A major scientific drive is to characterize the protein-coding genome, which is a primary basis for studying human health. But the fundamental question remains of what has been missed in previous analyses. Over the past decade, the translation of non-canonical open reading frames (ncORFs) has been observed across human cell types and disease states1–3, with major implications for biomedical science. However, a key gap in knowledge has been which ncORFs produce small microproteins or alternative protein molecules that contribute to the human proteome. Here we report the collaborative efforts of the TransCODE Consortium4 to produce a consensus landscape of protein-level evidence for ncORFs. We show that about 25% of a set of 7,264 ncORFs gives rise to detectable peptides in a large-scale analysis of 95,520 proteomics experiments. We develop an annotation framework for ncORF-encoded microproteins as human proteins and codify the new conceptual model of ‘peptideins’ as microproteins that have indeterminate potential as functional proteins. To probe the biological implications of peptideins, we create an evolutionary analysis approach, termed ORF relative branch length (ORBL), and determine that evolutionary constraint is common and associates with observation of ncORF-derived peptides. We then characterize a pan-essential cellular phenotype for one peptidein from the OLMALINC long non-coding RNA. Overall, we generate public research tools supported by GENCODE and PeptideAtlas and advance biomedical discovery for understudied components of the human proteome. A large-scale proteomics analysis of the dark proteome by the TransCODE Consortium reveals many translated non-canonical open reading frames to encode microproteins and peptideins.

Cell Painting images offer valuable insights into a cell's state and enable many biological applications, but publicly available arrayed datasets only include hundreds of genes perturbed. The JUMP Cell Painting Consortium perturbed roughly 75% of the protein-coding genome in human U-2 OS cells, generating a rich resource of single-cell images and extracted features. These profiles capture the phenotypic impacts of perturbing 15,243 human genes, including overexpressing 12,609 genes (using open reading frames) and knocking out 7,975 genes (using CRISPR-Cas9). Here we mitigated technical artifacts by rigorously evaluating data processing options and validated the dataset's robustness and biological relevance. Analysis of phenotypic profiles revealed previously undiscovered gene clusters and functional relationships, including those associated with mitochondrial function, cancer and neural processes. The JUMP Cell Painting genetic dataset is a valuable resource for exploring gene relationships and uncovering previously unknown functions.

