Myxofibrosarcoma (MFS) and undifferentiated pleomorphic sarcoma (UPS) are highly genetically complex soft tissue sarcomas. Up to 50% of patients develop distant metastases, but current systemic therapies have limited efficacy. MFS and UPS have recently been shown to commonly harbor copy number alterations or mutations in the tumor suppressor genes RB1 and TP53. As these alterations have been shown to engender dependence on the oncogenic protein Skp2 for survival of transformed cells in mouse models, we sought to examine its function and potential as a therapeutic target in MFS/UPS. Comparative genomic hybridization and next-generation sequencing confirmed that a significant fraction of MFS and UPS patient samples (n = 94) harbor chromosomal deletions and/or loss-of-function mutations in RB1 and TP53 (88% carry alterations in at least one gene; 60% carry alterations in both). Tissue microarray analysis identified a correlation between absent Rb and p53 expression and positive expression of Skp2. Downregulation of Skp2 or treatment with the Skp2-specific inhibitor C1 revealed that Skp2 drives proliferation of patient-derived MFS/UPS cell lines deficient in both Rb and p53 by degrading p21 and p27. Inhibition of Skp2 using the neddylation-activating enzyme inhibitor pevonedistat decreased growth of Rb/p53-negative patient-derived cell lines and mouse xenografts. These results demonstrate that loss of both Rb and p53 renders MFS and UPS dependent on Skp2, which can be therapeutically exploited and could provide the basis for promising novel systemic therapies for MFS and UPS.

Significance:

Loss of both Rb and p53 renders myxofibrosarcoma and undifferentiated pleomorphic sarcoma dependent on Skp2, which could provide the basis for promising novel systemic therapies.

See related commentary by Lambert and Jones, p. 2437

Myxofibrosarcoma (MFS) and undifferentiated pleomorphic sarcoma (UPS; formerly malignant fibrous histiocytoma; ref. 1) comprise one of the most common histologic groups of adult soft tissue sarcomas. Histologically, MFS and UPS are fibroblastic or myofibroblastic tumors displaying cellular pleomorphism (2), and differ only in the myxoid stromal content of the tumor, with MFS containing a higher proportion of myxoid stroma. MFS and UPS are also genetically indistinguishable based on multiplatform molecular analyses (3), suggesting that these two subtypes represent a spectrum of the same disease entity. UPS in its current definition may also include high-grade pleomorphic sarcomas that lack a specific lineage of differentiation.

After resection of MFS/UPS, which most commonly arises from the extremities or trunk, over 50% of patients develop distant metastases, usually to the lung (4). Unfortunately, the current treatment for unresectable or metastatic disease, anthracycline-based chemotherapy, provides only limited benefit. Newer strategies, including anti-platelet-derived growth factor alpha (PDGFRα) therapy combined with doxorubicin and immune checkpoint inhibitors, are not much better; phase II clinical trials indicate response rates below 30% (5–8).

Developing targeted therapies for MFS/UPS has been challenging because these tumors characteristically harbor vast numbers of copy number alterations and do not contain a defining fusion product or gene mutation. However, a recent analysis by The Cancer Genome Atlas (TCGA) found that the most common copy number alterations in MFS/UPS involve RB1 and TP53, which encode the Rb and p53 tumor suppressors, respectively (3). Furthermore, although the overall mutational burden in MFS/UPS is low relative to other cancers, TP53 and RB1 are also two of the three most commonly mutated genes in MFS/UPS (3).

The high percentage of MFS/UPS tumors with loss of function of Rb and p53 makes targeting the associated pathways an attractive possibility. One potential strategy arises from prior observations in mouse models that tumors deficient in either Rb or both Rb and p53 require the Skp2 oncogene for survival (9, 10). Skp2 promotes cell-cycle progression by facilitating the degradation of the cell-cycle checkpoint inhibitors p21 and p27. Skp2 forms a cullin–RING E3 ubiquitin ligase complex along with cullin-1 and Skp1 to promote p21 and p27 degradation in the S phase (11–13). In an Rb+/− loss-of-heterozygosity mouse model of retinoblastoma, the prototypical Rb-deficient cancer, Skp2 germline deletion was protective against tumor formation (9). Targeting Skp2 also suppresses tumorigenesis in p53/PTEN-deficient cells by inducing senescence via Atf4, p27, and p21 (14). Skp2 deletion also blocks prostate cancer and pituitary tumorigenesis in Rb/p53 doubly deficient mouse models (10). The underlying mechanism involves accumulation of p27, which is normally degraded by Skp2 and two other p27 ubiquitin ligases that are also p53 target genes; in the absence of Rb and p53, p27 accumulation blocks cell division. Finally, there is also evidence that Skp2 plays an oncogenic role in MFS. Skp2 inhibition decreased transwell invasion of MFS cells in vitro, and pharmacologic inhibition via the proteasome inhibitor bortezomib inhibited MFS xenograft growth in vivo (15). The Rb and/or p53 deficiency of the cell lines used in this study was not examined.

We hypothesize that MFS/UPS cells that are deficient in both Rb and p53 require Skp2 for survival, and thus may be responsive to pharmacologic agents targeting the Skp2 complex. To test our hypothesis, we assessed Rb, p53, and Skp2 expression in a panel of MFS/UPS patient tissue samples, examined the effects of SKP2 knockdown on proliferation and cell-cycle regulation in MFS/UPS patient-derived cell lines, and tested the in vitro and in vivo efficacy of pharmacologic Skp2 inhibition.

