Von Hippel-Lindau Expression Research Articles

Genotype-phenotype associations in Von-Hippel Lindau Syndrome: implications for screening

Abstract Background Von Hippel-Lindau (VHL) is an autosomal dominant genetic disease with an estimated incidence of 1:36,000. The type of germline VHLI alteration has been shown to impact downstream VHL expression and clinical phenotype. However, current screening practices do not differ based on VHL genomic subtype. We aim to assess genotype-phenotype association among an institutional cohort of VHL patients. Methods We conducted a retrospective cohort study of 69 patients (27 families) at the University of Utah and Huntsman Cancer Institute from 1998- December 2023. 41 patients had documented germline testing in our system to define VHL type. VHL Type 1 was defined as those with nonsense mutations, deletions, and duplications and Type 2 as those with missense and splice mutations. Mean follow-up was 86- and 59-months for VHL type 1 and 2 respectively. We then evaluated the association of VHL type with risk of pheochromocytoma (Pheo), renal cancer (RCC), and hemangioblastoma (HB). Results We identified 24 unique VHL alterations among 41 patients. 83% (29/41) patients had missense mutations, 7.5% (3/41) had nonsense mutations, 17% (6/41) had splice mutations, and 3% (3/41) had a deletion. The median age at VHL diagnosis was 43 years old for type 1 (range 19-76) and 38 for VHL type 2 (range 4-67). VHL Type 2 was more common, 85% (35/41). All VHL Type 1 patients (6/6) developed RCC and HB. No patients with VHL Type 1 developed PheoPara. Of the 35 patients diagnosed with VHL Type 2, 20% (7/35) were diagnosed with PheoPara, 9% (3/35) with RCC, 31% (n = 11) with HB, and 37% (n = 13) were asymptomatic. No VHL Type 1 patients were asymptomatic. Conclusions VHL type 2 comprised the majority of VHL diagnoses in our cohort. Individuals diagnosed with VHL type 2 have the highest penetrance for: hemangioblastoma (hb), Pheo-Para, and renal cell carcinoma (RCC). In our study, 37% of VHL type 2 participants did not demonstrate any VHL manifestation. VHL type 1 had a high prevalence for RCC and HB, with a notable absence of PheoPara manifestation. However, majority of these patients (75%) were below the age of 20, and thus likely did not develop a manifestation yet. This cohort add to evidence supporting utilizing genetic VHL Types to personalize surveillance guidelines. Validation in larger cohorts with longer follow-up suggest that VHL type 1 patients could forgo pheo/para screening.

Clinical implications of activation of the LIMD1-VHL-HIF1α pathway during head-&-neck squamous cell carcinoma development.

Background & objectives Given the importance of the role of hypoxia induced pathway in different cancers including head-and-neck squamous cell carcinoma (HNSCC), this study delved into elucidating the molecular mechanism of hypoxia-inducible factor-1α (HIF1α) activation in HNSCC. Additionally, it analyzes the alterations of its regulatory genes [von Hippel-Lindau (VHL) and LIM domain containing 1 (LIMD1)] and target gene vascular endothelial growth factor (VEGF) in head-and-neck lesions at different clinical stages in relation with human papillomavirus (HPV) infection. Methods Global mRNA expression profiles of HIF1α, VHL, LIMD1 and VEGF were evaluated from public datasets of HNSCC, followed by validation of their expression (mRNA/protein) in an independent set of HPV+ve/-ve HNSCC samples of different clinical stages. Results A diverse expression pattern of the HIF1α pathway genes was observed, irrespective of HPV infection, in the datasets. In validation in an independent set of HNSCC samples, high mRNA expressions of HIF1α/VEGF were observed particularly in HPV positive samples. However, VHL/LIMD1 mRNA expression was low in tumours regardless of HPV infection status. In immunohistochemical analysis, high/medium (H/M) expression of HIF1α/VEGF was observed in basal/parabasal layers of normal epithelium, with significantly higher expression in tumours, especially in HPV-positive samples. Conversely, high cytoplasmic VHL expression in these layers gradually decreased with the progression of HNSCC, regardless of HPV infection. A similar trend was noted in LIMD1 expression (nuclear/cytoplasmic) during the disease development. The methylation pattern of VHL and LIMD1 promoters in the basal/parabasal layers of normal epithelium correlated with their expression, exhibiting a gradual increase with the progression of HNSCC. The H/M expression of HIF1α/VEGF proteins and reduced VHL expression was associated with poor clinical outcomes. Interpretation & conclusions The results of this study showed differential regulation of the LIMD1-VHL-HIF1α pathway in HPV positive and negative HNSCC samples, illustrating the molecular distinctiveness of these two groups.

