Pml nuclear body disruption cooperates in APL pathogenesis and impairs DNA damage repair pathways in mice.

A hallmark of acute promyelocytic leukemia (APL) is altered nuclear architecture, with disruption of promyelocytic leukemia (PML) nuclear bodies (NBs) mediated by the PML-retinoic acid receptor α (RARα) oncoprotein. To address whether this phenomenon plays a role in disease pathogenesis, we generated a knock-in mouse model with NB disruption mediated by 2 point mutations (C62A/C65A) in the Pml RING domain. Although no leukemias developed in PmlC62A/C65A mice, these transgenic mice also expressing RARα linked to a dimerization domain (p50-RARα model) exhibited a doubling in the rate of leukemia, with a reduced latency period. Additionally, we found that response to targeted therapy with all-trans retinoic acid in vivo was dependent on NB integrity. PML-RARα is recognized to be insufficient for development of APL, requiring acquisition of cooperating mutations. We therefore investigated whether NB disruption might be mutagenic. Compared with wild-type cells, primary PmlC62A/C65A cells exhibited increased sister-chromatid exchange and chromosome abnormalities. Moreover, functional assays showed impaired homologous recombination (HR) and nonhomologous end-joining (NHEJ) repair pathways, with defective localization of Brca1 and Rad51 to sites of DNA damage. These data directly demonstrate that Pml NBs are critical for DNA damage responses, and suggest that Pml NB disruption is a central contributor to APL pathogenesis.

Preclinical Evaluation of Vemurafenib as Therapy for BRAFV600E Mutated Sarcomas.

The BRAFV600E mutation, which in melanoma is targetable with vemurafenib, is also found in sarcomas and we here evaluate the therapeutic potential in sarcoma cell lines. METHODS: Four sarcoma cell lines harboring the BRAFV600E mutation, representing liposarcomas (SA-4 and SW872), Ewing sarcoma (A673) and atypical synovial sarcoma (SW982), were treated with vemurafenib and the effects on cell growth, apoptosis, cell cycle progression and cell signaling were determined. RESULTS: Vemurafenib induced a strong cytostatic effect in SA-4 cells, mainly due to cell cycle arrest, whereas only moderate levels of apoptosis were observed. However, a high dose was required compared to BRAFV600E mutated melanoma cells, and removal of vemurafenib demonstrated that the continuous presence of drug was required for sustained growth inhibition. A limited growth inhibition was observed in the other three cell lines. Protein analyses demonstrated reduced phosphorylation of ERK during treatment with vemurafenib in all the four sarcoma cell lines confirming that the MAPK pathway is active in these cell lines, and that the pathway can be inhibited by vemurafenib, but also that these cells can proliferate despite this. CONCLUSIONS: These findings indicate that vemurafenib alone would not be an efficient therapy against BRAFV600E mutated sarcomas. However, further investigations of combination with other drugs are warranted.

Monogenic and polygenic determinants of sarcoma risk: an international genetic study.

