The most common fusion oncoprotein in the most common pediatric solid tumor, pediatric low-grade glioma (pLGG), is KIAA1549::BRAF. Although MAPK inhibitors show early promise, they often fail to achieve durable cures, highlighting the need to develop new therapeutics for KIAA1549::BRAF-driven tumors. In this study we leverage genome-scale anti-transformation CRISPR screens to uncover KIAA1549::BRAF-specific genetic vulnerabilities. Across all genes, POMT1 and POMT2, the two members of the heterodimeric Protein O-mannosyltransferase (POMT) complex emerge as the strongest KIAA1549::BRAF-specific dependencies. We demonstrate that KIAA1549 is a direct substrate of this complex, and that glycosylation of KIAA1549::BRAF by POMT is necessary for its maturation through the secretory pathway and subsequent oncogenic signaling. These data highlight for the first time a new route to directly target KIAA1549::BRAF independent of the MAPK pathway. This work also uncovered an unexpected and previously unrecognized role for KIAA1549 in driving KIAA1549::BRAF oncogenic signaling. The prevailing dogma has been that BRAF fusions exert their transforming activity through truncation and removal of N-terminal regulatory domains, thereby rendering the BRAF kinase domain constitutively active. We demonstrate that BRAF fusion partners, including KIAA1549, contain domains that facilitate membrane localization, thereby enhancing BRAF signaling. These findings challenge the traditional view of BRAF fusion biology and establish a role for fusion partners in shaping oncogenic potential. Sean Misek, Anna Borgenvik, Daniel Christen, Gloria Kyrila, Alexander Zhang, Sarah Reel, Michelle Boisvert, Kelly Cai, Kevin Zhou, Elizabeth Gonzales, Lorena Lazo de la Vega, Timothy Ragnoni, Nicole Persky, Tanaz Abid, Esteban Miglietta, Sergey Vakhrushev, Michael Stumpe, Jacquelyn Jones, Seth Malinowski, Lobna Elsadek, Aaron Fultineer, Kira Tang, Antonio Maldera, Hyesung Jeon, Sangita Pal, Todd Golub, William Hahn, Eric Fischer, Jesse Boehm, Jörn Dengjel, Henrik Clausen, Nada Jabado, Till Milde, Anne Carpenter, Beth Cimini, Keith Ligon, Katherine Janeway, Michael Eck, David Root, David Jones, Timothy Phoenix, Rameen Beroukhim, Hiren Jitendra Joshi, Tilman Tilman, Adnan Halim, Pratiti Bandopadhayay. O-mannosylation and protein maturation checkpoints represent therapeutic opportunities in BRAF fusion protein oncogenesis [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Fusion-Positive Cancer: From Discovery to Therapy; 2026 Jan 13-15; Philadelphia PA. Philadelphia (PA): AACR; Cancer Res 2026;86(1_Suppl):Abstract nr B026.