Abstract 2559: Genotype-phenotype associations in von hippel-lindau syndrome: Implications for screening

Abstract Introduction and Objectives: Von Hippel-Lindau (VHL) is an autosomal dominant genetic disease. The type of VHL alteration has been shown to impact downstream VHL expression and clinical phenotype. However, current screening practices do not differ based on VHL genetic subtype. We aim to define the genotype-phenotype association among an institutional cohort of VHL patients. Methods: We conducted a retrospective cohort study of 69 patients (27 families) with von Hippel-Lindau at the Huntsman Cancer Institute from 1998-2023. 55% (38/69) patients have documented VHL variants for review. We evaluated the association of VHL type with risk of pheochromocytoma (Pheo), renal cancer (RCC), and hemangioblastoma (HB). t test and Fisher’s exact test were used to assess differences in clinicopathologic characteristics by VHL Type. P-value < 0.05 was considered significant. Results: We identified 24 unique VHL alterations among 38 patients. 66% (25/38) of patients had missense mutations, 10% (4/38) nonsense mutations, 21% (8/38) splice mutations, and 3% (1/38) deletion/duplication (Table 1). The median age at VHL diagnosis was 31 years old (range 4-76). VHL Type 2 was more common, 87% (33/38). All VHL Type 1 patients (5/5) developed RCC and HB. No patients with VHL Type 1 developed PheoPara. Of the VHL Type 2 patients, 39% (13/33) have not developed any VHL-associated clinical manifestations. Of the 33 patients diagnosed with VHL Type 2, 18% (6/33) were diagnosed with PheoPara, 30% (10/33) with RCC, and 48% (16/33) with HB. Conclusion: VHL type 2 comprised the majority of VHL diagnoses in our cohort with a notable absence of PheoPara phenotype. VHL Type 1 had higher penetrance and a higher prevalence for RCC and hemangioblastoma. With validation in larger cohorts, this cohort supports utilizing VHL genetic subtypes to personalize surveillance. Additional VHL testing for the remainder of the cohort is ongoing. Clinicopathologic characteristics of von Hippel-Lindau Syndrome cohort with known genetic mutations All (n=38) Type 1 (n=5) Type 2 (n=33) p-value AgeMean (Median) 33.1 (31) 42 (28) 31.7 (31) 0.38 White, n (%) 92% (n=35) 5 (100%) 30 (91%) 1 Hispanic, n (%) 8% (n=3) - 3 (9%) 1 VHL alteration, n (%) - Missense 25 (66%) - 25 (76%) Nonsense 4 (10%) 4 (80%) - Splice site 8 (21%) - 8 (24%) Deletion/Duplication 1 (3%) 1 (20%) - Penetrance, n (%) 25 (66%) 5 (100%) 20 (61%) 0.14 Phenotype, n (%) PheoPara 6 (16%) - 6 (18%) 0.57 RCC 15 (39%) 5 (100%) 10 (30%) 0.006 Hemangioblastoma 21 (55%) 5 (100%) 16 (48%) 0.26 Citation Format: Nicole Murray, Colton Leavitt, Noah Shepard, Zera Gonzales, Jiaming Li, Brock O'Neil, Christopher Dechet, Bogdana Schmidt, Benjamin L. Maughan, Kristen Pauley, Anne Naumer, Wendy Kohlmann, Alejandro Sanchez. Genotype-phenotype associations in von hippel-lindau syndrome: Implications for screening [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2559.

Critical role of VHL/BICD2/STAT1 axis in crystal-associated kidney disease

Nephrolithiasis is highly prevalent and associated with the increased risk of kidney cancer. The tumor suppressor von Hippel-Lindau (VHL) is critical for renal cancer development, however, its role in kidney stone disease has not been fully elucidated until now. Here we reported VHL expression was upregulated in renal epithelial cells upon exposure to crystal. Utilizing Vhl+/mu mouse model, depletion of VHL exacerbated kidney inflammatory injury during nephrolithiasis. Conversely, overexpression of VHL limited crystal-induced lipid peroxidation and ferroptosis in a BICD2-depdendent manner. Mechanistically, VHL interacted with the cargo adaptor BICD2 and promoted its K48-linked poly-ubiquitination, consequently resulting in the proteasomal degradation of BICD2. Through promoting STAT1 nuclear translocation, BICD2 facilitated IFNγ signaling transduction and enhanced IFNγ-mediated suppression of cystine/glutamate antiporter system Xc−, eventually increasing cell sensitivity to ferroptosis. Moreover, we found that the BRAF inhibitor impaired the association of VHL with BICD2 through triggering BICD2 phosphorylation, ultimately causing severe ferroptosis and nephrotoxicity. Collectively, our results uncover the important role of VHL/BICD2/STAT1 axis in crystal kidney injury and provide a potential therapeutic target for treatment and prevention of renal inflammation and drug-induced nephrotoxicity.

VHL L169P Variant Does Not Alter Cellular Hypoxia Tension in Clear Cell Renal Cell Carcinoma.

