Metabolism-driven in vitro/in vivo disconnect of an oral ERɑ VHL-PROTAC.

Targeting the estrogen receptor alpha (ERα) pathway is validated in the clinic as an effective means to treat ER+ breast cancers. Here we present the development of a VHL-targeting and orally bioavailable proteolysis-targeting chimera (PROTAC) degrader of ERα. In vitro studies with this PROTAC demonstrate excellent ERα degradation and ER antagonism in ER+ breast cancer cell lines. However, upon dosing the compound in vivo we observe an in vitro-in vivo disconnect. ERα degradation is lower in vivo than expected based on the in vitro data. Investigation into potential causes for the reduced maximal degradation reveals that metabolic instability of the PROTAC linker generates metabolites that compete for binding to ERα with the full PROTAC, limiting degradation. This observation highlights the requirement for metabolically stable PROTACs to ensure maximal efficacy and thus optimisation of the linker should be a key consideration when designing PROTACs.

Multi-ancestry genome-wide association study of kidney cancer identifies 63 susceptibility regions.

Here, in a multi-ancestry genome-wide association study meta-analysis of kidney cancer (29,020 cases and 835,670 controls), we identified 63 susceptibility regions (50 novel) containing 108 independent risk loci. In analyses stratified by subtype, 52 regions (78 loci) were associated with clear cell renal cell carcinoma (RCC) and 6 regions (7 loci) with papillary RCC. Notably, we report a variant common in African ancestry individuals ( rs7629500 ) in the 3' untranslated region of VHL, nearly tripling clear cell RCC risk (odds ratio 2.72, 95% confidence interval 2.23-3.30). In cis-expression quantitative trait locus analyses, 48 variants from 34 regions point toward 83 candidate genes. Enrichment of hypoxia-inducible factor-binding sites underscores the importance of hypoxia-related mechanisms in kidney cancer. Our results advance understanding of the genetic architecture of kidney cancer, provide clues for functional investigation and enable generation of a validated polygenic risk score with an estimated area under the curve of 0.65 (0.74 including risk factors) among European ancestry individuals.

VHL loss reprograms the immune landscape to promote an inflammatory myeloid microenvironment in renal tumorigenesis.

Clear cell renal cell carcinoma (ccRCC) is characterized by dysregulated hypoxia signaling and a tumor microenvironment (TME) highly enriched in myeloid and lymphoid cells. Loss of the von Hippel Lindau (VHL) gene is a critical early event in ccRCC pathogenesis and promotes stabilization of HIF. Whether VHL loss in cancer cells affects immune cells in the TME remains unclear. Using Vhl WT and Vhl-KO in vivo murine kidney cancer Renca models, we found that Vhl-KO tumors were more infiltrated by immune cells. Tumor-associated macrophages (TAMs) from Vhl-deficient tumors demonstrated enhanced in vivo glucose consumption, phagocytosis, and inflammatory transcriptional signatures, whereas lymphocytes from Vhl-KO tumors showed reduced activation and a lower response to anti-programmed cell death 1 (anti-PD-1) therapy in vivo. The chemokine CX3CL1 was highly expressed in human ccRCC tumors and was associated with Vhl deficiency. Deletion of Cx3cl1 in cancer cells decreased myeloid cell infiltration associated with Vhl loss to provide a mechanism by which Vhl loss may have contributed to the altered immune landscape. Here, we identify cancer cell-specific genetic features that drove environmental reprogramming and shaped the tumor immune landscape, with therapeutic implications for the treatment of ccRCC.

Von-Hippel Lindau protein amyloid formation. The role of GST-tag.

In the last decade, much attention was given to the study of physiological amyloid fibrils. These structures include A-bodies, which are the nucleolar fibrillar formations that appear in the response to acidosis and heat shock, and disassemble after the end of stress. One of the proteins involved in the biogenesis of A-bodies, regardless of the type of stress, is Von-Hippel Lindau protein (VHL). Known also as a tumor suppressor, VHL is capable to form amyloid fibrils both in vitro and in vivo in response to the environment acidification. As with most amyloidogenic proteins fusion with various tags is used to increase the solubility of VHL. Here, we first performed AFM-study of fibrils formed by VHL protein and by VHL fused with GST-tag (GST-VHL) at acidic conditions. It was shown that formed by full-length VHL fibrils are short heterogenic structures with persistent length of 2400 nm and average contour length of 409 nm. GST-tag catalyzes VHL amyloid fibril formation, superimpose chirality, increases length and level of hierarchy, but decreases rigidity of amyloid fibrils. The obtained data indicate that tagging can significantly affect the fibrillogenesis of the target protein.

