Cross-Species Functional Genomic Screens Identify Novel Therapeutic Targets in Malignant Peripheral Nerve Sheath Tumors

Brittany Turner-Ivey; Amanda M Prechtl; Stephen T Guest; Shannon W Doutt; Jody F Longo; Elizabeth Garrett-Mayer; Steven L Carroll

doi:10.1016/j.ajpath.2025.05.019

. 2025 Jun 19;195(11):2176–2196. doi: 10.1016/j.ajpath.2025.05.019

Cross-Species Functional Genomic Screens Identify Novel Therapeutic Targets in Malignant Peripheral Nerve Sheath Tumors

Brittany Turner-Ivey ^∗, Amanda M Prechtl ^∗, Stephen T Guest ^†, Shannon W Doutt ^∗,^‡, Jody F Longo ^∗, Elizabeth Garrett-Mayer ^§, Steven L Carroll ^∗,^∗

PMCID: PMC12597697 PMID: 40543704

Abstract

As oncogenic pathways are highly conserved in vertebrates, genetically engineered mouse models can potentially be used to identify therapeutic targets relevant to rare human cancers such as malignant peripheral nerve sheath tumors (MPNSTs). To test this, genome-scale shRNA screens designed to identify genes driving proliferation and survival were performed in five MPNST cultures derived from myelin protein zero-glial growth factor beta 3 (P₀-GGFβ3) mice and three human MPNST cell lines. Several hundred gene hits mediating proliferation and survival were identified in human and mouse MPNST cells, many of which have been implicated in proliferation and survival in other cancers and/or mediate the pathogenesis of other cancer types. These hits and their associated signaling pathways extensively overlapped in human and mouse MPNST cells. A drug discovery pathway based on the Drug-Gene Interaction Database was developed to identify hits encoding druggable targets. Five druggable targets were selected for validation, with four of the five agents tested (the DNA polymerase α1 inhibitor clofarabine, the DNA nucleotidylexotransferase inhibitor cordycepin, the BCL6 inhibitor 79-6, and the lysophosphatidic acid receptor 1/3 inhibitor Ki16425) proving effective against human MPNST cells. Clofarabine was especially effective, potently reducing cell numbers at low nanomolar concentrations and inducing a senescent phenotype, possibly via the p53/p21 pathway. These results demonstrate the utility of cross-species functional oncogenomics for the discovery of novel therapeutic targets relevant to human MPNSTs and suggest that clofarabine warrants further evaluation for its therapeutic potential.

Graphical abstract

Malignant peripheral nerve sheath tumors (MPNSTs) are aggressive spindle cell neoplasms that arise from Schwann cells or their progenitors.¹ In addition to being the most common malignancy occurring in association with neurofibromatosis type 1 (NF1), MPNSTs arise at sites of previous radiotherapy and sporadically in the general population. In all three settings, the prognosis for individuals with MPNSTs is poor, with 5-year disease-free survival rates of 34% to 60%.2, 3, 4, 5, 6, 7 This is because the radiotherapeutic and chemotherapeutic regimens currently used to treat these tumors are ineffective, leaving complete surgical resection as the only efficacious means of treating an MPNST. Unfortunately, by the time an MPNST becomes clinically evident, complete surgical resection is often impossible because of extensive local invasion or metastasis.

The development of effective treatments for MPNSTs has been hampered by the limited understanding of their pathogenesis. Individuals with NF1 carry one wild-type and one mutated copy of the NF1 gene, which encodes neurofibromin, a GTPase-activating protein that inhibits Ras signaling. Benign plexiform neurofibromas develop when an inactivating mutation of the remaining functional NF1 allele occurs in a cell in the Schwann cell lineage, resulting in a loss of neurofibromin expression and hyperactivation of multiple Ras proteins and Ras-regulated signaling pathways.⁸^,⁹ Subsequent deletion of the CDKN2A/B locus triggers the progression of plexiform neurofibromas into atypical neurofibromatous neoplasms of uncertain biologic potential.10, 11, 12 Atypical neurofibromatous neoplasms of uncertain biologic potential transform into MPNSTs when mutations occur in additional tumor suppressor genes, such as TP53,13, 14, 15, 16 RB1,¹⁷^,¹⁸ and PTEN,¹⁹^,²⁰ and genes encoding polycomb repressive complex 2 components (SUZ12, EED²¹^,²²). However, these mutations are variably present in MPNSTs, and it is likely that other mutations that promote MPNST pathogenesis remain undiscovered.

Previous attempts to develop chemotherapeutic regimens for MPNSTs have largely focused on signaling pathways known to be dysregulated in these neoplasms. Because dysregulated Ras signaling is common to plexiform neurofibromas, atypical neurofibromatous neoplasms of uncertain biologic potential, and MPNSTs, attempts have been made to treat these tumors via Ras inhibition.²³ Unfortunately, Ras has proven difficult to target therapeutically in MPNSTs.⁸^,⁹ As a result, most subsequent studies have focused on targeting downstream mediators of Ras signaling, such as the Raf/mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase and phosphatase and tensin homolog/phosphatidylinositol 3-kinase/AKT/mammalian target of rapamycin effector pathways in both neurofibromas and MPNSTs. This approach has yielded mixed results; while MEK inhibitors are effective in treating neurofibromas, their effectiveness in MPNSTs has been moderate and variable.²⁴^,²⁵ Sorafenib, which targets Raf and several receptor tyrosine kinases, produced no responses in plexiform neurofibromas²⁶ or MPNSTs.²⁷ The mammalian target of rapamycin complex 1 inhibitor rapamycin, alone and in combination with MEK inhibitors, was promising in vitro,²⁸ but had only brief, temporary effects on MPNSTs in vivo.²⁹^,³⁰

Another major barrier to identifying new therapeutic targets in MPNSTs is the fact that these neoplasms are rare cancers, which makes it difficult to obtain the large number of specimens required to identify the full spectrum of driver gene mutations that occur in MPNSTs. In other rare cancer types, limitations such as these have led some investigators to use genetically engineered mouse (GEM) models of rare cancers for driver gene and therapeutic agent discovery. This approach, known as cross-species comparative oncogenomics, is based on the concept that oncogenic pathways are highly conserved among vertebrates.31, 32, 33, 34, 35, 36 This led us to ask whether functional genomic screens of mouse MPNST cells could identify new therapeutic targets relevant to human MPNSTs. To address this question, human MPNST cells were partnered with MPNST cells from myelin protein zero-glial growth factor beta 3 (P₀-GGFβ3) mice, a GEM model in which overexpression of the Schwann cell mitogen neuregulin-1 induces the formation of plexiform neurofibromas that progress to become MPNSTs at a high frequency.³⁷^,³⁸ The goals of this initial study were to identify genes required for the proliferation and survival of human and mouse MPNST cells, to determine whether human and mouse MPNST cell proliferation and survival is dependent on analogous genes and signaling pathways, to develop a pipeline for identifying genes that encode proteins that are druggable with existing therapeutic agents, and to validate MPNST responsiveness to candidate therapeutic agents identified with this pipeline.

Materials and Methods

Reagents and Antibodies

An anti-p38 (number 9212) antibody was purchased from Cell Signaling Technology (Danvers, MA). Anti–glyceraldehyde-3-phosphate dehydrogenase mouse monoclonal antibody (RDI-TRK5G4-6C5) was obtained from Fitzgerald Industries International (Acton, MA), and anti-p53 rabbit polyclonal antibody (number A300-247A) was purchased from Fortis Life Sciences (Waltham, MA). Volasertib (number S2235), rigosertib (number S1362), and methazolamide (number S4039) were purchased from Selleckchem (Houston, TX). Cordycepin (number C3394), clofarabine (number C7495), and the BCL6 inhibitor 79-6 (number 197445) were purchased from Sigma Aldrich (Burlington, MA).

Cell Culture

The sources of the human cell lines used in this study39, 40, 41 and the establishment of early passage P₀-GGFβ3 MPNST cultures³⁸ have been previously described. The mouse tumors from which the early-passage cultures were derived were graded using previously described criteria.⁴² A390 was World Health Organization grade IV, A426 was World Health Organization grade IV, A494 was World Health Organization grade III, and A496 was World Health Organization grade III; sufficient tissue was not available to assign a grade to B288. None of the mouse MPNSTs had the histologic features of an epitheloid MPNST. The identity of the human cell lines was confirmed by short tandem repeat analyses. Human and mouse MPNST cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 10 μg/mL streptomycin, and 10 IU/mL penicillin. Cells were regularly tested for Mycoplasma using MycoAlert assays.

