Introduction: Immune-mediated inflammatory diseases (IMIDs) constitute a clinically heterogeneous group with a disruption of the immune system that provokes inflammation of any organ system on a chronic basis. Previous observational investigations have demonstrated an association between a person's or their family's medical background of IMIDs and a higher likelihood of developing Hodgkin's lymphoma (HL). Nevertheless, the exact causal connection between specific IMIDs and HL remains ambiguous. Therefore, the investigation aims to explore the causal effects between IMIDs and HL. Methods: A two-sample Mendelian randomization (TSMR) approach was employed in this study, using publicly available genome-wide association study summary statistics. The inverse variance weighted (IVW) method was the primary approach used for the Mendelian randomization (MR) analysis. Furthermore, supplementary statistical approaches such as MR-Egger, simple mode, weighted median, and MR-PRESSO were employed. In order to identify potential heterogeneity and pleiotropy, several tests were conducted, including Cochran's Q test, the MR-Egger regression test, and the MR-PRESSO test. Moreover, the leave-one-out method was implemented to examine the reliability of the MR results. Results: Based on the IVW analysis the findings revealed a positive causal link at risk for multiple sclerosis (MS) and HL [odds ratio (OR) = 1.281; 95% CI = 1.087-1.510, p = 0. 003] and a negative causal link at risk for type 1 diabetes (T1D) and HL (OR = 0.853; 95% CI = 0.776-0.937, p = 0.001). However, no significant causal relationship was found between the risk of HL and the other eight IMIDs, including inflammatory bowel diseases (OR = 1.034; 95%CI = 0.910-1.176, p = 0.606), systemic lupus erythematosus (OR = 1.041; 95%CI = 0.944-1.149, p = 0.422), Crohn's disease (OR = 0.952; 95%CI = 1.840-1.078, p = 0.436), ulcerative colitis (OR = 1.042; 95%CI = 0.874-1.242, p = 0.644), primary sclerosing cholangitis (OR = 1.061; 95%CI = 0.937-1.202, p = 0.349), primary biliary cirrhosis (OR = 1.046; 95%CI = 0.918-1.192, p = 0.495), psoriasis (OR = 0.989; 95%CI = 0.954-1.026, p = 0.565), celiac disease (OR = 1.198; 95%CI = 0.911-1.574, p = 0.195). Figure 1 displayed a detailed visual representation of the findings obtained in the study. No indication was made of any potential heterogeneity or horizontal pleiotropy. Besides, no outliers were discovered in the MR using MR-PRESSO. In addition, the leave-one-out analysis indicates that the MR estimates are not driven by any single SNP, suggesting that the observed associations are robust and reliable. Conclusions: The present investigation emphasizes an increased susceptibility of MS to the risk of HL and a potential protective effect of T1D against the risk of HL. In light of these findings, patients with MS receiving immunosuppressive therapy are highly suggested to undergo periodic physical evaluations and lymphoma surveillance. Further investigations are warranted to explore the potential mechanisms of MS and T1D on the development of HL.

