CRISPR/Cas9 screening reveals that ASB2 inhibits p53-dependent ferroptosis to mediates Lenvatinib resistance in hepatocellular carcinoma

doi:10.21203/rs.3.rs-2444654/v1

Download PDF

Research Article

CRISPR/Cas9 screening reveals that ASB2 inhibits p53-dependent ferroptosis to mediates Lenvatinib resistance in hepatocellular carcinoma

https://doi.org/10.21203/rs.3.rs-2444654/v1

This work is licensed under a CC BY 4.0 License

Version 1

posted

You are reading this latest preprint version

Background

Lenvatinib is in a first-line therapy for advanced hepatocellular carcinoma (HCC), but its resistance is one of the main obstacles to treatment failure. The molecular mechanism of Lenvatinib resistance has not been well explored.

Methods

Genome-wide CRISPR/Cas9 knockout screening system was developed and bioinformatic analysis was used to identify key genes associated with Lenvatinib resistance in HCC. Whole transcriptome sequencing including coding and non-coding RNAs has also been performed in Lenvatinib resistance and sensitive HCC cells. Co-immunoprecipitation, confocal localization, western blot, immunofluorescence and other experiments were employed to assess the role of ASB2 in Lenvatinib resistance.

Results

ASB2 was found to be significantly increased at the mRNA and protein levels in Lenvatinib resistant HCC cells. ASB2 knockdown inhibited HCC Lenvatinib resistance cell proliferation, invasion and migration. Mechanistically, ASB2 activated NF-κB pathway by promoting IκBα ubiquitination and inhibited ferroptosis by promoting P53 ubiquitination and then mediated Lenvatinib resistance in HCC. Interestingly, NOTCH1 was shown to transcriptionly promote ASB2 expression and regulate NF-κB as well as ferroptosis pathways to induce Lenvatinib resistance in HCC. In further clinical translation, we found that Venetoclax could bind to ASB2 through a virtual screen of protein potential binding small molecules, and confirmed that Venetoclax and Lenvatinib combined significantly inhibited the progression of HCC, and the efficacy was better than Lenvatinib alone in vitro and vivo.

Conclusion

This study reveals that ASB2 which was transcriptionly promoted by NOTCH1, activated NF-κB pathway by promoting IκBα ubiquitination and inhibited ferroptosis by promoting P53 ubiquitination and mediated HCC Lenvatinib-resistance based on CRISPR/Cas9 screening. Venetoclax can potentially inhibit the function of ASB2 and the combination of Venetoclax and Lenvatinib can significantly inhibit the progression of HCC, which provides new targets and specific strategies for the treatment of HCC Lenvatinib-resistance, bringing new hope and benefits to HCC patients.

Lenvatinib

Genome-wide CRISPR/Cas9 library

ASB2

Ferroptosis

NF-κB

Hepatocellular carcinoma (HCC) is the third leading cause of cancer-related death worldwide, with a high incidence in Africa and the Asia-Pacific region [1]. Hepatitis C virus (HCV) and hepatitis B virus (HBV) infection, heavy alcohol consumption and non-alcoholic fatty liver disease are the main risk factors for HCC [2]. Surgical resection, local ablation, chemotherapy, molecular targeted therapy and liver transplantation are the main treatment options for HCC [3–4]. However, most patients with HCC are unresectable and the prognosis remains poor.

Lenvatinib, an oral multi-kinase inhibitor, has been recommended as first-line therapy for patients with unresectable HCC since 2019 in the United States, Japan, the European Union, and China. Although Lenvatinib performed poorly in terms of overall survival (OS), it was highly effective in improving objective response rate (ORR) and progression-free survival (PFS) [5]. More importantly, Lenvatinib is well tolerated compared with Sorafenib [5]. However, the efficacy and duration of treatment with Lenvatinib are also limited by acquired or intrinsic resistance [6]. Therefore, exploring potential biomarkers and molecular mechanisms of drug resistance may help elucidate strategies to improve the efficacy of Lenvatinib in the treatment of HCC.

High-throughput forward genetic screening methods are widely used to investigate the molecular mechanisms underlying specific cellular phenotype, including drug resistance in malignancies. In particular, RNA interference (RNAi) using shRNA libraries or clusters of regularly spaced short palindromic repeats of CRISPR-associated nuclease Cas9 (CRISPR/Cas9) is an effective tool and biological process for lose-of-function screening in a wide range of signaling pathways [7–8]. In the present study, a genome-wide CRISPR/Cas9 knockout screening system was established and bioinformatic analysis was used to identify critical genes associated with Lenvatinib resistance. Here, we present the full results of this screen, which included a list of genes that promote or repress HCC Lenvatinib resistance. This study reveals that Ankyrin repeat and SOCS box containing 2 (ASB2), which was transcriptionly promoted by NOTCH1, activated NF-κB pathway by promoting IκBα ubiquitination and inhibited ferroptosis by promoting P53 ubiquitination and mediated HCC Lenvatinib-resistance based on CRISPR/Cas9 screening. Venetoclax can potentially inhibit the function of ASB2 and the combination of Venetoclax and Lenvatinib can significantly inhibit the progression of HCC, which provides new targets and specific strategies for the treatment of HCC Lenvatinib-resistance.

Establishment of Lenvatinib resistant HCC cells

96-well plates containing HEP3B and HCCLM3 cells were seeded and treated with varying doses of lenvatinib. CCK-8 was used to assess cell viability after 72 hours of incubation. After that, Lenvatinib concentrations just below their IC50 were added to 6 well palates with 1× 10⁴ HEP3B and HCCLM3cells per well. Lenvatinib dosages were gradually increased over the subsequent weeks. Over the course of four months, lenvatinib of 30 uM-resistant HEP3B and HCCLM3 cell lines were established (HEP3B-LR and HCCLM3-LR). Following their establishment, Lenvatinib was consistently added to the culture of these resistant cell lines.