Patient samples

We screened samples collected under the Institutional Review Board (IRB)-approved (protocol no. 02-060) at Memorial Sloan Kettering Cancer Center (MSKCC) from February 2002 to March 2011. All patients enrolled on this protocol provided written informed consent to the research use of their samples. All studies employing patient samples were conducted in accordance with the Declaration of Helsinki. Inclusion criteria included a diagnosis of UPS or high-grade MFS, no prior treatment with chemotherapy or radiation, and documentation of consent for tissue banking. We included only high-grade MFS, similar to prior studies (16), to ensure greater group uniformity (all UPS is high-grade by definition), and because the focus of our study is on patients with the highest risk of developing systemic disease (4). Each diagnosis of MFS or UPS was reviewed and confirmed by a sarcoma pathologist (N. Agaram). All patients with available banked RNA-quality tissue were included in the study (n = 64 MFS, 30 UPS). Clinical data for these patients, including date of diagnosis, date of surgery, time to local and/or distant recurrence, and survival, were obtained from a prospectively maintained database.

Copy number and mutation analysis

Copy number variations were analyzed using array comparative genomic hybridization (CGH). DNA was prepared and analyzed on Agilent 244K or 1M arrays as described previously (17). For each gene of interest, copy number change was categorized as −2, −1, 0, 1, or 2 (deep deletion, shallow deletion, no change, gain, or amplification, respectively).

For mutational analysis, DNA was extracted from frozen banked tumor tissue and matched frozen whole blood or normal fat or muscle using the DNeasy Blood and Tissue Kits (Qiagen). For tumors with DNA of adequate quantity and quality (n = 80), we performed next-generation sequencing (NGS) targeted to RB1 and TP53 using the Illumina HiSeq Exome Variant Detection Pipeline at the MSKCC Integrated Genomics Operations core facility. The full pipeline is available at https://github.com/soccin/BIC-variants_pipeline and the post-processing code is at https://github.com/soccin/Variant-PostProcess.

The seven MFS and UPS cell lines were also analyzed using MSK-IMPACT (Memorial Sloan Kettering-Integrated Mutation Profiling of Actionable Cancer Targets), a next-generation targeted sequencing assay that identifies mutations, copy number alterations, and structural rearrangements in a panel of 468 genes (18, 19). Array CGH data are available from the GEO database (accession no. GSE142573) and sequencing data from dbGaP (accession no. phs001982.v1.p1).

Tissue microarray analysis and IHC

Two tissue microarrays (TMA) were constructed using an automated arrayer (ATA-27; Beecher Instruments, Sun Prairie, WI), to include 61 myxofibrosarcomas and 50 undifferentiated pleomorphic sarcomas. From each sample, triplicate cores were used, each measuring 0.6 mm in diameter. For IHC, antibodies to Rb, p53, Skp2, caspase-3, p21, and p27 were used (Supplementary Table S1). Skp2, caspase-3, Rb, and p53 staining was performed using the Ventana XT platform with a standard streptavidin–biotin immunoperoxidase method and 3,3-diaminobenzidine (DAB) detection system. p21 and p27 staining was performed on the BOND RX automated platform (Leica Biosystems) using a standard protocol with ER2 heat retrieval solution (30 minutes), primary antibody incubation (30 minutes), and a Bond Polymer Refine Detection Kit-DAB (Leica Biosystems, Catalog No. #DS9800). IHC staining was evaluated by a pathologist (N. Agaram) and scored as present or absent.

Statistical analysis

Association between copy number alterations and IHC staining for the same genes were assessed using Fisher exact tests and summarized as odds, as were associations in IHC staining between genes. Unpaired two-tailed Student t tests were used to compare groups for independent samples.

Cell culture

After obtaining IRB approval, seven primary high-grade MFS and UPS cell lines (8000S, 91002B, 8500, 4746, 9172, 2734, and 3672-3) were derived from fresh human tumor samples. All patient-derived cell lines were authenticated by comparison of array CGH or shallow whole-exome sequencing data with corresponding data from the source tumor tissue to validate that the cell lines were representative of the original tumor. As nontumor comparisons, we used pre-adipocyte stem cells (ASC) derived from liposuction samples (L090310), a generous gift from Dr. Jeffrey M. Gimble, and Simpson Golabi Behmel syndrome (SGBS) cells, a generous gift from Dr. Martin Wabitsch. KEL FIB cells and umbilical cord-derived human mesenchymal stem cells were obtained from the ATCC. Cells were confirmed as mycoplasma-negative by biochemical test (MycoAlert Plus; Lonza) prior to use in assays at the following passages: 8000S, 18 to 24; 4746, 8 to 12; ASCs, 5 to 10; SGBS, 10 to 20; KEL FIB, 2 to 6; MSCs, 5 to 10.

