Development of a PAK4-targeting PROTAC for renal carcinoma therapy: concurrent inhibition of cancer cell proliferation and enhancement of immune cell response

Summary

Background

Finding the oncogene, which was able to inhibit tumor cells intrinsically and improve the immune answers, will be the future direction for renal cancer combined treatment. Following patient sample analysis and signaling pathway examination, we propose p21-activated kinase 4 (PAK4) as a potential target drug for kidney cancer. PAK4 exhibits high expression levels in patient samples and plays a regulatory role in the immune microenvironment.

Methods

Utilizing AI software for peptide drug design, we have engineered a specialized peptide proteolysis targeting chimera (PROTAC) drug with selectivity for PAK4. To address challenges related to drug delivery, we developed a nano-selenium delivery system for efficient transport of the peptide PROTAC drug, termed PpD (PAK4 peptide degrader).

Findings

We successfully designed a peptide PROTAC drug targeting PAK4. PpD effectively degraded PAK4 with high selectivity, avoiding interference with other homologous proteins. PpD significantly attenuated renal carcinoma proliferation in vitro and in vivo. Notably, PpD demonstrated a significant inhibitory effect on tumor proliferation in a fully immunocompetent mouse model, concomitantly enhancing the immune cell response. Moreover, PpD demonstrated promising tumor growth inhibitory effects in mini-PDX and PDO models, further underscoring its potential for clinical application.

Interpretation

This PAK4-targeting peptide PROTAC drug not only curtails renal cancer cell proliferation but also improves the immune microenvironment and enhances immune response. Our study paves the way for innovative targeted therapies in the management of renal cancer.

Funding

This work is supported by Research grants from non-profit organizations, as stated in the Acknowledgments.

Research in context.

Evidence before this study

The lack of novel targeted therapies and immunotherapy efficacy is the main challenge in renal cell carcinoma (RCC) therapy. P21-activated kinase 4 (PAK4) is known to inhibiting the proliferation of cancer cells and concurrent enhancing the immune microenvironment by boosting the killing capacity of T cells, and has been identified as a “kill two birds with one stone” tumor target. Up to now, due to the highly conservatism among PAK family members, PAK4 inhibitors could not completely inhibit PAK4 protein function. Proteolysis-targeting chimera (PROTAC) drug, which is not only inhibits target protein function but also induces protein degradation, has emerged as a promising approach.

Added value of this study

Here, we utilizing the PAK4 protein structure and AI software for peptide drug design. We engineered a peptide PROTAC drug with selectivity for PAK4 protein degradation, termed PpD (PAK4 peptide degrader). Effectively degraded PAK4 with high selectivity, avoiding interference with other homologous proteins. Notably, PpD not only significant inhibition of renal carcinoma proliferation in patient-derived xenograft (PDX) and patient-derived organoid (PDO) models, but also exhibited potent synergistic effects with PD-1 blockade immunotherapy in vivo.

Implications of all the available evidence

By integrating multiple perspectives in druggable protein target analysis, we provided a De novo design of PAK4 peptide PROTAC. PpD provided a dual therapeutic advantage by simultaneously inhibiting renal cancer cell proliferation and enhancing immune cell response, being a potential synergistic therapy to clinical treatment, and it is not limited in renal cancer.

Introduction

Renal cell carcinoma (RCC) ranks among the top 10 most prevalent cancers globally and has exhibited a substantial increase in incidence in recent years.¹ Clear cell RCC (ccRCC) accounts for 70–80%. Early-stage RCC can be managed through surgical intervention, while roughly 40% of surgically resected patients eventually experience recurrence.² Nonetheless, a deficiency in effective treatment options persists for individuals experiencing recurrent and metastatic renal cell carcinoma (mRCC).³ The development of immunotherapy combined with targeted therapy for ccRCC has made good progress.^4,5 Unfortunately, there was up to 70% of patients with no response to combined therapy.6, 7, 8 A critical focal point in the advancement of combined therapy for ccRCC revolves around affecting tumor cells intrinsically and potentiating the immune answers.⁹ Hence, the pivotal strategy for ccRCC may lie in the careful selection of target proteins. These target proteins should not only be associated with tumor proliferation but also possess the capacity to modulate the immune environment.

The p21-activated kinase 4 (PAK4) is an onco protein, belongs to the serine/threonine (Ser/Thr) protein kinases family, and is frequently overexpressed in various human cancers and consistently associated with unfavorable prognoses.10, 11, 12, 13, 14, 15, 16, 17, 18 First, PAK4 amplification/mutation occurs among cancers and always related to higher grade/stage and shorter survival. Second, PAK4 plays a pivotal role in cancer malignant process by facilitating abnormal phosphorylation of substrates, such as upregulation of specific oncogenic pathways STAT3, ERK/MAPK, and PI3K/AKT et al.19, 20, 21 Additionally, concerning the suppression of the immune microenvironment, PAK4 holds significant influence in modulating tumor cell immune evasion. This is achieved through the activation of the Wnt/β-catenin signaling pathway.¹⁰ As such, we propose that targeting PAK4 in RCC could yield dual benefits: inhibiting the proliferation of cancer cells and concurrently enhancing the immune microenvironment by boosting the killing capacity of T cells against RCC cells, thus it potentially improves the immune response and enables a more effective immunotherapeutic strategy against RCC.

In recent years, several PAK4 inhibitors have been developed and entered into clinical trials.^15,22, 23, 24, 25 The PF-3758309 (a representative ATP-competitive inhibitor for group I p21 activated kinases), which inhibits both PAK1 and PAK4, failed in phase I clinical trial due to undesirable pharmacokinetic (PK) characteristics.²² GNE 2861 is a representative inhibitor for group II p21 activated kinases (IC₅₀ values are 7.5 nM, 126 nM, and 36 nM for PAK4, PAK5, and PAK6, respectively).²⁶ The KPT-9274 (a representative PAK4 allosteric inhibitor) was designed as a PAK4/NAMPT dual-targeting inhibitor.²⁵ KPT-9274 is now under phase I clinical trial (NCT02702492). Despite these advancements, the absence of highly selective PAK4-targeted drugs in clinical use highlights the imperative for further development of novel therapeutics targeting PAK4. Proteolysis-targeting chimera (PROTAC) drug design has emerged as a promising approach, as it not only inhibits target protein function but also induces protein degradation.^27,28 The PAKs all have an N-terminal GTPase-binding domain and a conserved C-terminal kinase domain, and drug-resistant mutations were always in protein kinase domains, and further considering the different PAKs have distinct modes of regulation, recognize specificity substrates, and ultimately control unique cellular processes to exert diverse functions.^18,29 The interaction regions between PAKs and other proteins vary. When designing selective PAK4 inhibitors, it is crucial to avoid the N-terminal structural region and ATP/substrate binding region. Instead, focus should be directed towards selecting the protein interaction interface. Here we report our design of a peptide PROTAC drug to efficiently degrade PAK4 without affecting other homologous kinases, and avoiding the PAK4 kinase domains. Utilizing the PAK4 protein structure from the Protein Data Bank (PDB) database and with AI software (Rosetta) assistance, we devised a peptide sequence specifically targeting PAK4. For E3 ligase selecting, we choosed MDM2 for PROTAC drug design. At present, the choice of E3 enzymes in PROTAC drugs primarily centers around VHL, CRBN, and MDM2. However, it is well-documented that the VHL gene undergoes mutations in approximately 40% of ccRCC cases, leading to its loss of function.^30,31 Therefore, the design of PROTAC drugs for renal cancer therapy should ideally circumvent the use of VHL. Conversely, MDM2 exhibits significantly higher expression levels in ccRCC compared to normal cells.32, 33, 34 The selection of MDM2 as an E3 ligase for PROTAC drug design leveraging its heightened expression in cancer cells. This choice aimed to achieve a degree of selective degradation within cancer cells. To overcome challenges related to poor cell-penetrating ability and in vivo stability of the peptide PROTAC, we employed a nano-selenium delivery system. Notably, nano-selenium holds a significant advantage over other nanosystems due to its FDA-approved status as a food additive, ensuring its safety as it can be metabolized and utilized by the human body.³⁵ Selenium nanoparticles can be conjugated to peptides via sulfhydryl groups on cysteine. The selenium–sulfur bond bears similarity to the disulfide bond and is susceptible to alteration within an intracellular redox environment. By integrating AI-assisted design to create a highly specific peptide and incorporating the efficient delivery of nano-selenium, we present a highly efficient PAK4-targeting PROTAC drug, referred to as PAK4 peptide degrader (PpD) in this context.