In the current era of tumor genome sequencing, single amino acid missense variants in the von Hippel-Lindau (VHL) tumor suppressor gene are frequently identified in clear cell renal carcinoma (ccRCC). Due to the incomplete knowledge of the structural architecture of VHL protein, the functional significance of many missense mutations cannot be assigned. L169P is one such missense mutation identified in the case of aggressive, metastatic ccRCC. Here, we characterized the biochemical activity, transcriptomic hypoxia signature and biological functions of the L169P variant. Lentiviral vector expressing either wildtype (WT) or L169P VHL were used to transduce two VHL-deficient human ccRCC cell lines, 786-O and RCC4. The stability of the VHL protein and the expression level of VHL, HIF1α and HIF2α were analyzed. The impact of restoring L169P or WT VHL on the hypoxia gene expression program in 786-O cells was assessed by mRNA sequencing (RNAseq) and computed hypoxic scores. The impact of restoring VHL expression on the growth of ccRCC models was assessed in cell cultures and in chorioallantoic membrane (CAM) xenografts. In the 786-O cells, the protein stability of L169P VHL was comparable to WT VHL. No obvious difference in the capability of degrading HIF1α and HIF2α was observed between WT and L169P VHL in the 786-O or RCC4 cells. The hypoxic scores were not significantly different in the 786-O cells expressing either wildtype or L169P VHL. From the cellular function perspective, both WT and L169P VHL slowed cell proliferation in vitro and in vivo. The L169P VHL variant is comparable to WT VHL in terms of protein stability, ability to degrade HIF1α factors and ability to regulate hypoxia gene expression, as well as in the suppression of ccRCC tumor cell growth. Taken together, our data indicate that the L169P VHL variant alone is unlikely to drive the oncogenesis of sporadic ccRCC.

Cancer-cell-secreted miR-204-5p induces leptin signalling pathway in white adipose tissue to promote cancer-associated cachexia

Cancer-associated cachexia is a multi-organ weight loss syndrome, especially with a wasting disorder of adipose tissue and skeletal muscle. Small extracellular vesicles (sEVs) serve as emerging messengers to connect primary tumour and metabolic organs to exert systemic regulation. However, whether and how tumour-derived sEVs regulate white adipose tissue (WAT) browning and fat loss is poorly defined. Here, we report breast cancer cell-secreted exosomal miR-204-5p induces hypoxia-inducible factor 1A (HIF1A) in WAT by targeting von Hippel-Lindau (VHL) gene. Elevated HIF1A protein induces the leptin signalling pathway and thereby enhances lipolysis in WAT. Additionally, exogenous VHL expression blocks the effect of exosomal miR-204-5p on WAT browning. Reduced plasma phosphatidyl ethanolamine level is detected in mice lack of cancer-derived miR-204-5p secretion in vivo. Collectively, our study reveals circulating miR-204-5p induces hypoxia-mediated leptin signalling pathway to promote lipolysis and WAT browning, shedding light on both preventive screenings and early intervention for cancer-associated cachexia.

Von Hippel-Lindau-targeting microRNA-143-3p attenuates mitochondrial abnormality via AMPK/PGC-1α axis in Parkinson's disease

Parkinson's disease (PD) is the second most common neurodegenerative disease characterized by selective loss of dopaminergic neurons. We previously found that inhibition of von Hippel-Lindau (VHL) can alleviate dopaminergic neuron degeneration in PD models via regulation of mitochondrial homeostasis, however, the disease-related alterations of VHL and the regulatory mechanisms of VHL level in PD need to be further investigated. In this study, we found that the levels of VHL were markedly increased in multiple cell models of PD and identified microRNA-143-3p (miR-143-3p) as a promising candidate for regulating VHL expression involved in PD. miR-143-3p directly bound to the 3′untranslated region of human VHL mRNA and inhibited its translation, and exerted neuroprotective effects by improving cell viability, apoptosis and tyrosine hydroxylase abnormality. Furthermore, we demonstrated that miR-143-3p exerted neuroprotection by attenuating mitochondrial abnormality via AMP-activated protein kinase (AMPK)/peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) axis, and AMPK inhibitor abolished the beneficial effects of miR-143-3p on the cell model of PD. Therefore, we identify the dysregulated VHL and miR-143-3p in PD, and propose the therapeutic potential of miR-143-3p to alleviate PD by improving mitochondrial homeostasis via AMPK/PGC-1α axis.

Co-targeting BCL-XL and BCL-2 by PROTAC 753B eliminates leukemia cells and enhances efficacy of chemotherapy by targeting senescent cells.

BCL-XL and BCL-2 are key anti-apoptotic proteins and validated cancer targets. 753B is a novel BCL-XL/BCL-2 proteolysis targeting chimera (PROTAC) that targets both BCL-XL and BCL-2 to the von Hippel-Lindau (VHL) E3 ligase, leading to BCLX L/BCL-2 ubiquitination and degradation selectively in cells expressing VHL. Because platelets lack VHL expression, 753B spares on-target platelet toxicity caused by the first-generation dual BCL-XL/BCL-2 inhibitor navitoclax (ABT-263). Here, we report pre-clinical single-agent activity of 753B against different leukemia subsets. 753B effectively reduced cell viability and induced dose-dependent degradation of BCL-XL and BCL-2 in a subset of hematopoietic cell lines, acute myeloid leukemia (AML) primary samples, and in vivo patient-derived xenograft AML models. We further demonstrated the senolytic activity of 753B, which enhanced the efficacy of chemotherapy by targeting chemotherapy-induced cellular senescence. These results provide a pre-clinical rationale for the utility of 753B in AML therapy, and suggest that 753B could produce an added therapeutic benefit by overcoming cellular senescence-induced chemoresistance when combined with chemotherapy.