DECIPHER Library Screening and Analysis

DECIPHER pooled lentiviral shRNA libraries were obtained from Cellecta (Mountain View, CA). The screens described here used all available DECIPHER modules, which includes Human Module 1 (Signaling Pathway Targets; queries 5043 mRNAs), Human Module 2 (Disease-Associated Targets; queries 5412 mRNAs), Human Module 3 (Cell Surface, Extracellular and DNA Binding Targets; queries 4922 mRNAs), Mouse Module 1 (Signaling Pathway Targets; queries 4625 mRNAs), and Mouse Module 2 (Disease-Associated Targets; queries 4520 mRNAs); a mouse equivalent of Human Module 3 has not been developed. Collectively, the human modules target 15,377 genes and the mouse modules target 9145 genes. Each module includes a minimum of five to six shRNAs targeting each mRNA. Each module also contains several internal controls, including shRNAs targeting PSMA1 (proteasome 20S subunit α1), RPL30 (ribosomal protein L30), and luciferase. Each of these control shRNAs is replicated with five different barcodes and, in a properly performing experiment, replicates with the different barcodes will all produce the same phenotype. Library screens were performed and analyzed as previously described.⁴³

Celigo Proliferation Assay

An imaging cytometer (Nexcelom Bioscience, Lawrence, MA) was used to perform proliferation assays. Cells were plated in 96-well plates at 2000 cells/well in 100 μL of Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 10 μg/mL streptomycin, and 10 IU/mL penicillin and allowed to adhere overnight. The following day, 100 μL of Hoescht 33342 was added to each well and incubated for 30 minutes at 37°C. Wells were then imaged using programmed autofocusing to gather day 1 baselines; direct cell counting (blue; excitation: 377/50 nm; emission: 470/22 nm) was used to assess the number of stained nuclei, and bright-field imaging was used to assess cell morphology. Plates were then imaged at days 3 and 5. Proliferation curves were calculated using GraphPad Prism version 9.5.0 (GraphPad Software, Boston, MA).

Immunoblots

Immunoblots were performed as previously described.³⁹

Senescence-Associated β-Galactosidase Staining

MPNST cells were plated at 50,000 cells/well in 6-well plates. A 3-day 150 nmol/L clofarabine treatment started the following day. Cells were fixed with 4% paraformaldehyde at room temperature for 5 minutes and then incubated overnight at 37°C without CO₂ in 1 mL of β-galactosidase staining solution [1 mg/mL 5-bromo-4-chloro-3-indolyl β-D-galactopyranoside in 40 mmol/L citric acid/40 mmol/L sodium phosphate (pH 6.0)/5 mmol/L potassium ferrocyanide/5 mmol/L potassium ferricyanide/150 mmol/L NaCl₂/2 mmol/L MgCl₂]. The stain was then removed, and cells were overlain with 70% glycerol. Bright-field images were collected on a Revolve microscope (ECHO, San Diego, CA).

Gene Set Enrichment Analysis

Gene set enrichment analysis (GSEA) was performed using the FunRich version 3.1.4 bioinformatic platform (https://www.funrich.org, last accessed March 31, 2025). Input genes used for GSEA were the genes from each human cell line and mouse early-passage culture with gene-level depletion scores that were in the top 90^th quantile or Bushman Laboratory Genes that were in the top 90^th quantile. To identify biological processes and gene-centered signaling pathways that included genes with gene-level depletion scores consistently in the top 90th quantile, integrated functional analyses were performed using the Enrich-KG version 1.0 bioinformatic platform (https://maayanlab.cloud/enrichr-kg, last accessed March 31, 2025). Input genes used for integrated functional analyses were the gene hits shared by at least two of three human cell lines and at least three of five mouse early passage cultures. An integrated functional analysis was also run on four genes identified as therapeutic targets (DNTT, PLK1, BCL6, and POLA1) to identify the disease gene network and gene ontology represented by these essential genes.

Results

Genome-Scale shRNA Screens Identify Genes Required for the Proliferation and Viability of Human and Mouse MPNST Cells

To identify genes required for the proliferation and/or viability of MPNST cells, genome-scale shRNA dropout screens were performed on human- and P₀-GGFβ3 (GEM)–derived MPNST cells. Two human NF1-associated MPNST cell lines (S462, T265-2c), a sporadic human MPNST cell line (2XSB⁴¹), and five early-passage P₀-GGFβ3 mouse MPNST cultures (A390, A426, A494, A496, and B288) were transduced with DECIPHER pooled lentiviral shRNA libraries. Portions of each transduced culture were harvested after 3 days of selection (day 0) and also five to seven population doublings later (Figure 1A). Subsequently, the abundance of individual shRNAs at each time point was determined by performing massively parallel sequencing of amplified shRNA-associated barcode sequences. After determining the abundance of the barcodes for each gene at both time points, these levels were compared to identify changes in their representation.

Genome-scale identification of genes responsible for mediating growth and survival of human and mouse MPNST cells. A: Workflow schematic demonstrating transduction of cells at low multiplicity of infection (MOI) with a genome-scale library of shRNA-expressing lentiviral vectors, each tagged with a unique barcode sequence, followed by selection for puromycin resistance. A representative sample of the transduced cell population is harvested at day 3 to provide a reference baseline, and remaining cells are harvested after five to seven population doublings. The lentiviral barcodes are amplified from both cell populations and sequenced to quantify how often individual barcodes are encountered at each time point. B: Potential outcomes expected in these screens. If a cell is transduced with a lentivirus expressing an shRNA targeting a transcript that is not required for proliferation or survival, the barcode integrated into the cell's DNA is replicated during proliferation and is present at an increased level five to seven doublings later compared with the baseline. However, if a cell is transduced with a shRNA targeting a gene required for proliferation, the cell proliferates slower than the rest of the population, resulting in decreased abundance of the barcode. If the cell is transduced with a shRNA targeting a gene required for survival, the cell dies, which also results in decreased abundance or loss of the bar code after five to seven population doublings.

In successful dropout screens, the abundance of shRNAs that target genes essential for proliferation or viability are reduced over the course of population doublings compared with the larger, unaffected population of cells.44, 45, 46 This leads to an increase in their gene-level depletion scores relative to those of the shRNAs targeting the much larger set of genes not essential for proliferation or survival (Figure 1B). To determine whether the results of these shRNA screens were consistent with this expectation, the distribution of the fold change in gene-level depletion scores was examined in each human MPNST cell line and early-passage P₀-GGFβ3 MPNST culture. In all of the human lines (Figure 2A) and mouse early-passage cultures (Figure 2B), the mean value of the log2 fold changes was 1.09, with maximum and minimum values of 10.5 and –1.4, respectively. This indicated that, as expected, most shRNAs in the human and mouse libraries targeted genes that were not essential for the proliferation and/or viability of these MPNST cells. Notably, however, these curves also demonstrated the presence of a collection of genes with increased gene-level depletion scores (Figure 2).

Identification of genes involved in mediating growth and survival of human and mouse MPNST cells. The distribution of the log2 gene-level depletion score fold changes for each human MPNST cell line (A) and each mouse P₀-GGFβ3 MPNST culture (B) shows that the mean value of the log2 fold depletion score is 1.09, with maximum and minimum values of 10.5 and –1.4, respectively. This indicates that most of the shRNAs in the library targeted genes that did not result in a significant fold change under these experimental conditions. **Boxed areas** highlight the population of genes with the greatest gene-level depletion scores.

As a quality control measure, the gene-level depletion scores for genes encoding ribosomal components were compared with those encoding matrix metalloproteases, with the expectation that inactivation of key ribosomal proteins would significantly impair cellular growth and viability, whereas the loss of matrix metalloproteases would not. As expected, the average gene-level depletion scores for ribosomal proteins were significantly higher than those for matrix metalloproteases in both human (Figure 3A) and mouse (Figure 3B) MPNST cells. These findings further indicated that the dropout screens appropriately identified genes required for proliferation and survival.

