Epigenetic Reprogramming of Autophagy Drives Mutant IDH1 Glioma Progression and Response to Radiation.

Mutant isocitrate dehydrogenase 1 (mIDH1; IDH1 R132H ) exhibits a gain of function mutation enabling 2-hydroxyglutarate (2HG) production. 2HG inhibits DNA and histone demethylases, inducing epigenetic reprogramming and corresponding changes to the transcriptome. We previously demonstrated 2HG-mediated epigenetic reprogramming enhances DNA-damage response and confers radioresistance in mIDH1 gliomas harboring p53 and ATRX loss of function mutations. In this study, RNA-seq and ChIP-seq data revealed human and mouse mIDH1 glioma neurospheres have downregulated gene ontologies related to mitochondrial metabolism and upregulated autophagy. Further analysis revealed that the decreased mitochondrial metabolism was paralleled by a decrease in glycolysis, rendering autophagy as a source of energy in mIDH1 glioma cells. Analysis of autophagy pathways showed that mIDH1 glioma cells exhibited increased expression of pULK1-S555 and enhanced LC3 I/II conversion, indicating augmented autophagy activity. This dependence is reflected by increased sensitivity of mIDH1 glioma cells to autophagy inhibition. Blocking autophagy selectively impairs the growth of cultured mIDH1 glioma cells but not wild-type IDH1 (wtIDH1) glioma cells. Targeting autophagy by systemic administration of synthetic protein nanoparticles packaged with siRNA targeting Atg7 (SPNP-siRNA-Atg7) sensitized mIDH1 glioma cells to radiation-induced cell death, resulting in tumor regression, long-term survival, and immunological memory, when used in combination with IR. Our results indicate autophagy as a critical pathway for survival and maintenance of mIDH1 glioma cells, a strategy that has significant potential for future clinical translation. One Sentence Summary: The inhibition of autophagy sensitizes mIDH1 glioma cells to radiation, thus creating a promising therapeutic strategy for mIDH1 glioma patients. Graphical abstract: Our genetically engineered mIDH1 mouse glioma model harbors IDH1 R132H in the context of ATRX and TP53 knockdown. The production of 2-HG elicited an epigenetic reprogramming associated with a disruption in mitochondrial activity and an enhancement of autophagy in mIDH1 glioma cells. Autophagy is a mechanism involved in cell homeostasis related with cell survival under energetic stress and DNA damage protection. Autophagy has been associated with radio resistance. The inhibition of autophagy thus radio sensitizes mIDH1 glioma cells and enhances survival of mIDH1 glioma-bearing mice, representing a novel therapeutic target for this glioma subtype with potential applicability in combined clinical strategies.

Characterizing nascent transcription patterns of PROMPTs, eRNAs, and readthrough transcripts in the ENCODE4 deeply profiled cell lines.

Arising as co-products of canonical gene expression, transcription-associated lincRNAs, such as promoter upstream transcripts (PROMPTs), enhancer RNAs (eRNAs), and readthrough (RT) transcripts, are often regarded as byproducts of transcription, although they may be important for the expression of nearby genes. We identified regions of nascent expression of these lincRNA in 16 human cell lines using Bru-seq techniques, and found distinctly regulated patterns of PROMPT, eRNA, and RT transcription using the diverse biochemical approaches in the ENCODE4 deeply profiled cell lines collection. Transcription of these lincRNAs was influenced by sequence-specific features and the local or 3D chromatin landscape. However, these sequence and chromatin features do not describe the full spectrum of lincRNA expression variability we identify, highlighting the complexity of their regulation. This may suggest that transcription-associated lincRNAs are not merely byproducts, but rather that the transcript itself, or the act of its transcription, is important for genomic function.

Targeting DNA Repair and Survival Signaling in Diffuse Intrinsic Pontine Gliomas to Prevent Tumor Recurrence.

