Targeting PAK4 inhibits Ras-mediated signaling and multiple oncogenic pathways in high-risk Rhabdomyosarcoma

Abstract

Rhabdomyosarcoma (RMS) is the most prevalent pediatric soft tissue sarcoma. Multimodal treatment including surgery and traditional chemotherapy with radiotherapy have contributed to improvements in overall survival rates. However, patients with recurrent or metastatic disease have 5-year survival rates of less than 30%. One reason for the lack of therapeutic advancement is identification and targeting of critical signaling nodes. p21-activated kinases (PAK) are a family of serine/threonine kinases downstream of multiple critical tumorigenic receptor tyrosine kinase (RTK) receptors and oncogenic regulators, including IGFR and RAS signaling, that significantly contribute to aggressive malignant phenotypes. Here we report that RMS cell lines and tumors exhibit enhanced PAK4 expression levels and activity, which are further activated by growth factors involved in RMS development. Molecular perturbation of PAK4 in multiple RMS models in vitro and in vivo resulted in inhibition of RMS development and progression. Fusion-positive and -negative RMS models were sensitive to two PAK4 small molecule inhibitors, PF-3758309 and KPT-9274, which elicited significant anti-tumor and anti-metastatic potential in several primary and metastatic in vivo models, including a relapsed RMS PDX model. Transcriptomic analysis of PAK4 targeted tumors revealed inhibition of the RAS-GTPase, Hedgehog, and Notch pathways, along with evidence of activation of anti-tumor immune response signatures. This PAK4 targeting gene signature showed prognostic significance for sarcoma patients. Overall, our results show for the first time that PAK4 is a novel and viable therapeutic target for the treatment of high-risk RMS.

Introduction

Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in children and adolescents with an incidence rate of 4.5 cases per million children (1). Although multimodal therapy including surgery, radiation, and cytotoxic chemotherapy has improved 5-year survival rates to almost 70%, prognosis for patients with relapsed or metastatic disease remains dismal at 20-30% (1), indicating a dire need for novel therapeutic targets. RMS is classified into embryonal (ERMS) and alveolar (ARMS) based upon molecular and histological features. About 70% of RMS are ERMS, while the rest are comprised of ARMS or fusion positive tumors that frequently contain the PAX3/7-FOXO1 chromosomal translocation, which creates a novel fusion protein (2,3). Recently, whole exome sequencing of RMS xenografts identified mutations in the RAS/NF1 pathways in approximately 45% of cases, predominantly within the fusion-negative (ERMS) population (4) and 75% of high-risk patients were found to have RAS pathway mutations (5,6). These studies have also shown that overall 93% of RMS cases carry alteration in the receptor tyrosine kinase/RAS/phosphoinositide 3-kinase (PI3K) axis (4). This clearly shows the importance and the need to further study the significance of RAS signaling in RMS. Thus, there have been several attempts to therapeutically target RAS, but to date no direct inhibitor of RAS mutants has been approved by FDA. This is partly due to the difficulty to directly target RAS, for the lack of deep hydrophobic pockets for binding of small molecule inhibitors (7). Very recently, promising preclinical results with AMG510, a direct inhibitor targeting the mutant KRAS G12C (8) have prompted a phase I and II clinical trial (NCT03600883) with ongoing recruitments and results are awaited with excitement. Moreover, attempts involving clinical trials to target downstream RAS effectors such as ERK/MAPK and PI3K/AKT (9) as single agents have not been successful. This highlights the necessity to probe into unexplored RAS effectors in RMS.

A group of serine/threonine kinases, namely p21-activated kinases (PAKs) are one such class of effectors. PAKs occupy a hub through which many oncogenic signaling pathways intersect. Initially identified as an effector of Rho GTPases, PAKS play a central role in the reorganization of the cytoskeleton and depolymerization (10) and consequently, cellular architecture, migration and adhesion (11,12). More recently they have been shown to regulate several cellular pathways including growth-factor and steroid-receptor mediated signaling, energy homeostasis, transcription, and mitosis (13,14). This central role of PAKs allows the linking of several key signaling pathways, which include the Ras-ERK, Wnt/β-catenin and estrogen receptor, androgen receptor pathways (15) making them potential therapeutic targets. Additionally, as recently reported PAK signaling plays a critical role in acquired resistance. In BRAF mutant melanoma, MEK inhibitor resistant cell lines and clinical samples show activation of PAK signaling that can be targeted to synergize with MEK inhibition (16).

So far six isoforms of PAKs have been identified in humans, divided in to two families, based on domain structure and regulation mechanisms. Group 1 PAKS comprising of PAK1-3, and Group 2 PAKs comprising of PAK4–6 (17,18). These latter PAKs are activated independent of GTPases (19,20). From the first group, PAK1, by virtue of being discovered first, has been most extensively studied among the PAKs and has been found to be overexpressed in several cancers, including breast, ovarian, bladder, colorectal, lungs and in T-cell lymphoma, glioblastoma (14,21,22). Among the group 2, PAK4 overexpression and/or hyperactivation has been shown to be associated with tumorigenesis in several malignancies such as prostate, breast, gastric, lung, ovarian, melanoma, and pancreatic cancer (15,23,24).

Interestingly, PAK4 has been demonstrated to be essential for Ras-mediated transformation. As reported by Callow et al., inactive PAK4 can inhibit, or significantly attenuate Ras transformation of NIH3T3 and rat intestinal epithelial (RIE) cells, and abolish anchorage-independent growth of Ras mutant HCT116 colon cancer cells (25). In addition, overexpression of wild type or activated PAK4 in NIH3T3 when injected in athymic mice were sufficient for tumor generation. Histological analysis of these tumors showed morphological features consistent with sarcoma transformation (26). Furthermore, PAK4 was reported to be required for oncogenic transformation of MDA-MB-231 breast cancer cells (23). Besides these oncogenic functions, PAK4 has also been noted to contribute to chemoresistance and a role in cancer stem cell phenotypes (14,27).

