Synthetic essentiality of TRAIL/TNFSF10 in VHL-deficient renal cell carcinoma

Abstract

Clear cell renal cell carcinoma (ccRCC) is the most common and aggressive subtype of kidney cancer. Loss of von Hippel-Lindau (VHL) and the consequent activation of hypoxia-inducible factor-α (HIFα, especially HIF2α) plays an essential role in ccRCC initiation and progression. The approved HIF2α inhibitor belzutifan faces the challenge of resistance, presenting an opportunity of co-targeting HIF2α and another vulnerability. This study elucidates the synthetic essentiality of TRAIL (tumor necrosis factor-related apoptosis-inducing ligand) in VHL-deficient ccRCC, uncovering a novel reciprocal regulation between HIF2α and TRAIL. TRAIL was identified as a direct transcriptional target of HIF2α and paradoxically found to be crucial for cell proliferation, primarily by activating the p38 MAPK pathway and facilitating G1/S phase transition. Depletion of endogenous TRAIL or inhibition of HIF2α with belzutifan sensitizes ccRCC cells to recombinant TRAIL, presenting a promising avenue for combination therapy to overcome both TRAIL resistance and belzutifan resistance in treating ccRCC.

Keywords: HIF2α; TRAIL; VHL; belzutifan; renal cell carcinoma; synthetic essentiality.

Figure 1.. TRAIL is a HIF2α target and a candidate synthetic essential gene.

A. Venn diagram between HIF2α-upregulated genes (logFC(Ctrl/sgEPAS1) > 1.5, FDR < 0.05) and DepMap preferentially essential genes (gene effect < 0, Z-score < −1) in 786O. B. Bubble plot of the 57 overlapped genes. The bubbles’ size and color reflect the false discovery rate (FDR) and Z-score, respectively. TRAIL has lowest Z-score and is highlighted. C. Relative mRNA expression of EPAS1 and TNFSF10 in control and HIF2α-knockout 786O sublines (n=3), measured with qRT-PCR. D. Immunoblotting of HIF2α and TRAIL in control and HIF2α-knockout 786O sublines. E. Relative mRNA expression of TNFSF10 in normal (n=161) and tumor tissues (n=532) of TCGA KIRC patient’s cohort. F. TRAIL protein expression in normal (n=84) and tumor tissues (n=110) of CPTAC KIRC patient’s cohort. G. Scatter plot of TNFSF10 and EPAS1 levels in the TCGA KIRC cohort. Spearman correlation coefficient ρ and P value are marked. H. HIF2α and input ChIP-Seq signals in the TNFSF10 locus with the promoter region chr3:172,242,513 – 172,243,623 highlighted in the red box. I. ChIP-qPCR result for IgG and anti- HIF2α at the TNFSF10 promoter (n=3). In C and I, data represent mean ± SEM. Unpaired t-test for C and I, Mann-Whitney test for E and F, **P<0.01, ***P<0.001, ****P<0.0001.

Figure 2.. TRAIL depletion attenuates ccRCC proliferation in vitro and tumor growth in vivo.

A. qRT-PCR result showing relative mRNA expression of TNFSF10 of 786O-dishTRAIL cells under doxycycline off or on (2 μg/ml, 24 hours) conditions (n=3). B. Immunoblotting of TRAIL of 786O-dishTRAIL cells under doxycycline off or on (2 μg/ml, 48 hours) conditions. C. Cell proliferation curves of 786O-dishTRAIL cells under doxycycline off or on (2 μg/ml) conditions over a 96-hour period. Each data point was calculated from the cell confluency images (n=6). D. Clonogenic assay showing numbers of colonies (diameter of colony > 0.2cm) of 786O-dishTRAIL cells under doxycycline off or on (2 μg/ml, 10 days) conditions (n=3). E. Spider plots showing normalized bioluminescence imaging (BLI) signals for nude mice orthotopically injected with 786Odish-TRAIL cells and fed with doxycycline diet (Dox on, n=5) and control diet (Dox off, n=4). F. Representative BLI images (day 0 and 35) and normalized BLI signals (day 35) for Dox off and Dox on groups. In A, C, D and F, error bars represent SEM. Unpaired t-test for A, C and D, Mann-Whitney test for F, **P<0.01, ****P<0.0001.