Comparison of gene-level depletion scores for ribosomal proteins and matrix metalloproteases in human MPNST cell lines and early-passage P₀-GGFβ3 MPNST cultures. A: Box plots for each human MPNST cell line, showing gene-level depletion scores for genes encoding ribosomal proteins (**blue boxed areas**) and genes encoding matrix metalloproteases (MMPs; **red boxed areas**). Each dot represents the gene-level depletion score for an individual ribosomal protein- or MMP-encoding gene. B: Box plots for each early-passage P₀-GGFβ3 MPNST culture, showing gene-level depletion scores for genes encoding ribosomal proteins (**blue boxed areas**) and genes encoding MMPs (**red boxed areas**). For both human and mouse MPNST cells, note that the shRNA screens resulted in a significant increase in the average gene-level depletion scores for ribosomal protein genes compared with the scores for MMP genes.

To identify the genes whose knockdown had the greatest effect on the proliferation and viability of human MPNST cells, the gene-level depletion scores from each screen were ranked, and those in the top 90^th quantile (hits) were selected for subsequent analyses. This ranking identified 1526, 1518, and 1523 gene hits in S462, T265-2c, and 2XSB cells, respectively (Supplemental Table S1). As an additional quality check, the hits identified in the human MPNST cell screens were examined to determine whether they included members of a set of 684 core essential genes (the CEG2 gene set); in previous large-scale shRNA and clustered regularly interspaced short palindromic repeats (CRISPR) screens,⁴⁷^,⁴⁸ these CEGs were shown to be required for the proliferation and/or survival of multiple cell lines derived from several different types of human cancer. A total of 13.8% to 18.3% of the genes identified as hits in human MPNST cells were members of the CEG2 gene set (Table 1 and Supplemental Table S1 for the identity of these genes). These findings further indicate that the human MPNST cell shRNA screens appropriately identified genes promoting proliferation and survival. They also demonstrate that the proliferation and survival of the screened human MPNST cells is dependent on many of the same genes that are required for growth and viability in other types of human cancer.

Table 1.

Characteristics of Hits in Human and Mouse MPNST Cell Screens

Species	Cells	Hits that are CEGs, % (n/total)	Hits shared with other cells of the same species, % (n/total)	Hits that are BLC genes, % (n/total)	Mouse hits that are hits in human lines, % (n/total)	Hits that are members of druggable genome, % (n/total)
Human	S462	18.3 (279/1526)	59.4 (907/1526)	15.1 (231/1526)	NA	17.4 (266/1526)
Human	T265-2c	13.8 (210/1518)	71.0 (1078/1518)	15.7 (239/1518)	NA	15.4 (235/1523)
Human	2XSB	14.6 (223/1523)	74.8 (1139/1523)	15.6 (237/1523)	NA	17.8 (270/1518)
Mouse	A390	16.0 (132/826)	66.1 (546/826)	22.6 (187/826)	34.3 (283/826)	28.0 (231/826)
Mouse	A426	13.8 (110/798)	52.0 (415/798)	20.9 (167/798)	29.9 (239/798)	24.8 (198/798)
Mouse	A494	17.6 (142/807)	69.0 (577/807)	23.4 (189/807)	36.2 (292/807)	28.1 (227/807)
Mouse	A496	19.3 (156/808)	67.7 (547/808)	24.0 (194/808)	35.4 (286/808)	27.0 (218/808)
Mouse	B288	15.0 (125/833)	59.9 (499/833)	23.6 (197/833)	34.6 (288/833)	35.8 (298/833)

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BLC, Bushman Lab Cancer; CEG, core essential gene; NA, not applicable.

The hits from each human MPNST cell line were compared with one another to identify the subset of hits that were shared by more than one line (Figure 4A). This comparison identified numerous genes that were required for the growth and/or viability of more than one human MPNST cell line, with 59.4% to 74.8% of the hits identified in each cell line being shared with at least one other human line (Table 1). A total of 32.9% of the genes identified as hits in these three cell lines (1054 of 3202 total hits) were hits in at least two of the three lines (Supplemental Table S2). Of these 1054 genes, 310 (29.4%) were hits in all three lines, with 744 genes (70.6%) being hits in two of the three lines. This indicates that the proliferation and/or survival of these three human MPNST cell lines is dependent on many of the same genes.

Human MPNST cell lines and mouse P₀-GGFβ3 MPNST early-passage cultures require many of the same genes for proliferation and survival. A: Venn diagram of the hits in three human MPNST cell lines, demonstrating the overlap in the genes that are required for proliferation and survival in the three lines that were screened. B: Venn diagram of the hits in early-passage P₀-GGFβ3 MPNST cell lines, demonstrating the overlap in the genes that are required for proliferation and survival in the five early-passage cultures that were screened. C: Bar graphs indicating the number of hits in individual mouse P₀-GGFβ3 MPNST early-passage cultures that were also hits in each human cell line that we screened. The identity of each P₀-GGFβ3 MPNST early-passage culture is indicated above the bars; the human MPNST cell lines that were compared with the mouse cells are indicated on the x axis. Numbers on each bar indicate the number of hits shared between mouse and human cells. D–H: Venn diagrams illustrating the number of hits that are common to mouse A390 (D), A426 (E), A494 (F), A496 (G), and B288 (H) MPNST cells and three human MPNST cell lines (T265-2c, S462, and 2XSB cells).

To similarly identify the genes whose knockdown had the greatest effect on the proliferation and viability of mouse MPNST cells, the gene-level depletion scores from each screen were ranked, and those in the top 90^th quantile (hits) were selected for subsequent analyses. In the early passage P₀-GGFβ3 MPNST cultures, there were 828, 798, 807, 808, and 833 hits identified in A390, A426, A494, A496, and B288 cells, respectively (Supplemental Table S3). A total of 13.8% to 19.3% of the hits identified in the screens were members of the CEG2 gene set (Table 1 and Supplemental Table S3 for the identity of these genes). To distinguish recurring mouse MPNST hits from those unique to an individual culture, the hits from each mouse early-passage MPNST culture were compared with one another (Figure 4B). A total of 52.0% to 69.0% of the hits identified in each early-passage culture were shared with at least one other mouse MPNST culture (Table 1). A total of 477 of the 2316 total hits (20.9%) were hits in most (three or more) of the P₀-GGFβ3 MPNST early-passage cultures (Supplemental Table S4). Of these 477 genes, 52.4% (250 genes) were hits in three lines, 30.8% (147 genes) were hits in four lines, and 16.8% (80 genes) were hits in all five lines. Thus, like the human MPNST cell lines, different P₀-GGFβ3 MPNSTs are dependent on many of the same genes for proliferation and/or survival.

Comparison of the Human and Mouse MPNST Cell Hits Demonstrates that They Target Many of the Same Genes and Biologic Pathways

Because the ultimate goal of this study was to determine whether screens of GEM MPNST cells could identify useful therapeutic targets in human MPNSTs, the human and mouse MPNST hit lists and the biological pathways containing these genes were compared to determine how similar they were. Because the human shRNA libraries queried more genes than the mouse libraries, it was not possible to determine whether all of the hits in human MPNST cells were also hits in mouse MPNST cells. Instead, the number and percentage of the hits in each P₀-GGFβ3 MPNST early-passage culture that were also hits in human MPNST cells was examined. A comparison of the hits in each P₀-GGFβ3 culture to the hits in each human MPNST cell line showed that 29.9% to 36.2% of the hits identified in mouse MPNST cells were also hits in at least one human MPNST cell line (Table 1 and Figure 4, C–H). Although this was lower than the percentage of recurring human (59.4% to 74.8%) and mouse (52.0% to 69.0%) MPNST cell hits that were identified in the initial human-human and mouse-mouse comparisons (Table 1), these human-mouse comparisons nonetheless demonstrated that a large fraction of the hits identified in the mouse MPNST cell screens were shared with human MPNST cells.

Next, the biological pathways that were impacted by the hits identified in these screens were identified and compared to determine how similar those pathways were in human and mouse MPNST cells. The initial step was to perform GSEAs on the hits from each human MPNST cell line (Supplemental Table S1). A comparison of the results of each GSEA showed that these analyses identified four biological pathways that were essential in all three human MPNST cell lines (Supplemental Figure S1): gene expression, translation, 3′-untranslated region–mediated translational regulation, and GTP hydrolysis/joining of the 60S ribosomal subunit. These GSEAs also identified six additional biological pathways that were essential for proliferation and survival in two of the three human lines. These shared pathways were DNA replication, ubiquitin-dependent degradation of cyclin D, cell cycle checkpoints, autodegradation of cadherin 1 (Cdh1) by Cdh1:anaphase promoting complex/cyclosome (APC/C), metabolism of proteins, and metabolism of RNA (Supplemental Figure S1). Notably, several of the shared pathways mediated cell cycle control, which further indicated that the screens appropriately identified genes involved in proliferation.

