MDM2-Dependent Rewiring of Metabolomic and Lipidomic Profiles in Dedifferentiated Liposarcoma Models.

Dedifferentiated liposarcoma (DDLPS) is an aggressive mesenchymal cancer marked by amplification of MDM2, an inhibitor of the tumor suppressor TP53. DDLPS patients with higher MDM2 amplification have lower chemotherapy sensitivity and worse outcome than patients with lower MDM2 amplification. We hypothesized that MDM2 amplification levels may be associated with changes in DDLPS metabolism. Six patient-derived DDLPS cell line models were subject to comprehensive metabolomic (Metabolon) and lipidomic (SCIEX 5600 TripleTOF-MS) profiling to assess associations with MDM2 amplification and their responses to metabolic perturbations. Comparing metabolomic profiles between MDM2 higher and lower amplification cells yielded a total of 17 differentially abundant metabolites across both panels (FDR < 0.05, log2 fold change < 0.75), including ceramides, glycosylated ceramides, and sphingomyelins. Disruption of lipid metabolism through statin administration resulted in a chemo-sensitive phenotype in MDM2 lower cell lines only, suggesting that lipid metabolism may be a large contributor to the more aggressive nature of MDM2 higher DDLPS tumors. This study is the first to provide comprehensive metabolomic and lipidomic characterization of DDLPS cell lines and provides evidence for MDM2-dependent differential molecular mechanisms that are critical factors in chemoresistance and could thus affect patient outcome.

Prospective Evaluation of the Concordance of Commercial Circulating Tumor DNA Alterations with Tumor-Based Sequencing across Multiple Soft Tissue Sarcoma Subtypes.

Soft tissue sarcomas (STS) are diverse tumors with heterogenous alterations. Platforms to detect circulating tumor DNA (ctDNA) have rapidly increased in popularity as they may avoid invasive biopsy morbidity. However, ctDNA profiling concordance with standard solid tumor comprehensive genomic profiling (CGP) is poorly characterized. Here, we report the outcomes of a single-institution experience comparing mutational results from commercial ctDNA and solid tumor CGP in advanced STS subjects. We identified STS subjects who had undergone solid tumor based CGP in four distinct cohorts: Dedifferentiated liposarcoma (DDLPS), leiomyosarcoma (LMS), undifferentiated pleomorphic sarcoma (UPS), and gastrointestinal stromal tumor (GIST). Subjects with radiographically measurable tumor were profiled using a commercial ctDNA CGP panel. Overlapping genes/exons on both biopsy panels were analyzed. Twenty-four subjects completed both ctDNA and solid tumor CGP. ctDNA was detected in 18/24 subjects. Subject level concordance rates in all overlapping genes were: LMS = 4/6; UPS = 2/6; DDLPS = 1/6; GIST = 0/6. Copy number alterations were notably poorly concordant. For subjects with short variant alterations and detectable tumor fractions, concordance with solid tumor CGP was 76% (13/17). LMS subjects had the highest median tumor fraction and concordance. No correlation was seen between tumor fraction or radiographic tumor volume largely driven by low estimated tumor fraction. A limitation of the study is that only targeted sequencing was performed. However, given the poor concordance in commonly altered genes, ctDNA panels in sarcoma cannot be broadly applied. Further, more extensive studies will need to be performed.

Inhibition of histone deacetylase 2 reduces MDM2 expression and reduces tumor growth in dedifferentiated liposarcoma.

Dedifferentiated liposarcoma (DDLPS) is a highly morbid mesenchymal tumor characterized and driven by genomic amplification of the MDM2 gene. Direct inhibition of MDM2 has shown promise pre-clinically, but has yet to be validated in clinical trials. Early in vitro studies have demonstrated that pan-histone deacetylase (HDAC) inhibition may have anti-MDM2 effects. Here we present in silico, in vitro, and mouse xenograft studies that suggest that specifically targeting HDAC2 reduces MDM2 expression and has anti-tumor affects in DDLPS. Two independent datasets, The Cancer Genome Atlas (TCGA; n = 58) and the Memorial Sloan-Kettering Cancer Center Dataset (MSKCC; n = 63), were used to identify the co-expression between class I HDACs and MDM2, and their clinical impact. HDAC2 was highly co-expressed with MDM2 (TCGA: Spearman's coefficient = 0.29, p = 0.03; MSKCC: Spearman's coefficient = 0.57, p < 0.001). As both a continuous and dichotomous predictor, elevated HDAC2 expression was associated with worsened disease-free survival in the TCGA (Continuous: Hazard-ratio (HR) 1.7; 95% Confidence Interval (95%CI) 0.97-2.9; p = 0.06; Dichotomous: HR 7.1, 95%CI 2.5-19.8, p < 0.001) and distant recurrence-free survival in the MSKCC (Continuous: HR 2.2; 95%CI 1.1-4.8; p = 0.04; Dichotomous: HR 2.8, 95%CI 1.2-6.4, p = 0.02). In vitro, treatment of DDLPS cell lines with the HDAC inhibitors MI-192 (HDAC2/3 inhibitor) or romidepsin (HDAC1/2 inhibitor) reduced MDM2 expression and induced apoptosis. In a murine DDLPS xenograft model, romidepsin reduced tumor growth and lowered tumor MDM2 expression. RNA-sequencing of romidepsin treated mouse tumors demonstrated markers of TP53 reactivation. Taken together, our data supports the hypothesis that targeting HDAC2 may represent a potential strategy to modulate MDM2 expression in DDLPS.

