p53 modulates kinase inhibitor resistance and lineage plasticity in NF1-related MPNSTs.

Malignant peripheral nerve sheath tumors (MPNSTs) are chemotherapy resistant sarcomas that are a leading cause of death in neurofibromatosis type 1 (NF1). Although NF1-related MPNSTs derive from neural crest cell origin, they also exhibit intratumoral heterogeneity. TP53 mutations are associated with significantly decreased survival in MPNSTs, however the mechanisms underlying TP53-mediated therapy responses are unclear in the context of NF1-deficiency. We evaluated the role of two commonly altered genes, MET and TP53, in kinome reprograming and cellular differentiation in preclinical MPNST mouse models. We previously showed that MET amplification occurs early in human MPNST progression and that Trp53 loss abrogated MET-addiction resulting in MET inhibitor resistance. Here we demonstrate a novel mechanism of therapy resistance whereby p53 alters MET stability, localization, and downstream signaling leading to kinome reprogramming and lineage plasticity. Trp53 loss also resulted in a shift from RAS/ERK to AKT signaling and enhanced sensitivity to MEK and mTOR inhibition. In response to MET, MEK and mTOR inhibition, we observed broad and heterogeneous activation of key differentiation genes in Trp53-deficient lines suggesting Trp53 loss also impacts lineage plasticity in MPNSTs. These results demonstrate the mechanisms by which p53 loss alters MET dependency and therapy resistance in MPNSTS through kinome reprogramming and phenotypic flexibility.

BISCUIT: an efficient, standards-compliant tool suite for simultaneous genetic and epigenetic inference in bulk and single-cell studies.

Data from both bulk and single-cell whole-genome DNA methylation experiments are under-utilized in many ways. This is attributable to inefficient mapping of methylation sequencing reads, routinely discarded genetic information, and neglected read-level epigenetic and genetic linkage information. We introduce the BISulfite-seq Command line User Interface Toolkit (BISCUIT) and its companion R/Bioconductor package, biscuiteer, for simultaneous extraction of genetic and epigenetic information from bulk and single-cell DNA methylation sequencing. BISCUIT's performance, flexibility and standards-compliant output allow large, complex experimental designs to be characterized on clinical timescales. BISCUIT is particularly suited for processing data from single-cell DNA methylation assays, with its excellent scalability, efficiency, and ability to greatly enhance mappability, a key challenge for single-cell studies. We also introduce the epiBED format for single-molecule analysis of coupled epigenetic and genetic information, facilitating the study of cellular and tissue heterogeneity from DNA methylation sequencing.

NF1 deficiency drives metabolic reprogramming in ER+ breast cancer.

OBJECTIVE: NF1 is a tumor suppressor gene and its protein product, neurofibromin, is a negative regulator of the RAS pathway. NF1 is one of the top driver mutations in sporadic breast cancer such that 27 % of breast cancers exhibit damaging NF1 alterations. NF1 loss-of-function is a frequent event in the genomic evolution of estrogen receptor (ER)+ breast cancer metastasis and endocrine resistance. Individuals with Neurofibromatosis type 1 (NF) - a disorder caused by germline NF1 mutations - have an increased risk of dying from breast cancer [1-4]. NF-related breast cancers are associated with decreased overall survival compared to sporadic breast cancer. Despite numerous studies interrogating the role of RAS mutations in tumor metabolism, no study has comprehensively profiled the NF1-deficient breast cancer metabolome to define patterns of energetic and metabolic reprogramming. The goals of this investigation were (1) to define the role of NF1 deficiency in estrogen receptor-positive (ER+) breast cancer metabolic reprogramming and (2) to identify potential targeted pathway and metabolic inhibitor combination therapies for NF1-deficient ER + breast cancer. METHODS: We employed two ER+ NF1-deficient breast cancer models: (1) an NF1-deficient MCF7 breast cancer cell line to model sporadic breast cancer, and (2) three distinct, Nf1-deficient rat models to model NF-related breast cancer [1]. IncuCyte proliferation analysis was used to measure the effect of NF1 deficiency on cell proliferation and drug response. Protein quantity was assessed by Western Blot analysis. We then used RNAseq to investigate the transcriptional effect of NF1 deficiency on global and metabolism-related transcription. We measured cellular energetics using Agilent Seahorse XF-96 Glyco Stress Test and Mito Stress Test assays. We performed stable isotope labeling and measured [U-13C]-glucose and [U-13C]-glutamine metabolite incorporation and measured total metabolite pools using mass spectrometry. Lastly, we used a Bliss synergy model to investigate NF1-driven changes in targeted and metabolic inhibitor synergy. RESULTS: Our results revealed that NF1 deficiency enhanced cell proliferation, altered neurofibromin expression, and increased RAS and PI3K/AKT pathway signaling while constraining oxidative ATP production and restricting energetic flexibility. Neurofibromin deficiency also increased glutamine influx into TCA intermediates and dramatically increased lipid pools, especially triglycerides (TG). Lastly, NF1 deficiency alters the synergy between metabolic inhibitors and traditional targeted inhibitors. This includes increased synergy with inhibitors targeting glycolysis, glutamine metabolism, mitochondrial fatty acid transport, and TG synthesis. CONCLUSIONS: NF1 deficiency drives metabolic reprogramming in ER+ breast cancer. This reprogramming is characterized by oxidative ATP constraints, glutamine TCA influx, and lipid pool expansion, and these metabolic changes introduce novel metabolic-to-targeted inhibitor synergies.