Introduction As a member of the protein arginine N-methyltransferase family, protein arginine methyltransferase 1 (PRMT1) plays a crucial regulatory role in the etiology of malignant neoplasms. PRMT1-mediated protein methylation promoted the maintenance of acute myeloid leukemia, which revealed the promising potency of PRMT1 inhibitors. Recent study suggested that dysregulation of PRMT5 promotes Richter's transformation in chronic lymphocytic leukemia (CLL). However, the effects of PRMT1 in the tumorigenesis and progression of CLL still remained ill-defined. Hence, the aim of this study was to investigate the clinical significance and mechanisms of PRMT1 underlying the development of CLL. Methods Peripheral blood samples were collected from 79 newly diagnosed CLL patients (47 males and 32 females; age range 39-85 years, median 63 years) in Shandong Provincial Hospital CLL (SPHCLL) cohort with informed consent. CRISPR-Cas9 technology was used to stably knockout PRMT1 in CLL cells. A label free quantitative proteomics analysis was implemented to reveal the protein methylation mediated by PRMT1. Assessment of cell viability, apoptosis and cell cycle were analyzed by cell counting kit-8, annexin V-PE/7AAD and PI/ RNase staining, respectively. This study was approved by the Medical Ethics Committee of Shandong Provincial Hospital. Results This study examined the expression of PRMT1 in SPHCLL and GEO databases, and discovered the upregulation of PRMT1 mRNA in CLL cells. Aberrantly elevated expression of PRMT1 was observed in a cohort of newly diagnosed CLL patients than healthy donors in mRNA level (donors vs. CLL patients, 0.07±0.06 vs. 0.20 ±0.21, p=0.013). Furthermore, increased expression of PRMT1 was correlated with inferior prognosis in two long-term follow-up cohorts of CLL patients (HR=2.80, p=0.002, and HR=1.551, p=0.009). To elucidate the functional significance of PRMT1 in CLL, we established stable PRMT1 knockdown cells using lentiviral shRNAs and PRMT1 knockout cells using CRISPR/Cas9 technology. The suppression of PRMT1 remarkably inhibited cell proliferation, induced cell apoptosis and blocked cell cycle at G1/S phase in CLL cells. Furthermore, it was observed that C7280948, a selective inhibitor of PRMT1, resulted in the defective proliferation of CLL primary cells in a dose-dependent manner. We subsequently performed pre-clinical investigations of C7280948 in the orthotopic CLL xenograft murine model. Notably, the administration of C7280948 significantly diminished the leukemia burden and induced the lessened degree of splenomegaly in comparison to the control group (Fig. 1A). Moreover, a reduction in the population of CLL cells was detected in bone marrow and spleen of mice treated with C7280948. To decipher the role of PRMT1 in the pathogenesis of CLL, the label free quantitative proteomics analysis was conducted on PRMT1-deficent and control MEC1 cells. Knockdown of PRMT1 diminished asymmetric dimethylarginine of some proteins in CLL cells. In accordance with the results of proteomics analysis, microtubule associated serine/threonine kinase 1 (MAST1) was selected as a candidate target for further investigation. The methylation site of MAST1 protein was identified as R802 (Fig. 1B). Increased expression of MAST1 was correlated with inferior prognosis of CLL patients ( p<0.001), indicating the essential role of MAST1 in the progression of CLL. These results supported the hypothesis that PRMT1 interacted with MAST1 and enhanced the activity of MAST1 via methylating this oncoprotein in CLL cells. Conclusion The present study provides robust evidence for the oncogenic function of PRMT1 in the pathogenesis of CLL, highlighting the potential therapeutic efficacy of the selective PRMT1 inhibitor C7280948. Collectively, the targeted inhibition of PRMT1 exhibits promising prospects for the management of CLL patients.

Introduction Chronic lymphocytic leukemia (CLL) is the most common adult leukemia in the Western world, and although the advent of novel targeted drugs has greatly improved the prognosis of CLL patients, CLL remains incurable at present. Improving the risk stratification of CLL patients and finding new CLL therapeutic targets are urgent issues to be addressed. Methods We performed untargeted metabolomic analysis using LC-MS on sera from CLL patients and healthy controls (HC). Statistical analysis was used to screen for differential metabolites (DMs). Differentially expressed genes (DEGs) were also screened for enrichment pathway enrichment relying on transcriptome data analysis through the GEO database. Finally, DMs and DEGs were