Dependence on the 26S proteasome is an Achilles’ heel for triple-negative breast cancer (TNBC) and multiple myeloma (MM). The therapeutic proteasome inhibitor, bortezomib, successfully targets MM but often leads to drug-resistant disease relapse and fails in breast cancer. Here we show that a 26S proteasome-regulating kinase, DYRK2, is a therapeutic target for both MM and TNBC. Genome editing or small-molecule mediated inhibition of DYRK2 significantly reduces 26S proteasome activity, bypasses bortezomib resistance, and dramatically delays in vivo tumor growth in MM and TNBC thereby promoting survival. We further characterized the ability of LDN192960, a potent and selective DYRK2-inhibitor, to alleviate tumor burden in vivo. The drug docks into the active site of DYRK2 and partially inhibits all 3 core peptidase activities of the proteasome. Our results suggest that targeting 26S proteasome regulators will pave the way for therapeutic strategies in MM and TNBC.
SUMMARYExtrachromosomal circular DNA (ecDNA) is an important driver of aggressive tumor growth, promoting high oncogene copy number, intratumoral heterogeneity, accelerated evolution of drug resistance, enhancer rewiring, and poor outcome. ecDNA has been reported in medulloblastoma (MB), the most common malignant pediatric brain tumor, but the ecDNA landscape and its association with specific MB subgroups, its impact on enhancer rewiring, and its potential clinical implications, are not known. We assembled a retrospective cohort of 468 MB patient samples with available whole genome sequencing (WGS) data covering the four major MB subgroups WNT, SHH, Group 3 and Group 4. Using computational methods for the detection and reconstruction of ecDNA1, we find ecDNA in 82 patients (18%) and observe that ecDNA+ MB patients are more than twice as likely to relapse and three times as likely to die of disease. In addition, we find that individual medulloblastoma tumors often harbor multiple ecDNAs, each containing different amplified oncogenes along with co-amplified non-coding regulatory enhancers. ecDNA was substantially more prevalent among 31 analyzed patient-derived xenograft (PDX) models and cell lines than in our patient cohort. By mapping the accessible chromatin and 3D conformation landscapes of MB tumors that harbor ecDNA, we observe frequent candidate “enhancer rewiring” events that spatially link oncogenes with co-amplified enhancers. Our study reveals the frequency and diversity of ecDNA in a subset of highly aggressive tumors and suggests enhancer rewiring as a frequent oncogenic mechanism of ecDNAs in MB. Further, these results demonstrate that ecDNA is a frequent and potent driver of poor outcome in MB patients.
Extrachromosomal circular DNA (ecDNA) is an important driver of particularly aggressive human cancers. However, the prevalence of ecDNA, and its role in tumor development and progression in the different molecular subgroups of medulloblastoma (MB), remain unknown. To answer these questions, we have assembled a multi-institutional retrospective cohort of 472 MB patients with available whole genome sequencing (WGS) data, drawing from three cancer genomic data repositories and covering all MB subgroups (WNT, SHH, Group 3 and Group 4). Using recent computational methods to detect and reconstruct ecDNA, we find ecDNA in 66 patients (14%) and observe that the presence of ecDNA is associated with significantly poorer outcomes. By subgroup, ecDNA was found in 0/24 WNT (0%), 22/109 SHH (20%), 15/107 Group 3 (14%) and 20/181 Group 4 (11%) patients. Affected genomic loci harbor up to hundredfold amplification of oncogenes including MYC, MYCN, TERT, and other novel putative oncogenes. We further analyzed 24 patient-derived xenograft (PDX) and four cell line models of MB tumors. ecDNA was substantially more frequent in patient-derived models (17 of 29, 59%) than in our patient cohort. To elucidate the functional regulatory landscapes of ecDNAs in MB, we generated transcriptional (RNA-seq), accessible chromatin (ATAC-seq), and chromatin interaction (Hi-C) profiles of 6 MB tumor samples. In each case, we identify regulatory interactions that cross fusion breakpoints on the ecDNA, representing potential “enhancer rewiring” events which may contribute to transcriptional activation of co-amplified oncogenes. To test this hypothesis, we are currently conducting in-vitro CRISPRi screens targeting regulatory regions on the ecDNA of a MB cell line to determine whether these enhancers promote proliferation. In summary, our study analyzes the frequency, diversity and functional relevance of ecDNA across MB subgroups and provides strong justification for continued mechanistic studies of ecDNA in MB with the potential to uncover new therapeutic approaches.