Therapeutic resistance remains a major obstacle to successful clinical management of diffuse intrinsic pontine glioma (DIPG), a high-grade pediatric tumor of the brain stem. In nearly all patients, available therapies fail to prevent progression. Innovative combinatorial therapies that penetrate the blood-brain barrier and lead to long-term control of tumor growth are desperately needed. We identified mechanisms of resistance to radiotherapy, the standard of care for DIPG. On the basis of these findings, we rationally designed a brain-penetrant small molecule, MTX-241F, that is a highly selective inhibitor of EGFR and PI3 kinase family members, including the DNA repair protein DNA-PK. Preliminary studies demonstrated that micromolar levels of this inhibitor can be achieved in murine brain tissue and that MTX-241F exhibits promising single-agent efficacy and radiosensitizing activity in patient-derived DIPG neurospheres. Its physiochemical properties include high exposure in the brain, indicating excellent brain penetrance. Because radiotherapy results in double-strand breaks that are repaired by homologous recombination (HR) and non-homologous DNA end joining (NHEJ), we have tested the combination of MTX-241F with an inhibitor of Ataxia Telangiectasia Mutated to achieve blockade of HR and NHEJ, respectively, with or without radiotherapy. When HR blockers were combined with MTX-241F and radiotherapy, synthetic lethality was observed, providing impetus to explore this combination in clinically relevant models of DIPG. Our data provide proof-of-concept evidence to support advanced development of MTX-241F for the treatment of DIPG. Future studies will be designed to inform rapid clinical translation to ultimately impact patients diagnosed with this devastating disease.

Cdc73 protects Notch-induced T-cell leukemia cells from DNA damage and mitochondrial stress.

ABSTRACT: Activated Notch signaling is highly prevalent in T-cell acute lymphoblastic leukemia (T-ALL), but pan-Notch inhibitors showed excessive toxicity in clinical trials. To find alternative ways to target Notch signals, we investigated cell division cycle 73 (Cdc73), which is a Notch cofactor and key component of the RNA polymerase-associated transcriptional machinery, an emerging target in T-ALL. Although we confirmed previous work that CDC73 interacts with NOTCH1, we also found that the interaction in T-ALL was context-dependent and facilitated by the transcription factor ETS1. Using mouse models, we showed that Cdc73 is important for Notch-induced T-cell development and T-ALL maintenance. Mechanistically, chromatin and nascent gene expression profiling showed that Cdc73 intersects with Ets1 and Notch at chromatin within enhancers to activate expression of known T-ALL oncogenes through its enhancer functions. Cdc73 also intersects with these factors within promoters to activate transcription of genes that are important for DNA repair and oxidative phosphorylation through its gene body functions. Consistently, Cdc73 deletion induced DNA damage and apoptosis and impaired mitochondrial function. The CDC73-induced DNA repair expression program co-opted by NOTCH1 is more highly expressed in T-ALL than in any other cancer. These data suggest that Cdc73 might induce a gene expression program that was eventually intersected and hijacked by oncogenic Notch to augment proliferation and mitigate the genotoxic and metabolic stresses of elevated Notch signaling. Our report supports studying factors such as CDC73 that intersect with Notch to derive a basic scientific understanding on how to combat Notch-dependent cancers without directly targeting the Notch complex.

Histone H3 K27M-mediated regulation of cancer cell stemness and differentiation in diffuse midline glioma.

Therapeutic resistance remains a major obstacle to preventing progression of H3K27M-altered Diffuse Midline Glioma (DMG). Resistance is driven in part by ALDH-positive cancer stem cells (CSC), with high ALDH1A3 expression observed in H3K27M-mutant DMG biopsies. We hypothesized that ALDH-mediated stemness and resistance may in part be driven by the oncohistone itself. Upon deletion of H3K27M, ALDH1A3 expression decreased dramatically and was accompanied by a gain in astrocytic marker expression and a loss of neurosphere forming potential, indicative of differentiation. Here we show that the oncohistone regulates histone acetylation through ALDH1A3 in a Wnt-dependent manner and that loss of H3K27M expression results in sensitization of DMGs to radiotherapy. The observed elevated Wnt signaling in H3K27M-altered DMG likely stems from a dramatic suppression of mRNA and protein expression of the Wnt inhibitor EYA4 driven by the oncohistone. Thus, our findings identify EYA4 as a bona fide tumor suppressor in DMG that upon suppression, results in aberrant Wnt signaling to orchestrate stemness and differentiation. Future studies will explore whether overexpression of EYA4 in DMG can impede growth and invasion. In summary, we have gained mechanistic insight into H3K27M-mediated regulation of cancer stemness and differentiation, which provides rationale for exploring new therapeutic targets for DMG.