Pilocytic astrocytomas (PAs) are low-grade gliomas (LGGs) and predominantly affect children under 14 years of age. PAs are driven by aberrant activation of the RAS/RAF/MEK/ERK axis, primarily by BRAF alterations. As a key activator of this pathway, the Ser/Thr-kinase BRAF plays a crucial role in promoting cellular survival and proliferation and is one of the most frequently mutated kinases in cancer. While the most prevalent mutation, BRAFV600E, is commonly associated with hairy cell leukemia, melanoma, and thyroid carcinomas, the KIAA1549::BRAF fusion is the hallmark oncogene of LGGs, particularly PAs. Further, due to advancements in precision oncology diagnostics, this fusion is also increasingly identified in other tumor types. Various breakpoint variants of the KIAA1549::BRAF fusion gene were reported, with the most common involving KIAA1549 exons 1-16 and BRAF exons 9-18. The oncogenic potential of KIAA1549::BRAF is attributed to the loss of exons encoding the autoinhibitory domains of BRAF, although there is increasing evidence that the fusion partners of RAF oncoproteins can influence their functionality and signalling. Despite the high frequency of KIAA1549::BRAF fusions, the function, topology, and localization of KIAA1549, an enigmatic putative transmembrane protein, remains unclear. In contrast to the cytoplasmic localisation of wild-type BRAF, point mutants and insertion or deletion mutants, KIAA1549::BRAF fusions retain the single transmembrane domain of the KIAA1549 portion, localizing it to the plasma membrane. This localization requires trafficking through the ER/Golgi system during maturation. Additionally, using TAILS mass spectrometry and genomic approaches, we demonstrate that KIAA1549 and KIAA1549::BRAF fusions are cleaved at a specific cleavage motif in the extracellular portion of the protein. Mutation of the cleavage motif slows down cell growth compared to non-cleavage impaired fusions. Known protease inhibitors can target this cleavage event, offering a promising strategy for drug development. Apart from a very recently approved RAF inhibitor (RAFi), there are very little options available for directly targeting KIAA1549::BRAF fusion proteins. This is due to the fact that the RAFi developed for BRAFV600E are ineffective against BRAF fusions and probably also due to our limited understanding of the influence of KIAA1549. Consequently, our data could support the development of urgently needed targeted therapies for this BRAF fusion protein. Daniel Christen, Sean Misek, Anna Borgenvik, Gloria Kyrila, Alexander Zhang, Sarah Reel, Michelle Boisvert, Kelly Cai, Kevin Zhou, Elizabeth M Gonzales, Amy Goodale, Esteban Miglietta, Jacquelyn Jones, Seth Malinowski, Lobna Elsadek, Merve Ozdemir, Zachary Eisenbies, Joohee Lee, April Apfelbaum, Jenna Robinson, Antonio Maldera, Daniel Bondeson, Jason Kwon, Mounica Vallurupalli, Sangita Pal, Todd Golub, William Hahn, Eric Fischer, Jesse Boehm, Jörn Dengjel, Henrik Clausen, Nada Jabado, Till Milde, Beth Cimini, Keith Ligon, Kathrine Janeway, Michael Eck, David Root, David Jones, Timothy N Phoenix, Rameen Beroukhim, Hiren Jitendra Joshi, Tilman Brummer, Adnan Halim, Pratiti Bandopadhayay. Molecular insights into KIAA1549::BRAF fusion proteins: Implications for targeted therapy in pediatric low-grade gliomas [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Discovery and Innovation in Pediatric Cancer— From Biology to Breakthrough Therapies; 2025 Sep 25-28; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2025;85(18_Suppl_2):Abstract nr A032.

Inactivation of the VHL gene stabilizes HIF2α, which drives clear-cell renal cell carcinoma (ccRCC). The HIF2α inhibitor belzutifan is approved for ccRCC treatment, but de novo and acquired resistance are common. HIF2α, bound to ARNT, transcriptionally activates many genes. We performed CRISPR-mediated gene activation screens in HIF2α-dependent ccRCC lines treated with a belzutifan analog to identify HIF2α-responsive genes that confer cell-autonomous belzutifan resistance when not downregulated. Sustaining the expression of the HIF2α target gene CCND1, encoding cyclin D1, promoted HIF2α independence/belzutifan resistance. This activity requires CDK4/6 activation by cyclin D1 but is not solely due to phosphorylation of the canonical cyclin D1 target, pRB. Indeed, ccRCC lines lacking all three pRB family members remained at least partially HIF2α-dependent. In this context, however, a kinase-defective cyclin D1 variant partially overrode belzutifan's antiproliferative effects, suggesting that ccRCC promotion by cyclin D1 requires the phosphorylation of pRB paralogs and one or more kinase-independent cyclin D1 activities. SIGNIFICANCE:We discovered that cyclin D1 is the key target of HIF2 driving the cell-autonomous proliferation of VHL-mutant kidney cancers and that cyclin D1 has targets beyond pRB in this setting. These findings have implications for treating kidney cancer with HIF2 inhibitors, alone or in combination with CDK4/6 inhibitors.