In this study, we have further validated PAK4 as a promising target in renal cancer. Patient microarray analysis confirmed elevated PAK4 expression in ccRCC compared to adjacent tissues, and PAK4 knockdown in ccRCC cells effectively inhibited their proliferation both in vitro and in vivo, providing a robust foundation for developing PAK4 degraders in RCC. To assess the therapeutic effect of our PAK4-targeting peptide degrader (PpD) on ccRCC, we conducted extensive experiments to verify efficient PAK4 degradation and its therapeutic efficacy at both cellular and animal levels. To investigate the potential of PpD in modulating the immune microenvironment alongside its anti-proliferative effects on renal cancer cells, we conducted additional experiments assessing its efficacy in fully immunocompetent mice. The PpD exhibited significant inhibition of tumor proliferation and concomitantly promoted the infiltration of CD8⁺ T cells. Comparable to PD-1 antibody treatment, PpD exhibited similar efficacy to PD-1 antibody in fully immunocompetent mice. These results indicate that PpD holds promise for enhancing the immune microenvironment in addition to its direct anti-cancer effects. Moreover, we have proved the universality of PpD by demonstrating its excellent therapeutic effects in patient-derived xenograft (mini-PDX) and patient-derived organoid (PDO) models.

In summary, our designed PAK4-targeted degradation drug, PpD, exhibits potent PAK4 degradation ability, providing a dual therapeutic advantage by simultaneously inhibiting renal cancer cell proliferation and increasing CD8⁺T cells infiltration in TEM. This study introduces a PAK4-targeting degrader as well as establishes, the potential of targeting PAK4 to enhance immunoresponse in ccRCC, highlighting the significance of selecting crucial targets that not only affect cancer cell proliferation but also modulate the immune response.

Methods

Fluorescence polarization (FP)

FP could be used to detect protein–protein interaction. Both Rhodamine (CAS: 81-88-9) and Cy5.5 (CAS: 2260669-63-2) utilized in this study are carboxy fluorograms, containing a carboxy group that can chemically interact with the amino group present on the peptide. PpD was labeled with rhodamine (excitation: 535 nm, emission: 580 nm) for FP analysis. FP experiments were performed in black 386-well plates with a Tecan Ultra plate reader. To detect the interaction between PpD and PAK4/MDM2, the concentration of PAK4 or MDM2 was reduced from 10 μM to 5 nM at equal dilutions. To detect the ability of PpD to induce trimer complex formation of PAK4/PpD/MDM2, 20 nM PAK4 protein and 20 nM PpD peptide were first incubated for 30 min. Then, the concentration of MDM2 was reduced from 20 μM to 5 nM at equal dilutions.

Gene set enrichment analysis

The Gene Set Enrichment Analysis (GSEA) was performed as described previously.³⁶ 611 patients RNA-seq data with ccRCC were download from the TCGA database (September, 2022). PAK4 high-expression group and PAK4 low-expression group were set up, and submitted to GSEA 2.0 software for the hallmark gene sets analysis. Kyoto Encyclopedia of Genes and Genomes (KEGG) and Reactome analysis³⁷ were appiled and performed by Omicsmart Co. (Guangzhou, China). PpD treatment 786-O RNA-seq no filter data was analyzed using R version 4.3.3 (2024-02-29 ucrt), MsigDb (v2023.2) was choose for HALLMARK, and the cut value was p.adj < 0.05.

Western blotting

The Cell Fraction Kit (Cell Signaling Technology, #9038, Shanghai, China) was employed to separate cellular proteins into distinct compartments, including cytoplasmic, membrane/organelle, and nuclear/cytoskeletal fractions. A total of 30 μg of whole protein was separated using 10% SDS-PAGE (#P2012, NCM Biotech, Shanghai, China). The following primary antibodies were used: anti-PAK1 (ABclonal, A19608, 1:1000, Shanghai, China, RRID:AB_2862697), anti-PAK2 (ABclonal, A4553, 1:1000, Shanghai, China, RRID:AB_2863294), anti-PAK3 (ABclonal, A3363, 1:1000, Shanghai, China RRID:AB_2863043), anti-PAK4 (#1 absin, abs146701, 1:1000, Shanghai, China, RRID:AB_3076408; #2 Cell Signaling Technology, #62690, 1:1000, Shanghai, China, RRID:AB_2827508), anti-p53 (Santa Cruz, sc-71817, 1:1000, Shanghai, China, RRID:AB_1126977), anti-MEK (ABclonal, A4868, 1:1000, Shanghai, China, RRID:AB_2863368), anti-AIF (Cell Signaling Technology, #5318, 1:1000, Shanghai, China, RRID:AB_10634755), anti-Histone H3 (Cell Signaling Technology, #4499T, 1:1000, Shanghai, China, RRID:AB_10544537), anti-FLAG (Abclonal AE092, 1:1000, Shanghai, China, RRID:AB_2940847), Anti-MYC (Abclonal AE070, 1:1000, Shanghai, China, RRID:AB_2863795), anti-MDM2 (Proteintech, 19058-1-AP, 1:1000, Wuhan, China, RRID:AB_2878572), anti-Vinculin (Abclonal, #A2752, 1:1000, Shanghai, China, RRID:AB_2863020)and anti-β-actin (Abclonal, #AC004, 1:1000, Wuhan, China, RRID:AB_2737399). For secondary antibodies, anti-mouse IgG (Beijing Zhongshan, #ZB-2305; 1:2000; Beijing, China, RRID:AB_2747415) and anti-rabbit IgG (Beijing Zhongshan, #ZB2301; 1:2000; Beijing, China, RRID:AB_2747412) were utilized.

Cell viability tests by cell counting Kit-8 (CCK-8)

The CCK-8 kit was purchased from NCM Biotech. CCK-8 was used to detect cell viability. 786-O, Caki-1, ACHN cells were plated at a density of 5 × 10³ cells/well on 96-well plates, and five independent samples were seeded for each PpD drug concentration (0, 31.3, 62.5, 125, 250, 500, 1000, 2000 nM), KPT-9274 (CAS: 1643913-93-2, 0, 3.12, 6.25, 12.5, 25, 50, 100, 200 μM), PF-3758309 (CAS: 898044-15-0, 0, 3.12, 6.25, 12.5, 25, 50, 100, 200 μM), Nultin 3 (CAS: 548472-68-0, 0, 3.125, 6.25, 12.5, 25, 50 μM). After 48 h, cells were incubated with CCK-8 regent for 4 h, then the absorbance values were captured at 450 nM. Cell viability rate was calculated as the average OD value in PpD or Se-group/average OD value in group × 100%.

Cell lines and cell culture

Human kidney cell lines HK-2 (RRID:CVCL_0302), 786-O (RRID:CVCL_1051), Caki-1 (RRID:CVCL_0234), OS-RC-2 (RRID:CVCL_1626), ACHN (RRID:CVCL_1067), and Renca (RRID:CVCL_2174) were purchased the from the American Type Culture Collection (ATCC, Manassas, VA, USA). The cell lines were grown in RPIM 1640 medium (Gibco-Thermo Fisher Scientific, Inc., Waltham, MA, USA), supplemented with 10% (v/v) fetal bovine serum (FBS; Gibco-Thermo Fisher Scientific, Inc., Waltham, MA, USA) at 37 °C and 5% CO₂ in a humidified incubator.

Drug treatment

HK-2, 786-O, Caki-1, OS-RC-2, and ACHN cells were seeded in 6-cm dishes, after 24 h, cells were treated with different PpD drug concentrations (0, 31.3, 62.5, 125, 250, 500, 1000, 2000 nM) for 48 h, then PAK4 protein level was detected by western blotting in whole cell lysate (WCLs). For PAK4 protein degradation rate in RCC cells, cells were treated with cycloheximide (CHX, CAS: 66-81-9) for the indicated times (0, 2, 4, 6, 8, 10 h), with or without 250 nM PpD drug treatment. For the PAK4 specificity degradation of PpD drug, 786-O, ACHN, and Caki-1 cells were treated with PpD drug (0, 125, and 250 nM) or KPT-9274 (0, 10, and 20 μM) for 48 h, PAK1, PAK2, PAK3, and PAK4 protein level were all detected in WCLs.

Clone and clonogenic assay

Caki-1,OS-RC-2, and 786-O renal cells were seeded in each well of a 6-well plate (1000 cells/well), cultured in a medium containing PpD drug (0, 125, 250, 500 nM) for 5 days, and triple independent samples were seeded for each PpD drug concentration. Crystal violet (CAS: 603-48-5) was used to stain cells. The colony formation capacity of the cells was tested by 2-dimensional culture and the number of clones was counted and plotted.