CRISPR/Cas9 sequencing and data analysis

The following steps were taken to create the CRISPR Screen II sequencing library. The library was created using a two-step PCR technique, with various reaction numbers chosen in accordance with the various sample types. Before and after the sgRNA sequence in the region, general primers were created, and high-fidelity enzymes were used to build the reaction system. The target fragment was obtained in the first step using PCR, and the number of cycles was typically kept between 18 and 22 cycles. In order to obtain the target fragments, PCR was carried out in a second step, adding primers (Index) and adapters (Adapter), and the number of cycles was typically controlled within 8–12 cycles. The glue is cut and cycled to obtain a target band following the two-step PCR. Following library purification, the concentration of the library was measured using qubit 3.0 (Invitrogen, Carlsbad, CA, USA), and the library fragment size was determined using 2200 (AgilentTechnologies, Palo Alto, USA). It was planned to use Nano QC (Illumina, San Diego, USA). According to Illumina's instrument instructions, DNA libraries with various index markers were mixed, and 2x150bp dual-end sequencing (PE) was carried out (Illumina, San Diego,USA). The HiSeq instrument's HiSeq Control Software (HCS) + OLB + Gapeline-1.6 (Illumina) was utilized for base detection and image analysis. Finally, GENEWIZ bioinformatics analysts examined the raw sequencing data. Using an internal script based on the upstream and downstream 5bp bases, target sgRNAs (19bp–21bp) were extracted. The extracted sgRNAs were then quality-controlled to produce clean reads. Using the MAGeCK software, the clean reads were compared to the sgRNA library, and the results were scored. MAGeCK software was used to compare the outcomes of differential gene analysis and GSEA enrichment analysis.

Whole transcriptome sequencing

The RNA total was taken out of cell samples. Short sequences can be sequenced on the Illumina platform, and rRNA can be removed to produce complete RNA sequences. mRNA can be randomly broken by using metal ions and choosing the right conditions, and a small fragment of about 300 bp can then be isolated by magnetic bead screening. The mRNA served as a template for the reverse synthesis of one-strand cDNA under the control of reverse transcriptase. In the two-strand synthesis, dUTP was substituted for dTTP in the dNTPs reagent, resulting in the bases A/U/C/G in the second strand of the cDNA. The double-stranded cDNA's 3' end receives an A base to connect the Y-shaped connector. The final library was purified and amplified by PCR. Libraries were pooled in accordance with the demands of effective concentration and anticipated data volume. Finally, sequencers of the corresponding models were chosen for on-machine sequencing based on the sequencing read length.

RNA extraction and qRT-PCR

TRIzol reagent (Invitrogen, USA) was used to extract total RNA from tissues and cells in accordance with the manufacturer's instructions. We converted RNA into cDNA using a reverse transcription kit from Takara, Japan. Table S1 includes a list of all primer sequences. The levels of mRNA expression were normalized using the internal control (GAPDH).

Cell transfection

In HEP3B-LR and HCCLM3-LR cells, we created shASB2-lentivirus packaging (Genechem, China). A suitable amount of virus and Polybrene (Sigma-Aldrich, USA) were added to 2×10⁵ cells/mL, and the mixture was thoroughly stirred. Exactly the same volume of fresh medium was used to dilute the polybrene after 4 hours of incubation at 37°C. After creating the sh3-ASB2 stable transfected cell line, we repeated the above transfection procedure using the sh3-ASB2 lentivirus, followed by a second round of puromycin screening, and finally obtained co-transfected cell lines. The shRNA and siRNA sequences used in this study are listed in Table S1. Western blotting and qRT-PCR were used to assess the effectiveness of the transfection.

Cell proliferation assay

We carried out the EdU experiment to measure cell proliferation using the Cell-Light 5-ethynyl-2'-deoxyuridine (EdU) DNA Cell Proliferation Kit (RiboBio, China). In 24-well plates, 2.5 × 10⁵ HEP3B-LR and HCCLM3-LR cells were plated before being cultured for 24 hours. After the cell lines had been exposed to a 50 mMol/L EdU solution for two hours, they were fixed with 4% paraformaldehyde. We applied DAPI seal and Apollo Dye Solution to the cell lines in accordance with the manufacturer's instructions. Under an Olympus FSX100 microscope, EdU cell lines were photographed and counted (Olympus, Japan).

Transwell assay

According to the manufacturer's instructions, we seeded 2×10⁴HEP3B-LR and HCCLM3-LR cells in 200 uL of serum-free medium in the upper chamber. For the invasion test, the matrix mixture was put inside the Transwell chamber (Corning, USA). A 10% FBS solution was added to the culture medium in the bottom cavity to act as a chemical attractant for cancer cells. Cells in the upper compartment were incubated for 24 hours before being fixed with methanol and stained for 15 minutes with crystal violet (Wobixin Inc., China). Imaging was used to see the cells, and cell counting was done.

Scratch wound experiment

After 48 hours of the transfecting process, when the cell confluence had reached about 90%, the wounds were created with a 200 ul pipette tip, and the cells were rinsed with PBS to remove debris and free-floating cells. The medium was supplemented, and the culturing plates underwent a 37°C incubation. The survey was given to wound healing at various times. Additionally, images of scrape lines were taken.

Immunofluorescence

Paraffin-embedded samples were sectioned at a thickness of 4 mm. By pressure cooking for 3 minutes in 0.01 mol/L citrate buffer, antigen retrieval was accomplished. Immunofluorescence tissue was permeabilized with 0.05 % Triton X-100 (Sigma-Aldrich, USA) in PBS for 5 minutes after being fixed with 4% paraformaldehyde (Wobixin Inc., China) for 20 min at room temperature. Samples were incubated with antibodies specific for GPX4 (1:200; Abcam, UK) and SLC7A11 (1:200; Abcam, UK) overnight at 4°C. Thereafter, samples were incubated with Alexa Fluor or HRP-conjugated secondary antibodies (Abcam, UK) for 1 h at room temperature. The nuclei were counterstained with DAPI (Sigma-Aldrich, USA), and laser scanning confocal microscopy was used to capture images (Zeiss,Germany).

Western blotting

RIPA buffer solution (Sigma-Aldrich, USA) was used to extract proteins from cells. After being lysed with SDS-polyacrylamide gels, the proteins were then transferred to PVDF membranes. Primary antibodies against ASB2, IκBα, P65, P-P65, SLC7A11, GPX4 were employed (Abcam, UK). GAPDH was assigned into the control. A peroxidase-conjugated secondary antibody (CST, Sigma-Aldrich, USA) was used with enhanced chemiluminescence (ECL; American Hot Fisher). The HA-ubiquitin was incubated with the total protein, and the products after incubation were analyzed by western blotting.