BRD4 Inhibitor GNE-987 Exerts Anticancer Effects by Targeting Super-Enhancer-Related Gene LYL1 in Acute Myeloid Leukemia.

Background AML (acute myeloid leukemia) is a common hematological malignancy in children with poor treatment effects and poor prognosis. Recent studies have shown that as a novel BRD4 (bromodomain containing 4) PROTACs (proteolysis targeting chimeras) degrader, GNE-987 can slow down the growth of various tumors and increase apoptosis, with promising clinical prospects. However, the function and molecular mechanism of GNE-987 in AML remain unclear. This study is aimed at investigating the therapeutic effect of GNE-987 on AML and its underlying mechanism. Methods The association between BRD4 and AML was assessed by studying public databases. After GNE-987 was added to AML cells, cell proliferation slowed down, the cycle was disturbed, and apoptosis increased. Western blotting was used to detect BRD2 (bromodomain containing 2), BRD3 (bromodomain containing 3), BRD4, and PARP (poly ADP-ribose polymerase) proteins. The effect of GNE-987 on AML cells was analyzed in vivo. RNA-seq (RNA sequencing) and ChIP-seq (chromatin immunoprecipitation sequencing) validated the function and molecular pathways of GNE-987 in processing AML. Results BRD4 expression was significantly elevated in pediatric AML samples compared with healthy donors. GNE-987 inhibited AML cell proliferation by inhibiting the cell cycle and inducing apoptosis. BRD2, BRD3, and BRD4 were consistent with decreased VHL (Von Hippel Lindau) expression in AML cells. In an AML xenograft model, GNE-987 significantly reduced the hepatosplenic infiltration of leukemia cells and increased the mouse survival time. Based on analysis of RNA-seq and ChIP-seq analyses, GNE-987 could target multiple SE- (super-enhancer-) related genes, including LYL1 (lymphoblastic leukemia 1), to inhibit AML. Conclusions GNE-987 had strong antitumor activity in AML. GNE-987 could effectively inhibit the expression of SE-related oncogenes including LYL1 in AML. Our results suggested that GNE-987 had broad prospects in the treatment of AML.

Short isoform thymic stromal lymphopoietin reduces inflammation and aerobic glycolysis of asthmatic airway epithelium by antagonizing long isoform thymic stromal lymphopoietin

BackgroundUp-regulation of aerobic glycolysis has been reported as a characterization of asthma and facilitates airway inflammation. We has been previously reported that short isoform thymic stromal lymphopoietin (sTSLP) could reduce inflammation in asthmatic airway epithelial cells. Here we wanted to investigate whether the inhibition of sTSLP on asthma is related to aerobic glycolysis.MethodsAsthmatic model was established in challenging Male BALB/c mice and 16-HBE (human bronchial epithelial) cell line with house dust mite (HDM). Indicators of glycolysis were assessed to measure whether involve in sTSLP regulating airway epithelial cells inflammation in asthmatic model in vivo and in vitro.ResultssTSLP decreased inflammation of asthmatic airway and aerobic glycolysis in mice. HDM or long isoform thymic stromal lymphopoietin (lTSLP) promoted HIF-1α expression and aerobic glycolysis by miR-223 to target and inhibit VHL (von Hippel-Lindau) expression 16-HBE. Inhibition of aerobic glycolysis restrained HDM- and lTSLP-induced inflammatory cytokines production. sTSLP along had almost no potential to alter aerobic glycolysis of 16-HBE. But sTSLP decreased LDHA (lactate dehydrogenase A) and LD (Lactic acid) levels in BALF, and HIF-1α and LDHA protein levels in airway epithelial cells of asthma mice model. lTSLP and sTSLP both induced formation of TSLPR and IL-7R receptor complex, and lTSLP obviously facilitated phosphorylation of JAK1, JAK2 and STAT5, while sTSLP induced a little phosphorylation of JAK1 and STAT5.ConclusionWe identified a novel mechanism that lTSLP could promote inflammatory cytokines production by miR-223/VHL/HIF-1α pathway to upregulate aerobic glycolysis in airway epithelial cells in asthma. This pathway is suppressed by sTSLP through occupying binding site of lTSLP in TSLPR and IL-7R receptor complex.

Multi-Omics Profiling to Assess Signaling Changes upon VHL Restoration and Identify Putative VHL Substrates in Clear Cell Renal Cell Carcinoma Cell Lines.

