Characterization of the pVHL Interactome in Human Testis Using High-Throughput Library Screening

Functional impairment of the von Hippel-Lindau (pVHL) tumor suppressor is causative of a familiar increased risk to develop cancer. As E3 substrate recognition particle, pVHL marks for degradation the hypoxia inducible factor 1α (HIF-1α) in normoxic conditions, thus acting as a key regulator of both acute and chronic cell adaptation to hypoxia. Further evidence showed pVHL to also play relevant roles in microtubules stabilization, participate in the formation of the extracellular matrix, as well as to regulate cell senescence and apoptosis. Male mice model carrying VHL gene conditional knockout present significative abnormalities in testis development paired with defects in spermatogenesis and infertility, indicating that pVHL exerts testis-specific roles, at least in mice. Here, we describe 55 novel interactors of the human pVHL obtained by testis-tissue library screening. We show that pVHL interacts with multiple human proteins directly involved in spermatogenesis and reproductive metabolism, suggesting that, in addition to its role in cancer formation, pVHL may be pivotal in the correct gonads development also in human.

MTHFD2 Expression Association with Bladder Cancer and Screening of its Potential Inhibitor

BackgroundBladder cancer is the most common urological malignancy. Genes of folate mediated 1 carbon metabolism are found to be highly up regulated in tumor cells and promotes tumor cell proliferation. Rationale and aim of the studyThe aim of the current study was to determine the expression of MTHFD2 gene and the impact of intronic SNP rs1667627 on MTHFD2 expression Furthermore, determination of potential ligand based inhibitors against MTHFD2. Methods & ResultsSemi-quantitative expression analysis and sanger sequencing were used for this purpose. Moreover, structure based virtual ligand library screening in order to find plausible inhibitors.MTHFD2 expression was significantly increased with tumor stage progression both in low and high-grade bladder cancer. However, the relative fold change difference in low grade bladder cancer in correlation with the tumor stage progression was more dramatic. Contrary to the TCGA dataset analysis, increased MTHFD2 expression was observed in papillary bladder cancer tumor. G to A transition in the intronic variant rs1667627 SNP was determined in tumor tissues as compared to control. Virtual ligand based library screening against the three dimensional MTHFD2 protein lead to identification of a plausible inhibitor MCULE-8027924848 that displayed lower binding free energy as compared to already documented LY345899. ConclusionMTHFD2 might be used as low-grade bladder cancer biomarker since its expression level changes drastically with tumor progression. Further, experimental studies are required to establish the potential mode of inhibition of MCULE-8027924848 ligand.

The Disulfiram/Copper Complex Induces Autophagic Cell Death in Colorectal Cancer by Targeting ULK1

Colorectal cancer (CRC) is highly prevalent worldwide, but there has been limited development of efficient and affordable treatment. Induced autophagy has recently been recognized as a novel therapeutic strategy in cancer treatment, and disulfiram (DSF), a well-known antialcohol drug, is also found to inhibit tumor growth in various malignancies. Recently, DSF has been reported to induce excessive autophagy in oral squamous cells; however, little is known about whether it can induce autophagy and suppress proliferation in CRC. In this study, we investigate the effect of DSF with copper (DSF/Cu) on CRC both in vitro and in vivo and find that the combination significantly inhibits CRC cell viability and mainly induces autophagy instead of apoptosis. Furthermore, we use whole genome CRISPR library screening and identify a new mechanism by which DSF triggers autophagy by ULK1. Overall, these findings provide a potential CRC treatment.

Large Screening Identifies ACE2 Positively Correlates With NF-κB Signaling Activity and Targeting NF-κB Signaling Drugs Suppress ACE2 Levels

Coronaviruses SARS-CoV-2 infected more than 156 million people and caused over 3 million death in the whole world, therefore a better understanding of the underlying pathogenic mechanism and the searching for more effective treatments were urgently needed. Angiotensin-converting enzyme 2 (ACE2) was the receptor for SARS-CoV-2 infection. In this study, we found that ACE2 was an interferon-stimulated gene (ISG) in human cell lines. By performing an ISG library screening, we found that ACE2 levels were positively regulated by multiple ISGs. Interestingly, ACE2 levels were highly correlated with ISGs-induced NF-κB activities, but not IFNβ levels. Furthermore, using an approved clinical durgs library, we found two clinical drugs, Cepharanthine and Glucosamine, significantly inhibited ACE2 level, IFNβ level, and NF-κB signaling downstream TNFα and IL6 levels. Our finding suggested the possible inhibitory effects of Cepharanthine and Glucosamine during SARS-CoV-2 infection and the subsequent inflammatory cytokine storm.

