Combining the Tyrosine Kinase Inhibitor Cabozantinib and the mTORC1/2 Inhibitor Sapanisertib Blocks ERK Pathway Activity and Suppresses Tumor Growth in Renal Cell Carcinoma

Abstract

Current treatment approaches for renal cell carcinoma (RCC) face challenges in achieving durable tumor responses due to tumor heterogeneity and drug resistance. Combination therapies that leverage tumor molecular profiles could offer an avenue for enhancing treatment efficacy and addressing the limitations of current therapies. To identify effective strategies for treating RCC, we selected ten drugs guided by tumor biology to test in six RCC patient-derived xenograft (PDX) models. The multitargeted tyrosine kinase inhibitor (TKI) cabozantinib and mTORC1/2 inhibitor sapanisertib emerged as the most effective drugs, particularly when combined. The combination demonstrated favorable tolerability and inhibited tumor growth or induced tumor regression in all models, including two from patients who experienced treatment failure with FDA-approved TKI and immunotherapy combinations. In cabozantinib-treated samples, imaging analysis revealed a significant reduction in vascular density, and single-nucleus RNA sequencing (snRNA-seq) analysis indicated a decreased proportion of endothelial cells in the tumors. SnRNA-seq data further identified a tumor subpopulation enriched with cell-cycle activity that exhibited heightened sensitivity to the cabozantinib and sapanisertib combination. Conversely, activation of the epithelial-mesenchymal transition pathway, detected at the protein level, was associated with drug resistance in residual tumors following combination treatment. The combination effectively restrained ERK phosphorylation and reduced expression of ERK downstream transcription factors and their target genes implicated in cell-cycle control and apoptosis. This study highlights the potential of the cabozantinib plus sapanisertib combination as a promising treatment approach for patients with RCC, particularly those whose tumors progressed on immune checkpoint inhibitors and other TKIs.

Significance: The molecular-guided therapeutic strategy of combining cabozantinib and sapanisertib restrains ERK activity to effectively suppress growth of renal cell carcinomas, including those unresponsive to immune checkpoint inhibitors.

Figure 1.

Study design and PDX relative tumor volume curves. A, Heat map summary of the treatment response for the single drug tests across six PDX lines. B, Schematic illustration of the study design. C, Line plots depicting the relative tumor volumes of mice treated with vehicle, cabozantinib, sapanisertib, and the combination (cabozantinib plus sapanisertib) in 6 PDX lines. Relative tumor volume is calculated by normalizing the tumor volume to its initial value at day 0 (expressed as 100%). Each treatment regimen included a specific number of mice: RESL5 and RESL4 models had four mice per regimen, and RESL3 and RESL12 models had three mice per regimen. In the case of RESL10, the control group comprised five mice, while the other groups had four mice each. RESL11 consisted of four mice in the control group and three mice in each of the other groups. D, Bodyweight changes (compared to day 0) for the six PDX lines (mice) treated with vehicle (CT; n = 17), cabozantinib (Cab; n = 19), sapanisertib (Sap; n = 17), and the combination (Cab+Sap; n = 17). P values were derived from the Wilcoxon test. E, Western blot analysis of pAKT (Ser473), total AKT, pERK1/2 (Thr202/Tyr204), total ERK1/2, and total GAPDH using untreated PDX tumor samples of RESL4, RESL5, and RESL10.

Figure 2.

H&E images for the PDX models. A, Representative H&E images for the six PDX models. B, Representative H&E images for tumors in mice treated with vehicle, cabozantinib, sapanisertib, and the combination (cabozantinib plus sapanisertib) in PDX lines RESL5 and RESL10. Red arrows, clear cells; green arrows, sarcomatoid cells; blue arrows, vessels.

Figure 3.

Immunofluorescence staining for tumor cells and vasculature in PDX lines RESL5 and RESL10. A, Representative IF images of untreated tumor samples stained with anti-CA9 antibody (green), anti-Ki67 antibody (red), and DAPI (blue) for PDX lines RESL5 and RESL10. Scale bars, 100 μm. B, Representative immunofluorescent images of tumor samples stained with anti-CD31 antibody (red) and anti-CA9 antibody (green) in mice treated with vehicle, cabozantinib, sapanisertib, and the combination (cabozantinib plus sapanisertib) in PDX lines RESL5 and RESL10. Three independent experiments were performed with similar results. Scale bars, 100 μm. C, Bar plot showing the normalized CD31 expression from immunofluorescent images for mice treated with vehicle, cabozantinib, sapanisertib, and the combination in PDX lines RESL5 and RESL10. ****, P < 0.0001 using Student t test.

Figure 4.