Tumor-infiltrating lymphocytes

The immune-related signatures of 28 infiltrating lymphocytes (TILs) were analyzed using the TISIDB platform and TIMER2 web, the webs could integrate repository portal for tumor immune system interactions.^38,39 28 TILs, gene expression, copy number, methylation, and mutation were viewed and downloaded on the page. For each cancer type, the relative abundance of TILs was inferred by using gene set variation analysis (GSVA) based on gene expression profile.

Reactions and KEGG pathways enrichment analysis

After 786-O cells being treated with PpD drug, RNA extraction, library construction and sequencing were constructed by Genedenovo company (Guangzhou, China). Reactome enrichment and KEGG pathway enrichment analysis were appiled between the control group and the PpD drug treatment group. The calculating formula is the same as that in Gene Ontology (GO) analysis. The calculated p-value was gone through FDR Correction, taking FDR ≤ 0.05 as a threshold. Reactions meeting this condition were defined as significantly enriched Reactions in DEGs.

Immunohistochemical analysis

Tissue microarrays (#HKidE180Su03) were purchased from Shanghai Outdo BioTeck Co. Ltd (Shanghai, China). HKidE180Su03 included 90 patient with ccRCC tumor tissues and 90 adjacent renal tissues. GTVisionTM⁺ Detection System/Mo&Rb (#GK600510, Shanghai) was employed to perform immunohistochemistry (IHC) staining and immunohistochemical staining scores were determined as previously described. For IHC primary-antibody information: PAK4 (absin, abs146701, 1:200, Shanghai, China, RRID:AB_3076408), and Ki67 (#9449, Cell Signaling Technology, 1:300, Beijing, China, RRID:AB_2797703). IHC intensity was scored as follows: 4 for highly positive; 3 for positive; 2 for minimally positive; 1 for negative. The staining percentage of the relative number of cells stained was graded as follows: 0 for 0%; 1 for ≤25%; 2 for 25–50%; 3 for 50–75%; 4 for ≥75%. Multiplying the intensity and percentage scores was IHC score for each section.

Transfection and establishment of PAK4 knock out stable clone cells

PAK4 sgRNAs were subcloned into the RCC cells, cells were transfected with packaging vectors (delta-8.9 and VSVG), PAK4 knock out (KO) cells were selected with puromycin (CAS: 58-58-2). The sgRNA sequences for human PAK4 KO were 5′-CACCGGGACGAGTTTGAGAACATGT-3′ and 5′-AAACACATGTTCTCAAACTCGTCCC-3′. The PAK4 was detected by western blotting. Plasmids: Flag-MDM2 (human), myc-PAK4 (human), Flag-MDM2^Δ51-62(human), myc-PAK4^Δ475-482(human), and His-Ub (human) were purchased from MiaoLing (Hubei, China), and plasmids transfected using lipofectamine 2000 reagent (#11668-019, invitrogen, USA).

In vivo experimental therapy in mouse models

All mice were raised in the Animal Center of Xi’an Jiaotong University Health Science Center in an SPF environment. In 786-O xenograft therapy experiment, BALB/c 4-week-old nude mice (female) were randomly separated into 4 groups (5 mice/group), and then 2 × 10⁶ 786-O cells were subcutaneously injected into the right shoulder. After 3 days, the mice were treated with Control, Se (CAS: 7782-49-2), KPT-9274 (2.5 mg/kg), or PpD (2.5 mg/kg) every 2 days by abdominal injection. The determination of drug concentration at the animal level hinges upon the drug’s onset concentration at the cellular level, as well as its metabolism and pharmacokinetic properties. At the cellular level, the IC50 of PpD is approximately 300–500 nM, demonstrating its efficacy in inhibiting the proliferation of renal cancer cells at 2uM. Concurrently, our pharmacokinetic (PK) experiments reveal a half-life of 9.6 h for the PpD drug within the body. Hence, we selected a concentration of 2.5 mg/kg. Calculating based on the mouse blood volume of 2 mL, intravenous injection would yield a blood concentration of 5 uM. However, given our intraperitoneal administration approach, there will be some loss in drug absorption. Considering this, along with the impact of the drug’s half-life, we estimate that 2.5 mg/kg constitutes an effective dose. In ACHN-cell derived xenograft experiment, BALB/c 4-week-old nude mice (female) were randomly separated into 2 groups (5 mice/group). 2 × 10⁶ sg-control ACHN cells and 2 × 10⁶ sg-PAK4 ACHN cells were subcutaneously injected into the right shoulder as planted. In ACHN-cell derived xenograft therapy experiment, BALB/c 4-week-old nude mice (female) were randomly separated into 4 groups (5 mice/group), and 2 × 10⁶ ACHN cells were subcutaneously injected into the right shoulder as planted. After 3 days, the mice were treated with PBS (Control), nano-selenium with peptide control (Se), PF-3758309 (5 mg/kg), or PpD (5 mg/kg) every 2 days by abdominal injection. In the therapy experiment, BALB/c 4-week-old nude mice (female) were randomly separated into groups, 0.4 × 10⁶ Renca cells were subcutaneously injected into the right shoulder, and mice were treated with Control, Se, PpD (2.5 mg/kg), PD-1 (2.5 mg/kg), PpD combined PD-1 (internal, 2.5 mg/kg), every 2 days by abdominal injection. For tumor TILs sorting, mice were sacrificed. Kinetics of tumor formation was estimated by measuring tumor size and volume every 3 days with the body weight of the mice. Tumor volume was calculated using the following equation: tumor volume (V) = length × width/2. The animals were euthanized at the end of the experiment. Mice heart, liver, spleen, renal, and tumor tissue sections were fixed in 4% paraformaldehyde and embedded in paraffin for H&E staining. Furthermore, PAK4, Ki67, β-catenin (Cell Signaling Technology, Inc., #9562, 1:100, Shanghai, China, RRID:AB_331149), and Phospho-β-Catenin^Ser675 (Cell Signaling Technology, Inc., #4176T, 1:100, Shanghai, China, RRID:AB_1903923), immunohistochemical staining were conducted in tumor tissue sections.

Tumor infiltrating lymphocytes analysis

10 days after the treatment of Renca tumors with indicated compounds (Control, Se, PpD, PD-1 mAb, PpD plusPD-1 mAb), CD8⁺T cells and CD4⁺T cells in TILs were isolated and stained. Cells were acquired and analyzed by FACSCalibur™ flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA). Antibodies information: PE anti-mouse CD3 antibody (#100206, Biolegend, RRID:AB_312663); PerCP anti-mouse CD4 antibody (#100432, Biolegend, RRID:AB_893323); APC anti-mouse CD8b antibody (#126614, Biolegend, RRID:AB_2562775). CD8⁺T cells and CD4⁺T cells in TILs gating: gate cells exclude dead cells (Live/DeadTM fixable green dead cell stain kit) and debris based on cells size, live cells gate based on Live-Dead NIR negative cells, then gate CD3⁺ cells, then gate CD3⁺CD4⁺ cells and CD3⁺CD8⁺ cells.

ATP-based tumor chemosensitivity assay (ATP-TCA)

After obtained informed consent form from patients with ccRCC, tumor tissues (3 patient’s information in Supplementary Table S1) were washed to remove nontumor tissues and necrotic tumor tissue, then single tumor cells were separated from fresh tumor tissues, mixed with 0, 1.25, 2.5, 5.0, 10.0, 20.0 μM concentrations of drugs (PpD, KPT-9274, and PF-3758309), and inoculated into 96-well plates for 6 days. Then, cell viability in each plate hole was measured separately.⁴⁰ The anti-tumor effects of different drug were calculated.

Mini patient derived xenograft (mi-PDX)

Mini-PDX model was established followed by the protocol describing in Zhang et al.’s article.⁴¹ Briefly as: informed consent form was obtained from patients with ccRCC, ccRCC tumor tissues (3 patient’s information in Supplementary Table S2) were washed to remove nontumor tissues and necrotic tumor tissue, then cells were harvested and digested into single cells. Finally, cells were filled into OncoVee® capsules, and capsules were implanted subcutaneously via a small skin incision with one capsule per mouse (4-week old nu/nu mouse). The mice were treated with Control, Se, PF-3758309 (2.5 mg/kg), KPT-9274 (2.5 mg/kg), or PpD (2.5 mg/kg) every 2 days by abdominal injection for 7 days. Tumor cell proliferation rate was evaluated.