First-in-Class NADH/Ubiquinone Oxidoreductase Core Subunit S7 (NDUFS7) Antagonist for the Treatment of Pancreatic Cancer.

Pancreatic cancer cells adapt to nutrient-scarce metabolic conditions by increasing their oxidative phosphorylation reserve to survive. Here, we present a first-in-class small-molecule NDUFS7 antagonist that inhibits oxidative phosphorylation (OXPHOS) for the treatment of pancreatic cancer. The lead compound, DX2-201, suppresses the proliferation of a panel of cell lines, and a metabolically stable analogue, DX3-213B, shows significant efficacy in a syngeneic model of pancreatic cancer. Exome sequencing of six out of six clones resistant to DX2-201 revealed a pV91M mutation in NDUFS7, providing direct evidence of its drug-binding site. In combination studies, DX2-201 showed synergy with multiple metabolic modulators, select OXPHOS inhibitors, and PARP inhibitors. Importantly, a combination with 2-deoxyglucose overcomes drug resistance in vivo. This study demonstrates that an efficacious treatment for pancreatic cancer can be achieved through inhibition of OXPHOS and direct binding to NDUFS7, providing a novel therapeutic strategy for this hard-to-treat cancer.

The ENCODE4 long-read RNA-seq collection reveals distinct classes of transcript structure diversity.

The majority of mammalian genes encode multiple transcript isoforms that result from differential promoter use, changes in exonic splicing, and alternative 3' end choice. Detecting and quantifying transcript isoforms across tissues, cell types, and species has been extremely challenging because transcripts are much longer than the short reads normally used for RNA-seq. By contrast, long-read RNA-seq (LR-RNA-seq) gives the complete structure of most transcripts. We sequenced 264 LR-RNA-seq PacBio libraries totaling over 1 billion circular consensus reads (CCS) for 81 unique human and mouse samples. We detect at least one full-length transcript from 87.7% of annotated human protein coding genes and a total of 200,000 full-length transcripts, 40% of which have novel exon junction chains. To capture and compute on the three sources of transcript structure diversity, we introduce a gene and transcript annotation framework that uses triplets representing the transcript start site, exon junction chain, and transcript end site of each transcript. Using triplets in a simplex representation demonstrates how promoter selection, splice pattern, and 3' processing are deployed across human tissues, with nearly half of multi-transcript protein coding genes showing a clear bias toward one of the three diversity mechanisms. Evaluated across samples, the predominantly expressed transcript changes for 74% of protein coding genes. In evolution, the human and mouse transcriptomes are globally similar in types of transcript structure diversity, yet among individual orthologous gene pairs, more than half (57.8%) show substantial differences in mechanism of diversification in matching tissues. This initial large-scale survey of human and mouse long-read transcriptomes provides a foundation for further analyses of alternative transcript usage, and is complemented by short-read and microRNA data on the same samples and by epigenome data elsewhere in the ENCODE4 collection.

KMT2D links TGF-ß signaling to noncanonical activin pathway and regulates pancreatic cancer cell plasticity.

Although KMT2D, also known as MLL2, is known to play an essential role in development, differentiation, and tumor suppression, its role in pancreatic cancer development is not well understood. Here, we discovered a novel signaling axis mediated by KMT2D, which links TGF-ß to the activin A pathway. We found that TGF-ß upregulates a microRNA, miR-147b, which in turn leads to post-transcriptional silencing of KMT2D. Loss of KMT2D induces the expression and secretion of activin A, which activates a noncanonical p38 MAPK-mediated pathway to modulate cancer cell plasticity, promote a mesenchymal phenotype, and enhance tumor invasion and metastasis in mice. We observed a decreased KMT2D expression in human primary and metastatic pancreatic cancer. Furthermore, inhibition or knockdown of activin A reversed the protumoral role of KMT2D loss. These findings support a tumor-suppressive role of KMT2D in pancreatic cancer and identify miR-147b and activin A as novel therapeutic targets.

KLIPP - a precision CRISPR approach to target structural variant junctions in cancer.