Cells were grown in a 1:1 mixture of high-glucose DMEM and F12 medium with 10% FBS, 2 mmol/L l-glutamine, 100 units/mL penicillin, and 100 μg/mL streptomycin, and were maintained in a 37°C incubator with 5% CO2. Pevonedistat was obtained from Cayman Chemical and C1 from Calbiochem. Both were dissolved in DMSO for in vitro experiments.

siRNA transfection and lentiviral shRNA infection

Human ON-TARGETplus siRNA (Supplementary Table S2) and nontargeting control siRNA (catalog D-001810) were obtained from Dharmacon. Cells were plated at 1.5 × 105 cells/well onto six-well plates, incubated with 2 μL of DharmaFECT (Dharmacon) and 20 nmol/L siRNA in a mixture of 400 μL of Opti-MEM and 1.6 mL of culture media for 24 hours, and replated in standard cell culture media for experiments.

Human pLKO.1 shRNA lentiviral vectors were obtained from Thermo Fisher Scientific (Supplementary Table S3). Virus was generated in HEK293FT cells (from ATCC) as described previously (20). For lentiviral infection, cells were incubated overnight at 37°C in complete medium containing viral particles and 10 μg/mL polybrene. Following a 24-hour incubation with standard cell culture media, infected cells were selected using puromycin at 0.25 to 1 μg/mL in cell culture media as described previously.

Cell proliferation and apoptosis

Cell proliferation was assessed using the CyQUANT Cell Proliferation Assay Kit (Thermo Fisher Scientific) as described previously (21). Briefly, cells were plated at 2 × 103 cells/well in sextuplicate onto 96-well plates, treated or incubated for a specified duration, and then frozen at −80°C. On the day of analysis, cells were thawed at room temperature, then lysed, and stained with a CyQUANT lysis buffer/GR dye mixture. Fluorescence (excitation 480 nm/emission 520 nm) was measured using the SpectraMax M4 plate reader and SoftMax Pro v5.4.1 software (Molecular Devices). BrdUrd incorporation was assessed using BrdU Cell Proliferation Kit (Cell Signaling Technology). Apoptosis was assessed using the Muse Annexin V and Dead Cell Assay Kit (Millipore Sigma) following the manufacturer's protocol. Briefly, cells were plated at 1 × 104 to 2 × 104 cells/well in triplicate onto six-well plates, and treated or incubated for a specified duration. On the day of analysis, cells were collected as single-cell suspensions, stained with Muse reagent, and analyzed using the Muse Cell Analyzer (Millipore Sigma).

Western blotting

Cells were lysed in high-concentration SDS lysis buffer (2.5% SDS, 62.5 mmol/L Tris-HCl pH 6.8, 10% glycerol) and immediately boiled for 3 minutes to denature proteins. Xenograft tissue was lysed in RIPA buffer (Thermo Fisher Scientific) containing protease and phosphatase inhibitors. Protein concentration was quantified using the DC Protein Assay (Bio-Rad Laboratories). Cell lysates were run on 4% to 12% gradient Bis-Tris gels (Thermo Fisher Scientific), then transferred to PVDF (Immobilon, EMD Millipore). Membranes were blocked with Starting Block T20 (Thermo Fisher Scientific) and incubated with primary antibodies diluted in Starting Block T20, followed by secondary antibodies diluted in Tris-buffered saline with Tween 20 (TBST) with 5% milk. Commercial antibodies and dilutions are listed in Supplementary Table S1.

Real-time qPCR

RNA was extracted from cells using the RNeasy Kit (Qiagen). cDNA was prepared as described previously (20). Briefly, the SuperScript III First-Strand Synthesis System (Thermo Fisher Scientific) was used to synthesize cDNA from 400 ng of RNA according to manufacturer instructions. Next, 0.5 μL of cDNA reaction was mixed with the corresponding TaqMan Gene Expression Assay primers (Supplementary Table S4) and TaqMan Universal PCR Master Mix (Thermo Fisher Scientific), then amplified using the Viia 7 Real-Time PCR System (Thermo Fisher Scientific). Relative expression was quantified using the comparative CT method (22), with 18S as the internal reference gene.

Animal experiments

All animal experiments were performed with approval of the MSKCC Institutional Animal Care and Usage Committee (IACUC protocol No. #02-09-024) and the assistance of the MSKCC Antitumor Assessment (ATA) core facility. For xenograft drug treatment experiments, 8000S cells (2.5 × 106 cells) and 4746 cells (6 × 106 cells) were mixed in a 1:1 PBS and Matrigel mixture (LEDV-free; Corning) mixture and injected subcutaneously into NSG mice (female, age 6–8 weeks). Drug treatment began once tumors reached approximately 100 mm3 by caliper measurement. Pevonedistat (Active Biochem) was dissolved in distilled deionized water containing 5% DMSO, 30% PEG300, and 5% Tween-80 and administered at 50 mg/kg twice a day subcutaneously.

Loss of RB1 and TP53 is common in MFS and UPS

To confirm the relevance of Skp2 as a potential therapeutic target to MFS/UPS, we first examined the prevalence of RB1 and TP53 deletions and mutations in a panel of 94 untreated primary tumor tissue samples, of which, 64 were high-grade MFS and 30 were UPS. The median tumor size was 8.6 cm. At a median follow-up of 36 months, both for all patients and for the 58 surviving patients, 18 patients (19%) developed a local recurrence, 38 (40%) developed distant metastases, and 26 (28%) died of disease. Forty-two patients (45%) did not develop either local or distant recurrence and had no evidence of disease at last follow-up. Using CGH arrays, we identified shallow or deep deletions of RB1 in 64 samples (68%), and shallow or deep deletions of TP53 in 47 samples (50%). Of the 80 samples with sufficient quality and quantity of paired normal and tumor DNA for NGS, loss-of-function mutations were identified in RB1 in 9 samples (11%) and in the TP53 gene in 27 samples (33%; Fig. 1A). The majority of samples (56; 60%) carried alterations in both genes (Fig. 1A).