Supplementary Table S1 STARS Scores

Supplementary Table S3 Gene Sets

Supplementary Table S2 RNAseq

Fusions between protein-coding genes are common oncogenic drivers across cancers, typically pairing a proto-oncogene with partner that does not independently drive cancer. In all therapeutically actionable fusions, the proto-oncogene is the drug target, the contributions to oncogenicity of the fusion partner have largely been ignored. We studied the role of BRAF fusion partners and found that they are necessary for transformation. In the setting of KIAA1549::BRAF, the most common fusion protein across brain tumors, we found that KIAA1549 is necessary for the oncogenicity of KIAA1549::BRAF and engenders a striking and specific dependency on the protein O-mannosyltransferase complex (POMT1/2). Specifically, we show that genetic silencing or pharmacologic inhibition of the protein O-mannosyltransferase complex (POMT1/2) reverses fusion-induced transformation, thereby representing a novel and MAPK independent therapeutic target. Furthermore, POMT1/2 is required to glycosylate and enable maturation of the K::B fusion protein. These findings represent a proof-of-concept for targeting the partners in oncogenic fusions as a potential cancer therapeutic strategy. ### Competing Interest Statement PB has received grant funding from Novartis Institute of Bio-medical Research, and has served on paid advisory boards for QED Therapeutics, and Day One Biopharmaceuticals. RB consults for and owns equity in Scorpion Therapeutics, Kar-yoverse Therapeutics, and LOH Therapeutics. WCH is a consultant for Thermo Fischer, Solasta Ventures, KSQ Ther-apeutics, Frontier Medicines, Jubilant Therapeutics, RAPPTA Therapeutics, Serinus Biosciences, Kestral Therapeutics, Crane Biotherapeutics, Function Oncology, Recursion Pharma, and Weaver Biotherapetics and Perceptive. DER receives research funding from members of the Functional Genomics Consortium (Abbvie, BMS, Jannsen, Merck, Vir), and is a director of Addgene, Inc. TM is advisory board member for Ipsen Pharma GmbH. SAM is a consultant for Karyoverse Therapeutics and is a consultant for and owns equity in LOH Therapeutics. ESF is a founder, scientific advisory board (SAB) member, and equity holder of Civetta Therapeutics, Proximity Therapeutics, Neomorph, Inc. (also board of direc-tors), StelexisBiosciences, Inc., Anvia Therapeutics, Inc. (also board of directors) and CPD4, Inc. (also board of directors). He is an equity holder and SAB member for Avilar Therapeu-tics, PhotysTherapeutics, and Ajax Therapeutics and an equity holder in Lighthorse Therapeutics. E.S.F. is a consultant to Novartis, EcoR1 capital, Odyssey and Deerfield. The Fischer lab receives or has received research funding from Deerfield, Novartis, Ajax, Interline, Bayer and Astellas. TB received speaker honoraria from Pierre Fabre and the European Society for Medical Oncology (ESMO)

Supplemental Figures S1-S6 and Legends

Fusions between protein-coding genes are common oncogenic drivers across cancers, typically pairing a proto-oncogene with a partner that does not independently drive cancer. In all therapeutically actionable fusions, the proto-oncogene is the drug target; the contributions to oncogenicity of the fusion partner have largely been ignored. KIAA1549::BRAF (K::B) is the most common fusion protein across brain tumors, and particularly in pediatric gliomas. We found that KIAA1549 is necessary for oncogenicity of K::B, in contrast to the notion that the BRAF kinase domain alone is enough for transformation. We therefore performed genome-wide CRISPR/Cas9 anti-transformation screens in isogenic neural stem cell models with K::B overexpression to identify genetic dependencies specific to this oncogenic driver. We found that the BRAF fusion partner KIAA1549 engenders a striking and specific dependency on the protein O-mannosyltransferase complex (POMT1/2). Specifically, we show that genetic silencing or pharmacologic inhibition of the POMT1/2 complex reverses K::B transformation, thereby representing a novel therapeutic target beyond the MAPK pathway. Furthermore, POMT1/2 is required to glycosylate and enable maturation of K::B, reflecting the native biology of KIAA1549 but representing a non-canonical process for BRAF. These findings represent a proof-of-concept for targeting the partners in oncogenic fusions as a potential cancer therapeutic strategy. Anna Borgenvik, Sean Misek, Daniel Christen, Gloria Kyrila, Alexander Zhang, Sarah Reel, Michelle Boisvert, Kelly Cai, Kevin Zhou, Elizabeth M Gonzales, Lorena Lazo de la Vega, Amy Goodale, Esteban Miglietta, Jacquelyn Jones, Seth Malinowski, Lobna Elsadek, Merve Ozdemir, Zachary Eisenbies, Joohee Lee, April Apfelbaum, Jenna Robinson, Antonio Maldera, Daniel Bondeson, Jason Kwon, Mounica Vallurupalli, Sangita Pal, Todd Golub, William Hahn, Eric Fischer, Jesse Boehm, Jörn Dengjel, Henrik Clausen, Nada Jabado, Till Milde, Beth A Cimini, Keith Ligon, Kathrine Janeway, Michael Eck, David Root, David Jones, Timothy N Phoenix, Rameen Beroukhim, Hiren Jitendra Joshi, Tilman Brummer, Adnan Halim, Pratiti Bandopadhayay. O-mannosylation represents a therapeutic opportunity in BRAF fusion protein oncogenesis [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Discovery and Innovation in Pediatric Cancer— From Biology to Breakthrough Therapies; 2025 Sep 25-28; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2025;85(18_Suppl_2):Abstract nr B019.