Molecular mechanism of infectious spleen and kidney necrosis virus in manipulating the hypoxia-inducible factor pathway to augment virus replication.

Infectious spleen and kidney necrosis virus (ISKNV), a member of the genus Megalocytivirus in the family Iridoviridae, can infect over 50 fish species and cause significant economic losses in Asia. Our previous study showed that hypoxia triggers the hypoxia-inducible factor pathway (HIF-pathway), leading to increased replication of ISKNV through promoting the upregulation of viral hypoxic response genes like orf077r. This study delved into the molecular mechanism of how ISKNV manipulates the HIF-pathway to enhance its replication. In vitro and in vivo experiments confirmed that ISKNV infection activated the HIF-pathway, which in turn promoted ISKNV replication. These findings suggest that ISKNV actively manipulates the HIF-pathway. Co-immunoprecipitation experiments revealed that the ISKNV-encoded protein VP077R interacts with the Von Hippel-Lindau (VHL) protein at the HIF-binding region, competitively inhibiting the interaction of HIF-1α with VHL. This prevents HIF degradation and activates the HIF-pathway. Furthermore, VP077R interacts with factor-inhibiting HIF (FIH), recruiting FIH and S-phase kinase-associated protein 1 (Skp1) to form an FIH - VP077R - Skp1 complex. This complex promotes FIH protein degradation via ubiquitination, further activating the HIF-pathway. These findings indicated that ISKNV takes over the HIF-pathway by releasing two "brakes" on this pathway (VHL and FIH) via VP077R, facilitating virus replication. We speculate that hypoxia initiates a positive feedback loop between ISKNV VP077R and the HIF pathway, leading to the outbreak of ISKNV disease. This work offers valuable insights into the complex interactions between the environment, host, and virus.

VHL mutation drives human clear cell renal cell carcinoma progression through PI3K/AKT-dependent cholesteryl ester accumulation.

BACKGROUND: Cholesteryl ester (CE) accumulation in intracellular lipid droplets (LDs) is an essential signature of clear cell renal cell carcinoma (ccRCC), but its molecular mechanism and pathological significance remain elusive. METHODS: Enabled by the label-free Raman spectromicroscopy, which integrated stimulated Raman scattering microscopy with confocal Raman spectroscopy on the same platform, we quantitatively analyzed LD distribution and composition at the single cell level in intact ccRCC cell and tissue specimens in situ without any processing or exogenous labeling. Since we found that commonly used ccRCC cell lines actually did not show the CE-rich signature, primary cancer cells were isolated from human tissues to retain the lipid signature of ccRCC with CE level as high as the original tissue, which offers a preferable cell model for the study of cholesterol metabolism in ccRCC. Moreover, we established a patient-derived xenograft (PDX) mouse model that retained the CE-rich phenotype of human ccRCC. FINDINGS: Surprisingly, our results revealed that CE accumulation was induced by tumor suppressor VHL mutation, the most common mutation of ccRCC. Moreover, VHL mutation was found to promote CE accumulation by upregulating HIFα and subsequent PI3K/AKT/mTOR/SREBPs pathway. Inspiringly, inhibition of cholesterol esterification remarkably suppressed ccRCC aggressiveness in vitro and in vivo with negligible toxicity, through the reduced membrane cholesterol-mediated downregulations of integrin and MAPK signaling pathways. INTERPRETATION: Collectively, our study improves current understanding of the role of CE accumulation in ccRCC and opens up new opportunities for treatment. FUNDING: This work was supported by National Natural Science Foundation of China (No. U23B2046 and No. 62027824), National Key R&D Program of China (No. 2023YFC2415500), Fundamental Research Funds for the Central Universities (No. YWF-22-L-547), PKU-Baidu Fund (No. 2020BD033), Peking University First Hospital Scientific and Technological Achievement Transformation Incubation Guidance Fund (No. 2022CX02), and Beijing Municipal Health Commission (No. 2020-2Z-40713).

