ORIGINAL ARTICLE CHK1 Inhibition in Soft-Tissue Sarcomas: Biological and Clinical Implications
A Laroche-Clary1,2*, C Lucchesi1,2, C Rey1,2*, S Verbeke1,2*, A Bourdon1,2, V Chaire1,2, M-P Algéo4, S Cousin1,2, M Toulmonde1,2, V Vélasco 2,3, J Shutzman5, A Savina5, F Le Loarer2,3 A Italiano1,2,4
1
INSERM ACTION U1218, Institute Bergonié, Bordeaux, France; 2Sarcoma Unit, Institute
Bergonié, Bordeaux, France; 3Department of Pathology, Institut Bergonié, Bordeaux, France; 4
University of Bordeaux, France
*These authors have contributed equally to the work
Corresponding author: Antoine ITALIANO, MD, PhD, Institut Bergonié 229 cours de l’Argonne, 33076 Bordeaux cedex, France E-mail:
[email protected]
© The Author 2018. Published by Oxford University Press on behalf of the European Society for Medical Oncology. All rights reserved. For permissions, please email:
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ABSTRACT Background: Inhibition of ChK1 appears as a promising strategy for selectively potentiate the efficacy of chemotherapeutic agents in G1 checkpoint-defective tumor cells such as those that lack functional p53 protein. The p53 pathway is commonly dysregulated in softtissue sarcomas (STS) through mutations affecting TP53 or MDM2 amplification. GDC-0575 is a selective ATP-competitive inhibitor of CHK1. Methods: We have performed a systematic screening of a panel of 10 STS cell lines by combining the treatment of GDC-0575 with chemotherapy. Cell proliferation, cell death and cell cycle analysis were evaluated with high throughput assay. In vivo experiments were performed by using TP53-mutated and TP53 wild-type patient-derived xenograft models of STS. Clinical activity of GDC-0575 combined with chemotherapy in patients with TP53mutated and TP53 wild-type STS was also assessed. Results: We found that GDC-0575 abrogated DNA damage-induced S and G2–M checkpoints, exacerbated DNA double-strand breaks and induced apoptosis in STS cells. Moreover, we observed a synergistic or additive effect of GDC-0575 together with gemcitabine in vitro and in vivo in TP53-proficient but not TP53-deficient sarcoma models. In a phase 1 study of GDC-0575 in combination with gemcitabine, two patients with metastatic TP53-mutated STS had an exceptional, long-lasting response despite administration of a very low dose of gemcitabine whereas one patient with wild-type TP53 STS had no clinical benefit. Genetic profiling of samples from a patient displaying secondary resistance after 1 year showed loss of one preexisting loss-of-function mutation in the helical domain of DNA2. Conclusion: We provide the first pre-clinical and clinical evidence that potentiation of chemotherapy activity with a CHK1 inhibitor is a promising strategy in TP53-deficient STS and deserves further investigation in the phase 2 setting. Keywords: sarcoma, TP53, CHK1, GDC-0575, gemcitabine
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INTRODUCTION There is a compelling unmet medical need for effective therapy for patients with recurrent metastatic STS. Doxorubicin-based chemotherapy is considered the standard first-line therapy. Gemcitabine is also an active drug in STS as a single agent or in combination with other cytotoxic agents such as docetaxel or dacarbazine and represent a standard of care according to current guidelines (1). The gene encoding CHK1, an essential kinase that is regulated by ATR and required for the G2/M DNA damage checkpoint, is part of the molecular CINSARC signature that predicts metastasis in individuals with non-translocation-related STS (2). The major function of CHK1 is to coordinate the DNA damage response (DDR) and cell cycle checkpoint response. Indeed, the ATR-CHK1 pathway recognizes a broad spectrum of DNA abnormalities ranging from UV light, to DNA replication inhibition, virus infection, interstrand DNA crosslinking and DSB end resection (reviewed in ref. 3). We and others have demonstrated that TP53 pathway abrogation appears as a pivotal event in soft tissue sarcoma oncogenesis (4). Tumor cells lacking a functional TP53 pathway are unable to arrest in the G1 phase of the cell division cycle. TP53-deficient tumor cells maintain their ability to arrest in the S and G2-phases of the cell division cycle due to CHK1 activity. However, they are compromised in their ability to sustain these arrests (5). Several studies performed in other tumor models have shown that ChK1 inhibitors potentiates the effects of DNA damage induced by DNA-damaging chemotherapeutic agents. In particular, the anti-tumor activity of CHK1 inhibitors have been shown to be more profound when combined with chemotherapeutic regimens including gemcitabine in particular in tumor cells with nonfunctional TP53 (6, 7).