Cell State of Origin Impacts Development of Distinct Endometriosis-Related Ovarian Carcinoma Histotypes.

Clear cell ovarian carcinoma (CCOC) and endometrioid ovarian carcinoma (ENOC) are ovarian carcinoma histotypes, which are both thought to arise from ectopic endometrial (or endometrial-like) cells through an endometriosis intermediate. How the same cell type of origin gives rise to two morphologically and biologically different histotypes has been perplexing, particularly given that recurrent genetic mutations are common to both and present in nonmalignant precursors. We used RNA transcription analysis to show that the expression profiles of CCOC and ENOC resemble those of normal endometrium at secretory and proliferative phases of the menstrual cycle, respectively. DNA methylation at the promoter of the estrogen receptor (ER) gene (ESR1) was enriched in CCOC, which could potentially lock the cells in the secretory state. Compared with normal secretory-type endometrium, CCOC was further defined by increased expression of cysteine and glutathione synthesis pathway genes and downregulation of the iron antiporter, suggesting iron addiction and highlighting ferroptosis as a potential therapeutic target. Overall, these findings suggest that while CCOC and ENOC arise from the same cell type, these histotypes likely originate from different cell states. This "cell state of origin" model may help to explain the presence of histologic and molecular cancer subtypes arising in other organs. SIGNIFICANCE: Two cancer histotypes diverge from a common cell of origin epigenetically locked in different cell states, highlighting the importance of considering cell state to better understand the cell of origin of cancer.

Formation of templated inclusions in a forebrain α-synuclein mouse model is independent of LRRK2.

Leucine-rich repeat kinase 2 (LRRK2) and α-synuclein share enigmatic roles in the pathobiology of Parkinson's disease (PD). LRRK2 mutations are a common genetic cause of PD which, in addition to neurodegeneration, often present with abnormal deposits of α-synuclein in the form of Lewy-related pathology. As Lewy-related pathology is a prominent neuropathologic finding in sporadic PD, the relationship between LRRK2 and α-synuclein has garnered considerable interest. However, whether and how LRRK2 might influence the accumulation of Lewy-related pathology remains poorly understood. Through stereotactic injection of mouse α-synuclein pre-formed fibrils (PFF), we modeled the spread of Lewy-related pathology within forebrain regions where LRRK2 is most highly expressed. The impact of LRRK2 genotype on the formation of α-synuclein inclusions was evaluated at 1-month post-injection. Neither deletion of LRRK2 nor G2019S LRRK2 knockin appreciably altered the burden of α-synuclein pathology at this early timepoint. These observations fail to provide support for a robust pathophysiologic interaction between LRRK2 and α-synuclein in the forebrain in vivo. There was, however, a modest reduction in microglial activation induced by PFF delivery in the hippocampus of LRRK2 knockout mice, suggesting that LRRK2 may contribute to α-synuclein-induced neuroinflammation. Collectively, our data indicate that the pathological accumulation of α-synuclein in the mouse forebrain is largely independent of LRRK2.