In the mouse MPNST early-passage cultures, GSEAs of the hits from each line established that these hits targeted several essential biological pathways in most (three or more) of the cultures that were screened (Figure 5). These shared processes included APC/C-mediated degradation of cell cycle proteins, regulation of mitotic cell cycle, mitotic M-M/G₁ phases, S phase, and M/G₁ transition. A comparison of the biological pathways identified in most of the human MPNST cell lines and most of the mouse MPNST cultures identified two biological pathways common to human and mouse cells—cell cycle checkpoints and metabolism of RNA (Figure 5). As in the human lines, the mouse hits targeted several biological pathways relevant to the cell cycle, including cyclin E–associated events during G₁/S transition, mitotic G₁-G₁/S phases, G₁/S transition, and regulation of APC/C activators between G₁/S and early anaphase (Figure 5).

Pathway analyses demonstrate cross-species overlap in the signaling pathways required for MPNST proliferation and survival. A–E: Gene set enrichment analysis was performed using genes with gene-level depletion scores identified in the top 90^th quantile as the input gene list. Results are presented for mouse A390 (A), A426 (B), A494 (C), A496 (D), and B288 (E) MPNST cells. Only statistically significant enriched functional pathways are shown. Biologic pathways that were hits in most mouse MPNST cells are in **blue boxed areas**. Biologic pathways that were hits in most of both the human and mouse MPNST cells are in **red boxed areas**. Pathways that are both relevant to the cell cycle and hits in human and mouse MPNST cells are in **green boxed areas**. APC/C, anaphase-promoting complex/cyclosome; Cdh, cadherin 1.

To further verify that the shRNA screens appropriately selected for genes essential for proliferation and/or survival, integrated functional analyses were also performed using the Enrich-KG bioinformatic platform. The hits that were common to at least two of three human MPNST cell lines and three of five mouse P₀-GGFβ3 MPNST cultures were used as inputs for these analyses. As part of the integrated functional analyses, an assessment of Gene Ontology biological processes was performed. This assessment demonstrated that the human and mouse hits were linked to key proliferative and prosurvival processes, including mitotic sister chromatid segregation, regulation of the mitotic cell cycle, cellular responses to DNA damage stimuli, DNA metabolic processes, and DNA repair (Figure 6A); they were also linked to transcription factors regulating the cell cycle (E2F1, E2F3, E2F4, MYC, and TP53), apoptosis (MYC, TP53), and senescence (TP53). An examination of the relationship of these hits to disease gene networks showed that they were linked to several cancer types, including hematopoietic malignancies, lymphomas, osteosarcomas, and malignant peripheral nerve sheath tumors (Figure 6B).

Gene set enrichment analyses identified candidate MPNST therapeutic agents. A: Integrated functional analysis demonstrates overlapping biological processes using the Enrich-KG bioinformatic platform. This analysis was performed using the hits that occurred in most human and mouse MPNST cells as the input. Lavender circles indicate Gene Ontology (GO) biological processes 2021, and yellow circles indicate TRRUST Transcription Factors 2019. The dark blue circles indicate four of the genes encoding proteins that were selected for therapeutic intervention. B: The disease relationship between selected hits occurring in most human and mouse MPNST cells was analyzed with disease gene networks (DisGeNETs) to demonstrate that these genes are linked to several cancer types, including MPNSTs. KEGG, Kyoto Encyclopedia of Genes and Genomes.

The Proliferation and Survival of Human and Mouse MPNST Cells Is Dependent on Multiple Genes Previously Implicated in the Pathogenesis of Other Cancer Types

The observation that multiple gene hits were linked to several cancer types raised the question of whether the human and mouse MPNST cell hits included genes previously implicated in the pathogenesis of other types of human cancer. To determine whether this was the case, these hits were compared with the Bushman Lab Cancer (BLC) Gene List version 5 (http://www.bushmanlab.org/links/genelists, last accessed June 15, 2021). This list, which includes 2480 cancer-associated genes that perform myriad functions, was compiled from multiple sources, including CANgenes,⁴⁹ CIS,⁵⁰ the Atlas of Genetics and Cytogenetics in Oncology and Haematology,⁵⁰ the Sanger gene list,⁵¹ and the Vogelstein cancer gene list.⁵² Notably, the BLC Gene List is largely distinct from the CEG2 gene set, with only 100 genes shared between the two lists (Supplemental Figure S2A). Results showed that 15.1% to 15.7% of the hits identified in each human line were BLC genes (Table 1 and Supplemental Table S1). Furthermore, 34.5% of the hits occurring in two or more of the three human MPNST cell lines were BLC genes (Supplemental Table S2). The cancer-associated genes that were hits in two or more human MPNST cell lines included several genes that have previously been identified as potential therapeutic targets in MPNSTs, such as CDK4,53, 54, 55, 56 MYC,³⁸^,⁵⁷ LCK,⁵⁸ RRAS2,⁹ AURKA,⁵⁹^,⁶⁰ ERBB3,⁶¹ PLK1,⁶² and SGK1.⁶³ These hits also included NUMA1⁴¹ and BMPR1A,⁶⁴ two genes previously found to be mutated in some MPNSTs, and VCP, a gene encoding a neurofibromin-interacting protein.⁶⁵

Performing the same comparison with the mouse MPNST hits demonstrated that an even higher percentage of the mouse hits (20.9% to 24.0%) (Table 1) were BLC genes. Among the genes that were hits in most mouse MPNST early-passage cultures, 22.5% (108/480 genes) were BLC genes (Supplemental Table S4). Interestingly, the mouse MPNST screens identified a distinct set of BLC genes previously implicated in the pathogenesis of human MPNSTs. These included Bcl2,⁶⁶ Yap1,⁶⁷ Notch3,⁶⁸^,⁶⁹ and Rac1.⁷⁰^,⁷¹ In keeping with the experimental design of the shRNA screens described here, the mouse BLC hits also included multiple genes involved in proliferation (Ccnb1, Pcna, and Ccnd1), DNA synthesis and repair (Pola1, Polq), and survival (Bcl2, Bcl6).

A comparison of the BLC gene hits in mouse and human MPNST cells showed that similar numbers of these hits (Supplemental Figure S2B), like the CEG hits (Supplemental Figure S2C), were identified in human and mouse MPNST cells. To better understand the significance of the BLC hits, the biological pathways impacted by the hits occurring in most human and mouse MPNST cells were compared to determine how similar those pathways were in human and mouse MPNST cells and to gain further insight into the processes promoting proliferation and survival in MPNST cells. Pathway analyses of BLC gene hits identified in individual mouse MPNST cultures showed striking similarities, with these cultures repeatedly showing dependence on several pathways regulated by receptor tyrosine kinases as well as Arf GTPase 6 (Arf6), sphingosine-1-phosphate, class I phosphatidylinositol 3-kinase, mammalian target of rapamycin, plasma membrane estrogen receptor, serine/threonine kinase 11 (LKB1), and proteoglycan syndecan-mediated signaling (Figures 7 and 8). Many of these same pathways were also implicated in the proliferation and survival of the human MPNST cells (Supplemental Figure S3). Overall, 18 biological pathways were shared by all three human MPNST lines (Supplemental Figure S4A), and 14 were common to all five mouse MPNST cultures (Supplemental Figure S4B). A comparison of the 18 human pathways and the 14 mouse pathways identified 13 biological pathways that were shared by all three human MPNST cell lines and all five mouse cultures (Supplemental Figure S4C). These pathways included several involved in erbB (Internalization of ErbB1, ErbB1 Downstream Signaling, and ErbB Receptor Signaling Network) and insulin/insulin-like growth factor-1 signaling (Insulin Pathway, Insulin-Like Growth Factor-1 Pathway), as well as the platelet-derived growth factor receptor β signaling pathway, the sphingosine-1-phosphate pathway, and the urokinase-type plasminogen activator and urokinase-type plasminogen activator receptor mediated signaling pathway. Several intracellular signaling pathways were also shared, including the mammalian target of rapamycin signaling pathway, Arf6 signaling events and downstream pathway, class I phosphatidylinositol 3-kinase signaling events, and focal adhesion kinase signaling events.