As alluded to, while the oncogenic role of PAKs has been studied in several malignancies, there is a still a glaring gap in knowledge regarding their role in sarcomas. Our work in Ewing Sarcoma was one of the first studies to report that PAKs are critical regulators of aggressive phenotypes in pediatric sarcomas. We noted that activation of PAK signaling promotes metastatic phenotypes in vitro and metastasis in vivo in Ewing sarcoma (28). A very recent study has shown that guanine nucleotide exchange factor T (GEFT) is correlated with metastatic progression in RMS and in general with a worse prognosis, the underlying mechanism of which is through the Rac1/Cdc42-mediated pathway (29).

Therefore, there is a strong scientific rationale that inhibition of PAKs may be an effective therapeutic strategy in high-risk RMS. To date, there have been no studies investigating the prospects of therapeutically targeting PAKs for the treatment of RMS. PAKs have become a significant target for therapeutic development by pharmaceutical companies (30,31). Recent studies in lung cancer and melanoma have demonstrated the therapeutic importance of inhibiting PAKs (32-35). Out of these targeted inhibitors, PF-3758309 was evaluated in clinical trials (NCT00932126), but the trial was terminated due to poor bioavailability and undesirable pharmacokinetic characteristics. A second compound, KPT-9274, a dual inhibitor of PAK4 and NAMPT, is presently in clinical trials for refractory/relapsed hematologic and solid tumors (NCT02702492). Hence, in this work we attempt to address the validity to test understudied effectors of oncogenic signaling pathways, in this case PAK4. In conjunction with our PAK4-KD and pharmaceutical targeting of PAK4 in vivo models, our results provide extremely strong pre-clinical evidence that targeting PAK4 can be a potential therapeutic approach for advanced RMS.

Materials and Methods

Reagents:

KPT-9274, was provided by Karyopharm Therapeutics Inc. (Newton, MA) was reconstituted in dimethyl sulfoxide (DMSO) and further diluted in culture media. PF-3758309 was procured from MedChemExpress (#HY-13007). Propidium Iodide Flow Cytometry Cell Kit (#ab139418) was procured from Abcam. Dead Cell Apoptosis Assay kit (#V13242) was procured from ThermoFisher. BrdU Cell Proliferation Kit (#6813) was obtained from Cell Signaling Technology. Following antibodies were used for Western Blot and/or immunostaining: PAK4 from ProteinTech (#14685-1-AP), HIF-1α from Novus Biological, and b-Actin (clone C4) from Millipore; AXL (Tyr702) (#D12B2), b-catenin (#9582), S675-phospho-b-Catenin (#4176), S474-phospho-PAK4 (#3241), and Vinculin (#13901) from Cell Signaling.

Cell lines, cell culture and stable cell generation:

Human Rhabdomyosarcoma cell lines were procured from Children’s Oncology Group (COG). Cells were grown in RPMI media supplemented with 20% FBS. Cells were grown in a humidified atmosphere containing 5% CO2 at 37°C. TCCC-RMS40, a patient derived xenograft cell line was generated from tumor biopsy from local recurrence 6 months after completion of chemotherapy from 8 year old male with left parameningeal embryonal rhabdomyosarcoma (stage 3 group III) complicated by intracranial extension. To generate stably transfected knockdown cells, RMS cells were seeded in wells of 6-well plate at 60% density. Lipofectamine 3000 was used to transfect DNA and Lipofectamine RNAiMAX (Invitrogen, Carlsbad, CA, USA) was used for siRNAs according to manufacturer’s instruction. A final concentration of 10nM siRNA pool was used for each transfection. Stable clones were generated by selecting under puromycin. All cell lines were tested for mycoplasma contamination every six months by MycoAlert™ Mycoplasma Detection Kit from Lonza. No cell line was used for more than ten passages after thawing from frozen stocks. All cell lines were authenticated at the Cell Line Characterization core of MD Anderson Cancer Center as recently as July 22, 2019.

Rhabdomyosarcoma Patient Samples

Patient samples were obtained under the approval of H-32129, which was approved by the Institutional Review Boards for Baylor College of Medicine and Affiliated Institutions (IRB00002649). The BCM IRB has approved a waiver of consent/HIPAA authorization and has determined that all requirements are met by this protocol in order to grant the waiver. Informed written consent was not needed from patients. Patient clinical annotations are outlined in Supplemental Table S1.

Migration and Invasion Assay

Migration/Invasion assays were analyzed in a 24-well Boyden chamber. For Invasion 30 μg/ml collagen coated wells were used. Cells were suspended in 100 μl serum-free media and lower chamber was filled with media containing 10% FBS. After incubation at 37°C for the 24 hours, cells were fixed and stained with 0.1% crystal violet. Random fields were quantified using ImageJ.

Western Blot Analysis

Briefly, total cell lysates were isolated in 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM EDTA, 1% Triton X-100 lysis buffer supplemented with protease inhibitors (Complete mini, Roche, Indianapolis, IN, USA) and phosphatase inhibitors. Proteins were separated on 4%–12% Bis-Tris (Invitrogen) or TGX (BioRad) gels, and transferred to polyvinylidene difluoride (PVDF) membranes. Blots were incubated sequentially overnight with primary and secondary antibodies. Immunoreactive signals were developed with ECL kit (Thermo Scientific, Waltham, MA, USA) or by LiCor-Odyssey system.

Analysis of Cell Cycle

Briefly, cells were fixed in ice-cold 70% ethanol at 4°C overnight, followed by PBS wash next day. Cells were re-suspended in 1X buffer with propidium iodide along with RNaseA and incubated for 15 min at room temperature. Analysis was performed on a BD LSRII Analyzer. Statistical modelling was done with FlowJo software to fit the curves. (v10). A total of 10,000 cells were analyzed for each sample.

Cell Growth Assay

Growth assays were assessed by plating 1000 cells per well in triplicate per day for 4 days in a 96-well plate unless otherwise indicated. Cell proliferation was detected two ways. In the first method, through the addition of the colorimetric reagent Cell Counting Kit-8 (CCK-8) (Dojindo Laboratories, Kumamoto, Japan), according to the manufacturer’s instructions (Cell counting kit-8 (CCK-8) assay kit; Dojindo Laboratories). In the second method, CellTiter-Glo Luminescent Cell Viability Assay (Promega) was used to measure cell viability. Assays were done in triplicates.