Degree of MDM2 Amplification Affects Clinical Outcomes in Dedifferentiated Liposarcoma.

BACKGROUND: Dedifferentiated liposarcomas (DDLPS) are mesenchymal tumors associated with universally poor response to treatment. Genomic amplification of murine double minute 2 (MDM2) is used as a diagnostic biomarker; however, no established biomarkers exist to guide DDLPS treatment. In the largest study of its kind, we report that the extent of MDM2 amplification, not simply the presence of MDM2 amplification, may be biologically important to the actions of DDLPS. PATIENTS AND METHODS: The distribution of MDM2 amplification in DDLPS was assessed using data from a commercial sequencing laboratory (n = 642) and The Cancer Genome Atlas (n = 57). Data from two retrospective clinical trials (n = 15, n = 16) and one prospective clinical trial (n = 25) were used to test MDM2's utility as a clinical biomarker. in vitro and in vivo assessments were conducted in DDLPS cell lines. RESULTS: Genomic MDM2 amplification follows a highly reproducible log-normal distribution. In patients with DDLPS treated with complete tumor resection, elevated MDM2 was associated with shortened time to recurrence as measured by genomic amplification (p = .003) and mRNA expression (p = .04). In patients requiring systemic therapy, higher MDM2 amplification was associated with reduced overall survival (p = .04). Doxorubicin treatment of DDLPS cells in vitro demonstrated variable sensitivity based on baseline MDM2 levels, and doxorubicin treatment elevated MDM2 expression. In vivo, treatment with doxorubicin followed by an MDM2 inhibitor improved doxorubicin sensitivity. CONCLUSION: MDM2 amplification levels in DDLPS follow a reproducible distribution and are associated with clinical outcomes and drug sensitivity. These results suggest that a prospective study of MDM2 as a predictive biomarker in DDLPS is warranted. IMPLICATIONS FOR PRACTICE: No validated biomarkers exist for treatment selection in dedifferentiated liposarcoma (DDLPS). Although murine double minute 2 (MDM2) is currently used for diagnosis, the clinical relevance of MDM2 amplification has yet to be fully assessed. This study found that MDM2 amplification follows a predictable distribution in DDLPS and correlates with clinical and biological outcomes. These data suggests that MDM2 amplification may be a useful biomarker in DDLPS.

Loss of TXNIP enhances peritoneal metastasis and can be abrogated by dual TORC1/2 inhibition.

Peritoneal metastasis (PM) is a debilitating consequence of multiple cancers. As cancer cells lose tonic signaling related to attachment dependence, critical morphologic shifts result in alteration of the transcriptome. Identifying key genes associated with this transformation may lead to targeted therapies for this devastating complication. TC71, CHLA9, PANC1, YOU and HEYA8 cell lines were grown as tumor spheroids in polyHEMA coated plates. Temporal profiling of transcriptomic alterations over 72 hrs was used to develop a comprehensive PM model. We identified transcriptomic outliers using Gaussian mixtures model clustering to identify drivers of spheroid formation. Outliers were validated in The Cancer Genome Atlas (TCGA) and an ovarian tissue microarray (TMA) and by modulation in ovarian cancer models in vitro and in peritoneal xenograft models. Outlier analysis of PM genes identified the gene TXNIP and the TORC signaling as central to PM. Ovarian cancer spheroids isolated from patient ascites had significantly higher TXNIP than their attached counterparts (p = 0.047). TXNIP levels predicted progression-free (log-rank p = 0.026) survival in stage 1/2 ovarian cancer and overall survival (log rank p = 0.047) in stage 3/4 ovarian cancer. In vitro, TXNIP silencing was associated with increased mTOR signaling and enhanced spheroid development which could be overcome by TAK228, a TORC1/2 inhibitor. Similarly, in vivo peritoneal xenograft models of carcinomatosis were prevented by TAK228. PM is driven by TXNIP-associated TORC1/2 signaling. This work provides the first evidence that TORC1/2 inhibition may prevent PM.