Bushman pathway analysis in mouse MPNST cells demonstrates significant overlap in common biological pathways. Results are presented for mouse A390 (A) and A426 (B) MPNST cells. Only statistically significant enriched functional pathways are shown. Biological pathways that were common in all mouse MPNST lines are in **green boxed areas** (ErbB1 internalization and signaling). Pathways common to four of five lines are in **blue boxed areas** (insulin and insulin-like growth factor-1 pathways; Figure 8 for the results from other mouse MPNST cells). Arf6, Arf GTPase 6; LKB1, serine/threonine kinase 11; mTOR, mammalian target of rapamycin; PI3K, phosphatidylinositol 3-kinase; S1P1, sphingosine-1-phosphate; TRAIL, TNF superfamily member 10; VEGF, vascular endothelial growth factor; VEGFR, VEGF receptor.

Multiple Hits in Human and Mouse MPNST Cells Encode Proteins Targetable with Existing Therapeutic Agents

To identify potential therapeutic targets in MPNSTs, a drug discovery pipeline was developed on the basis of the human and mouse MPNST cell shRNA screens (Figure 9A). The initial step in this pipeline was to identify the subset of hits in human and mouse MPNST cells that encoded proteins that were druggable with existing agents. To achieve this, the 2316 genes that were hits in one or more of the five P₀-GGFβ3 MPNST early-passage cultures were screened using the Drug-Gene Interaction Database⁷² (https://dgidb.org, last accessed December 31, 2021), and the hits that were members of the druggable genome were identified. This search identified 680 genes that were both members of the druggable genome and a hit in at least one of the mouse MPNST cultures (Supplemental Table S5). The 3203 genes that were hits in the three human MPNST cell lines were similarly screened using the Drug-Gene Interaction Database. This search identified 558 genes that were hits in at least one of the human MPNST cell lines and members of the druggable genome (Supplemental Table S6).

Development of a drug discovery pipeline. A: The pipeline used to identify genes encoding druggable targets began by identifying these genes with the Drug Gene Interaction Database (DGIdb). The pipeline further filtered candidate druggable genes by determining which of these genes were hits in at least three of five mouse MPNST cultures and hits in one or more human MPNST cell lines. The Medical University of South Carolina (MUSC) Drug Discovery Core was then consulted to determine which candidate therapeutic agents were commercially available. B: Venn diagram showing the overlap between candidate therapeutic targets identified in human and mouse MPNST cells.

On the basis of the expectation that druggable genome hits occurring repeatedly in mouse MPNST cells were most likely to encode useful therapeutic targets in human MPNSTs, the next step in the pipeline was to identify the druggable genome hits that occurred in most of the mouse MPNST cells that were screened (Figure 9A). This analysis identified 138 druggable genome hits that occurred in three or more P₀-GGFβ3 MPNST cultures (Supplemental Table S5). These candidate targets were then further filtered by asking which ones were also a hit in one or more of the three human MPNST cell lines (Supplemental Table S7). The 65 genes remaining after these filtering steps represented the initial pool of candidate therapeutic targets (Figure 9B).

The Medical University of South Carolina Drug Discovery Core was consulted to identify commercially available drugs that inhibited proteins encoded by the genes identified with the drug discovery pipeline. After identifying drugs that were commercially available, additional criteria were applied to prioritize therapeutic agents for validation. Agents that effectively inhibit enzymes previously implicated in proliferation and/or survival and inhibitors that affected gene regulation relevant to proliferation and/or survival were prioritized. The resulting list of inhibitors was then further narrowed by evaluating the previously demonstrated clinical significance of these inhibitors, including whether they acted at concentrations that were clinically achievable, the effectiveness of these inhibitors against other human cancer types, and evidence implicating the inhibitor's target in the pathogenesis of other human cancer types. On the basis of these criteria, five inhibitors were chosen for initial validation of the drug discovery pipeline (Table 2). These inhibitors were 79-6 (targets BCL6), methazolamide (targets carbonic anhydrase 1), Ki16425 [targets lysophosphatidic acid receptor 3 (LPAR3) as well as LPAR1], clofarabine [targets the catalytic subunit of DNA polymerase α1 (POLA1)], and cordycepin [targets DNA nucleotidylexotransferase (DNTT)]. Notably, three of the five therapeutic targets [and polo like kinase 1 (PLK1), a kinase previously identified as a therapeutic target in MPNSTs⁶²^,⁷³] that were hits in human and mouse MPNST cells are linked to the regulation of key cell cycle and DNA repair functions (Figure 6A).

Table 2.

Selected Target Gene Functions and Clinical Relevance

Gene	Function	Clinical significance	Inhibitor
LPAR3	G-protein–coupled receptor; LPA receptor	Implicated in tumor proliferation, invasiveness, and metastasis in breast and other cancers	Ki16425
BCL6	B-cell lymphoma 6 protein; transcription repressor	Implicated in hematologic and solid tumors, including B-ALL, CML, breast, and NSCLC	79-6 (CID5721353)
DNTT	DNA nucleotidylexotransferase; specialized DNA polymerase	Associated with ALL, CML, other leukemias, and metastatic breast cancer	Cordycepin
POLA1	DNA polymerase α; the enzyme responsible for initiating DNA synthesis	TCGA data show moderate to strong positivity in most malignancies	Clofarabine
CA1	Carbonic anhydrase 1; involved in cellular homeostasis	CA1 knockdown enhances malignant potential of prostate tumors; autoantibodies detected in leukemia and gastric cancer	Methazolamide

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ALL, acute lymphoblastic leukemia; B-ALL, B-cell ALL; CML, chronic myeloid leukemia; LPA, lysophosphatidic acid; NSCLC, non–small-cell lung cancer; TCGA, The Cancer Genome Atlas.

Validation of Therapeutic Targets in Human MPNST Cells

To directly test the sensitivity of MPNST cells to these candidate therapeutic agents, the human MPNST cell lines used for the shRNA screens (T265-2c, S462, and 2XSB cells) were challenged with vehicle or a range of concentrations of each inhibitor that was centered on the concentration of inhibitor that inhibited the response by 50% (IC₅₀) previously defined in other types of human cancer cells. Cell numbers were quantified for each condition 1, 3, and 5 days after plating using an imaging cytometer. Cell numbers were normalized to the number of cells in the wells receiving vehicle at day 1 and then compared to assess the effect each inhibitor exerted on cell numbers. As an initial assessment of whether these experimental conditions appropriately identified drug-induced decreases in cell numbers, multiple human MPNST cell lines were challenged with 0.01 to 1 μmol/L concentrations of the highly potent PLK1 inhibitors rigosertib (Supplemental Figure S5) and volasertib (Supplemental Figure S6). Both of these agents decreased cell numbers in all lines tested in a concentration-dependent manner.

The POLA1 inhibitor clofarabine was tested over a concentration range of 5 to 500 nmol/L. Compared with vehicle, clofarabine reduced the number of cells in all three MPNST cell lines in a concentration-dependent manner, with a reduction in cell numbers already evident by day 3 and being even more pronounced after 5 days of treatment (Figure 10A). Although clofarabine potently decreased cell numbers in all of the lines that were tested, S462 cells were more sensitive to this drug than were T265-2c and 2XSB cells. In keeping with this, calculation of the IC₅₀ for each cell line demonstrated that S462 cells had the lowest IC₅₀ for clofarabine among the three lines initially tested (Figure 10, B and C).

Validation of candidate therapeutic targets in human MPNST cell lines. A: Heat maps demonstrating cell numbers in cultures of human 2XSB, T265-2, and S462 MPNST cells challenged with vehicle or varying concentrations of the DNA polymerase α1 inhibitor clofarabine, the BCL6 inhibitor 79-6, and the DNA nucleotidylexotransferase inhibitor cordycepin. Cell numbers, assessed 1, 3, and 5 days after plating using an imaging cytometer, were normalized to the number of cells present at day 1 in cultures challenged with vehicle (1.00). Blue shading indicates lower cell numbers, and red shading indicates higher cell numbers. Numbers on each cell of the map indicate the normalized cell numbers measured under each condition. B: Micromolar IC₅₀ values calculated for clofarabine, cordycepin, and 79-6 in each cell line. C: Graphical representation of the log₂ micromolar IC₅₀ values for each inhibitor (indicated on the x axis) in 2XSB, S462, and T265 cells.