Co-immunoprecipitation

The HEP3B-LR and HCCLM3-LR cells were planted in a 100 mm culture dish. When the cell density reached about 70%, lysate without SDS was added to lyse the cells. Mix the lysate and centrifuge for supernatant; then add 5 × loading buffer, and 95 °C heat for 5 min. The remaining superoxide was transferred to the new EP tube, and the primary antibody was added. The mixture was inverted and incubated overnight at 4 °C, and the agar-sugar beads were added and incubated for 2 hours at 4 °C. Precipitation was collected by centrifuging at 4 °C, 14000 rpm, and the supernatant was discarded. Precooling PBS was used to wash the precipitation for several times. Precipitation was added 2× loading buffer, and denaturized for 5 minutes at 95 °C. The samples were detected by western blot.

Fluorescence probe assay of Reactive oxygen species (ROS)

HEP3B-LR and HCCLM3-LR cells were seeded in a 8-well plate (Corning), cultured at 37°C in 5% CO2 incubate overnight. Remove the medium and add 200 ul diluted in serum-free DMEM medium. The shASB2 group and the shNC group of HEP3B-LR and HCCLM3-LR were respectively placed in a 5% CO2 incubator and cultured at 37°C overnight. Wash the medium twice in 200 ul of HBSS after removing it. HBSS should be removed before adding 200 ul of diluted 1 umol/l Liperfluo. The solution was incubated at 37 °C for 30 min in a 5 % CO₂ incubator. With 200 ul of HBSS, wash twice after removing the solution. using a laser confocal fluorescence microscope to observe (excitation wavelength: 488 nm, emission wavelength: 500-550 nm).

Virtual screening

For structure-based VS(SBVS), MOE's dock module was utilized. The DrugBank approved drug molecules were chosen as the VS library. The SBVS flowchart for small molecules is shown in Figure 1. Each compound was prepared using MOE's Wash module. The binding site was defined by the Site Finder module in MOE and the binding site was chosen around the residues ASP420, MET448 GLY451, CYS452, ASP453, GLY454, GLU455, TYR515, VAL516, GLY517, VAL519, LEU554, ARG555, ARG557, LYS558, GLY561, LYS562, ILE565 and TYR583). After that all compounds were ranked by the flexible docking with the “induced fit” protocol. Prior to docking, the implicit solvation model of Reaction Field (R-field) and the force field of AMBER12: EHT were chosen. At a pH of 7 and a temperature of 300 K, the QuickPrep module optimized the protein's protonation state and the hydrogens' orientation. The docked poses for flexible docking were ranked first by London dG scoring, then a force field refinement was done on the top 10 poses, followed by a rescoring of GBVI/WSA dG, and the best ranked pose was kept. Finally, the top 100 molecules were selected as potential hits.

Animal models

Our animal testing was authorized by Nanjing Medical University's animal care committee. All procedures adhered to the relevant ethical guidelines for institutions performing animal experiments. All of the mice used in our studies were kept in the Experimental Animal Center at Nanjing Medical University under SPF conditions. Mice were sacrificed by cervical dislocation.

Prior to inoculation or injection, five-week-old nude BALB/c mice were randomly assigned to the following groups (n = 4 per group). To create the subcutaneous model, mice (3.0×10⁶ cells/mouse) were subcutaneously injected with HCCLM3-shNC, HEP3B-shNC, HCCLM3-shASB2, and HEP3B-shASB2 cells. On day 20, subcutaneous tumors for the xenograft model were removed and dissected.

The injection of Hepa1-6 cells was subcutaneously made into C57BL/6 mice. Carcinoma transplanted tumor model mice fell to four groups, PBS, Lenvatinib (MCE, USA), Venetoclax (MCE, USA), Lenvatinib + Venetoclax with 4 mice in the respective group. On the first day and every two days after that, 100 ul of Venetoclax was injected intraperitoneally; 200 ul of Lenvatinib was administered by oral gavage dissolved in 3 mM HCL per day. Before and after the experiment, the mice in each of the aforementioned mouse models had their daily activities, spirits, and diets monitored. Vernier calipers were used to measure the tumor's long diameter (mm) and short diameter (m) every four days. The tumor growth curve was then plotted after calculating the mice's tumor volume (V) using the formula V= ab²/2. On day 20, the mice were killed, and tumor samples were examined for further research.

Statistical analysis

GraphPad Prism 8.0 (GraphPad, USA) was used for our statistical analyses, and a P value of 0.05 or lower was regarded as statistically significant. While categorical variables were compared using the chi-square test, continuous variables were compared independently between the two groups using the t-test.

Analysis of CRISPR/Cas9 screening in HCC Lenvatinib-resistance cells

We established HCC Lenvatinib-resistance cell lines and performed CRISPR/Cas9 Screen on the model so that we could identify the key regulatory factors regulating HCC Lenvatinib-resistance. We confirmed resistance to Lenvatinib of HCC cell lines by analysis of IC50(Figure S1C). Using CRISPR/Cas9 high-throughput screening technology for positive or negative screening in tumor cells, candidate gene-directed RNA (gRNA) can be enriched to search for target genes of interest. The complete process of this technology includes the following steps: lentiviral library construction, virus infection of cells within the library, experimental screening of cells, genomic DNA extraction and second-generation sequencing library construction, second generation sequencing and bioinformatics analysis (Figure 1A). For PCR amplification products without any sequencing adapter sequence, after passing the quality inspection, take 100~500ng DNA fragments and go through End Repair, 5'Phosphorylation and dA-Tailing, Adapter Ligation, Clean Up, PCR Enrichment and Clean Up and other steps to construct and form a high-throughput sequencing library on the Illumina platform (Figure 1B). After obtaining raw data with sufficient sequencing depth, bioinformatic analysis was performed, which consists of the following three main stages. The detailed analysis process was shown in Figure 1C. We used volcano plots to show genes with significant differences in positive and negative screening results (Figure 1D-1E). GO analysis revealed that the positive group was mainly enriched in ERBB, cell development etc., while the negative group was mainly enriched in fatty acid, immune response etc. (Figure 1F, Figure S1A). KEGG analysis indicated that the positive group was mainly enriched in signaling by RANBP2, KRAS etc., while the negative group was mainly enriched in NF-κB ,TP53 etc. (Figure 1G, Figure S1B ). These results suggested that CRISPR/Cas9 screening technology indeed provides a large number of reliable differential genes for promoting or inhibiting HCC Lenvatinib-resistance, which can be targeted for subsequent studies.