analyzed together to explore CLL metabolic alteration pathways. In this study, we targeted the gene ENPP2. Peripheral blood samples from 82 CLL patients were collected at the Department of Hematology in Shandong Provincial Hospital. Expressions levels of ENPP2 in CLL patients were determined by quantitative RT-PCR. In addition, cell viability and apoptosis were assessed by cell counting kit-8 and annexin V-PE/7AAD staining. Results A total of 52 differential metabolites were annotated, 40 of them were up-regulated and 12 were down-regulated. There were significant differences in sphingolipids (SP), glycerolipids (GL), glycerophospholipids (GP), and fatty acids (FA) levels in CLL patients compared to health controls. Fifty-two differential metabolites between samples from CLL patients and HC were analyzed by pathway analysis using metaboanalyst v5.0. KEGG enrichment showed that differential metabolites in CLL patients and HC were mainly annotated and enriched in human diseases, metabolism, and organismal systems (Figure 1A). Furthermore, we obtained differential expression information from the GEO database for CLL patients with HC. A total of 539 DEGs were screened, of which 279 were up-regulated and 260 were down-regulated. A total of 257 KEGG pathways were enriched. In the current work, a joint analysis of the two histological data was performed to validate each other. A total of 20 KEGG pathways were repeatedly obtained from metabolomics and transcriptomics data-based pathway analysis, including pathways in cancer, insulin resistance, long-term depression, regulation of lipolysis in adipocytes metabolic pathways, glycerophospholipid metabolism, glycerolipid metabolism, sphingolipid metabolism, arachidonic acid metabolism, ether lipid metabolism, inositol phosphate metabolism, choline metabolism in cancer, phospholipase D signaling pathway. A PPI network was constructed for the identified significantly different genes, from which we also screened for the gene ENPP2, a secreted lysophospholipase D, that we desired to investigate further (Figure 1B). We validated the difference in ENPP2 expression between CLL patients and healthy donors in the GEO database and its guiding effect on CLL prognosis. The results showed that elevated expression of ENPP2 in CLL patients and relatively high expression in CLL patients implied a worse prognosis. In addition, to validate the role of ENPP2 in CLL, we did the further investigation in CLL patients in our own center. Aberrantly increased expression of ENPP2 were detected in CLL primary cells and cell line (MEC1) at mRNA compared with normal B cells. To investigate the biological processes of ENPP2 involving in CLL progression, functional assays were performed. The survival rate of CLL cells treated with ENPP2 inhibitor PF-8380 was significantly decreased, while apoptosis was increased. In addition, we performed a combination of ibrutinib with PF8380, which showed a further decrease in cell viability and showed better anti-CLL effects. Conclusion Taken together, the present work provides evidence that the identified metabolic biomarkers can be used for CLL diagnosis and screening. The combined analysis of metabolomics and transcriptomics data provides a comprehensive understanding of the metabolic pathways in CLL. Besides, this study was the first investigation on the role of ENPP2 in CLL. ENPP2 expression was increased in CLL cells, and the ENPP2 inhibitor PF-8380 inhibited cell proliferation and induced apoptosis, with therapeutic potential. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Introduction Complex heterogeneity is a hallmark of chronic lymphocytic leukemia (CLL) that challenges oncological research and optimal treatment regimens. Although genetic markers with prognostic values have been developed for risk assessment in CLL, the existing prognostic models are far from satisfaction. Single-cell RNA sequencing (scRNA-seq) technologies have made it possible to illustrate the genetic heterogeneity and cell differentiation status at the single-cell level. A prognostic model associated with CLL cell differentiation status was established to refine the risk assessment of CLL patients. Methods Primary CLL cells from 90 patients and CD19+ B cells from 20 healthy donors were collected with informed consents according to the Declaration of Helsinki. Expression levels of SORL1 mRNA and protein in CLL cells were determined by quantitative RT-PCR and western blotting. Cell viability was measured by the CCK-8 assay. Cell cycle and apoptosis were analyzed by PI and Annexin V-PE/7AAD staining. Fatty acid oxidation