Soft Agar Colony Formation Assay:

Soft agar colony formation assay was done in 6 well plates with 0.5% agarose in 1X media as base layer. RMS cells (or stable clones) were seeded at the density of twenty to fifty thousand in a 6-well plate dissolved in the top layer of 0.35% agarose in 1X media over the bottom layer. Media from the top was refreshed every three days. After 14 to 21 days, cells were stained with crystal violet and methanol. Images of colonies were taken with a scanner and quantified using ImageJ. All experiments were done in triplicate.

Relative Quantification of Gene Expression:

Total RNA was harvested using Trizol extraction method. The qScript cDNA SuperMix (QuantaBio, Beverly, MA, USA) was used to synthesize cDNA from 200 ng of total RNA from each sample. Relative quantifications of mRNA expression of genes were examined by quantitative RT-PCR (qRT-PCR) with iTaq Universal SYBR Green supermix (Bio-Rad). Reactions were performed on a StepOnePlus™ System. All reactions were run in triplicate. Melting curve analysis verified that all primers yielded a single PCR product. Gene expressions were normalized to 18S to yield a 2^−ΔΔCt value. All primers were purchased from Sigma-Aldrich. Primer sequences are included in supplementary table S2.

In Vivo Xenograft Assay

All animal experiments were conducted according to institutional animal care and use committee protocols after approval was obtained from the BCM Institutional Review Board (BCM Animal Protocol AN-5225). KPT-9274 was administered through gavage while PF-3758309 by subcutaneous administration.

Immunostaining and Immunohistochemistry

Tissue micro-array slides with 18 human rhabdomyosarcoma sample, along with 1 sample each of smooth muscle, skeletal muscle and cardiac muscle tissue was obtained from US Biomax Inc (#SO751). Formalin-fixed, paraffin-embedded tissue sections were deparaffinized followed by rehydration by taking the slides through a graded xylene and ethanol series. VECTASTAIN Elite ABC kit following the manufacturer's instructions was used for visualization. For control, IgG isotype was used instead of primary antibody, wherever indicated. ImageJ based analysis of immunostaining is described in supplemental materials.

Reverse Phase Protein Array

Reverse phase protein array assays were carried out as described previously (28,36).

Gene Ontology Analysis

Gene ontology analysis was done using Thomson Reuters Metacore™ program for genes that had a differential expression of 1.5-fold and P-values, as evaluated by two tailed t-test, P<0.05.

RNA-Sequencing Library Preparation and Sequencing

RNA samples underwent quality control assessment using the RNA tape on Tapestation 4200 (Agilent) and were quantified with Qubit Fluorometer (Thermo Fisher). The RNA libraries were prepared and sequenced at the University of Houston Seq-N-Edit Core per standard protocols. RNA libraries were prepared with QIAseq Stranded Total RNA library Kit (Qiagen) using 500 ng input RNA. mRNA was enriched with Oligo-dT probes attached to Pure mRNA beads (Qiagen). RNA was fragmented, reverse transcribed into cDNA, and ligated with Illumina sequencing adaptors. The size selection for libraries was analyzed using the DNA 1000 tape Tapestation 4200 (Agilent). The prepared libraries were pooled and sequenced using NextSeq 500 (Illumina); generating ~17million 2×76 bp paired-end reads per samples.

Total RNA-Seq analysis

Paired-end sequencing reads were trimmed using the trimGalore software. Detail description of the analysis is described in the supplemental materials.

Statistical Analysis

A P-value <0.05 was considered significant was used to calculate the level of statistical significance. PAK1 and PAK4 expression data was pulled from the Schafer Welle mixed rhabdomyosarcoma dataset in R2: Genomics Analysis and Visualization Platform (http://r2.amc.nl) with 30 Rhabdomyosarcoma samples and 26 normal muscle samples. Expression levels were graphed. All statistical significance tests were performed using GraphPad Prism.

Results

Elevated PAK4 level in RMS cell lines, PDX and disease correlation

p21-activated kinases (PAKs) are potential central signaling nodes integrating pathways critical to growth for both fusion positive and negative RMS tumors, including growth factor receptor signaling and Ras-activation (Fig. 1A). We initially evaluated the transcript expression of both PAK1 and PAK4, using the Schafer Welle mixed rhabdomyosarcoma dataset with the bioinformatics program: R2 (http://r2.amc.nl). We noted significantly increased expression of PAK4 (Fig. 1B), but not PAK1 in both embryonal and alveolar RMS compared to normal human muscle tissue. Analysis of other PAK members revealed that PAK3 was the only other PAK significantly upregulated in RMS tumors compared to all the other PAKs (supplementary Fig. S1A), but with a much wider variation in expression in the RMS samples.

Figure 1: PAK4 Expression and Activation Pathway in Rhabdomyosarcoma:

A) Schematic showing PAK4 as a major node of signaling primarily within the RAS-RAC pathway in both fusion positive and fusion negative RMS. (RTK- Receptor Tyrosine Kinases) B) PAK4 expression in normal muscle and subgroups of RMS in individual clinical samples evaluated using the R2 dataset C) PAK4 levels in sections of ERMS and ARMS compared to normal muscle tissue in a tumor micro array by immunostaining with anti-PAK4 antibody and IgG as isotype control. D) Quantification of PAK4 levels in the tumor micro array. E) Western blot for PAK4 protein levels from patient and patient derived xenograft tumor samples. F) PAK4 phosphorylated (S474) and total protein level in established RMS cell lines in comparison to non-malignant skeletal muscle myoblast cells (SK-MM). p <0.05 was calculated with ordinary one-way Anova (** <0.01) compared to normal control.

