Phosphatidylinositol-3-kinase (PI3K)/Akt Signaling is ... · 2Division of Translational Pathology,...

Phosphatidylinositol-3-kinase (PI3K)/Akt Signaling is Functionally Essential in

Myxoid Liposarcoma

Marcel Trautmann1,2 §, Magdalene Cyra1,2 #, Ilka Isfort1,2, Birte Jeiler1,2, Arne Krüger1,2,

Inga Grünewald1,2, Konrad Steinestel1,3, Bianca Altvater4, Claudia Rossig4,5, Susanne

Hafner6, Thomas Simmet6, Jessica Becker7, Pierre Åman8, Eva Wardelmann1,

Sebastian Huss1, and Wolfgang Hartmann1,2 §

1Gerhard-Domagk-Institute of Pathology, Münster University Hospital, Germany;

2Division of Translational Pathology, Gerhard-Domagk-Institute of Pathology, Münster

University Hospital, Germany;

3Institute of Pathology and Molecular Pathology, Bundeswehrkrankenhaus Ulm,

Germany;

4Department of Pediatric Hematology and Oncology, University Children´s Hospital

Münster, Germany;

5Cells in Motion Cluster of Excellence (EXC 1003 – CiM), University of Münster,

Germany;

6Institute of Pharmacology of Natural Products & Clinical Pharmacology, Ulm University,

Germany;

7Institute of Human Genetics, University of Bonn, School of Medicine & University

Hospital Bonn, Germany;

8Sahlgrenska Cancer Center, University of Gothenburg, Sweden

Authors contributed equally

Running title: PI3K/Akt signaling in myxoid liposarcoma

Key words: myxoid liposarcoma, FUS-DDIT3, PI3K, PIK3CA, BKM120, Buparlisib

§ Correspondence should be addressed to:

Marcel Trautmann & Wolfgang Hartmann; Division of Translational Pathology,

Gerhard-Domagk-Institute of Pathology, Münster University Hospital, Germany

Phone: +49 (0) 251-83-57623 and -58479; Fax: +49 (0) 251-83-57559

E-mail: [email protected]; [email protected]

The authors declare no potential conflicts of interest.

on March 23, 2020. © 2019 American Association for Cancer Research. mct.aacrjournals.org Downloaded from

Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on February 20, 2019; DOI: 10.1158/1535-7163.MCT-18-0763

http://mct.aacrjournals.org/

PI3K/Akt signaling in myxoid liposarcoma

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ABSTRACT

Myxoid liposarcoma is an aggressive soft tissue tumor characterized by a specific

reciprocal t(12;16) translocation resulting in expression of the chimeric FUS-DDIT3

fusion protein, an oncogenic transcription factor. Similar to other

translocation-associated sarcomas, myxoid liposarcoma are characterized by a low

frequency of somatic mutations, albeit a subset of myxoid liposarcoma has previously

been shown to be associated with activating PIK3CA mutations. The present study was

performed to assess the prevalence of PI3K/Akt signaling alterations in myxoid

liposarcoma and the potential of PI3K-directed therapeutic concepts.

In a large cohort of myxoid liposarcoma, key components of the PI3K/Akt

signaling cascade were evaluated by next-generation sequencing, fluorescence in situ

hybridization and immunohistochemistry. In three myxoid liposarcoma cell lines, PI3K

activity was inhibited by RNAi and the small molecule PI3K inhibitor BKM120

(Buparlisib) in vitro. A myxoid liposarcoma cell line based avian chorioallantoic

membrane model was applied for in vivo confirmation.

In total, 26.8% of myxoid liposarcoma cases displayed activating alterations in

PI3K/Akt signaling components, with PIK3CA gain-of-function mutations representing

the most prevalent finding (14.2%). Immunohistochemistry pointed to PI3K/Akt activation

in a far larger subgroup of myxoid liposarcoma, implying alternative mechanisms of

pathway activation. PI3K-directed therapeutic interference showed that myxoid

liposarcoma cell proliferation and viability significantly depended on PI3K-mediated

signals in vitro and in vivo.

Our preclinical study underlines the elementary role of PI3K/Akt signals in myxoid

liposarcoma tumorigenesis and provides a molecularly based rationale for a

PI3K-targeted therapeutic approach which may be particularly effective in tumors

carrying activating genetic alterations in PI3K/Akt signaling components.





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INTRODUCTION

Myxoid liposarcoma (MLS) account for approximately 5-10% of all soft tissue sarcomas,

representing about 20% of all malignant adipocytic tumors (1). MLS constitute the most

frequent liposarcoma subtype in patients below the age of 20 years. A high rate of local

recurrence and particular propensity to develop distant metastases is reported in

approximately 40% of MLS patients (2). MLS represent a morphological spectrum

ranging from myxoid tumors with low cellularity to hypercellular, round cell sarcomas

associated with an aggressive clinical course (3). Genetically, 95% of MLS are

characterized by a chromosomal t(12;16)(q13;p11) translocation, juxtaposing the FUS

and DDIT3 genes. The remaining 5% display an alternative chromosomal

t(12;22)(q13;q12) rearrangement leading to an EWSR1-DDIT3 gene fusion (4). The

resulting FUS-DDIT3 and EWSR1-DDIT3 fusion proteins have been shown to play an

essential role in MLS tumorigenesis, acting as pathogenic transcriptional (dys-)

regulators (5-8). Current therapeutic approaches in high-grade MLS complement radical

surgery and adjuvant radiation and/or conventional chemotherapy based on

anthracyclines and ifosfamide, recently supplemented by agents such as Trabectedin or

Eribulin (9-11). Although MLS display a higher chemosensitivity than other liposarcoma

subtypes, the substantial frequency of recurrence and metastasis in MLS underlines the

urgent need for novel, biology-guided therapeutic strategies. In principle, counteracting

the effects of the aberrant FUS-DDIT3 transcription factor represents the most promising

strategy to selectively target MLS tumor cells; however, the therapeutic interference with

(chimeric) transcription factors in vivo represents a particular challenge.