Patient-derived organoids (PDO)

For PDO, an informed consent form was obtained from a 53-year-old patient with ccRCC. Tumor tissues were washed and digested at 37 °C for 50 min. Dissociated tissues were spun down for 10 min and resuspended in cell medium. Dissociated cell clusters were centrifuged again. Cell clusters were resuspended in BME buffer on ice and plated as a 300 μL drop within a 12 mm, 0.4 μm inner Transwell chamber. The drop was solidified by a 30-min incubation at 37 °C and 5% CO₂ with 1 mL of renal cancer organoid medium. The growth state of organoids was monitored on day 1, 3, 10. Hematoxylin & eosin (H&E) staining was performed on tumor organoids to reveal cancer nuclear division pattern and atypia. Renal cancer organoids were cultured with the medium containing drugs as planned for 72 h. The concentrations of 3 drugs (PpD, KPT-9274 and PF-3758309) were 0, 0.0032, 0.016, 0.08, 0.4, 2, 10 μM, and cell viability was tested by cellter-blue assay.

Statistical analysis

The Graph Pad Prism version 6.0 software (GraphPad, USA) was used to perform One-way ANOVA analysis, Pearson’s correlation analysis, linear regression analysis, spearman analysis and Student’s t-test to analyze differences between groups. p value less than 0.05 was considered statistically significant.

Ethical approval

All experimental procedures involving animals were conducted by institutional guidelines and were approved by the Laboratory Animal Center and Biomedical Ethics Committee of Xi’an Jiaotong University (approval number: 2020-256). The experiments on renal primary tumors from patients were performed under the supervision of the Ethics Committee of the First Affiliated Hospital of Xi’an Jiaotong University (approval number: 2020-G165).

Role of funders

The funding source had no role in study design, data collection and interpretation, analysis, writing of this report, or in the decision to submit the paper for publication.

Results

PAK4 as an effective therapeutic target in renal cancer

To investigate the clinical relevance of PAK4 protein in patients with RCC, we evaluated PAK4 protein levels in a human tissue microarray (TMA), each consisting of 90 samples with ccRCC, including both benign and tumor tissues. Our analysis showed a significant difference in PAK4 protein expression between benign and tumor tissues (Fig. 1a and b). We further assessed the dependence of renal cancer cell proliferation on PAK4 protein by deletion of PAK4 (Fig. 1c). As shown in Supplementary Fig. S1a–e, PAK4 knockout significantly inhibited the proliferation of renal carcinoma cells. In accordance with this observation, consistent loss of PAK4 protein (PAK4 knock out ACHN cells) resulted in a significant reduction in tumor growth in the cell-derived xenograft experiment (Fig. 1d–h). To explore the oncogenic mechanism of PAK4 in ccRCC, we downloaded the protein data of patients with ccRCC in TCGA database, and analyzed PAK4-related signaling pathways in ccRCC. As a result, PAK4 has a strong positive correlation with the expression of a key oncogenic factor mTOR (Fig. 1i, R = 0.57, p < 0.00001, spearman), indicating the involvement of PAK4 in the occurrence and progression of ccRCC. Specifically, further GSEA hallmark gene sets analysis showed that PAK4 expression is associated with the cell cycle G₂/M checkpoint pathway (Fig. 1j) and E2F signaling pathway (Fig. 1k) during cell proliferation. These data suggest PAK4 emerges as a feasible and potential target for the treatment of renal cancer.

Fig. 1.

Exploration and validation of PAK4 as a therapeutic target in renal cell carcinoma. (a) Representative IHC images of PAK4 expression in ccRCC and benign renal tissues. (b) Quantification of PAK4 IHC staining scores in benign renal tissues (n = 72) and ccRCC tissues (n = 72). Statistical analysis was performed using Student’s t-test between the two groups, with error bars representing ± standard deviation (SD). ∗∗∗∗ indicates p < 0.00001. (c) Construction and validation of stable PAK4 knockout cell lines. (d) Images of excised tumors at the end of the experiment in both ACHN and sg-PAK4 ACHN xenograft animal models. (e) Tumor growth curves of ACHN and sg-PAK4 ACHN xenografts in BALB/c nude mice (n = 5 per group). Statistical analysis was conducted using Student’s t-test between the two groups, with error bars indicating ±SD. ∗ represents p < 0.05; ns indicates no statistical significance. (f) Average tumor weight excised from each group of mice at the end of the experiment. (g) Representative histopathology (H&E) and PAK4 IHC images of tumors at the end of the experiment. (h) Statistical analysis of IHC scores for PAK4 in tumors between the control group and sg-PAK4 group. (i) Correlation analysis of PAK4 and mTOR mRNA expression in The Cancer Genome Atlas (TCGA) database. (j) Impact of PAK4 overexpression on G₂M checkpoint pathway. (k) Impact of PAK4 overexpression on E2F targets pathway.

PAK4 peptide PROTAC drug design and nano selenium delivery system construction

To achieve the goal of cancer treatment by targeting PAK4 in renal cancer, we have developed a peptide-based PAK4 PROTAC drug (Fig. 2a). The design of this drug involved selecting the most suitable PAK4-targeting peptide antagonist and integrating it with an E3 ligase-recruiting element via a flexible linker to create an effective PAK4 PROTAC degrader. To begin, we utilized the AI-assisted program Rosetta, developed by Baker’s Group, to design a new binding peptide derived from the crystal structure of PAK4. We chose the structure of PAK4 with an 11 amino acids peptide (PDB: 2q0n) for further design and optimization. Through virtual amino acid screening at crucial amino acid sites with Rosetta,⁴² In essence, mutations were introduced at individual amino acid positions based on the original peptide sequence bound with PAK4. Predictions were made regarding alterations in the binding energy of amino acids post-mutation. By mutating the amino acid at each position to a higher affinity amino acid, we obtained a peptide with high affinity to PAK4. The new designed high-affinity PAK4-binding peptide sequence: RQLRKGKFFSE was deduced by synthesizing the mutation outcomes at each position, as depicted in Fig. 2b and c. For the construction of the PAK4 PROTAC drug, we employed MDM2 as the E3 ligase, based on our previous publication.⁴³ We incorporated a flexible linker sequence-GGSGG between the PAK4 targeting sequence and MDM2 targeting sequence,⁴⁴ leading to the final designed PAK4 PROTAC sequence: RQLRKGKFFSEGGSGGTSFEQFWAWLWP. Nano selenium can react with sulfhydryl groups on cysteine and are released under REDOX conditions in cells. To facilitate in vivo delivery of the PAK4 peptide PROTAC, we introduced a cysteine amino acid to the sequence, allowing for conjugation with a nano selenium system through selenium-thiol bonds. Nano-selenium serves as a carrier for the PAK4 PROTAC drug, ensuring effective delivery to the target site. In conclusion, the culmination of our efforts has resulted in the final designed PAK4 PROTAC peptide sequence: CRQLRKGKFFSEGGSGGTSFEQFWAWLWP. This innovative approach holds great promise in advancing targeted therapy for renal cancer, and its potential to selectively degrade PAK4 provides new avenues for more effective and precise treatment strategies.

Fig. 2.

PAK4 peptide PROTAC drug design and nano selenium delivery system construction. (a) The complex structure of PAK4 with a leading peptide drug (PDB: 2q0n) used for drug design. (b) Amino acids of the PAK4 binding peptide ranked individually based on computationally predicted frequency. Wild-type peptide residues used in protein ensemble generation are shown in red. The dashed line indicates a typical cutoff for selecting the top 5 amino acid choices at each position. (c) Sequence logos showing the predicted sequence mutations for the PAK4 binding peptide. (d) Binding affinities between PAK4 PROTAC peptide and PAK4/MDM2 were assessed using fluorescence polarization. (e) Detection of the ability of PAK4 PROTAC peptide to induce PAK4/MDM2/PAK4 PROTAC peptide complex formation using fluorescence polarization. (f) Hydrodynamic diameter distributions and transmission electron microscopy (TEM) analysis of the nano-selenium with PpD complex. The hydrodynamic diameter of the PpD complex is 78.14 ± 3.14 nm. Scale bar in TEM analysis: 50 nm. (g) Surface zeta potential of the PpD complex in PBS at pH 7.4. The surface charge of the PpD complex is 44.2 ± 5.8 mV. (h) Serum resistance of the PpD complex tested in PBS containing 10% fetal bovine serum. The half-life of the PpD complex is 11.7 h. (i) Confocal micrographs of ACHN cells incubated with 200 nM PpD complex labeled with Cy5.5 (red). Cell nucleus was stained with DAPI (blue). All images were acquired with the same excitation wavelength and detector gain settings. Scale bar: 50 μm.

