Ewing sarcoma usually expresses the EWS/FLI fusion transcription factor oncoprotein. EWS/FLI regulates myriad genes required for Ewing sarcoma development. EWS/FLI binds GGAA-microsatellite sequences in vivo and in vitro. These sequences provide EWS/FLI-mediated activation to reporter constructs, suggesting that they function as EWS/FLI-response elements. We now demonstrate the critical role of an EWS/FLI-bound GGAA-microsatellite in regulation of the gene as well as for Ewing sarcoma proliferation and anchorage-independent growth. Clinically, genomic GGAA-microsatellites are highly variable and polymorphic. Current data suggest that there is an optimal "sweet-spot" GGAA-microsatellite length (of 18-26 GGAA repeats) that confers maximal EWS/FLI-responsiveness to target genes, but the mechanistic basis for this remains unknown. Our biochemical studies, using recombinant Δ22 (a version of EWS/FLI containing only the FLI portion), demonstrate a stoichiometry of one Δ22-monomer binding to every two consecutive GGAA-repeats on shorter microsatellite sequences. Surprisingly, the affinity for Δ22 binding to GGAA-microsatellites significantly decreased, and ultimately became unmeasureable, when the size of the microsatellite was increased to the sweet-spot length. In contrast, a fully functional EWS/FLI mutant (Mut9, which retains approximately half of the EWS portion of the fusion) showed low affinity for smaller GGAA-microsatellites but instead significantly increased its affinity at sweet-spot microsatellite lengths. Single-gene ChIP and genome-wide ChIP-sequencing (ChIP-seq) and RNA-seq studies extended these findings to the in vivo setting. Together, these data demonstrate the critical requirement of GGAA-microsatellites as EWS/FLI activating response elements in vivo and reveal an unexpected role for the EWS portion of the EWS/FLI fusion in binding to sweet-spot GGAA-microsatellites.
EWS/FLI is the pathognomic fusion oncoprotein that drives Ewing sarcoma. The amino-terminal EWS portion coordinates transcriptional regulation and the carboxy-terminal FLI portion contains an ETS DNA-binding domain. EWS/FLI acts as an aberrant transcription factor, orchestrating a complex mix of gene activation and repression, from both high affinity ETS motifs and repetitive GGAA-microsatellites. Our overarching hypothesis is that executing multi-faceted transcriptional regulation requires EWS/FLI to use distinct molecular mechanisms at different loci. Many attempts have been made to map distinct functions to specific features of the EWS domain, but described deletion mutants are either fully active or completely “dead” and other approaches have been limited by the repetitive and disordered nature of the EWS domain. Here, we use transcriptomic approaches to show an EWS/FLI mutant, called DAF, previously thought to be nonfunctional, displays context-dependent and partial transcriptional activity but lacks transforming capacity. Using transcriptomic and phenotypic anchorage-independent growth profiles of other EWS/FLI mutants coupled with reported EWS/FLI localization data, we have mapped the critical structure-function requirements of the EWS domain for EWS/FLI-mediated oncogenesis. This approach defined unique classes of EWS/FLI response elements and revealed novel structure-function relationships required for EWS/FLI activation at these response elements.
Minimally invasive robotic-assisted surgeries have been increasingly used as a first-line of treatment for patients undergoing oncologic surgeries. In-situ tissue identification is critical to guide tissue resection and assist decision-making. Traditional intraoperative histopathologic analysis of frozen tissue sections can be time-consuming and present logistical challenges which interrupt surgical workflows. We report the development and implementation of a laparoscopic, drop-in version of the MasSpec Pen device integrated into the da Vinci Xi Surgical system for in vivo tissue analysis in a robotic-assisted porcine surgery. We evaluated the performance of the drop-in MasSpec Pen during surgery by introducing the device into the animal upper gastrointestinal system and performing in vivo analyses of the stomach and liver, including charred and bloody tissues after electrocauterization. The molecular profiles obtained included ions tentatively identified as metabolites and lipids typically observed with MasSpec Pen analysis, without causing observable tissue damage. Statistical classifiers built to distinguish porcine liver and stomach tissues using the in vivo data yielded an overall tissue identification accuracy of 98% (n = 53 analyses). The results provide evidence that the drop-in MasSpec Pen developed can be used to acquire mass spectra in vivo during a robotic-assisted surgery and might be used as an in vivo tissue assessment tool to help guide surgical resections and streamline surgical workflows.
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The role of cutaneous viral infections in the development of non-melanoma skin cancer (NMSC), including cutaneous squamous cell carcinoma (SCC), among chronic lymphocytic leukemia (CLL) and blood and marrow transplant (BMT) patients is not established. CLL (n = 977) and BMT (n = 3587) patients treated at the Moffitt Cancer Center were included in a retrospective cohort study. Human papillomavirus (HPV) and human polyomavirus (HPyV) DNA were examined in a subset of incident SCC tumors. Five-year cumulative incidence of NMSC was 1.42% in both BMT (n = 31 NMSCs) and CLL (n = 18 NMSCs) cohorts. Of the nine SCC tumors examined from each cohort, 22.2% and 33.3% were positive for viral DNA in the transplant (HPV 65, MCV) and CLL (HPV 38, HPV 15, HPyV6) cohort, respectively. Enhanced skin cancer screening of BMT/CLL patients should be conducted to better capture incident NMSCs and examine the role of viral infections in these tumors.
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