Upstream open reading frames: new players in the landscape of cancer gene regulation.

The translation of RNA by ribosomes represents a central biological process and one of the most dysregulated processes in cancer. While translation is traditionally thought to occur exclusively in the protein-coding regions of messenger RNAs (mRNAs), recent transcriptome-wide approaches have shown abundant ribosome activity across diverse stretches of RNA transcripts. The most common type of this kind of ribosome activity occurs in gene leader sequences, also known as 5' untranslated regions (UTRs) of the mRNA, that precede the main coding sequence. Translation of these upstream open reading frames (uORFs) is now known to occur in upwards of 25% of all protein-coding genes. With diverse functions from RNA regulation to microprotein generation, uORFs are rapidly igniting a new arena of cancer biology, where they are linked to cancer genetics, cancer signaling, and tumor-immune interactions. This review focuses on the contributions of uORFs and their associated 5'UTR sequences to cancer biology.

Deep learning to decode sites of RNA translation in normal and cancerous tissues.

The biological process of RNA translation is fundamental to cellular life and has wide-ranging implications for human disease. Yet, accurately delineating the variation in RNA translation represents a significant challenge. Here, we develop RiboTIE, a transformer model-based approach to map global RNA translation. We find that RiboTIE offers unparalleled precision and sensitivity for ribosome profiling data. Application of RiboTIE to normal brain and medulloblastoma cancer samples enables high-resolution insights into disease regulation of RNA translation.

Translation of non-canonical open reading frames as a cancer cell survival mechanism in childhood medulloblastoma.

A hallmark of high-risk childhood medulloblastoma is the dysregulation of RNA translation. Currently, it is unknown whether medulloblastoma dysregulates the translation of putatively oncogenic non-canonical open reading frames (ORFs). To address this question, we performed ribosome profiling of 32 medulloblastoma tissues and cell lines and observed widespread non-canonical ORF translation. We then developed a stepwise approach using multiple CRISPR-Cas9 screens to elucidate non-canonical ORFs and putative microproteins implicated in medulloblastoma cell survival. We determined that multiple lncRNA-ORFs and upstream ORFs (uORFs) exhibited selective functionality independent of main coding sequences. A microprotein encoded by one of these ORFs, ASNSD1-uORF or ASDURF, was upregulated, associated with MYC-family oncogenes, and promoted medulloblastoma cell survival through engagement with the prefoldin-like chaperone complex. Our findings underscore the fundamental importance of non-canonical ORF translation in medulloblastoma and provide a rationale to include these ORFs in future studies seeking to define new cancer targets.

What Can Ribo-Seq, Immunopeptidomics, and Proteomics Tell Us About the Noncanonical Proteome?

Ribosome profiling (Ribo-Seq) has proven transformative for our understanding of the human genome and proteome by illuminating thousands of noncanonical sites of ribosome translation outside the currently annotated coding sequences (CDSs). A conservative estimate suggests that at least 7000 noncanonical ORFs are translated, which, at first glance, has the potential to expand the number of human protein CDSs by 30%, from ∼19,500 annotated CDSs to over 26,000 annotated CDSs. Yet, additional scrutiny of these ORFs has raised numerous questions about what fraction of them truly produce a protein product and what fraction of those can be understood as proteins according to conventional understanding of the term. Adding further complication is the fact that published estimates of noncanonical ORFs vary widely by around 30-fold, from several thousand to several hundred thousand. The summation of this research has left the genomics and proteomics communities both excited by the prospect of new coding regions in the human genome but searching for guidance on how to proceed. Here, we discuss the current state of noncanonical ORF research, databases, and interpretation, focusing on how to assess whether a given ORF can be said to be "protein coding."

What can Ribo-seq and proteomics tell us about the non-canonical proteome?