Abstract Inter-individual variability in drug response is a key challenge in precision oncology. Despite recent breakthroughs in targeting RAS-mutant cancers, clinical responses remain heterogeneous. To systematically dissect the molecular determinants of drug sensitivity and resistance, we implemented a high-throughput single-cell multi-omic profiling framework using a PRISM pool: a barcoded co-culture platform of ∼400 human cancer cell lines spanning diverse lineages and genetic backgrounds. We treated the PRISM pool with two RAS inhibitors (BI-2865 and RMC-6236), as well as a negative control (DMSO) and a positive control (Panobinostat), and collected cells at early time points (3h and 12h) to capture initial drug response dynamics. We employed a modified 10x Genomics Flex protocol enabling simultaneous capture of the whole transcriptome, a targeted proteome (∼320-plex Proteintech panel), and PRISM identity via expressed DNA barcodes. The prototype Sequencing by Expansion (SBX) platform was leveraged to generate ∼230 billion reads, enabling the analysis of 315,547 high-quality single cells. Drug treatments induce diverse and cell line-specific shifts in transcriptional and proteomic states. Applying Non-negative Matrix Factorization (NMF) on each cell line’s single-cell transcriptome data and then performing hierarchical clustering, we identified 20 drug-responsive, recurrently co-expressed gene meta-programs (MPs), condensing 1831 underlying programs related to cell cycle, growth, structure, and stress response. Multiple MP dynamics are associated with drug sensitivity and often stratified by genetic background. Notably, cell lines that exhibited a delayed stress-response MP (non-responsive at 3 hours but catching up at 12 hours) exhibited high drug sensitivity. To identify key protein players in drug response, we conducted differential expression (DE) analysis for each cell line comparing drug treatments and controls, identifying recurrently significant DE proteins whose altered expression was also significantly associated with cell viability. Importantly, these protein changes frequently lacked a corresponding significant change in the expression of their encoding RNA transcripts, underscoring the power of multi-modal profiling to more comprehensively illuminate functional mechanisms driving drug response. Our study establishes a scalable paradigm for linking genotype, transcriptome, and proteome to pharmacologic phenotype at single-cell resolution across genetically diverse human models. These data, enabled with massively high-throughput sequencing using SBX, provide a rich resource for mechanistic discovery and rational design of combination therapies targeting the RAS pathway. Citation Format: Houlin Yu, Guoping Wang, Stephanie Yaung, Heejo Choi, Megan Rogers-Peckham, Michael Kartje, Matthew Rees, Paul Lund, Brian Haas, Charlotte Yang, Laura Doherty, Lacey McGee, Kendall Berg, Cynthia Cech, Salka Barrett, Anasha Arryman, Joanne Leadbetter, Taylor Lehmann, John Mannion, Chen Zhao, Marc Prindle, Melud Nabavi, Carolyn Morrison, Peter Smibert, Kit Nazor, Todd Golub, Catarina D. Campbell, Jennifer Roth, Niall Lennon, Victoria Popic, Dean Procter, Kokoris Mark, Aziz Al'Khafaji. Single-cell multiomic drug response profiling of PRISM-multiplexed cancer cell lines sequenced with SBX [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 495.

A central challenge in cancer research is to identify the secreted factors that sustain tumor cell survival. This is best exemplified in Hodgkin lymphoma, where malignant cells constitute a minor fraction of the tumor and rely on signals from the microenvironment for survival. Using genome-wide transcriptional profiling with spatial and single-cell resolution, we show that the neighborhood around malignant cells forms a distinct niche of 31 non-malignant cell types, enriched in helper T cells and myeloid cells, but depleted of plasma cells. Moreover, our spatial analysis nominates IL13 as a candidate survival factor. Recombinant IL13 augments malignant cell growth in vitro, and genome-wide loss-of-function screens across >1000 human cancer cell lines identify IL4R and IL13RA1, heterodimeric components of the IL13 receptor, as uniquely essential in Hodgkin lymphoma. Importantly, blocking antibodies phenocopy genetic inactivation. Our findings provide a biological rationale for testing IL13-directed therapies, which are already FDA-approved, in Hodgkin lymphoma. Hodgkin Reed Sternberg (HRS) cells and their surrounding microenvironment in Hodgkin lymphoma remain poorly characterized. Here, the authors perform genome-wide transcriptional profiling with spatial and single-cell resolution to explore the cellular and molecular composition of the Hodgkin lymphoma microenvironment and used machine learning to identify IL13 as a potential HRS cell survival factor.

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.