A Novel Humanized Immune Stroma PDX Cancer Model for Therapeutic Studies.

Standard preclinical human tumor models lack a human tumor stroma. However, as stroma contributes to therapeutic resistance, the lack of human stroma may make current models less stringent for testing new therapies. To address this, using patient-derived tumor cells, patient derived cancer-associated mesenchymal stem/progenitor cells, and human endothelial cells, we created a Human Stroma-Patient Derived Xenograft (HS-PDX) tumor model. HS-PDX, compared to the standard PDX model, demonstrate greater resistance to targeted therapy and chemotherapy, and better reflect patient response to therapy. Furthermore, HS-PDX can be grown in mice with humanized bone marrow to create humanized immune stroma patient-derived xenograft (HIS-PDX) models. The HIS-PDX model contains human connective tissues, vascular and immune cell infiltrates. RNA sequencing analysis demonstrated a 94-96% correlation with primary human tumor. Using this model, we demonstrate the impact of human tumor stroma on response to CAR-T cell therapy and immune checkpoint inhibitor therapy. We show an immunosuppressive role for human tumor stroma and that this model can be used to identify immunotherapeutic combinations to overcome stromally mediated immunosuppression. Combined, our data confirm a critical role for human stoma in therapeutic response and indicate that HIS-PDX can be an important tool for preclinical drug testing. Statement of Significance: We developed a tumor model with human stromal, vascular, and immune cells. This model mirrors patient response to chemotherapy, targeted therapy, and immunotherapy, and can be used to study therapy resistance.

Formation of templated inclusions in a forebrain α-synuclein mouse model is independent of LRRK2.

Leucine-rich repeat kinase 2 (LRRK2) and α-synuclein share enigmatic roles in the pathobiology of Parkinson's disease (PD). LRRK2 mutations are a common genetic cause of PD which, in addition to neurodegeneration, often present with abnormal deposits of α-synuclein in the form of Lewy-related pathology. As Lewy-related pathology is a prominent neuropathologic finding in sporadic PD, the relationship between LRRK2 and α-synuclein has garnered considerable interest. However, whether and how LRRK2 might influence the accumulation of Lewy-related pathology remains poorly understood. Through stereotactic injection of mouse α-synuclein pre-formed fibrils (PFF), we modeled the spread of Lewy-related pathology within forebrain regions where LRRK2 is most highly expressed. The impact of LRRK2 genotype on the formation of α-synuclein inclusions was evaluated at 1-month post-injection. Neither deletion of LRRK2 nor G2019S LRRK2 knockin appreciably altered the burden of α-synuclein pathology at this early timepoint. These observations fail to provide support for a robust pathophysiologic interaction between LRRK2 and α-synuclein in the forebrain in vivo. There was, however, a modest reduction in microglial activation induced by PFF delivery in the hippocampus of LRRK2 knockout mice, suggesting that LRRK2 may contribute to α-synuclein-induced neuroinflammation. Collectively, our data indicate that the pathological accumulation of α-synuclein in the mouse forebrain is largely independent of LRRK2.

PGC-1ß maintains mitochondrial metabolism and restrains inflammatory gene expression.

Metabolic programming of the innate immune cells known as dendritic cells (DCs) changes in response to different stimuli, influencing their function. While the mechanisms behind increased glycolytic metabolism in response to inflammatory stimuli are well-studied, less is known about the programming of mitochondrial metabolism in DCs. We used lipopolysaccharide (LPS) and interferon-ß (IFN-ß), which differentially stimulate the use of glycolysis and oxidative phosphorylation (OXPHOS), respectively, to identify factors important for mitochondrial metabolism. We found that the expression of peroxisome proliferator-activated receptor gamma co-activator 1ß (PGC-1ß), a transcriptional co-activator and known regulator of mitochondrial metabolism, decreases when DCs are activated with LPS, when OXPHOS is diminished, but not with IFN-ß, when OXPHOS is maintained. We examined the role of PGC-1ß in bioenergetic metabolism of DCs and found that PGC-1ß deficiency indeed impairs their mitochondrial respiration. PGC-1ß-deficient DCs are more glycolytic compared to controls, likely to compensate for reduced OXPHOS. PGC-1ß deficiency also causes decreased capacity for ATP production at steady state and in response to IFN-ß treatment. Loss of PGC-1ß in DCs leads to increased expression of genes in inflammatory pathways, and reduced expression of genes encoding proteins important for mitochondrial metabolism and function. Collectively, these results demonstrate that PGC-1ß is a key regulator of mitochondrial metabolism and negative regulator of inflammatory gene expression in DCs.