Ribosome profiling (Ribo-seq) has proven transformative for our understanding of the human genome and proteome by illuminating thousands of non-canonical sites of ribosome translation outside of the currently annotated coding sequences (CDSs). A conservative estimate suggests that at least 7,000 non-canonical open reading frames (ORFs) are translated, which, at first glance, has the potential to expand the number of human protein-coding sequences by 30%, from ∼19,500 annotated CDSs to over 26,000. Yet, additional scrutiny of these ORFs has raised numerous questions about what fraction of them truly produce a protein product and what fraction of those can be understood as proteins according to conventional understanding of the term. Adding further complication is the fact that published estimates of non-canonical ORFs vary widely by around 30-fold, from several thousand to several hundred thousand. The summation of this research has left the genomics and proteomics communities both excited by the prospect of new coding regions in the human genome, but searching for guidance on how to proceed. Here, we discuss the current state of non-canonical ORF research, databases, and interpretation, focusing on how to assess whether a given ORF can be said to be "protein-coding". In brief: The human genome encodes thousands of non-canonical open reading frames (ORFs) in addition to protein-coding genes. As a nascent field, many questions remain regarding non-canonical ORFs. How many exist? Do they encode proteins? What level of evidence is needed for their verification? Central to these debates has been the advent of ribosome profiling (Ribo-seq) as a method to discern genome-wide ribosome occupancy, and immunopeptidomics as a method to detect peptides that are processed and presented by MHC molecules and not observed in traditional proteomics experiments. This article provides a synthesis of the current state of non-canonical ORF research and proposes standards for their future investigation and reporting. Highlights: Combined use of Ribo-seq and proteomics-based methods enables optimal confidence in detecting non-canonical ORFs and their protein products.Ribo-seq can provide more sensitive detection of non-canonical ORFs, but data quality and analytical pipelines will impact results.Non-canonical ORF catalogs are diverse and span both high-stringency and low-stringency ORF nominations.A framework for standardized non-canonical ORF evidence will advance the research field.

Shining a light on the dark proteome: Non-canonical open reading frames and their encoded miniproteins as a new frontier in cancer biology.

In the decades following the discovery that genes encode proteins, scientists have tried to exhaustively and comprehensively characterize the human genome. Recent advances in computational methods along with transcriptomic and proteomic techniques have now shown that historically non-coding genomic regions may contain non-canonical open reading frames (ncORFs), which may encode functional miniproteins or otherwise exert regulatory activity through coding-independent functions. Increasingly, it is clear that these ncORFs may play critical roles in major human diseases such as cancer. In this review, we summarize the history and current progress of ncORF research and explore the known functions of ncORFs and the miniproteins they may encode. We particularly highlight the emerging body of evidence supporting a role for ncORFs and miniproteins contributions in cancer. Finally, we provide a blueprint for high-priority areas of future research for ncORFs in cancer, focusing on ncORF detection, functional characterization, and therapeutic intervention.

Translation of non-canonical open reading frames as a cancer cell survival mechanism in childhood medulloblastoma.

A hallmark of high-risk childhood medulloblastoma is the dysregulation of RNA translation. Currently, it is unknown whether medulloblastoma dysregulates the translation of putatively oncogenic non-canonical open reading frames. To address this question, we performed ribosome profiling of 32 medulloblastoma tissues and cell lines and observed widespread non-canonical ORF translation. We then developed a step-wise approach to employ multiple CRISPR-Cas9 screens to elucidate functional non-canonical ORFs implicated in medulloblastoma cell survival. We determined that multiple lncRNA-ORFs and upstream open reading frames (uORFs) exhibited selective functionality independent of the main coding sequence. One of these, ASNSD1-uORF or ASDURF, was upregulated, associated with the MYC family oncogenes, and was required for medulloblastoma cell survival through engagement with the prefoldin-like chaperone complex. Our findings underscore the fundamental importance of non-canonical ORF translation in medulloblastoma and provide a rationale to include these ORFs in future cancer genomics studies seeking to define new cancer targets.

Evolutionary origins and interactomes of human, young microproteins and small peptides translated from short open reading frames.

All species continuously evolve short open reading frames (sORFs) that can be templated for protein synthesis and may provide raw materials for evolutionary adaptation. We analyzed the evolutionary origins of 7,264 recently cataloged human sORFs and found that most were evolutionarily young and had emerged de novo. We additionally identified 221 previously missed sORFs potentially translated into peptides of up to 15 amino acids-all of which are smaller than the smallest human microprotein annotated to date. To investigate the bioactivity of sORF-encoded small peptides and young microproteins, we subjected 266 candidates to a mass-spectrometry-based interactome screen with motif resolution. Based on these interactomes and additional cellular assays, we can associate several candidates with mRNA splicing, translational regulation, and endocytosis. Our work provides insights into the evolutionary origins and interaction potential of young and small proteins, thereby helping to elucidate this underexplored territory of the human proteome.