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.

The diversity of CRISPR systems, coupled with scientific ingenuity, has led to an explosion of applications; however, to test newly-described innovations in their model systems, researchers typically embark on cumbersome, one-off cloning projects to generate custom reagents that are optimized for their biological questions. Here, we leverage Golden Gate cloning to create the Fragmid toolkit, a modular set of CRISPR cassettes and delivery technologies, along with a web portal, resulting in a combinatorial platform that enables scalable vector assembly within days. We further demonstrate that multiple CRISPR technologies can be assessed in parallel in a pooled screening format using this resource, enabling the rapid optimization of both novel technologies and cellular models. These results establish Fragmid as a robust system for the rapid design of CRISPR vectors, and we anticipate that this assembly approach will be broadly useful for systematic development, comparison, and dissemination of CRISPR technologies.

Abstract The epidermal growth factor receptor, EGFR, is frequently activated in lung cancer and glioblastoma by genomic alterations including missense mutations. The different mutation spectra in these diseases are reflected in divergent responses to EGFR inhibition: significant patient benefit in lung cancer, but limited in glioblastoma. Here, we report a comprehensive mutational analysis of EGFR function. We performed saturation mutagenesis of EGFR and assessed function of ~22,500 variants in a human EGFR-dependent lung cancer cell line. This approach revealed enrichment of erlotinib-insensitive variants of known and unknown significance in the dimerization, transmembrane, and kinase domains. Multiple EGFR extracellular domain variants, lacking approved targeted therapies, were sensitive to dacomitinib. Strikingly, at least two glioblastoma patients with G598V dimerization domain mutations showed responses to dacomitinib treatment together with subsequent off-target resistance in one case. In summary, this comprehensive screen reveals novel functional EGFR variants and suggests broader clinical investigation of EGFR inhibition for cancers harboring extracellular domain mutations. Citation Format: Tikvah Hayes, Elisa Aquilanti, Erica Kim, Nicole Persky, Lisa Brenan, Amy Goodale, Ted Sharpe, Doug Alan, Robert Shue, Lior Golomb, Brianna Silverman, Yvonne Li, Lindsay Westlake, Federica Piccioni, Andrew Cherniack, Xiaoping Yang Yang, David Root, J. Kevin Hicks, Andrew Chi, Jorg Dietrich, Daniel Cahill, Cory Johannessen, Tracy Batchelor, Matthew Meyerson. Deep mutational scanning of EGFR reveals potential domain-specific TKI sensitivities in lung cancer and glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1220.