Current cancer therapies typically give rise to dose-limiting normal tissue toxicity. We have developed KLIPP, a precision cancer approach that specifically kills cancer cells using CRISPR/Cas9 technology. The approach consists of guide RNAs that target cancer-specific structural variant junctions to nucleate two parts of a dCas9-conjugated endonuclease, Fok1, leading to its activation. We show that KLIPP causes induction of DNA double strand breaks (DSBs) at the targeted junctions and cell death. When cancer cells were grown orthotopically in mice, activation of Fok1 at only two junctions led to the disappearance of tumor cells in 7/11 mice. This therapeutic approach has high specificity for tumor cells and is independent of tumor-specific drivers. Individualized translation of KLIPP to patients would be transformative and lead to consistent and simplified cancer treatment decisions.

Cdc73 protects Notch-induced T-cell leukemia cells from DNA damage and mitochondrial stress.

Activated Notch signaling is highly prevalent in T-cell acute lymphoblastic leukemia (T-ALL) but pan-Notch inhibitors were toxic in clinical trials. To find alternative ways to target Notch signals, we investigated Cell division cycle 73 (Cdc73), which is a Notch cofactor and component of transcriptional machinery, a potential target in T-ALL. While we confirmed previous work that CDC73 interacts with NOTCH1, we also found that the interaction in T-ALL was context-dependent and facilitated by the lymphoid transcription factor ETS1. Using mouse models, we showed that Cdc73 is important for Notch-induced T-cell development and T-ALL maintenance. Mechanistically, Cdc73, Ets1, and Notch intersect chromatin at promoters and enhancers to activate oncogenes and genes that are important for DNA repair and oxidative phosphorylation. Consistently, Cdc73 deletion in T-ALL cells induced DNA damage and impaired mitochondrial function. Our data suggests that Cdc73 might promote a gene expression program that was eventually intersected by Notch to mitigate the genotoxic and metabolic stresses of elevated Notch signaling. We also provide mechanistic support for testing inhibitors of DNA repair, oxidative phosphorylation, and transcriptional machinery. Inhibiting pathways like Cdc73 that intersect with Notch at chromatin might constitute a strategy to weaken Notch signals without directly targeting the Notch complex.

GRHL2-controlled gene expression networks in luminal breast cancer.

Grainyhead like 2 (GRHL2) is an essential transcription factor for development and function of epithelial tissues. It has dual roles in cancer by supporting tumor growth while suppressing epithelial to mesenchymal transitions (EMT). GRHL2 cooperates with androgen and estrogen receptors (ER) to regulate gene expression. We explore genome wide GRHL2 binding sites conserved in three ER⍺/GRHL2 positive luminal breast cancer cell lines by ChIP-Seq. Interaction with the ER⍺/FOXA1/GATA3 complex is observed, however, only for a minor fraction of conserved GRHL2 peaks. We determine genome wide transcriptional dynamics in response to loss of GRHL2 by nascent RNA Bru-seq using an MCF7 conditional knockout model. Integration of ChIP- and Bru-seq pinpoints candidate direct GRHL2 target genes in luminal breast cancer. Multiple connections between GRHL2 and proliferation are uncovered, including transcriptional activation of ETS and E2F transcription factors. Among EMT-related genes, direct regulation of CLDN4 is corroborated but several targets identified in other cells (including CDH1 and ZEB1) are ruled out by both ChIP- and Bru-seq as being directly controlled by GRHL2 in luminal breast cancer cells. Gene clusters correlating positively (including known GRHL2 targets such as ErbB3, CLDN4/7) or negatively (including TGFB1 and TGFBR2) with GRHL2 in the MCF7 knockout model, display similar correlation with GRHL2 in ER positive as well as ER negative breast cancer patients. Altogether, this study uncovers gene sets regulated directly or indirectly by GRHL2 in luminal breast cancer, identifies novel GRHL2-regulated genes, and points to distinct GRHL2 regulation of EMT in luminal breast cancer cells. Video Abstract.

Zinc Finger MYND-Type Containing 8 (ZMYND8) Is Epigenetically Regulated in Mutant Isocitrate Dehydrogenase 1 (IDH1) Glioma to Promote Radioresistance.