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We have reported recently that the CHK1 inhibitor GDC-0575 can be safely combined with gemcitabine with promising clinical activity in several solid tumor types (8). We describe here the first pre-clinical and clinical assessment of the therapeutic potential of CHK1 inhibition alone and in combination with gemcitabine in STS. METHODS
Cells and cell culture The STS cell lines used in this study were derived from human STS surgical specimens after obtaining written, informed patient consent (Table 1) and Institut Bergonié IRB approval. Each cell line was characterized by array comparative genomic hybridization to verify that its genomic profile was still representative of the originating tumor sample. Cells were grown in RPMI medium 1640 (Sigma Life Technologies, Saint Louis, MO) in the presence of 10% fetal calf serum (Dutscher, France) in flasks. Cells were maintained at 37 °C in a humidified atmosphere containing 5% CO2.
Reagents GDC-0575 (CHK1 inhibitor) was supplied by Genentech (San Francisco, USA), and Gemcitabine was purchased from Sigma (Sigma-Aldrich Chimie, Saint-Quentin-Fallavier, France).
Cell viability Antiproliferative and cytotoxic effects of GDC-0575 and gemcitabine were first determined on 10 cell lines using Cytation 3 technology (Colmar, France). Briefly, cells were seeded in 384-well plates and were then exposed to GDC-0575 and/or gemcitabine for 72 h. 4
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Cells were then marked with propidium iodide (PI) and Syto 24 fluorochromes for 30 min. Quantitative fluorescence and cell imaging were performed with Cytation 3 at λ = 617 nm for PI and 521 nm for Syto 24. Experiments were realized in triplicates.
Cell cycle analysis The cell cycle distribution was studied by examining DNA content using fluorescenceactivated cell sorting (FACS) and analyzed using Cell Quest Pro software (BD Biosciences, San Jose, CA, USA). 2x105 cells were seeded in 6-well plates, and after 24 h, the cells were treated for 24 and 48 h with two different concentrations of GDC-0575 and/or gemcitabine, centrifuged at 1500 g for 5 min, and washed twice with PBS. The cells were then fixed with 70% ethanol at 4 °C overnight. Following ethanol removal, the cells were washed twice with PBS. Next, 300 µl of a PI and ribonuclease-containing solution were added to the cells and then analyzed by FACS. The data were analyzed with FlowJo v.7.6.3 software, and the results were expressed in terms of percentage of cells in a given phase of the cell cycle.
Apoptosis For apoptosis assessment, 1.5x105 cells were seeded in 6-well plates. After 24 h, cells were treated with two concentrations of GDC-0575 and/or gemcitabine for 72 h and exposed to FITC-Annexin V and PI according to the manufacturer’s protocol (BD Biosciences, Erembodegem, Belgium). This allows us to distinguish Annexin V-positive cells in early apoptosis from Annexin V- and PI-positive cells in late apoptosis. Cells were analyzed by flow cytometry using FL1 for Annexin V and FL2 for PI. Flow cytometry (FACScan; BD Biosciences) data were analyzed with FlowJo v.7.6.3 software.
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Western blot Treated and control cells were harvested in 60 µL of radio-immunoprecipitation assay (RIPA) lysis buffer (Harlow and Lane, 2006). The lysate was centrifuged (13 000 rpm, 15 min, 4°C), and the supernatant was stored at -80°C. Equal amounts of total protein (30 µg) were electrophoresed on 12% sodium dodecyl sulfate (SDS) polyacrylamide gels and transferred onto polyvinylidene difluoride (PVDF) membranes. The blots were probed overnight at 4°C with an anti-phospho-CHK1 (S296, ab79758, 1/1000 Abcam), and an antiCHK1 (Ab47574, 1/500, Abcam) primary antibody diluted in PBST (100 mM phosphate, 27 mM KCl, 1.37 M NaCl, pH 7.4 after 1X dilution; 0.2% Tween-20) with 5% BSA. The horseradish peroxidase-conjugated secondary antibody (Santa Cruz Biotechnology) was diluted 1:5000. Bound antibodies were visualized on Fusion Fx7 imaging system (Fisher Bioblock Scientific, Waltham, MA, USA) using the ImmobilonTM Western enhanced chemiluminescence detection kit (Millipore Corporation, Billerica, MA, USA). The resulting bands were analyzed and quantified using ImageJ® 1.49g software (National Institutes of Health, Bethesda, MD, USA).
