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EWS fusion oncoproteins underlie several human malignancies including Desmoplastic Small Round Cell Tumor (DSRCT), an aggressive cancer driven by EWS-WT1 fusion proteins. Here we combine chromatin occupancy and 3D profiles to identify EWS-WT1-dependent gene regulation networks and target genes. We show that EWS-WT1 is a powerful chromatin activator controlling an oncogenic gene expression program that characterizes primary tumors. Similar to wild type WT1, EWS-WT1 has two isoforms that differ in their DNA binding domain and we find that they have distinct DNA binding profiles and are both required to generate viable tumors that resemble primary DSRCT. Finally, we identify candidate EWS-WT1 target genes with potential therapeutic implications, including CCND1, whose inhibition by the clinically-approved drug Palbociclib leads to marked tumor burden decrease in DSRCT PDXs in vivo. Taken together, our studies identify gene regulation programs and therapeutic vulnerabilities in DSRCT and provide a mechanistic understanding of the complex oncogenic activity of EWS-WT1.

The impact of variations in the three-dimensional structure of the genome has been recognized, but solid cancer tissue studies are limited. Here, we performed integrated deep Hi-C sequencing with matched whole-genome sequencing, whole-genome bisulfite sequencing, 5-hydroxymethylcytosine (5hmC) sequencing and RNA sequencing across a cohort of 80 biopsy samples from patients with metastatic castration-resistant prostate cancer. Dramatic differences were present in gene expression, 5-methylcytosine/5hmC methylation and in structural variation versus mutation rate between A and B (open and closed) chromatin compartments. A subset of tumors exhibited depleted regional chromatin contacts at the AR locus, linked to extrachromosomal circular DNA (ecDNA) and worse response to AR signaling inhibitors. We also identified topological subtypes associated with stark differences in methylation structure, gene expression and prognosis. Our data suggested that DNA interactions may predispose to structural variant formation, exemplified by the recurrent TMPRSS2-ERG fusion. This comprehensive integrated sequencing effort represents a unique clinical tumor resource.

UNLABELLED: The comprehensive genomic analysis of the head and neck squamous cell carcinoma (HNSCC) oncogenome revealed the frequent loss of p16INK4A (CDKN2A) and amplification of cyclin D1 genes in most human papillomavirus-negative HNSCC lesions. However, cyclin-dependent kinase 4 and 6 (CDK4/6) inhibitors have shown modest effects in the clinic. The aberrant activation of the PI3K/mTOR pathway is highly prevalent in HNSCC, and recent clinical trials have shown promising clinical efficacy of mTOR inhibitors (mTORi) in the neoadjuvant and adjuvant settings but not in patients with advanced HNSCC. By implementing a kinome-wide CRISPR/Cas9 screen, we identified cell-cycle inhibition as a synthetic lethal target for mTORis. A combination of mTORi and palbociclib, a CDK4/6-specific inhibitor, showed strong synergism in HNSCC-derived cells in vitro and in vivo. Remarkably, we found that an adaptive increase in cyclin E1 (CCNE1) expression upon palbociclib treatment underlies the rapid acquired resistance to this CDK4/6 inhibitor. Mechanistically, mTORi inhibits the formation of eIF4G-CCNE1 mRNA complexes, with the consequent reduction in mRNA translation and CCNE1 protein expression. Our findings suggest that mTORi reverts the adaptive resistance to palbociclib. This provides a multimodal therapeutic option for HNSCC by cotargeting mTOR and CDK4/6, which in turn may halt the emergence of palbociclib resistance. SIGNIFICANCE: A kinome-wide CRISPR/Cas9 screen identified cell-cycle inhibition as a synthetic lethal target of mTORis. A combination of mTORi and palbociclib, a CDK4/6-specific inhibitor, showed strong synergistic effects in HNSCC. Mechanistically, mTORis inhibited palbociclib-induced increase in CCNE1.

BACKGROUND: Neurotrophic tyrosine receptor kinase (NTRK) gene fusions are rare oncogenic drivers prevalent in 0.3% of solid tumors. They are most common in salivary gland cancer (2.6%), thyroid cancer (1.6%), and soft-tissue sarcoma (1.5%). Currently, there are 2 US Food and Drug Administration-approved targeted therapies for NTRK gene fusions: larotrectinib, approved in 2018, and entrectinib, approved in 2019. To date, the real-world uptake of tyrosine receptor kinase inhibitor (TRKi) use for NTRK-positive solid tumors in academic cancer centers remains largely unknown. OBJECTIVE: To describe the demographics, clinical and genomic characteristics, and testing and treatment patterns of patients with NTRK-positive solid tumors treated at US academic cancer centers. METHODS: This was a retrospective chart review study conducted in academic cancer centers in the United States. All patients diagnosed with an NTRK fusion-positive (NTRK1, NTRK2, NTRK3) solid tumor (any stage) and who received cancer treatment at participating sites between January 1, 2012, and July 1, 2023, were included in this study. Patient demographics, clinical characteristics, genomic characteristics, NTRK testing data, and treatment patterns were collected from electronic medical records and analyzed using descriptive statistics as appropriate. RESULTS: In total, 6 centers contributed data for 55 patients with NTRK-positive tumors. The mean age was 49.3 (SD = 20.5) years, 51% patients were female, and the majority were White (78%). The median duration of time from cancer diagnosis to NTRK testing was 85 days (IQR = 44-978). At the time of NTRK testing, 64% of patients had stage IV disease, compared with 33% at cancer diagnosis. Prevalent cancer types in the overall cohort included head and neck (15%), thyroid (15%), brain (13%), lung (13%), and colorectal (11%). NTRK1 fusions were most common (45%), followed by NTRK3 (40%) and NTRK2 (15%). Across all lines of therapy, 51% of patients (n = 28) received a TRKi. Among TRKi-treated patients, 71% had stage IV disease at TRKi initiation. The median time from positive NTRK test to initiation of TRKi was 48 days (IQR = 9-207). TRKis were commonly given as first-line (30%) or second-line (48%) therapies. Median duration of therapy was 610 (IQR = 182-764) days for TRKi use and 207.5 (IQR = 42-539) days for all other first-line therapies. CONCLUSIONS: This study reports on contemporary real-world NTRK testing patterns and use of TRKis in solid tumors, including time between NTRK testing and initiation of TRKi therapy and duration of TRKi therapy.

Neuroblastoma is the most common extracranial malignant tumor of childhood, accounting for 15% of all pediatric cancer deaths. Despite significant advances in our understanding of neuroblastoma biology, five-year survival rates for high-risk disease remain less than 50%, highlighting the importance of identifying novel therapeutic targets to combat the disease. MYCN amplification is the most frequent and predictive molecular aberration correlating with poor outcome in neuroblastoma. N-Myc is a short-lived protein primarily due to its rapid proteasomal degradation, a potentially exploitable vulnerability in neuroblastoma. AF1q is an oncoprotein with established roles in leukemia and solid tumor progression. It is normally expressed in brain and sympathetic neurons and has been postulated to play a part in neural differentiation. However, no role for AF1q in tumors of neural origin has been reported. In this study, we found AF1q to be a universal marker of neuroblastoma tumors. Silencing AF1q in neuroblastoma cells caused proteasomal degradation of N-Myc through Ras/ERK and AKT/GSK3β pathways, activated p53 and blocked cell cycle progression, culminating in cell death via the intrinsic apoptotic pathway. Moreover, silencing AF1q attenuated neuroblastoma tumorigenicity in vivo signifying AF1qs importance in neuroblastoma oncogenesis. Our findings reveal AF1q to be a novel regulator of N-Myc and potential therapeutic target in neuroblastoma.