Figure 1.

Loss of RB1 and TP53 is common in MFS and UPS. A, Copy number alterations and mutations were assessed using array CGH and NGS, respectively, for 94 untreated primary MFS and UPS tumor samples. Each vertical rectangle indicates an individual sample. B, A TMA including 88 of 94 samples was immunostained for Rb, p53, and Skp2. Fisher exact test was used to calculate the P value for association between Rb and p53 double negativity and Skp2 positivity. C, Representative slides showing negative and positive staining for Rb and Skp2 on the TMA. Scale bars, 1 mm.

Figure 1.

Loss of RB1 and TP53 is common in MFS and UPS. A, Copy number alterations and mutations were assessed using array CGH and NGS, respectively, for 94 untreated primary MFS and UPS tumor samples. Each vertical rectangle indicates an individual sample. B, A TMA including 88 of 94 samples was immunostained for Rb, p53, and Skp2. Fisher exact test was used to calculate the P value for association between Rb and p53 double negativity and Skp2 positivity. C, Representative slides showing negative and positive staining for Rb and Skp2 on the TMA. Scale bars, 1 mm.

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We compared results from our cohort to MFS/UPS samples in three other datasets: the MSK-IMPACT dataset from MSKCC (19), the GENIE dataset from the Dana Farber Cancer Institute (DFCI; ref. 23), and the sarcoma TCGA (which shares 20 patients with our cohort; ref. 3), and found similarly high rates of RB1 and TP53 copy number alterations and/or mutations in all datasets (Supplementary Figs. S1A and S1B). MSK-IMPACT identifies only deep, not shallow, copy number deletions, likely contributing to the lower rate of called copy number alterations for both genes.

For both Rb and p53, DNA copy number correlated with IHC staining among the 88 samples for which paraffin-embedded tissue was available (Supplementary Figs. S2A and S2B), confirming that DNA and protein measurements were reliable across platforms. We next examined whether Rb and p53 expression negatively correlate with that of Skp2 at the protein level. On the TMA, negative staining for both Rb and p53 was significantly associated with positive staining for Skp2 (Fig. 1B), with 42 samples (48%) staining negative for Rb, negative for p53, and positive for Skp2 (P = 0.018, Fisher exact test). Representative images from the TMA are shown in Fig. 1C.

Rb and p53 are lost or mutated, and Skp2 expression is high in patient-derived MFS/UPS cell lines

Towards examining the function of Skp2 in Rb/p53-deficient MFS/UPS patient-derived cell lines, we first characterized seven cell lines (five MFS and two UPS) in terms of copy number variations, mRNA expression, and protein levels of Rb, p53, and Skp2. Five cell lines had shallow or deep deletions in RB1, three had shallow or deep deletions in TP53, and four had gain or amplification of SKP2 by both array CGH and MSK-IMPACT. One MFS cell line (9172) had both a missense mutation and a shallow copy deletion in RB1. Of the four cell lines without TP53 deletions, three had truncating mutations detected on MSK-IMPACT. The remaining cell line, 8500, had a partial deep deletion of TP53 detected on array CGH but not MSK-IMPACT (Fig. 2A). Compared with nontumor mesenchymal cell lines, most MFS and UPS cell lines expressed lower levels of RB1 and TP53 mRNA and higher levels of SKP2 mRNA (Fig. 2B). RB1 mRNA expression was low to undetectable in four cell lines, whereas 9172, 2734, and 3672-3 retained RB1 mRNA. All cell lines expressed low or undetectable levels of TP53 mRNA, and all cell lines except 2734 expressed higher levels of SKP2 mRNA relative to the nontumor mesenchymal cell lines. Protein expression of Rb, Skp2, and p53 showed similar patterns (Fig. 2C). None of the cell lines expressed p53 protein, but 2734 and 3672-3 had detectable RB1 mRNA and Rb protein. Thus, these two cell lines provided an Rb-wild type (Rb-WT)/p53-deficient comparison for the in vitro phenotypes of Rb- and p53-deficient MFS/UPS.

Figure 2.

Rb and p53 are lost and Skp2 expression is high in patient-derived MFS/UPS cell lines. A, Copy number alterations as determined by array CGH (aCGH) and MSK-IMPACT. RB1 and TP53 mutations called by MSK-IMPACT are also shown on the right. B, mRNA expression levels as measured by qPCR with the comparative CT method in four nontumor mesenchymal cell lines [L090310 (normal ASCs), SGBS (pre-adipocytes), KEL FIB (keloid fibroblasts), and MSC (mesenchymal stem cells)] and seven MFS and UPS cell lines. 18S was used as the internal reference gene, and L090310 was used as the reference control sample. Error bars, SD. C, Western blot analysis for Skp2, Rb, and p53 in protein lysates of the same cell lines as above. Histone H3 was used as a loading control. Throughout the figure, MFS is indicated in purple and UPS in green.

Figure 2.