rate was assessed with Fatty Acid Oxidation Complete Assay Kit. Red oil O staining was performed to evaluate the lipid deposition in CLL cells. Single cell sequencing data were analyzed by the Seurat package and Monocle package in R4.0.5. Results The scRNA-seq profile revealed remarkable intra- and inter-tumoral heterogeneity of CLL. According to the differentiation status, this study reconstructed the evolutionary history of CLL cells, which reflected the molecular classification and risk subgroups of CLL. At the key point of cell differentiation, CLL cells were divided into two subsets with progressive and indolent genetic signatures respectively. Genes differentially expressed between aggressive and indolent subsets were identified. Through univariate and multivariate analysis, four genes (SORL1, SGK1, CYBB, RPL22L1) with significant prognostic values were selected to construct a risk model named SSCR-Score. The risk score was calculated and validated in two independent validation cohorts (Fig 1A). SSCR-Score has revealed the positive prognostic value of SORL1 in CLL. Increased expression of SORL1 was associated with prolonged overall survival and improved time to first treatment in CLL. Among patients with ZAP70 (-) or IGHV mutated status, the expression levels of SORL1 were up-regulated. Although increased expression of SORL1 was associated with favorable prognosis, the biological mechanism of SORL1 still remained ill-defined. This study demonstrated that overexpression of SORL1 restrained cell proliferation, induced cell apoptosis and G2/M cell cycle arrest in CLL cells. In accordance with the RNA sequencing profile, overexpression of SORL1 suppressed abnormal lipid reposition and fatty acid oxidation in CLL cells. On a molecular level, SORL1 down-regulated the expression of lipid-metabolism related genes, including lipoprotein lipase (LPL), carnitine palmitoyltransferase 1A (CPT1A) and carnitine palmitoyltransferase 2 (CPT2). To illustrate the SORL1-mediated regulation of LPL, immunofluorescence staining was performed. Results showed that SORL1 accelerated the degradation of LPL. After the inhibition of lysosome function, accelerated degradation of LPL was not observed in CLL cells overexpressing SORL1. Rescue experiments substantiated that SORL1 impeded progression of CLL through downregulating the activity of LPL which promoted lipid metabolism. Immuno-coprecipitation experiment showed that anti-SORL1 antibody precipitated LPL protein. Additionally, 6-Shohaol induced overexpression of SORL1 in CLL cells. It exhibited potent anti-CLL efficacy by suppressing cell proliferation, promoting cell apoptosis and G2/M cell cycle arrest in CLL cells (Fig 1B). Conclusion CLL presented remarkable heterogeneity which reflected different biological subclasses and risk groups. Through identifying genes differentially expressed between aggressive and indolent cell subsets, a novel prognostic model named SSCR-Score was developed to optimize risk stratification of CLL patients. Moreover, SORL1 restrained progression of CLL by inhibiting lipid metabolism regulated by LPL in CLL cells. SORL1 may serve as a potential strategy for the development of novel therapeutic target in CLL. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Introduction: APG115 is an orally active, selective, potent small molecule inhibitor of the MDM2-p53 interaction, which destabilizes MDM2-p53 complex and promotes p53 activation. Previous studies reported that APG115 promoted cell apoptosis in several types of cancer. However, its role and mechanism in the development of CLL are still unclear. Methods: Peripheral blood samples from de novo CLL patients were collected with informed consents at the Department of Hematology in Shandong Provincial Hospital Affiliated to Shandong University. The effects of APG115 on cell proliferation, apoptosis and cycle were detected by CCK8, Annexin V-PE/7AAD staining and PI/RNase staining respectively. Related protein expression of p53 pathway in CLL cell lines and primary cells were detected by Western blot. Protein interactions were verified by COIP. Immunofluorescence was applied to detect protein localization. Results: To study the role of APG115, we added APG115 into CLL cell lines and primary cells to select appropriate concentration according to IC50. The cells viability of gradually decreased along with the extension of time or concentration augment, which suggested that CLL cells were time-dose-dependent on APG115. In addition, the protein levels of MDM2, p53, PUMA and p21 increased under the treatment of APG115, indicating that APG115 relieved