In line with the situation in other translocation-associated soft tissue and bone

sarcomas driven by specific gene fusions, e.g. Ewing sarcoma, synovial sarcoma or

alveolar rhabdomyosarcoma (12-14), MLS are genomically “stable” tumors with few

mutations beyond the driving gene fusion. However, two recent studies described

activating gain-of-function mutations in the PIK3CA gene encoding the catalytic PI3K

subunit in a subset of MLS. Demicco and colleagues provided evidence of PTEN losses

occurring as further structural alteration of crucial PI3K/Akt pathway components (12,

15). Beyond that, it has been reported that EGFR, MET, VEGFR1, RET and PDGFRB

signaling sustained by autocrine/paracrine transduction and receptor tyrosine kinase

(RTK) cross-talk led to an increase in PI3K-dependent signals (16, 17). The PI3K/Akt





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cascade is an elementary hub in the signal transduction of diverse RTK stimuli involving

different growth-controlling regulators such as GSK-3β as well as the cell cycle effectors

Cyclin D1 and p21WAF (18-21).

Overall, previously published results indicate a fundamental importance of

PI3K/Akt signals as specific liability of MLS tumor cells, accomplished either by genetic

PIK3CA and/or PTEN alterations or complex signaling networks (8, 12, 15-17, 22) that

all involve an aberrant activation of PI3K activity. The present preclinical study was

conducted to explore the functional importance of PI3K/Akt signals in MLS

tumorigenesis, and to establish a molecularly targeted strategy employing small

molecule PI3K inhibitors.





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MATERIALS AND METHODS

Tumor specimens and tissue microarray (TMA)

MLS tumor specimens were selected from the archive of the Gerhard-Domagk-Institute

of Pathology (Münster University Hospital, Germany). All diagnoses were reviewed by

three experienced pathologists (EW, SH, WH) based on morphological criteria, DDIT3

break-apart fluorescence in situ hybridization (FISH) or reverse transcriptional PCR

(RT-PCR) analysis in accordance to the current WHO classification of tumours of Soft

Tissue and Bone (1). Scoring of the round cell component (3, 23) and TMA

sampling/construction was performed as described before (8). In total, 56 MLS tumor

specimens were included (24/42.9% female, 32/57.1% male). Median patients age at

diagnosis was 48 years (range 24-73 years of age). Median tumor size was 10 cm

(range 1.5-29 cm). Clinicopathological characteristics of the cohort are summarized in

Table 1 and Supplementary Table S1. The study was approved by the Ethics review

board of the University of Münster (2015-548-f-S), conducted in accordance with

recognized ethical guidelines (Declaration of Helsinki, 1975) and written informed

consent from patients was obtained.

Immunohistochemistry (IHC)

Following primary antibodies were applied: phospho-Akt (S473, monoclonal rabbit, D9E,

1:50, #4060), Cyclin D1 (monoclonal rabbit, 92G2, 1:50, #2978), phospho-GSK-3α/β

(S21/9, polyclonal rabbit, 1:50, #9331), p21 (monoclonal rabbit, 12D1, 1:1000, #2947)

(all purchased from Cell Signaling Technology). IHC staining was performed with a

BenchMark ULTRA Autostainer (VENTANA/Roche) on 3 μm tissue sections as

described before (8). IHC results were evaluated by a semi-quantitative approach (H-

score; ranging from 0-300) determining the percentage of cells at each staining intensity

level, using following formula: [1 × (% cells 1+) + 2 × (% cells 2+) + 3 × (% cells 3+)].

Immunoreactivity was assessed defining the staining intensity (0, negative; 1+, weak;

2+, moderate; 3+, strong) in the positive control (breast cancer; no special type) as

strong. TMA tissue cores with H-score >50 were considered positive. The discriminatory

threshold (positive = semi-quantitative H-score >50) was pre-specified without prior

analyses of the clinical course. Loss of PTEN protein was analyzed by IHC as previously

described (8, 15). The IHC readers were blinded to the outcome data.





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Fluorescence in situ hybridization (FISH)

PIK3CA gene amplification was examined by FISH analyses using the SPEC

PIK3CA/CEN 3 Dual Color probe (ZytoVision). At least 40 cells of each MLS tumor

specimen were assessed. PIK3CA gene amplification was defined by a

PIK3CA/centromere 3 (CEN3) ratio ≥2.0.

Next-generation sequencing (NGS) / Sanger sequencing

A customized GeneRead DNAseq panel (Mix-n-Match V2, Qiagen) was applied to

assess the complete exonic region of 23 genes that are frequently mutated in various

cancer types (summarized in Supplementary Table S2). Target enrichment was

performed by means of the GeneRead DNAseq PCR V2 Kit (Qiagen). All end repair,

A-addition and DNA barcode ligation steps were processed applying the GeneRead

DNA Library I Core Kit (Qiagen). Amplification of adapter-ligated DNA was performed

using the GeneRead DNA I Amp Kit (Qiagen). NGS was conducted with the Illumina

MiSeq system. The Quantitative Multiplex FFPE Reference Standard (Horizon

Discovery, #HD200) was applied as isogenic quality control (11 somatic mutations

verified at 0.8-24.5% allelic frequency in genomic DNA) for routine performance

monitoring and evaluation of NGS workflow integrity (pre-analytical DNA extraction,

NGS workflow and post-analytical bioinformatics). NGS data was analyzed by means of

the CLC Biomedical Genomics Workbench software (CLC bio, Qiagen). Validation by

Sanger sequencing was performed using the BigDye Terminator v3.1 Cycle Sequencing

Kit (Life Technologies).