Rb and p53 are lost and Skp2 expression is high in patient-derived MFS/UPS cell lines. A, Copy number alterations as determined by array CGH (aCGH) and MSK-IMPACT. RB1 and TP53 mutations called by MSK-IMPACT are also shown on the right. B, mRNA expression levels as measured by qPCR with the comparative CT method in four nontumor mesenchymal cell lines [L090310 (normal ASCs), SGBS (pre-adipocytes), KEL FIB (keloid fibroblasts), and MSC (mesenchymal stem cells)] and seven MFS and UPS cell lines. 18S was used as the internal reference gene, and L090310 was used as the reference control sample. Error bars, SD. C, Western blot analysis for Skp2, Rb, and p53 in protein lysates of the same cell lines as above. Histone H3 was used as a loading control. Throughout the figure, MFS is indicated in purple and UPS in green.

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Skp2 knockdown decreases proliferation and increases apoptosis in Rb/p53-deficient MFS and UPS

To test the hypothesis that Rb/p53-deficient cells require Skp2 for survival, we used two siRNAs to knock down Skp2 in both Rb/p53-deficient and Rb-WT/p53-deficient cells. Skp2 knockdown in the Rb/p53-deficient 9100 2B and 8000S cell lines significantly decreased proliferation (by ∼35% and ∼50%, respectively; Fig. 3A) and increased apoptosis (Fig. 3B and C). By comparison, Skp2 knockdown in the Rb-WT/p53-deficient 2734 and 3672-3 had much smaller effects on proliferation (Fig. 3A) and apoptosis (Fig. 3B and C). As expected, Skp2 knockdown increased protein levels of both of its degradation targets, p21 and p27, in Rb/p53-deficient cells (Fig. 3C). Interestingly, Skp2 knockdown increased levels of p27, but not p21, in Rb-WT/p53-deficient cells. Skp2 knockdown also slightly decreased proliferation and increased apoptosis in normal ASCs (L090310) (Supplementary Figs. S3A–S3C) but did not appreciably increase protein levels of either p21 or p27 (Supplementary Fig. S3C). We also confirmed our findings using knockdown via lentiviral shRNA. Three anti-Skp2 shRNAs decreased proliferation and increased apoptosis to a greater degree in Rb/p53-deficient 8000S cells than in the Rb-WT/p53-deficient 2734 cells or normal ASCs (Supplementary Figs. S3D–S3F).

Figure 3.

Skp2 knockdown increases p21 and p27 expression, decreases proliferation, and induces apoptosis of Rb/p53-deficient MFS/UPS cell lines. A, Proliferation of two Rb/p53-deficient and two Rb-WT/p53-deficient cell lines in which Skp2 was knocked down using two different siRNAs, assessed by CyQUANT assay. B, Apoptosis as measured by Annexin V staining on posttransfection day 7. C, Western blot analysis for cleaved caspase-3, Skp2, p21, and p27 in two Rb/p53-deficient and two Rb-WT/p53-deficient cell lines on posttransfection day 7. Vinculin was used as a loading control. Error bars, SD. *, P < 0.05; **, P < 0.01; ***, P < 0.001; NS, not significant, P ≥ 0.05.

Figure 3.

Skp2 knockdown increases p21 and p27 expression, decreases proliferation, and induces apoptosis of Rb/p53-deficient MFS/UPS cell lines. A, Proliferation of two Rb/p53-deficient and two Rb-WT/p53-deficient cell lines in which Skp2 was knocked down using two different siRNAs, assessed by CyQUANT assay. B, Apoptosis as measured by Annexin V staining on posttransfection day 7. C, Western blot analysis for cleaved caspase-3, Skp2, p21, and p27 in two Rb/p53-deficient and two Rb-WT/p53-deficient cell lines on posttransfection day 7. Vinculin was used as a loading control. Error bars, SD. *, P < 0.05; **, P < 0.01; ***, P < 0.001; NS, not significant, P ≥ 0.05.

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Silencing of Skp2 using siRNA also induced cell-cycle arrest, as indicated by a significant decrease in BrdUrd incorporation at day 3 and phosphorylation of histone H3 compared with control (scramble or nontransfected) cells at days 3 and 5 (Supplementary Figs. S4A and S4B). In addition, consistent with a previous report demonstrating the requirement for Skp2 to activate Akt and enhance glycolysis (24), we also found that Skp2 knockdown led to reduced phospho-Akt at day 5 (Supplementary Fig. S4B). These results show that Skp2 is required for proliferation and survival in Rb/p53-deficient MFS/UPS cells, but is dispensable for both Rb-WT/p53-deficient MFS/UPS cells and normal ASCs.

p21 and p27 inhibit proliferation and induce apoptosis in the absence of Skp2

To assess the importance of p27 and p21 in mediating the effects of Skp2 knockdown on Rb/p53-deficient cells, we knocked down each protein in combination with Skp2 using siRNA in 8000S cells. Concomitant knockdown of p27 and/or p21 with Skp2 increased proliferation (Fig. 4A) compared with Skp2 knockdown alone and rescued cells from apoptosis (Fig. 4B and C). Interestingly, knockdown of either p21 or both p21 and p27 resulted in a greater restoration of proliferation and reduction of apoptosis compared with knockdown of p27 alone (Fig. 4AC). Although this may be a result of the knockdown potency of the p21 siRNA being much higher than that of the p27 siRNA, these results suggest that the reduction of p21 and p27 mediate Skp2's promotion of proliferation in Rb/p53-deficient MFS/UPS, and that p21 may be the major checkpoint inhibitor to overcome in Rb/p53-deficient MFS/UPS tumorigenesis.