the inhibition of MDM2 on p53 and activated downstream apoptotic pathways. Meanwhile, the APG115 induced cell apoptotic elevation and cell cycle arrest in G0/G1 phase. Subsequently, aimed to explore whether APG115 affects the ibrutinib sensitivity in CLL cells, the cell viability of combination group under both APG115 and ibrutinib was obviously suppressed than groups with single drug intervention. Immunofluorescence images revealed that p53 promoted MCL-1 into cytoplasm under the action of APG115. However, on the basis of ibrutinib intervention, APG115 facilitated the degradation of MCL-1 by p53. Furthermore, the invasion range of CLL cells in the combination group was smaller than that in the single agent group, and the invasion range of CLL cells in the single agent group was lower than that in the control group in vivo(Figure A). After injection of medication, both the monotherapy group and the combination group were lower than the control group, but there was no significant difference between the monotherapy group and the combination group. The survival rate of combination group was higher than that of single agent group (Figure B). Immunohistochemistry of the dissected spleens of mice showed that the malignancy of the cells in the single agent group was lower than that in the control group, while that in the combination group was lower than that in the monotherapy group. Finally, ibrutinib-resistant cell line was established to study the effect of APG115 on chemoresistance. After adding APG115, it was found that the survival rate of drug-resistant strain was lower than that of sensitive strain. It was proved that p53 induced the ubiquitination process when combined with MCL-1 to reduce the inhibitory effect of apoptosis via CO-IP. After APG115 was added, the apoptosis rate of drug-resistant strains was higher than that of sensitive strains. When APG115 was added on the basis of ibrutinib, the difference in apoptosis rate of drug-resistant strains was greater than that of sensitive strains. Conclusions: In summary, this study first revealed anticancer effects of the novel MDM2-p53 inhibitor APG115 in CLL. Promisingly, APG115 is expected to be a potential agent to improve the effects of BTK inhibitor treatment in CLL. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Bromodomain-containing protein 9 (BRD9) functions as a key component of the chromatin remodeling complex in regulation of gene transcription, which is widely implicated in tumorigenicity. Although venetoclax has transformed the paradigm of CLL treatment, some patients are still unable to achieve or maintain clinical remission, indicating the medical need of novel therapeutic target or combinatory regimen. Hence, this study was performed to explore the regulatory mechanism and potential clinical utility of BRD9 in CLL. At first, aberrantly elevated BRD9 expression was frequently observed in CLL patients (n=94) than healthy donors (n=17) both in mRNA and protein level (donors vs. CLL patients, 0.94 ± 0.41 vs. 10.96 ± 2.01, p=0.03). Furthermore, BRD9 overexpression was identified as the independent prognostic factor in CLL (HR=3.55, p<0.001). The exceptionally upregulation of BRD9 was intensely correlated with inferior prognosis in CLL patients, including overall survival (HR=3.246, p=0.02) and time to first treatment (HR=2.326, p=0.005). To investigate the functional significance of BRD9 in CLL, stable BRD9 knockdown cells using lentiviral shRNAs were established. The suppression of BRD9 remarkably impeded cell proliferation, increased cell apoptosis and induced G1/S phase blockage in CLL. What's more, I-BRD9, a selective inhibitor of BRD9, resulted in a defective proliferation in CLL primary cells in a time- and dose-dependent manner. We subsequently performed pre-clinical investigations of I-BRD9 in orthotopic CLL xenograft murine model. Notably, I-BRD9 treatment showed obviously reduced leukemia burden and manifested splenomegaly to a lesser extent than control (Figure 1A), which was further confirmed by H&E and Ki67 staining of spleen. Additionally, I-BRD9 could significantly prolong the lifespan (p=0.02) and delay disease progression of the CLL xenograft mice (p<0.01). Also, a reduction of CLL cells in bone marrow and spleen drawn from I-BRD9 treated mice was corroborated by using flow cytometry. To decipher the specific contribution of BRD9 in CLL pathogenesis, ATAC-seq and CHIP-seq were employed to monitor BRD9-deficent and control cells. Knockdown of BRD9 diminished global ChIP signal and chromatin accessibility near transcriptional start sites. Integrated analysis of mRNA