BRCA1/2 Functional Loss Defines a Targetable Subset in Leiomyosarcoma.

BACKGROUND: Soft-tissue sarcomas (STS) describe a heterogeneous group of mesenchymal tumors with limited treatment options. Targeted therapies exist for BRCA1/2 gene alterations, but their prevalence and role have not been fully described in STS. Here, we present the largest effort to characterize the frequency of homologous recombination (HR) DNA repair pathway alterations in STS subtypes and highlight the unique nature of leiomyosarcoma (LMS). MATERIALS AND METHODS: DNA sequencing data were analyzed for HR pathway alterations for 1,236 patients with STS. DNA sequencing data from an additional 1,312 patients were used to confirm the prevalence of HR pathway alterations in LMS. Four uterine LMS (uLMS) patients with functional BRCA2 loss were evaluated for response to poly (ADP-ribose) polymerase (PARP) inhibition. RESULTS: In an unselected STS study population, BRCA2 alterations were identified in 15 (1%) patients, and homozygous BRCA2 loss was detected in 9 (<1%). However, subset analysis revealed that these BRCA2 alterations were concentrated in uLMS as compared with any other STS subtype. Notably, 10% of uLMS tumors had a BRCA2 alteration. We further report that PARP inhibitors had demonstrated durable clinical benefit in four uLMS patients with BRCA2 loss. CONCLUSION: HR pathway alterations are rare in most STS. However, we identify uLMS to be enriched for BRCA2 loss and report the positive outcomes of a series of patients treated with PARP inhibitors. Our data suggest that patients with uLMS should be considered for somatic BRCA2 profiling. Prospective trials are necessary to confirm the efficacy of PARP inhibition in uLMS. IMPLICATIONS FOR PRACTICE: Soft-tissue sarcomas are a highly morbid, diverse set of tumors with limited treatment options. This study identifies an increased prevalence of functional BRCA1/2 loss in patients with uterine leiomyosarcoma (uLMS). It also presents four patients with uLMS and BRCA2 loss who achieved durable clinical benefit from poly (ADP-ribose) polymerase inhibition. These data suggest that patients with uLMS in particular should be screened for BRCA1/2 alterations and may benefit from treatment targeted to these alterations.

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.

Multiscale-omic assessment of EWSR1-NFATc2 fusion positive sarcomas identifies the mTOR pathway as a potential therapeutic target.

Sarcomas harboring EWSR1-NFATc2 fusions have historically been categorized and treated as Ewing sarcoma. Emerging evidence suggests unique molecular characteristics and chemotherapy sensitivities in EWSR1-NFATc2 fusion positive sarcomas. Comprehensive genomic profiles of 1024 EWSR1 fusion positive sarcomas, including 14 EWSR1-NFATc2 fusions, were identified in the FoundationCore® database. Additional data from the Gene Expression Omnibus, the Genomics of Drug Sensitivity in Cancer and The Cancer Genome Atlas datasets were included for analysis. EWSR1-NFATc2 fusion positive sarcomas were genomically distinct from traditional Ewing sarcoma and demonstrated upregulation of the mTOR pathway. We also present a case of a 58-year-old male patient with metastatic EWSR1-NFATc2 fusion positive sarcoma who achieved 47 months of disease stabilization when treated with combination mTOR and VEGF inhibition. EWSR1-NFATc2 fusion positive sarcomas are molecularly distinct entities with overactive mTOR signaling; which may be therapeutically targetable. These findings support the use of precision medicine in the Ewing family of tumors.

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.

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.

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.

Social overcrowding as a chronic stress model that increases adiposity in mice.

Stress is a widely recognized risk factor for psychiatric and metabolic disorders. A number of animal models utilizing various stressors have been developed to facilitate our understanding in the pathophysiology of stress-related dysfunctions. The most commonly used chronic stress paradigms include the unpredictable chronic mild stress paradigm, the social defeat paradigm and the social deprivation paradigm. Here we assess the potential of social crowding as an alternative chronic stress model to study the effects on affective behaviors and metabolic disturbances. Ten-week-old male C57BL/6 mice were housed in groups of four (control) or eight (social crowding; SC) in standard cage for 9 weeks. Exploration, anxiety- and depressive-like behaviors were assessed in the open field test, the elevated T-maze, the novelty-suppressed feeding test and the forced swim test. SC mice exhibited a modest anxiety-like phenotype without change in depressive-like behaviors. Nine weeks of social crowding did not affect the body weight, but robustly increased adiposity as determined by increased mass of fat depots. Consistent with the increased fat content, serum leptin was markedly elevated in the SC mice. Specific changes in gene expression were also observed in the hypothalamus and the white adipose tissue following SC housing. Our study demonstrates the potential of social crowding as an alternative model for the study of stress-related metabolic and behavioral dysfunctions.

Metabolic Effects of Social Isolation in Adult C57BL/6 Mice.

Obesity and metabolic dysfunction are risk factors for a number of chronic diseases, such as type 2 diabetes, hypertension, heart disease, stroke, and certain forms of cancers. Both animal studies and human population-based and clinical studies have suggested that chronic stress is a risk factor for metabolic disorders. A good social support system is known to exert positive effects on the mental and physical well-being of an individual. On the other hand, long-term deprivation of social contacts may represent a stressful condition that has negative effects on health. In the present study, we investigated the effects of chronic social isolation on metabolic parameters in adult C57BL/6 mice. We found that individually housed mice had increased adipose mass compared to group-housed mice, despite comparable body weight. The mechanism for the expansion of white adipose tissue mass was depot-specific. Notably, food intake was reduced in the social isolated animals, which occurred around the light-dark phase transition periods. Similarly, reductions in heat generated and the respiratory exchange ratio were observed during the light-dark transitions. These phase-specific changes due to long-term social isolation have not been reported previously. Our study shows social isolation contributes to increased adiposity and altered metabolic functions.