Furthermore, staining for PAK4 by immunohistochemistry on a tissue microarray (TMA) of primary patient tumor specimens showed high expression of total PAK4 compared to non-malignant tissue specimens (Fig. 1C). A comparison of the alveolar and embryonic RMS with the normal muscle samples on this TMA showed PAK4 to be to be significantly higher in both ARMS and ERMS samples (Fig. 1D). Subsequently, we confirmed higher expression of PAK4 using direct patient biopsy/surgery samples as well as patient derived xenograft (PDX) tumor samples by Western blotting (Fig. 1E and supplementary Fig. S1B). Secondary to these results, we compared the protein expression of total and phosphorylated PAK4 across a panel of fusion positive and negative RMS cell lines to primary human skeletal muscles cells (Fig. 1F). We observed that both phosphorylated and total PAK4 protein levels were elevated in the tumor cell lines albeit with some heterogeneity among the cell lines with respect to the level of this elevation. RD, which is a fusion negative cell line, harboring mutations in NRAS, p53, and c-Myc amplification, shows higher PAK4 phosphorylation compared to other cell lines tested. The convergence of these dysregulated pathways on the regulation of PAK4 can be a potential explanation for these findings.

To further investigate the importance of PAK4 mediated signaling, we first looked at activation of PAK4 downstream of major growth factor receptor-mediated signaling in RMS by Western blot in two cell lines, RD and RH30 (supplementary Fig. S1C). Densitometric quantification (supplementary Fig. S1D) of the western indicated an increase in activation of PAK4 levels for both cell lines with basal fibroblast growth factor 2 (bFGF2) stimulation within 15 minutes, while stimulation with insulin-like growth factor 2 (IGF2) resulted in increase in PAK4 activation in RD cell line only. Concomitantly we saw an increase in phospho-β-catenin over its total and phospho-ERK1/2 over total ERK1/2. this increase was significant for phospho-ERK1/2 over total ERK1/2 (supplementary Fig. S1D).

PAK4 contributes to RMS tumorigenic and metastatic phenotypes in vitro

To evaluate the functional significance of PAK4, we took multiple molecular and pharmacological approaches to attenuate PAK4 levels and/or activity in RMS tumor models. We first generated PAK4 knockdown (KD) in two RMS cell lines, RH30 and RD, using two independent shRNAs against PAK4. Knockdown was confirmed by Western blot (supplementary Fig. S2A and S2B). No discernable off-target effects were noted, as we did not observe any effects on PAK1 (supplementary Fig. S2C). Using fusion negative and positive RMS cell lines models, RH30 (Fig. 2A) and RD (Fig. 2B), multiple clones of both of which showed a significant reduction in cell viability when compared to the control scrambled. In addition, we observed significant inhibition in migratory and invasive potential in RH30 (Fig. 2C and 2D) as represented by microscopic images (supplementary Fig. S2D) and in RD (Fig. 2E and 2F; and S2E). To further ascertain the role of PAK4 in tumorigenesis we assessed anchorage independent growth via soft agar colony formation assay in RH30 and RD cell line and all KD clones demonstrated significantly fewer colony formation under these conditions, compared to the scramble-controls (Fig. 2G and supplementary Fig. S2F). A complementary, reciprocal experiment with stable overexpression of full-length PAK4 in the RH30 cell lines showed a significant increase in the number of soft agar colonies (Fig. 2H and supplementary Fig. S2G).

Figure 2: Knockdown of PAK4 inhibits RMS growth and metastatic potential in vitro.

A) Growth curve of RH30-PAK4-KD cells. Stable clones with either scrambled control (scr) Clone C1 or clones from two different shRNA (shRNA clone A2 and shRNA clone E2) were allowed to grow for 72 hours and monitored for viability every 24 hours. B) Growth curve of RD-PAK4-KD clones over 3 days. Boyden chamber assay shows PAK4 is involved in C) RH30-migration D) RH30-invasion E) RD-migration and F) RD invasion. G) Soft agar colony formation assay in RD stable clones with scrambled control (Scr clone A3) compared to sh-PAK4 A5 and E7 KD clones. H) Overexpression of PAK4 induced more colony formation in RH30 cells evaluated by soft agar colony formation assay in 6 wells, quantified by ImageJ analysis. Data shown are means ± SD of 3 determinations. Statistical significance of p <0.05 was calculated with two-way Anova with Dunnett’s multiple correction (* <0.05, ** <0.01, *** <0.001, **** <0.0001) compared to corresponding control with ns indicating non-significant.

Decreased PAK4 expression inhibits tumor growth and metastatic potential in vivo

After characterizing the phenotypic contributions of PAK4 in vitro, we investigated the role of PAK4 knockdown (KD) on primary and metastatic tumor development in vivo using orthotropic and tail vein murine models. Upon orthotopic injection into the gastrocnemius muscle of NSG mice, RH30 shPAK4 cells resulted in a significant decrease in tumor growth compared to scrambled control cells as seen by either mean growth rate of each arm (Fig. 3A) or for individual mice in each group (supplementary Fig. S3A) . We observed similar findings with RD PAK4 KD cells (Fig. 3B and supplementary Fig. S3B). Tumors samples showed a significant increase in cleaved Caspase-3 staining in the RH30-PAK4 knocked down tumors (Fig. 3C and supplementary Fig. S3C). On termination of experiment, in the residual tumor, we did not find any significant difference in PAK4 staining (Fig. S3D).

Figure 3: Knockdown of PAK4 inhibits tumor growth and metastatic potential in vivo.

A) RH30 cells and B) RD cells (0.5x10⁶) stably transduced with either scramble (control) or shRNA targeting PAK4 were injected in the gastrocnemius muscle of NSG mice (n=6) and tumor volume was measured twice a week. Statistical significance of p <0.05 was calculated with two-way Anova with Dunnett’s multiple correction (*<0.05, ** <0.01, *** <0.001, **** <0.0001) compared to corresponding control. C) Image J based quantification of 3 random samples from each group of scrambled control and PAK4 KD tumors stained with cleaved Caspase-3. p=0.002 calculated by two-way ANOVA. D) Outline of tail vein injection for a lung metastatic model where RMS cells either control clones with scrambled DNA or KD clones with shRNA targeting PAK4 were injected via tail vein and allowed to colonize distant organs, which were evaluated after three weeks of growth. E) Metastatic colonies in liver visualized by hematoxylin and eosin staining in the two cohorts from RH30. The images were taken at 10x using the Leica DMi8 microscope and LASX software to stitch one entire image. F) Gross metastatic load between control and KD clones in terms of liver weight and percentage area covered in by metastatic lesions in liver sections of RH30 clones. G) Metastatic colonies in liver in RD tumors with scrambled and shRNA clones. H) Gross metastatic load between control and KD clones in terms of liver weight and percentage area for RD metastatic lesions. *p <0.05 was regarded as significant and calculated by unpaired t test.