As shown in Supplementary Fig. S2a and b, the PAK4 PROTAC peptide with high purity was synthesized by solid-phase peptide synthesis. Fluorescence polarization experiments between PAK4/MDM2 with PAK4 PROTAC were performed to detect the binding affinity. Firstly, PAK4 PROTAC was labeled with rhodamine. PAK4 (Supplementary Fig. S3a–c) and MDM2 (Supplementary Fig. S3d–f) were expressed by E.coli and the results of purified proteins are shown in Supplementary Fig. S3. As shown in Fig. 2d, the PAK4 PROTAC peptide is composed of a high-affinity sequence for the targeting protein - PAK4 and MDM2. Also, PAK4 PROTAC could effectively induce trimer complex formation (Fig. 2e). The fundamental principle underlying fluorescence polarization lies in its correlation with the size and molecular weight of proteins, enabling the detection of protein–protein interactions. Our initial step involved labeling luciferin on the PAK4 protein. Given that the peptide induces trimer formation, we observed an increase in fluorescence polarization values, where the X-coordinate indicated the PAK4 peptide PROTAC drug concentration. With incremental drug dosage, a corresponding rise in trimer induction occurred, allowing for the characterization of the degree of PROTAC-induced trimer formation. In our fluorescence polarization experiment, the protein concentration of labeled fluorescence typically remains below 100 nM. Before the main experiment, a preliminary test affirmed the effective detection of polarization values at 20 nM. Consequently, we opted for a concentration of 20 nM for our detection purposes. It’s important to note that alternative concentrations remain viable and do not exert any influence on the outcomes.

To overcome the disadvantages of short half-life and poor cell-penetrating ability of peptide drug, we constructed a nano selenium system to deliver PAK4 PROTAC drug. As shown in Fig. 2f, nano selenium - PAK4 PROTAC peptide (referred to as PpD hereafter) showed 78.14 nm size as detected by TEM and a particle size analyzer. And the surface zeta potential detection of PpD proved that PpD has a positive surface charge to enter cells (Fig. 2g). Serum stability detection of PpD proved that PpD has a sufficient half-life for cancer therapy in vivo (Fig. 2h). Confocal analysis of ACHN cells incubated with 200 nM Cy 5.5 labeled PpD proved that this complex could enter renal carcinoma cells in a time-dependent manner (Fig. 2i). In addition, 10 mM GSH was used to mimic the intracellular REDOX environment to test whether nano selenium could release the peptide PAK4 PROTAC drug. As shown in Supplementary Fig. S4, peptide PAK4 PROTAC drug was released under 10 mM GSH. In summary, our results highlight the successful synthesis of the PAK4 PROTAC peptide and its high affinity towards PAK4 and MDM2 to form a trimer complex.

PpD showed potent and selective PAK4 degradation ability in RCC cells

The putative PAK4 degradation ability of PpD was tested at different concentrations in the PAK4 positive RCC cell lines with a 24 h treatment time. PpD effectively reduced the protein level of PAK4 in 786-O and Caki-1 cells with a DC₅₀ ∼ 200 nM (Fig. 3a). We next evaluated the ability of PpD to inhibit cancer cell growth, with free PAK4 PROTAC peptide (refer to as control) and nano selenium-mutation peptide control (refer to as Se) included as negative controls (Fig. 3b). As we employed selenium nanoparticles as the delivery system, it was imperative to demonstrate that the selenium nanoparticles could facilitate the efficacy of the peptide PROTAC, while concurrently ruling out the impacts of the nano-delivery system and non-specific peptide cytotoxicity. To achieve this, we utilized Se nanoparticles conjugated mutation PAK4 peptide PROTAC drug (Se) as a reference to exclude toxicity associated with the nano delivery system. Additionally, a free peptide group (Control) referred to a peptide without a nano-delivery system, thereby demonstrating the necessity of nano-delivery systems. Our data demonstrated that PpD is highly potent in the inhibition of cell growth in the tested three cell lines and achieves IC50 values of 305 and 523 nM in the 786-O, and Caki-1 cells, respectively (Fig. 3b). To evaluate the effect of PpD on PAK4 half-life, western blotting analysis in cells treated with cycloheximide (CHX) was performed. The mechanism of action of PROTAC drugs involves accelerating protein degradation. To demonstrate that PpD does not inhibit the protein synthesis of PAK4 but rather accelerates its degradation, we treated cells with CHX to specifically inhibit protein synthesis, observing only protein degradation. The results showed that the PpD drug induced degradation of PAK4 protein effectively accelerated the degradation of PAK4 (Fig. 3c and d). To test the selectivity of PpD for PAK family proteins, western blotting assays were performed and showed that PpD failed to affect the protein levels of other members in the PAK family, including PAK1-3, at the concentrations of 0.5 and 1 μM (Fig. 3e). Also in ACHN cells, PpD showed similar effects (Supplementary Fig. S5a–e). Colony formation assay further demonstrated the inhibitory effect of PpD on RCC cell proliferation (Supplementary Fig. S6a and b). Additionally, we investigated the phosphorylation status of β-catenin, a component in the signaling pathway downstream of PAK4, following PpD treatment. The results demonstrated that PpD treatment effectively inhibits downstream signaling, as shown in Supplementary Fig. S7a and b.

Fig. 3.

PpD demonstrates potent and selective PAK4 degradation ability in a MDM2 dependent degration manner. (a) Western blotting assay analysis of PAK4 protein levels in 786-O and Caki-1 cell lines treated with indicated concentrations of PpD for 48 h. (b) Cell viability assay of ccRCC cells after treatment with indicated concentrations of PpD, Se-mutation peptide (Se), and free PAK4 PROTAC peptide (Control) for 48 h. Se and control groups showed no toxicity in 786-O and Caki-1 cells, while PpD demonstrated a dose-dependent growth inhibition in 786-O and Caki-1 cells, with IC₅₀ values of 305.9 nM and 532.2 nM, respectively. (c, d) Western blotting assay for detecting PAK4 protein degradation after treatment with cycloheximide (CHX) for different time points (0, 2, 4, 6, 8, 10 h), with or without 500 nM PpD in 786-O and Caki-1 cells. The signal intensity of PAK4 protein normalized to β-actin was quantified for each time point. (e) Specificity of PpD towards PAK4 in RCC cell lines. After treatment with 0, 250, or 500 nM PpD for 48 h, western blotting analyses of PAK1, PAK2, PAK3, and PAK4 in whole cell lysates (WCLs) from 786-O and Caki-1 cells were performed. (f) Detection of the binding ability of PpD to induce PAK4 and MDM2. IB analysis of WCLs and anti-Flag immunoprecipitated (IP) from 293T cells transfected with the indicated plasmids, with or without PpD drug treatment. (g) Detection of the binding ability of PpD to induce PAK4^Δ475-482 and MDM2. (h) PpD enhances PAK4 ubiquitination ability detection.

We assessed the mRNA level of PAK4 after PpD treatment, confirming its independence from transcription (Supplementary Fig. S8a). Moreover, the PAK4 protein decreasing phenomenon was detectable from 1000 nM in HK-2 cells much worse than the effect in cancer cells, demonstrating the safety of PpD in normal cells (Supplementary Fig. S8b). However, PAK4 inhibitor KPT-9274 showed no effect on protein levels of PAK4 demonstrated that the mechanism of action of PROTAC drugs is different from that of inhibitors (Supplementary Fig. S8c). In addition, PpD induced PAK4 degradation leading to cytoplasm PAK4 protein level sharply decreasing (Supplementary Fig. S8d).

The mechanism of action of PROTAC drugs involves enhancing the ubiquitination of target proteins by inducing their affinity for E3 ligase. To elucidate the mechanism of action of PpD, we initially demonstrated the proteasome dependency of PpD’s effect by employing a proteasome inhibitor that effectively countered the impact of PpD (Supplementary Fig. S9a and b). In further support of PpD’s mechanism, a co-immunoprecipitation (co-IP) assay was conducted to assess whether PpD could augment the binding between PAK4 and MDM2. Transfecting 293T cells with Flag-MDM2 and myc-PAK4 plasmids revealed a significant enhancement in the affinity between PAK4 and MDM2 in the presence of PpD (see Fig. 3f). Subsequent experiments involving the deletion of binding sites in either PAK4 (myc-PAK4^Δ475-482) or MDM2 (Flag-MDM2^Δ51-62) demonstrated that PpD failed to enhance PAK4 protein degradation, providing compelling evidence for the specificity of PpD (see Fig. 3g and Supplementary Fig. S10). We conducted a IP assay in 786-O cells to test if PpD increase ubiquitination level of PAK4. As shown in Fig. 3h, after PpD treatment, the ubiquitination level of PAK4 was highly increased.