In silico tools to predict the deleterious impact of detected gene variants

The functional context of NGS-detected variants in the coding regions was predicted

using following in silico tools: PolyPhen-2 (v2.2.2r398) (24), Protein Variation Effect

Analyzer (PROVEAN; v1.1.3) (25), Sorting Intolerant From Tolerant (SIFT; Ensembl 66)

(26), Mutation Assessor (release 3) (27) and Combined Annotation Dependent

Depletion (CADD; v.1.3) (28). In brief, the PolyPhen-2 method utilizes evolutionary and

physical comparative considerations to predict amino acid substitutions on protein

structure and function. Scoring ranges from 0 (neutral) to 1 (deleterious) and functional

significance is categorized into benign, possibly damaging, and probably damaging. The





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web-based PROVEAN algorithm classified each single nucleotide variant (SNV) as

either neutral or deleterious (cut-off -2.5). SIFT was used with the default settings

classifying variants from 0 (damaging) to 1 (tolerated). CADD is a trained method to

differentiate 14.7 million high-frequency human-derived alleles for objective integration

of diverse annotations into a single measure (C score). Scoring correlates with allelic

diversity, annotations of pathogenicity, disease severity, experimentally measured

regulatory effects and complex trait associations. Calculated C scores rank a pathogenic

variant relative to all possible substitutions of the human genome. To increase the

prediction accuracy and the level of confidence, a combination of in silico tools based on

evolutionary information, protein structure and functional parameters was used. We

combined the individual output from ≥3 of 5 in silico prediction tools and defined a single

consensus outcome summarized in Supplementary Table S3.

Cell culture and cell lines

The human MLS cell lines MLS402-91 (FUS-DDIT3 exon 7-2; type 1), MLS2645-94

(FUS-DDIT3 exon 5-2; type 2), and MLS1765-92 (FUS-DDIT3 exon 13-2; type 8) were

contributed by Pierre Åman (29). For the purpose of cell line authentication, presence of

the MLS specific FUS-DDIT3 fusion gene transcripts was confirmed by RT-PCR and

Sanger Seqeuncing. All monolayer cell cultures were maintained in Roswell Park

Memorial Institute medium 1640 (RPMI), supplemented with 10% fetal bovine serum

(FBS; Life Technologies) and incubated under standard condition (humidified

atmosphere, 5% CO2, 37°C). Cells were passaged for a maximum of 20-30 culturing

cycles and mycoplasma testing was performed quarterly by standardized PCR. To study

the biological effects of treatment with the class I PI3K inhibitor BKM120 (30, 31) and

the prototypic pan-PI3K inhibitor LY294002 (32, 33), MLS cells were cultured in medium

supplemented with 2% FBS. For experimental PI3K modulation, MLS402-91 cells were

transfected with pCMV2-Tag 2A plasmids encoding either the mutant ΔH1047R PIK3CA

(Addgene #16639) or wild type PIK3CA (Addgene #16643) coding sequence (34).

Immunoblotting

Following primary antibodies were used: Akt (monoclonal rabbit, C67E7, 1:1000,

#4691), phospho-Akt (S473, monoclonal rabbit, D9E, 1:1000, #4060), Cyclin D1

(monoclonal rabbit, 92G2, 1:1000, #2978), GSK-3β (monoclonal rabbit, 27C10, 1:1000,





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#9315), phospho-GSK-3β (S9, polyclonal rabbit, 1:1000, #9336), PI3K p110α (rabbit,

1:1000, #4255), p21 (monoclonal rabbit, 12D1, 1:1000, #2947) (all purchased from Cell

Signaling Technology), and β-actin (loading control; monoclonal mouse, AC15,

1:10.000, #A5441 Sigma-Aldrich). Secondary antibody labeling (Bio-Rad Laboratories)

and immunoblot development was performed using an enhanced chemiluminescence

detection kit (SignalFire ECL Reagent; Cell Signaling Technology) and the Molecular

Imager ChemiDoc system (Image Lab Software; Bio-Rad Laboratories).

Cell viability assay (MTT)

MTT cell proliferation assays (Roche) were performed as described before (35-37). In

brief, 2.5×103 MLS402-91, MLS2645-94, and MLS1765-92 cells were seeded in 96-well

cell culture plates (100 µl of medium supplemented with 2% FBS) and exposed to

increasing concentrations of the class I PI3K inhibitor BKM120 (1.25-5 µmol/L) and the

prototypic pan-PI3K inhibitor LY294002 (3.125-50 µmol/L) for 72 h. DMSO was included

as vehicle control.

In vivo efficacy of BKM120 and LY294002 in MLS chick chorioallantoic membrane

(CAM) studies

For in vivo confirmation, we used the chick chorioallantoic membrane (CAM) model as

previously reported and validated for anticancer agents (8, 38, 39). Due to its

immunodeficiency and vascular supply, the CAM enables the transplantation of human

cancer cells and the subsequent development of solid tumor xenografts in a

3-dimensional in vivo microenvironment. The CAM model matches the 3R

recommendations to reduce mammalian animal experiments and is regarded as

reproducible, reliable, and effective (40).