Lurbinectedin Inhibits the EWS-WT1 Transcription Factor in Desmoplastic Small Round Cell Tumor.

Desmoplastic small round cell tumor (DSRCT) is a rare pediatric sarcoma with poor overall survival. This tumor is absolutely dependent on the continued expression and activity of its defining molecular lesion, the EWS-WT1 transcription factor. Unfortunately, the therapeutic targeting of transcription factors is challenging, and there is a critical need to identify compounds that inhibit EWS-WT1. Here we show that the compound lurbinectedin inhibits EWS-WT1 by redistributing the protein within the nucleus to the nucleolus. This nucleolar redistribution interferes with the activity of EWS-WT1 to reverse the expression of over 70% of the transcriptome. In addition, the compound blocks the expression of the EWS-WT1 fusion protein to inhibit cell proliferation at the lowest GI50 ever reported for this compound in any cell type. The effects occur at concentrations that are easily achievable in the clinic and translate to the in vivo setting to cause tumor regressions in multiple mice in a xenograft and PDX model of DSRCT. Importantly, this mechanism of nucleolar redistribution is also seen with wild-type EWSR1 and the related fusion protein EWS-FLI1. This provides evidence for a "class effect" for the more than 18 tumors driven by EWSR1 fusion proteins. More importantly, the data establish lurbinectedin as a promising clinical candidate for DSRCT.

Rare cases of medulloblastoma with hypermutation.

BACKGROUND: Medulloblastoma is the most common malignant brain tumor of childhood and is considered a tumor with low mutational burden (~1 Mut/Mb). Therefore, though the medulloblastoma genomes have been extensively characterized in literature, reports on potential hypermutations and underlying mutagenic processes in medulloblastomas are limited. AIM: In this report, we studied the landscape of mutational burden in primary and recurrent medulloblastoma. Furthermore, we wanted to understand the differences in underlying mutagenic mechanisms in medulloblastoma with low and high mutational burdens. METHODS: Fifty-three primary and recurrent medulloblastoma genomic sequence were downloaded from the European Genome Archive as BAM files. Thirty-three cases were obtained from formalin-fixed paraffin-embedded tissues from pathology diagnostic archives of Spectrum Health and Cooperative Human Tissue Network. Somatic mutations were called using Mutect2, following best practices guidelines for Genome Analysis Toolkit V4. Mutational signatures were analyzed using deconstructSigs. RESULTS: We identified nine medulloblastoma cases with high mutational burden (>5 Mut/Mb). Of them, five cases met the criteria of hypermutation (>10Mut/Mb), two of the five tumors had canonical mutations in the POLE proof-reading domain, where a large proportion of mutations in these tumor genomes contributed to signature 10. The hypermutated cases also demonstrated mutational signatures 14, 15, and 21, indicating the role of mis match repair deficiency in their mutagenesis. Of the four known molecular subgroups in medulloblastoma-SHH, WNT, Group 3, and Group 4-both the POLE-mutated cases belonged to the SHH subgroup. This report identifies rare cases of hypermutation in medulloblastoma driven by defects in DNA repair mechanisms. CONCLUSION: Hypermutation in medulloblastoma can impact therapeutic decisions, especially at recurrence in otherwise fatal high risk SHH-medulloblastomas. A defect in DNA repair leading to SHH -medulloblastoma is yet another important mechanism that should be further investigated in the genesis of these tumors. Therefore, this report provides important scientific and clinical rationale for future research looking for incidence of hypermutation in large cohorts of medulloblastoma patients.

Evaluation of whole-genome DNA methylation sequencing library preparation protocols.