Proteome-scale discovery of protein degradation and stabilization effectors.

Targeted protein degradation and stabilization are promising therapeutic modalities because of their potency, versatility and their potential to expand the druggable target space1,2. However, only a few of the hundreds of E3 ligases and deubiquitinases in the human proteome have been harnessed for this purpose, which substantially limits the potential of the approach. Moreover, there may be other protein classes that could be exploited for protein stabilization or degradation3-5, but there are currently no methods that can identify such effector proteins in a scalable and unbiased manner. Here we established a synthetic proteome-scale platform to functionally identify human proteins that can promote the degradation or stabilization of a target protein in a proximity-dependent manner. Our results reveal that the human proteome contains a large cache of effectors of protein stability. The approach further enabled us to comprehensively compare the activities of human E3 ligases and deubiquitinases, identify and characterize non-canonical protein degraders and stabilizers and establish that effectors have vastly different activities against diverse targets. Notably, the top degraders were more potent against multiple therapeutically relevant targets than the currently used E3 ligases cereblon and VHL. Our study provides a functional catalogue of stability effectors for targeted protein degradation and stabilization and highlights the potential of induced proximity screens for the discovery of new proximity-dependent protein modulators.

Targeting MYC induces lipid droplet accumulation by upregulation of HILPDA in clear cell renal cell carcinoma.

Metabolic reprogramming is critical during clear cell renal cell carcinoma (ccRCC) tumorigenesis, manifested by accumulation of lipid droplets (LDs), organelles that have emerged as new hallmarks of cancer. Yet, regulation of their biogenesis is still poorly understood. Here, we demonstrate that MYC inhibition in ccRCC cells lacking the von Hippel Lindau (VHL) gene leads to increased triglyceride content potentiating LD formation in a glutamine-dependent manner. Importantly, the concurrent inhibition of MYC signaling and glutamine metabolism prevented LD accumulation and reduced tumor burden in vivo. Furthermore, we identified the hypoxia-inducible lipid droplet-associated protein (HILPDA) as the key driver for induction of MYC-driven LD accumulation and demonstrated that conversely, proliferation, LD formation, and tumor growth are impaired upon its downregulation. Finally, analysis of ccRCC tissue as well as healthy renal control samples postulated HILPDA as a specific ccRCC biomarker. Together, these results provide an attractive approach for development of alternative therapeutic interventions for the treatment of this type of renal cancer.

Belzutifan for von Hippel-Lindau Disease: Pancreatic Lesion Population of the Phase 2 LITESPARK-004 Study.

PURPOSE: Primary analysis of the ongoing, single-arm, phase 2 LITESPARK-004 study (NCT03401788) showed clinically meaningful antitumor activity in von Hippel-Lindau (VHL) disease-associated renal cell carcinoma (RCC) and other neoplasms with belzutifan treatment. We describe results of belzutifan treatment for VHL disease-associated pancreatic lesions [pancreatic neuroendocrine tumors (pNET) and serous cystadenomas]. PATIENTS AND METHODS: Adults with VHL diagnosis based on germline VHL alteration, ≥1 measurable RCC tumor, no renal tumor >3 cm or other VHL neoplasm requiring immediate surgery, Eastern Cooperative Oncology Group performance status of 0 or 1, and no prior systemic anticancer treatment received belzutifan 120 mg once daily. End points included objective response rate (ORR), duration of response (DOR), progression-free survival (PFS), and linear growth rate (LGR) in all pancreatic lesions and pNETs per RECIST version 1.1 by independent review committee, and safety. RESULTS: All 61 enrolled patients (100%) had ≥1 pancreatic lesion and 22 (36%) had ≥1 pNET measurable at baseline. Median follow-up was 37.8 months (range, 36.1-46.1). ORR was 84% [51/61; 17 complete responses (CR)] in pancreatic lesions and 91% (20/22; 7 CRs) in pNETs. Median DOR and median PFS were not reached in pancreatic lesions or pNETs. After starting treatment, median LGR for pNETs was -4.2 mm per year (range, -7.9 to -0.8). Eleven patients (18%) had ≥1 grade 3 treatment-related adverse event (AE). No grade 4 or 5 treatment-related AEs occurred. CONCLUSIONS: Belzutifan continued to show robust activity and manageable safety in VHL disease-associated pNETs.