Whole transcriptome results of HCC Lenvatinib-resistance cells

In this study, we further performed whole-transcriptome sequencing including coding and non-coding RNAs of HCCLM3-LR and HEP3B-LR cell lines. For mRNAs, heat map and volcano map showed differential genes in susceptible and resistant groups, respectively (Figure 2A-2B). Venn diagrams compared the differential genes common to and unique to different comparison combinations (Figure 2C). GO analysis revealed that the different genes in HCCLM3-LR/LS was mainly enriched in Developmental process, Chromosome, Molecular function etc., while the different genes in HEP3B-LR/LS was mainly enriched in Metabolic process, Intracellular, Protein binding etc. (Figure 2D-2E). KEGG analysis indicated that the different genes in HCCLM3-LR/LS was mainly enriched in Apoptosis, Alcoholism, Toll like receptor signaling pathway etc., while the different genes in HEP3B-LR/LS was mainly enriched in Systemic lupus erythematosus, Alcoholism, Necroptosis etc. (Figure 2F-2G).

For lncRNAs, heat map and volcano map showed differential genes in susceptible and resistant groups, respectively(Figure S2A-2B).Venn diagrams compared the differential genes common to and unique to different comparison combinations (Figure S2C).GO analysis revealed that the different genes in HCCLM3-LR/LS was mainly enriched in Phosphorus metabolic process, Mitochondrial membrane, NADH dehydrogenase(ubiquinone) activity etc., while the different genes in HEP3B-LR/LS was mainly enriched in Regulation of metabolic process, Non-membrane bounded organelle, Molecular function etc (Figure S2D-S2E). KEGG analysis indicated that the different genes in HCCLM3-LR/LS was mainly enriched in Thermogenesis, Oxidative phosphorylation, Chemokine signaling pathway etc., while the different genes in HEP3B-LR/LS was mainly enriched in Necroptosis, Focal adhesion, TNF signaling pathway etc. (Figure S2F-S2G).

For circRNAs, volcano map showed differential genes in susceptible and resistant groups, respectively (Figure S3A). GO analysis revealed that the different genes in HCCLM3-LR/LS was mainly enriched in Cellular metabolic process, Intracellular, Protein binding etc, while the different genes in HEP3B-LR/LS was mainly enriched in Metabolic process, Organelle, Molecular function etc(Figure S3B). KEGG analysis indicated that the different genes in HCCLM3-LR/LS was mainly enriched in Ubiquitin mediated proteolysis, Lysine degradation, Autophagy etc., while the different genes in HEP3B-LR/LS was mainly enriched in Protein process in endoplasmic reticulum, Cellular senescence, Cell cycle etc (Figure S3C). According to the prediction results of miRNA binding sites, the regulatory network of miRNA-circRNA was constructed (Figure S3D).

For miRNAs, we demonstrated the first base bias of known miRNAs ranging in length from 18 to 30nt in four cells (HCCLM3-LR/LS, HEP3B-LR/LS), respectively (Figure 3A). Heat map and volcano map showed differential genes in susceptible and resistant groups, respectively(Figure 3B-3C). GO analysis revealed that the different genes in HCCLM3-LR/LS was mainly enriched in Biological process, Intracellular, Molecular function etc., while the different genes in HEP3B-LR/LS was mainly enriched in Cellular process, Intracellular, Molecular function etc (Figure 3D,3F). KEGG analysis indicated that the different genes in HCCLM3-LR/LS was mainly enriched in VEGF signaling pathway, Mitophagy, Apoptosis, while the different genes in HEP3B-LR/LS was mainly enriched in VEGF, MAPK pathway,etc. (Figure 3E, 3G).

We also performed a joint analysis of the above RNAs and showed the KEGG analysis diagram. miRNA-mRNA joint showed that the different genes in HCCLM3-LR/LS was mainly enriched in Alcoholism, Tyrosine metabolism, Metobolic pathways etc., while the different genes in HEP3B-LR/LS was mainly enriched in MAPK signaling pathway, MicroRNAs in cancer, P53 signaling pathway etc (Figure S4A). circRNA-mRNA joint showed that the different genes in HCCLM3-LR/LS was mainly enriched in TGF-βsignaling pathway, Hippo signaling pathway, Cell cycle etc., while the different genes in HEP3B-LR/LS was mainly enriched in Serotonergic synapse, RAS signaling pathway, Hippo signaling pathway etc (Figure 4). LncRNA-mRNA joint showed that the different genes in HCCLM3-LR/LS was mainly enriched in TNF signaling pathway, P53 signaling pathway, Ferroptosis etc., while the different genes in HEP3B-LR/LS was mainly enriched in TNF signaling pathway, Focal adhesion, Autophagy etc (Figure S4C). LncRNA-miRNA-mRNA joint showed that the different genes in HCCLM3-LR/LS was mainly enriched in MicroRNAs in cancer, MAPK signaling pathway, P53 signaling pathway etc, while the different genes in HEP3B-LR/LS was mainly enriched in Metabolic pathways, Histidine metabolism, Serotonergic synapse etc., (Figure S4D). CircRNA-miRNA-mRNA joint showed that the different genes in HCCLM3-LR/LS was mainly enriched in MicroRNAs in cancer, MAPK signaling pathway, P53 signaling pathway etc., while the different genes in HEP3B-LR/LS was mainly enriched in Metabolic pathways, Histidine metabolism, Serotonergic synapse etc. (Figure S4E). Whole transcriptome sequencing systematically studies various RNA molecules and their interactions, providing a rich database for HCC Lenvatinib-resistance.

ASB2 facilitated the progressioninHCC Lenvatinib-resistance cells

Based on the cross-analysis of HCCLM3-LR and HEP3B-LR cell lines, we found that ASB2 was involved in the resistance of both cell lines, so it has the potential to be further studied. Three shRNAs against ASB2 (shASB2) were developed to silence ASB2 in HCCLM3-LR and HEP3B-LR cells, which was confirmed by qRT-PCR and western blotting and sh3-ASB2 was selected for further experiment (Figure 4A-4B). shASB2 inhibited proliferation in HCCLM3-LR and HEP3B-LR cells, as per the results of EdU assays (Figure 4C). Transwell and wound healing assays showed that shASB2 significantly hindered the invasion and migration functions of cancer cells (Figure 4D-4E). These results demonstrated that ASB2 promoted the progression of HCC Levatinib-resistant cells in vitro.