The BCL6 inhibitor 79-6 was tested over a concentration range of 1 to 100 μmol/L. Cell numbers were decreased in all three MPNST cell lines challenged with 79-6, with the drug exerting its effects in a concentration-dependent manner (Figure 10A). Although 79-6 was effective against all three cell lines, 2XSB cells were slightly more responsive to this drug, demonstrating a lower IC₅₀ value for 79-6 in comparison to T265-2c and S462 cells (Figure 10, B and C). The sensitivity of these three human MPNST cell lines to BCL6 inhibition was consistent with the recent demonstration that knockdown of BCL6 similarly reduced cell numbers in these same three MPNST cell lines. In those earlier experiments, MPNST lines were transduced with lentiviral vectors expressing BCL6 shRNAs, and it was found that BCL6 shRNAs markedly reduced cell numbers in comparison to cells transduced with a nonsense shRNA.⁴³

The DNTT inhibitor cordycepin was also tested at concentrations ranging from 1 to 100 μmol/L. Cordycepin reduced cell numbers in all three human MPNST cell lines in a concentration-dependent manner (Figure 10A). The IC₅₀ for cordycepin was highly similar in 2XSB and S462 cells, with both of these lines demonstrating IC₅₀s that were lower than was observed in T265-2c cells (Figure 10, A and B).

In contrast to the results obtained with clofarabine, 79-6, and cordycepin, a significant reduction in cell numbers was not seen in 2XSB, T265-2c, and S462 cells challenged with the carbonic anhydrase 1 inhibitor methazolamide (Supplemental Figure S7). The LPAR 1/3 inhibitor Ki16425 did reduce cell numbers in 2XSB, T265-2c, and S462 but only at higher concentrations (Supplemental Figure S7).

To determine whether the three inhibitors that were most effective against T265-2c, S462, and 2XSB cells also effectively inhibited the proliferation of other MPNST cell lines, two additional sporadic MPNST cell lines (HS.Sch2, STS-26T) and an additional NF1-associated cell line (ST88.14) were challenged with the same concentrations of 79-6, clofarabine, and cordycepin used in the experiments described above. All three inhibitors reduced cell numbers in these three additional human MPNST cell lines (Figure 11A). The BCL6 inhibitor 79-6 was even more effective against ST88.14, HS.Sch2, and STS-26T cells than in the three lines originally tested (compare Figure 11, B and C with Figure 10, B and C). 79-6 was particularly effective against HS.Sch2 cells, with STS-26T and ST88.14 cells having IC₅₀s that was higher than seen in HS.Sch2 cells (but still lower than observed in 2XSB, S462, and T265-2c cells). Likewise, cordycepin reduced cell numbers in all three lines, showing the greatest effectiveness against ST88.14 cells (Figure 10A); the IC₅₀s calculated for ST88.14, HS.Sch, and STS-26T cells were similar to those observed in the three lines that were initially tested (Figure 11, B and C). The POLA1 inhibitor clofarabine had the greatest effect on the proliferation of these cell lines; at low nanomolar doses, all cell lines showed a pronounced decrease in growth (Figure 11). These results thus further validated the targets identified by the drug discovery pipeline and provided additional evidence that these gene products are consistently important for the proliferation and/or survival of human MPNST cell lines.

Validation of candidate therapeutic targets in additional MPNST cell lines. A: Heat maps demonstrating cell numbers in cultures of human ST88-14, Hs.Sch2, and STS-26T MPNST cells challenged with vehicle or varying concentrations of the DNA polymerase α1 inhibitor clofarabine, the BCL6 inhibitor 79-6, and the DNA nucleotidylexotransferase inhibitor cordycepin. Cell numbers, assessed 1, 3, and 5 days after plating using an imaging cytometer, were normalized to the number of cells present at day 1 in cultures challenged with vehicle (1.00). Blue shading indicates lower cell numbers, and red shading indicates higher cell numbers. Numbers on each cell indicate the normalized cell numbers measured under each condition. B: Micromolar IC₅₀ values calculated for clofarabine, cordycepin, and 79-6 in each cell line. C: Graphical representation of the log₂ micromolar IC₅₀ values for each inhibitor (indicated on the x axis) in ST88-14, Hs.Sch2, and STS-26T cells.

Clofarabine Induces Senescence in MPNST Cells

An initial microscopic examination of human 2XSB and S462 MPNST cells treated with clofarabine suggested that this drug both reduced cell numbers and induced morphologic changes. To further assess the latter observation, 2XSB (Figure 12A) and S462 (Figure 12B) cells were treated with clofarabine for 3 days, fixed, and then stained with phalloidin and DAPI to identify possible changes in the actin cytoskeleton and nuclear morphology, respectively. Both 2XSB and S462 cells displayed an enlarged and flattened shape reminiscent of the changes associated with the induction of senescence. To determine whether these cells were entering a senescent state in response to clofarabine treatment, 2XSB (Figure 12C) and S462 (Figure 12D) cells were treated for 3 days with clofarabine and then stained for senescence-associated β-galactosidase activity. These stains demonstrated the presence of senescence-associated β-galactosidase activity in 2XSB and S462 cells treated with clofarabine but not in vehicle controls. Quantification of senescence-associated β-galactosidase staining confirmed a statistically significance increase in senescence-associated β-galactosidase staining relative to control cultures in 2XSB (Figure 12E) and S462 (Figure 12F) cells treated with clofarabine. Cellular senescence can be induced by activation of either the p53/p21 or p38/p16/Rb pathways. To determine whether either of these pathways was activated by clofarabine treatment, S462 cells were treated with vehicle or clofarabine for 12, 24, or 48 hours. Lysates of these cells were then immunoblotted and probed for p53 and p38. In S462 cells treated with clofarabine, an increase in p53 expression was evident after 12 hours of treatment, and a further increase in p53 levels was present after 24 hours of treatment; a similar increase in p53 expression was not seen in cultures receiving vehicle (Figure 12G). In contrast, there was no evidence of an increase in p38 expression in S462 cells treated with clofarabine.

Discussion

Cross-species comparative oncogenomics is an emerging approach to novel driver gene and therapeutic target discovery in cancers,³⁴^,³⁵ as demonstrated by an increase in the number of publications using this approach over the past two decades. For instance, the gene NEDD9 was initially identified as a melanoma metastasis gene through comparative oncogenomics.⁷⁴ Moreover, this approach was used to validate the tumor suppressive function of DLC1⁷⁵ and the protumorigenic role of the transcription factor Yap in liver cancer⁷⁶ and to support the use of a preclinical mouse model investigating the role of tumor-initiating cells when mouse mammary tumors were shown to have gene expression patterns similar to those of human claudin-low tumors.³³ Comparative oncogenomics is potentially particularly well suited for identifying driver genes and therapeutic targets in rare nervous system tumors. A review of NF mouse models revealed that their tumors are comparable to the human tumors from which they are derived. A DECIPHER pooled shRNA library screen identified fibroblast growth factor receptor and the serine/threonine protein phosphatase 2A as top hits and potential therapeutic targets for those experiencing diffuse intrinsic pontine gliomas.⁷⁷ As another example, a comparison of 147 zebrafish MPNSTs to human MPNSTs effectively separated passenger mutations from driver mutations.³⁶ This was possible because of the highly disorganized nature of gene locations in zebrafish compared with human, which allowed the removal or reduction of passenger gene candidates by comparing copy number alterations in genes found in zebrafish and human tumors.

In this study, cross-species functional genomics was applied in the form of large-scale shRNA screens to human MPNST cells and early-passage MPNST cultures from P₀-GGFβ3 mice, a GEM model in which overexpression of the growth factor neuregulin-1 drives the pathogenesis of plexiform neurofibromas that subsequently progress to become MPNSTs with high penetrance.³⁷^,³⁸ Neuregulin-1 overexpression in P₀-GGFβ3 mice promotes peripheral nervous system neoplasia predominantly via its effects on Nf1-regulated signaling cascades⁴² and MPNSTs in these mice have tumor suppressor gene mutations that parallel those seen in their human counterparts.³⁸^,⁴² However, comprehensive genomic and transcriptomic analyses of a large series of P₀-GGFβ3 MPNSTs also indicate that these GEM tumors are diverse, which suggests that they are potentially well suited for the discovery of therapeutic targets unique to different tumor subtypes (J.F.L. and S.L.C., unpublished data, 2022).