Seven days after fertilization, MLS402-91 and MLS1765-92 myxoid liposarcoma cells

(1-1.5x106 cells/egg; dissolved in medium/matrigel 1:1, v/v) were xenografted onto the

CAM and incubated with 60% relative humidity at 37°C. Topical treatment with the class

I PI3K inhibitor BKM120 (1-2 µmol/L), the prototypic pan-PI3K inhibitor LY294002

(10 µmol/L) or vehicle control (0.2% DMSO in NaCl 0.9%) was initiated on day 8 and

recapitulated for two consecutive days. Three days after treatment initiation,

tumor-bearing CAM xenografts were imaged, explanted and fixed (5% PFA). Tumor

volume (mm3) was calculated according to the formula: TV= length (mm) x width² (mm)





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x π/6. The in vivo studies were directly approved by an Institutional Animal Care and

Use Committee (84-02-04-2016-A195) and conducted in accordance with recognized

standards of the National and European Union guidelines.

Compounds

The class I PI3K inhibitor BKM120 (Buparlisib/NVP-BKM120; C18H21F3N6O2;

CAS#: 944396-07-0) (30, 31) was purchased from Selleck Chemicals and dissolved in

dimethyl sulfoxide (DMSO; Sigma-Aldrich). The prototypic pan-PI3K inhibitor LY294002

(C19H17NO3; CAS#: 154447-36-6) (32, 33) was purchased from Cell Signaling

Technology and dissolved in DMSO. Final DMSO concentration did not exceed

0.2% (v/v) for all in vitro and in vivo experiments.





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RESULTS

Expression of PI3K/Akt signaling components in human MLS tissue specimens

and MLS cell lines

To determine the involvement of PI3K/Akt signaling in MLS tumorigenesis, expression of

phospho-Akt (S473), phospho-GSK-3α/β (S21/9), p21 and Cyclin D1 was analyzed in a

set of 56 MLS tissue specimens by immunohistochemistry (Figure 1A-B and

Supplementary Table S1). Based on the calculated H-score, 62.0% of MLS tumor

specimens were positive for phosphorylated Akt (S473) and 38.5% for phosphorylated

GSK-3α/β (S21/9). No significant Akt and/or GSK-3β phosphorylation levels were

detected in 23.2% of the cases. 32.7% of MLS specimens were positive for p21, while

expression of Cyclin D1 was shown in 48.1% (Figure 1A and 1B). Loss of PTEN protein

expression was demonstrated in 5/56 (8.9%) MLS tissue specimens (Supplementary

Table S1). Phosphorylation and expression levels of PI3K/Akt signaling components did

not correlate with tumor size, FUS-DDIT3 translocation subtype, patients’ age and/or

gender. In accordance with the immunohistochemical results in MLS tissue specimens,

protein expression (PI3K p110α, p21 and Cyclin D1) and phosphorylation levels

(phospho-Akt S473 and phospho-GSK-3β S9) were demonstrated in total protein

extracts of MLS402-91 (FUS-DDIT3 type 1), MLS2645-94 (FUS-DDIT3 type 2), and

MLS1765-92 (FUS-DDIT3 type 8) cell lines (Figure 1C, upper panel). Expression of the

specific FUS-DDIT3 fusion transcript was confirmed in all three MLS cell lines by

RT-PCR (Figure 1C, lower panel). These findings provide evidence that activation of

PI3K/Akt signaling is a common feature of MLS.

Mutational profiling of MLS

As pathogenic PIK3CA mutations can be responsible for constitutive activation of the

PI3K/Akt signaling cascade, we examined the entire PIK3CA coding region by targeted

NGS followed by Sanger sequencing for validation purposes. In 12 out of 56 (21.4%)

MLS cases, genetic alterations in PIK3CA (most frequently H1047R) could be detected

(Figure 2A, Supplementary Table S1). Overall, 55.6% (5/9) of the observed variants in

the PIK3CA coding region (N345K, C378Y, C901F, H1047R and N1068fs) were

predicted to have a deleterious impact by ≥3 independent in silico tools (summarized in

Figure 2B and Supplementary Table S3). No PIK3CA gene mutations were identified in





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all three MLS cell lines. PIK3CA gene amplification was examined by FISH and could be

excluded for all analyzed MLS cases. Additionally, the coding sequence of AKT1 and

PTEN was analyzed. Few deleterious variants were identified in both genes: AKT1

(E17K and W80R) and PTEN (V119F and I253fs). Notably, round cell MLS (3/6; 50.0%)

were more likely than myxoid MLS tumors (5/13; 38.5%) to harbor a deleterious

mutation in the PIK3CA gene. In contrast, all deleterious mutations in AKT1 (E17K and

W80R) and PTEN (V119F and I253fs) were exclusively identified in myxoid tumors. In

Kaplan-Meier correlations, MLS patients with round cell morphology (Figure 2C, left

panel; **p=0.0014) and activating alterations in PI3K/Akt signaling components

(Figure 2C, right panel; **p=0.0071) displayed a significantly reduced overall/event free

survival. In addition, all coding exons of 20 further cancer-associated genes were

analyzed with another 11 actionable non-synonymous mutations detected in 10 MLS

cases (summarized in Supplementary Table S2 and S3): CTNNB1 (P321H), ERBB2

(R138W), FGFR3 (T311M), GNAQ (M59L), GNAS (P533R), and TP53 (V272G) (allelic

frequency: 10.5-90.3%). In summary, 28 non-synonymous somatic variants were

identified in 25/56 (44.6%) MLS tumor specimens. The overall concordance between all

5 in silico prediction tools was 52.2% and independent of the deleterious/damaging or

neutral/tolerated status. At least 4 out of 5 in silico predictions were in agreement in

34.8% of variants and in only 13.0% of variants was the prediction based on 3 out of 5

in silico tools (summarized in Supplementary Table S3). Correlating the (global)

presence of gene mutations with MLS tumor morphology, no significant trend

concerning the mutational burden could be determined comparing the round cell to the

myxoid MLS subtype which was evaluated as a well-established prognostically relevant

histologic parameter (1, 3, 23). In total, gene mutations were detected in 9/21 (42.8%)

round cell MLS versus 16/35 (45.7%) myxoid tumors. Overall, 85.7% of MLS patients

with alterations in PIK3CA, AKT1 or PTEN were positive for phosphorylated Akt (S473)

and/or GSK-3α/β (S21/9) as examined by immunohistochemistry and regarded as

indicators of an activation of PI3K/Akt signaling. In the set of PIK3CA/AKT1/PTEN wild

type MLS patients, the fraction of tumors immunohistochemically displaying either

phosphorylation of Akt and/or GSK-3β was substantially lower (69.4%).





