Myxoid liposarcoma (MLS) is an aggressive soft-tissue tumor characterized by a specific reciprocal t(12;16) translocation resulting in expression of the chimeric FUS-DDIT3 fusion protein, an oncogenic transcription factor. Similar to other translocation-associated sarcomas, MLS is characterized by a low frequency of somatic mutations, albeit a subset of MLS has previously been shown to be associated with activating PIK3CA mutations. This study was performed to assess the prevalence of PI3K/Akt signaling alterations in MLS and the potential of PI3Kdirected therapeutic concepts. In a large cohort of MLS, key components of the PI3K/Akt signaling cascade were evaluated by next generation seqeuncing (NGS), fluorescence in situ hybridization (FISH), and immunohistochemistry (IHC). In three MLS cell lines, PI3K activity was inhibited by RNAi and the small-molecule PI3K inhibitor BKM120 (buparlisib) in vitro. An MLS cell line-based avian chorioallantoic membrane model was applied for in vivo confirmation. In total, 26.8% of MLS cases displayed activating alterations in PI3K/Akt signaling components, with PIK3CA gain-of-function mutations representing the most prevalent finding (14.2%). IHC suggested PI3K/Akt activation in a far larger subgroup of MLS, implying alternative mechanisms of pathway activation. PI3K-directed therapeutic interference showed that MLS cell proliferation and viability significantly depended on PI3K-mediated signals in vitro and in vivo. Our preclinical study underlines the elementary role of PI3K/Akt signals in MLS tumorigenesis and provides a molecularly based rationale for a PI3K-targeted therapeutic approach which may be particularly effective in the subgroup of tumors carrying activating genetic alterations in PI3K/Akt signaling components.

The diagnosis of giant cell-rich lesions of bone can be challenging if radiological findings are ambiguous and tissue of the biologically deciding component is underrepresented in biopsy specimens. The frequent association of giant cell tumor of bone (GCT) and chondroblastoma (CB) with (secondary) aneurysmal bone cysts (ABC) may further impede correct classification. The present study evaluates the potentials and limitations of mutation-specific histone H3.3 and DOG1 immunohistochemistry, Sanger-/next generation sequencing (NGS) and FISH analysis in the differential diagnosis of 23 GCT, 14 CB and 19 ABC. All morphologically typical GCT and CB harbored mutations in the H3F3A or H3F3B gene, respectively. These were, except for one uncommon G34L mutation in a GCT, reliably and specifically detected by mutation-specific H3.3 G34W or H3.3 K36M immunohistochemistry and DNA sequencing. In the diagnostic substantiation of CB, DOG1 staining was less sensitive compared to H3.3 K36M immunohistochemistry. 47% of ABC specifically showed translocations of the USP6 gene, while mutations in H3F3A/B were absent.Based on the results of this study, we conclude that mutation-specific H3.3 immunohistochemistry (selectively complemented with NGS-based DNA sequencing) and USP6 FISH analysis enable a reliable diagnostic distinction of GCT, CB and ABC of morphologically and radiologically difficult cases.

Burkitt's lymphoma (BL) is a highly aggressive form of B-cell non-Hodgkin's lymphoma. The clinical outcome in children with BL has improved over the last years but the prognosis for adults is still poor, highlighting the need for novel treatment strategies. Here, we report that the combinational treatment with the Smac mimetic BV6 and TRAIL triggers necroptosis in BL when caspases are blocked by zVAD.fmk (TBZ treatment). The sensitivity of BL cells to TBZ correlates with MLKL expression. We demonstrate that necroptotic signaling critically depends on MLKL, since siRNA-induced knockdown and CRISPR/Cas9-mediated knockout of MLKL profoundly protect BL cells from TBZ-induced necroptosis. Conversely, MLKL overexpression in cell lines expressing low levels of MLKL leads to necroptosis induction, which can be rescued by pharmacological inhibitors, highlighting the important role of MLKL for necroptosis execution. Importantly, the methylation status analysis of the MLKL promoter reveals a correlation between methylation and MLKL expression. Thus, MLKL is epigenetically regulated in BL and might serve as a prognostic marker for treatment success of necroptosis-based therapies. These findings have crucial implications for the development of new treatment options for BL.