Aberrant expression of the E26 transformation-specific (ETS) transcription factors characterizes numerous human malignancies. Many of these proteins, including EWS:FLI1 and EWS:ERG fusions in Ewing sarcoma (EwS) and TMPRSS2:ERG in prostate cancer (PCa), drive oncogenic programs via binding to GGAA repeats. We report here that both EWS:FLI1 and ERG bind and transcriptionally activate GGAA-rich pericentromeric heterochromatin. The respective pathogen-like HSAT2 and HSAT3 RNAs, together with LINE, SINE, ERV, and other repeat transcripts, are expressed in EwS and PCa tumors, secreted in extracellular vesicles (EVs), and are highly elevated in plasma of patients with EwS with metastatic disease. High human satellite 2 and 3 (HSAT2,3) levels in EWS:FLI1- or ERG-expressing cells and tumors were associated with induction of G2/M checkpoint, mitotic spindle, and DNA damage programs. These programs were also activated in EwS EV-treated fibroblasts, coincident with accumulation of HSAT2,3 RNAs, proinflammatory responses, mitotic defects, and senescence. Mechanistically, HSAT2,3-enriched cancer EVs induced cGAS-TBK1 innate immune signaling and formation of cytosolic granules positive for double-strand RNAs, RNA-DNA, and cGAS. Hence, aberrantly expressed ETS proteins derepress pericentromeric heterochromatin, yielding pathogenic RNAs that transmit genotoxic stress and inflammation to local and distant sites. Monitoring HSAT2,3 plasma levels and preventing their dissemination may thus improve therapeutic strategies and blood-based diagnostics.

The high incidence of epithelial malignancies in HIV-1 infected individuals is associated with co-infection with oncogenic viruses, such as high-risk human papillomaviruses (HR HPVs), mostly HPV16. The molecular mechanisms underlying the HIV-1-associated increase in epithelial malignancies are not fully understood. A collaboration between HIV-1 and HR HPVs in the malignant transformation of epithelial cells has long been anticipated. Here, we delineated the effects of HIV-1 reverse transcriptase on the in vitro and in vivo properties of HPV16-infected cervical cancer cells. A human cervical carcinoma cell line infected with HPV16 (Ca Ski) was made to express HIV-1 reverse transcriptase (RT) by lentiviral transduction. The levels of the mRNA of the E6 isoforms and of the factors characteristic to the epithelial/mesenchymal transition were assessed by real-time RT-PCR. The parameters of glycolysis and mitochondrial respiration were determined using Seahorse technology. RT expressing Ca Ski subclones were assessed for the capacity to form tumors in nude mice. RT expression increased the expression of the E6*I isoform, modulated the expression of E-CADHERIN and VIMENTIN, indicating the presence of a hybrid epithelial/mesenchymal phenotype, enhanced glycolysis, and inhibited mitochondrial respiration. In addition, the expression of RT induced phenotypic alterations impacting cell motility, clonogenic activity, and the capacity of Ca Ski cells to form tumors in nude mice. These findings suggest that HIV-RT, a multifunctional protein, affects HPV16-induced oncogenesis, which is achieved through modulation of the expression of the E6 oncoprotein. These results highlight a complex interplay between HIV antigens and HPV oncoproteins potentiating the malignant transformation of epithelial cells.

AbstractRNA-binding proteins (RBPs) are emerging as a novel class of therapeutic targets in cancer, including in leukemia, given their important role in posttranscriptional gene regulation, and have the unexplored potential to be combined with existing therapies. The RBP insulin-like growth factor 2 messenger RNA-binding protein 3 (IGF2BP3) has been found to be a critical regulator of MLL-AF4 leukemogenesis and represents a promising therapeutic target. Here, we study the combined effects of targeting IGF2BP3 and menin-MLL interaction in MLL-AF4-driven leukemia in vitro and in vivo, using genetic inhibition with CRISPR-Cas9-mediated deletion of Igf2bp3 and pharmacologic inhibition of the menin-MLL interaction with multiple commercially available inhibitors. Depletion of Igf2bp3 sensitized MLL-AF4 leukemia to the effects of menin-MLL inhibition on cell growth and leukemic initiating cells in vitro. Mechanistically, we found that both Igf2bp3 depletion and menin-MLL inhibition led to increased differentiation in vitro and in vivo, seen in functional readouts and by gene expression analyses. IGF2BP3 knockdown had a greater effect on increasing survival and attenuating disease than pharmacologic menin-MLL inhibition with small molecule MI-503 alone and showed enhanced antileukemic effects in combination. Our work shows that IGF2BP3 is an oncogenic amplifier of MLL-AF4-mediated leukemogenesis and a potent therapeutic target, providing a paradigm for targeting leukemia at both the transcriptional and posttranscriptional level.

Neurotrophic receptor tyrosine kinases (NTRKs) belong to the receptor tyrosine kinase (RTK) family. NTRKs are responsible for the activation of multiple downstream signaling pathways that regulate cell growth, proliferation, differentiation, and apoptosis. NTRK-associated mutations often result in oncogenesis and lead to aberrant activation of downstream signaling pathways including MAPK, JAK/STAT, and PLCγ1. This study characterizes the NACC2-NTRK2 oncogenic fusion protein that leads to pilocytic astrocytoma and pediatric glioblastoma. This fusion joins the BTB domain (Broad-complex, Tramtrack, and Bric-a-brac) domain of NACC2 (Nucleus Accumbens-associated protein 2) with the transmembrane helix and tyrosine kinase domain of NTRK2. We focus on identifying critical domains for the biological activity of the fusion protein. Mutations were introduced in the charged pocket of the BTB domain or in the monomer core, based on a structural comparison of the NACC2 BTB domain with that of PLZF, another BTB-containing protein. Mutations were also introduced into the NTRK2-derived portion to allow comparison of two different breakpoints that have been clinically reported. We show that activation of the NTRK2 kinase domain relies on multimerization of the BTB domain in NACC2-NTRK2. Mutations which disrupt BTB-mediated multimerization significantly reduce kinase activity and downstream signaling. The ability of these mutations to abrogate biological activity suggests that BTB domain inhibition could be a potential treatment for NACC2-NTRK2-induced cancers. Removal of the transmembrane helix leads to enhanced stability of the fusion protein and increased activity of the NACC2-NTRK2 fusion, suggesting a mechanism for the oncogenicity of a distinct NACC2-NTRK2 isoform observed in pediatric glioblastoma.

T(8;21)(q22;q22), which generates the AML1-ETO fusion oncoprotein, is a common chromosomal abnormality in acute myeloid leukemia (AML) patients. Despite having favorable prognosis, 40% of patients will relapse, highlighting the need for innovative models and application of the newest technologies to study t(8;21) leukemogenesis. Currently, available AML1-ETO mouse models have limited utility for studying the pre-leukemic stage because AML1-ETO produces mild hematopoietic phenotypes and no leukemic transformation. Conversely, overexpression of a truncated variant, AML1-ETO9a (AE9a), promotes fully penetrant leukemia and is too potent for studying pre-leukemic changes. To overcome these limitations, we devised a germline-transmitted Rosa26 locus AE9a knock-in mouse model that moderately overexpressed AE9a and developed leukemia with long latency and low penetrance. We observed pre-leukemic alterations in AE9a mice, including skewing of progenitors towards granulocyte/monocyte lineages and replating of stem and progenitor cells. Next, we performed single-cell RNA sequencing to identify specific cell populations that contribute to these pre-leukemic phenotypes. We discovered a subset of common myeloid progenitors that have heightened granulocyte/monocyte bias in AE9a mice. We also observed dysregulation of key hematopoietic transcription factor target gene networks, blocking cellular differentiation. Finally, we identified Sox4 activation as a potential contributor to stem cell self-renewal during the pre-leukemic stage.

The majority of oncogenic drivers are intracellular proteins, constraining their immunotherapeutic targeting to mutated peptides (neoantigens) presented by individual human leukocyte antigen (HLA) allotypes1. However, most cancers have a modest mutational burden that is insufficient for generating responses using neoantigen-based therapies2,3. Neuroblastoma is a paediatric cancer that harbours few mutations and is instead driven by epigenetically deregulated transcriptional networks4. Here we show that the neuroblastoma immunopeptidome is enriched with peptides derived from proteins essential for tumorigenesis. We focused on targeting the unmutated peptide QYNPIRTTF discovered on HLA-A*24:02, which is derived from the neuroblastoma-dependency gene and master transcriptional regulator PHOX2B. To target QYNPIRTTF, we developed peptide-centric chimeric antigen receptors (PC-CARs) through a counter panning strategy using predicted potentially cross-reactive peptides. We further proposed that PC-CARs can recognize peptides on additional HLA allotypes when presenting a similar overall molecular surface. Informed by our computational modelling results, we show that PHOX2B PC-CARs also recognize QYNPIRTTF presented by HLA-A*23:01, the most common non-A2 allele in people with African ancestry. Finally, we demonstrate potent and specific killing of neuroblastoma cells expressing these HLAs in vitro and complete tumour regression in mice. These data suggest that PC-CARs have the potential to expand the pool of immunotherapeutic targets to include non-immunogenic intracellular oncoproteins and allow targeting through additional HLA allotypes in a clinical setting.