The DNA-incorporating nucleoside analogs azacytidine (AZA) and decitabine (DEC) have clinical efficacy in blood cancers, yet the precise mechanism by which these agents kill cancer cells has remained unresolved - specifically, whether their anti-tumor activity arises from conventional DNA damage or DNA hypomethylation via DNA methyltransferase 1 (DNMT1) inhibition. This incomplete mechanistic understanding has limited their broader therapeutic application, particularly in solid tumors, where early clinical trials showed limited efficacy. Here, through the assessment of drug sensitivity in over 600 human cancer models and comparison to a non-DNA-damaging DNMT1 inhibitor (GSK-3685032), we establish DNA hypomethylation, rather than DNA damage, as the primary killing mechanism of AZA and DEC across diverse cancer types. In further support of an epigenetic killing mechanism, CRISPR drug modifier screens identified a core set of chromatin regulators, most notably the histone deubiquitinase USP48, as AZA and DEC protective factors. We show that USP48 is recruited to newly hypomethylated CpG islands and deubiquitinates non-canonical histones, establishing USP48 as a key molecular link between the two components of epigenetic gene regulation: DNA methylation and chromatin modification. Furthermore, loss of USP48, which occurs naturally through biallelic deletions in human cancers, sensitized both hematologic and solid tumors to DNMT1 inhibition in vitro and in vivo. Our findings elucidate the epigenetic mechanism of action of AZA and DEC and identify a homeostatic link between DNA methylation and chromatin state, revealing new therapeutic opportunities for DNMT1 inhibitors in solid tumors.

Cancer genome alterations often lead to vulnerabilities that can be used to selectively target cancer cells. Various inhibitors of such synthetic lethal targets have been approved by the FDA or are in clinical trials, highlighting the potential of this approach1-3. Here we analysed large-scale CRISPR knockout screening data from the Cancer Dependency Map and identified a new synthetic lethal target, PELO, for two independent molecular subtypes of cancer: biallelic deletion of chromosomal region 9p21.3 or microsatellite instability-high (MSI-H). In 9p21.3-deleted cancers, PELO dependency emerges from biallelic deletion of the 9p21.3 gene FOCAD, a stabilizer of the superkiller complex (SKIc). In MSI-H cancers, PELO is required owing to MSI-H-associated mutations in TTC37 (also known as SKIC3), a critical component of the SKIc. We show that both cancer subtypes converge to destabilize the SKIc, which extracts mRNA from stalled ribosomes. In SKIc-deficient cells, PELO depletion induces the unfolded protein response, a stress response to accumulation of misfolded or unfolded nascent polypeptides. Together, our findings indicate PELO as a promising therapeutic target for a large patient population with cancers characterized as MSI-H with deleterious TTC37 mutations or with biallelic 9p21.3 deletions involving FOCAD.

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)

Programmable control of gene expression in specific cell types is essential for both basic discovery and therapeutic intervention, yet current strategies lack scalability across diverse cellular contexts. Here, we introduce SPICE (Splicing Proportions In Cell types), an integrated experimental and computational framework that harnesses alternative RNA splicing as a programmable modality for cell type-specific gene regulation. To power SPICE, we constructed a massively parallel reporter assay (MPRA) comprising 46,372 human-derived sequences and profiled exon skipping across 43 cell lines spanning 10 lineages, uncovering widespread cell type-specific exon skipping. Using this data, we trained deep learning models that both predict splicing in unseen contexts and generate synthetic sequences with programmed, cell type-specific splicing patterns. Leveraging these models, we further engineered sequences that selectively splice in cells harboring oncogenic splicing factor mutations, demonstrating translational potential. SPICE provides a generalizable strategy for dissecting splicing regulation and engineering alternative splicing as a gene expression regulatory layer for research and therapeutic applications.

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.

Macrophages in the tumor microenvironment exert potent anti-tumorigenic activity through phagocytosis. Yet therapeutics that enhance macrophage phagocytosis have not improved outcomes in clinical trials for patients with acute myeloid leukemia (AML) or myelodysplastic syndrome (MDS). To systematically identify regulators of phagocytosis, we performed genome-scale CRISPR knockout screens in human leukemia cells co-cultured with human monocyte-derived macrophages. Surprisingly, we found that whereas the classic "don't eat me" signal CD47 inhibited mouse macrophages, it did not inhibit phagocytosis by human macrophages. In contrast, the O-linked glycosylation and sialylation pathways were strong negative regulators of phagocytosis. In AML, the cell surface O-linked glycoprotein CD43 was the major effector of the O-linked glycosylation and sialylation pathways. Genetic deletion or antibody blockade of CD43 enhanced macrophage phagocytosis. This work highlights the importance of using human platforms to identify immune checkpoints, and nominates CD43 as a glyco-immune regulator of human macrophage phagocytosis.