ASB2activated NF-κB pathway by promoting IκBα ubiquitination to mediate HCC Lenvatinib-resistance

The above results raised our curiosity about the reason why ASB2 promotes HCC Lenvatinib-resistance. We enriched the CRISPR/Cas9 results and surprisingly found differential promoting genes were enriched NF-κB pathway (Figure 1G ). Continued activation of NF-κB is thought to be associated with cancer resistance, including Sorafenib resistance in HCC [9-10]. So we wanted to explore how does ASB2 activate NF-κB and promote HCC Lenvatinib-resistance. STRING analysis predicted that ASB2 protein was associated with NF-κB pathway-related proteins(Figure S5). Based on the fact that ASB2 is a ubiquitinase, we hypothesized that ASB2 activates the NF-κB pathway by promoting the ubiquitination and degradation of IκBα. After transfection with ASB2 plasmid, the ubiquitination level of IκBα increased, and the expression of IκBα decreased in 293T cells (Figure 5A). We further examined the effect of ASB2 knockdown on IκBα ubiquitination in HCCLM3-LR and HEP3B-LR cells and found that the ubiquitination of IκBα was decreased by ASB2 knockdown and the expression of IκBα was increased (Figure 5B).To verify the interaction between ASB2 and IκBα, we performed co-immunoprecipitation experiments. As shown in Figure 5C, ASB2 and IκBα interacted with each other in 293 T cells. We observed co-localization of ASB2 and IκBα through immunofluorescence experiments in 293 T cells(Figure 5C-5D). In addition ,western blot confirmed that the expression of P-P65 was down-regulated in shASB2 group but P65 was not differentially expressed, suggesting that the NF-κB pathway was inhibited (Figure 5E).These results verified ASB2 promoted the ubiquitination of IκBα, activating NF-κB pathway in HCC Lenvatinib-resistance cells.

ASB2 inhibitedferroptosis by promoting P53 ubiquitination to mediate HCC Lenvatinib-resistance

CRISPR/Cas9 enrichment results also revealed differential gene enrichment in the fatty acid pathway (Figure 1F). Ferroptosis is a common type of fatty acid metabolism [11-12]. We found that ASB2 production was significantly negatively correlated with GPX4 in HCC via GEPIA database (Figure S6). Therefore, we tried to investigate that whether ASB2 mediates ferroptosis to promote drug resistance. To prove our hypothesis, we first examined the ROS content in shASB2 and shNC groups. The ROS probe suggested that the ROS content in shASB2 group was significantly higher than that of the control group (Figure 6A). Electron micrographs showed that the mitochondria of shASB2 group were pyknotic, smaller in size, higher in electron density, and expanded in cristae, consistent with the characteristics of ferroptosis (Figure 6B). So how does ASB2 affect ferroptosis deserves further study. Ubibrowser predicted that P53 may be degraded by ASB2 ubiquitination (Figure S7). Recent studies have found that P53 acts as a positive regulator of ferroptosis by promoting ROS production. P53 directly regulates the metabolic versatility of cells by favoring mitochondrial respiration, leading to ROS-mediated ferroptosis[13-14]. After transfection with ASB2 plasmid, the ubiquitination level of P53 increased in 293T cells (Figure 6C). We detected the effect of ASB2 knockdown on P53 ubiquitination and results showed that the ubiquitination of P53 was decreased by ASB2 knockdown and the expression of P53 was increased in HCCLM3-LR and HEP3B-LR cells (Figure 6D). Western blot confirmed that ferroptosis-related proteins including GPX4 and SLC7A11 proteins were all down-regulated in shASB2 group, indicating ferroptosis increased and iron inducers-erastin was used as a control (Figure 6E). These results confirmed ASB2 promoted the ubiquitination of P53, inhibiting ferroptosis pathway in HCC Lenvatinib-resistance cells.

NOTCH1 transcriptionally regulated the expression of ASB2 inHCC Lenvatinib-resistance cells

The above studies confirmed the downstream pathway of ASB2, so who regulates the expression of ASB2? Previous study indicated that NOTCH1 could induced ASB2 expression to promote protein ubiquitination by forming non-canonical E3 ligase complexes [15]. Therefore, we hypothesized that NOTCH1 activates the expression of ASB2 through transcriptional regulation and then participates in NF-κB and ferroptosis pathways leading to persistent drug resistance in HCC Lenvatinib resistance cells. Results showed that the addition of NOTCH1 inhibitor (LY3039478, iNOTCH1) decreased the proliferation, invasion and migration in HCCLM3-LR and HEP3B-LR cells (Figure S8). Both ASB2 and ferroptosis genes including GPX4 and SLC7A11 in mRNA level were significantly reduced after the addition of NOTCH1 inhibitor (Figure 7A). Western blot results indicated that in iNOTCH1 group, ferroptosis-related proteins were all down-regulated and the coexistence of iNOTCH1 and shASB2 further enhanced ferroptosis (Figure 7B). The expression of P-P65 was down-regulated in iNOTCH1 group but P65 was not differentially expressed, and the expression of IκBα was increased (Figure 7B).The ROS probe suggested that the ROS content in iNOTCH1 group was significantly higher than that of the control group (Figure 7C). Electron microscopic observation showed that the mitochondria of iNOTCH1 group were pyknotic, small in size, and expanded in cristum, which were consistent with the characteristics of ferroptosis (Figure 7D). The ChIP data from GSE39263 showed that NOTCH1 had a significant peak in the upstream of ASB2, suggesting that there was an obvious binding site between NOTCH1 and ASB2 (Figure 7E).

Knocked down of ASB2 inhibited the growth of HCC Lenvatinib-resistance tumors in vivo

To explore the relevance of ASB2 to HCC growth in vivo, the shASB2 and shNC HCCLM3-LR and HEP3B-LR cells were injected into the axilla of nude mice, respectively. Results revealed that the down-regulated expression of ASB2 significantly inhibited the growth of HCCLM3-LR and HEP3B-LR compared to the shNC group (Figure 8A-8C). Immunofluorescence results showed that shASB2 decreased the expression of SLC7A11 and GPX4 in tumor tissues, while the group of shNC showed increased the expression of SLC7A11 and GPX4 (Figure 8D). These evidences indicated that knocked down of ASB2 promoted ferroptosis and inhibited the growth of HCC Lenvatinib-resistance tumors in vivo.

Venetoclax acting as ASB2 inhibitor decreased the progression of HCC Lenvatinib-resistance tumors in vivo

As there is no readily available drug to inhibit ASB2, we used virtual screening to predict small molecule compounds that might inhibit ASB2. A total of 100 small molecule compounds with potential binding to ASB2 were screened, and the first 20 were shown (Figure S89). After evaluating it, we found that Venetoclax can bind to ASB2 and has a potential inhibitory function using virtual screening (Figure 8E). According to the results of Edu, transwell and wound healing assays, Venetoclax significantly inhibited the proliferation, invasion and migration of HCCLM3-LR and HEP3B-LR cells (Figure S10). In vivo experiments confirmed that Venetoclax inhibited the growth of HCCLM3-LR and HEP3B-LR compared to the PBS group. The combination of Venetoclax and Lenvatinib significantly inhibited the progression of HCC, and the efficacy was better than Lenvatinib alone (Figure 8F-8H). These results demonstrate that Venetoclax as an ASB2 inhibitor can reduce the progression of HCC Lenvatinib-resistant tumors.