Therapeutic Targeting of Hypoxia-Inducible Factors in Cancer.

In the realm of cancer therapeutics, targeting the hypoxia-inducible factor (HIF) pathway has emerged as a promising strategy. This study delves into the intricate web of HIF-associated mechanisms, exploring avenues for future anticancer therapies. Framing the investigation within the broader context of cancer progression and hypoxia response, this article aims to decipher the pivotal role played by HIF in regulating genes influencing angiogenesis, cell proliferation, and glucose metabolism. Employing diverse approaches such as HIF inhibitors, anti-angiogenic therapies, and hypoxia-activated prodrugs, the research methodologically intervenes at different nodes of the HIF pathway. Findings showcase the efficacy of agents like EZN-2968, Minnelide, and Acriflavine in modulating HIF-1α protein synthesis and destabilizing HIF-1, providing preliminary proof of HIF-1α mRNA modulation and antitumor activity. However, challenges, including toxicity, necessitate continued exploration and development, as exemplified by ongoing clinical trials. This article concludes by emphasizing the potential of targeted HIF therapies in disrupting cancer-related signaling pathways.

The protein tyrosine phosphatase PPH-7 is required for fertility and embryonic development in C. elegans at elevated temperatures.

Post-translational modifications are key in the regulation of activity, structure, localization, and stability of most proteins in eukaryotes. Phosphorylation is potentially the most studied post-translational modification, also due to its reversibility and thereby the regulatory role this modification often plays. While most research attention was focused on kinases in the past, phosphatases remain understudied, most probably because the addition and presence of the modification is more easily studied than its removal and absence. Here, we report the identification of an uncharacterized protein tyrosine phosphatase PPH-7 in C. elegans, a member of the evolutionary conserved PTPN family of phosphatases. Lack of PPH-7 function led to reduction of fertility and embryonic lethality at elevated temperatures. Proteomics revealed changes in the regulation of targets of the von Hippel-Lindau (VHL) E3 ligase, suggesting a potential role for PPH-7 in the regulation of VHL.

VHL suppresses autophagy and tumor growth through PHD1-dependent Beclin1 hydroxylation.

The Von Hippel-Lindau (VHL) protein, which is frequently mutated in clear-cell renal cell carcinoma (ccRCC), is a master regulator of hypoxia-inducible factor (HIF) that is involved in oxidative stresses. However, whether VHL possesses HIF-independent tumor-suppressing activity remains largely unclear. Here, we demonstrate that VHL suppresses nutrient stress-induced autophagy, and its deficiency in sporadic ccRCC specimens is linked to substantially elevated levels of autophagy and correlates with poorer patient prognosis. Mechanistically, VHL directly binds to the autophagy regulator Beclin1, after its PHD1-mediated hydroxylation on Pro54. This binding inhibits the association of Beclin1-VPS34 complexes with ATG14L, thereby inhibiting autophagy initiation in response to nutrient deficiency. Expression of non-hydroxylatable Beclin1 P54A abrogates VHL-mediated autophagy inhibition and significantly reduces the tumor-suppressing effect of VHL. In addition, Beclin1 P54-OH levels are inversely correlated with autophagy levels in wild-type VHL-expressing human ccRCC specimens, and with poor patient prognosis. Furthermore, combined treatment of VHL-deficient mouse tumors with autophagy inhibitors and HIF2α inhibitors suppresses tumor growth. These findings reveal an unexpected mechanism by which VHL suppresses tumor growth, and suggest a potential treatment for ccRCC through combined inhibition of both autophagy and HIF2α.

Belzutifan, HIF-2α Inhibitor, and Clear Cell Renal Cell Carcinoma With Somatic Von-Hippel-Lindau Loss-of-Function Mutation.

The Von-Hippel-Lindau (VHL) gene, acting as a tumor suppressor, plays a crucial role in the tumorigenesis of clear cell renal cell carcinoma (ccRCC). Approximately 90% of individuals with advanced ccRCC exhibit somatic mutations in the VHL gene. Belzutifan, orally administered small-molecule inhibitor of hypoxia-induced factor-2α, has demonstrated promising efficacy in solid tumors associated with germline loss-of-function mutations in VHL, including ccRCC. However, its impact on cases with somatic or sporadic VHL mutations remains unclear. Here, we present 2 cases where belzutifan monotherapy was employed in patients with advanced ccRCC and somatic loss-of-function mutations in VHL. Both patients exhibited a swift and sustained response, underscoring the potential role of belzutifan as a viable option in second or subsequent lines of therapy for individuals with somatic VHL mutations. Despite both patients experiencing a pulmonary crisis with respiratory compromise, their rapid response to belzutifan further emphasizes its potential utility in cases involving pulmonary or visceral crises. This report contributes valuable insights into the treatment landscape for advanced ccRCC with somatic VHL mutations.