To investigate the role of PAK4 in contributing towards RMS metastatic potential, we performed tail vein injections to model tumor dissemination and allowed for cells to circulate, colonize, and grow colonies for 3 weeks (Fig. 3D). At the end of 3 weeks, compared to control RH30 cells, RH30 shPAK4 cells resulted in lower metastatic burden as we observed fewer metastatic foci from H&E staining (Fig. 3E) along with a significant decrease in overall liver weights and surface area covered by metastatic lesions in the liver sections (Fig. 3F) . Similar inhibition of metastatic potential was observed in mice injected with RD PAK4 KD cells (Fig. 3G and 3H).

Pharmacological inhibition of PAK4 leads to regression of tumorigenic properties in vitro

After demonstrating that knockdown of PAK4 has potent anti-tumor and metastatic properties in RMS, we investigated the efficacy of small molecule inhibitors of PAK4 in order to provide additional pre-clinical assessment for the therapeutic targeting of this kinase in RMS. We utilized two independent PAK4 small molecule inhibitors, KPT-9274, an allosteric inhibitor of PAK4 and NAMPT (Karyopharm Therapeutics) and PF-3758309 (Pfizer), a potent ATP-competitive, pyrrolopyrazole inhibitor of PAK4 (supplementary Fig. S4A).

We first assessed the effect of KPT-9274 and PF-3758309 on proliferation of several RMS cell lines in vitro. A dose dependent reduction in cell proliferation was observed with both drugs at low nanomolar concentrations. For KPT-9274, the IC₅₀ ranged from 40-80 nmol/L for the multiple cell lines studied, whereas for PF-3758309 the IC₅₀ ranged from 8.4 to 33.7 nmol/L (supplementary Fig. S4B). As seen in Figures 1C and 1D, normal tissue and myoblast cells have very low PAK4 expression and several studies have shown very little toxicity of KPT-9274 on normal tissues (37,38). To further address this we investigated the effect of KPT-9274 and PF-3758309 on normal skeletal muscle myoblast (SKMM) cells. We observed (Fig. S4B) the IC₅₀ were 8-10 times higher for both PAK4 inhibitors on the non-malignant cells. Overexpression of PAK4 in RH30 cells increased the IC₅₀ from 80 to 95 nmol/L under KPT-9274 treatment and from 33.7 to 45 nmol/L under PF-3758309 treatment. Overexpression of PAK4 in RD cells (supplementary Fig. S4C), on the other hand, were significant (p <0.001) for both small molecule inhibitors, with the IC₅₀ increasing from 75.5 to 118 nmol/L with KPT-9274 treatment and also with PF-3758309, by increasing the IC₅₀ from 8.5 to 23.5 nmol/L (supplementary Fig. S4B). To evaluate potential off-target effects we evaluated PAK1 expression for RMS cells treated with KPT-9274 and PF-3758309 under similar conditions as above and observed no effect on PAK1 levels (Fig. 4A).

Figure 4: Pharmacological Inhibition of PAK4 in vitro:

A) Off target effect on PAK1 was investigated. β-Actin was used for loading control. Numbers indicate fold change with respect to vehicle treated control, after normalization with β-Actin. B) Cell death, primarily via apoptosis and secondarily via late apoptosis/necrosis induced in RD and RH30 cells treated with KPT-9274 at 100 and 200 nM concentrations, and with PF-3758309 at 100 nM concentration for 72 hours. Flow cytometry analysis for apoptosis using Annexin-V FITC for apoptotic cell and 7-AAD for dead cell population is shown. Cells are represented as a percentage of the total population. Data shown is representative of three or more repeats. C) Cell cycle analysis of RMS cells, untreated or treated for 3 days with 100 nM KPT-9274 or 100 nM PF-3758309. Cells were treated for 72 h followed by staining with propidium iodide (PI). DNA content was quantified by flow cytometry. Analysis was done using FlowJo software with Dean-Jen Fox statistical model. Data is represented as the percentage of cells in the G0/G1, S and G2/M phase of the cell cycle. For each sample 10,000 cells were acquired. Data shown are means ± SEM. D) Boyden chamber assays of motility and invasion with RD and E) RH30 treated with 100 nM KPT-9274 or 100 nM PF-3758309 are summarized as fold change, compared to untreated control. Analysis includes all experimental replicates. *p <0.05 was regarded as significant and calculated by unpaired t test. F) Densitometric quantification of band intensities from Western blot to show effect on PAK4 activation and expression level and its downstream targets in RD and RH30 cell lines when treated with KPT-9274 or 100 nM PF-3758309 for 24 hours. The levels of phosphorylated and total PAK4, β-Catenin, and AKT along with PARP cleavage were determined by Western blot. Band intensities were normalized to untreated control after adjusting for loading variation with respective β-actin bands. *p <0.05 was regarded as significant and calculated by one-way ANOVA. All comparisons are done with the untreated or vehicle control in that specific group.

To evaluate for the mechanism for this reduction in cell proliferation, we first looked at induction of apoptosis by flow cytometry. Treatment for 72 hours with either drugs resulted in significant increase in apoptotic cells in both RD and RH30 cells (Fig. 4B). Percentage of apoptotic cells in the RD cell line increased from 1.4% with vehicle treatment to 4% and 4.7% with 100nM and 200nM KPT-9274, respectively. Treatment of 100nM of PF-3758309 had the most effect by raising distribution of apoptotic cells to 5.7%. In the RH30 cell line, KPT-9274 increased apoptotic cells significantly from 0.1% to 4.8% at a concentration of 100nM and to 5.5 at 200nM. Treatment with PF-3758309 resulted in an increase to 2.2% (Fig. 4B and S4D). Thus, both drugs significantly induced apoptosis at nanomolar concentrations. Accordingly, investigating the effect of the two PAK4 inhibitors on cell proliferation rate via BrdU incorporation, we found KPT caused a significant reduction at 200 nM for both cell lines, while RH30 had a significant reduction in BrdU incorporation when treated with 200nM PF-3758309 (supplementary Fig. S4E).