Detailed comparisons of the hits in human and mouse MPNST cells and the biologic pathways that include these hits demonstrated that there was considerable overlap in the human and mouse genes that were required for proliferation and survival. Furthermore, the responses of human MPNST cells to four of the five drugs selected for validation demonstrates that the approach described here is effective for identifying novel therapeutic agents that are effective against human MPNSTs. However, the hits that were identified were not completely identical between human and mouse MPNST cells. Thus, there are potentially some species-specific differences in the genes required for proliferation and survival in individual mouse and human MPNSTs. Given this, it is critically important that cross-species functional screens, such as the ones described here, include human cancer cell lines as a gold standard when prioritizing candidate therapeutic agents for validation. Alternatively, the presence of hits that are unique to individual human and mouse MPNST cells may indicate that there are molecular subtypes of MPNSTs that differ in some of the genes that are required for proliferation and survival.

The possibility that the list of candidate therapeutic targets could be further narrowed by excluding CEGs was considered because the CEG gene set includes genes that are required for basal metabolism in both neoplastic and nonneoplastic cells. An examination of the collections of hits from mouse and human MPNST cells confirmed that these hits included genes involved in basal metabolism (eg, GAPDH); targeting proteins encoded by these genes would likely not be feasible because of the toxic effects that they would have on nonneoplastic cells. However, the CEG2 gene set also includes several genes whose products are being effectively targeted in other human cancer types, such as c-Myc (MYC), polo-like kinase 1 (PLK1), and aurora kinase B (AURKB). It was concluded that it would be inappropriate to globally exclude all CEGs as candidate therapeutic targets and that it would be more prudent to instead consider each of these genes on a case-by-case basis.

The genomic makeup of MPNSTs is highly heterogeneous, with large numbers of duplications and deletions. In this regard, it is notable that three genes that were prioritized as therapeutic targets (POLA1, DNTT, and BCL6) play a role in DNA replication or transcription, which are significantly altered in MPNSTs. The POLA1 inhibitor clofarabine was the standout target identified in the validation studies described here. POLA1 is a component of the Pol α complex, which initiates DNA replication at origins of replication on both leading and lagging strands. The Pol α complex is composed of four subunits: the catalytic subunit POLA1, the regulatory subunit POLA2, and the primase subunits PRIM1 and PRIM2. In keeping with the structure of this complex, PRIM1 and PRIM2 were hits in both the human and mouse MPNST cell screens, and POLA2 was identified as a hit in one mouse MPNST culture. Recently, it has been shown that the Pol α complex is involved in DNA-damage response signaling triggered by stalled replication forks, maintenance of telomeres, and epigenetic regulation. Given the extensive genetic damage in human MPNSTs, it makes sense that POLA1 would play a critical role in these neoplasms.

Inhibition of POLA1 leads to the phenomenon of replication catastrophe, whereby cells generate single-strand DNA faster than they can protect it, which results in massive genomic instability and ultimately cell death.⁷⁸ Interestingly, POLA1 mutations coincide with resistance to CD437, a retinoid-like small molecule that induces apoptosis specifically in cancer cells. Silencing of POLA1 sensitizes non–small-cell lung cancer and colorectal cancer cell lines to checkpoint kinase 1 (CHK1) inhibitors,⁷⁹ suggesting that low levels of POLA1 could be a biomarker for cancers that are susceptible to CHK1 targeted therapy. Furthermore, the relationship between POLA1 and ATR checkpoint kinase (ATR)/CHK1 has been described as synthetically lethal, representing another novel approach to cancer therapy. These observations suggest that combinatorial therapies simultaneously targeting POLA1 and CHK1 or ATR are worth investigating in MPNSTs.

In addition to reducing cell numbers, clofarabine profoundly alters the morphology of the actin cytoskeleton of MPNST cells and triggers cellular senescence in S462 cells that is associated with increased p53 expression. Curiously, however, whole-exome sequencing of S462 cells indicates that these cells have loss of heterozygosity at the TP53 locus, with a p.R110P mutation in the remaining TP53 allele. The effect of this mutation is incompletely understood. However, because some p53 mutations that are severely compromised for transcriptional activation are still capable of inducing senescence,⁸⁰ we cannot rule out that the p.R110P mutated TP53 in S462 cells induces senescence. Alternatively, clofarabine may induce senescence by another TP53-independent pathway.

The contribution of senescence to cancer progression and therapeutic responses is complicated and disputed. Traditionally, cellular senescence has been viewed as a protective measure that cells take to oppose tumor progression; however, recent publications have disputed this concept. At present, there are two major trains of thought regarding the therapeutic significance of senescence in cancer. Some investigators think that senescence offers a therapeutic advantage because it can sensitize cells to other inhibitors used in combinatorial treatments. Other investigators instead emphasize that senescent cells secrete cytokines, growth factors, proteases, and other factors (the senescence-associated secretory phenotype), thereby influencing the tumorigenic potential of surrounding cells in a manner that is not therapeutically beneficial. It is not yet clear what effect clofarabine-induced senescence will have on MPNSTs in vivo. Future studies administering clofarabine alone and in combination with other inhibitors will be essential for elucidating how clofarabine-induced senescence affects therapeutic responses in MPNSTs in the clinical setting.

The utility of these cross-species functional screens for identifying useful therapeutic targets in human MPNSTs is further supported by the fact that these screens also identified two therapeutic targets, LPAR3 and erb-b2 receptor tyrosine kinase 3 (ERBB3), that were previously shown to be essential for the proliferation and/or survival of human MPNST cells. LPAR3, which was identified as a hit in most of mouse and human MPNST cell lines that were screened, is one of two LPA receptors expressed by MPNST cells (LPAR1 and LPAR3).⁸¹ LPAR1 and LPAR3 play fundamentally different roles in MPNSTs; knockdown of LPAR1 reduces the migration, but not the proliferation, of LPA-treated MPNST cells, whereas knockdown of LPAR3 inhibits only proliferation. ERBB3 is commonly overexpressed in human MPNST cell lines, and ERBB3 knockdown inhibits cell proliferation.⁶¹ Furthermore, ERBB3 regulates calcium-calmodulin signaling in MPNST cells. Treatment of MPNST cells with the inhibitors of key nodes in the calcium-calmodulin signaling pathway (the calmodulin inhibitor trifluoperazine and the PIM kinase inhibitor AZD1208) also inhibits proliferation and survival. Interestingly, trifluoperazine activates multiple Src family kinases, including LCK, and inhibition of these kinases with saracatinib blocks trifluoperazine-induced phosphorylation of ERBB3. Notably, LCK was also a hit in both the human and mouse MPNST screens.

It is likely that screens such as those described here will ultimately be able to identify sensitivities specific to MPNSTs that have loss of NF1, CDKN2A, TP53, or components of the polycomb repressor complex 2. However, identifying such sensitivities will be dependent on screening larger numbers of human or mouse MPNST cells with these mutations and comparing the results of those screens with one another to narrow down the field of candidate genes specifically affected by these mutations and to then identify therapeutic agents that can exploit these sensitivities.

In summary, three clinically available drugs were identified that reproducibly and potently inhibit proliferation and/or survival in multiple human MPNST cell lines. The approach described here demonstrates that integrating functional screens from human cells with corresponding mouse models has enormous translational potential. This approach is particularly well suited to rare tumor types where large numbers of tumors and cell lines for comprehensive analyses are unobtainable. Mining the data from the cross-species oncogenomic comparison reported here has only begun, and it is likely that this data set includes several more clinically useful therapeutic targets in MPNSTs that await validation. It will also be interesting to determine whether the gene hits that are unique to specific cells are indicative of the presence of distinct molecular subtypes of MPNSTs. We have already established early-passage cultures from 120 independently arising P₀-GGFβ3 MPNSTs, which greatly exceeds the number of human MPNST cell lines currently in existence. Because tumorigenesis in this GEM model is driven by overexpression of a growth factor rather than specific tumor suppressor gene mutations, P₀-GGFβ3 MPNSTs are likely to be molecularly heterogeneous. Initial whole-exome sequencing, RNA sequencing, and array comparative genomic hybridization analyses of 46 P₀-GGFβ3 MPNSTs are consistent with this expected heterogeneity (J.F.L. and S.L.C., unpublished data. 2022), suggesting that these cultures will be useful for identifying molecular subtypes of mouse MPNSTs potentially relevant to their human counterparts. The use of early-passage cultures of P₀-GGFβ3 MPNSTs also offers advantages over other models, such as the use of established cell lines, because early-passage cultures have not selected for clones that most advantageously grow under culture conditions.