The inactivation of von Hippel–Lindau (VHL) is critical for clear cell renal cell carcinoma (ccRCC) and VHL syndrome. VHL loss leads to the stabilization of hypoxia-inducible factor α (HIFα) and other substrate proteins, which, together, drive various tumor-promoting pathways. There is inadequate molecular characterization of VHL restoration in VHL-defective ccRCC cells. The identities of HIF-independent VHL substrates remain elusive. We reinstalled VHL expression in 786-O and performed transcriptome, proteome and ubiquitome profiling to assess the molecular impact. The transcriptome and proteome analysis revealed that VHL restoration caused the downregulation of hypoxia signaling, glycolysis, E2F targets, and mTORC1 signaling, and the upregulation of fatty acid metabolism. Proteome and ubiquitome co-analysis, together with the ccRCC CPTAC data, enlisted 57 proteins that were ubiquitinated and downregulated by VHL restoration and upregulated in human ccRCC. Among them, we confirmed the reduction of TGFBI (ubiquitinated at K676) and NFKB2 (ubiquitinated at K72 and K741) by VHL re-expression in 786-O. Immunoprecipitation assay showed the physical interaction between VHL and NFKB2. K72 of NFKB2 affected NFKB2 stability in a VHL-dependent manner. Taken together, our study generates a comprehensive molecular catalog of a VHL-restored 786-O model and provides a list of putative VHL-dependent ubiquitination substrates, including TGFBI and NFKB2, for future investigation.

Downregulation of lncRNA APCDD1L-AS1 due to DNA hypermethylation and loss of VHL protein expression promotes the progression of clear cell renal cell carcinoma

Background: The current studies only indicated that long non-coding RNA (lncRNA) APCDD1L-AS1, as a novel lncRNA, may play a role in oral squamous cell carcinoma and lung cancer. However, its potential role in clear cell renal cell carcinoma (ccRCC) and its possible mechanism of action remain vague.Methods: TCGA-KIRC and GEO data and qRT-PCR and pyrosequencing results of clinical specimens were used to identify the expression level and DNA methylation status of APCDD1L-AS1. The effects of APCDD1L-AS1 overexpression on ccRCC growth and metastasis were determined by function experiments. Western blot and Tandem mass tags (TMT) were utilized to explore the relationship between APCDD1L-AS1 and VHL expression and its downstream underlying mechanisms.Results: The expression of APCDD1L-AS1 was downregulated in ccRCC. Decreased APCDD1L-AS1 expression was related to higher tumor stage and histological grade and shorter RFS (Relapse-free survival). Besides, APCDD1L-AS1 overexpression restrained the growth and metastasis of ccRCC cells in vitro and in vivo. Moreover, reduced APCDD1L-AS1 expression could be caused by DNA hypermethylation and loss of von Hippel Lindau (VHL) protein expression. Furthermore, the dysregulation of histones expression caused by APCDD1L-AS1 overexpression may be one of the important mechanisms to suppress the progression of ccRCC.Conclusion: APCDD1L-AS1 was able to inhibit the progression of ccRCC, and its decreased expression could be caused by DNA hypermethylation and loss of VHL protein expression. Therefore, APCDD1L-AS1 may serve as a new therapeutic target in the treatment of ccRCC.

Identification of the feature of immune cells infiltration in inherited renal carcinoma with von Hippel-Lindau syndrome

Objective: To investigate the feature of immune cells infiltration in inherited renal carcinoma with von Hippel-Lindau (VHL) syndrome and their relationship with clinicopathological characteristics and prognosis. Methods: The samples were collected from patients with VHL syndrome renal carcinoma who were diagnosed and treated surgically at the Department of Urology, Peking University First Hospital from 2010 to 2019. RNA-Seq was performed on 6 pairs of VHL syndrome renal carcinoma and adjacent normal tissues. To identify the specific infiltrated immune cells, RNA-Seq data was converted into the infiltration data of 14 types of immune cells using the TIP tool. Immunohistochemical staining was used to verify the expression of the markers of these specific infiltrated immune cells in the paraffin sections of 54 paired VHL syndrome renal carcinoma and adjacent normal tissues, and to analyze their relationship with clinicopathological characteristics and prognosis. Results: Compared with adjacent normal tissues, CD4 Naive infiltration level was significantly down-regulated (0.289±0.009 vs 0.200±0.012,P<0.001) and CD4 Memory infiltration level was significantly up-regulated (0.123±0.014 vs 0.222±0.016,P<0.001) in VHL syndrome renal carcinoma. Immunohistochemical staining results showed that CD45RA (a CD4 Naive cell marker) expression was significantly reduced (50.9±1.9 vs 15.6±0.9,P<0.001) and CD45RO (a CD4 Memory cell marker) expression was significantly increased (22.2±1.1 vs 80.8±4.3,P<0.001) in VHL syndrome renal carcinoma. Besides, lower CD45RA expression and higher CD45RO expression were associated with higher histological grade, advanced tumor stage and shorter disease-free survival (all P<0.01). In addition, CD45RA expression was positively correlated with VHL expression (r=0.693 3, P<0.000 1) and CD45RO expression was negatively correlated with VHL expression (r=-0.609 0, P<0.000 1). Conclusions: This study found that CD4 Naive and CD4 Memory cells may be differentially infiltrated immune cells in VHL syndrome renal carcinoma, and their infiltration levels were associated with the expression of VHL and the prognosis of patients.

