Damon A. Hofman

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.

BACKGROUND: Immunotherapy in high-risk neuroblastoma (HR-NBL) does not live up to its full potential due to inadequate (adaptive) immune engagement caused by the extensive immunomodulatory capacity of HR-NBL. We aimed to tackle one of the most notable immunomodulatory processes in neuroblastoma (NBL), absence of major histocompatibility complex class I (MHC-I) surface expression, a process greatly limiting cytotoxic T cell engagement. We and others have previously shown that MHC-I expression can be induced by cytokine-driven immune modulation. Here, we aimed to identify tolerable pharmacological repurposing strategies to upregulate MHC-I expression and therewith enhance T cell immunogenicity in NBL. METHODS: Drug repurposing libraries were screened to identify compounds enhancing MHC-I surface expression in NBL cells using high-throughput flow cytometry analyses optimized for adherent cells. The effect of positive hits was confirmed in a panel of NBL cell lines and patient-derived organoids. Compound-treated NBL cell lines and organoids were cocultured with preferentially expressed antigen of melanoma (PRAME)-reactive tumor-specific T cells and healthy-donor natural killer (NK) cells to determine the in vitro effect on T cell and NK cell cytotoxicity. Additional immunomodulatory effects of histone deacetylase inhibitors (HDACi) were identified by transcriptome and translatome analysis of treated organoids. RESULTS: Drug library screening revealed MHC-I upregulation by inhibitor of apoptosis inhibitor (IAPi)- and HDACi drug classes. The effect of IAPi was limited due to repression of nuclear factor kappa B (NFκB) pathway activity in NBL, while the MHC-I-modulating effect of HDACi was widely translatable to a panel of NBL cell lines and patient-derived organoids. Pretreatment of NBL cells with the HDACi entinostat enhanced the cytotoxic capacity of tumor-specific T cells against NBL in vitro, which coincided with increased expression of additional players regulating T cell cytotoxicity (eg, TAP1/2 and immunoproteasome subunits). Moreover, MICA and MICB, important in NK cell cytotoxicity, were also increased by entinostat exposure. Intriguingly, this increase in immunogenicity was accompanied by a shift toward a more mesenchymal NBL cell lineage. CONCLUSIONS: This study indicates the potential of combining (immuno)therapy with HDACi to enhance both T cell-driven and NKcell-driven immune responses in patients with HR-NBL.

TP53 nonsense mutations in cancer produce truncated inactive p53 protein. We show that 5-FU metabolite 5-Fluorouridine (FUr) induces full-length p53 in human tumor cells carrying R213X nonsense mutant TP53. Ribosome profiling visualized translational readthrough at the R213X premature stop codon and demonstrated that FUr-induced readthrough is less permissive for canonical stop codon readthrough compared to aminoglycoside G418. FUr is incorporated into mRNA and can potentially base-pair with guanine, allowing insertion of Arg tRNA at the TP53 R213X UGA premature stop codon and translation of full-length wild-type p53. We confirmed that full-length p53 rescued by FUr triggers tumor cell death by apoptosis. FUr also restored full-length p53 in TP53 R213X mutant human tumor xenografts in vivo. Thus, we demonstrate a novel strategy for therapeutic rescue of nonsense mutant TP53 and suggest that FUr should be explored for treatment of patients with TP53 nonsense mutant tumors.

Cancer continues to be a major global health challenge, accounting for 10 million deaths annually worldwide.Since the inception of genome-wide cancer sequencing studies 20 years ago, a core set of ~700 oncogenes and tumor suppressor genes has become the basis for cancer research.However, this research has been based largely on an understanding that the human genome encodes ~19 500 protein-coding genes.Complementing this genomic landscape, recent advances have described numerous microproteins which are now poised to redefine our understanding of oncogenic processes and open new avenues for therapeutic intervention.This review explores the emerging evidence for microprotein involvement in cancer mechanisms and discusses potential therapeutic applications, with an emphasis on highlighting recent advances in the field. Microproteins emerge as a new area of study in cell biologyCancer arises as a result of an intricate interplay of genetic, epigenetic, and environmental forces, and often lacks a single, canonical genetic driver [1,2].Despite significant advancements in our understanding of cancer biology, gaps in our knowledge remain, particularly in recognizing and characterizing non-traditional molecular players.Recently, the study of microproteins has emerged as a promising area of research in cancer biology.These proteins, typically defined as being <100 amino acids long, have historically been overlooked due to technical limitations and biases in genome annotation [3][4][5].However, a newfound appreciation for microproteins raises a pivotal question: are we missing key molecular players in cancer biology?Advances in next-generation sequencing techniques, such as ribosome profiling, as well as enhanced sensitivity of mass spectrometry (MS)-based proteomics, have revealed extensive translation of short open reading frames (sORFs) into microproteins across the human genome, challenging previous assumptions about our genome's protein-coding potential.These sORFs can originate from diverse genomic contexts, including regions previously thought to be noncoding, such as 5 and 3 untranslated regions of mRNAs or long non-coding RNAs (lncRNAs).Additionally, alternative ORFs within known protein-coding genes but translated in a different reading frame can give rise to novel microproteins [6][7][8].Recent large-scale efforts have implicated microproteins in a wide range of biological functions, including cell survival, DNA repair, and gene regulation [7,[9][10][11][12][13][14][15][16][17][18][19].As the dysregulation of these basic cellular functions underpins cancer cell biology, the identification of microproteins in these processes suggests that they could also play crucial roles in oncogenesis.Despite increasing awareness of microproteins' functional significance, the field of microprotein research still grapples with substantial challenges in determining the mechanisms by which these small proteins influence cancer development.Several key questions remain.(i) Are Highlights Microproteins, historically overlooked, are emerging as key players in cancer biology.Advances in genomics and proteomics enable systematic identification of cancer-associated microproteins.Microproteins contribute to cancer pathogenesis, are an important source of cryptic cancer antigens, and may offer novel therapeutic targets.Harnessing the potential of microproteins could revolutionize cancer diagnostics, therapeutics, and immunotherapy.

Abstract Acquisition of oncogenic mutations with age is believed to be rate limiting for carcinogenesis. However, the incidence of leukemia in children is higher than in young adults. Here we compare somatic mutations across pediatric acute myeloid leukemia (pAML) patient-matched leukemic blasts and hematopoietic stem and progenitor cells (HSPC), as well as HSPCs from age-matched healthy donors. HSPCs in the leukemic bone marrow have limited genetic relatedness and share few somatic mutations with the cell of origin of the malignant blasts, suggesting polyclonal hematopoiesis in patients with pAML. Compared with normal HSPCs, a subset of pAML cases harbored more somatic mutations and a distinct composition of mutational process signatures. We hypothesize that these cases might have arisen from a more committed progenitor. This subset had better outcomes than pAML cases with mutation burden comparable with age-matched healthy HSPCs. Our study provides insights into the etiology and patient stratification of pAML. Significance: Genome-wide analysis of pAML and patient-matched HSPCs provides new insights into the etiology of the disease and shows the clinical potential of these analyses for patient stratification. This article is highlighted in the In This Issue feature, p. 403