BACKGROUND: With rapidly dropping sequencing cost, the popularity of whole-genome DNA methylation sequencing has been on the rise. Multiple library preparation protocols currently exist. We have performed 22 whole-genome DNA methylation sequencing experiments on snap frozen human samples, and extensively benchmarked common library preparation protocols for whole-genome DNA methylation sequencing, including three traditional bisulfite-based protocols and a new enzyme-based protocol. In addition, different input DNA quantities were compared for two kits compatible with a reduced starting quantity. In addition, we also present bioinformatic analysis pipelines for sequencing data from each of these library types. RESULTS: An assortment of metrics were collected for each kit, including raw read statistics, library quality and uniformity metrics, cytosine retention, and CpG beta value consistency between technical replicates. Overall, the NEBNext Enzymatic Methyl-seq and Swift Accel-NGS Methyl-Seq kits performed quantitatively better than the other two protocols. In addition, the NEB and Swift kits performed well at low-input amounts, validating their utility in applications where DNA is the limiting factor. RESULTS: The NEBNext Enzymatic Methyl-seq kit appeared to be the best option for whole-genome DNA methylation sequencing of high-quality DNA, closely followed by the Swift kit, which potentially works better for degraded samples. Further, a general bioinformatic pipeline is applicable across the four protocols, with the exception of extra trimming needed for the Swift Biosciences's Accel-NGS Methyl-Seq protocol to remove the Adaptase sequence.

Gut Microbiota Dysbiosis Is Associated with Elevated Bile Acids in Parkinson's Disease.

The gut microbiome can impact brain health and is altered in Parkinson's disease (PD). The vermiform appendix is a lymphoid tissue in the cecum implicated in the storage and regulation of the gut microbiota. We sought to determine whether the appendix microbiome is altered in PD and to analyze the biological consequences of the microbial alterations. We investigated the changes in the functional microbiota in the appendix of PD patients relative to controls (n = 12 PD, 16 C) by metatranscriptomic analysis. We found microbial dysbiosis affecting lipid metabolism, including an upregulation of bacteria responsible for secondary bile acid synthesis. We then quantitatively measure changes in bile acid abundance in PD relative to the controls in the appendix (n = 15 PD, 12 C) and ileum (n = 20 PD, 20 C). Bile acid analysis in the PD appendix reveals an increase in hydrophobic and secondary bile acids, deoxycholic acid (DCA) and lithocholic acid (LCA). Further proteomic and transcriptomic analysis in the appendix and ileum corroborated these findings, highlighting changes in the PD gut that are consistent with a disruption in bile acid control, including alterations in mediators of cholesterol homeostasis and lipid metabolism. Microbially derived toxic bile acids are heightened in PD, which suggests biliary abnormalities may play a role in PD pathogenesis.

Mithramycin induces promoter reprogramming and differentiation of rhabdoid tumor.

Rhabdoid tumor (RT) is a pediatric cancer characterized by the inactivation of SMARCB1, a subunit of the SWI/SNF chromatin remodeling complex. Although this deletion is the known oncogenic driver, there are limited effective therapeutic options for these patients. Here we use unbiased screening of cell line panels to identify a heightened sensitivity of rhabdoid tumor to mithramycin and the second-generation analogue EC8042. The sensitivity of MMA and EC8042 was superior to traditional DNA damaging agents and linked to the causative mutation of the tumor, SMARCB1 deletion. Mithramycin blocks SMARCB1-deficient SWI/SNF activity and displaces the complex from chromatin to cause an increase in H3K27me3. This triggers chromatin remodeling and enrichment of H3K27ac at chromHMM-defined promoters to restore cellular differentiation. These effects occurred at concentrations not associated with DNA damage and were not due to global chromatin remodeling or widespread gene expression changes. Importantly, a single 3-day infusion of EC8042 caused dramatic regressions of RT xenografts, recapitulated the increase in H3K27me3, and cellular differentiation described in vitro to completely cure three out of eight mice.

Detection of Chemotherapy-resistant Pancreatic Cancer Using a Glycan Biomarker, sTRA.