In the future, it will be beneficial to expand these screens to test a larger and more diverse group of human and mouse MPNST cells, including lines with intact NF1 genes. This will allow the systematic identification of distinct MPNST subtypes and the identification of appropriate therapeutic targets specific for each subtype. To identify the optimal therapeutic targets, it will be necessary to partner shRNA and/or CRISPR screens with large-scale screens of candidate drugs identified in these screens. Nonetheless, because many therapeutic compounds identified with cell culture screens fail when tested in animal models, it should be recognized that these in vitro screens must be followed by in vivo screens. This is particularly essential when dealing with rare cancers such as MPNSTs; because only a limited pool of participants is available for clinical trials, every effort must be made to thoroughly vet candidate therapeutics before they advance into clinical trials.

Disclosure Statement

None declared.

Footnotes

Supported by the National Institute of Neurological Diseases and Stroke grants R01 NS048353 and R01 NS109655 (S.L.C.); the National Cancer Institute grants R01 CA122804 (S.L.C.) and F30 CA247139 (S.W.D.); and the Department of Defense grants X81XWH-09-1-0086 and W81XWH-12-1-0164 (S.L.C.).

Current address of E.G.-M.: American Society of Clinical Oncology, Alexandria, VA.

Supplemental material for this article can be found at https://doi.org/10.1016/j.ajpath.2025.05.019.

Supplemental Data

Supplemental Figure S1

Pathway analyses demonstrate overlap in the signaling pathways required for MPNST proliferation and survival. A–C: Gene set enrichment analysis was performed using genes with gene-level depletion scores identified in the top 90th quantile as the input gene list. A–C: Results are presented for human 2XSB (A), S462 (B), and T265-2c (C) MPNST cells. Only statistically significant enriched functional pathways are shown. Biologic pathways that were hits in all three human MPNST cells are black boxed areas. Pathways that are common between 2XSB and S462 cell lines are in red, teal, and light purple boxed areas. Pathways in common between S462 and T265-2c are in orange, dark purple, and yellow boxed areas. APC/C, anaphase-promoting complex/cyclosome; Cdh1, cadherin 1; UTR, untranslated region.

mmc1.pdf^{(3.4MB, pdf)}

Supplemental Figure S2

Bushman Lab and core essential gene 2 (CEG2) gene list comparisons. A: Venn diagram illustrating the overlap in Bushman Lab and CEG2 gene lists. Only a small subset of genes (100) was found to be common between the two gene lists. B: Bar graph showing the number of Bushman Lab genes identified as hits in each individual human and mouse cell line. Note that despite a smaller shRNA DECIPHER library, the mouse cell lines yielded a comparable number of Bushman gene hits compared with the human cell lines. C: Bar graph showing the number of CEG2 genes identified as hits in both human and mouse cell lines.

mmc2.pdf^{(836.4KB, pdf)}

Supplemental Figure S3

Pathway analyses of Bushman genes identified in human MPNST cell lines demonstrate similarities in signaling pathways. Results are presented for human 2XSB (A), S462 (B), and T265 (C) MPNST cells. Only statistically significant enriched functional pathways are shown. Biological pathways that were common in all three human MPNST lines are in green boxed areas (ErbB1 internalization and signaling). Pathways common in two of three lines are in blue boxed areas (insulin and insulin-like growth factor-1 pathways). Arf6, Arf GTPase6; LKB1, serine/threonine kinase 11; mTOR, mammalian target of rapamycin; PDGFR, platelet-derived growth factor receptor; PI3K, phosphatidylinositol 3-kinase; S1P1, sphingosine-1-phosphate; TRAIL, TNF superfamily member 10; VEGF, vascular endothelial growth factor; VEGFR, VEGF receptor.

mmc3.pdf^{(7.2MB, pdf)}

Supplemental Figure S4

Bushman pathway analysis in mouse and human MPNST cell lines show striking overlap in common biological pathways. A: Venn diagram showing 18 common Bushman pathways shared among 2XSB, S462, and T265 human MPNST cell lines. B: Venn diagram of Bushman pathways shared among all mouse (A390, A426, A494, A496, and B288) MPNST lines show 14 pathways in common. C: Venn diagram of all common biological pathways in human and mouse MPNST lines. Results show 13 Bushman pathways are shared between mouse and human MPNST lines, including ErbB1 internalization and downstream signaling events, insulin and insulin-like growth factor (IGF) pathway, and phosphatidylinositol 3-kinase (PI3K) signaling events (indicated in red). Arf6, Arf GTPase 6; mTOR, mammalian target of rapamycin; PDGFR, platelet-derived growth factor receptor; S1P1, sphingosine-1-phosphate.

mmc4.pdf^{(1.7MB, pdf)}

Supplemental Figure S5

Multiple human MPNST cell lines show reduced proliferation with the polo-like kinase 1 (PLK1) inhibitor rigosertib. A–C: Nine different human MPNST cell lines were treated with 0.01 to 1 μmol/L concentrations of rigosertib. Most of the cell lines tested showed a decrease in cell numbers in a concentration-dependent manner. ∗P ≤ 0.05, ∗∗P ≤ 0.005, and ∗∗∗P ≤ 0.0005.

mmc5.pdf^{(1.1MB, pdf)}

Supplemental Figure S6

Multiple human MPNST cell lines show reduced proliferation with the polo-like kinase 1 (PLK1) inhibitor volasertib. A and B: Five different MPNST human cell lines were treated with increasing concentrations of volasertib. Results indicate reduced cell numbers in all cell lines treated in a dose-dependent manner. ∗P ≤ 0.05, ∗∗P ≤ 0.005.

mmc6.pdf^{(834.7KB, pdf)}

Supplemental Figure S7

Human MPNST cell proliferation following treatment with the lysophosphatidic acid receptor 1/3 inhibitor Ki16425 and carbonic anhydrase 1 (CA1) inhibitor methazolamide. A: Human MPNST cell lines were treated with Ki16425 over the course of 7 days. Results indicate a decrease in cell number among all three human MPNST cell lines (T265-2c, 2XSB, and S462) at higher doses of the Ki16425 inhibitor. B: Proliferation of human MPNST cells with methazolamide treatment over 7 days. Results demonstrate no effect on cell proliferation following inhibition of CA1.

mmc7.pdf^{(625.5KB, pdf)}

Supplemental Table S1

mmc8.xlsx^{(124.2KB, xlsx)}

Supplemental Table S2

mmc9.xlsx^{(48.1KB, xlsx)}

Supplemental Table S3

mmc10.xlsx^{(99KB, xlsx)}

Supplemental Table S4

mmc11.xlsx^{(31.3KB, xlsx)}

Supplemental Table S5

mmc12.xlsx^{(39.5KB, xlsx)}

Supplemental Table S6

mmc13.xlsx^{(32.6KB, xlsx)}

Supplemental Table S7

mmc14.xlsx^{(18.6KB, xlsx)}

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplemental Figure S1

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PERMALINK

Cross-Species Functional Genomic Screens Identify Novel Therapeutic Targets in Malignant Peripheral Nerve Sheath Tumors

Brittany Turner-Ivey

Amanda M Prechtl

Stephen T Guest

Shannon W Doutt

Jody F Longo

Elizabeth Garrett-Mayer

Steven L Carroll

Abstract

Graphical abstract

Materials and Methods

Reagents and Antibodies

Cell Culture

DECIPHER Library Screening and Analysis

Celigo Proliferation Assay

Immunoblots

Senescence-Associated β-Galactosidase Staining

Gene Set Enrichment Analysis

Results

Genome-Scale shRNA Screens Identify Genes Required for the Proliferation and Viability of Human and Mouse MPNST Cells

Figure 1.

Figure 2.

Figure 3.

Table 1.

Figure 4.

Comparison of the Human and Mouse MPNST Cell Hits Demonstrates that They Target Many of the Same Genes and Biologic Pathways

Figure 5.

Figure 6.

The Proliferation and Survival of Human and Mouse MPNST Cells Is Dependent on Multiple Genes Previously Implicated in the Pathogenesis of Other Cancer Types

Figure 7.

Figure 8.

Multiple Hits in Human and Mouse MPNST Cells Encode Proteins Targetable with Existing Therapeutic Agents

Figure 9.

Table 2.

Validation of Therapeutic Targets in Human MPNST Cells

Figure 10.

Figure 11.

Clofarabine Induces Senescence in MPNST Cells

Figure 12.

Discussion

Disclosure Statement

Footnotes

Supplemental Data

References

Associated Data

Supplementary Materials

ACTIONS

PERMALINK

RESOURCES

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