Cancer cell genomes carry a variety of genetic aberrations, which accumulate from congenital and acquired mutations and are triggered by successive clonal expansions [16]. Identifying genes that drive tumor evolution can shed light on cancer occurrence and progression [17]. Large-scale genomic screening is a powerful tool for detecting mutated genes responsible for various cancers [16]. Functional genomic screening using CRISPR can reveal phenotypic changes after drug therapy or other stimuli, thereby identifying new targets for cancer therapy [18]. Chune Yu et al. reported that a genome-wide CRISPR-Cas9 knockdown screen identified GRB7 as a driver of MEK inhibitor resistance in KRAS mutant colon cancer [19].Qianqian Wang et al. identified MDM2 as an essential gene and potential therapeutic target of WT-p53-RTK Non-small cell lung adenocarcinoma(NSCLC) by genome-wide CRISPR/Cas9 screening. RG7388 alone or in combination with pemetrexed resulted in significant tumor suppression [20]. Xue Sui et al. identified a novel cisplatin-resistant esophageal oncogene, ERCC8, based on genome-scale CRISPR/Cas9 screening [21]. In Lu Yonggang’s study, they performed a genome-wide CRISPR/Cas9 knockout library screen using the HCC cell line Huh7 [22]. They identified two key resistance genes, neurofibrin 1 (NF1) and bi-specific phosphatase 9 (DUSP9), and demonstrated that they induce Lenvatinib resistance by activating PI3K/AKT and MEK/ERK signaling pathways in vivo and in vitro. They also screened out trametinib, a small-molecule pathway inhibitor that can be used to reverse Lenvatinib resistance in HCC [22]. Shanzhou Huang et al. identified six genes associated with Lenvatinib resistance in HCC HepG2 and Huh7 cell lines, including DUSP4, CCBL1, DHDH, CNTN2, NOS3 and TNF. It was found that DUSP4 mRNA and protein levels were significantly decreased in Lenvatinib-resistant HCC cells. DUSP4 knockdown enhanced HCC cell survival, cell proliferation, and migration during Lenvatinib treatment in vitro and in vivo, while modulating p-ERK and P-MEK levels. This finding suggests that DUSP4 deficiency induces Lenvatinib resistance. Interestingly, DUSP4-deficient induced resistance to Lenvatinib was abrogated by the MEK inhibitor Selumetinib, implying that resistance requires MEK phosphorylation and DUSP4-dependent ERK activation [23]. Although our study was not the first globally reported use of CRISPR/Cas9 screening to find HCC Lenvatinib-resistant genes,the cell lines (HCCLM3 and HEP3B) we used are different from others. Another highlight of our study was that whole transcriptome sequencing was used to detect the differential genes and enrichment pathways in the Lenvatinib-resistant and sensitive groups, which provided a complete database for the study of Lenvatinib-resistant in HCC.

In the present study, ASB2 was found to be significantly increased at the mRNA and protein levels in Lenvatinib resistant HCC cells. ASB2 knockdown inhibited HCC Lenvatinib resistance cell proliferation,invasion and migration. Mechanistically, ASB2 activated NF-κB pathway by promoting IκBα ubiquitination and mediated Lenvatinib resistance in HCC. ASB2 encodes a member of the ankyrin repeat and suppressor of cytokine signalling (SOCS) box-containing (ASB) protein family. ASB2 is expressed regionally specifically in the heart as early as the formation of the cardiac crescent at early developmental stages. ASB2 ubiquitinates SMAD9 instead of SMAD1 or SMAD5 and targets SMAD9 for proteasomal degradation to avoid excessive accumulation of SMAD9 in the embryo for normal cardiac development [24]. June Ho Shin et al. found that AHR regulates the expression of ASB2, while ASB2 regulates the ubiquitination and proteasome degradation of filament protein A, thereby regulating NK cell migration [25]. However, there are few reports about ASB2 in tumor. It has been reported that FLI1 regulates both the classical NF-κB pathway at the transcriptional level, and the alternative NF-κB pathway, via ASB2 in germinal center B-cell (GCB) diffuse large B-cell lymphoma (DLBCL) [26]. FLI1 and ASB2 inhibition represents a potential novel therapeutic approach for GCB DLBCL. The escape of cancer cells from host immunosurveillance involves a shift in immune responses, including an imbalance in Th1 and Th2 cells. A Th1-dominated immune response predicts positive outcomes in colorectal cancer. Spinner Camille A et al. found that ASB2 promotes a Th2 phenotype, which in turn is associated with tumor progression in a mouse model of colitis [27]. Our study enriches the relationship between ASB2 and NF-κB pathway in cancer especially drug resistance.

Our study is the first to link ASB2 to ferroptosis. Ferroptosis is an intracellular form of iron-dependent cell death, which is different from apoptosis, necrosis and autophagy [28–29]. Erastin-induced cell death, also known as ferroptosis, is characterized by an imbalance of redox status and elevated intracellular ROS levels. Loss of redox state balance in biological systems is the cause of many diseases [30]. Emerging evidence suggests that ferroptosis plays a key role in cancer treatment by interfering with target molecules and is involved in cancer development. In addition, ferroptosis has been reported to be used to overcome resistance to targeted therapies. Sorafenib has been identified as an inhibitor of several oncogenic kinases and is the first systemic therapy approved for patients with advanced HCC. However, its use is limited by acquired resistance, resulting in poor prognosis. Metallothionein-1g (MT-1G) is a functional isomer of metallothionein-1. Activation of the transcription factor nuclear factor erythrocyte 2-related factor 2 (NRF2) is critical for the induction of MT-1G expression after Sorafenib treatment.MT-1G up-regulation can limit GSH consumption and lipid peroxidation, which contribute to Sorafenib induced ferroptosis [31]. However, there are very few studies on Lenvatinib-resistant and ferroptosis in HCC. Norifumi Iseda et al reported that NRF2/FGFR4 silenced HCC cells showed sensitivity to Lenvatinib and high lipid ROS levels. The efficacy of Lenvatinib in recurrent HCC lesions with low FGFR4 expression was significantly lower than those with high FGFR4 expression. Patients with FGFR4-positive HCC had significantly longer progression-free survival than patients with FGFR4-negative HCC. Lenvatinib is involved in the sensitivity of HCC to Lenvatinib by inhibiting FGFR4 and NRF2 to induce ferroptosis [32]. Our study innovatively discovered the ASB2 inhibited ferroptosis by promoting P53 ubiquitination to mediate HCC Lenvatinib-resistance, providing a new target for the treatment of HCC.