Discovery of a novel, highly potent EZH2 PROTAC degrader for targeting non-canonical oncogenic functions of EZH2.

Aberrant expression of EZH2, the main catalytic subunit of PRC2, has been implicated in numerous cancers, including leukemia, breast, and prostate. Recent studies have highlighted non-catalytic oncogenic functions of EZH2, which EZH2 catalytic inhibitors cannot attenuate. Therefore, proteolysis-targeting chimera (PROTAC) degraders have been explored as an alternative therapeutic approach to suppress both canonical and non-canonical oncogenic activity. Here we present MS8847, a novel, highly potent EZH2 PROTAC degrader that recruits the E3 ligase von Hippel-Lindau (VHL). MS8847 degrades EZH2 in a concentration-, time-, and ubiquitin-proteasome system (UPS)-dependent manner. Notably, MS8847 induces superior EZH2 degradation and anti-proliferative effects in MLL-rearranged (MLL-r) acute myeloid leukemia (AML) cells compared to previously published EZH2 PROTAC degraders. Moreover, MS8847 degrades EZH2 and inhibits cell growth in triple-negative breast cancer (TNBC) cell lines, displays efficacy in a 3D TNBC in vitro model, and has a pharmacokinetic (PK) profile suitable for in vivo efficacy studies. Overall, MS8847 is a valuable chemical tool for the biomedical community to investigate canonical and non-canonical oncogenic functions of EZH2.

Utilizing a novel model of PANoptosis-related genes for enhanced prognosis and immune status prediction in kidney renal clear cell carcinoma.

Kidney renal clear cell carcinoma (KIRC) is the most common histopathologic type of renal cell carcinoma. PANoptosis, a cell death pathway that involves an interplay between pyroptosis, apoptosis and necroptosis, is associated with cancer immunity and development. However, the prognostic significance of PANoptosis in KIRC remains unclear. RNA-sequencing expression and mutational profiles from 532 KIRC samples and 72 normal samples with sufficient clinical data were retrieved from the Cancer Genome Atlas (TCGA) database. A prognostic model was constructed using differentially expressed genes (DEGs) related to PANoptosis in the TCGA cohort and was validated in a Gene Expression Omnibus (GEO) cohorts. Incorporating various clinical features, the risk model remained an independent prognostic factor in multivariate analysis, and it demonstrated superior performance compared to unsupervised clustering of the 21 PANoptosis-related genes alone. Further mutational analysis showed fewer VHL and more BAP1 alterations in the high-risk group, with alterations in both genes also associated with patient prognosis. The high-risk group was characterized by an unfavorable immune microenvironment, marked by reduced levels of CD4 + T cells and natural killer cells, but increased M2 macrophages and regulatory T cells. Finally, the risk model was predictive of response to immune checkpoint blockade, as well as sensitivity to sunitinib and paclitaxel. The PANoptosis-related risk model developed in this study enables accurate prognostic prediction in KIRC patients. Its associations with the tumor immune microenvironment and drug efficacy may offer potential therapeutic targets and inform clinical decisions.

Design, synthesis, and evaluation of VHL-based EZH2 degraders for breast cancer.

EZH2 (enhancer of zeste homolog 2) is one of the most important histone methyltransferases (HMTs), and overexpression of EZH2 can lead to proliferation, migration and angiogenesis of tumor cells. But most of EZH2 inhibitors are only effective against some hematologic malignancies and have poor efficacy against solid tumors. Here, we report the design, synthesis, and evaluation of highly potent proteolysis targeting chimeric (PROTACs) small molecules targeting EZH2. We developed a potent and effective EZH2 degrader P4, which effectively induced EZH2 protein degradation and inhibited breast cancer cell growth. Further studies showed that P4 can significantly decrease the degree of H3K27me3 in MDA-MB-231 cell line, induce apoptosis and G0/G1 phase arrest in Pfeiffer and MDA-MB-231 cell lines. Therefore, P4 is a potential anticancer molecule for breast cancer treatment.