BACKGROUND: Sarcomas are rare, phenotypically heterogeneous cancers that disproportionately affect the young. Outside rare syndromes, the nature, extent, and clinical significance of their genetic origins are not known. We aimed to investigate the genetic basis for bone and soft-tissue sarcoma seen in routine clinical practice. METHODS: In this genetic study, we included 1162 patients with sarcoma from four cohorts (the International Sarcoma Kindred Study [ISKS], 966 probands; Project GENESIS, 48 probands; Asan Bio-Resource Center, 138 probands; and kConFab, ten probands), who were older than 15 years at the time of consent and had a histologically confirmed diagnosis of sarcoma, recruited from specialist sarcoma clinics without regard to family history. Detailed clinical, pathological, and pedigree information was collected, and cancer diagnoses in probands and relatives were independently verified. Targeted exon sequencing using blood (n=1114) or saliva (n=48) samples was done on 72 genes (selected due to associations with increased cancer risk) and rare variants were stratified into classes approximating the International Agency for Research on Cancer (IARC) clinical classification for genetic variation. We did a case-control rare variant burden analysis using 6545 Caucasian controls included from three cohorts (ISKS, 235 controls; LifePool, 2010 controls; and National Heart, Lung, and Blood Institute Exome Sequencing Project [ESP], 4300 controls). FINDINGS: The median age at cancer diagnosis in 1162 sarcoma probands was 46 years (IQR 29-58), 170 (15%) of 1162 probands had multiple primary cancers, and 155 (17%) of 911 families with informative pedigrees fitted recognisable cancer syndromes. Using a case-control rare variant burden analysis, 638 (55%) of 1162 sarcoma probands bore an excess of pathogenic germline variants (combined odds ratio [OR] 1·43, 95% CI 1·24-1·64, p<0·0001), with 227 known or expected pathogenic variants occurring in 217 individuals. All classes of pathogenic variants (known, expected, or predicted) were associated with earlier age of cancer onset. In addition to TP53, ATM, ATR, and BRCA2, an unexpected excess of functionally pathogenic variants was seen in ERCC2. Probands were more likely than controls to have multiple pathogenic variants compared with the combined control cohort group and the LifePool control cohort (OR 2·22, 95% CI 1·57-3·14, p=1·2 × 10(-6)) and the cumulative burden of multiple variants correlated with earlier age at cancer diagnosis (Mantel-Cox log-rank test for trend, p=0·0032). 66 of 1162 probands carried notifiable variants following expert clinical review (those recognised to be clinically significant to health and about which patients should be advised), whereas 293 (25%) probands carried variants with potential therapeutic significance. INTERPRETATION: About half of patients with sarcoma have putatively pathogenic monogenic and polygenic variation in known and novel cancer genes, with implications for risk management and treatment. FUNDING: Rainbows for Kate Foundation, Johanna Sewell Research Foundation, Australian National Health and Medical Research Council, Cancer Australia, Sarcoma UK, National Cancer Institute, Liddy Shriver Sarcoma Initiative.

Functional genomics analysis reveals a MYC signature associated with a poor clinical prognosis in liposarcomas.

Liposarcomas, which are malignant fatty tumors, are the second most common soft-tissue sarcomas. Several histologically defined liposarcoma subtypes exist, yet little is known about the molecular pathology that drives the diversity in these tumors. We used functional genomics to classify a panel of diverse liposarcoma cell lines based on hierarchical clustering of their gene expression profiles, indicating that liposarcoma gene expression profiles and histologic classification are not directly correlated. Boolean probability approaches based on cancer-associated properties identified differential expression in multiple genes, including MYC, as potentially affecting liposarcoma signaling networks and cancer outcome. We confirmed our method with a large panel of lipomatous tumors, revealing that MYC protein expression is correlated with patient survival. These data encourage increased reliance on genomic features in conjunction with histologic features for liposarcoma clinical characterization and lay the groundwork for using Boolean-based probabilities to identify prognostic biomarkers for clinical outcome in tumor patients.

Characterization of liposarcoma cell lines for preclinical and biological studies.

Liposarcoma cell lines represent in vitro models for studying disease mechanisms at the cellular level and for preclinical evaluation of novel drugs. To date there are a limited number of well-characterized models available. In this study, nine immortal liposarcoma cell lines were evaluated for tumor-forming ability, stem cell- and differentiation potential, and metastatic potential, with the aim to generate a well-characterized liposarcoma cell line panel. Detailed stem cell and differentiation marker analyses were also performed. Five of the liposarcoma cell lines were tumorigenic, forming tumors in mice. Interestingly, tumor-forming ability correlated with high proliferative capacity in vitro. All the cell lines underwent adipocytic differentiation, but the degree varied. Surprisingly, the expression of stem cell and differentiation markers did not correlate well with function. Overall, the panel contains cell lines suited for in vivo analyses (LPS141, SA-4, T778, SW872, and LISA-2), for testing novel drugs targeting cancer stem cells (LPS141) and for studying tumor progression and metastasis (T449 and T778).

Noninvasive Detection of ctDNA Reveals Intratumor Heterogeneity and Is Associated with Tumor Burden in Gastrointestinal Stromal Tumor.

