Osalmid sensitizes clear cell renal cell carcinoma to navitoclax through a STAT3/BCL-XL pathway

Abstract

Clear cell renal cell carcinoma (ccRCC) is a common and lethal urinary malignancy characterized by its resistance to apoptosis. Despite the emerging treatment options available for ccRCC, only a small proportion of patients achieve long-term survival benefits. Previous studies have demonstrated that inducing tumor cell senescence, followed by treatment using senolytics, represents a potential strategy for triggering tumor cell apoptosis. However, it remains unclear whether this strategy is suitable for the treatment of ccRCC. Using the whole-genome CRISPR screening database Dependency Map portal (DepMap), we identified ribonucleotide reductase family member 2 (RRM2), which catalyzes the conversion of ribonucleotides to deoxyribonucleotides (dNTPs), as an essential targetable gene for ccRCC. Herein, we report that the combination of the choleretic drug osalmid targeting RRM2 and the senolytic compound navitoclax targeting BCL-XL represents a novel therapeutic approach for ccRCC. Furthermore, we have validated this approach across a panel of human ccRCC cells with different genetic backgrounds and multiple preclinical models, including cell line-derived xenografts (CDX), patient-derived xenografts (PDX), and patient-derived organoids (PDO). Mechanistically, osalmid-mediated inhibition of dNTPs generation induces cellular senescence in ccRCC, concomitant with STAT3 activation and upregulation of BCL-XL, thus rendering these cells vulnerable to navitoclax, which targets the BCL-2 protein family.

Keywords: Apoptosis; BCL-XL; Cellular senescence; Clear cell renal cell carcinoma; RRM2.

Figure 1.. Osalmid targets ccRCC essential gene RRM2.

A, Targetable essential genes were found by analyzing the overlap of genes up-regulated in ccRCC tumor tissues in TCGA database (log₂ Fold Change > 1.2) and genes essential for ccRCC cell lines proliferation (CERE scores < −0.75) in DepMap database. B, Essential genes of ccRCC cell lines ranked by CERE scores from low to high produced by analysis of CRISPR Screens in DepMap. RRM2 is marked by a short line on the right side. C, CERE scores of RRM2 in multiple ccRCC cell lines produced by analysis of CRISPR Screens in DepMap. D, Relative dNTP content of ccRCC cells treated with vehicle control or 50μM osalmid for 96h. dNTP was absolutely quantified by LC/MS, then relatively quantified to the control (n=2 biological replicates). Statistical significance was determined by two-way analysis of variance (ANOVA).

Figure 2.. Osalmid induces ccRCC cellular senescence.

A, Long term colony formation assay of ccRCC cell lines and the renal epithelial cell line treated with indicated concentration of osalmid for 96h. B, Representative images of β-gal staining performed on ccRCC cell lines and the renal epithelial cell line treated with vehicle control or 50μM osalmid for 96h. The bottom panel showed quantification of β-gal positive rate (n=3 biological replicates). C, Senescence markers protein expression detected by western blots in ccRCC cells treated with vehicle control or 50μM osalmid for 96h. VINCULIN was used as the loading control. D, Senescence associated secretory phenotype genes expression detected by qPCR in ccRCC cell lines treated with vehicle control or 50μM osalmid treated for 96h (n=3 biological replicates). Relative expression was normalized by β-ACTIN. Statistical significance was determined by two-way analysis of variance (ANOVA).

Figure 3.. Osalmid treatment sensitizes ccRCC cells to senolytic treatment.

A, Simplified schematic of the experimental design, generated by bioRENDER. B, Long term colony formation assays of ccRCC cell lines treated with indicated concentration of navitoclax for 48h in the presence or absence of 96h 50μM osalmid pretreatment. C, Cell viability assessed in ccRCC cell lines after treatment with different concentrations of navitoclax for 48h in the presence or absence of 96h 50μM osalmid pretreatment. Each dots represents a biological replicate (n=3). D, Representative images of in-situ caspase 3/7 staining performed on ccRCC cells treated with vehicle control or 5μM navitoclax for 4h in the presence or absence of 96h 50μM osalmid pretreatment. Caspase 3/7 was detected by green fluorescent labeled probe. E, PARP protein expression detected by western blots in ccRCC cells treated with vehicle control, osalmid, navitoclax, or both drugs combined. VINCULIN was used as the loading control. F, Long term colony formation assays of ccRCC cell lines treated with vehicle control or 5μM navitoclax with or without 20μM Z-VAD-FMK for 48h in the presence or absence of 96h 50μM osalmid pretreatment.

Figure 4.. STAT3 regulated BCL-XL drives sensitivity to senolytic therapy.

A, Long term colony formation assay of ccRCC cell lines treated with indicated concentration of BCL-XL specific inhibitor A-1331852 and navitoclax analogue ABT-737 for 24h in the presence or absence of 96h 50μM osalmid pretreatment. B, Long term colony formation assay of empty vector or BCL-XL overexpressed 786-O treated with indicated concentration of navitoclax for 48h in the presence or absence of 96h 50μM osalmid pretreatment. C, BCL-2 family protein expression detected by western blots in ccRCC cells treated with vehicle control or 50μM osalmid for 96h. ACTIN was used as the loading control. D, STAT3 protein expression detected by western blots in ccRCC cells treated with vehicle control or 50μM osalmid for 96h. ACTIN was used as the loading control. E, Representative photographs of immunohistochemistry staining for p-STAT3 and BCL-XL expression in 786-O derived subcutaneous tumor xenografts treated with vehicle control or osalmid. F, Simplified schematic of the molecular mechanism and workflow, generated by bioRENDER.

Figure 5.. Osalmid and navitoclax combined therapy suppresses ccRCC growth in multiple preclinical models.

A, Representative photographs of 786-O derived subcutaneous tumors grown in BALB/c nude mice treated with vehicle control, osalmid (150mg/kg/d), navitoclax (75mg/kg/d) or both drugs combined. (n=4 mice per group). B, Volumes and weights of 786-O derived subcutaneous tumors in Figure 5A. Statistical significance was determined by unpaired t-test. C, Representative photographs of ccRCC patient derived subcutaneous tumors grown in BALB/c nude mice treated with vehicle control or osalmid (150mg/kg/d) combined with navitoclax (75mg/kg/d) (n=5 mice per group). D, Growth curves and weights of ccRCC patient derived subcutaneous tumors in Figure 5C. Statistical significance was determined by unpaired t-test. E, Representative photographs of immunohistochemistry staining for cleaved caspase-3 expression in subcutaneous tumors in Figure 5A and Figure 5C. F, Representative photographs and corresponding viability of ccRCC patient derived organoids treated with control, 50μM osalmid, 2.5μM navitoclax or both drugs combined for 5 days (n=5 biological replications per group). Statistical significance was determined by unpaired t-test.