Figure 4.

Knockdown of p21 and/or p27 restores proliferation and prevents apoptosis in Rb/p53-deficient 8000S cells after Skp2 knockdown. A, Proliferation following knockdown of Skp2, p21, and/or p27, as measured by CyQUANT assay. siSCR, scramble control. All three panels are derived from the same experiment. B, Apoptosis measured using Annexin V staining on posttransfection day 9. C, Western blot analysis for Skp2, p27, p21, and cleaved caspase-3 on posttransfection day 7. Vinculin was used as a loading control. Error bars, SD. **, P < 0.01; ***, P < 0.001.

Figure 4.

Knockdown of p21 and/or p27 restores proliferation and prevents apoptosis in Rb/p53-deficient 8000S cells after Skp2 knockdown. A, Proliferation following knockdown of Skp2, p21, and/or p27, as measured by CyQUANT assay. siSCR, scramble control. All three panels are derived from the same experiment. B, Apoptosis measured using Annexin V staining on posttransfection day 9. C, Western blot analysis for Skp2, p27, p21, and cleaved caspase-3 on posttransfection day 7. Vinculin was used as a loading control. Error bars, SD. **, P < 0.01; ***, P < 0.001.

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Pevonedistat, an inhibitor of Skp2 complex formation, reduces proliferation and increases apoptosis in Rb/p53-deficient MFS/UPS

Given the antiproliferative and proapoptotic effects of Skp2 knockdown in Rb/p53-deficient MFS/UPS cell lines, we next investigated whether pharmacologic inhibition of the Skp2 interaction with cullin1 could promote the self-degradation of Skp2 and produce similar effects. Pevonedistat (MLN4924), a neddylation-activating enzyme (NAE) inhibitor, which blocks formation of the Skp2 complex by inhibiting the addition of NEDD8 to cullin-1 (21), was used to promote the self-degradation of Skp2. Pevonedistat is currently in phase I clinical trials in both hematologic and advanced solid malignancies, and has an acceptable toxicity profile in patients with cancer (21, 22, 25).

Similar to Skp2 knockdown, pevonedistat treatment decreased proliferation in a dose-dependent manner in 4 Rb-deficient MFS/UPS cell lines, with IC50s ranging from 66 to 197 nmol/L (Fig. 5A). Normal ASCs (L090310) were less sensitive to pevonedistat than the Rb/p53-deficient MFS/UPS lines, with an IC50 of 458 nmol/L. Pevonedistat also increased apoptosis in a dose-dependent manner and increased p21 and p27 protein levels in the Rb/p53-deficient 8000S and 91002B cell lines (Fig. 5B and C). Similar to Skp2 knockdown, pevonedistat treatment decreased BrdUrd incorporation at 6 hours, consistent with induction of cell-cycle arrest (Supplementary Fig. S4C). To further confirm that inhibition of the Skp2 interaction with p27 and p21 is toxic to Rb/p53-deficient MFS/UPS cells, we investigated the effects of the compound C1, which occupies the substrate-binding pocket in the Skp2–Cks1 complex, preventing the interaction of Skp2 with p27 (26). Similar to pevonedistat and genetic silencing of Skp2, C1 inhibited proliferation, induced apoptosis, and increased expression of p21 and p27 in 8000S cells (Supplementary Figs. S5A–S5C).

Figure 5.

Pevonedistat decreases proliferation and induces apoptosis in Rb/p53-deficient MFS/UPS. A, Proliferation of four Rb/p53-deficient MFS/UPS cell lines and normal ASCs (L090310) after treatment with varying doses of pevonedistat, as measured by CyQUANT assay on day 4. B, Apoptosis as measured by Annexin V staining in Rb/p53-deficient 8000S and 9100 2B cells treated for 3 days with pevonedistat at 0, 100, or 333 nmol/L. C, Western blot analyses for Nedd8-cullin1, Skp2, p27, p21, and cleaved caspase-3 in cells treated with pevonedistat at 0, 100, or 333 nmol/L for 6 or 72 hours. Error bars, SD. ***, P < 0.001.

Figure 5.

Pevonedistat decreases proliferation and induces apoptosis in Rb/p53-deficient MFS/UPS. A, Proliferation of four Rb/p53-deficient MFS/UPS cell lines and normal ASCs (L090310) after treatment with varying doses of pevonedistat, as measured by CyQUANT assay on day 4. B, Apoptosis as measured by Annexin V staining in Rb/p53-deficient 8000S and 9100 2B cells treated for 3 days with pevonedistat at 0, 100, or 333 nmol/L. C, Western blot analyses for Nedd8-cullin1, Skp2, p27, p21, and cleaved caspase-3 in cells treated with pevonedistat at 0, 100, or 333 nmol/L for 6 or 72 hours. Error bars, SD. ***, P < 0.001.