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.

Abstract BACKGROUND: Large-scale genomic studies such as The Cancer Genome Atlas (TCGA) and Pan-Cancer Analysis of Whole Genomes (PCAWG) show that the breast cancer (BrCa) genome is dominated by structural variation (SV) rather than single base pair mutations, producing a fertile environment for gene fusions. In this study, we implement a rigorous, expression-based approach to create a comprehensive landscape of fusion RNAs in metastatic breast cancer (MBC). We find fusion RNAs—many of which are novel involving known oncogenes—are surprisingly common in the advanced setting and credential their use as base-editing therapeutic targets. METHODS: Two retrospective cohorts of MBC RNA-sequencing data were analyzed— Dana-Farber Cancer Institute CCPM (n = 252 cases, 276 specimens), MichiganCSER (n = 171 cases, 190 specimens)—with a Fusion MetaCaller that integrates 5 unsupervised fusion-finding algorithms. Fusion RNAs identified in at least 2 callers (High-Confidence) and absent in RNA-seq from normal tissue (Cancer-Specific) were classified as HCCS-Fusions. Further removal of common artifactual fusions was performed using public databases. Expression of each HCCS-Fusion was quantified using a supervised method (FusionInspector)—expressed HCCS-Fusions were defined as having a Fusion Fragment Per Million (FFPM) value > 0.1 and at least 10% of read counts mapping to the fusion breakpoint versus the flanking 5’ or 3’ partners’ exons. Normalized gene-level expression abundances were calculated to correlate transcriptomic features (gene expression, PAM50) with fusion RNAs. Recurrent, potentially pathogenic fusion RNAs were annotated using OncoKB and outlier expressed fusions (Q3 FFPM + [1.5 X IQR]) were interrogated. RESULTS: The frequency of HCCS-Fusions differed between subtypes with basal BrCa harboring the most per tumor followed by Her2, LumB and LumA—with a median HCCS-Fusion count of 13, 12, 7, 4 respectively. 64.5% of cases harbored a HCCS-Fusion in an OncoKB cancer-related gene. The most recurrent fusions involving a cancer-related gene were 5’ ESR1 fusions (14 cases)—all with in-frame breakpoints near exon6/7, disrupting ESR1’s ligand binding domain. 13 of 14 ESR1 fusions were called in Luminal B (LumB) metastases (Fisher’s exact enrichment p < 0.005 vs other subtypes)—defining an ESR1 fusion frequency of 6.5% in LumB disease. Beyond ESR1, we identify recurrent, low-frequency (2-4 cases) in-frame kinase fusions involving FGFR2, ADK, TLK2, PRKCA, BRAF, CHKA, CSNK1D, NEK11, TNIK—some potentially targetable with FDA-approved small molecule inhibitors—as well as recurrent, predicted loss-of-function fusion RNAs in NF1, MSI2, USP32, PTEN, and CDH1. Lastly, 33.6% of cases harbored at least one outlier expressed fusion RNA; including highly expressed in-frame fusions involving known BrCa mediators such as ERBB2, BRCA1, ARID1B, RPS6KB1/2, PIK3R3, AXIN1, TGFB1/2, FOXP1, PAK1, and CREBBP. CONCLUSIONS: Taken together, these results demonstrate that fusion RNAs in MBC—some recurrent, many highly expressed and unique to individual tumors—are common. We create the most comprehensive catalog of ESR1 fusions in MBC, better define their frequency, discover their enrichment in LumB-like tumors, and will discuss clinicopathologic and transcriptomic features associated with ESR1 fusion positive disease. We identify druggable fusions that would likely be missed by current testing standards, find recurrent loss-of-function fusion RNAs, and show that over one-third of metastatic cases harbor at least one outlier expressed fusion—many of which involve BrCa-related genes. In summary, we propose that fusion RNAs are a driving and perhaps overlooked mechanism of tumor evolution in therapy-resistant disease and postulate fusion transcripts present a compelling therapeutic opportunity in MBC. Preliminary data targeting fusion RNA breakpoints using a novel RNA base-editing approach will be discussed. Citation Format: Nolan Priedigkeit, Alinés Lebrón-Torres, Janny Liao, Jean-Baptiste Alberge, Stefania Morganti, Jakob Weiss, Jorge Gomez Tejeda Zanudo, Albert Grinshpun, Melissa Hughes, Karla Helvie, Kerry Sendrick, Kyleen Nguyen, Sarah Strauss, Janet Files, Maxwell Lloyd, Nikhil Wagle, Chip Stewart, Eric Winer, Bruce Johnson, Yvonne Li, Rinath Jeselsohn, Sara Tolaney, Daniel Abravanel, Nancy Lin, Heather Parsons, Gad Getz, Steffi Oesterreich, Adrian Lee, Todd Golub. Characterization and proposed therapeutic exploitation of fusion RNAs in metastatic breast cancers [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr GS03-09.