Interestingly, NOTCH1 was shown to transcriptional promote ASB2 expression and regulate NF-κB as well as ferroptosis pathways to induce Lenvatinib resistance in HCC.Wu Wei et al. demonstrated for the first time that NOTCH1 can activate the transcription of ASB2, which then stimulates activation of NF-κB in T cell acute lymphoblastic leukemia (T-ALL) cells. ASB2 exerts its effects by inducing degradation and dissociation of IκBα from NF-κB in T-ALL cells. Moreover, specific suppression of ASB2 expression can promote apoptosis and inhibit proliferation of T-ALL cells [33]. Our study enriches the NOTCH1-ASB2 axis in cancer.

In further clinical translation, we found that Venetoclax could bind to ASB2 through a virtual screen of protein potential binding small molecules,and confirmed that Venetoclax and Lenvatinib combined significantly inhibited the progression of HCC, and the efficacy was better than Lenvatinib alone in vitro and vivo.The small-molecule "BH3 (BCL-2 homeodomain 3) -mimetic" drug Venetoclax (Venclexta™) is currently approved by the U.S. Food and Drug Administration (FDA) for the treatment of 17p deletion in patients with relapsed chronic lymphocytic leukemia (CLL) [34]. Recent studies have shown that Venetoclax could be used to treat other cancers. Lok Sheau W et al demonstrated that combining Venetoclax with endocrine therapy has a tolerable safety profile and elicits notable activity in ER and BCL2-positive metastatic breast cancer [35]. Perimenis Philippos et al found that Birinapant and AT-406 can synergise with BCL-2 inhibitor to reduce viability of adenocarcinoma cells with high BCL-2 expression [36]. Therefore, the combination of Venetoclax and Lenvatinib is expected to enhance the anti-HCC effect and alleviate the resistance of Lenvatinib in the future.

In summary, the present study reveals that ASB2 activates NF-κB pathway by promoting IκBα ubiquitination and inhibits ferroptosis by promoting P53 ubiquitination and mediates HCC Lenvatinib-resistance based on CRISPR/Cas9 screening. Venetoclax can potentially inhibit the function of ASB2 and the combination of Venetoclax and Lenvatinib can significantly inhibit the progression of HCC, which provides new targets and specific strategies for the treatment of HCC Lenvatinib-resistance, bringing new hope and benefits to HCC patients (Fig. 9).

Ethics approval and consent to participate

The Ethics Committee of Nanjing Medical University approved this study and gave their consent for the experiment to be carried out.

Consent for publication

All the authors agree to the content of the paper and are being listed as a co-author of the paper

Availability of data and material

The corresponding author will provide the datasets used or analyzed during the current study upon reasonable request.

Competing interests

The authors declare no competing interests.

Acknowledgements

Not applicable.

Funding

We are grateful for the grants from the National Natural Science Foundation of China (No.82070676) and Nanjing Medical University Science and Technology Development Fund (NMUB20210212).

Author contributions

There are 4 first authors in this manuscript and they have equally contributed to this project. Dr. GQS, ZQC, YLW and HSC were responsible for designing and performing the experiments. Dr. TH, CL, DWR, MRD, HYL, XL, GSS contributed to performing part of experiment. Furthermore, we have 4 corresponding authors in this manuscript. Dr. JK, YXX, TTF, and WWT have contributed to data interpretation, editing and critical revision of the manuscript. Dr. JK, YXX, TTF, and WWT have contributed to study design and critical revision of the manuscript. All authors read and approved the final manuscript.