PURPOSE: Mutant isocitrate dehydrogenase 1 (mIDH1) alters the epigenetic regulation of chromatin, leading to a hypermethylation phenotype in adult glioma. This work focuses on identifying gene targets epigenetically dysregulated by mIDH1 to confer therapeutic resistance to ionizing radiation (IR). EXPERIMENTAL DESIGN: We evaluated changes in the transcriptome and epigenome in a radioresistant mIDH1 patient-derived glioma cell culture (GCC) following treatment with an mIDH1-specific inhibitor, AGI-5198. We identified Zinc Finger MYND-Type Containing 8 (ZMYND8) as a potential target of mIDH1 reprogramming. We suppressed ZMYND8 expression by shRNA knockdown and genetic knockout (KO) in mIDH1 glioma cells and then assessed cellular viability to IR. We assessed the sensitivity of mIDH1 GCCS to pharmacologic inhibition of ZMYND8-interacting partners: HDAC, BRD4, and PARP. RESULTS: Inhibition of mIDH1 leads to an upregulation of gene networks involved in replication stress. We found that the expression of ZMYND8, a regulator of DNA damage response, was decreased in three patient-derived mIDH1 GCCs after treatment with AGI-5198. Knockdown of ZMYND8 expression sensitized mIDH1 GCCs to radiotherapy marked by decreased cellular viability. Following IR, mIDH1 glioma cells with ZMYND8 KO exhibit significant phosphorylation of ATM and sustained γH2AX activation. ZMYND8 KO mIDH1 GCCs were further responsive to IR when treated with either BRD4 or HDAC inhibitors. PARP inhibition further enhanced the efficacy of radiotherapy in ZMYND8 KO mIDH1 glioma cells. CONCLUSIONS: These findings indicate the impact of ZMYND8 in the maintenance of genomic integrity and repair of IR-induced DNA damage in mIDH1 glioma. See related commentary by Sachdev et al., p. 1648.

miRNA analysis reveals novel dysregulated pathways in amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease. Its complex pathogenesis and phenotypic heterogeneity hinder therapeutic development and early diagnosis. Altered RNA metabolism is a recurrent pathophysiologic theme, including distinct microRNA (miRNA) profiles in ALS tissues. We profiled miRNAs in accessible biosamples, including skin fibroblasts and whole blood and compared them in age- and sex-matched healthy controls versus ALS participants with and without repeat expansions to chromosome 9 open reading frame 72 (C9orf72; C9-ALS and nonC9-ALS), the most frequent ALS mutation. We identified unique and shared profiles of differential miRNA (DmiRNA) levels in each C9-ALS and nonC9-ALS tissues versus controls. Fibroblast DmiRNAs were validated by quantitative real-time PCR and their target mRNAs by 5-bromouridine and 5-bromouridine-chase sequencing. We also performed pathway analysis to infer biological meaning, revealing anticipated, tissue-specific pathways and pathways previously linked to ALS, as well as novel pathways that could inform future research directions. Overall, we report a comprehensive study of a miRNA profile dataset from C9-ALS and nonC9-ALS participants across two accessible biosamples, providing evidence of dysregulated miRNAs in ALS and possible targets of interest. Distinct miRNA patterns in accessible tissues may also be leveraged to distinguish ALS participants from healthy controls for earlier diagnosis. Future directions may look at potential correlations of miRNA profiles with clinical parameters.

Divergent Molecular and Cellular Responses to Low and High-Dose Ionizing Radiation.

Cancer risk after ionizing radiation (IR) is assumed to be linear with the dose; however, for low doses, definite evidence is lacking. Here, using temporal multi-omic systems analyses after a low (LD; 0.1 Gy) or a high (HD; 1 Gy) dose of X-rays, we show that, although the DNA damage response (DDR) displayed dose proportionality, many other molecular and cellular responses did not. Phosphoproteomics uncovered a novel mode of phospho-signaling via S12-PPP1R7, and large-scale dephosphorylation events that regulate mitotic exit control in undamaged cells and the G2/M checkpoint upon IR in a dose-dependent manner. The phosphoproteomics of irradiated DNA double-strand breaks (DSBs) repair-deficient cells unveiled extended phospho-signaling duration in either a dose-dependent (DDR signaling) or independent (mTOR-ERK-MAPK signaling) manner without affecting signal magnitude. Nascent transcriptomics revealed the transcriptional activation of genes involved in NRF2-regulated antioxidant defense, redox-sensitive ERK-MAPK signaling, glycolysis and mitochondrial function after LD, suggesting a prominent role for reactive oxygen species (ROS) in molecular and cellular responses to LD exposure, whereas DDR genes were prominently activated after HD. However, how and to what extent the observed dose-dependent differences in molecular and cellular responses may impact cancer development remain unclear, as the induction of chromosomal damage was found to be dose-proportional (10-200 mGy).