BackgroundCyclin E1 (CCNE1) is a potential predictive marker and therapeutic target in tubo-ovarian high-grade serous carcinoma (HGSC). Smaller studies have revealed unfavorable associations for CCNE1 amplification and CCNE1 overexpression with survival, but to date no large-scale, histotype-specific validation has been performed. The hypothesis was that high-level amplification of CCNE1 and CCNE1 overexpression, as well as a combination of the two, are linked to shorter overall survival in HGSC.MethodsWithin the Ovarian Tumor Tissue Analysis consortium, amplification status and protein level in 3029 HGSC cases and mRNA expression in 2419 samples were investigated.ResultsHigh-level amplification (>8 copies by chromogenic in situ hybridization) was found in 8.6% of HGSC and overexpression (>60% with at least 5% demonstrating strong intensity by immunohistochemistry) was found in 22.4%. CCNE1 high-level amplification and overexpression both were linked to shorter overall survival in multivariate survival analysis adjusted for age and stage, with hazard stratification by study (hazard ratio [HR], 1.26; 95% CI, 1.08-1.47, p = .034, and HR, 1.18; 95% CI, 1.05-1.32, p = .015, respectively). This was also true for cases with combined high-level amplification/overexpression (HR, 1.26; 95% CI, 1.09-1.47, p = .033). CCNE1 mRNA expression was not associated with overall survival (HR, 1.00 per 1-SD increase; 95% CI, 0.94-1.06; p = .58). CCNE1 high-level amplification is mutually exclusive with the presence of germline BRCA1/2 pathogenic variants and shows an inverse association to RB1 loss.ConclusionThis study provides large-scale validation that CCNE1 high-level amplification is associated with shorter survival, supporting its utility as a prognostic biomarker in HGSC.

FGFR3-TACC3 represents an oncogenic fusion protein frequently identified in glioblastoma, lung cancer, bladder cancer, oral cancer, head and neck squamous cell carcinoma, gallbladder cancer, and cervical cancer. Various exon breakpoints of FGFR3-TACC3 have been identified in cancers; these were analyzed to determine the minimum contribution of TACC3 for activation of the FGFR3-TACC3 fusion protein. While TACC3 exons 11 and 12 are dispensable for activity, our results show that FGFR3-TACC3 requires exons 13-16 for biological activity. A detailed analysis of exon 13, which consists of 8 heptads forming a coiled coil, further defined the minimal region for biological activity as consisting of 5 heptads from exon 13, in addition to exons 14-16. These conclusions were supported by transformation assays of biological activity, examination of MAPK pathway activation, analysis of disulfide-bonded FGFR3-TACC3, and by examination of the Endoglycosidase H-resistant portion of FGFR3-TACC3. These results demonstrate that clinically identified FGFR3-TACC3 fusion proteins differ in their biological activity, depending upon the specific breakpoint. This study further suggests the TACC3 dimerization domain of FGFR3-TACC3 as a novel target in treating FGFR translocation driven cancers.

The most common somatic event in primary prostate cancer is a fusion between the androgen-related TMPRSS2 gene and the ERG oncogene. Tumors with these fusions, which occur early in carcinogenesis, have a distinctive etiology. A smaller subset of other tumors harbor fusions between TMPRSS2 and members of the ETS transcription factor family other than ERG. To assess the genomic similarity of tumors with non-ERG ETS fusions and those with fusions involving ERG, this study derived a transcriptomic signature of non-ERG ETS fusions and assessed this signature and ERG-related gene expression in 1,050 men with primary prostate cancer from three independent population-based and hospital-based studies. Although non-ERG ETS fusions involving ETV1, ETV4, ETV5, or FLI1 were individually rare, they jointly accounted for one in seven prostate tumors. Genes differentially regulated between non-ERG ETS tumors and tumors without ETS fusions showed similar differential expression when ERG tumors and tumors without ETS fusions were compared (differences explained: R2 = 69-77%), including ETS-related androgen receptor (AR) target genes. Differences appeared to result from similarities among ETS tumors rather than similarities among non-ETS tumors. Gene sets associated with ERG fusions were consistent with gene sets associated with non-ERG ETS fusions, including fatty acid and amino acid metabolism, an observation that was robust across cohorts.ImplicationsConsidering ETS fusions jointly may be useful for etiologic studies on prostate cancer, given that the transcriptome is profoundly impacted by ERG and non-ERG ETS fusions in a largely similar fashion, most notably genes regulating metabolic pathways.

Not available.

Oncogenic fusions formed through chromosomal rearrangements are hallmarks of childhood cancer that define cancer subtype, predict outcome, persist through treatment, and can be ideal therapeutic targets. However, mechanistic understanding of the etiology of oncogenic fusions remains elusive. Here we report a comprehensive detection of 272 oncogenic fusion gene pairs by using tumor transcriptome sequencing data from 5190 childhood cancer patients. We identify diverse factors, including translation frame, protein domain, splicing, and gene length, that shape the formation of oncogenic fusions. Our mathematical modeling reveals a strong link between differential selection pressure and clinical outcome in CBFB-MYH11. We discover 4 oncogenic fusions, including RUNX1-RUNX1T1, TCF3-PBX1, CBFA2T3-GLIS2, and KMT2A-AFDN, with promoter-hijacking-like features that may offer alternative strategies for therapeutic targeting. We uncover extensive alternative splicing in oncogenic fusions including KMT2A-MLLT3, KMT2A-MLLT10, C11orf95-RELA, NUP98-NSD1, KMT2A-AFDN and ETV6-RUNX1. We discover neo splice sites in 18 oncogenic fusion gene pairs and demonstrate that such splice sites confer therapeutic vulnerability for etiology-based genome editing. Our study reveals general principles on the etiology of oncogenic fusions in childhood cancer and suggests profound clinical implications including etiology-based risk stratification and genome-editing-based therapeutics.

The CBFA2T3-GLIS2 (C/G) fusion is a product of a cryptic translocation primarily seen in infants and early childhood and is associated with dismal outcome. Here, we demonstrate that the expression of the C/G oncogenic fusion protein promotes the transformation of human cord blood hematopoietic stem and progenitor cells (CB HSPCs) in an endothelial cell coculture system that recapitulates the transcriptome, morphology, and immunophenotype of C/G acute myeloid leukemia (AML) and induces highly aggressive leukemia in xenograft models. Interrogating the transcriptome of C/G-CB cells and primary C/G AML identified a library of C/G-fusion-specific genes that are potential targets for therapy. We developed chimeric antigen receptor (CAR) T cells directed against one of the targets, folate receptor α (FOLR1), and demonstrated their preclinical efficacy against C/G AML using in vitro and xenograft models. FOLR1 is also expressed in renal and pulmonary epithelium, raising concerns for toxicity that must be addressed for the clinical application of this therapy. Our findings underscore the role of the endothelial niche in promoting leukemic transformation of C/G-transduced CB HSPCs. Furthermore, this work has broad implications for studies of leukemogenesis applicable to a variety of oncogenic fusion-driven pediatric leukemias, providing a robust and tractable model system to characterize the molecular mechanisms of leukemogenesis and identify biomarkers for disease diagnosis and targets for therapy.