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RNA interference (RNAi)-mediated depletion of PIK3CA affects PI3K/Akt signaling

activity and cell viability in MLS cells

To confirm the biological importance and to analyze the functional role of PI3K/Akt

signaling by a non-pharmacological approach, three established MLS cell lines

(MLS402-91, MLS2645-94 and MLS1765-92) were transfected with two different

pre-validated siRNAs (to exclude unspecific off-target effects) directed against human

PIK3CA. Consistently, specific knockdown of PIK3CA led to reduced phosphorylation

levels of Akt (S473) and GSK-3β (S9), combined with diminished Cyclin D1 downstream

target expression (Figure 3A). In MTT assays, all MLS cell lines displayed a significant

reduction of cell viability (***p<0.001) in comparison with non-targeting control siRNA

(Figure 3B). These results imply that PI3K/Akt-mediated signals may be of major

functional relevance in the pathogenic context of MLS.

BKM120 reduces MLS cell viability in vitro

Our results suggested that aberrant PI3K/Akt signaling might be a therapeutic target in

MLS. We therefore evaluated the biological effects of pharmacological PI3K inhibition. In

MTT assays, three MLS cell lines were exposed to increasing concentrations

(1.25-5 µmol/L) of the class I PI3K inhibitor BKM120. Cell viability and growth was

significantly suppressed in all three MLS cell lines with IC50 values ranging from

1.01 to 1.84 µmol/L (BKM120), indicating a dose-dependent mode of action (Figure 3C,

Table 2). Analogue experiments with the prototypic pan-PI3K inhibitor LY294002

(3.125-50 µmol/L) showed comparable effects with IC50 values ranging from 9.57 to

12.02 µmol/L (Supplementary Figure S1A)

BKM120 inhibits PI3K/Akt signal transduction activity in vitro

To examine the functional basis of PI3K/Akt suppression, we analyzed the specific

effects of treatment with the class I PI3K inhibitor BKM120 on the activation status of

PI3K/Akt signaling components by immunoblotting. The growth-inhibitory effects were

associated with a significant dose-dependent reduction of phosphorylation levels for Akt

(S473) and GSK-3β (S9) in all three MLS cell lines (Figure 3D). Analogue effects were

observed with the prototypic pan-PI3K inhibitor LY294002 (Supplementary Figure S1B).





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BKM120 reduces MLS cell viability by induction of apoptosis

Performing flow cytometric analyses, poly-adenosine diphosphate (ADP)-ribose

polymerase (cPARP; Asp214) cleavage was employed as a marker of apoptosis. After

treatment with increasing concentrations of BKM120 (1-2 µmol/L; 72 h), all three MLS

cell lines showed a significantly and dose-dependently increased rate of apoptosis

(Figure 3E and F).

In vivo efficacy of PI3K inhibition in CAM models of MLS

Anti-tumor efficacy of the class I PI3K inhibitor BKM120 on tumor growth was tested in a

previously established in vivo model of human MLS (8). We xenografted MLS402-91

and MLS1765-92 cells onto chick CAMs to initiate MLS tumor formation (n≥5 eggs per

group). A significant reduction of MLS tumor volume through topical administration of

BKM120 (1-2 µmol/L) compared to the DMSO vehicle-treated control group (Figure 3G)

was demonstrated for two different MLS cell lines. Analogue experiments with the

prototypic pan-PI3K inhibitor LY294002 (10 µmol/L) resulted in comparable effects

(Supplementary Figure S1C). Collectively, these results showed that inhibition of the

PI3K/Akt signaling cascade impairs the maintenance of MLS tumors in vivo, further

supporting the concept that PI3K/Akt pathway activation represents a novel, molecularly

based target for the therapeutic intervention in advanced high-grade MLS patients.

Mutational PIK3CA status in MLS cells alters the apoptotic effects in response to

BKM120 treatment

To assess the biological effects of ΔH1047R-mutated PIK3CA in response to BKM120

treatment, MLS402-91 cells transfected with pCMV2-Tag 2A plasmid DNA encoding

different PI3K p110 catalytic subunit alpha variants were exposed to BKM120 (2 µmol/L;

DMSO was employed as vehicle control). Significantly increased rates of apoptosis were

detected applying two different markers of apoptosis: (i) caspase 3/7 activity (Figure 4A)

and (ii) poly-adenosine diphosphate (ADP)-ribose polymerase (cPARP; Asp214)

cleavage (Figure 4B). Consistently, MLS402-91 cells expressing ΔH1047R-mutated

PIK3CA showed increased rates of apoptosis upon treatment with BKM120 compared to

cells expressing wild type PIK3CA.