We selected MDM2 as the E3 ligase of choice, given its well-established role with p53, a pivotal substrate known for its significance in cancer. Consequently, we examined p53 protein levels following PpD treatment, focusing on two cell lines harboring the wild-type p53 gene. As shown in Supplementary Fig. S11a and b, while PpD induced a noticeable increase in p53 protein levels in Caki-1, no such effect was observed in the other cell lines. This observation strongly suggests that the primary impact of PpD involves inducing PAK4 degradation to exert its therapeutic influence on cancer, rather than significantly elevating p53 protein levels. To explore potential synergistic effects between the PAK4 inhibitor and MDM2 inhibitor, we co-administered KPT-9274 and nutlin-3. Our findings, illustrated in the Supplementary Fig. S12a and b, indicate the absence of a synergistic effect between KPT-9274 and nutlin-3. This further supports the notion that our drug primarily operates by inducing PAK4 degradation.

The significant inhibitory effect of PpD on tumor growth in RCC xenograft models

The anticancer activity of PpD was further evaluated in a mouse 786-O xenograft model, as shown schematically in Fig. 4a. KPT-9274 was used as the positive compound due to its high PAK4 inhibitory activity, while Se-mutation peptide (Se) as a negative control. The mice were treated with low dose of each drug (2.5 mg/kg, i.g., qd). The tumor growth inhibition was analyzed from the tumor volume and tumor weight. As outlined in Fig. 4b–d, the tumor weight after the PpD treatment was significantly lighter than compared with the vehicle group and Se group (p < 0.005, p < 0.05, respectively. One-way ANOVA analysis was used to do statistical test), and the relative tumor volume also significantly decreased compared with the vehicle group and Se group (p < 0.05, p < 0.05, respectively. One-way ANOVA analysis). There was no observable difference in tumor volume measurements between the PpD and KPT-9274 groups. However, upon completion of the experiment, the tumor weight of PpD treatment group was smaller than that of KPT-9274 group (Fig. 4c). The results of IHC analysis showed that the expression of Ki67 and PAK4 protein in both KPT-9274 and PpD group was significantly reduced compared with the vehicle group and Se group (One-way ANOVA analysis). Simultaneously, we aimed to assess the impact of PpD on the downstream signaling pathway of PAK4 in animal experiments. We substantiated the inhibition of PAK4 downstream signaling in vivo by detecting β-catenin and phosphorylated β-catenin (Fig. 4e–i).

Fig. 4.

PpD inhibits tumor growth in the 786-O xenograft model. (a) Schematic of tumor-bearing BALB/c nude mice treated with indicated drugs. 20 BALB/c nude mice were randomly separated into 4 groups and treated with Control (PBS, n = 5), Se (Se-mutation peptide nanoparticles, n = 5), PpD (2.5 mg/kg, n = 5), or KPT-9274 (2.5 mg/kg, n = 5) every 3 days. The tumor volume and mice weight of BALB/c nude mice were measured and recorded every 3 days until sacrifice. (b) Photos of 786-O tumors excised at the end of the experiment after different drug treatments. (c) Average tumor weight excised from each group of mice at the end of drug treatment. The data were presented as the mean ± SD values (n = 5). Statistical analysis of data was calculated using One-way ANOVA analysis among 4 groups, error bars indicated ±SD, n = 5; ∗ represents p < 0.05; ∗∗ represents p < 0.01. (d) Tumor growth curves of ACHN xenografts of BALB/c nude mice in different groups (n = 5 per group). Statistical analysis of data was calculated using One-way ANOVA analysis among 4 groups, error bars indicated ±SD, n = 5; ∗ represents p < 0.05; ns represents no statistical significance. (e) Histopathological analysis of the excised tumors from Control, Se, KPT-9274, and PpD groups, using H&E staining assay for pathological diagnosis, and IHC assay for Ki67 staining (tumor cell growth marker), PAK4 staining (PAK4 protein level in tumor cells), β-catenin and phospho-β-Catenin^Ser675 staining (PAK4 protein derectly downstream). Statistical analysis of IHC scores about Ki67 (f), PAK4 (g), β-catenin (h) and phospho-β-Catenin^Ser675 (i) on 786-O tumors after different drugs treatment. Statistical analysis of data was calculated using One-way ANOVA analysis among groups, error bars indicated ±SD, n = 5; ∗ represents p < 0.05; ∗∗ represents p < 0.01; ∗∗∗ represents p < 0.001; ∗∗∗∗ represents p < 0.0001; ns represents no statistically significance. (j) Photos of tumors excised at the end of tumor-bearing BALB/c nude mice experiment in 786-O sg-control, 786-O sg-control (with 5.0 mg/kg PpD treatment), 786-O sg-PAK4-3, and 786-O sg-PAK4-3 (with 5.0 mg/kg PpD treatment). (k) Tumor weight excised from each group of mice at the end experiment and (l) tumor growth curve of 786-O xenografts from BALB/c nude mice in groups (n = 5 per group). The data were presented as the mean ± SD values (n = 5). Statistical analysis of data was calculated using One-way ANOVA analysis among groups, ∗ represents p < 0.05; ∗∗ represents p < 0.01, ns represents no statistical significance.

To further validate the inhibitory effect of PpD on ccRCC in vivo, we considered and compared a 5.0 mg/kg dose with a PAK4 knock-out 786-O cell xenograft model. The consistent loss of PAK4 protein in PAK4 knock-out 786-O cells resulted in a significant reduction in tumor growth in the 786-O-derived xenograft experiment. As predicted, the 5.0 mg/kg dose of PpD treatment group demonstrated a significantly inhibitory effect on both tumor volume and weight (see Fig. 4j–l, One-way ANOVA analysis). Importantly, PpD exhibited no effect on 786-O renal cancer tumor growth after PAK4 knockdown, further underscoring the drug specificity of PpD.

The inhibitory effect of PpD on renal cancer was also tested in ACHN-xenograft model (Supplementary Fig. S13). A significant reduction in proliferation was observed in ACHN tumors after PpD treatment, further confirming the tumor-inhibitory effects of PpD in vivo (Supplementary Fig. S13a–d). Additionally, IHC analysis of PAK4 protein levels showed a significant reduction in the PAK4 protein level in the PpD treatment group compared to the vehicle and Se groups (Supplementary Fig. S13e–g).

In conclusion, the results obtained from the xenograft models of both 786-O and ACHN cells consistently demonstrate the potent tumor growth inhibitory effects of PpD in vivo. The results establish PpD as a promising candidate for ccRCC therapy, and beyond ccRCC.

PpD induced immune cell response and prolong survival for RCC

The rationale for selecting PAK4 as a target for drug development in renal cancer is multifaceted. Firstly, PAK4 exhibits high expression levels in renal cancer and is closely associated with several pivotal carcinogenic signaling pathways, as previously discussed. Secondly, PAK4 has demonstrated its role in regulating the immune microenvironment. This indicates that targeting PAK4 in renal cancer could not only suppress cancer cell proliferation but also improve immune microenvironment, potentially resulting in enhanced outcomes for patients.

To further explore the immune correlates of PAK4 in cancers, the relationship between PAK4 expression levels and the abundance of 28 tumor-infiltrating lymphocytes (TILs) was analyzed using the TISIDB database, which integrates information on tumor-immune system interactions. The analysis revealed that PAK4 expression was highly correlated with multiple types of TILs in ccRCC (Supplementary Fig. S14a). To investigate the effects of PpD on immune-related pathways, 786-O RNA-seq analysis was performed after PpD treatment, 1118 up-expression genes and 518 down-expression genes in treatment group with FDR ≤ 0.05 and expression fold-change ≥2 (Supplementary Fig. S14b), 759 genes (46.4%) were annotated in KEGG. KEGG pathway enrichment analysis of differentially expressed genes (DEGs) showed the involvement of immune-related signaling pathways such as the TNF signaling pathway, NOD-like receptor signaling pathway, and cytokine–cytokine receptor interaction (Fig. 5a). Reactome results revealed that 11 enrichment signalings were directly to tumor cell immune therapy genes expression, such as: expression of IFN-induced genes signaling, Interferon alpha/beta/gamma signaling (Fig. 5b). Meanwhile, we found that the immune system signaling enrichment 277 genes, that account 19% percent (277/1636) of total DEGs. GSEA analysis further corroborated the impact of PpD on immune-related pathways in renal cancer (Supplementary Fig. S14c and d). Our results underscore the potential of PpD to bolster anti-tumor immune responses, based on three different methods of analysis of 786-O RNA-seq results. These insights lay the foundation for future studies exploring the immunomodulatory characteristics of PpD and its potential in synergistic application with immunotherapies for the treatment of renal cancer.

Fig. 5.