Abstract CC09-01: Targeting BCL-XL/BCL-2 by PROTAC 753b effectively eliminates AML cells and enhances efficacy of chemotherapy by targeting senescent cells

Abstract BCL-XL and BCL-2 are key anti-apoptotic proteins facilitating cancer survival, senescence and chemoresistance. 753b is a novel BCL-XL/BCL-2 proteolysis targeting chimera (PROTAC) that targets both BCL-XL and BCL-2 to the Von Hippel-Lindau (VHL) E3 ligase, sparing platelets that lack VHL expression. Here we studied the efficacy of 753b and its senolytic activity in leukemia. We first evaluated the sensitivity of genetically diverse 17 leukemia cell lines to BCL-XL/2 dual inhibitor ABT-263, BCL-XL PROTAC DT2216 and BCL-XL/2 PROTAC 753b. 753b induced time-dependent BCL-XL degradation within 6 hours, at concentrations lower than that of DT2216. BCL-XL was degraded by 753b in all 17 cell lines tested; BCL-2 was partially degraded in 12/17 cell lines. 753b treatment (24hrs) caused a dose-dependent reduction of viability in AML cell lines by CellTiter-Glo (CTG) assay, with IC50 ranging from 0.06-27umol/L. Six AML (KG-1, Kasumi-1, OCI-AML3, U937, TF-1; MPN-AML HEL-92) and three T-ALL (Jurkat, PF832, CCRF-CEM) were sensitive to 753b with average IC50 812nM and to ABT263 (average IC50 2,232 nM). Recent findings indicate that chemoresistance in AML is associated with chemotherapy (Ara-C)-induced senescence (Duy et al., Cancer Discovery 2021). In vitro, Ara-C induced cellular senescence (SnCs) in AML cell lines Molm14 and Kasumi-1, as manifested by increased cell size, induction of senescence-associated β-galactosidase activity, upregulation of cycle regulator proteins (p16, p21, p53) and mRNA expression of senescence-associated secretory phenotype (IL-6, IL-8, IL-1β).Conversely, 753b combined with Ara-C largely reversed chemotherapy-induced SnCs phenotype and facilitated induction of cell death in senescent AML cells. To explore the senolytic mechanism of 753b, we FACS-sorted viable leukemia cells into senescence-high or -low populations based on C12-FDG (fluorogenic substrate di-β-D-galactopyranoside). Consistent with prior data in normal HSC (Chang et al., Nat. Med. 2016), C12-FDG high population expressed higher levels of BCL-XL/2, representing therapeutic target for 753b. To study pre-clinical activity of 753b in primary AML, we exposed freshly isolated blasts from 16 AML patients to concentration range of 753b. 753b potently reduced viability of primary AML with median IC50 values of 228 nmol/L, ranging between 18 - 2,291 nmol/L (13/16 with IC50 below 450 nmol/L). Consistent with cell line data, 753b was more potent degrader of BCL-XL and apoptosis inducer compared with DT2216. BCL-2 degradation was seen in 3 out of 5 samples tested. 753b at 1 umol/L induced apoptotic cell death in both, bulk AML blasts and CD34+ stem/progenitor cells (mean specific apoptosis: 63.9% vs 52.4%, n=7). In summary, BCL-XL/2 PROTAC 753b reduced viability by inducing apoptosis in AML cell lines and in the majority of primary AML samples. Targeting BCL-XL effectively eliminated chemotherapy-induced senolytic cells. In vivo experiments in the cell line- and patient-derived xenografts of 753b combined with chemotherapy are ongoing and will be presented. Citation Format: Yannan Jia, Qi Zhang, Weiguo Zhang, Michael Andreeff, Nitin Jain, Helen Ma, Peiyi Zhang, Guangrong Zheng, Daohong Zhou, Marina Konopleva. Targeting BCL-XL/BCL-2 by PROTAC 753b effectively eliminates AML cells and enhances efficacy of chemotherapy by targeting senescent cells [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr CC09-01.