Extrachromosomal circular DNA (ecDNA) is an important driver of aggressive cancers, including medulloblastoma (MB), the most common malignant pediatric brain tumor. Our study’s aim is to better understand how ecDNA containing cells can potentiate malignant growth. EcDNA’s role in the development of treatment resistance and association with poor outcomes is hypothesized to arise from its contribution to intra-tumoral heterogeneity and its potential to promote oncogene dependency switching. To analyze the intra-tumoral distribution of ecDNA, we have now simultaneously analyzed the accessible chromatin and gene expression in single cells of a SHH medulloblastoma (MB) patient using multiome single-cell ATAC-seq and gene expression (10X Genomics). Whole genome sequencing (WGS) of this tumor previously revealed a heterozygous somatic TP53 mutation and two distinct ecDNAs: a 3.2Mbp amplicon comprising 3 regions of chr1 and another 4.5Mbp amplicon comprising 23 segments originating from chr7 and chr17. We then used multimodal analysis to describe the tumor cell types, gene expression, variant signatures and estimate ecDNA copy number in the medulloblastoma tumor sample. We identified 12 distinct clusters in the human tumor, 5 of which were determined to be normal non-tumor [OSC1] cells, as identified by specific cell type markers, and 7 of which were determined to be tumor cells. Enrichment of ecDNA was restricted to only one of these tumor clusters. In addition, we also performed the same multiome single-cell analyses in an orthotopic xenograft mouse model derived from this SHH MB patient tumor. In the PDX, 17 clusters were identified, all of which were determined to be tumor cells and enriched for ecDNA. Our preliminary results indicate that tumor cells with ecDNA in the human tumor (particularly the ecDNA enriched cluster) almost exclusively account for [OSC2] the cells in the corresponding PDX, emphasizing the aggressiveness of ecDNA containing cells.
Ependymoma (EPN) is an aggressive pediatric tumor that occurs throughout the central nervous system. The two most aggressive molecular subgroups of EPN are the supratentorial ZFTA-fusion associated group (ST-EPN-ZFTA) and the posterior fossa group A (PF-EPN-A). Although the molecular characteristics underlying the tumorigenesis of these subgroups have been extensively studied, these tumors remain difficult to treat. Hence, innovative therapeutic approaches are urgently needed. Here, we used genome-wide chromosome conformation capture (Hi-C), complemented with CTCF (insulators) and H3K27ac (active enhancers) ChIP-seq, as well as gene expression and whole-genome DNA methylation profiling in primary and relapsed EPN tumors and cell lines, to identify chromosomal rearrangements and regulatory mechanisms underlying aberrant expression of genes that are essential for EPN tumorigenesis. By integrating these heterogenous data types, we have observed the formation of new topologically associated domains (‘neo-TADs’) caused by intra- and inter-chromosomal structural variants in both tumors. In addition, we observed 3D chromatin complexes of regulatory elements, and the replacement of CTCF insulators by DNA hyper-methylation in PF-EPN-A tumors. These tumor-specific 3D genome conformations can be associated with the transcriptional upregulation of nearby genes. Through inhibition experiments we validated that these newly identified genes, including RCOR2, ITGA6, LAMC1, and ARL4C, are highly essential for the survival of patient-derived EPN cell lines in a disease subgroup-specific manner. Thus, our study identifies novel potential therapeutic vulnerabilities in EPN and extends our ability to reveal tumor-dependency genes and pathways by oncogenic 3D genome conformations even in tumors that lack known genetic alterations.
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