PURPOSE: A subset of pancreatic ductal adenocarcinomas (PDACs) is highly resistant to systemic chemotherapy, but no markers are available in clinical settings to identify this subset. We hypothesized that a glycan biomarker for PDACs called sialylated tumor-related antigen (sTRA) could be used for this purpose. EXPERIMENTAL DESIGN: We tested for differences between PDACs classified by glycan expression in multiple systems: sets of cell lines, organoids, and isogenic cell lines; primary tumors; and blood plasma from human subjects. RESULTS: The sTRA-expressing models tended to have stem-like gene expression and the capacity for mesenchymal differentiation, in contrast to the nonexpressing models. The sTRA cell lines also had significantly increased resistance to seven different chemotherapeutics commonly used against pancreatic cancer. Patients with primary tumors that were positive for a gene expression classifier for sTRA received no statistically significant benefit from adjuvant chemotherapy, in contrast to those negative for the signature. In another cohort, based on direct measurements of sTRA in tissue microarrays, the patients who were high in sTRA again had no statistically significant benefit from adjuvant chemotherapy. Furthermore, a blood plasma test for the sTRA glycan identified the PDACs that showed rapid relapse following neoadjuvant chemotherapy. CONCLUSIONS: This research demonstrates that a glycan biomarker could have value to detect chemotherapy-resistant PDAC in clinical settings. This capability could aid in the development of stratified treatment plans and facilitate biomarker-guided trials targeting resistant PDAC.

Examining the molecular mechanisms contributing to the success of an invasive species across different ecosystems.

Invasive species provide an opportune system to investigate how populations respond to new environments. Baby's breath (Gypsophila paniculata) was introduced to North America in the 1800s and has since spread throughout the United States and western Canada. We used an RNA-seq approach to explore how molecular processes contribute to the success of invasive populations with similar genetic backgrounds across distinct habitats. Transcription profiles were constructed from seedlings collected from a sand dune ecosystem in Petoskey, MI (PSMI), and a sagebrush ecosystem in Chelan, WA (CHWA). We assessed differential gene expression and identified SNPs within differentially expressed genes. We identified 1,146 differentially expressed transcripts across all sampled tissues between the two populations. GO processes enriched in PSMI were associated with nutrient starvation, while enriched processes in CHWA were associated with abiotic stress. Only 7.4% of the differentially expressed transcripts contained SNPs differing in allele frequencies of at least 0.5 between populations. Common garden studies found the two populations differed in germination rate and seedling emergence success. Our results suggest the success of G. paniculata in these two environments is likely due to plasticity in specific molecular processes responding to different environmental conditions, although some genetic divergence may be contributing to these differences.

Loss of microRNA-21 leads to profound stromal remodeling and short survival in K-Ras-driven mouse models of pancreatic cancer.

The microenvironment of pancreatic cancer adenocarcinoma (PDAC) is highly desmoplastic with distinct tumor-restraining and tumor-promoting fibroblast subpopulations. Re-education rather than indiscriminate elimination of these fibroblasts has emerged as a new strategy for combination therapy. Here, we studied the effects of global loss of profibrotic noncoding regulatory microRNA-21 (miR-21) in K-Ras-driven p53-deleted genetically engineered mouse models of PDAC. Strikingly, loss of miR-21 accelerated tumor initiation via mucinous cystic neoplastic lesions and progression to locally advanced invasive carcinoma from which animals precipitously succumbed at an early age. The absence of tumor-restraining myofibroblasts and a massive infiltrate of immune cells were salient phenotypic features of global miR-21 loss. Stromal miR-21 activity was required for induction of tumor-restraining myofibroblasts in in vivo isograft transplantation experiments. Low miR-21 expression negatively correlated with a fibroblast gene expression signature and positively with an immune cell gene expression signature in The Cancer Genome Atlas PDAC data set (n = 156) mirroring findings in the mouse models. Our results exposed an overall tumor-suppressive function of miR-21 in in vivo PDAC models. These results have important clinical implications for anti-miR-21-based inhibitory therapeutic approaches under consideration for PDAC and other cancer types. Mechanistic dissection of the cell-intrinsic role of miR-21 in cancer-associated fibroblasts and other cell types will be needed to inform best strategies for pharmacological modulation of miR-21 activity to remodel the tumor microenvironment and enhance treatment response in PDAC.

Desmoplastic small round cell tumor is dependent on the EWS-WT1 transcription factor.