Additionally, to further account for the reduction in the number of tumor cells we looked at the stages of the cell cycle by flow cytometry (Fig. 4C and supplementary Fig. S4F). In RH30 cells, treatment with either the KPT-9274 or PF-3758309 resulted in a significant increase of cells in G1 with a concomitant decrease of cells in S phase, indicating a block in G1-S progression. In the RD cell line, like RH30, KPT-9274 induced a G1-S arrest, by significantly increasing cells in G1, while reducing the percentage in S phase. However, treatment with PF-3758309 seemed to have no effect on the distribution of cells in the G1 phase, but reduced cells in S phase with an increase in G2, indicating a G2/M arrest. This difference in stages of arrest could be attributed to the fact that KPT-9274 is a dual inhibitor of NAMPT and PAK4, and some of observations are possibly due to inhibition of NAMPT or a combination of blocking both targets.

Recurrence and metastasis remain a therapeutic challenge in treating RMS patients. Indeed, PAK4 signaling has been implicated in cytoskeletal alterations and signaling pathways associated with enhanced metastatic phenotypes. Thus, we were interested in assessing the effect of inhibiting PAK4 on phenotypic properties associated with metastasis. Using the Boyden trans-well assay, we probed the effect of KPT-9274 on invasive and migratory properties. Our results show that KPT-9274 significantly inhibited cell motility for both cell lines (Fig. 4D, 4E, and supplementary Fig. S5A), while PF-3758309 had a significant inhibitory effect on the motility of RD cells (Fig. 4D), but not on RH30 cells (Fig. 4E). Treatment with either of the two drugs induced a significant inhibition in invasive properties for both cell lines tested, with greater reduction in invasive properties noted with PF-3758398 treatment.

Besides the phenotypic properties, we investigated the ramifications on downstream signaling secondary to PAK4 targeting. As noted in Figure 4F, in vitro treatment with KPT-9274 and PF-3758309 confirmed a reduction of PAK4 activity via decreased PAK4 phosphorylation and decreased activation of the downstream target β-catenin as shown by representative Western blot (supplementary Fig. S5B) from two independent studies (Fig. 4F). Besides the perturbations in signaling, we also noted a significant increase in PARP cleavage in RH30 cells, thus confirming induction of apoptosis. While in RD cells, we noticed a cleavage product at molecular weight 55-60kD, which is indicative of necrosis (39,40). Thus, pharmacological inhibition of PAK4 leads to potent anti-tumor and metastatic properties in vitro through downregulating critical signaling pathways involved in tumor development and progression.

Pharmacologic inhibition of PAK4 leads to regression of tumor growth and metastatic progression

Results confirming significant anti-tumor and anti-metastatic effects in vitro, lead us to evaluate the pre-clinical testing of KPT-9274 against RMS primary and metastatic growth in vivo, using orthotropic and metastatic tail vein mouse models. For orthotropic models, RMS cells were injected into the gastrocnemius muscle of NSG mice. As shown in the schematic (Fig. 5A), once a tumor was palpable mice were randomized to receive either KPT-9274 or vehicle by oral gavage, and administration was continued until the endpoint of tumor burden was reached for one of the cohorts. KPT-9274 treatment resulted in a significant regression in tumor growth with RH30 cell lines (Fig. 5B) as compared to the control cohorts. To confirm that this observed anti-tumorigenic effect of KPT-9274 is indeed through inhibition of PAK4, we performed western blot and IHC for PAK4 from tumor samples from the RH30 in vivo studies. PAK4 protein level attenuation was observed in the tumors from animals treated with KPT-9274 compared to the control tumors (Fig. 5C&D and supplementary Fig. S6A). We also looked at inhibition of β-catenin activation (S675) as a direct downstream target of PAK4 (supplementary Fig. S6B) and inhibition at the level of protein expression for both activated and total β-catenin was observed. To assess for apoptosis in these tissues, we stained for cleaved Caspase-3 (CC-3) in three random tumor samples from each group and there was a significant increase in CC-3 positive cells in the KPT treated group (Fig. 5E). Similar anti-tumorigenic growth result was observed with RD cell lines as well (Fig, 5F and S6C) when treated with KPT-9274. This reduction was not associated with any apparent toxicity, and there was no significant weight loss for animals receiving KPT-9274 when compared to animals receiving vehicle control (Fig. S6D). The xenograft experiments were repeated with similar results in both RH30 (Fig. S6E) with no significant effect on body weight (Fig. S6F).

Figure 5: Pharmacological Targeting of PAK4 in vivo Inhibits Tumor Growth and Progression:

A) Schematic of experiment showing experimental outline. NSG mice were injected with RMS cells in the gastrocnemius muscle and monitored for tumor growth. As soon as palpable tumor developed, mice were randomized and received vehicle (placebo) or KPT-9274 at the indicated concentration, via gavage administration, twice, daily, 5 days/week. B) Tumor volume was measured twice a week for RH30 cells injected in NSG mice (n=6) C) Tumor samples at the end of the experiment were collected, lysed and subjected to Western blot analysis to confirm PAK4 inhibition in the treated group. D) Assessment of PAK4 level from immunostained slides of treated and control mice tumor. P-value calculated as unpaired t-test showing significant reduction in treated mice. E) Cleaved Caspase-3 staining on three random tumor samples from each of vehicle treated and KPT-9274 treated mice with RH30 cells. P-value calculated as two-way ANOVA. F) Tumor volume measurement in NSG mice (n=6) injected with RD cells randomized in two cohorts, placebo, and KPT-9274 at the indicated concentration, via gavage administration, twice, daily, 5 days/week. G) Schematic showing metastatic model with tail vein injection in NSG mice, followed by randomization in placebo and treated group. H) Gross metastatic load shown by photographs of livers from mice receiving vehicle control or KPT-9274 with white arrows indicating metastatic nodule. I) Hematoxylin and Eosin staining of the liver samples showing hyper nucleated tumor nodules (indicated by black arrows) specifically in the control group. Images are taken at 10x magnification.