The endosomal sorting complexes required for transport (ESCRT) system is an ancient and ubiquitous membrane scission machinery that catalyzes the budding and scission of membranes. ESCRT-mediated scission events, exemplified by those involved in the budding of HIV-1, are usually directed away from the cytosol ("reverse topology"), but they can also be directed toward the cytosol ("normal topology"). The ESCRT-III subunits CHMP1B and IST1 can coat and constrict positively curved membrane tubes, suggesting that these subunits could catalyze normal topology membrane severing. CHMP1B and IST1 bind and recruit the microtubule-severing AAA+ ATPase spastin, a close relative of VPS4, suggesting that spastin could have a VPS4-like role in normal-topology membrane scission. Here, we reconstituted the process in vitro using membrane nanotubes pulled from giant unilamellar vesicles using an optical trap in order to determine whether CHMP1B and IST1 are capable of membrane severing on their own or in concert with VPS4 or spastin. CHMP1B and IST1 copolymerize on membrane nanotubes, forming stable scaffolds that constrict the tubes, but do not, on their own, lead to scission. However, CHMP1B-IST1 scaffolded tubes were severed when an additional extensional force was applied, consistent with a friction-driven scission mechanism. We found that spastin colocalized with CHMP1B-enriched sites but did not disassemble the CHMP1B-IST1 coat from the membrane. VPS4 resolubilized CHMP1B and IST1 without leading to scission. These observations show that the CHMP1B-IST1 ESCRT-III combination is capable of severing membranes by a friction-driven mechanism that is independent of VPS4 and spastin.

'Intracranial mesenchymal tumor, FET-CREB fusion-positive' occurs primarily in children and young adults and has previously been termed intracranial angiomatoid fibrous histiocytoma (AFH) or intracranial myxoid mesenchymal tumor (IMMT). Here we performed genome-wide DNA methylation array profiling of 20 primary intracranial mesenchymal tumors with FET-CREB fusion to further study their ontology. These tumors resolved into two distinct epigenetic subgroups that were both divergent from all other analyzed intracranial neoplasms and soft tissue sarcomas, including meningioma, clear cell sarcoma of soft tissue (CCS), and AFH of extracranial soft tissue. The first subgroup (Group A, 16 tumors) clustered nearest to but independent of solitary fibrous tumor and AFH of extracranial soft tissue, whereas the second epigenetic subgroup (Group B, 4 tumors) clustered nearest to but independent of CCS and also lacked expression of melanocytic markers (HMB45, Melan A, or MITF) characteristic of CCS. Group A tumors most often occurred in adolescence or early adulthood, arose throughout the neuroaxis, and contained mostly EWSR1-ATF1 and EWSR1-CREB1 fusions. Group B tumors arose most often in early childhood, were located along the cerebral convexities or spinal cord, and demonstrated an enrichment for tumors with CREM as the fusion partner (either EWSR1-CREM or FUS-CREM). Group A tumors more often demonstrated stellate/spindle cell morphology and hemangioma-like vasculature, whereas Group B tumors more often demonstrated round cell or epithelioid/rhabdoid morphology without hemangioma-like vasculature, although robust comparison of these clinical and histologic features requires future study. Patients with Group B tumors had inferior progression-free survival relative to Group A tumors (median 4.5 vs. 49 months, p = 0.001). Together, these findings confirm that intracranial AFH-like neoplasms and IMMT represent histologic variants of a single tumor type ('intracranial mesenchymal tumor, FET-CREB fusion-positive') that is distinct from meningioma and extracranial sarcomas. Additionally, epigenomic evaluation may provide important prognostic subtyping for this unique tumor entity.

Gene fusions involving the neurotrophic receptor tyrosine kinase genes NTRK1, NTRK2, and NTRK3, are well established oncogenic drivers in a broad range of pediatric and adult tumors. These fusions are also important actionable markers, predicting often dramatic response to FDA approved kinase inhibitors. Accurate interpretation of the clinical significance of NTRK fusions is a high priority for diagnostic laboratories, but remains challenging and time consuming given the rapid pace of new data accumulation, the diversity of fusion partners and tumor types, and heterogeneous and incomplete information in variant databases and knowledgebases. The ClinGen NTRK Fusions Somatic Cancer Variant Curation Expert Panel (SC-VCEP) was formed to systematically address these challenges and create an expert-curated resource to support clinicians, researchers, patients and their families in making accurate interpretations and informed treatment decisions for NTRK fusion-driven tumors. We describe a system for NTRK fusion interpretation (including compilation of key elements and annotations) developed by the NTRK fusions SC-VCEP. We illustrate this stepwise process on examples of LMNA::NTRK1 and KANK1::NTRK2 fusions. Finally, we provide detailed analysis of current representation of NTRK fusions in public fusion databases and the CIViC knowledgebase, performed by the NTRK fusions SC-VCEP to determine existing gaps and prioritize future curation activities.

The small arginine-rich protein protamine condenses complete genomic DNA into the sperm head. Here, we applied its high RNA binding capacity for spontaneous electrostatic assembly of therapeutic nanoparticles decorated with tumour-cell-specific antibodies for efficiently targeting siRNA. Fluorescence microscopy and DLS measurements of these nanocarriers revealed the formation of a vesicular architecture that requires presence of antibody-protamine, defined excess of free SMCC-protamine, and anionic siRNA to form. Only these complex nanoparticles were efficient in the treatment of non-small-cell lung cancer (NSCLC) xenograft models, when the oncogene KRAS was targeted via EGFR-mediated delivery. To show general applicability, we used the modular platform for IGF1R-positive Ewing sarcomas. Anti-IGR1R-antibodies were integrated into an antibody-protamine nanoparticle with an siRNA specifically against the oncogenic translocation product EWS/FLI1. Using these nanoparticles, EWS/FLI1 knockdown blocked in vitro and in vivo growth of Ewing sarcoma cells. We conclude that these antibody-protamine-siRNA nanocarriers provide a novel platform technology to specifically target different cell types and yet undruggable targets in cancer therapy by RNAi.

The Hedgehog (Hh) family of lipid-modified signaling proteins directs embryonic tissue patterning and postembryonic tissue homeostasis, and dysregulated Hh signaling drives familial and sporadic cancers. Hh ligands bind to and inhibit the tumor suppressor Patched and allow the oncoprotein Smoothened (SMO) to accumulate in cilia, which in turn activates the GLI family of transcription factors. Recent work has demonstrated that endogenous cholesterol and oxidized cholesterol derivatives (oxysterols) bind and modulate SMO activity. Here we discuss the myriad sterols that activate or inhibit the Hh pathway, with emphasis on endogenous 24(S),25-epoxycholesterol and 3β,5α-dihydroxycholest-7-en-6-one, and propose models of sterol regulation of SMO. Synthetic inhibitors of SMO have long been the focus of drug development efforts. Here, we discuss the possible utility of steroidal SMO ligands or inhibitors of enzymes involved in sterol metabolism as cancer therapeutics.

The tropomyosin receptor kinase (TRK) family of proteins is encoded by neurotrophic tyrosine receptor kinase (NTRK) genes and has a role in the development and normal functioning of the nervous system. NTRK gene fusions have been identified as oncogenic drivers in a wide range of tumors in both adult and pediatric patients. There has recently been a paradigm shift in cancer treatment toward biomarker-based targeted therapies, as an increasing number of actionable targets are being identified across different tumors and/or tumor histologies. These targeted agents offer greater comparative effectiveness and safety vs historical nontargeted standard therapies. The development of drugs that specifically target oncogenic drivers of cancer has led to the emergence of screening technologies to identify the patients most likely to benefit from targeted therapy. This review describes the role of NTRK gene fusions in cancer and outlines the epidemiology of NTRK gene fusions, the therapeutic benefits of targeting TRK fusions with small molecule inhibitors, and recommendations for NTRK gene fusion testing in adult and pediatric patients with cancer, in order to guide treatment decisions.

Despite recent advances in therapeutic approaches, patients with MLL-rearranged leukemia still have poor outcomes. Here, we find that the RNA-binding protein IGF2BP3, which is overexpressed in MLL-translocated leukemia, strongly amplifies MLL-Af4-mediated leukemogenesis. Deletion of Igf2bp3 significantly increases the survival of mice with MLL-Af4-driven leukemia and greatly attenuates disease, with a minimal impact on baseline hematopoiesis. At the cellular level, MLL-Af4 leukemia-initiating cells require Igf2bp3 for their function in leukemogenesis. At the molecular level, IGF2BP3 regulates a complex posttranscriptional operon governing leukemia cell survival and proliferation. IGF2BP3-targeted mRNA transcripts include important MLL-Af4-induced genes, such as those in the Hoxa locus, and the Ras signaling pathway. Targeting of transcripts by IGF2BP3 regulates both steady-state mRNA levels and, unexpectedly, pre-mRNA splicing. Together, our findings show that IGF2BP3 represents an attractive therapeutic target in this disease, providing important insights into mechanisms of posttranscriptional regulation in leukemia.