Desmoplastic small round cell tumor (DSRCT) is a rare and aggressive soft-tissue malignancy with a poor overall survival and no effective therapeutic options. The tumor is believed to be dependent on the continued activity of the oncogenic EWS-WT1 transcription factor. However, the dependence of the tumor on EWS-WT1 has not been well established. In addition, there are no studies exploring the downstream transcriptional program across multiple cell lines. In this study, we have developed a novel approach to selectively silence EWS-WT1 without impacting either wild-type EWSR1 or WT1. We show a clear dependence of the tumor on EWS-WT1 in two different cell lines, BER and JN-DSCRT-1. In addition, we identify and validate important downstream target pathways commonly dysregulated in other translocation-positive sarcomas, including PRC2, mTOR, and TGFB. Surprisingly, there is striking overlap between the EWS-WT1 and EWS-FLI1 gene signatures, despite the fact that the DNA-binding domain of the fusion proteins, WT1 and FLI1, is structurally unique and classified as different types of transcription factors. This study provides important insight into the biology of this disease relative to other translocation-positive sarcomas, and the basis for the therapeutic targeting of EWS-WT1 for this disease that has limited therapeutic options.

CDK9 Blockade Exploits Context-dependent Transcriptional Changes to Improve Activity and Limit Toxicity of Mithramycin for Ewing Sarcoma.

There is a need to develop novel approaches to improve the balance between efficacy and toxicity for transcription factor-targeted therapies. In this study, we exploit context-dependent differences in RNA polymerase II processivity as an approach to improve the activity and limit the toxicity of the EWS-FLI1-targeted small molecule, mithramycin, for Ewing sarcoma. The clinical activity of mithramycin for Ewing sarcoma is limited by off-target liver toxicity that restricts the serum concentration to levels insufficient to inhibit EWS-FLI1. In this study, we perform an siRNA screen of the druggable genome followed by a matrix drug screen to identify mithramycin potentiators and a synergistic "class" effect with cyclin-dependent kinase 9 (CDK9) inhibitors. These CDK9 inhibitors enhanced the mithramycin-mediated suppression of the EWS-FLI1 transcriptional program leading to a shift in the IC50 and striking regressions of Ewing sarcoma xenografts. To determine whether these compounds may also be liver protective, we performed a qPCR screen of all known liver toxicity genes in HepG2 cells to identify mithramycin-driven transcriptional changes that contribute to the liver toxicity. Mithramycin induces expression of the BTG2 gene in HepG2 but not Ewing sarcoma cells, which leads to a liver-specific accumulation of reactive oxygen species (ROS). siRNA silencing of BTG2 rescues the induction of ROS and the cytotoxicity of mithramycin in these cells. Furthermore, CDK9 inhibition blocked the induction of BTG2 to limit cytotoxicity in HepG2, but not Ewing sarcoma cells. These studies provide the basis for a synergistic and less toxic EWS-FLI1-targeted combination therapy for Ewing sarcoma.

Population Genomics in Wild Tomatoes-The Interplay of Divergence and Admixture.

Hybridization between closely related plant species is widespread, but the outcomes of hybridization are not fully understood. This study investigates phylogenetic relationships and the history of hybridization in the wild tomato clade (Solanum sect. Lycopersicon). We sequenced RNA from individuals of 38 different populations and, by combining this with published data, build a comprehensive genomic data set for the entire clade. The data indicate that many taxa are not monophyletic and many individuals are admixed due to repeated hybridization. The most polymorphic species, Solanum peruvianum, has two genetic and geographical subpopulations, while its sister species, Solanum chilense, has distinct coastal populations and reduced heterozygosity indicating a recent expansion south following speciation from S. peruvianum circa 1.25 Ma. Discontinuous populations west of 72° are currently described as S. chilense, but are genetically intermediate between S. chilense and S. peruvianum. Based upon molecular, morphological, and crossing data, we test the hypothesis that these discontinuous "S. chilense" populations are an example of recombinational speciation. Recombinational speciation is rarely reported, and we discuss the difficulties in identifying it and differentiating between alternative demographic scenarios. This discovery presents a new opportunity to understand the genomic outcomes of hybridization in plants.