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DISCUSSION Myxoid liposarcomas are malignant tumors of adipocyte origin with a high rate of local

recurrence and particular propensity to develop distant metastases. High histological

grade, defined as round cell component >5% is the primary predictor of an unfavorable

clinical outcome (3, 23). While there is an established role for conventional cytotoxic and

radiotherapy therapies in MLS (10), molecularly targeted therapeutic protocols are not

implemented so far. Almost all MLS are characterized by a FUS-DDIT3 gene fusion,

encoding an oncogenic transcription factor with the potential to transform mesenchymal

stem cells to form MLS in mice (5). As in other sarcomas driven by specific gene

fusions, e.g. Ewing sarcoma, synovial sarcoma or alveolar rhabdomyosarcoma (12-14),

MLS are genomically “stable” tumors with a low mutational burden. However, two recent

studies described recurrent PIK3CA gene mutations in a subset of 14-18% MLS cases

beyond the pathognomonic FUS-DDIT3 gene fusion (12, 15). Additionally, PTEN loss

and IGF-IR overexpression have been described which both represent alternative

mechanisms of PI3K/Akt signal transduction activation (8, 15, 41). Beyond that, it has

been reported that EGFR, MET, VEGFR1, RET and PDGFRB signaling sustained by

autocrine/paracrine transduction and RTK cross-talk involving the extensive tumor

vasculature of MLS led to an additional increase in PI3K/Akt signaling pathway activity

(16, 17). Both, IGF-IR overexpression and PIK3CA mutations appear to be

overrepresented in the round cell variant of MLS (15, 41). Given the fact that (i) round

cell-ness, (ii) presence of an activating PIK3CA mutation and (iii) IGF-IR overexpression

are associated with an unfavorable clinical course, overactivation of PI3K/Akt signals

may represent a crucial factor for a more aggressive biology of MLS (3, 12, 15, 41).

Unfortunately, the limited size of our MLS tissue set and the retrospective nature of our

analysis prevented a definitive evaluation of the contribution of each of these factors to

MLS prognosis; however, this interesting issue might be worth to be systematically

analyzed in future prospective trials.

We previously provided substantial evidence that therapeutic interference with a

cell-autonomous stimulation loop involving IGF-IR-dependent signals may represent a

highly effective approach guiding therapeutic benefit which is specifically founded in the

tumor´s biology involving FUS-DDIT3-mediated overexpression of IGF-II (8). However,

this therapeutic strategy applies only to those tumors in which PI3K/Akt signaling is not





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activated through mutations of central components as PIK3CA, AKT1 and/or PTEN. We

therefore hypothesized that central pathway interference through PI3K inhibition might

constitute a rational therapeutic alternative since it biologically covers different modes of

pathogenic PI3K/Akt activation.

Our mutational study, identifying activating gain-of-function PIK3CA mutations in

14.2% of MLS concordantly confirms published results (12, 15), while genomic

amplifications of PIK3CA could be excluded. In line with data presented by Barretina

and colleagues (12), we identified PIK3CA mutations to be associated with a negative

prognostic impact. Notably, round cell MLS (50.0%) were more likely than myxoid MLS

tumors (38.5%) to harbor a deleterious mutation in the PIK3CA gene. Additionally, we

here detected deleterious AKT1 (E17K and W80R) and PTEN (V119F and I253fs)

alterations which were bioinformatically predicted to be of functional relevance; these

were exclusively detected in myxoid tumors. In agreement with Demicco et al. (15), we

found 8.9% of the analyzed cases to be negative for PTEN, pointing either to a genetic

loss or loss of expression e.g. through promotor hypermethylation (8, 42, 43). All

deleterious mutations found in PIK3CA, AKT1 and PTEN were mutually exclusive,

resulting in a total fraction of 26.8% of MLS tumors with evidence of an activated

PI3K/Akt signaling pathway caused by genetic/epigenetic alterations. The few additional

mutations in classic oncogenic drivers detected in our genomic screen were randomly

spread among all MLS cases and appear to be secondary passenger alterations rather

than crucial oncogenic drivers in MLS tumorigenesis. Given further kinase signaling

input in MLS e.g. via the IGF-IR system as well as the effector loop discussed by Negri

and colleagues (8, 17), it is not surprising that the fraction of tumors

immunohistochemically displaying either phosphorylation of Akt and/or GSK-3β (both

regarded as indicators of an activation of PI3K/Akt signaling) was substantially higher

than 26.8%. With regard to the prognostic impact of PIK3CA mutations as shown in our

study and the previous works published by Barretina and Demicco (12, 15) it appears

improbable that these alterations represent mere passenger mutations, while it remains

to be elucidated in which way aberrant PI3K signaling contributes to MLS pathogenesis

which essentially depends on the FUS-DDIT3 gene fusion. Given our findings, it

appeared rational to extend the study to preclinically evaluate the therapeutic potential

of a PI3K/Akt-directed approach, which up to now has not been assessed in the context

of MLS.





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Considering the therapeutic accessibility of PI3K/Akt signaling, PI3K enzymes

themselves represent the central hub of the signaling cascade as they serve as

integrators and (enhancing) distributors of diverse signaling cues, and the development

of inhibitory molecules is certainly most advanced for PI3K as therapeutic target.

Combining pharmacological studies and RNAi-based approaches, our study

convincingly shows in vitro that proliferation and viability of MLS cells significantly

depend on PI3K-mediated signals. Importantly, we were able to show that expression of

the ΔH1047R-mutated PIK3CA variant in MLS402-91 was associated with an increased

sensitivity to BKM120. Consistent with our in vitro findings, administration of BKM120

and LY294002 to xenografted MLS402-91 or MLS1765-92 cells led to a significant

suppression of MLS tumor growth in vivo. While these findings are consistent and imply

a potential therapeutic role of PI3K inhibitory approaches in MLS, we are aware of the

limitations of the employed avian CAM model as an in vivo tool in the preclinical transfer.