Genome-scale CRISPR-Cas9 knockout screening in hepatocellular carcinoma with lenvatinib resistance

Lenvatinib is the first target drug approved for advanced hepatocellular carcinoma (HCC). However, the development of drug resistance is common, and the mechanisms of lenvatinib resistance and resistant targets in HCC are poorly understood. By using CRISPR/Cas9 library screening, we screened out two key resistance genes, neurofibromin 1(NF1), and dual specificity phosphatase 9 (DUSP9), as critical drivers for lenvatinib resistance in HCC. With RNAi knockdown and CRISPR/Cas9 knockout models, we further clarified the mechanisms by which NF1 loss reactivates the PI3K/AKT and MAPK/ERK signaling pathways, while DUSP9 loss activates the MAPK/ERK signaling pathways, thereby inactivating FOXO3, followed by degradation of FOXO3, finally induced lenvatinib resistance. We also screened out trametinib, a small molecule pathway inhibitor for MEK, that can be used to reverse resistance induced by NF1 and DUSP9 loss in HCC cells. Trametinib was still able to halt HCC growth even when NF1 was knocked out in mice. Collectively, the findings indicate that NF1 and DUSP9 takes critical role in lenvatinib resistance and may be novel specific targets and predictive markers for lenvatinib resistance in HCC.

The Molecular Landscape and Essential Pathways Involved in Bone Marrow-Mediated Carfilzomib Resistance of Multiple Myeloma

Background Multiple myeloma (MM) remains an incurable malignancy, with most patients relapsing and dying from the disease. Anti-myeloma drugs, such as proteasome inhibitors (PIs) bortezomib and carfilzomib (CFZ), have considerably improved prognosis in myeloma. Despite these advances, disease heterogeneity, early relapse and treatment resistance still pose major challenges in MM treatment. Understanding the mechanisms that mediate PI resistance provide a key to targeting both, PI-resistant minimal residual disease that drives relapsed MM after prolonged PI-containing frontline therapy, as well as PI-refractory, aggressive advanced MM. While key mechanisms of the in vitro-generated PI resistance in MM have been revealed in cell line models, we lack understanding of PI resistance in vivo, where in particular clonal heterogeneity and the tumor microenvironment (TME) within the bone marrow (BM) add additional levels of complexity. Therefore, the aim of our study was to analyze the molecular landscape and changes occurring during MM progression under CFZ treatment in vivo and to identify key molecular processes contributing to CFZ-resistance of MM cells in the presence of stromal cells in vitro, to ultimately identify new molecular pathways and develop innovative treatment strategies in PI-resistant MM. Methods The NSG mice intrafemorally engrafted with human RPMI-8226 cells were either untreated or treated long-term with 4 mg/kg CFZ (intravenously) until they became drug resistant. At this point, CFZ naïve and CFZ-resistant cells were isolated and processed for single-cell RNA sequencing (scRNA-seq, 10x Genomics) with the aim to characterize a transcriptional CFZ-resistance signature in refractory cells. To investigate the role of the TME as well as the importance of cell-cell interactions in CFZ-resistance in vitro, we performed two independent genome-wide CRISPR/Cas9 library screenings. In the first one, Brunello library transduced RPMI-8226 cells were co-cultured with human stromal cells (HS5) and treated with CFZ to identify CFZ sensitivity/resistance candidate genes. In the second experiment, Brunello library and synthetic Notch (synNotch) receptor transduced HS5 cells were co-cultured with synNotch ligand transduced RPMI-8226 cells to identify genes that are essential for establishing cell-cell contacts between stromal and MM cells. Subsequent functional analysis of the highest-ranking CFZ sensitivity/resistance candidates in the RPMI-8226+HS5 co-culture included shRNA-silencing, single-gene knockouts, viability assays, cell cycle analysis and protein synthesis analysis using the SUnSET assay. Results ScRNA-seq analysis of CFZ-refractory RPMI-8226 cells growing in the BM of NSG mice showed a different transcriptional landscape, compared to CFZ-naïve cells isolated from the BM of untreated mice. The unsupervised clustering analysis, using UMAP, revealed that cells exposed to CFZ show distinct populations with a strong increase in the OXPHOS and protein folding capacity as well as down-regulation of several genes involved in proliferation and apoptosis, when compared to naïve cells. The CRISPR/Cas9 library screening where RPMI-8226 cells were co-cultured with HS5 cells and exposed to CFZ revealed several CFZ sensitivity candidates at the cut-off of false discovery rate (FDR) < 0.01 and fold change above 1.5-fold. Those genes are involved in cytokine signaling, cell growth, invasion, metastasis and quality control of translational elongation. At the same time, the CRISPR/Cas9 library screening, where synNotch receptor transduced HS5 cells were co-cultured with synNotch ligand transduced RPMI-8226 cells revealed gene candidates at the cut-off of FDR < 0.01 and fold change greater than 1.5-fold, which mediate stronger or weaker cell-cell interaction. Those genes are particularly involved in cytokine signaling and mitochondrial metabolism. Conclusion In conclusion, MM cells that acquired CFZ-resistance upon cell-cell contact with certain cell types within the TME, such as stromal cells, differ significantly from CFZ-naïve cells. CFZ-resistance, caused by cell-cell contact with stromal cells, is presumably mediated via decreased proliferative as well as protein synthesis capacity of MM cells. Therefore, stimulation of MM cells to proliferate and synthesize more proteins may be a key to targeting CFZ-resistance in vivo. Disclosures No relevant conflicts of interest to declare.