Journey of Von Hippel-Lindau (VHL) E3 ligase in PROTACs design: From VHL ligands to VHL-based degraders.

The scientific community has shown considerable interest in proteolysis-targeting chimeras (PROTACs) in the last decade, indicating their remarkable potential as a means of achieving targeted protein degradation (TPD). Not only are PROTACs seen as valuable tools in molecular biology but their emergence as a modality for drug discovery has also garnered significant attention. PROTACs bind to E3 ligases and target proteins through respective ligands connected via a linker to induce proteasome-mediated protein degradation. The discovery of small molecule ligands for E3 ligases has led to the prevalent use of various E3 ligases in PROTAC design. Furthermore, the incorporation of different types of linkers has proven beneficial in enhancing the efficacy of PROTACs. By far more than 3300 PROTACs have been reported in the literature. Notably, Von Hippel-Lindau (VHL)-based PROTACs have surfaced as a propitious strategy for targeting proteins, even encompassing those that were previously considered non-druggable. VHL is extensively utilized as an E3 ligase in the advancement of PROTACs owing to its widespread expression in various tissues and well-documented binders. Here, we review the discovery of VHL ligands, the types of linkers employed to develop VHL-based PROTACs, and their subsequent modulation to design advanced non-conventional degraders to target various disease-causing proteins. Furthermore, we provide an overview of other E3 ligases recruited in the field of PROTAC technology.

Inhibition of VHL by VH298 Accelerates Pexophagy by Activation of HIF-1α in HeLa Cells.

Autophagy is a pivotal biological process responsible for maintaining the homeostasis of intracellular organelles. Yet the molecular intricacies of peroxisomal autophagy (pexophagy) remain largely elusive. From a ubiquitin-related chemical library for screening, we identified several inhibitors of the Von Hippel-Lindau (VHL) E3 ligase, including VH298, thereby serving as potent inducers of pexophagy. In this study, we observed that VH298 stimulates peroxisomal degradation by ATG5 dependently and escalates the ubiquitination of the peroxisomal membrane protein ABCD3. Interestingly, the ablation of NBR1 is similar to the curtailed peroxisomal degradation in VH298-treated cells. We also found that the pexophagy induced by VH298 is impeded upon the suppression of gene expression by the translation inhibitor cycloheximide. Beyond VHL inhibition, we discovered that roxadustat, a direct inhibitor of HIF-α prolyl hydroxylase, is also a potent inducer of pexophagy. Furthermore, we found that VH298-mediated pexophagy is blocked by silencing HIF-1α. In conclusion, our findings suggest that VH298 promotes pexophagy by modulating VHL-mediated HIF-α transcriptional activity.

FOXA2 activates HIF2α expression to promote tumor progression and is regulated by the E3 ubiquitin ligase VHL in renal cell carcinoma.

Renal cell carcinoma (RCC) is a frequent malignancy of the urinary system with high mortality and morbidity. However, the molecular mechanisms underlying RCC progression are still largely unknown. In this study, we identified FOXA2, a pioneer transcription factor, as a driver oncogene for RCC. We show that FOXA2 was commonly upregulated in human RCC samples and promoted RCC proliferation, as evidenced by assays of cell viability, colony formation, migratory and invasive capabilities, and stemness properties. Mechanistically, we found that FOXA2 promoted RCC cell proliferation by transcriptionally activating HIF2α expression in vitro and in vivo. Furthermore, we found that FOXA2 could interact with VHL (von Hippel‒Lindau), which ubiquitinated FOXA2 and controlled its protein stability in RCC cells. We showed that mutation of lysine at position 264 to arginine in FOXA2 could mostly abrogate its ubiquitination, augment its activation effect on HIF2α expression, and promote RCC proliferation in vitro and RCC progression in vivo. Importantly, elevated expression of FOXA2 in patients with RCC positively correlated with the expression of HIF2α and was associated with shorter overall and disease-free survival. Together, these findings reveal a novel role of FOXA2 in RCC development and provide insights into the underlying molecular mechanisms of FOXA2-driven pathological processes in RCC.