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Pevonedistat inhibits tumor growth and induces apoptosis in Rb/p53-deficient MFS and UPS xenografts

Given the promising antitumor effects of Skp2 inhibition in vitro, we tested the clinical grade neddylation inhibitor pevonedistat in vivo using subcutaneous xenograft models of Rb/p53-deficient MFS (8000s) and UPS (4746). We injected 2.5 × 106 8000S cells or 6 × 106 4746 cells into the flanks of NSG mice, and treated mice with either vehicle control or pevonedistat 50 mg/kg twice daily subcutaneously once tumors reached approximately 100 mm3 in size by caliper measurements. In both xenograft models, pevonedistat treatment significantly inhibited tumor growth relative to vehicle control; tumor size remained relatively stable over the treatment period in mice treated with pevonedistat, compared with the growth in mice treated with vehicle control (Fig. 6A). Western blots of xenograft lysates harvested from mice treated with pevonedistat revealed less cullin-1 neddylation and higher p21 and p27 protein levels compared with xenograft lysates harvested from control mice (Fig. 6B). Greater p21 and p27 expression after pevonedistat treatment was also evident by IHC staining; p21 appeared exclusively on tumor cells, whereas p27 was mostly expressed on the stromal component and a minor population of positive tumor cells (Fig. 6C; Supplementary Fig. S6A). The inhibitor also increased levels of cleaved caspase-3 compared with vehicle-treated xenografts, as seen on both Western blot analysis and IHC staining (Fig. 6B and C; Supplementary Fig. S6A). Pevonedistat caused no weight loss in the first 14 days of treatment, although it led to a statistically significant but small amount of weight loss at later time points in mice bearing 8000S xenografts (11%, Supplementary Fig. S6B); no other significant toxicities were observed.

Figure 6.

Pevonedistat inhibits growth and increases p21, p27, and apoptosis in Rb/p53-deficient MFS and UPS xenografts. The 8000S (MFS) or 4746 (UPS) cells were injected subcutaneously into single flanks of NSG mice (n = 10). When tumors reached 100 mm3 in size by caliper measurement, mice were treated with either vehicle control (n = 5) or pevonedistat 50 mg/kg (n = 5; twice daily subcutaneous injection). A, Growth of 8000s (left) and 4746 (right) tumors were measured using calipers. B, Western blot analyses for Nedd8-cullin1, Skp2, p27, p21, and cleaved caspase-3 in 8000s (left) and 4746 (right) tumors. Vinculin served as a loading control. C, 8000s xenograft tumor tissue sections stained with hematoxylin and eosin (H&E) or antibodies for p27, p21, or cleaved caspase-3. Scale bars in 40× images, 50 μm; in 10×, 200 μm. Error bars, SD. ***, P < 0.001.

Figure 6.

Pevonedistat inhibits growth and increases p21, p27, and apoptosis in Rb/p53-deficient MFS and UPS xenografts. The 8000S (MFS) or 4746 (UPS) cells were injected subcutaneously into single flanks of NSG mice (n = 10). When tumors reached 100 mm3 in size by caliper measurement, mice were treated with either vehicle control (n = 5) or pevonedistat 50 mg/kg (n = 5; twice daily subcutaneous injection). A, Growth of 8000s (left) and 4746 (right) tumors were measured using calipers. B, Western blot analyses for Nedd8-cullin1, Skp2, p27, p21, and cleaved caspase-3 in 8000s (left) and 4746 (right) tumors. Vinculin served as a loading control. C, 8000s xenograft tumor tissue sections stained with hematoxylin and eosin (H&E) or antibodies for p27, p21, or cleaved caspase-3. Scale bars in 40× images, 50 μm; in 10×, 200 μm. Error bars, SD. ***, P < 0.001.

Close modal

Motivated by the need for targeted therapies for MFS and UPS, we examined their dependence on Skp2, recently shown to be required for the transformation and survival of RB1- and TP53-deletion-derived tumors in mice (9, 10, 14). Although MFS and UPS are genetically complex and lack a defining chromosomal alteration or gene mutation, we found that loss of Rb and p53 due to chromosomal deletions or loss-of-function mutations in the RB1 and TP53 genes are common in MFS and UPS. Together, our results show that loss of Rb and p53 renders MFS/UPS cells reliant on Skp2, representing, to our knowledge, the first demonstration of this conditional dependence in Rb and p53-deficient human cancer. Our in vitro experiments suggest that Skp2 plays multiple important roles in promoting proliferation, driving cell-cycle progression, and inhibiting apoptosis in Rb/p53-deficient MFS/UPS cells. Importantly, patient-derived xenograft experiments show that growth of Rb/p53-deficient MFS/UPS tumors is prevented by the neddylation inhibitor pevonedistat, which indirectly interferes with Skp2 function, suggesting the translational relevance of these findings. Further, our TMA data correlating lack of Rb and p53 expression with Skp2 expression provide a basis for identifying patients with Skp2-dependent tumors.

We found that siRNA knockdown of p21 and/or p27 in the absence of Skp2 restored proliferation and reduced apoptosis in Rb/p53-deficient MFS/UPS cells. The role of p27 in Skp2-mediated oncogenesis has been well-described in many other cancers (10, 25, 27–32). There is also evidence for a role of p21 in Skp2's tumorigenic effects and in Rb-deficient tumorigenesis. p21 is a well-described target of Skp2 (11), and inactivation of the p21 gene accelerates tumor development in Rb+/− mice (33). Interestingly, knockdown of p21 and/or p27 in MFS/UPS cells with functional Skp2 did not seem to further increase proliferation, which suggests that in these tumors Skp2 is already maximally suppressing p21 and p27 function.