The MCL1 gene is frequently amplified in cancer and codes for the antiapoptotic protein myeloid cell leukemia 1 (MCL1), which confers resistance to the current standard of care. Therefore, MCL1 is an attractive anticancer target. Here we describe BRD-810 as a potent and selective MCL1 inhibitor and its key design principle of rapid systemic clearance to potentially minimize area under the curve-driven toxicities associated with MCL1 inhibition. BRD-810 induced rapid cell killing within 4 h in vitro but, in the same 4-h window, had no impact on cell viability or troponin I release in human induced pluripotent stem cell-derived cardiomyocytes, even at suprapharmacologic concentrations. In vivo BRD-810 induced efficacy in xenograft hematological and solid tumor models despite the short residence time of BRD-810 in plasma. In totality, our data support the hypothesis that short-term inhibition of MCL1 with BRD-810 can induce apoptosis in tumor cells while maintaining an acceptable safety profile. We, therefore, intend to advance BRD-810 to clinical trials. Rauh et al. developed a selective MCL1 inhibitor that is efficacious in hematological and solid tumors and has the advantage of limited cardiotoxicity because of more rapid clearance of the drug in vivo.

Copper is an essential co-factor for all organisms, and yet it becomes toxic if concentrations exceed a threshold maintained by evolutionarily conserved homeostatic mechanisms. The precise mechanisms through which excess copper induces cell death, are still poorly characterized. Our recent discoveries illuminated that copper ionophores targeted to the mitochondria induce a new form of regulated cell death, distinct from other forms of regulated cell death (i.e apoptosis, ferroptosis and necroptosis) which is metabolism dependent. We term this new mechanism of cell death "cuproptosis." Chemical genomic approaches and thorough biochemical validation studies established that cuproptosis is regulated by mitochondrial ferredoxin 1 (FDX1) and protein lipoylation, a conserved lysine post-translational modification regulating only 4 key mitochondrial enzymes. Here we describe our current understanding of how copper targets both lipoylated and Fe-S cluster proteins, the regulation of this processes by FDX1 and the unique cellular and biochemical characteristics of cuproptosis. PT is sponsored by NIH Research Project Grant, NCI R011R01CA279550-01 and Office of Naval Research (ONR), N00014-23-1-2465.

Liquid biopsies enable early detection and monitoring of diseases such as cancer, but their sensitivity remains limited by the scarcity of analytes such as cell-free DNA (cfDNA) in blood. Improvements to sensitivity have primarily relied on enhancing sequencing technology ex vivo. We sought to transiently augment the level of circulating tumor DNA (ctDNA) in a blood draw by attenuating its clearance in vivo. We report two intravenous priming agents given 1 to 2 hours before a blood draw to recover more ctDNA. Our priming agents consist of nanoparticles that act on the cells responsible for cfDNA clearance and DNA-binding antibodies that protect cfDNA. In tumor-bearing mice, they greatly increase the recovery of ctDNA and improve the sensitivity for detecting small tumors.