO6 upregulation mitigated the role of miR-485-5p. LINC01224 knockdown decreased xenograft tumor growth. Conclusion YY1-induced LINC01224 regulates CRC development via modulating miR-485-5p/MYO6 axis.

YY1 and RTCB in mouse uterine decidualization and embryo implantation

As a multifunctional transcription factor, YY1 regulates the expression of many genes essential for early embryonic development. RTCB is an RNA ligase that plays a role in tRNA maturation and Xbp1 mRNA splicing. YY1 can bind in vitro to the response element in the proximal promoter of Rtcb and regulate Rtcb promoter activity. However, the in vivo regulation and whether these two genes are involved in the mother-fetal dialogue during early pregnancy remain unclear. In this study, we validated that YY1 bound in vivo to the proximal promoter of Rtcb in mouse uterus of early pregnancy. Moreover, via building a variety of animal models, our study suggested that both YY1 and RTCB might play a role in mouse uterus decidualization and embryo implantation during early pregnancy.

Distinct requirements for Pho, Sfmbt, and Ino80 for cell survival in Drosophila

The Drosophila proteins Pleiohomeotic (Pho) and its paralog Pho-like (Phol) are the homologs of the mammalian transcription factor YY1. Pho and Phol are subunits of the Polycomb group protein complex PhoRC and they are also stably associated with the INO80 nucleosome remodeling complex. Drosophila lacking both Pho and Phol arrest development as larvae with small misshaped imaginal discs. The basis of this phenotype is poorly understood. We find that in pho phol mutant animals cells retain the capacity to proliferate but show a high incidence of apoptotic cell death that results in tissue hypoplasia. Clonal analyses establish that cells stringently require Pho and Phol to survive. In contrast, the PhoRC subunit Sfmbt and the ATP-dependent nucleosome remodeling factor Ino80 are not essential for cell viability. Pho and Phol, therefore, execute their critical role for cell survival through mechanisms that do not involve Sfmbt function or INO80 nucleosome remodeling.

LINC00842 inactivates transcription co-regulator PGC-1α to promote pancreatic cancer malignancy through metabolic remodelling

The molecular mechanism underlying pancreatic ductal adenocarcinoma (PDAC) malignancy remains unclear. Here, we characterize a long intergenic non-coding RNA LINC00842 that plays a role in PDAC progression. LINC00842 expression is upregulated in PDAC and induced by high concentration of glucose via transcription factor YY1. LINC00842 binds to and prevents acetylated PGC-1α from deacetylation by deacetylase SIRT1 to form PGC-1α, an important transcription co-factor in regulating cellular metabolism. LINC00842 overexpression causes metabolic switch from mitochondrial oxidative catabolic process to fatty acid synthesis, enhancing the malignant phenotypes of PDAC cells. High LINC00842 levels are correlated with elevated acetylated- PGC-1α levels in PDAC and poor patient survival. Decreasing LINC00842 level and inhibiting fatty acid synthase activity significantly repress PDAC growth and invasiveness in mouse pancreatic xenograft or patient-derived xenograft models. These results demonstrate that LINC00842 plays a role in promoting PDAC malignancy and thus might serve as a druggable target.

Studies on the Role of the Transcription Factor Tcf21 in the Transdifferentiation of Parietal Epithelial Cells into Podocyte-Like Cells