Proteomic analysis of the treatment effects. A, Volcano plot showing differentially expressed proteins between tumors after combination treatment of cabozantinib plus sapanisertib compared with the vehicle-treated controls, mapping 97 upregulated proteins (red dots) and 213 downregulated proteins (blue dots). B, Dot plot showing the overrepresented gene sets in proteins downregulated (top) and upregulated (bottom) after combination treatment. C, Volcano plots showing differentially expressed proteins between tumors after combination treatment versus single-agent treatments. Left, combination-treated and cabozantinib-treated tumors, mapping 59 upregulated and 146 downregulated proteins. Right, the comparison between combination-treated and sapanisertib-treated tumors, mapping 42 upregulated and 154 downregulated proteins. D, Violin plots showing protein levels of IGF2BP3, ERO1B, and PYCR1 in the tumor and normal adjacent tissue (NAT) samples in the CPTAC ccRCC discovery cohort. E, Kaplan–Meier curves displaying progression-free survival probability for two groups of patients in the CPTAC ccRCC discovery cohort. The two groups of patients were selected on the basis of the IGF2BP3 protein levels in their primary tumors. High IGF2BP3 protein expression represents those with protein expression in the upper 35% quantile. Low IGF2BP3 protein expression represents those with protein expression in the bottom 35% quantile.

Figure 5.

Overview of snRNA-seq data. A, Uniform Manifold Approximation and Projection (UMAP) visualization of the human tumor cells, colored by cluster IDs. Left, all the human tumor cells from the eight samples. Right, the cells divided by sample. B, UMAP visualization of the mouse cells, colored by cell type. Left, all the mouse cells from the eight samples. Right, the cells divided by sample. C, Dot plot showing the expression of marker genes for different mouse cell types. D, Bar plot showing the percentage of different cell types in each of the eight samples.

Figure 6.

Pathway activities of tumor cell clusters in treated and control tumors. A, Top, Uniform Manifold Approximation and Projection (UMAP) visualization of the human tumor cells, colored by meta-cluster IDs and divided by sample. Bottom, box plot showing the number of marker genes per cluster for different clustering resolutions. B, Heat map showing the pathway activity of each meta-cluster. C, Left, heat map showing the pairwise correlation coefficient of meta-cluster cells divided by different samples. Both the x-axis and y-axis represent meta-cluster cells divided by sample. Right, heat map showing the pathway activities of meta-cluster cells divided by different samples, sharing the same y-axis as the left and the same x-axis as B. D, Bar plot showing the percentage of cells in MC12 (cycling tumor cells) in each sample. E, Line plot showing the percentage of cells in different meta-clusters in control and combo-treated samples, colored by meta-cluster ID. F, Box plot showing the EMT score distribution in control and combo-treated samples.

Figure 7.

Protein markers associated with treatment effect. A, Heat map showing the scaled protein/phosphorylation abundance of the key members in the PI3K–mTOR pathway and RTKs targeted by cabozantinib (that were detected in the proteomics and phosphoproteomics datasets) in the control tumor samples across the PDX lines. B, Heat map showing the scaled gene expression of the key members in the PI3K–mTOR pathway and RTKs targeted by cabozantinib in the control tumor samples across 6 PDX lines. The unscaled gene expression represents log₂ (TPM+1). C, Scatter plot showing the association between baseline MET protein level (left) or MET gene expression (right) with the tumor growth inhibition at day 28. D,MET (top) and HGF (bottom) gene expression in the human tumor cells in the snRNA-seq data of the RESL10 and RESL5 control tumor samples. E, Western blot of the phospho-MET (Tyr1234/1235), total c-MET, and total GAPDH in untreated RESL4, RESL5, and RESL10 tumor samples.

Figure 8.

Western blot analysis and mechanistic insights into pERK signaling. A, Representative Western blot analysis of pERK and ERK protein expression. Vinculin was used as the loading control. Ctrl, control; Cab+Sap, cabozantinib plus sapanisertib. B, Bar plots showing normalized densitometric analysis of pERK (n = 4). The phosphoprotein bands were normalized against the loading control for each lane. The ratio of phosphoprotein/total corresponding protein was calculated by dividing the density of phosphoprotein by the corresponding total protein intensity in the same experiment. Left graph, the relative expression of phosphoprotein. Right graph, the ratio of phosphoprotein/total corresponding protein. Data points represent mean ± SEM. Paired two-tailed Student t test. C, Bar plots displaying the scores of TF activities for selected TFs. Paired two-tailed Student t test. D, Line plot illustrating the normalized gene expression of selected ERK downstream genes in control and combo-treated samples. E, Bar plot representing the normalized gene expression of selected ERK downstream genes in control, cabozantinib-treated, sapanisertib-treated, and combo-treated samples. F, Illustration depicting the potential ERK signaling cascade following cabozantinib plus sapanisertib treatment. The combo treatment inhibits ERK kinase activity, resulting in decreased pERK levels. Consequently, the activities of downstream transcription factors (e.g., AP-1 and MYC) are inhibited due to the absence of phosphorylation by ERK. This leads to reduced transcription of their target genes, including those involved in cell proliferation and survival.