However, apart from a published patient derived xenograft (PDX) model which was not

available to us (44), establishment and propagation of cell line-based MLS

xenotransplants have not been successful to our knowledge which lead to our choice of

the (artificial) CAM system. It would, of course, be rational to try to transfer our findings

to a mammalian in vivo system and to elaborate on combinations of diverse therapeutic

agents including PI3K inhibitors. The results presented by Qi and colleagues showing

efficacy of the PI3K inhibitor PF-04691502 in a PIK3CA mutated MLS PDX model (44),

(44), support the relevance of our findings.

Given its particular dependence on kinase signals, MLS somehow resembles a

genetically different mesenchymal neoplasia as gastrointestinal stromal tumors (GIST)

or dermatofibrosarcoma protuberans (DFSP). These tumors are driven either by

activating mutations in the receptor tyrosine kinases KIT/PDGFRA or a

translocation-dependent overexpression of the PDGFR ligand PDGFB (45, 46).

However, PI3K-directed therapeutic approaches were not established in these tumors

as effective inhibition of the pathogenic signal and clinical benefit were specifically

realized through interference with the activated KIT and PDGFR RTKs employing the

tyrosine kinase inhibitor imatinib (46-48). In the context of MLS, the situation is more

complex, as the primary tumor-driving oncogene is the chimeric FUS-DDIT3

transcription factor, and only a subset of about 30% MLS cases is characterized by

additional activating alterations in central components of the PI3K/Akt signaling





- 17 -

pathway. However, given our previously reported finding of a FUS-DDIT3 driven

cell-autonomous stimulation of MLS cells involving an IGF-IR/-(PI3K/Akt) transactivation

loop, MLS appear to be a PI3K/Akt-dependent neoplasia in a much larger fraction.

Given the diversity of activation modes of PI3K/Akt signal transduction in MLS

tumorigenesis and regarding the fact that IGF-IR-driven tumors with higher probability

involve signaling cascades in parallel to PI3K/Akt, the identification of appropriate

predictive biomarkers once more becomes the crucial issue in the establishment of

molecularly targeted therapeutic approaches. In MLS, PIK3CA, AKT1 and PTEN

mutational testing as well as PTEN immunohistochemistry/FISH may help to identify

those patients which most probably may clinically benefit from a PI3K-directed

therapeutic approach. In patients without alterations in these components a (combined)

IGF-IR-directed therapeutic approach might be more beneficial as documented

IGF-IR-driven signals also involve PI3K/Akt-independent intracellular signaling pathways

as the MEK/ERK cascade (8, 41). Despite promising preclinical data, clinical trials

testing diverse PI3K inhibitory approaches in different solid tumors showed a limited

efficacy of monotherapy inhibition (49, 50). This may be due to insufficient target

inhibition which may be increased by optimized small molecule structures, but also to

the fact that in many solid tumors, activating mutations in components of the PI3K/Akt

signaling cascade occur as one genetic alteration among many others, entering the

discussion on driver and passenger mutations. In MLS, the situation differs from the vast

majority of cancers in so far as activating mutations in components of the PI3K/Akt

signaling cascade appear to represent the outstanding alteration apart from the

FUS-DDIT3 gene fusion against the background of genetic stability. This characteristic

feature of MLS underlines the potential of PI3K inhibitory approaches and should

promote systematic biomarker-guided clinical trials.

In conclusion, the results of this study imply that (apart from the pathognomonic

FUS-DDIT3 gene fusion) activation of PI3K/Akt signaling is an essential pattern inMLS

tumorigenesis which is realized, at least in part, by genetic alterations in PIK3CA, AKT1

or PTEN. Interference with PI3K-mediated signals viasmall molecule compounds is an

effective therapeuticstrategy for advanced high-grade MLS that could be exploited for

clinical benefit. The present preclinical testing ofa PI3K-targeted therapeutic approach





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indicates potent effects both in vitro and in vivo, qualifying the PI3K/Akt signaling

pathway as a molecularly based target in MLS cancer therapy.

ACKNOWLEDGEMENTS

The authors thank Charlotte Sohlbach and Inka Buchroth for excellent technical support.

The study was supported by grants from the Deutsche Forschungsgemeinschaft (DFG,

W. Hartmann and M. Trautmann; HA4441/2-1), the Wilhelm-Sander-Stiftung (W.

Hartmann, M. Trautmann and K. Steinestel; 2016.099.1) and the "Innovative Medical

Research" funding program of the University of Münster Medical School (IMF; M.

Trautmann; TR121716 and I-TR221611, M. Trautmann and S. Huss; I-HU121421).





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TABLES

Table 1. Clinicopathological characteristics of myxoid

liposarcoma patients and tumors (n=56)

Age (years)

Mean (±SD) 48.1 (±12.2)

Median (range) 48 (24-73)

<48 26 (46.4%)

≥48 30 (53.6%)

Type

Primary tumor 35 (62.5%)

Metastasis 9 (16.1%)

Recurrence 7 (12.5%)

ND 5 (8.9%)

Morphology

Myxoid 35 (62.5%)

Round cell 21 (37.5%)

Size (cm)

Mean (±SD) 10.6 (±5.7)

Median (range) 10.0 (1.5-29)

<10 18 (32.1%)

≥10 25 (44.6%)

ND 13 (23.2%)

Sex

Female 24 (42.9%)

Male 32 (57.1%)

FISH

DDIT3 break apart positive 56 (100%)

t(12;16) translocation type

FUS-DDIT3 (type 1; exon 7-2) 11 (19.6%)

FUS-DDIT3 (type 2; exon 5-2) 26 (46.4%)

ND 19 (33.9%)

SD, standard deviation; ND, not determined; FISH, fluorescence in situ hybridization.