Genomic analysis has recently identified multiple ESR1 gene translocations in estrogen receptor alpha-positive (ERα+) metastatic breast cancer (MBC) that encode chimeric proteins whereby the ESR1 ligand binding domain (LBD) is replaced by C-terminal sequences from many different gene partners. Here we functionally screened 15 ESR1 fusions and identified 10 that promoted estradiol-independent cell growth, motility, invasion, epithelial-to-mesenchymal transition, and resistance to fulvestrant. RNA sequencing identified a gene expression pattern specific to functionally active ESR1 gene fusions that was subsequently reduced to a diagnostic 24-gene signature. This signature was further examined in 20 ERα+ patient-derived xenografts and in 55 ERα+ MBC samples. The 24-gene signature successfully identified cases harboring ESR1 gene fusions and also accurately diagnosed the presence of activating ESR1 LBD point mutations. Therefore, the 24-gene signature represents an efficient approach to screening samples for the presence of diverse somatic ESR1 mutations and translocations that drive endocrine treatment failure in MBC. SIGNIFICANCE: This study identifies a gene signature diagnostic for functional ESR1 fusions that drive poor outcome in advanced breast cancer, which could also help guide precision medicine approaches in patients harboring ESR1 mutations.

PurposeHuman papillomavirus (HPV) plays a major role in oncogenesis and circular extrachromosomal DNA (ecDNA) is found in many cancers. However, the relationship between HPV and circular ecDNA in human cancer is not understood.Experimental designForty-four primary tumor tissue samples were obtained from a cohort of patients with HPV-positive oropharynx squamous cell carcinoma (OPSCC). Twenty-eight additional HPV oropharyngeal cancer (HPVOPC) tumors from The Cancer Genome Atlas (TCGA) project were analyzed as a separate validation cohort. Genomic, transcriptomic, proteomic, computational, and functional analyses of HPVOPC were applied to these datasets.ResultsOur analysis revealed circular, oncogenic DNA in nearly all HPVOPC, with circular human and human-viral hybrid ecDNA present in over a third of HPVOPC and viral circular DNA in remaining tumors. Hybrid ecDNA highly express fusion transcripts from HPV promoters and HPV oncogenes linked to downstream human transcripts that drive oncogenic transformation and immune evasion, and splice multiple, diverse human acceptors to a canonical SA880 viral donor site. HPVOPC have high E6*I expression with specific viral oncogene expression pattern related to viral or hybrid ecDNA composition.ConclusionsNonchromosomal circular oncogenic DNA is a dominant feature of HPVOPC, revealing an unanticipated link between HPV and ecDNA that leverages the power of extrachromosomal inheritance to drive HPV and somatic oncogene expression.

CBFB-MYH11 transcripts and KIT mutations predict relapse in AML. High-risk CBFB-MYH11 transcripts are associated with distinct transcriptional landscapes and upregulation of early hematopoiesis genes.

BackgroundSinonasal Undifferentiated Carcinoma (SNUC) is a rare and aggressive skull base tumor with poor survival and limited treatment options. To date, targeted sequencing studies have identified IDH2 and SMARCB1 as potential driver alterations, but the molecular alterations found in SMARCB1 wild type tumors are unknown.MethodsWe evaluated survival outcomes in a cohort of 46 SNUC patients treated at an NCI designated cancer center and identify clinical and disease variables associated with survival on Kaplan-Meier and Cox multivariate survival analysis. We performed exome sequencing to characterize a series of SNUC tumors (n = 5) and cell line (MDA8788-6) to identify high confidence mutations, copy number alterations, microsatellite instability, and fusions. Knockdown studies using siRNA were utilized for validation of a novel PGAP3-SRPK1 gene fusion.ResultsOverall survival analysis revealed no significant difference in outcomes between patients treated with surgery +/- CRT and CRT alone. Tobacco use was the only significant predictor of survival. We also confirmed previously published findings on IDH and SMARC family mutations and identified novel recurrent aberrations in the JAK/STAT and PI3K pathways. We also validated a novel PGAP3-SRPK1 gene fusion in the SNUC cell line, and show that knockdown of the fusion is negatively associated with EGFR, E2F and MYC signaling.ConclusionCollectively, these data demonstrate recurrent alterations in the SWI/SNF family as well as IDH, JAK/STAT, and PI3K pathways and discover a novel fusion gene (PGAP3-SRPK1). These data aim to improve understanding of possible driver mutations and guide future therapeutic strategies for this disease.

Human papillomavirus (HPV) is a ubiquitous human pathogen that can be cleared by host immunity. Nonetheless, a small percentage of the patients develop persistent infection with oncogenic HPV, which poses an increased risk of developing HPV-associated malignancy. Although cell-mediated immunity is a known systemic factor, local factors that influence persistent HPV infection have not been fully investigated. HPV-related head/neck cancers have a strong site preference for the oropharynx, suggesting the existence of unique local factors that promote HPV-induced oncogenesis. The human oropharynx often harbors anaerobic bacteria that produce a variety of byproducts, including butyrate. Because butyrate is a potent epigenetic modulator, it could be an environmental factor influencing the development of HPV-positive oropharyngeal malignancy. In this study, we showed that butyrate treatment changed the property of HPV16 E6/E7-immortalized keratinocytes. In vitro, the treatment increased the cells' migration ability, slowed the growth, and increased the genotoxic resistance. When implanted in the syngeneic mice, the treated keratinocytes survived longer and exhibited a different growth pattern. The survival advantage obtained after butyrate exposure may increase the susceptibility of HPV-infected oropharyngeal keratinocytes to further malignant transformation. These results suggest that fermentation products of tonsillar bacteria may play an important role in the long-term persistence of high-risk HPV infection, which is a critical risk factor for developing HPV-positive oropharyngeal malignancy.

PURPOSE: Although NRG1 fusions are oncogenic drivers across multiple tumor types including lung cancers, these are difficult to study because of their rarity. The global eNRGy1 registry was thus established to characterize NRG1 fusion-positive lung cancers in the largest and most diverse series to date. METHODS: From June 2018 to February 2020, a consortium of 22 centers from nine countries in Europe, Asia, and the United States contributed data from patients with pathologically confirmed NRG1 fusion-positive lung cancers. Profiling included DNA-based and/or RNA-based next-generation sequencing and fluorescence in situ hybridization. Anonymized clinical, pathologic, molecular, and response (RECIST v1.1) data were centrally curated and analyzed. RESULTS: Although the typified never smoking (57%), mucinous adenocarcinoma (57%), and nonmetastatic (71%) phenotype predominated in 110 patients with NRG1 fusion-positive lung cancer, further diversity, including in smoking history (43%) and histology (43% nonmucinous and 6% nonadenocarcinoma), was elucidated. RNA-based testing identified most fusions (74%). Molecularly, six (of 18) novel 5 partners, 20 unique epidermal growth factor domain-inclusive chimeric events, and heterogeneous 5/3 breakpoints were found. Platinum-doublet and taxane-based (post-platinum-doublet) chemotherapy achieved low objective response rates (ORRs 13% and 14%, respectively) and modest progression-free survival medians (PFS 5.8 and 4.0 months, respectively). Consistent with a low programmed death ligand-1 expressing (28%) and low tumor mutational burden (median: 0.9 mutations/megabase) immunophenotype, the activity of chemoimmunotherapy and single-agent immunotherapy was poor (ORR 0%/PFS 3.3 months and ORR 20%/PFS 3.6 months, respectively). Afatinib achieved an ORR of 25%, not contingent on fusion type, and a 2.8-month median PFS. CONCLUSION: NRG1 fusion-positive lung cancers were molecularly, pathologically, and clinically more heterogeneous than previously recognized. The activity of cytotoxic, immune, and targeted therapies was disappointing. Further research examining NRG1-rearranged tumor biology is needed to develop new therapeutic strategies.