Plasma membrane accumulation of phosphorylated mixed lineage kinase domain-like (MLKL) is a hallmark of necroptosis, leading to membrane rupture and inflammatory cell death. Pro-death functions of MLKL are tightly controlled by several checkpoints, including phosphorylation. Endocytosis and exocytosis limit MLKL membrane accumulation and counteract necroptosis, but the exact mechanisms remain poorly understood. Here, we identify linear ubiquitin chain assembly complex (LUBAC)-mediated M1 poly-ubiquitination (poly-Ub) as novel checkpoint for necroptosis regulation downstream of activated MLKL in human cells. Loss of LUBAC activity inhibits necroptosis, without affecting necroptotic signaling, but by preventing membrane accumulation of activated MLKL. Flotillin-1/2 act as putative necroptotic M1 poly-Ub targets that inhibit necroptosis suppression induced by LUBAC inhibition. Finally, we confirm LUBAC-dependent suppression of necroptosis in primary human pancreatic organoids. Our findings identify LUBAC as species-specific regulator of necroptosis which prevents MLKL membrane accumulation and pioneer primary human organoids to model necroptosis in near-physiological settings.

<p>Supplementary Figure S1: In vitro and in vivo evaluation of PI3K suppression (LY294002) in myxoid liposarcoma cell lines.</p>

<p>Supplementary Materials and Methods: (Flow cytometry, Apoptosis assay, RNA interference and Statistical analysis)</p>

<p>Supplementary Figure S1: In vitro and in vivo evaluation of PI3K suppression (LY294002) in myxoid liposarcoma cell lines.</p>

Introduction: Myxoid liposarcoma (MLS) is the second most common type of liposarcoma, accounting for 30-35% of all LS cases. Over 90% of tumors are characterized by a reciprocal translocation t (12; 16) (q13; p11), resulting in a pathogenic gene fusion. The chimeric FUS-DDIT3 fusion protein is suggested to play a crucial role in MLS tumorigenesis and progression, although the specific biological function and the mechanism of action remain to be defined. We compiled a comprehensive cohort of 105 well-characterized MLS tissue specimens to identify actionable genetic aberrations. Methods: Targeted next-generation sequencing (NGS) using the Illumina MiSeq platform was performed to examine the mutational status of 23 cancer-related genes (covering all exons) known to be frequently mutated across various neoplasms. Furthermore, we examined the amplification/deletion status and characterized the specific chromosomal FUS-DDIT3 rearrangements by FISH and RT-PCR. A multivariate analysis was conducted to investigate the prognostic significance of mutation/amplification/deletion status. Results: Targeted next-generation sequencing drives the potential to generate comprehensive genetic information including less frequent mutated genes relevant for actionable treatments and prognostic assessment. Besides PIK3CA, six additional genes showed at least five mutations, including AKT1, CTNNB1, EGFR, ERBB2, MET and PTEN. Several oncogenic mutations were detected which have not been reported in MLS previously. We demonstrated several gene amplification/deletion events in MLS. Conclusion: Our results indicate the occurrence of mutational aberrations besides the chromosomal FUS-DDIT3 hallmark. These appear not to be related to specific subtypes of FUS-DDIT3 fusion transcripts in terms of a molecular pattern. Molecular screening for actionable mutations might represent a rational tool for the implementation of innovative targeted therapeutic approaches in MLS. To our best knowledge, this study is the most extensive one to yield a detailed map of actionable genetic aberrations across a comprehensive cohort of &gt;100 well-characterized MLS tissue specimens. Moreover, it reveals several molecular alteration-specific targets for innovative therapy strategies. Citation Format: Marcel Trautmann, Arne Krüger, Birte Jeiler, Christian Bertling, Jasmin Menzel, Magdalene Cyra, Konrad Steinestel, Inga Grünewald, Pierre Åman, Eva Wardelmann, Sebastian Huss, Wolfgang Hartmann. Myxoid liposarcoma: A molecular and clinicopathological analysis by targeted next-generation sequencing and fluorescence in situ hybridization [abstract]. In: Proceedings of the AACR International Conference: New Frontiers in Cancer Research; 2017 Jan 18-22; Cape Town, South Africa. Philadelphia (PA): AACR; Cancer Res 2017;77(22 Suppl):Abstract nr B05.