Forner A, Reig M, Bruix J. Hepatocellular carcinoma. Lancet. 2018;391(10127):1301-14.
Kanwal F, Singal AG. Surveillance for Hepatocellular Carcinoma: Current Best Practice and Future Direction. Gastroenterology. 2019;157(1):54-64.
Kudo M. Targeted and immune therapies for hepatocellular carcinoma: Predictions for 2019 and beyond. World J Gastroenterol. 2019;25(7):789-807.
Vilchez V, Turcios L, Marti F, Gedaly R. Targeting Wnt/β-catenin pathway in hepatocellular carcinoma treatment. World J Gastroenterol. 2016;22(2):823-32.
Al-Salama ZT, Syed YY, Scott LJ. Lenvatinib: A Review in Hepatocellular Carcinoma. Drugs. 2019;79(6):665-74.
Fu R, Jiang S, Li J, Chen H, Zhang X. Activation of the HGF/c-MET axis promotes lenvatinib resistance in hepatocellular carcinoma cells with high c-MET expression. Med Oncol. 2020;37(4):24.
Wang T, Wei JJ, Sabatini DM, Lander ES. Genetic screens in human cells using the CRISPR-Cas9 system. Science. 2014;343(6166):80-4.
Chen S, Sanjana NE, Zheng K, Shalem O, Lee K, Shi X, et al. Genome-wide CRISPR screen in a mouse model of tumor growth and metastasis. Cell. 2015;160(6):1246-60.
Yu J, Wang N, Gong Z, Liu L, Yang S, Chen GG, et al. Cytochrome P450 1A2 overcomes nuclear factor kappa B-mediated sorafenib resistance in hepatocellular carcinoma. Oncogene. 2021;40(3):492-507.
Hu B, Xu Y, Li YC, Huang JF, Cheng JW, Guo W, et al. CD13 promotes hepatocellular carcinogenesis and sorafenib resistance by activating HDAC5-LSD1-NF-κB oncogenic signaling. Clin Transl Med. 2020;10(8):e233.
Fernández-Acosta R, Iriarte-Mesa C, Alvarez-Alminaque D, Hassannia B, Wiernicki B, Díaz-García AM, et al. Novel Iron Oxide Nanoparticles Induce Ferroptosis in a Panel of Cancer Cell Lines. Molecules. 2022;27(13).
Shi L, Liu Y, Li M, Luo Z. Emerging roles of ferroptosis in the tumor immune landscape: from danger signals to anti-tumor immunity. Febs j. 2022;289(13):3655-65.
Zhang W, Gai C, Ding D, Wang F, Li W. Targeted p53 on Small-Molecules-Induced Ferroptosis in Cancers. Front Oncol. 2018;8:507.
Ji H, Wang W, Li X, Han X, Zhang X, Wang J, et al. p53: A double-edged sword in tumor ferroptosis. Pharmacol Res. 2022;177:106013.
Nie L, Zhao Y, Wu W, Yang YZ, Wang HC, Sun XH. Notch-induced Asb2 expression promotes protein ubiquitination by forming non-canonical E3 ligase complexes. Cell Res. 2011;21(5):754-69.
Hanahan D, Weinberg RA. Hallmarks of cancer: the next generation. Cell. 2011;144(5):646-74.
Garraway LA, Lander ES. Lessons from the cancer genome. Cell. 2013;153(1):17-37.
Bester AC, Lee JD, Chavez A, Lee YR, Nachmani D, Vora S, et al. An Integrated Genome-wide CRISPRa Approach to Functionalize lncRNAs in Drug Resistance. Cell. 2018;173(3):649-64.e20.
Yu C, Luo D, Yu J, Zhang M, Zheng X, Xu G, et al. Genome-wide CRISPR-cas9 knockout screening identifies GRB7 as a driver for MEK inhibitor resistance in KRAS mutant colon cancer. Oncogene. 2022;41(2):191-203.
Wang Q, Li J, Zhu J, Mao J, Duan C, Liang X, et al. Genome-wide CRISPR/Cas9 screening for therapeutic targets in NSCLC carrying wild-type TP53 and receptor tyrosine kinase genes. Clin Transl Med. 2022;12(6):e882.
Sui X, Tang X, Wu X, Liu Y. Identification of ERCC8 as a novel cisplatin-resistant gene in esophageal cancer based on genome-scale CRISPR/Cas9 screening. Biochem Biophys Res Commun. 2022;593:84-92.
Lu Y, Shen H, Huang W, He S, Chen J, Zhang D, et al. Genome-scale CRISPR-Cas9 knockout screening in hepatocellular carcinoma with lenvatinib resistance. Cell Death Discov. 2021;7(1):359.
Huang S, Ma Z, Zhou Q, Wang A, Gong Y, Li Z, et al. Genome-Wide CRISPR/Cas9 Library Screening Identified that DUSP4 Deficiency Induces Lenvatinib Resistance in Hepatocellular Carcinoma. Int J Biol Sci. 2022;18(11):4357-71.
Min KD, Asakura M, Shirai M, Yamazaki S, Ito S, Fu HY, et al. ASB2 is a novel E3 ligase of SMAD9 required for cardiogenesis. Sci Rep. 2021;11(1):23056.
Shin JH, Moreno-Nieves UY, Zhang LH, Chen C, Dixon AL, Linde MH, et al. AHR Regulates NK Cell Migration via ASB2-Mediated Ubiquitination of Filamin A. Front Immunol. 2021;12:624284.
Sartori G, Napoli S, Cascione L, Chung EYL, Priebe V, Arribas AJ, et al. ASB2 is a direct target of FLI1 that sustains NF-κB pathway activation in germinal center-derived diffuse large B-cell lymphoma. J Exp Clin Cancer Res. 2021;40(1):357.
Spinner CA, Lamsoul I, Métais A, Febrissy C, Moog-Lutz C, Lutz PG. The E3 Ubiquitin Ligase Asb2α in T Helper 2 Cells Negatively Regulates Antitumor Immunity in Colorectal Cancer. Cancer Immunol Res. 2019;7(8):1332-44.
Cheah JH, Kim SF, Hester LD, Clancy KW, Patterson SE, 3rd, Papadopoulos V, et al. NMDA receptor-nitric oxide transmission mediates neuronal iron homeostasis via the GTPase Dexras1. Neuron. 2006;51(4):431-40.
Yagoda N, von Rechenberg M, Zaganjor E, Bauer AJ, Yang WS, Fridman DJ, et al. RAS-RAF-MEK-dependent oxidative cell death involving voltage-dependent anion channels. Nature. 2007;447(7146):864-8.
Dröge W. Free radicals in the physiological control of cell function. Physiol Rev. 2002;82(1):47-95.
Sun X, Niu X, Chen R, He W, Chen D, Kang R, et al. Metallothionein-1G facilitates sorafenib resistance through inhibition of ferroptosis. Hepatology. 2016;64(2):488-500.
Iseda N, Itoh S, Toshida K, Tomiyama T, Morinaga A, Shimokawa M, et al. Ferroptosis is induced by lenvatinib through fibroblast growth factor receptor-4 inhibition in hepatocellular carcinoma. Cancer Sci. 2022;113(7):2272-87.
Wu W, Nie L, Zhang L, Li Y. The notch pathway promotes NF-κB activation through Asb2 in T cell acute lymphoblastic leukemia cells. Cell Mol Biol Lett. 2018;23:37.
Stilgenbauer S, Eichhorst B, Schetelig J, Coutre S, Seymour JF, Munir T, et al. Venetoclax in relapsed or refractory chronic lymphocytic leukaemia with 17p deletion: a multicentre, open-label, phase 2 study. Lancet Oncol. 2016;17(6):768-78.
Lok SW, Whittle JR, Vaillant F, Teh CE, Lo LL, Policheni AN, et al. A Phase Ib Dose-Escalation and Expansion Study of the BCL2 Inhibitor Venetoclax Combined with Tamoxifen in ER and BCL2-Positive Metastatic Breast Cancer. Cancer Discov. 2019;9(3):354-69.
Perimenis P, Galaris A, Voulgari A, Prassa M, Pintzas A. IAP antagonists Birinapant and AT-406 efficiently synergise with either TRAIL, BRAF, or BCL-2 inhibitors to sensitise BRAFV600E colorectal tumour cells to apoptosis. BMC Cancer. 2016;16:624.

No competing interests reported.

Supplementary.docx

Download PDF

Version 1

posted

You are reading this latest preprint version

CRISPR/Cas9 screening reveals that ASB2 inhibits p53-dependent ferroptosis to mediates Lenvatinib resistance in hepatocellular carcinoma

Status:

Version 1

Abstract

Background

Methods

Results

Conclusion

Figures

Introduction

Materials And Methods

Co-immunoprecipitation

Results

Discussion

Conclusion

Declarations

References

Additional Declarations

Supplementary Files

Status:

Version 1