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Table 2. IC50

values for the class I PI3K inhibitor BKM120 and the prototypic pan-

PI3K inhibitor LY294002 analyzed in three myxoid liposarcoma cell lines

Compound

IC50

(µmol/L)

MLS402-91

(FUS-DDIT3 type 1)

MLS2645-94

(FUS-DDIT3 type 2)

MLS1765-92

(FUS-DDIT3 type 8)

BKM120

(Buparlisib) 1.46 ± 0.08 1.01 ± 0.13 1.84 ± 0.12

LY294002 9.57 ± 0.35 12.02 ± 1.86 11.96 ± 1.72

Cytotoxic effects on myxoid liposarcoma (MLS402-91, MLS2645-94 and MLS1765-92) cell viability were assessed in MTT assays (72 h). Results are represented as mean ± SEM of at least three independent experiments performed in quintuplicates.





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FIGURE LEGENDS

Figure 1. Activation of PI3K/Akt signaling in a comprehensive cohort of myxoid

liposarcoma tissue specimens (n=56) and cell lines.

(A) Immunohistochemical staining shows strong phosphorylation levels of Akt (S473)

and GSK-3α/β (S21/9) as well as increased expression levels of p21 and Cyclin D1 in a

representative case of MLS (original magnification: x20, inset x40). (B)

Immunohistochemical spectrum of MLS tissue specimens summarized as positive IHC

reactivity (H-score >50). (C) Immunoblotting results demonstrate expression and

phosphorylation levels of PI3K/Akt signaling components in total protein extracts of three

different MLS cell lines. Detection of FUS-DDIT3 fusion gene transcripts in MLS402-91

(type 1; exon 7-2), MLS2645-94 (type 2; exon 5-2), and MLS1765-92 (type 8; exon 13-2)

cells by RT-PCR (28S rRNA employed as loading reference; lower panel).

Figure 2. Mutational profile of myxoid liposarcoma.

(A) Detected variants in the coding regions of 23 cancer-associated genes. Mutations in

different genes (rows) are indicated for each MLS case (columns). In silico predictions

are illustrated as: green (neutral/tolerated) or red (deleterious/damaging).

Clinicopathological information is summarized according to the legend. (B) Distribution

of detected PIK3CA mutations annotated in the PI3K p110 catalytic subunit alpha

protein domains (red: deleterious/damaging). (C) In Kaplan-Meier correlations,

overall/event free survival is significantly reduced in MLS tumors with a round cell

content >5% compared to the myxoid subtype (**p=0.0014, left panel) and in association

with activating alterations in PI3K/Akt signaling components (**p=0.0071, right panel).

Figure 3. In vitro and in vivo evaluation of PI3K suppression (BKM120) in myxoid

liposarcoma cell lines.

(A) In three MLS cell lines, RNAi-mediated knockdown of PIK3CA reduced

phosphorylation levels of Akt (S473) and GSK-3β (S9). Expression of p21 was induced

and Cyclin D1 downstream target expression diminished. Efficient RNAi-mediated

depletion was confirmed by PI3K p110α immunoblotting for two different siRNA

molecules. (B) Significant reduction of cell viability (MTT assay) upon RNAi-mediated

depletion of PIK3CA in two MLS cell lines (***p<0.001). To exclude unspecific off-target





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effects, a second siRNA was equally tested. Experiments were performed in

quintuplicates and results are expressed as mean + SEM. (C) In MTT assays, viability of

MLS402-91, MLS2645-94, and MLS1765-92 cells was significantly reduced by

treatment with increasing concentrations of the class I PI3K inhibitor BKM120

(1.25-5 µmol/L). (D) BKM120 (0.5-1 µmol/L) suppressed phosphorylation levels of Akt

(S473) and GSK-3β (S9) in all three MLS cell lines. Changes in the PI3K p110 catalytic

subunit alpha protein levels were determined by immunoblotting. (E-F) In flow cytometric

analyses, significantly increased rates of apoptosis (cleaved PARP) were detected upon

treatment with BKM120 (1-2 µmol/L; DMSO employed as vehicle control). (G)

MLS402-91 and MLS1765-92 cells were xenografted on the CAM of fertilized chick

eggs. Tumor-bearing eggs were randomized and topically treated with BKM120 (n≥5) or

vehicle control (0.2% DMSO in NaCl 0.9%; n≥5). Significantly reduced tumor volumes

+ SEM in BKM120-treated (1-2 µmol/L) versus the DMSO vehicle control group and

representative CAM explants of MLS xenografts are shown (scale bar: 1 mm, **P<0.01;

*P<0.05).

Figure 4. In vitro evaluation of response to BKM120 depending on PIK3CA

mutational status in myxoid liposarcoma cells.

(A) Apoptotic rates of MLS402-91 cells expressing wild type or ΔH1047R-mutated

PIK3CA after treatment with the class I PI3K inhibitor BKM120 for 24 h. In ApoTox-Glo

analyses, significantly increased rates of apoptosis (caspase 3/7 activity; ***p<0.001)

were detected upon treatment with BKM120. (B) Employing cleaved PARP (cPARP) as

a second marker for apoptosis in flow cytometric analyses, the increase in apoptotic rate

upon treatment with BKM120 was more marked in MLS402-91 cells transfected with

ΔH1047R-mutated PIK3CA compared to the wild type PIK3CA control.




Published OnlineFirst February 20, 2019.Mol Cancer Ther Marcel Trautmann, Magdalene Cyra, Ilka Isfort, et al. Functionally Essential in Myxoid LiposarcomaPhosphatidylinositol-3-kinase (PI3K)/Akt Signaling is

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