Confocal microscopy Cells were seeded on coverslips and treated with one concentration of GDC-0575, gemcitabine, or a combination of the two drugs for 72 h. The slides were then washed twice with PBS, fixed in 4% formaldehyde, and incubated with anti-phospho-γH2ax monoclonal antibody (Cell Signaling, Leiden, Netherlands) overnight and then with goat anti-rabbit Alexa Fluor 488 antibody (Invitrogen, Paisley, United Kingdom). The slides were then counterstained using 4,6-diamidino-2-phenylindole (Hoechst).
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PDX generation IRB-approved informed consent to generate patient-derived murine xenografts (PDXs) was obtained from the relevant patients. Animal care and procedures were approved by the Institutional Animal Care and Use Committee Office of the University of Bordeaux, France. Tumor specimens from three patients with UPS were cut into 3 × 3 × 3 mm pieces that were transferred to RPMI. Tumor pieces were inserted into incisions on the right flank of 5 nude mice.
In vivo study Four- to five-week-old female Ragγ2C-/- mice were used. Induction of tumor xenografts was performed by subcutaneous injection of 0.2 ml cell suspensions containing 5x106 live cells (IB115, IB136) or by subcutaneous implantation of a UPS tumor fragment (PDX) into the right flank of the mice. This study followed French and European Union guidelines for animal experimentation (RD 1201/05, RD 53/2013 and 86/609/CEE, respectively). Mice were randomized into control and treatment groups (n = 6 in each group of treatment for IB115 and for IB136 and n = 5 for vehicle and gemcitabine groups and n = 6 for GDC-0575 and combination groups in the three PDX models) three weeks after the tumor became measurable (15 days after injection: day 1 of treatment). Mice were randomized into 4 groups: vehicle (0.9% NaCl), gemcitabine alone (30 mg/kg IP, one time per week at J1), GDC-0575 alone (25 mg/kg by oral gavage twice a week at J2 and J4), and both drugs. Further details are provided in the supplementary data.
Clinical trial
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NCT01564251 is an active multicenter phase I dose-escalation study evaluating the safety and pharmacokinetics of GDC-0575, a selective CHK1 inhibitor, as a single agent and in combination with gemcitabine in unselected patients with metastatic or advanced solid tumors without standard therapy options (see supplementary data).
Exome sequencing DNA extraction was performed with a QIAamp DNA mini kit (Qiagen, Courtaboeuf, France) as described by the manufacturer. The purity of the genomic DNA was measured with a NanoDrop 1000 apparatus (NanoDrop Products), and the quantity was estimated by a fluorescence-based method using a Qubit double-stranded DNA BR assay kit and a Qubit fluorometer (Life Technologies, Germany) according to the manufacturer’s instructions. Exome sequencing and bioinformatic analyses are described in the Supplementary Methods.
Statistical analysis Data were analyzed using Student’s t-test for comparison of two means and ANOVA followed by Tukey’s multiple comparison tests for more than two groups; all the experiments were repeated in duplicate or triplicate. Data are represented as mean ± SD and significant differences are indicated as *p<0.05, **p<0.01 and ***p<0.001. Analysis of progression-free survival was made using the log-rank test (Mantel-Cox test).
RESULTS Aberrant expression of CHK1 is associated with adverse outcome in complex genomics sarcomas 8
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By analyzing the expression profile of a series of 339 complex genomics sarcomas and 108 translocation-related sarcomas (2), we found that CHK1 was significantly associated with worse metastases-free survival in both subgroups (Supplementary Figure 1).