SEQing to find hidden medulloblastoma cells.

Minimal residual disease (MRD) assessment has revolutionized the clinical management of pediatric leukemias but has remained challenging to implement in pediatric brain tumors. In this issue of Cancer Cell, Liu et al. describe an approach to measuring MRD in pediatric medulloblastoma through the use of cell-free DNA in cerebrospinal fluid, with important prognostic implications.

Noncanonical open reading frames encode functional proteins essential for cancer cell survival.

Although genomic analyses predict many noncanonical open reading frames (ORFs) in the human genome, it is unclear whether they encode biologically active proteins. Here we experimentally interrogated 553 candidates selected from noncanonical ORF datasets. Of these, 57 induced viability defects when knocked out in human cancer cell lines. Following ectopic expression, 257 showed evidence of protein expression and 401 induced gene expression changes. Clustered regularly interspaced short palindromic repeat (CRISPR) tiling and start codon mutagenesis indicated that their biological effects required translation as opposed to RNA-mediated effects. We found that one of these ORFs, G029442-renamed glycine-rich extracellular protein-1 (GREP1)-encodes a secreted protein highly expressed in breast cancer, and its knockout in 263 cancer cell lines showed preferential essentiality in breast cancer-derived lines. The secretome of GREP1-expressing cells has an increased abundance of the oncogenic cytokine GDF15, and GDF15 supplementation mitigated the growth-inhibitory effect of GREP1 knockout. Our experiments suggest that noncanonical ORFs can express biologically active proteins that are potential therapeutic targets.

Safety and efficacy of gamma-secretase inhibitor nirogacestat (PF-03084014) in desmoid tumor: Report of four pediatric/young adult cases.

Systemic therapy for pediatric desmoid tumors has been challenged by a lack of high-quality clinical evidence and potential adverse effects. The gamma-secretase inhibitor nirogacestat has shown promising efficacy in adults. We report four cases of pediatric and young adult desmoid tumor patients (three with familial adenomatous polyposis [FAP] syndrome) who received nirogacestat on compassionate use. After a median of 13.5 months (range 6-18), three had durable benefit: a complete response (Case 1); a partial response (Case 2); stable disease (Case 3). The fourth had disease progression after a partial response. No patient experienced grade 3 or 4 adverse events.

An emerging role for endothelial barrier support therapy for congenital disorders of glycosylation.

Congenital disorders of glycosylation (CDGs) are clinically heterogeneous disorders defined by a decreased ability to modify biomolecules with oligosaccharides. Critical disruptions in protein recognition, interaction, binding, and anchoring lead to broad physiological effects. Patients present with endocrinopathy, immunodeficiency, hepatopathy, coagulopathy, and neurodevelopmental impairment. Patients may experience mortality/morbidity associated with shock physiology that is frequently culture negative and poorly responsive to standard care. Oedema, pleural and pericardial effusions, ascites, proteinuria, and protein-losing enteropathy are observed with an exaggerated inflammatory response. The negative serum protein steady state results from several mechanisms including reduced hepatic synthesis and secretion, increased consumption, and extravasation. Disruption of the glycocalyx, a layer of glycosylated proteins that lines the endothelium preventing thrombosis and extravasation, is a suspected cause of endothelial dysfunction in CDG patients. We performed a retrospective review of CDG patients admitted to our institution with acute illness over the past 2 years. Longitudinal clinical and laboratory data collected during the sick and well states were assessed for biomarkers of inflammation and efficacy of interventions. Six patients representing 4 CDG subtypes and 14 hospitalisations were identified. Acute D-dimer elevation, proteinuria, decreased serum total protein levels, coagulation proteins, and albumin were observed with acute illness. Infusion of fresh frozen plasma, and in some cases protein C concentrate, was associated with clinical and biomarker improvement. This was notable with intra-patient comparison of treated vs untreated courses. Use of endothelial barrier support therapy may reduce endothelial permeability by restoring both regulatory serum protein homeostasis and supporting the glycocalyx and is likely a critical component of care for this population.

Burkitt Lymphoma Presenting as Menorrhagia and a Vaginal Mass in an Adolescent.