Even though PF-3758309, another potent PAK4 inhibitor, did not successfully progress through clinical trials, as a proof of principle and to confirm that the anti-tumor activities seen in xenograft models can be attributed to blocking PAK4, we injected RH30 cells into the gastrocnemius muscle of NSG mice. Once tumors were palpable, mice were randomized to receive either PF-3758309 or vehicle by oral gavage, and administration was continued until the endpoint of tumor burden was reached for one of the cohorts. Again, we observed a significant reduction in tumor growth with PAK4 inhibition (Fig. S6G).

The PAK4 KD and PAK4 small molecule inhibition in vitro studies suggest a significant role for PAK4 in dictating metastatic phenotypes, thus providing the rationale to investigate pharmacological efficacy of KPT-9274 in targeting metastatic RMS in vivo. Using the RD cell line in a tail vein metastatic model we evaluated the efficacy of PAK4 inhibition by KPT-9274. Treatment of KPT-9274 was started two weeks post injection and continued for 3-4 weeks as highlighted in the schema (Fig. 5G). At the end of the treatment period we evaluated for the presence of metastatic disease. We observed the KPT-9274 treatment group had lower metastatic burden in liver compared to the vehicle treated group (Fig. 5H) by gross macroscopic visualization as well as by histological staining (Fig. 5I) with H&E.

Tumor growth is significantly impaired in patient-derived xenograft model by pharmacologic inhibition of PAK4

After demonstrating significant anti-tumor activity on established fusion positive and fusion negative RMS cell lines we tested the potential anti-tumor effects of KPT-9274 on a high-risk fusion negative patient derived xenograft (PDX) model. Cryopreserved tumor pieces from TCCCRMS40, derived from a relapsed RMS patient that also had high PAK4 protein expression, were implanted subcutaneously into NSG mice (Fig 6A). As evident in Figure 6B, KPT-9274 treatment dramatically inhibited tumor growth, with significant differences in tumor volumes (Fig. 6C) at the end of the study. The study was performed without any apparent toxicities or changes in body weight (Fig. 6D).

Figure 6: Pharmacological Inhibition of PAK4 by KPT-9274 Inhibits Patient-Derived Xenograft Growth:

A) Schematic of experimental design showing collection of tumor samples from patient, followed by implantation in mice. Once tumors were palpable, mice were randomized and received vehicle (n=5) or KPT-9274 (n=7) at the indicated concentration, via gavage administration, twice, daily, 5 days/week. B) Representative photomicrograph of tumors extracted at the end of the experiment. C) Quantification of tumor volumes from the two cohorts at the end of the experiment. P-value calculated as unpaired parametric t-test. D) Body weight of individual mice measured throughout the experiment, twice a week.

PAK4 inhibition leads to attenuation of molecular signatures involved in cell cycle and metastatic progression

In order to further elucidate the downstream signaling effects secondary to PAK4 inhibition, we performed transcriptomic and proteomic analysis of RH30 control and KPT-9274 treated tumors. Analysis of the transcriptomic data revealed several key cancer-associated pathways to be modulated in KPT treated tumors. As shown through the volcano plot, with KPT treatment, 628 were significantly up-regulated a fold change of 1.25 or higher (red dots) while 65 genes were significantly down-regulated showing fold change of 1/1.25 or lower (blue dots) (Fig. 7A). Gene Set Enrichment Analysis (GSEA) using the KEGG, REACTOME, GO, and HALLMARK pathway collections revealed multiple oncogenic pathway were enriched with a negative NES (Fig. 7B). Consistent with the mechanism of action of the KPT-9274 compound we noted GTPase and RAS signaling pathways were significantly inhibited (Fig. 7C). Further, two major pathways previously implicated in Rhabdomyosarcoma biology, namely Notch and Hedgehog signaling, were also significantly downregulated (Fig. 7C). String database was used to analyze for protein-protein interaction (PPI) in Notch signaling and PPI networks were visualized with Cytoscape software and color coded based on up-regulation (red) or down-regulation (blue) in KPT treated samples as compared to control (Fig. 7D). Finally, a network of pathways enriched via hypergeometric distribution (p-value<0.05) in the significantly differential genes was depicted using the Cytoscape software (Fig. 7E), with edges indicating the overlap between pathways.

Figure 7: Transcriptomic Analysis of KPT-9274 treated RMS tumors:

A) Volcano plot for KPT-9274 treated transcriptomic data. Each dot represents a gene. Significantly upregulated genes (p-value < 0.05 & fold change > 1.25) are marked in red; significantly downregulated genes (p-value < 0.05 & fold change < 1/1.25) are marked in blue. B) Enriched pathway via Gene Set Enrichment Analysis (GSEA) reveals down-regulation of oncogenic pathways. C) GSEA enrichment score plots for selected suppressed oncogenic pathways (Normalized Enrichment Score or NES <0) with Q-values (adjusted p-values using an optimized FDR approach). D) Protein-protein interaction networks within the NOTCH pathway evaluated using the String database E) A network of enriched immune system and oncogenic pathways was visualized using the Cytoscape software. Node sizes depicts the number of KPT-modulated genes in each corresponding pathway, whereas edge thickness portrays the number of KPT-modulated genes shared between node-associated pathways.

We further assessed the effects of the KPT-9274 treatment on RH30 cells using Reverse Phase Protein Array (RPPA) profiling. Hierarchical clustering of the differentially expressed proteins revealed distinct sets of proteins being modulated between the two treatment groups (supplementary Fig. S7A). Several of the oncogenic pathways determined using RNA-Seq were confirmed by the targeted proteomic profiling; specifically, we determined enrichment in the proteomic signature for cell cycle and migration, corroborating our functional in vitro studies (Fig. S7B). As a surrogate marker of PAK4 inhibition, β-catenin, a known downstream target of PAK4 was found to be inhibited. Among other groups of differentially expressed proteins revealed by hierarchical clustering, we found proteins involved in metastatic progression, such as AXL and HIF-1α being significantly diminished in the KPT-9274 treated tumors. Several of these key modulations were validated by Western blot from the KPT-9274 treated tumor lysates (Fig. S7C).

Discussion

While advancements in the genetic characterization have propelled our molecular insights into RMS, there are still significant deficiencies in the identification and implementation of effective therapeutic regimens for advanced stages of the disease (4).