Figure 3.. TRAIL activates p38 MAPK and facilitates G1/S progression in ccRCC cells.

A. Heatmap showing differential gene expression (|fold Change| > 1.5 and FDR < 0.05) between 786O-dishTRAIL treated with doxycycline (Dox on, 48 hours) and untreated controls (Dox off). TNFSF10 is among the downregulated genes by TRAIL knockdown. B. GSEA result showing MSigDB hallmark pathways (P < 0.05, FDR < 0.25) enriched to Dox-off condition. C. Representative flow cytometric plots of annexin-V-APC and DAPI staining on 786O-dishTRAIL under Dox on (48 hours) and Dox off conditions (n=4) and qualification of the percentage of apoptotic cells (red box). D-E. Representative flow cytometric plots for DNA content analysis using propidium iodide (PI) staining of 786O-dishTRAIL under Dox on (48 hours) and Dox off conditions (n=3), based on which the proportions of three cell cycle phases were quantitated. F. Immunoblotting showing changes of the indicated proteins and phosphorylations for 786O-dishTRAIL under Dox on and Dox off conditions for various durations (48, 72, 96 hours). G. Scatter plot of protein levels of TRAIL and p38 in ccRCC CPTAC data. Pearson correlation coefficient ρ and P value are indicated. H. Immunoblotting of p38 and phorpho-p38(Thr180/Tyr182) for 786O-dishTRAIL treated with 10μM necrostatin-1s (Nec-1s) for 1 hour or 48 hours of doxycycline. In C and E, error bars represent SEM. Unpaired t-test, *P < 0.05, ***P < 0.001, ^#P > 0.05.

Figure 4.. TRAIL is required to sustain HIF2α expression and activity in ccRCC cells.

A. Immunoblotting of HIF2α for 786O-dishTRAIL treated with doxycycline (Dox on) and untreated controls (Dox off) across three time points (48,72, and 96 hours). B. qRT-PCR result of relative mRNA expression of VEGFA and SLC2A1 for 786O-Ctrl, 786O-sgEPAS1, and 786O-dishTRAIL under Dox off and Dox on (96 hours) conditions (n=3). C. Immunoblotting of HIF2α for 786O treated with 25μM SB203580 for 1 hour or 6 hours. D. qRT-PCR result of relative mRNA expression of VEGFA, SLC2A1 and EPAS1 for 786O treated with 25μM SB203580 for 6 hours (n=3). In B and D, error bars represent SEM. Unpaired t-test, ***P < 0.001, ^#P > 0.05.

Figure 5.. TRAIL depletion re-localizes DR5 to the plasma membrane.

A. Immunofluorescence co-staining of TRAIL and DR5 in 786O. Scale bar 20μm. B. Immunofluorescence co-staining of TRAIL and lysosomal marker LAMP1 in 786O. Scale bar 20μm. C. Immunofluorescence co-staining of DR5 and LAMP1 in 786O. Scale bar 20μm. D. Representative flow cytometry plot and quantitation of mean fluorescence intensity (MFI) of DR5 signals on the cell surface in 786O-dishTRAIL cells under Dox on or Dox off conditions (48 hours, n=3). Error bars represent SEM. Unpaired t-test, ****P < 0.0001.

Figure 6.. HIF2α inhibitor synergizes with recombinant TRAIL to reduce 786O viability.

A. Normalized percentages of viable 786O-dishTRAIL cells treated with doxycycline and untreated control (48 hours) followed by treatment with two different concentrations of rTRAIL for 24 hours. Error bars represent SEM. Ordinary one-way ANOVA test with Tukey’s multiple comparison correction, ***P < 0.001, ****P < 0.0001. B. Immunoblotting of HIF2α, TRAIL, p-p38, and p38 in 786O treated with 2μM or 4μM belzutifan for 48 hours. C. Cell viability quantification of 786O under three treatments: incremental doses of rTRAIL (black), incremental doses of belzutifan (red), and combinations of rTRAIL and belzutifan at a fix ratio of 100 (cyan). The results were analyzed with CompuSyn software to construct FA-CI plot (right). CI<1 indicates synergism effect.