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.

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.

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.

The lncRNA landscape of breast cancer reveals a role for DSCAM-AS1 in breast cancer progression.

Molecular classification of cancers into subtypes has resulted in an advance in our understanding of tumour biology and treatment response across multiple tumour types. However, to date, cancer profiling has largely focused on protein-coding genes, which comprise <1% of the genome. Here we leverage a compendium of 58,648 long noncoding RNAs (lncRNAs) to subtype 947 breast cancer samples. We show that lncRNA-based profiling categorizes breast tumours by their known molecular subtypes in breast cancer. We identify a cohort of breast cancer-associated and oestrogen-regulated lncRNAs, and investigate the role of the top prioritized oestrogen receptor (ER)-regulated lncRNA, DSCAM-AS1. We demonstrate that DSCAM-AS1 mediates tumour progression and tamoxifen resistance and identify hnRNPL as an interacting protein involved in the mechanism of DSCAM-AS1 action. By highlighting the role of DSCAM-AS1 in breast cancer biology and treatment resistance, this study provides insight into the potential clinical implications of lncRNAs in breast cancer.

Modulation of long noncoding RNAs by risk SNPs underlying genetic predispositions to prostate cancer.

Long noncoding RNAs (lncRNAs) represent an attractive class of candidates to mediate cancer risk. Through integrative analysis of the lncRNA transcriptome with genomic data and SNP data from prostate cancer genome-wide association studies (GWAS), we identified 45 candidate lncRNAs associated with risk to prostate cancer. We further evaluated the mechanism underlying the top hit, PCAT1, and found that a risk-associated variant at rs7463708 increases binding of ONECUT2, a novel androgen receptor (AR)-interacting transcription factor, at a distal enhancer that loops to the PCAT1 promoter, resulting in upregulation of PCAT1 upon prolonged androgen treatment. In addition, PCAT1 interacts with AR and LSD1 and is required for their recruitment to the enhancers of GNMT and DHCR24, two androgen late-response genes implicated in prostate cancer development and progression. PCAT1 promotes prostate cancer cell proliferation and tumor growth in vitro and in vivo. These findings suggest that modulating lncRNA expression is an important mechanism for risk-associated SNPs in promoting prostate transformation.

Integrative Clinical Sequencing in the Management of Refractory or Relapsed Cancer in Youth.

IMPORTANCE: Cancer is caused by a diverse array of somatic and germline genomic aberrations. Advances in genomic sequencing technologies have improved the ability to detect these molecular aberrations with greater sensitivity. However, integrating them into clinical management in an individualized manner has proven challenging. OBJECTIVE: To evaluate the use of integrative clinical sequencing and genetic counseling in the assessment and treatment of children and young adults with cancer. DESIGN, SETTING, AND PARTICIPANTS: Single-site, observational, consecutive case series (May 2012-October 2014) involving 102 children and young adults (mean age, 10.6 years; median age, 11.5 years, range, 0-22 years) with relapsed, refractory, or rare cancer. EXPOSURES: Participants underwent integrative clinical exome (tumor and germline DNA) and transcriptome (tumor RNA) sequencing and genetic counseling. Results were discussed by a precision medicine tumor board, which made recommendations to families and their physicians. MAIN OUTCOMES AND MEASURES: Proportion of patients with potentially actionable findings, results of clinical actions based on integrative clinical sequencing, and estimated proportion of patients or their families at risk of future cancer. RESULTS: Of the 104 screened patients, 102 enrolled with 91 (89%) having adequate tumor tissue to complete sequencing. Only the 91 patients were included in all calculations, including 28 (31%) with hematological malignancies and 63 (69%) with solid tumors. Forty-two patients (46%) had actionable findings that changed their cancer management: 15 of 28 (54%) with hematological malignancies and 27 of 63 (43%) with solid tumors. Individualized actions were taken in 23 of the 91 (25%) based on actionable integrative clinical sequencing findings, including change in treatment for 14 patients (15%) and genetic counseling for future risk for 9 patients (10%). Nine of 91 (10%) of the personalized clinical interventions resulted in ongoing partial clinical remission of 8 to 16 months or helped sustain complete clinical remission of 6 to 21 months. All 9 patients and families with actionable incidental genetic findings agreed to genetic counseling and screening. CONCLUSIONS AND RELEVANCE: In this single-center case series involving young patients with relapsed or refractory cancer, incorporation of integrative clinical sequencing data into clinical management was feasible, revealed potentially actionable findings in 46% of patients, and was associated with change in treatment and family genetic counseling for a small proportion of patients. The lack of a control group limited assessing whether better clinical outcomes resulted from this approach than outcomes that would have occurred with standard care.

The landscape of antisense gene expression in human cancers.

High-throughput RNA sequencing has revealed more pervasive transcription of the human genome than previously anticipated. However, the extent of natural antisense transcripts' (NATs) expression, their regulation of cognate sense genes, and the role of NATs in cancer remain poorly understood. Here, we use strand-specific paired-end RNA sequencing (ssRNA-seq) data from 376 cancer samples covering nine tissue types to comprehensively characterize the landscape of antisense expression. We found consistent antisense expression in at least 38% of annotated transcripts, which in general is positively correlated with sense gene expression. Investigation of sense/antisense pair expressions across tissue types revealed lineage-specific, ubiquitous and cancer-specific antisense loci transcription. Comparisons between tumor and normal samples identified both concordant (same direction) and discordant (opposite direction) sense/antisense expression patterns. Finally, we provide OncoNAT, a catalog of cancer-related genes with significant antisense transcription, which will enable future investigations of sense/antisense regulation in cancer. Using OncoNAT we identified several functional NATs, including NKX2-1-AS1 that regulates the NKX2-1 oncogene and cell proliferation in lung cancer cells. Overall, this study provides a comprehensive account of NATs and supports a role for NATs' regulation of tumor suppressors and oncogenes in cancer biology.