Through extensive pre-clinical studies, our report provides evidence for the first time that PAK4 is a viable and novel therapeutic target for the treatment of advanced RMS. Using multiple in vitro and in vivo cell line and PDX models, in conjunction with molecular and pharmacological targeting of PAK4, we have demonstrated that knocking down or inhibition of PAK4 can reduce cell proliferation in vitro and delay or regress tumor growth.

Blocking PAK4 had a profound effect not just on proliferation but also on metastasis, as evidenced by diminished in vitro transwell assays and in vivo metastatic foci formation in mouse models. While KPT-9274 is a dual inhibitor of PAK4 and NAMPT, and therefore we cannot exclude some of the biological ramifications can be secondary to NAMPT inhibition, such as the effects seen on AKT inhibition could partially be contributed by NAMPT inhibition, with the latter being involved in energy metabolism. This is also relevant when we observe a G1 arrest in the RMS cells with both drugs but a G2/M arrest in RD cells with only PF-3758309 treatment. Although, a G1 arrest induced by PAK4 inhibition has been reported in breast and ovarian cancer via inhibition of cyclin D1 (41,42) through the PAK4/c-Src/EGFR pathway, or in pancreatic cancer (43) by downregulating cyclin D1 and upregulation of p21, we can not rule out the possibility that some of these effects are secondary to, or a combination of PAK4 and NAMPT inhibition.

However, we believe that the use of a complementary PAK4 inhibitor, PF-3758309, provides convincing evidence through both molecular perturbation that targeting PAK4 has significant anti-tumor and metastatic activity in RMS. Western blot and immunostaining of our KPT treated tumors also confirm PAK4 inhibition (Fig. 5C and D). Furthermore, our comprehensive transcriptomic analysis of KPT-9274 treated tumors leads to molecular signatures consistent with targeting Ras/small GTPases, again signifying target engagement. Additional studies into the role of NAMPT in RMS biology, which is unrelated to the focus of this work, are warranted. Therefore, we believe that our comprehensive studies targeting PAK4 in multiple tumor model systems, including a PDX derived from a relapsed fusion negative RMS patient, pave the way to a novel therapeutic strategy for high-risk RMS patients.

Mechanistically, our studies unveil several novel molecular insights into the role for PAK regulation in RMS. As mentioned above, besides modulation of Ras/GTPase downstream signaling, our comprehensive transcriptomic analysis identified pathways that were significantly modulated by PAK4 including Hedgehog, Notch and PI3K signaling, which corroborate previous studies that characterized critical roles for these pathways in RMS tumorigenesis (44) (45). Studies investigating the mechanism by which PAK4 regulates, or cross-talks with these pathways are in preparation and will provide additional insights into the interplay between these vital signaling cascades.

Interestingly, besides alterations in intrinsic signaling perturbations, our transcriptomic analysis of KPT-9274 treated tumors provided preliminary evidence for extrinsic tumor microenvironmental regulation dictated by PAK4 signaling as we identified enhanced immune modulation frequently associated with anti-tumor activity. Several tumor immunity related pathways were enriched with a positive Normalized Enrichment Score (NES) with KPT-9274 treatment (supplementary Fig. S8A). Specifically, we observed a significant upregulation in adaptive immunity, IFN-α and -Ɣ response, and pathways in antigen processing in KPT-9274 treated tumors (supplementary Fig. S8B and C).

Although validating this observation is beyond the scope of this manuscript, these findings are supported by the recent publication identifying PAK4 inhibition can alter the tumor immune microenvironment and further enhance anti-PD-1 efficacy and increased T cell infiltration in syngeneic melanoma murine models (46). However, the use of immunodeficient murine models limits our interpretation of the significance of these molecular alterations. We plan to utilize genetically engineered mouse models for RMS, and syngeneic murine RMS cell lines for future studies that can be used to further evaluate the role of PAK4 in the regulation of the tumor immune microenvironment and immune evasion.

Finally, besides observing the inhibitory effects on oncogenic pathways and activation of immune stimulatory signatures, we were able to evaluate the association between the KPT-9274 signature and survival in the TCGA-SARC patient cohort. Although this cohort does not include RMS patient information, the association was significant (p<0.05) and patients with transcriptome profiles closer to those of the KPT-treated tumors had a significantly better outcome than those with profiles closer to the RH30 control-treated cells (supplementary Fig. S8D).

Additionally, gene ontology analysis of the proteogenomic data indicates signatures associated with metastatic progression and myogenic differentiation, through AXL, and HIF-1α. Aside from its role in microenvironmental modulation in the context of tumor hypoxia, HIF1α has also been reported to be critical in driving therapy resistance and other metastatic properties (47,48). AXL is a receptor tyrosine kinase, found to be overexpressed in many cancer types and implicated for its role in proliferation and invasion and in epithelial-mesenchymal transition (EMT) processes (49). AXL has been identified as a “semi-direct” regulator that can negatively regulate muscle genes in the presence of a sensitizer and thus, its knockdown can promote myogenic differentiation (50). The ligand to AXL, growth arrest–specific 6 (GAS6) has been reported to play a role in tumor dormancy in the bone marrow (51) by downregulating AXL during ambient physiological conditions, but not under hypoxic conditions, thereby promoting metastasis. In another study in clear cell renal cell carcinoma, AXL was found to be activated under hypoxia by both HIF-1 and HIF-2 binding to the hypoxia-response element in the AXL proximal promoter (52). Thus, the GAS6/AXL signaling pathway is a critical player in metastasis and our RPPA data shows PAK4 is modulating this pathway. Further mechanistic studies are warranted to gain insights into the regulation of hypoxia-mediated functions by PAK4 in RMS.

Taken together, we are the first to demonstrate that PAK4 is a regulator of critical intrinsic, and potentially extrinsic mechanisms of RMS development and progression, and is a viable therapeutic target for the treatment of advanced stages of rhabdomyosarcoma.

Significance: These data demonstrate a novel oncogenic role for PAK4 in rhabdomyosarcoma and show that targeting PAK4 activity is a promising viable therapeutic option for advanced RMS.