Patients with non-supernumerary ring chromosome 7 syndrome have an increased incidence of hemangiomas, café-au-lait spots, and melanocytic nevi. The mechanism for the increased incidence of these benign neoplasms is unknown. We present the case of a 22-year-old man with ring chromosome 7 and multiple melanocytic nevi. Two nevi, one on the right ear and the other on the right knee, were biopsied and diagnosed as desmoplastic Spitz nevi. Upon targeted next-generation DNA sequencing, both harbored BRAF fusions. Copy number alterations and fluorescence in situ hybridization (FISH) for BRAF suggested that the fusions arose on the ring chromosome 7. Hence, one reason for increased numbers of nevi in patients with non-supernumerary ring chromosome 7 syndrome may be increased likelihood of BRAF fusions, due to the instability of the ring chromosome.

Non-genetic mechanisms have recently emerged as important drivers of cancer therapy failure1, where some cancer cells can enter a reversible drug-tolerant persister state in response to treatment2. Although most cancer persisters remain arrested in the presence of the drug, a rare subset can re-enter the cell cycle under constitutive drug treatment. Little is known about the non-genetic mechanisms that enable cancer persisters to maintain proliferative capacity in the presence of drugs. To study this rare, transiently resistant, proliferative persister population, we developed Watermelon, a high-complexity expressed barcode lentiviral library for simultaneous tracing of each cell's clonal origin and proliferative and transcriptional states. Here we show that cycling and non-cycling persisters arise from different cell lineages with distinct transcriptional and metabolic programs. Upregulation of antioxidant gene programs and a metabolic shift to fatty acid oxidation are associated with persister proliferative capacity across multiple cancer types. Impeding oxidative stress or metabolic reprogramming alters the fraction of cycling persisters. In human tumours, programs associated with cycling persisters are induced in minimal residual disease in response to multiple targeted therapies. The Watermelon system enabled the identification of rare persister lineages that are preferentially poised to proliferate under drug pressure, thus exposing new vulnerabilities that can be targeted to delay or even prevent disease recurrence.

Overlapping myelodysplastic/myeloproliferative neoplasms (MDS/MPN) are clonal hematopoietic disorders with features of myelodysplasia and myeloproliferation. The only well-characterized MDS/MPN in children is juvenile myelomonocytic leukemia, an aggressive disorder of infants and toddlers. The biochemical hallmark of this disease is hyperactivation of the Ras/MAPK signaling pathway caused by mutations in Ras pathway genes in more than 90% of patients. Translocations involving receptor tyrosine kinases have been identified in rare cases. Here, we report a 2-year-old patient who presented with MDS/MPN driven by a cytogenetically cryptic NUP98-NSD1 fusion, a translocation thought to exclusively occur in patients with acute myeloid leukemia.

Intracranial mesenchymal tumors with FET-CREB fusions are a recently described group of neoplasms in children and young adults characterized by fusion of a FET family gene (usually EWSR1, but rarely FUS) to a CREB family transcription factor (ATF1, CREB1, or CREM), and have been variously termed intracranial angiomatoid fibrous histiocytoma or intracranial myxoid mesenchymal tumor. The clinical outcomes, histologic features, and genomic landscape are not well defined. Here, we studied 20 patients with intracranial mesenchymal tumors proven to harbor FET-CREB fusion by next-generation sequencing (NGS). The 16 female and four male patients had a median age of 14 years (range 4-70). Tumors were uniformly extra-axial or intraventricular and located at the cerebral convexities (n = 7), falx (2), lateral ventricles (4), tentorium (2), cerebellopontine angle (4), and spinal cord (1). NGS demonstrated that eight tumors harbored EWSR1-ATF1 fusion, seven had EWSR1-CREB1, four had EWSR1-CREM, and one had FUS-CREM. Tumors were uniformly well circumscribed and typically contrast enhancing with solid and cystic growth. Tumors with EWSR1-CREB1 fusions more often featured stellate/spindle cell morphology, mucin-rich stroma, and hemangioma-like vasculature compared to tumors with EWSR1-ATF1 fusions that most often featured sheets of epithelioid cells with mucin-poor collagenous stroma. These tumors demonstrated polyphenotypic immunoprofiles with frequent positivity for desmin, EMA, CD99, MUC4, and synaptophysin, but absence of SSTR2A, myogenin, and HMB45 expression. There was a propensity for local recurrence with a median progression-free survival of 12 months and a median overall survival of greater than 60 months, with three patients succumbing to disease (all with EWSR1-ATF1 fusions). In combination with prior case series, this study provides further insight into intracranial mesenchymal tumors with FET-CREB fusion, which represent a distinct group of CNS tumors encompassing both intracranial myxoid mesenchymal tumor and angiomatoid fibrous histiocytoma-like neoplasms.

BackgroundNeurotrophic tyrosine receptor kinase (NTRK) gene fusions are oncogenic drivers in various tumor types. While NTRK gene fusions are predictive of benefit from tropomyosin receptor kinase inhibitors regardless of tumor type, the prognostic significance of NTRK gene fusions in a pan-tumor setting remains unclear.ObjectiveThis study evaluated the characteristics and prognosis of tropomyosin receptor kinase fusion cancer in the real-world setting.Patients and methodsThis retrospective study used a de-identified clinico-genomic database and included patients with cancer who had comprehensive genomic profiling between January 2011 and July 2018. Patients were classified as having cancer with NTRK gene fusions or NTRK wild-type genes. Patients were matched with a 1:4 ratio (NTRK fusion:NTRK wild-type) using the Mahalanobis distance method on demographic and clinical characteristics, including age and Eastern Cooperative Oncology Group performance status. Descriptive analysis of clinical and molecular characteristics was conducted. Kaplan-Meier estimator and Cox regression were used for overall survival analysis.ResultsMedian overall survival was 12.5 months (95% confidence interval 9.5-not estimable) and 16.5 months (95% confidence interval 12.5-22.5) in the NTRK gene fusion (n = 27) and NTRK wild-type cohorts (n = 107), respectively (hazard ratio 1.44; 95% confidence interval 0.61-3.37; p = 0.648). Co-occurrence of select targetable biomarkers including ALK, BRAF, ERBB2, EGFR, ROS1, and KRAS was lower in cancers with NTRK gene fusions than in NTRK wild-type cancers.ConclusionsAlthough the hazard ratio for overall survival suggested a higher risk of death for patients with NTRK gene fusions, the difference was not statistically significant. Co-occurrence of NTRK gene fusions and other actionable biomarkers was uncommon.

A subset of Spitz tumors harbor fusions of NTRK3 with ETV6, MYO5A, and MYH9. We evaluated a series of 22 melanocytic tumors in which an NTRK3 fusion was identified as part of the diagnostic workup. Tumors in which NTRK3 was fused to ETV6 occurred in younger patients were predominantly composed of epithelioid melanocytes and were classified by their histopathologic features as Spitz tumors. In contrast, those in which NTRK3 was fused to MYO5A were predominantly composed of spindled melanocytes arrayed in fascicles with neuroid features such as pseudo-Verocay bodies. To further investigate the effects of the fusion kinases ETV6-NTRK3 and MYO5A-NTRK3 in melanocytes, we expressed them in immortalized melanocytes and determined their subcellular localization by immunofluorescence. ETV6-NTRK3 was localized to the nucleus and diffusely within the cytoplasm and caused melanocytes to adopt an epithelioid cytomorphology. In contrast, MYO5A-NTRK3, appeared excluded from the nucleus of melanocytes, was localized to dendrites, and resulted in a highly dendritic cytomorphology. Our findings indicate that ETV6-NTRK3 and MYO5A-NTRK3 have distinct subcellular localizations and effects on cellular morphology.