Antiproliferative activity of GDC-0575 and gemcitabine in STS cell lines We studied the sensitivity of 10 STS cell lines to GDC-0575 and gemcitabine (Table 1). All of the cell lines were highly sensitive to gemcitabine, with IC50 values ranging between 0.01 and 13 nM. 9 out of 10 cell lines were sensitive to GDC-0575, with IC50 values ranging between 0.025 and 5.9 µM. IB133 was resistant to GDC-0575, (IC50= 41µM). All the cell lines had impaired TP53 signaling as a result of TP53 mutation/deletion or MDM2 amplification except for two lines (IB128 and IB140). The resistant IB133 cell line was the sole line characterized by a concomitant deletion of TP53 and MDM2. In the sensitive cell lines, the activity of GDC-0575 was associated with inhibition of the phosphorylation of Ser296 as assessed by western blotting (data not shown).
The combination of GDC-0575 and gemcitabine increases DNA damage By analyzing γ-H2AX expression, we found hat the combination of GDC-0575 and gemcitabine induced significantly higher levels of DNA damage (Figure 1).
GDC-0575 and gemcitabine act synergistically in STS cell lines We studied the effects of the combination of gemcitabine and GDC-0575 as previously described (9). Interestingly, we observed an additive (n=2) or synergistic effect (n=5) in 80% of the STS cell lines (Table 1). An antagonistic effect was observed in two leiomyosarcoma cell lines (IB134 and IB140).
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The combination of GDC-0575 and gemcitabine induces apoptosis and cell cycle arrest in STS cell lines We studied the effects of the combination of GDC-0575 and gemcitabine on apoptosis induction after 72 h of drug exposure as well as cell cycle effects after 24 and 48 h of treatment. We observed that the drug combination significantly increased the rate of apoptosis in comparison with the drugs alone (Figure 2). The effect of the drug combination on the cell cycle was variable. In two cell lines (IB115 and IB111), there was an increase in the S phase population with either, drug alone and the combination of the two drugs. However, in the IB136 cell line, there was an accumulation of cells in S phase following treatment with gemcitabine and an accumulation of cells in G1 phase following treatment with GDC-0575 alone or the drug combination.
The combination of GDC-0575 and gemcitabine reduces tumor growth in vivo To further validate the in vitro study, we performed in vivo studies to test the antitumor effects of the GDC-0575 and gemcitabine combination in two xenografts models (IB115: dedifferentiated liposarcoma and IB136: leiomyosarcoma) and two patients- derived xenografts models (one with TP53 mutation, one with wild-type TP53). After three weeks of treatment we observed a significant reduction of the tumor growth rate and progressionfree survival in the IB115, IB136 and P53mut PDX UPS models (Figure 3A, 3B and Supplementary figure 2) for the combination of drugs in comparison to single agent alone. There was no effect in the P53wt PDX UPS model (Figure 3C and D). No signs of toxicity were observed with the combination treatment. After three weeks of treatment, the mice were sacrificed. The effects of the drug combination on DNA damage were evaluated via γH2ax staining, and there was a good correlation between DNA breaks and 10
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treatment in the P53mut PDX model but no correlation in the other model (Supplementary Figure 4A and B).
Patients Three patients with advanced STS were included in the GP28153 study and treated with 500 mg/m² gemcitabine bi-weekly combined with GDC-0575 (60 mg) the day after chemotherapy infusion. Two patients had exceptional long-lasting tumor responses whereas one patient displayed no clinical benefit. Detailed case reports are presented in the supplementary data. Analysis of the TP53 mutational status allowed the identification of a TP53 mutation in the tumor cells of the two patients with response to treatment whereas no mutation was identified in the patient with progressive disease. Exceptional responses and secondary resistance to CHK1 inhibition in sarcoma patients To elucidate the molecular aberrations involved in the exceptional responses described above and in secondary resistance, we performed exome-based molecular profiling of Patient 1 and Patient 2. For Patient 1, a tumor sample gathered before initiation of treatment and a tumor sample obtained at occurrence of progression were sequenced. Copy number alteration profiling of all samples revealed complex patterns of alterations, consistent with complex genomics sarcomas. (Supplementary Figures 5 and 6). Detailed results are presented in the supplementary data. Of note, the comparative analysis of the secondary resistant and primary samples of patients did not allow the identification of a secondary mutation of CHK1. However, we observed, in particular, the disappearance in the secondary resistant sample of a loss-of-function mutation of the DNA2 gene which encodes a DNA replication helicase/nuclease (Supplementary Figure 7).