Expression of VHL, VEGF and HIF-1α in endolymphatic sac tumors

Objective: To investigate the expression of von Hippel-Lindau (VHL), vascular endothelial growth factor (VEGF) and hypoxia inducible factor-1α (HIF-1α) in endolymphatic sac tumor (ELST) and its clinical significance, and to analyze its association with VHL gene mutation. Methods: Twenty-four cases of ELST, which were surgically resected and diagnosed by pathological examination in Beijing Tongren Hospital Affiliated to Capital Medical University, Beijing, China during 2012-2020, were recruited as the ELST group, and 24 cases of otitis media diagnosed in the same hospital were selected as the control group. The expression of VHL, VEGF, and HIF-1α was assessed using EnVision immunohistochemical staining and compared between the ELST and control groups. Sanger sequencing was performed to detect the VHL mutation status in 24 ELSTs. The correlations among VHL, VEGF and HIF-1α expression were analyzed. The associations of VHL, VEGF and HIF-1α expression with age of onset, gender, tumor size, bone invasion and clinical stage in ELST were also analyzed. Results: The expression rate of VHL in the ELST group was significantly lower than that in the control group (P<0.05), but the expression rates of VEGF and HIF-1α in the ELST group were significantly higher than those in the control group (P<0.05). VHL expression was inversely correlated with VEGF and HIF-1α expression. The expression of VEGF and HIF-1α was associated with bone invasion and clinical stage (P<0.05), but the expression of VHL, VEGF and HIF-1α had no significant associations with the age of onset, gender, or tumor size of ELST (P>0.05). Conclusions: The expression of VHL is decreased while that of VEGF and HIF-1α increased in ELST. Expression of VHL is inversely correlated with that of VEGF and HIF-1α. The expression of VEGF and HIF-1α is correlated with bone invasion and clinical stage. Thus, VEGF and HIF-1α may be therapeutic targets of ELST.

Myoblast differentiation of C2C12 cell may related with oxidative stress.

Muscle is a contractile tissue responsible for maintaining posture and the movement of all parts of the body. Prolonged oxidizative stress can lead to the damage of cells, tissues, and organs. In this study, we investigated the possibility of oxidative stress in the process of myoblast differentiation of C2C12 cells. First, the myoblast differentiation model of C2C12 cells was constructed and verified by Giemsa staining. The expression of hypoxia inducible factor1-alpha (HIF1-α), hypoxia inducible factor1-beta (HIF1-β), Von Hippel-Lindau (VHL), lysyl oxidase (Lox), EGL-9 family hypoxia-inducible factor 1 (EGLN1), proline 4-hydroxylase alpha 1 (P4HA1) and heme oxygenase-1 (HOMX1) in the process of myoblast differentiation was verified by in vitro experiments and Gene Expression Omnibus (GEO) bioinformatic analysis. We found that with the increased expression of myogenic factor 5 (MYF5), myogenic differentiation 1 (MYOD1), and Desmin, myotube fusion became more obvious during the process of C2C12 cell differentiation. Both experimental and GEO analysis indicated that the expression of HIF1-α, HIF1-β, VHL, LOX, EGLN1 and P4HA1 increased, and the expression of HOMX1 decreased during myogenic differentiation. Therefore, we suggest that the myoblast differentiation of C2C12 cells may be related to oxidative stress. Their possible relationship was proposed, though further studies are needed.

Ginsenoside 20(S)-Rg3 upregulates tumor suppressor VHL gene expression by suppressing DNMT3A-mediated promoter methylation in ovarian cancer cells

To explore the mechanism by which ginsenoside 20(S)-Rg3 upregulates the expression of tumor suppressor von Hippel-Lindau (VHL) gene in ovarian cancer cells. Ovarian cancer cell line SKOV3 treated with 20(S)-Rg3 were examined for mRNA and protein levels of VHL, DNMT1, DNMT3A and DNMT3B by real-time PCR and Western blotting, respectively. The changes in VHL mRNA expression in SKOV3 cells in response to treatment with 5-Aza-CdR, a DNA methyltransferase inhibitor, were detected using real-time PCR. VHL gene promoter methylation was examined with methylation-specific PCR and VHL expression levels were determined with real-time PCR and Western blotting in non-treated or 20(S)-Rg3-treated SKOV3 cells and in 20(S)-Rg3-treated DNMT3A-overexpressing SKOV3 cells. VHL and DNMT3A protein levels were detected by immunohistochemistry in subcutaneous SKOV3 cell xenografts in nude mice. Treatment of SKOV3 cells with 20(S)-Rg3 significantly upregulated VHL and downregulated DNMT3A expressions at both the mRNA and protein levels (P < 0.05) and upregulated DNMT3B expression only at the mRNA level, but did not cause significant changes in either the mRNA or protein level of DNMT1. Treatment of the cells with 2 and 5 μmol/L 5-Aza-CdR obviously increased VHL mRNA expression by by over 3 folds (P < 0.05). 20(S)-Rg3 significantly decreased the methylation level in the promoter region of VHL gene, and this effect was abrogated by DNMT3A overexpression in the cells (P < 0.05). Immunohistochemisty showed a significantly increased VHL expression but a lowered DNMT3A expression in subcutaneous SKOV3 cell xenografts in 20 (S)-Rg3-treated nude mice. Ginsenoside 20(S)-Rg3 upregulates VHL expression in ovarian cancer cells by suppressing DNMT3A-mediated DNA methylation.