PpD modulates immune-related pathways and enhances CD8⁺T cells infiltration in TEM in vitro. (a) Top 20 KEGG pathways were analyzed from RNA-sequence results upon 786-O treated with PpD (250 nM) for 48 h. Gene percent represents the percentage of the DEGs in pathways to 759 DEGs (1636 genes with FDR ≤ 0.05 and expression fold-change ≥2, 759 genes were annotated in KEGG), respectively. The number showed in each bar represents the DEGs enrichmented in each pathway and the -log₁₀ (Qvalue) in brackets. The size of the barplots represents the number of DEGs annotated in KEGG in each pathways, and the gradient color of the barplots represents significance. (b) Results from RNA-sequence analysis after PpD treatment were analyzed for Reactome. Gene ratio: the percentage of DEGs enrichment in each pathway to the number genes annotated in Reactome in this pathway (1636 genes with FDR ≤ 0.05 and expression fold-change ≥2, and 930 genes were annotated in Reactome), respectively. The size of the bar represents the number of DEGs enrichment in the pathway, and the gradient color of the bar represents significance (the value of -log₁₀ (Qvalue)). (c) Schematic representation of tumor-bearing BALB/c mice treated with indicated drugs. 50 BALB/c mice were subcutaneously injected with 40 × 10⁴ Renca cells. After 2 weeks, mice were randomly separated into 5 groups and treated with control (PBS, n = 10, every 2 days), Se (Se-mutation peptide, n = 10, every 3 days), PpD (2.5 mg/kg, n = 10, every 3 days), PD-1 (2.5 mg/kg, n = 10, every 3 days), and PpD combined with PD-1 (2.5 mg/kg, n = 10, PpD was injected after mice were treated with PD-1 mAb for 3 days). The mice weight and tumor volume of BALB/c nude mice were recorded every 3 days. (d) Kaplan–Meier survival curves for each treatment group (Control, n = 10; Se, n = 10; PpD, n = 10; PD-1 mAb, n = 10; Combined therapy, n = 10). Statistical analysis of data was calculated using One-way ANOVA analysis among groups, Error bars indicate ±SD, n = 10; ∗∗ represents p < 0.01; ns represents no statistical significance. (e) Schematic representation of CD8⁺ T cells and CD4⁺ T cells detection in tumors excised from BALB/c mice. (f) Representative dot plots of CD8⁺ T cells and CD4⁺ T cells out of CD45+CD3+ TILs in renca syngeneic tumors. (g, h) Proportions of CD8⁺ T/CD4⁺ T cells out of CD45+CD3+ TILs in Renca syngeneic tumors treated with Control (n = 6), Se (n = 6); PpD (n = 6), PD-1 mAb (n = 6), or combined therapy (n = 6). Statistical analysis of data was calculated using One-way ANOVA analysis among groups, Error bars indicate ±SD, n = 6; ∗ represents p < 0.05; ns represents no statistical significance.

In order to further validate whether PAK4 targeted drugs can prolonging mice survival by regulating the immune microenvironment in vivo. Firstly, a renal cancer model with complete immunity was constructed (Fig. 5c). Mice were treated intraperitoneally (i.p.) with PpD, while PBS and Se-mutation peptide served as negative controls. The PpD reduced the mice tumor volume (Supplementary Fig. S14e) and prolonged mice survival (Fig. 5d, p < 0.01, One-way ANOVA). In fully immunocompetent mice, PpD demonstrated comparable efficacy to PD-1 antibody treatment. However, the combination of PD-1 antibody with PpD did not yield a synergistic effect. This observation suggests potential overlap in the immune pathways targeted by PAK4 and PD-1 antibodies. It is plausible that PpD may exhibit synergistic effects with other immunotherapies.

To assess the impact of PpD on T cell populations, CD8⁺ T cells and CD4⁺ T cells were analyzed by flow cytometry. Tumors were harvested and digested into single cells for analysis with flow cytometry after staining with different markers. The experiment was stopped before the mice died (10 days, Fig. 5e) to ensure fresh tumor tissue for fluorescence-activated cell sorting (FACS) analysis. As shown in Fig. 5f–h, although no appreciable difference was observed in the ratio of CD4⁺ T cells among the three groups, CD8⁺ T cells increased upon treatments with PpD (25.12%), and this level was significantly higher than the control (11.63%, p < 0.05, one-way ANOVA analysis). In regard to its influence on CD8⁺ T cells, PpD exhibited a comparable effect to PD-1 antibodies. This demonstrated that PpD could enhance the infiltration of CD8⁺ T cells into the tumor and synergize to inhibit tumor growth in vivo through a CD8⁺ T cell-dependent pathway.

The findings of this in vivo study provide strong evidence supporting the potential of PpD as an enhancer of immunotherapy for renal cancer. By promoting the infiltration of CD8⁺ T cells, PpD can improve the overall therapeutic response. These results open up new avenues for exploring combination therapies - targeting PAK4 and immunotherapy as a promising approach to enhance the efficacy of combine therapy for renal cancer treatment.

PpD Demonstrated Significant Efficacy Against Renal Cancer in Both Patient-derived Xenograft Models and Patient-derived Organoid Models

ATP-based tumor chemosensitivity Assay (ATP-TCA), patient-derived xenograft (PDX) and patient-derived organoid (PDO) models have emerged as valuable tools in drug development, allowing researchers to better verify the potential efficacy of drugs in a preclinical setting that closely mimics the clinical scenario. To capture the heterogeneity of patients with ccRCC and facilitate drug screening, the study utilized primary cells isolated from patients as well as mini-PDX models to test the efficacy of PpD (Fig. 6a). The patients’ information was shown in Supplementary Tables S1 and S2. The proliferation rate of ccRCC primary tumor cells was measured under different drug treatment conditions using the ATP-TCA. PpD demonstrated stable efficacy in susceptibility tests, with both the mean and median pooled T/C ratio (Tumor/Control ratio) being less than 50% in three patients (Fig. 6b). This indicates that PpD exhibited greater potency in inhibiting ccRCC primary tumor cells compared to the other tested drugs. Additionally, in the mini-PDX models, the analysis revealed that ccRCC primary tumor cells were more sensitive to PpD (IC₅₀ values 0.49, 4.44, and 0.27 μM for patient 1, 2, and 3, respectively) compared to KPT-9274 and PF-3758309 (varying IC₅₀ values for different patients, Fig. 6c). This suggests that PpD effectively inhibited tumor growth in these mini-PDX models. Moreover, primary ccRCC cells were isolated from a 53-year-old patient with T2N0M0 classification and established into a PDO model (Fig. 6d–g). In this PDO model, PpD effectively inhibited the growth of renal cancer primary cells (Fig. 6e–g, PpD IC₅₀ = 471.4 nM, KPT-9247 IC₅₀ = 986.2 nM, and PF-3758309 IC₅₀ = 519.4 nM), demonstrating its potent cancer-inhibitory ability in a context that closely resembles the patient’s tumor.

Fig. 6.

PpD demonstrates potency in renal cancer mini patient-derived xenograft (PDX) and patient-derived organoid (PDO) models. (a) Schematic diagram depicting the design of the tumor chemosensitivity assay (TCA) and mini PDX model to assess the anti-tumor effect of PpD and PAK4 inhibitors in renal cancer. (b) TCA assay showing the tumor cell growth inhibition of three patient with RCC primary cells treated with indicated concentrations (0, 0.016, 0.08, 0.4, 2, 10 μM) of PpD, KPT-9274, and PF-3758309 for 6 days. PpD demonstrates stable tumor cell growth inhibition in patient 1, 2, and 3-derived cells, with IC₅₀ values of 0.49 μM, 4.44 μM, and 0.27 μM, respectively. (c) Mini PDX assay showing the anti-tumor effect of PpD, KPT-9274, and PF-3758309 (2.5 mg/kg) in vivo. Tumor cell viability in OncoVee® capsules was detected to evaluate the antitumor capacity of PpD, KPT-9274, and PF-3758309 in patient-derived primary tumor cells. Statistical analysis of data was calculated using One-way ANOVA analysis among groups, Error bars indicate ±SD, n = 3; ∗ represents p < 0.05; ∗∗ represents p < 0.01; ∗∗∗ represents p < 0.0001; ns represents no statistical significance. (d) Schematic of patient tumor cells treated with drugs in 3D culture. (e) Representative pictures of clear cell ccRCC PDO cells after treated with 1 μM of PpD, 1 μM of KPT-9274, and 1 μM of PF-3758309 for 48 h, respectively. (f) IHC pictures of ccRCC PDO cells treated with 1 μM of PpD, 1 μM of KPT-9274, and 1 μM of PF-3758309 for 48 h, respectively. (g) Cell viability assay of ccRCC PDO cells treated with drugs for 48 h. PpD shows a dose-dependent growth inhibition in patient cells, with an IC₅₀ value of 471.4 nM.