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DISCUSSION Here, we show that the selective ATP-competitive inhibitor of CHK1, GDC-0575, markedly sensitizes STS cells to gemcitabine. As anticipated, GDC-0575 abrogated DNA damage-induced S and G2–M checkpoints, exacerbated DNA DSBs and induced cell death by apoptosis. The effect on the cell cycle was variable as observed in other tumor models with an increase in the S phase population (6) or in G1 (10). The two patients with TP53-mutated STS had a meaningful response despite the use of gemcitabine 3 times lower than that used in the routine setting. Although surprising, these clinical observations are supported by recent mechanistic data. Indeed, Koh et al. have shown that even sub-GI50 concentrations of gemcitabine and CHK1i are sufficient to yield synergistic growth inhibition in pancreatic tumor cells. In this setting, the authors showed that G2–M bypass is dispensable for synergy between gemcitabine and CHK1 inhibition. Indeed, in their proposed model, the immediate response of cells upon addition of CHK1i together with gemcitabine at low doses is not premature S-phase mitotic entry. Instead, this results in an increase in the S-phase population, prolonged interphase with enhanced DNA damage and replication stress (6), as we have observed in our in vitro experiments. Our study represents the first clinical evidence suggesting that “metronomic” gemcitabine represents an opportunity where the synergistic interaction between the antimetabolite and CHK1 inhibitor could be exploited (11). Several studies suggested a synergistic lethality between TP53 abrogation and CHK1 inhibition (5,7). To best model this concept, we used patient-derived xenografts from patients with undifferentiated pleomorphic sarcoma (UPS), the most frequent STS and also the most aggressive (12). UPS are associated with TP53 mutations in up to 30% of cases (13, 12
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14) and we assessed two different models, one with TP53 mutation and one wild type. Consistent with results observed in epithelial tumors (5,7), cotreatment of a TP53-mutant UPS with gemcitabine and GDC-0575 substantially delayed cancer progression and improved survival relative to chemotherapy alone. Strikingly, this synergy, which correlated with an increased proportion of cells with DNA damage, was not observed in TP53-WT UPS models, suggesting a crucial synthetic lethal relationship between CHK1 inhibition and TP53 deficiency. Importantly, we confirmed the clinical relevance of these findings by performing next-generation exome sequencing of tumor samples of the two patients who showed an exceptional response to GDC-0575 in combination with gemcitabine. Both patients had TP53 mutations whereas the patient with no clinical benefit of treatment had a wild-type TP53 tumor. Although the preferential sensitivity of TP53-deficient cells to G2-M phase checkpoint inhibition appears to be mediated at least partly through such a checkpoint short circuit, it remains possible that this synthetic lethal interaction is also attributable to distinct mechanisms, including direct genome-destabilizing effects in the S and G2 phases of the cell cycle. Therefore, we cannot exclude the possibility that the activity of CHK1 inhibitors in STS extends beyond the scope of TP53 deficiency to situations in which maintenance of genome integrity in S and G2 is challenged through other gene alterations. In this regard, the comparative analysis of the mutational landscape of tumor cells from patient 1 before treatment onset and at secondary resistance gives novel insights. Although the majority of variants, including TP53, were common to the pre-treatment and secondary resistant tumor samples, we identified a list of 6 gene mutations with significant differences between the pre-treatment and the secondary resistant samples. Mutation of DNA2 was associated with the highest significant change. This gene encodes for a highly conserved 13