Background/Aims: Podocyte differentiation is essential for proper blood filtration in the kidney. It is well known that transcription factors play an essential role to maintain the differentiation of podocytes. The present study is focused on the basic helix-loop-helix (bHLH) transcription factor Tcf21 (Pod1) which is essential for the development of podocytes in vivo. Since parietal epithelial cells (PECs) are still under debate to be progenitor cells which can differentiate into podocytes, we wanted to find out whether the expression of Tcf21 induces a transition of PECs into podocytes. Methods: We transfected PECs with Tcf21-GFP and analyzed the expression of PEC- and podocyte-specific markers. Furthermore, we performed ChIP-Seq analysis to identify new putative interaction partners and target genes of Tcf21. Results: By gene arrays analysis, we found that podocytes express high levels of Tcf21 in vivo in contrast to cultured podocytes and parietal epithelial cells (PECs) in vitro. After the expression of Tcf21 in PECs, we observed a downregulation of specific PEC markers like caveolin‑1, β-catenin and Pax2. Additionally, we found that the upregulation of Tcf21 induced multi-lobulation of cell nuclei, budding and a formation of micronuclei (MBM). Furthermore, a high number of PECs showed a tetraploid set of chromosomes. By qRT-PCR and Western blot analysis, we revealed that the transcription factor YY1 is downregulated by Tcf21. Interestingly, co-expression of YY1 and Tcf21 rescues MBM and reduced tetraploidy. By ChIP-Seq analysis, we identified a genome-wide Tcf21-binding site (CAGCTG), which matched the CANNTG sequence, a common E-box binding motif used by bHLH transcription factors. Using this technique, we identified additional Tcf21 targets genes that are involved in the regulation of the cell cycle (e.g. Mdm2, Cdc45, Cyclin D1, Cyclin D2), on the stability of microtubules (e.g. Mapt) as well as chromosome segregation. Conclusion: Taken together, we demonstrate that Tcf21 inhibits the expression of PEC-specific markers and of the transcription factor YY1, induces MBM as well as regulates the cell cycle suggesting that Tcf21 might be important for PEC differentiation into podocyte-like cells.

Effect of the transcription factor YY1 on the development of pancreatic endocrine and exocrine tumors: a narrative review

Pancreatic tumors are classified into endocrine and exocrine types, and the clinical manifestations in patients are nonspecific. Most patients, especially those with pancreatic ductal adenocarcinoma (PDAC), have lost the opportunity to receive for the best treatment at the time of diagnosis. Although chemotherapy and radiotherapy have shown good therapeutic results in other tumors, their therapeutic effects on pancreatic tumors are minimal. A multifunctional transcription factor, Yin-Yang 1 (YY1) regulates the transcription of a variety of important genes and plays a significant role in diverse tumors. Studies have shown that targeting YY1 can improve the survival time of patients with tumors. In this review, we focused on the mechanism by which YY1 affects the occurrence and development of pancreatic tumors. We found that a YY1 mutation is specific for insulinomas and has a role in driving the degree of malignancy. In addition, changes in the circadian network are a key causative factor of PDAC. YY1 promotes pancreatic clock progression and induces malignant changes, but YY1 seems to act as a tumor suppressor in PDAC and affects many biological behaviors, such as proliferation, migration, apoptosis and metastasis. Our review summarizes the progress in understanding the role of YY1 in pancreatic endocrine and exocrine tumors and provides a reasonable assessment of the potential for therapeutic targeting of YY1 in pancreatic tumors.

The DNA-helicase HELLS drives ALK− ALCL proliferation by the transcriptional control of a cytokinesis-related program

Deregulation of chromatin modifiers, including DNA helicases, is emerging as one of the mechanisms underlying the transformation of anaplastic lymphoma kinase negative (ALK−) anaplastic large cell lymphoma (ALCL). We recently identified the DNA-helicase HELLS as central for proficient ALK−ALCL proliferation and progression. Here we assessed in detail its function by performing RNA-sequencing profiling coupled with bioinformatic prediction to identify HELLS targets and transcriptional cooperators. We demonstrated that HELLS, together with the transcription factor YY1, contributes to an appropriate cytokinesis via the transcriptional regulation of genes involved in cleavage furrow regulation. Binding target promoters, HELLS primes YY1 recruitment and transcriptional activation of cytoskeleton genes including the small GTPases RhoA and RhoU and their effector kinase Pak2. Single or multiple knockdowns of these genes reveal that RhoA and RhoU mediate HELLS effects on cell proliferation and cell division of ALK−ALCLs. Collectively, our work demonstrates the transcriptional role of HELLS in orchestrating a complex transcriptional program sustaining neoplastic features of ALK−ALCL.