Molecular analysis of circulating tumor DNA (ctDNA) has a large potential for clinical application by capturing tumor-specific aberrations through noninvasive sampling. In gastrointestinal stromal tumor (GIST), analysis of KIT and PDGFRA mutations is important for therapeutic decisions, but the invasiveness of traditional biopsies limits the possibilities for repeated sampling. Using targeted next-generation sequencing, we have analyzed circulating cell-free DNA from 50 GIST patients. Tumor-specific mutations were detected in 16 of 44 plasma samples (36%) from treatment-naïve patients and in three of six (50%) patients treated with tyrosine kinase inhibitors. A significant association between detection of ctDNA and the modified National Institutes of Health risk classification was found. All patients with metastatic disease had detectable ctDNA, and tumor burden was the most important detection determinant. Median tumor size was 13.4 cm for patients with detectable mutation in plasma compared with 4.4 cm in patients without detectable mutation (P = 0.006). ctDNA analysis of a patient with disease progression on imatinib revealed that multiple resistance mutations were synchronously present, and detailed analysis of tumor tissue showed that these were spatially distributed in the primary tumor. Plasma samples taken throughout the course of treatment demonstrated that clonal evolution can be monitored over time. In conclusion, we have shown that detection of GIST-specific mutations in plasma is particularly feasible for patients with high tumor burden. In such cases, we have demonstrated that mutational analysis by use of liquid biopsies can capture the molecular heterogeneity of the whole tumor, and may guide treatment decisions during progression. Mol Cancer Ther; 17(11); 2473-80. ©2018 AACR.

Preclinical evaluation of potential therapeutic targets in dedifferentiated liposarcoma.

Sarcomas are rare cancers with limited treatment options. Patients are generally treated by chemotherapy and/or radiotherapy in combination with surgery, and would benefit from new personalized approaches. In this study we demonstrate the potential of combining personal genomic characterization of patient tumors to identify targetable mutations with in vitro testing of specific drugs in patient-derived cell lines. We have analyzed three metastases from a patient with high-grade metastatic dedifferentiated liposarcoma (DDLPS) by exome and transcriptome sequencing as well as DNA copy number analysis. Genomic aberrations of several potentially targetable genes, including amplification of KITLG and FRS2, in addition to amplification of CDK4 and MDM2, characteristic of this disease, were identified. We evaluated the efficacy of drugs targeting these aberrations or the corresponding signaling pathways in a cell line derived from the patient. Interestingly, the pan-FGFR inhibitor NVP-BGJ398, which targets FGFR upstream of FRS2, strongly inhibited cell proliferation in vitro and induced an accumulation of cells into the G0 phase of the cell cycle. This study indicates that FGFR inhibitors have therapeutic potential in the treatment of DDLPS with amplified FRS2.

Metabolic reprogramming of metastatic breast cancer and melanoma by let-7a microRNA.

Let-7 microRNAs (miRNAs) are highly conserved well-established promoters of terminal differentiation that are expressed in healthy adult tissues and frequently repressed in cancer cells. The tumor suppressive role of let-7 in a variety of cancers in vitro and in vivo has been widely documented and prompted these miRNAs to be candidate genes for miRNA replacement therapy. In this study we described a new role of let-7a in reprogramming cancer metabolism, recently identified as a new hallmark of cancer. We show that let-7a down-regulates key anabolic enzymes and increases both oxidative phosphorylation and glycolysis in triple-negative breast cancer and metastatic melanoma cell lines. Strikingly, the accelerated glycolysis coexists with drastically reduced cancer features. Moreover, let-7a causes mitochondrial ROS production concomitant with the up-regulation of oxidative stress responsive genes. To exploit these increased ROS levels for therapeutic purposes, we combined let-7a transfection with the chemotherapeutic drug doxorubicin. In both cancer types let-7a increased cell sensitivity to doxorubicin. Pre-treatment with N-acetyl cysteine (NAC) totally abolished this effect, indicating that the increased doxorubicin sensitivity of let-7a cells depends on the redox pathway. We thus have demonstrated that let-7a plays a prominent role in regulating energy metabolism in cancer cells, further expanding its therapeutic potential.

Liposarcoma Cells with Aldefluor and CD133 Activity have a Cancer Stem Cell Potential.

Aldehyde dehydrogenase (ALDH) has recently been shown to be a marker of cancer stem-like cells (CSCs) across tumour types. The primary goals of this study were to investigate whether ALDH is expressed in liposarcomas, and whether CSCs can be identified in the ALDHhigh subpopulation. We have demonstrated that ALDH is indeed expressed in 10 out of 10 liposarcoma patient samples. Using a liposarcoma xenograft model, we have identified a small population of cells with an inducible stem cell potential, expressing both ALDH and CD133 following culturing in stem cell medium. This potential CSC population, which makes up for 0,1-1,7 % of the cells, displayed increased self-renewing abilities and increased tumourigenicity, giving tumours in vivo from as few as 100 injected cells.