BACKGROUND: Menorrhagia is a common gynecologic complaint among adolescents, which rarely is secondary to malignancy. Burkitt lymphoma can mimic gynecologic malignancy, however it is rarely seen in adolescents. Burkitt lymphoma of the gynecologic tract requires early diagnosis and intervention for optimal outcomes. CASE: We report a case of a 15-year-old adolescent who had multiple admissions for menorrhagia that was thought to be secondary to anovulatory bleeding until pelvic ultrasound revealed a large 8-cm vaginal/cervical mass. Histologic examination of the biopsy specimen revealed Burkitt lymphoma, which was treated with chemotherapy leading to resolution of her menorrhagia. SUMMARY AND CONCLUSION: Burkitt lymphoma presenting as a vaginal/cervical mass is exceedingly rare, especially in the adolescent patient. Burkitt lymphoma is generally highly responsive to chemotherapy, and symptoms rapidly improve after initiation of treatment.

Clinically Integrated Sequencing Alters Therapy in Children and Young Adults With High-Risk Glial Brain Tumors.

PURPOSE: Brain tumors have become the leading cause of cancer-related mortality in young patients. Novel effective therapies on the basis of the unique biology of each tumor are urgently needed. The goal of this study was to evaluate the feasibility, utility, and clinical impact of integrative clinical sequencing and genetic counseling in children and young adults with high-risk brain tumors. PATIENTS AND METHODS: Fifty-two children and young adults with brain tumors designated by the treating neuro-oncologist to be high risk (> 25% chance for treatment failure; mean age, 10.2 years; range, 0 to 39 years) were enrolled in a prospective, observational, consecutive case series, in which participants underwent integrative clinical exome (tumor and germline DNA) and transcriptome (tumor RNA) sequencing and genetic counseling. Results were discussed in a multi-institutional brain tumor precision medicine teleconference. RESULTS: Sequencing revealed a potentially actionable germline or tumor alteration in 25 (63%) of 40 tumors with adequate tissue, of which 21 (53%) resulted in an impact on treatment or change of diagnosis. Platelet-derived growth factor receptor or fibroblast growth factor receptor pathway alterations were seen in nine of 20 (45%) glial tumors. Eight (20%) sequenced tumors harbored an oncogenic fusion isolated on RNA sequencing. Seventeen of 20 patients (85%) with glial tumors were found to have a potentially actionable result, which resulted in change of therapy in 14 (70%) patients. Patients with recurrent brain tumors receiving targeted therapy had a median progression-free survival (from time on therapy) of 4 months. CONCLUSION: Selection of personalized agents for children and young adults with highrisk brain tumors on the basis of integrative clinical sequencing is feasible and resulted in a change in therapy in more than two thirds of children and young adults with high-risk glial tumors.

Oncogenic Role of THOR, a Conserved Cancer/Testis Long Non-coding RNA.

Large-scale transcriptome sequencing efforts have vastly expanded the catalog of long non-coding RNAs (lncRNAs) with varying evolutionary conservation, lineage expression, and cancer specificity. Here, we functionally characterize a novel ultraconserved lncRNA, THOR (ENSG00000226856), which exhibits expression exclusively in testis and a broad range of human cancers. THOR knockdown and overexpression in multiple cell lines and animal models alters cell or tumor growth supporting an oncogenic role. We discovered a conserved interaction of THOR with IGF2BP1 and show that THOR contributes to the mRNA stabilization activities of IGF2BP1. Notably, transgenic THOR knockout produced fertilization defects in zebrafish and also conferred a resistance to melanoma onset. Likewise, ectopic expression of human THOR in zebrafish accelerated the onset of melanoma. THOR represents a novel class of functionally important cancer/testis lncRNAs whose structure and function have undergone positive evolutionary selection.

Precision medicine in pediatric oncology: Lessons learned and next steps.

The maturation of genomic technologies has enabled new discoveries in disease pathogenesis as well as new approaches to patient care. In pediatric oncology, patients may now receive individualized genomic analysis to identify molecular aberrations of relevance for diagnosis and/or treatment. In this context, several recent clinical studies have begun to explore the feasibility and utility of genomics-driven precision medicine. Here, we review the major developments in this field, discuss current limitations, and explore aspects of the clinical implementation of precision medicine, which lack consensus. Lastly, we discuss ongoing scientific efforts in this arena, which may yield future clinical applications.