The utilization of PDX and PDO models, along with testing on primary ccRCC cells from patients, provides robust evidence for the efficacy of PpD in inhibiting renal cancer. These models enhance the understanding of PpD’s potential therapeutic benefits and further validate its promise as an effective drug candidate for ccRCC treatment.

Pharmacokinetics (PK) and toxicity analysis of PpD

To assess PK properties of PpD, the PAK4 targeting peptide PROTAC was labeled with Cy5.5 to enable tracking in the body. The study evaluated the biodistribution of PpD in organs and tumors at different time points. Surprisingly, PpD showed favorable tumor-accumulating properties, possibly due to the enhanced permeability and retention effect (EPR) of nanoparticles (Fig. 7a). This nanoparticle system mediated accumulation was further confirmed through comparison the biodistribution between PpD and the free peptide PROTAC drug, with little free peptide PROTAC drug found in the tested organs (Fig. 7b). These results indicated that Se nanoparticles could efficiently accumulate in the tumor site and enhance the stability and retention time of PpD in the body. To further assess the druggability of PpD, PK analysis was performed using inductively coupled plasma mass spectrometry (ICP-MS) in a Balb/C mouse model. The results revealed a half-life (t_1/2) of 9.6 h, demonstrating a sufficiently long cycle time for PpD (Fig. 7c). This information is essential for designing appropriate dosing regimens and determining the optimal treatment schedule for potential clinical use.

Fig. 7.

Pharmacokinetics (PK) and biosafety analysis of PpD. (a) Biodistribution of PpD at different time points in 786-O xenograft models. (b) Comparison of biodistribution between free PAK4 PROTAC peptide and PpD in 786-O xenograft models. (c) Pharmacokinetics analysis of PpD in BALB/c nude mice by ICP-MS, demonstrating the drug’s distribution and clearance profile. (d) Biosafety evaluation of PpD after a 21-day treatment period. Serum biochemical analysis of ALT (alanine transaminase), AST (aspartate transaminase), ALP (alkaline phosphatase), γ-GT (gama-glutamyltransferase), and TBA (total bile acid) levels to assess potential organ toxicity. (e) Representative H&E staining photographs of heart, renal, liver, and spleen sections from mice after 21 days of treatment with the indicated concentrations of Control (PBS), Se nanoparticles, KPT-9274, and PpD, demonstrating no apparent toxicity in major organs. The scale bar represents 73 μm.

Furthermore, toxicity analysis of PpD was conducted by performing biochemical analysis of serum after PpD treatment. During drug treatment, no significant changes in body weight or observable morphological abnormalities were observed in the administration groups compared to the vehicle group (Supplementary Fig. S15a–f). Additionally, the analysis of liver function markers, including ALT, AST, ALP, γ-GT, and total bile acid (TBA), showed no evidence of toxicity in the heart, liver, spleen, and renal (Fig. 7d). Histological examination of these organs through H&E staining further supported the absence of toxicity (Fig. 7e). These findings collectively indicate that PpD exhibits a favorable safety profile.

The comprehensive PK and biosafety analysis of PpD provide valuable insights into its behavior in the body and its potential for further clinical evaluation. The favorable tumor-accumulating properties, extended half-life with Se nanoparticles, and absence of significant toxicity support the potential of PpD as a promising candidate for clinical development in the treatment of renal cancer.

Discussion

The development of targeted drugs for renal cancer and the enhancement of immunotherapy effectiveness are critical areas of research for future renal cancer therapies. PAK4, with its overexpression and increased activity, has been linked to the development and progression of various tumor malignancies by altering key oncogenic pathways, including those involved in immune cell exclusion, such as the PI3K/AKT and WNT/β-catenin pathways. Furthermore, Knocking out PAK4 or using KPT-9274 has been proven to improve the immune checkpoint blockade response rate and reverse the drug resistance caused by immune checkpoint blockade in melanoma,^10,12 glioblastoma,¹¹ pancreatic cancer,¹⁴ breast cancer,^15,16 prostate cancer,¹⁷ bladder cancer⁴⁵ and other tumors.⁴⁶ So the selective PAK4 PROTAC drug is an intriguing candidate to enhance the efficacy of immunotherapies like PD-1 antibodies in tumors. This makes PAK4 an attractive target for combination therapies to enhance the clinical applicability of immunotherapy. By targeting PAK4, potential therapeutic interventions can inhibit renal cancer cell proliferation directly.

In this study, we have successfully developed a potent and selective PAK4 PROTAC drug, PpD. PF-3758309, an ATP-competitive inhibitor, binds to the ATP-binding pocket of PAK4. It was the first PAK inhibitor tested in clinical trials but was terminated due to poor PAK4 selectivity, adverse events, and pharmacokinetic issues observed in phase I studies. In contrast, KPT-9274 is a PAK4/NAMPT dual inhibitor that induces PAK4 instability directly rather than inhibiting its kinase activity. The binding site to PAK4 is unknown. Our designed compound, PpD, differs in its mechanism. It does not bind to the ATP-binding pocket or substrate-binding site of PAK4; instead, it targets the 475–482 amino acids of PAK4, offering improved selectivity. Our experiments have shown that PpD effectively inhibits the growth of ccRCC cells and specifically degrades PAK4, making it a highly selective PAK4 degrader in ccRCC cells. In particular, PpD does not affect the homologous family proteins of PAK4 and is highly selective, and ACHN cells were occupied the PpD results in 786-O cells, indicated PpD may had the same effect like KPT-9274 in other tumors. Meanwhile, our data revealed that PpD-induced PAK4 deletion is associated with immune-related signaling pathways, such as TNF signaling pathway, interferon-gamma signaling pathway, and inflammatory response signaling pathway. This observation aligns with our findings that PpD treatment increasing CD8⁺ T cell in TEM, indicating its potential to improve the effectiveness of immunotherapy. Specially, PpD demonstrated comparable efficacy to PD-1 antibody treatment in fully immunocompetent mice model. However, the combination of PD-1 antibody with PpD did not produce a synergistic effect. This observation suggests a potential overlap in the immune pathways targeted by PAK4 and PD-1 antibodies. Consequently, it is plausible that PpD may elicit synergistic effects when combined with other immunotherapies.

In preclinical in vivo models, PpD demonstrated the ability to inhibit tumor growth more effectively than KPT-9247 and exhibited promising pharmacokinetic profiles. Our study highlights the significance of PpD as a promising drug candidate that can overcome the selectivity issue observed in previous PAK4 inhibitors, and represents an approach in combination therapy.

In conclusion, our research has addressed the challenges associated with PAK4 drug design and successfully developed PpD as a promising candidate for enhancing the effectiveness of immunotherapy in renal cancer (Fig. 8). These encouraging results from preclinical models, including TCA, mini PDX, and PDO models, support the potential of PpD for future clinical applications in ccRCC, and beyond ccRCC. The translation of PpD to human trials holds considerable promise and may pave the way for more effective and targeted therapies in renal cancer.

Fig. 8.

Schematic diagram of PpD for RCC therapy. PpD, a highly potent and selective PAK4 degrader, exerts its therapeutic effects through multiple mechanisms. Firstly, PpD targets and degrades PAK4 by hijacking the E3 ligase MDM2, leading to a reduction in PAK4 protein levels in the cytoplasm and nucleus. This action helps prevent the expression of various oncogenes, conferring additional strength to treatment. Additionally, PpD treatment induces a favorable tumor microenvironment by recruiting CD8⁺T cells into the tumor site. CD8⁺T cells infiltration enhances the sensitivity of ccRCC tumors to PD-1/PD-L1 blockage therapy and promotes tumor regression. In summary, PpD’s unique mechanism of action offers a promising approach for ccRCC therapy by inhibiting tumor growth, modulating the tumor microenvironment.

Contributors

Conceptualization, Methodology and Writing–Original Draft, S.X., B.H.M. and Y.L.J.; Investigation, S.X., B.H.M., K.W., Y.Z.F., and X.Y.F.; Visualization and Data Curation, S.X., B.H.M., C.Y., J.L.A., Z.X.W, J.N.C. and H.J.X.; Data verification: S.X., B.H.M., Y.L.J., Y.Z.F. Writing – Review & Editing, Y.L.J., Y.G, J.M. and Y.L.C.; Funding Acquisition, S.X., B.H.M., Y.L.J., L.L.; Supervision and Project Administration, L.L. All authors have agreed and approved the final version of the manuscript.

Data sharing statement

The data that support the findings of this study are available from the corresponding author (L.L.), upon reasonable request.

Declaration of interests

All other authors declare they have no competing interests.