In addition, we found that Skp2 knockdown produced much more dramatic increases in p21 in Rb/p53-deficient MFS/UPS cells compared with Rb-WT/p53-deficient cells. These suggest that Rb-WT cells are somehow more readily able to suppress p21 upregulation in the absence of Skp2, and may explain why they are less sensitive to Skp2 knockdown. The underlying mechanism is unclear, but a previous study showed that knockdown of Rb in mouse embryonic fibroblasts increased p21 expression, suggesting that Rb may negatively regulate p21 expression in certain contexts (34). In addition, other studies have shown that E2F1, a transcription factor inhibited by Rb, promotes the transcription of CDKN1A, the gene encoding p21 (35, 36). Of note, although p21 is classically considered to be downstream of the p53 pathway (37), p21 regulation in both Rb-negative and Rb-WT MFS/UPS cell lines must be p53-independent, because all of our cell lines lacked p53 expression.

We also found that the neddylation inhibitor pevonedistat was effective against Rb-deficient MFS/UPS, both in vitro and in vivo. Because pevonedistat prevents formation of all cullin-RING ligases, some of its effects may be independent of the Skp2-p21/27 axis. Other well-described targets of pevonedistat include Cdt1 (38), which promotes DNA replication by helping to assemble the replicative complex, and is then degraded during either the G1 phase or late mitosis by cullin-RING ligases to prevent DNA re-replication (39). Pevonedistat inhibits Cdt1 degradation to promote DNA re-replication, leading eventually to either apoptosis or senescence (38). Interestingly, Cdt1 is partially regulated by Skp2-containing cullin-RING ligases (38–40), so it is possible that its degradation also plays a role, alongside p21 and p27, in Skp2-mediated oncogenesis. Despite its broad range of molecular targets, pevonedistat has been well tolerated in phase I clinical trials (27, 28, 41).

In addition to pevonedistat, other pharmacologic strategies could be used to block Skp2 activity. Several groups have discovered compounds with more Skp2-specific activity, including inhibitors of Skp2-mediated p27 degradation (26) and another that prevents Skp2–Skp1 interactions (42). The series of compounds, including C1, that were specifically designed to inhibit the Skp2-p27 interaction were found to induce both p27 and p21 expression in metastatic melanoma cell lines without changing the protein levels of Skp2 or Cullin1. We also found that in MFS cells, the C1 compound, a direct inhibitor of Skp2-mediated p27 ubiquitylation, inhibited proliferation and induced apoptosis by inducing p27 and p21. These results suggest that the strategy of targeting the Skp2-p27 interaction more specifically might lead to effective therapies that could be safely administered at higher doses with less off-target toxicity, and warrants further study. However, none of these more Skp2-specific compounds has been tested in humans, and we were unable to deliver adequate concentrations of C1 to achieve therapeutic efficacy in our Rb/p53-deficient MFS xenograft mouse model.

Our results provide evidence that pevonedistat is effective in the preclinical setting against Rb/p53-deficient MFS and UPS. In addition, MSK-IMPACT could be used to identify MFS/UPS with RB1 and TP53 mutations or deletions that are likely to respond to anti-Skp2 therapy. Taken together, we would advocate testing pevonedistat in a phase I/II trial in patients with metastatic Rb- and p53-deficient MFS or UPS.

No potential conflicts of interest were disclosed.

Conception and design: G.Z. Li, T. Okada, Y.-M. Kim, L.-X. Qin, S. Singer

Development of methodology: G.Z. Li, T. Okada, Y. Shen, A.S. Martin, L.-X. Qin

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): G.Z. Li, T. Okada, N.P. Agaram, J. Rios, S. Singer

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): G.Z. Li, T. Okada, Y.-M. Kim, N.P. Agaram, F. Sanchez-Vega, Y. Shen, N. Tsubokawa, A.S. Martin, M.A. Dickson, L.-X. Qin, N.D. Socci, S. Singer

Writing, review, and/or revision of the manuscript: G.Z. Li, T. Okada, Y.-M. Kim, N.P. Agaram, F. Sanchez-Vega, M.A. Dickson, S. Singer

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): G.Z. Li, T. Okada, Y. Shen, M.A. Dickson, N.D. Socci, S. Singer

Study supervision: G.Z. Li, T. Okada, M.A. Dickson, S. Singer

This research was supported by the American College of Surgeons Resident Research Scholarship (to G.Z. Li), NCI SPORE in Soft Tissue Sarcoma P50 CA140146 (to S. Singer), Siskind Family Sarcoma Fund (to S. Singer), MFH Research Fund (to S. Singer), and NCI Cancer Center Support Grant P30 CA008748 (institutional). The authors would like to thank Jessica Moore for her editorial assistance with both the manuscript and figures. We also thank Marina Asher (Department of Pathology, MSKCC) for assistance with IHC, Elisa De Stanchina, PhD, Huiyong Zhao, MD, Qing Chang, MD, and Besnik Qeriqi (Antitumor Assessment Core, MSKCC) for assistance in the mouse experiments, Richard Cass, MD, for reading the manuscript, and Aihong Liu and the members of the Singer lab for help and discussion. Finally, we thank Xiaoliang Xu, MD, PhD, for his guidance and contributions to the initial formulation of this project.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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