Diagnostic accuracy of combined MUC5A, S100P, and von Hippel-Lindau in differentiating intrahepatic cholangiocarcinoma from metastatic pancreaticobiliary carcinomas presented as hepatic focal lesions

Background Sometimes differentiation between intrahepatic cholangiocarcinoma (ICC) and metastatic pancreaticobiliary carcinoma (mPBC) could be challenging, and it is of great importance owing to different treatment modalities. We aimed to measure the diagnostic accuracy of MUC5A, von Hippel-Lindau (VHL), and S100p in differentiating ICC from mPBC in hepatic focal lesions. Patients and methods The study included 70 cases of liver biopsies presented as hepatic focal lesions divided into two groups: group 1: 40 cases of mPBC and group 2: 30 cases of ICC. Results There was a highly statistically significant difference regarding MUC5A, S100P, and VHL expression between both groups (P&lt;0.001). mPBC showed MUC5A in 87.5%, S100P in 95%, and VHL in 5%. However, cases of ICC showed positive MUC5A in 10%, S100P in 29.6%, and VHL in 80%. Regarding the validity of diagnosing mPBC, S100P had the highest sensitivity (95%), whereas both panels of (MUC5A−/S100P+/VHL−) and (MUC5A+/S100P+/VHL−) showed the highest specificity and positive predictive values (100%). Regarding the validity for diagnosing ICC, VHL had the highest sensitivity (80%), whereas (MUC5A−/S100P−/VHL+) showed the highest specificity and positive predictive value (100%). Conclusion These results indicate the validity of combined MUC5A, S100P, and VHL as a highly sensitive and specific panel for differentiating ICC from mPBC.

The involvement of hypoxia-inducible factor 1α (HIF1α)-stabilising factors in steroidogenic acute regulatory (STAR) protein-dependent steroidogenesis in murine KK1 granulosa cells in vitro.

As a component of hypoxia-inducible factor1 (HIF1)-complexes, HIF1α regulates the expression of steroidogenic acute regulatory (STAR) protein in granulosa cells. However, severe hypoxia or exaggeratedly expressed HIF1α have detrimental effects. HIF1α is regulated by factor inhibiting HIF (FIH), prolyl hydroxylases (PHD1, 2, 3) and von Hippel-Lindau (VHL) suppressor protein. In this study, the expression of FIH, PHD1, 2, 3 and VHL was investigated in murine ovaries and immortalised KK1 granulosa cells. We found FIH, VHL and PHD2 transcripts predominantly in growing tertiary follicles. Functional aspects were assessed in KK1 cells exposed to decreasing O2 (20%, 10%, 1%), by determining HIF1α, FIH, VHL, PHD1-3 and STAR expression. The main findings indicated gradually increasing PHD2 under lowered O2. Functional blocking of PHDs revealed biphasic effects on STAR expression; concomitantly with increasing HIF1α, STAR expression, which was initially induced, decreased significantly when HIF1α was strongly stabilised. Finally, PHD2 in particular might act as a specific regulator of HIF1α and, thereby, of STAR availability in granulosa cells.

VHL gene methylation contributes to excessive erythrocytosis in chronic mountain sickness rat model by upregulating the HIF-2α/EPO pathway

AimsHypoxia-inducible factors (HIFs) play important roles in the pathogenesis of erythrocytosis in chronic mountain sickness (CMS). von Hippel-Lindau (VHL) is a key regulator of hypoxia that can direct the poly-ubiquitylation and degradation of HIFs. Epigenetic mechanisms are believed to contribute toward adaption to chronic hypoxia. Here, we investigated the contribution and mechanism of VHL methylation in rats with erythrocytosis in CMS. Main methodsThe methylation status of VHL was measured via bisulfite sequencing PCR, while VHL, DNMT1, DNMT3α, and DNMT3β expression were assessed using real-time reverse transcription PCR and western blotting. HIF-2α and EPO expression levels in bone marrow were determined via immunohistochemical staining, and erythroid hyperplasia in bone marrow sections were observed with hematoxylin and eosin staining. Key findingsWe found that chronic hypoxia triggered erythroid hyperplasia in the bone marrow and increased the quantity of peripheral red blood cells in CMS rats. Chronic hypoxia significantly induced methylation at the CpG site in the VHL promoter, decreased VHL expression, and increased HIF-2α and EPO expression. Chronic hypoxia increased DNMT3α and DNMT3β expression, consistent with the decrease in VHL expression. The DNA methyltransferase inhibitor 5-azacytidine reduced chronic hypoxia-induced erythroid proliferation in the bone marrow of rats with CMS by suppressing VHL methylation and DNMTs expression. SignificanceOur study suggests that VHL methylation contributes toward excessive erythrocytosis in CMS by upregulating the HIF-2α/EPO pathway in the bone marrow of rats. We demonstrated that the DNMT inhibitor 5-azacytidine can attenuate erythroid hyperplasia in the bone marrow by demethylating the VHL promoter.