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nuclease protein that plays a crucial role in replication, DNA damage repair, and homologous recombination (HR). DNA2 mediates the resection of the 5′ strand at DNA DSB ends, (15) an early step in HR, and also at stalled and regressed replication forks (15), and is overexpressed in several cancers. Loss of a tumor-suppressor gene such as TP53 leads to excessive replication stress, which results in replication-associated lesions, predominantly DNA double-strand breaks (DSBs). HR is one of the major mechanisms used by cancer cells to repair such replication-associated DSBs. Recent studies have shown that DNA2 inhibition results in decreased DSB end resection and HR, and sensitizes cancer cells to chemotherapy inducing DNA damage (16, 17). Interestingly, we found in the pre-treatment sample of patient 1 a loss-of-function mutation of DNA2 which is expected to result in decreased DNA double-strand break end resection and attenuation of DNA repair via homologous recombination. This mutation was not detected in the resistant sample, suggesting a potential synthetic lethal interaction between DNA2 depletion and CHK1 pathway disruption. Since the ATR/CHK1 pathway plays a central role in the response to a variety of cellular stresses, it is conceivable that other common characteristics in cancers, such as alterations of genes involved in DNA repair, will also predict responsiveness to CHK1 inhibition. Thus, the careful genetic characterization of patients enrolled in future clinical trials assessing CHK1 inhibitors will be crucial for improving the efficacy of this therapeutic strategy. Conflict of interest: Jennifer Schutzman and Ariel Savina are Roche/Genentech employees. The other authors have no conflicts of interest to declare. Financial support: Roche, SIRIC BRIO 2012-146
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FIGURE LEGENDS Figure 1: (A) IB111, IB115, 93T449 and IB136 cells were immunostained with anti -γH2AXspecific antibodies before and after treatment with gemcitabine at 50 nM, 3.3 nM, 100 nM, and 6.25 nM; GDC-0575 at 2.7 µM, 4 µM, 100 nM, and 25 µM; or both drugs in combination. (B) Quantification of P-H2AX punctae in IB115, IB111, IB136, and 93T449 cell lines. The experiments were performed in duplicate.
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Figure 2: Effect of the combination of gemcitabine and GDC-0575 on apoptosis (A) Annexin V FITC-A vs propidium iodide-A plots from the gated cells show the populations corresponding to viable and non-apoptotic (Annexin V–PI–), early (Annexin V+PI–), and late (Annexin V+PI+) apoptotic cells in the IB111 cell line. (B) Quantification of apoptotic cells after 72 h of treatment with GDC-0575 or Gemcitabine alone or a combination of the two drugs. Effect of gemcitabine and GDC-0575 combination on cell cycle progression in 4 STS cell lines: IB111, IB115, IB136, and 93T449. (C) Cell-cycle profile after 24 h of treatment with gemcitabine and/or GDC-0575 analyzed by PI incorporation and flow cytometry in the IB115 cell line. (D) Cell-cycle distribution was calculated from the flow cytogram.
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Figure 3: (A) Effect of the combination of gemcitabine and GDC-0575 on tumor growth in the P53mut UPS PDX model. (B) Kaplan-Meier survival curves for different mouse cohorts in the P53mut UPS PDX model. (C) Effect of the combination of gemcitabine and GDC-0575 on tumor growth in the P53wt UPS PDX model. (D) Kaplan-Meier survival curves for the 4 mouse cohorts in the P53wt UPS PDX model. A log-rank (Mantel-Cox) test was used to calculate P-values comparing the survival rates.
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TABLES Table 1. Anti-proliferative activity of GDC-0575 and gemcitabine in soft-tissue sarcoma cell lines TP53 mutational status*
MDM2 amplification status
IC50 GDC0575 (µM)
Dedifferentiated liposarcoma
Wild-type
Amplified
5.4
IB112
Leiomyosarcoma
Null
Normal
IB114
Myxofibrosarcoma
Wild-type
Dedifferentiated liposarcoma
IC50 Gemcitabine
Combination index
Comments
5
0.59
Synergistic
0.39
1.6
0.97
Additive
Gain
0.45
8.6
0.74
Synergistic
Wild-type
Amplified
4
3.3
0.48
Synergistic
Leiomyosarcoma
Wild-type
Normal
0.047
1.6
1.2
Antagonistic
Extra-skeletal osteosarcoma
Wild-type
Normal
0.18
8.4
0.85
Synergistic
IB133
Leiomyosarcoma
Exon 2-3 deleted
Deleted
41
undef
ND
ND
IB134
Leiomyosarcoma
S215R
Gain
0.14
5.5
1.38
Antagonistic
IB136
Leiomyosarcoma
Null
Gain
5.9
13
0.34
Synergistic
Well differentiated liposarcoma
Wild-type
Amplified
0.025
0.01
1.04
Additive
Histological subtype
IB140
(nM)
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Figure 1 232x191mm (150 x 150 DPI)
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Figure 2 262x190mm (150 x 150 DPI)
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Figure 3 236x146mm (150 x 150 DPI)
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