Tumor-specific elucidation of physical and functional oncoprotein interactions could improve tumorigenic mechanism characterization and therapeutic response prediction. Current interaction models and pathways, however, lack context specificity and are not oncoprotein specific. We introduce SigMaps as context-specific networks, comprising modulators, effectors and cognate binding-partners of a specific oncoprotein. SigMaps are reconstructed de novo by integrating diverse evidence sources-including protein structure, gene expression and mutational profiles-via the OncoSig machine learning framework. We first generated a KRAS-specific SigMap for lung adenocarcinoma, which recapitulated published KRAS biology, identified novel synthetic lethal proteins that were experimentally validated in three-dimensional spheroid models and established uncharacterized crosstalk with RAB/RHO. To show that OncoSig is generalizable, we first inferred SigMaps for the ten most mutated human oncoproteins and then for the full repertoire of 715 proteins in the COSMIC Cancer Gene Census. Taken together, these SigMaps show that the cell's regulatory and signaling architecture is highly tissue specific.

The recurring translocation t(1;19) (q23;p13) with TCF3-PBX1 rearrangements are uncommon in adult acute lymphoblastic leukemia (ALL), and their prognostic impact remains to be described in the era of modern chemotherapies. We investigated 427 adult patients with newly diagnosed pre-B ALL, 16 (4%) had t(1;19)(q23;p13) at diagnosis. All 16 patients achieved complete remission after induction with intensive chemotherapy, and with a median of 7-year follow-up, 2 relapsed. The 5-year cumulative incidence of relapse and overall survival rates were 14% and 82%, respectively. Our analysis showed that adult patients with t(1;19)(q23;p13) positive ALL had favorable prognosis with intensive chemotherapy regimens.

Acute myeloid leukemia (AML) harboring inv(16)(p13q22) expresses high levels of miR-126. Here we show that the CBFB-MYH11 (CM) fusion gene upregulates miR-126 expression through aberrant miR-126 transcription and perturbed miR-126 biogenesis via the HDAC8/RAN-XPO5-RCC1 axis. Aberrant miR-126 upregulation promotes survival of leukemia-initiating progenitors and is critical for initiating and maintaining CM-driven AML. We show that miR-126 enhances MYC activity through the SPRED1/PLK2-ERK-MYC axis. Notably, genetic deletion of miR-126 significantly reduces AML rate and extends survival in CM knock-in mice. Therapeutic depletion of miR-126 with an anti-miR-126 (miRisten) inhibits AML cell survival, reduces leukemia burden and leukemia stem cell (LSC) activity in inv(16) AML murine and xenograft models. The combination of miRisten with chemotherapy further enhances the anti-leukemia and anti-LSC activity. Overall, this study provides molecular insights for the mechanism and impact of miR-126 dysregulation in leukemogenesis and highlights the potential of miR-126 depletion as a therapeutic approach for inv(16) AML.

MLL gene rearrangements (MLLr) are a common cause of aggressive, incurable acute lymphoblastic leukemias (ALL) in infants and children, most of which originate in utero. The most common MLLr produces an MLL-AF4 fusion protein. MLL-AF4 promotes leukemogenesis by activating key target genes, mainly through recruitment of DOT1L and increased histone H3 lysine-79 methylation (H3K79me2/3). One key MLL-AF4 target gene is PROM1, which encodes CD133 (Prominin-1). CD133 is a pentaspan transmembrane glycoprotein that represents a potential pan-cancer target as it is found on multiple cancer stem cells. Here we demonstrate that aberrant PROM1/CD133 expression is essential for leukemic cell growth, mediated by direct binding of MLL-AF4. Activation is controlled by an intragenic H3K79me2/3 enhancer element (KEE) leading to increased enhancer-promoter interactions between PROM1 and the nearby gene TAPT1. This dual locus regulation is reflected in a strong correlation of expression in leukemia. We find that in PROM1/CD133 non-expressing cells, the PROM1 locus is repressed by polycomb repressive complex 2 (PRC2) binding, associated with reduced expression of TAPT1, partially due to loss of interactions with the PROM1 locus. Together, these results provide the first detailed analysis of PROM1/CD133 regulation that explains CD133 expression in MLLr ALL.

Marek's disease virus (MDV) encodes a basic-leucine zipper (BZIP) protein, Meq, which is considered the major MDV oncoprotein. It has been reported that the oncogenicity of Meq is associated with its interaction with C-terminal binding protein 1 (CtBP), which is also an interaction partner of Epstein-Barr virus encoded EBNA3A and EBNA3C oncoproteins. Since both EBNA3C and CtBP interact with histone deacetylase 1 (HDAC1) and HDAC2, we examined whether Meq shares this interaction with chicken HDAC1 (chHDAC1) and chHDAC2. Using confocal microscopy analysis, we show that Meq co-localizes with chHDAC1 and chHDAC2 in the nuclei of MDV lymphoblastoid tumor cells. In addition, immunoprecipitation assays demonstrate that Meq interacts with chHDAC1 and chHDAC2 in transfected cells and MDV lymphoblastoid tumor cells. Using deletion mutants, interaction domains were mapped to the N-terminal dimerization domain of chHDAC1 and chHDAC2, and the BZIP domain of Meq. Our results further demonstrate that this interaction mediates the degradation of chHDAC1 and chHDAC2 via the proteasome dependent pathway. In addition, our results show that Meq also induces the reduction of global ubiquitinated proteins through a proteasome dependent pathway. In conclusion, our results provide evidence that Meq interacts with chHDAC1 and chHDAC2, and induces their proteasome dependent degradation.

During early G1 phase, Rb is exclusively mono-phosphorylated by cyclin D:Cdk4/6, generating 14 different isoforms with specific binding patterns to E2Fs and other cellular protein targets. While mono-phosphorylated Rb is dispensable for early G1 phase progression, interfering with cyclin D:Cdk4/6 kinase activity prevents G1 phase progression, questioning the role of cyclin D:Cdk4/6 in Rb inactivation. To dissect the molecular functions of cyclin D:Cdk4/6 during cell cycle entry, we generated a single cell reporter for Cdk2 activation, RB inactivation and cell cycle entry by CRISPR/Cas9 tagging endogenous p27 with mCherry. Through single cell tracing of Cdk4i cells, we identified a time-sensitive early G1 phase specific Cdk4/6-dependent phosphorylation gradient that regulates cell cycle entry timing and resides between serum-sensing and cyclin E:Cdk2 activation. To reveal the substrate identity of the Cdk4/6 phosphorylation gradient, we performed whole proteomic and phospho-proteomic mass spectrometry, and identified 147 proteins and 82 phospho-peptides that significantly changed due to Cdk4 inhibition in early G1 phase. In summary, we identified novel (non-Rb) cyclin D:Cdk4/6 substrates that connects early G1 phase functions with cyclin E:Cdk2 activation and Rb inactivation by hyper-phosphorylation.

Aberrant alternative splicing (AS) is a hallmark of cancer and a potential target for novel anti-cancer therapeutics. Breast cancer-associated AS events are known to be linked to disease progression, metastasis, and survival of breast cancer patients. To identify altered AS programs occurring in metastatic breast cancer, we perform a global analysis of AS events by using RNA-mediated oligonucleotide annealing, selection, and ligation coupled with next-generation sequencing (RASL-seq). We demonstrate that, relative to low-metastatic, high-metastatic breast cancer cells show different AS choices in genes related to cancer progression. Supporting a global reshape of cancer-related splicing profiles in metastatic breast cancer we found an enrichment of RNA-binding motifs recognized by several splicing regulators, which have aberrant expression levels or activity during breast cancer progression, including SRSF1. Among SRSF1-regulated targets we found DCUN1D5, a gene for which skipping of exon 4 in its pre-mRNA introduces a premature termination codon (PTC), thus generating an unstable transcript degraded by nonsense-mediated mRNA decay (NMD). Significantly, distinct breast cancer subtypes show different DCUN1D5 isoform ratios with metastatic breast cancer expressing the highest level of the NMD-insensitive DCUN1D5 mRNA, thus showing high DCUN1D5 expression levels, which are ultimately associated with poor overall and relapse-free survival in breast cancer patients. Collectively, our results reveal global AS features of metastatic breast tumors, which open new possibilities for the treatment of these aggressive tumor types.