expression profiles of RNA-seq and differential signals of CHIP-seq, Nrf2 was selected as a candidate target for further investigation. The expression of Nrf2 and downstream targets (like HO-1, NOQ1 and SOD1) were significantly decreased in BRD9-silencing CLL cells (p<0.01). Furthermore, the existence of BRD9 binding site was identified in a 2kb region upstream of the NFE2L2 (encoding Nrf2) promoter through constructing truncations (p<0.05). In addition, the enrichment of H3K27ac modification in the binding site was further substantiated by CHIP-qPCR (p<0.01). The inhibitor of histone acetyltransferase reduced the enrichment of BRD9 in this region, indicating that BRD9 bound to NFE2L2 through recognizing the acetylated histone marks. What's more, the Hi-C data suggested a looping of the transcription factor complexes among the labeled regions which showed divergent CHIP signals between shControl and shBRD9 (Figure 1B). The GO analysis of differentially expressed genes obtained from RNA-seq demonstrated a mainly enrichment in apoptotic pathway, which prompted us to investigate the role of BRD9 in venetoclax sensitivity. Following venetoclax addition, CLL cells with BRD9 silenced presented markedly reduced viability than control cells, indicating BRD9 inhibition could potentially drive a therapeutic vulnerability to the venetoclax (p<0.05). Besides, supplementing I-BRD9 with venetoclax strikingly alleviated malignant proliferation and enhanced apoptosis on CLL primary cells than single agent (all combination index<0.6, p<0.05). Western blotting assays suggested the involvement of Nrf2/ARE signaling in venetoclax sensitivity induced by BRD9 inhibition. Taking all these data together, our study firstly credentials the oncogenic role of BRD9 in CLL leukemogenesis, suggesting the therapeutic synergy of combinatory venetoclax/I-BRD9 treatment for clinical utility. Targeting BRD9 displays promising clinical application in the context of extensive use of venetoclax in CLL patients. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

BACKGROUND:Chronic lymphocytic leukemia (CLL) is a heterogeneous B-cell malignancy that lacks specific biomarkers and drug targets. Competing endogenous RNAs (ceRNAs) play vital roles in oncogenesis and tumor progression by sponging microRNAs (miRNAs). Nevertheless, the regulatory mechanisms of survival-related ceRNA networks in CLL remain to be uncovered.METHODS:We included 865 de novo CLL patients to investigate RNA expression profiles and Illumina sequencing was performed on four CLL patients, two CLL cell lines and six healthy donors in our center. According to univariate Cox regression, LASSO regression as well as multivariate Cox regression analyses, we established a novel risk score model in CLL patients. Immune signatures were compared between the low- and high-risk groups with CIBERSORT and ESTIMATE program. Afterwards, we analyzed the relationship between differentially expressed miRNAs (DEmiRNAs) and IGHV mutational status, p53 mutation status and del17p. Based on the survival analyses and differentially expressed RNAs with targeting relationships, the lncRNA/circRNA-miRNA-mRNA ceRNA networks were constructed. In addition, the circRNA circ_0002078/miR-185-3p/TCF7L1 axis was verified and their interrelations were delineated by dual-luciferase reporter gene assay.RESULTS:Totally, 57 differentially expressed mRNAs (DEmRNAs) and 335 DEmiRNAs were identified between CLL patient specimens and normal B cells. A novel risk score model consisting of HTN3, IL3RA and NCK1 was established and validated. The concordance indexes of the model were 0.825, 0.719 and 0.773 in the training, test and total sets, respectively. The high-risk group was related to del(13q14) as well as shorter overall survival (OS). Moreover, we identified DEmiRNAs that related to cytogenetic abnormality of CLL patients, which revealed that miR-324-3p was associated with IGHV mutation, p53 mutation and del17p. The survival-related lncRNA/circRNA-miRNA-mRNA ceRNA networks were constructed to further facilitate the development of potential predictive biomarkers. Besides, the expression of circ_0002078 and TCF7L1 were significantly elevated and miR-185-3p was obviously decreased in CLL patients. Circ_0002078 regulated TCF7L1 expression by competing with TCF7L1 for miR-185-3p.CONCLUSIONS:The comprehensive analyses of RNA expression profiles provide pioneering insights into the molecular mechanisms of CLL. The novel risk score model and survival-related ceRNA networks promote the development of prognostic biomarkers and potential therapeutic vulnerabilities for CLL.