The identification of genetic and chemical perturbations with similar impacts on cell morphology can elucidate compounds' mechanisms of action or novel regulators of genetic pathways. Research on methods for identifying such similarities has lagged due to a lack of carefully designed and well-annotated image sets of cells treated with chemical and genetic perturbations. Here we create such a Resource dataset, CPJUMP1, in which each perturbed gene's product is a known target of at least two chemical compounds in the dataset. We systematically explore the directionality of correlations among perturbations that target the same protein encoded by a given gene, and we find that identifying matches between chemical and genetic perturbations is a challenging task. Our dataset and baseline analyses provide a benchmark for evaluating methods that measure perturbation similarities and impact, and more generally, learn effective representations of cellular state from microscopy images. Such advancements would accelerate the applications of image-based profiling of cellular states, such as uncovering drug mode of action or probing functional genomics. The CPJUMP1 Resource comprises Cell Painting images and profiles of 75 million cells treated with hundreds of chemical and genetic perturbations. The dataset enables exploration of their relationships and lays the foundation for the development of advanced methods to match perturbations.

The epidermal growth factor receptor, EGFR, is frequently activated in lung cancer and glioblastoma by genomic alterations including missense mutations. The different mutation spectra in these diseases are reflected in divergent responses to EGFR inhibition: significant patient benefit in lung cancer, but limited in glioblastoma. Here, we report a comprehensive mutational analysis of EGFR function. We perform saturation mutagenesis of EGFR and assess function of ~22,500 variants in a human EGFR-dependent lung cancer cell line. This approach reveals enrichment of erlotinib-insensitive variants of known and unknown significance in the dimerization, transmembrane, and kinase domains. Multiple EGFR extracellular domain variants, not associated with approved targeted therapies, are sensitive to afatinib and dacomitinib in vitro. Two glioblastoma patients with somatic EGFR G598V dimerization domain mutations show responses to dacomitinib treatment followed by within-pathway resistance mutation in one case. In summary, this comprehensive screen expands the landscape of functional EGFR variants and suggests broader clinical investigation of EGFR inhibition for cancers harboring extracellular domain mutations.

Abstract Since the KIAA1549::BRAF fusion was discovered as the most common driver of pediatric low-grade glioma (pLGG), it has been hypothesized that the fusion induces oncogenicity through BRAF activation as a result of KIAA1549 replacing the BRAF negative regulatory N-terminus. This led to the rapid translation of MAPK pathway inhibitors into the clinical setting. Despite most tumors exhibiting promising initial responses, some tumors are not sensitive and about half of responsive tumors grow back after treatment cessation. Therefore, strategies that result in sustained tumor responses are desperately needed. We have recently performed genome-scale CRISPR/Cas9 screens across isogenic neural stem cell models transduced to express pLGG-associated oncogenes (KIAA1549::BRAF, BRAFV600E, and multiple FGFR1 and MYB family alterations) to generate a dependency map of genetic vulnerabilities associated with expression of these oncogenes. We also included normal neural stem cells to allow identification of genetic dependencies specifically induced by the expression of each oncogene. Surprisingly, through these efforts, we have discovered KIAA1549::BRAF expressing cells to harbor striking and specific dependency on multiple members of an enzymatic complex that exerts its activity outside of the MAPK signaling axis*. Interestingly, this enzymatic complex has been described to modify only a few substrates, including KIAA1549, suggesting specificity. We have now validated this dependency across other isogenic models of KIAA1549::BRAF-expressing cells. Finally, our KIAA1549::BRAF-expressing models exhibit preferential sensitivity to a tool compound that targets our novel enzyme, suggesting novel therapeutic potential for KIAA1549::BRAF. These findings highlight a MAPK pathway-independent avenue for therapeutically targeting the most frequent genomic alterations in pediatric brain tumors. Consequently, we also propose that the fusion partner KIAA1549 is instrumental for the aberrant BRAF signaling driving pLGGs. (* we are currently in the process of working with our IP offices to ensure that we can disclose the names of the genes and proteins at the ISPNO meeting)