KDM6A Loss Recruits Tumor-Associated Neutrophils and Promotes Neutrophil Extracellular Trap Formation in Pancreatic Cancer.

Lysine (K)-specific demethylase 6A (KDM6A) is a frequently mutated tumor suppressor gene in pancreatic ductal adenocarcinoma (PDAC). However, the impact of KDM6A loss on the PDAC tumor immune microenvironment is not known. This study used a genetically engineered, pancreas-specific Kdm6a knockout (KO) PDAC mouse model and human PDAC tissue samples to demonstrate that KDM6A loss correlates with increased tumor-associated neutrophils and neutrophil extracellular traps (NET) formation, which are known to contribute to PDAC progression. Genome-wide bromouridine sequencing analysis to evaluate nascent RNA synthesis showed that the expression of many chemotactic cytokines, especially CXC motif chemokine ligand 1 (CXCL1), was upregulated in KDM6A KO PDAC cells. KDM6A-deficient PDAC cells secreted higher levels of CXCL1 protein, which in turn recruited neutrophils. Furthermore, in a syngeneic orthotopic mouse model, treatment with a CXCL1 neutralizing antibody blocked the chemotactic and NET-promoting properties of KDM6A-deficient PDAC cells and suppressed tumor growth, confirming CXCL1 as a key mediator of chemotaxis and PDAC growth driven by KDM6A loss. These findings shed light on how KDM6A regulates the tumor immune microenvironment and PDAC progression and suggests that the CXCL1-CXCR2 axis may be a candidate target in PDAC with KDM6A loss. SIGNIFICANCE: KDM6A loss in pancreatic cancer cells alters the immune microenvironment by increasing CXCL1 secretion and neutrophil recruitment, providing a rationale for targeting the CXCL1-CXCR2 signaling axis in tumors with low KDM6A.

CDK12 regulates co-transcriptional splicing and RNA turnover in human cells.

The cyclin-dependent kinase CDK12 has garnered interest as a cancer therapeutic target as DNA damage response genes are particularly suppressed by loss of CDK12 activity. In this study, we assessed the acute effects of CDK12 inhibition on transcription and RNA processing using nascent RNA Bru-seq and BruChase-seq. Acute transcriptional changes were overall small after CDK12 inhibition but over 600 genes showed intragenic premature termination, including DNA repair and cell cycle genes. Furthermore, many genes showed reduced transcriptional readthrough past the end of genes in the absence of CDK12 activity. RNA turnover was dramatically affected by CDK12 inhibition and importantly, caused increased degradation of many transcripts from DNA damage response genes. We also show that co-transcriptional splicing was suppressed by CDK12 inhibition. Taken together, these studies reveal the roles of CDK12 in regulating transcription elongation, transcription termination, co-transcriptional splicing, and RNA turnover.

Transcription and genome integrity.

Transcription can cause genome instability by promoting R-loop formation but also act as a mutation-suppressing machinery by sensing of DNA lesions leading to the activation of DNA damage signaling and transcription-coupled repair. Recovery of RNA synthesis following the resolution of repair of transcription-blocking lesions is critical to avoid apoptosis and several new factors involved in this process have recently been identified. Some DNA repair proteins are recruited to initiating RNA polymerases and this may expediate the recruitment of other factors that participate in the repair of transcription-blocking DNA lesions. Recent studies have shown that transcription of protein-coding genes does not always give rise to spliced transcripts, opening the possibility that cells may use the transcription machinery in a splicing-uncoupled manner for other purposes including surveillance of the transcribed genome.

EWS::FLI1 and HOXD13 Control Tumor Cell Plasticity in Ewing Sarcoma.