Post-transcriptional gene regulation, including that by RNA binding proteins (RBPs), has recently been described as an important mechanism in cancer. We had previously identified a set of RBPs that were highly dysregulated in B-cell acute lymphoblastic leukemia (B-ALL) with MLL translocations, which carry a poor prognosis. Here, we sought to functionally characterize these dysregulated RBP genes by performing a focused CRISPR dropout screen in B-ALL cell lines, finding dependencies on several genes including EIF3E, EPRS and USO1. Validating our findings, CRISPR/Cas9-mediated disruption of USO1 in MLL-translocated B-ALL cells reduced cell growth, promoted cell death, and altered the cell cycle. Transcriptomic analysis of USO1-deficient cells revealed alterations in pathways related to mTOR signaling, RNA metabolism, and targets of MYC. In addition, USO1-regulated genes from these experimental samples were significantly and concordantly correlated with USO1 expression in primary samples collected from B-ALL patients. Lastly, we found that loss of Uso1 inhibited colony formation of MLL-transformed in primary bone marrow cells from Cas9-EGFP mice. Together, our findings demonstrate an approach to performing focused sub-genomic CRISPR screens and highlight a putative RBP vulnerability in MLL-translocated B-ALL, thus identifying potential therapeutic targets in this disease.

Simple Summary: Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer in the world, with a heterogeneous characteristic. We firstly systematically screened the critical gene ubiquitin-specific protease 14 (USP14) in HNSCC from the DUB library after building a risk signature. Our data suggested that USP14 regulated heat shock transcription factor 1 (HSF1) stabilization by its deubiquitination and increased its downstream proteins, such as heat shock protein 60 (HSP60), HSP70, and HSP90, to promote HNSCC proliferation and lung metastasis in vivo and in vitro, and the overexpression of HSF1 reversed the inhibitory effect of USP14 depletion in vivo. Forty-five tissue samples from patients with HNSCC cancer were collected to determine the correlation of USP14 and HSF1 expression in the diagnosis of HNSCC, which is a novel mechanism of USP14 as a carcinogenic gene in HNSCC. Our results emphasized that USP14 and its downstream HSF1 have therapeutic potential in targeting HNSCC. The ubiquitin-proteasome system is a pivotal intracellular proteolysis process in posttranslational modification. It regulates multiple cellular processes. Deubiquitinating enzymes (DUBs) are a stabilizer in proteins associated with tumor growth and metastasis. However, the link between DUBs and HNSCC remains incompletely understood. In this study, therefore, we identified USP14 as a tumor proliferation enhancer and a substantially hyperactive deubiquitinase in HNSCC samples, implying a poor prognosis prediction. Silencing USP14 in vitro conspicuously inhibited HNSCC cell proliferation and migration. Consistently, defective USP14 in vivo significantly diminished HNSCC tumor growth and lung metastasis compared to the control group. Luciferase assays indicated that HSF1 was downstream from USP14, and an evaluation of the cellular effects of HSF1 overexpression in USP14-dificient mice tumors showed that elevated HSF1 reversed HNSCC growth and metastasis predominantly through the HSF1-HSP pathway. Mechanistically, USP14 encouraged HSF1 expression by deubiquitinating and stabilizing HSF1, which subsequently orchestrated transcriptional activation in HSP60, HSP70, and HSP90, ultimately leading to HNSCC progression and metastasis. Collectively, we uncovered that hyperactive USP14 contributed to HNSCC tumor growth and lung metastasis by reinforcing HSF1-depedent HSP activation, and our findings provided the insight that targeting USP14 could be a promising prognostic and therapeutic strategy for HSNCC. [ABSTRACT FROM AUTHOR]

BackgroundDesmoplastic small round cell tumor (DSRCT) is a rare, highly aggressive, translocation-associated soft-tissue sarcoma that primarily affects children, adolescents, and young adults, with a striking male predominance. It is characterized by t(11;22) generating a novel EWSR1-WT1 fusion gene. Secondary genomic alterations are rarely described.MethodsTumor tissue from 83 DSRCT patients was assayed by hybrid-capture based comprehensive genomic profiling, FoundationOne® Heme next generation sequencing analysis of 406 genes and RNA sequencing of 265 genes. Tumor mutation burden was calculated from a minimum of 1.4 Mb sequenced DNA. Microsatellite instability status was determined by a novel algorithm analyzing 114 specific loci.ResultsComprehensive genomic profiling identified several genomically-defined DSRCT subgroups. Recurrent genomic alterations were most frequently detected in FGFR4, ARID1A, TP53, MSH3, and MLL3 genes. With the exception of FGFR4, where the genomic alterations predicted activation, most of the alterations in the remaining genes predicted gene inactivation. No DSRCT were TMB or MSI high.ConclusionsIn summary, recurrent secondary somatic alterations in FGFR4, ARID1A, TP53, MSH3, and MLL3 were detected in 82% of DSRCT, which is significantly greater than previously reported. These alterations may have both prognostic and therapeutic implications.

Chromosomal instability in cancer consists of dynamic changes to the number and structure of chromosomes1,2. The resulting diversity in somatic copy number alterations (SCNAs) may provide the variation necessary for tumour evolution1,3,4. Here we use multi-sample phasing and SCNA analysis of 1,421 samples from 394 tumours across 22 tumour types to show that continuous chromosomal instability results in pervasive SCNA heterogeneity. Parallel evolutionary events, which cause disruption in the same genes (such as BCL9, MCL1, ARNT (also known as HIF1B), TERT and MYC) within separate subclones, were present in 37% of tumours. Most recurrent losses probably occurred before whole-genome doubling, that was found as a clonal event in 49% of tumours. However, loss of heterozygosity at the human leukocyte antigen (HLA) locus and loss of chromosome 8p to a single haploid copy recurred at substantial subclonal frequencies, even in tumours with whole-genome doubling, indicating ongoing karyotype remodelling. Focal amplifications that affected chromosomes 1q21 (which encompasses BCL9, MCL1 and ARNT), 5p15.33 (TERT), 11q13.3 (CCND1), 19q12 (CCNE1) and 8q24.1 (MYC) were frequently subclonal yet appeared to be clonal within single samples. Analysis of an independent series of 1,024 metastatic samples revealed that 13 focal SCNAs were enriched in metastatic samples, including gains in chromosome 8q24.1 (encompassing MYC) in clear cell renal cell carcinoma and chromosome 11q13.3 (encompassing CCND1) in HER2+ breast cancer. Chromosomal instability may enable the continuous selection of SCNAs, which are established as ordered events that often occur in parallel, throughout tumour evolution.

Women with dense breasts have an increased lifetime risk of malignancy that has been attributed to a higher epithelial density. Quantitative proteomics, collagen analysis, and mechanical measurements in normal tissue revealed that stroma in the high-density breast contains more oriented, fibrillar collagen that is stiffer and correlates with higher epithelial cell density. microRNA (miR) profiling of breast tissue identified miR-203 as a matrix stiffness-repressed transcript that is downregulated by collagen density and reduced in the breast epithelium of women with high mammographic density. Culture studies demonstrated that ZNF217 mediates a matrix stiffness- and collagen density-induced increase in Akt activity and mammary epithelial cell proliferation. Manipulation of the epithelium in a mouse model of mammographic density supported a causal relationship between stromal stiffness, reduced miR-203, higher levels of the murine homolog Zfp217, and increased Akt activity and mammary epithelial proliferation. ZNF217 was also increased in the normal breast epithelium of women with high mammographic density, correlated positively with epithelial proliferation and density, and inversely with miR-203. The findings identify ZNF217 as a potential target toward which preexisting therapies, such as the Akt inhibitor triciribine, could be used as a chemopreventive agent to reduce cancer risk in women with high mammographic density.

The dominant paradigm for HPV carcinogenesis includes integration into the host genome followed by expression of E6 and E7 (E6/E7). We explored an alternative carcinogenic pathway characterized by episomal E2, E4, and E5 (E2/E4/E5) expression. Half of HPV positive cervical and pharyngeal cancers comprised a subtype with increase in expression of E2/E4/E5, as well as association with lack of integration into the host genome. Models of the E2/E4/E5 carcinogenesis show p53 dependent enhanced proliferation in vitro, as well as increased susceptibility to induction of cancer in vivo. Whole genomic expression analysis of the E2/E4/E5 pharyngeal cancer subtype is defined by activation of the fibroblast growth factor receptor (FGFR) pathway and this subtype is susceptible to combination FGFR and mTOR inhibition, with implications for targeted therapy.