PURPOSE: Propagation of Ewing sarcoma requires precise regulation of EWS::FLI1 transcriptional activity. Determining the mechanisms of fusion regulation will advance our understanding of tumor progression. Here we investigated whether HOXD13, a developmental transcription factor that promotes Ewing sarcoma metastatic phenotypes, influences EWS::FLI1 transcriptional activity. EXPERIMENTAL DESIGN: Existing tumor and cell line datasets were used to define EWS::FLI1 binding sites and transcriptional targets. Chromatin immunoprecipitation and CRISPR interference were employed to identify enhancers. CUT&RUN and RNA sequencing defined binding sites and transcriptional targets of HOXD13. Transcriptional states were investigated using bulk and single-cell transcriptomic data from cell lines, patient-derived xenografts, and patient tumors. Mesenchymal phenotypes were assessed by gene set enrichment, flow cytometry, and migration assays. RESULTS: We found that EWS::FLI1 creates a de novo GGAA microsatellite enhancer in a developmentally conserved regulatory region of the HOXD locus. Knockdown of HOXD13 led to widespread changes in expression of developmental gene programs and EWS::FLI1 targets. HOXD13 binding was enriched at established EWS::FLI1 binding sites where it influenced expression of EWS::FLI1-activated genes. More strikingly, HOXD13 bound and activated EWS::FLI1-repressed genes, leading to adoption of mesenchymal and migratory cell states that are normally suppressed by the fusion. Single-cell analysis confirmed that direct transcriptional antagonism between HOXD13-mediated gene activation and EWS::FLI1-dependent gene repression defines the state of Ewing sarcoma cells along a mesenchymal axis. CONCLUSIONS: Ewing sarcoma tumors are comprised of tumor cells that exist along a mesenchymal transcriptional continuum. The identity of cells along this continuum is, in large part, determined by the competing activities of EWS::FLI1 and HOXD13. See related commentary by Weiss and Bailey, p. 4360.

Myotubularin-related phosphatase 5 is a critical determinant of autophagy in neurons.

Autophagy is a conserved, multi-step process of capturing proteolytic cargo in autophagosomes for lysosome degradation. The capacity to remove toxic proteins that accumulate in neurodegenerative disorders attests to the disease-modifying potential of the autophagy pathway. However, neurons respond only marginally to conventional methods for inducing autophagy, limiting efforts to develop therapeutic autophagy modulators for neurodegenerative diseases. The determinants underlying poor autophagy induction in neurons and the degree to which neurons and other cell types are differentially sensitive to autophagy stimuli are incompletely defined. Accordingly, we sampled nascent transcript synthesis and stabilities in fibroblasts, induced pluripotent stem cells (iPSCs), and iPSC-derived neurons (iNeurons), thereby uncovering a neuron-specific stability of transcripts encoding myotubularin-related phosphatase 5 (MTMR5). MTMR5 is an autophagy suppressor that acts with its binding partner, MTMR2, to dephosphorylate phosphoinositides critical for autophagy initiation and autophagosome maturation. We found that MTMR5 is necessary and sufficient to suppress autophagy in iNeurons and undifferentiated iPSCs. Using optical pulse labeling to visualize the turnover of endogenously encoded proteins in live cells, we observed that knockdown of MTMR5 or MTMR2, but not the unrelated phosphatase MTMR9, significantly enhances neuronal degradation of TDP-43, an autophagy substrate implicated in several neurodegenerative diseases. Our findings thus establish a regulatory mechanism of autophagy intrinsic to neurons and targetable for clearing disease-related proteins in a cell-type-specific manner. In so doing, our results not only unravel novel aspects of neuronal biology and proteostasis but also elucidate a strategy for modulating neuronal autophagy that could be of high therapeutic potential for multiple neurodegenerative diseases.

Induction of Genes Implicated in Stress Response and Autophagy by a Novel Quinolin-8-yl-nicotinamide QN523 in Pancreatic Cancer.

Using a cytotoxicity-based phenotypic screen of a highly diverse library of 20,000 small-molecule compounds, we identified a quinolin-8-yl-nicotinamide, QN519, as a promising lead. QN519 represents a novel scaffold with drug-like properties, showing potent in vitro cytotoxicity in a panel of 12 cancer cell lines. Subsequently, lead optimization campaign generated compounds with IC50 values < 1 µM. An optimized compound, QN523, shows significant in vivo efficacy in a pancreatic cancer xenograft model. QN523 treatment significantly increased the expression of HSPA5, DDIT3, TRIB3, and ATF3 genes, suggesting activation of the stress response pathway. We also observed a significant increase in the expression of WIPI1, HERPUD1, GABARAPL1, and MAP1LC3B, implicating autophagy as a major mechanism of action. Due to the lack of effective treatments for pancreatic cancer, discovery of novel agents such as the QN series of compounds with unique mechanism of action has the potential to fulfill a clear unmet medical need.