A genetic basis for sex differences in Xp11 translocation renal cell carcinoma.

Xp11 translocation renal cell carcinoma (tRCC) is a rare, female-predominant cancer driven by a fusion between the transcription factor binding to IGHM enhancer 3 (TFE3) gene on chromosome Xp11.2 and a partner gene on either chromosome X (chrX) or an autosome. It remains unknown what types of rearrangements underlie TFE3 fusions, whether fusions can arise from both the active (chrXa) and inactive X (chrXi) chromosomes, and whether TFE3 fusions from chrXi translocations account for the female predominance of tRCC. To address these questions, we performed haplotype-specific analyses of chrX rearrangements in tRCC whole genomes. We show that TFE3 fusions universally arise as reciprocal translocations and that oncogenic TFE3 fusions can arise from chrXi:autosomal translocations. Female-specific chrXi:autosomal translocations result in a 2:1 female-to-male ratio of TFE3 fusions involving autosomal partner genes and account for the female predominance of tRCC. Our results highlight how X chromosome genetics constrains somatic chrX alterations and underlies cancer sex differences.

Kinase-mediated RAS signaling via membraneless cytoplasmic protein granules.

Receptor tyrosine kinase (RTK)-mediated activation of downstream effector pathways such as the RAS GTPase/MAP kinase (MAPK) signaling cascade is thought to occur exclusively from lipid membrane compartments in mammalian cells. Here, we uncover a membraneless, protein granule-based subcellular structure that can organize RTK/RAS/MAPK signaling in cancer. Chimeric (fusion) oncoproteins involving certain RTKs including ALK and RET undergo de novo higher-order assembly into membraneless cytoplasmic protein granules that actively signal. These pathogenic biomolecular condensates locally concentrate the RAS activating complex GRB2/SOS1 and activate RAS in a lipid membrane-independent manner. RTK protein granule formation is critical for oncogenic RAS/MAPK signaling output in these cells. We identify a set of protein granule components and establish structural rules that define the formation of membraneless protein granules by RTK oncoproteins. Our findings reveal membraneless, higher-order cytoplasmic protein assembly as a distinct subcellular platform for organizing oncogenic RTK and RAS signaling.

Proteogenomic Characterization Reveals Therapeutic Vulnerabilities in Lung Adenocarcinoma.

To explore the biology of lung adenocarcinoma (LUAD) and identify new therapeutic opportunities, we performed comprehensive proteogenomic characterization of 110 tumors and 101 matched normal adjacent tissues (NATs) incorporating genomics, epigenomics, deep-scale proteomics, phosphoproteomics, and acetylproteomics. Multi-omics clustering revealed four subgroups defined by key driver mutations, country, and gender. Proteomic and phosphoproteomic data illuminated biology downstream of copy number aberrations, somatic mutations, and fusions and identified therapeutic vulnerabilities associated with driver events involving KRAS, EGFR, and ALK. Immune subtyping revealed a complex landscape, reinforced the association of STK11 with immune-cold behavior, and underscored a potential immunosuppressive role of neutrophil degranulation. Smoking-associated LUADs showed correlation with other environmental exposure signatures and a field effect in NATs. Matched NATs allowed identification of differentially expressed proteins with potential diagnostic and therapeutic utility. This proteogenomics dataset represents a unique public resource for researchers and clinicians seeking to better understand and treat lung adenocarcinomas.

Tracing Oncogene Rearrangements in the Mutational History of Lung Adenocarcinoma.

Mutational processes giving rise to lung adenocarcinomas (LADCs) in non-smokers remain elusive. We analyzed 138 LADC whole genomes, including 83 cases with minimal contribution of smoking-associated mutational signature. Genomic rearrangements were not correlated with smoking-associated mutations and frequently served as driver events of smoking-signature-low LADCs. Complex genomic rearrangements, including chromothripsis and chromoplexy, generated 74% of known fusion oncogenes, including EML4-ALK, CD74-ROS1, and KIF5B-RET. Unlike other collateral rearrangements, these fusion-oncogene-associated rearrangements were frequently copy-number-balanced, representing a genomic signature of early oncogenesis. Analysis of mutation timing revealed that fusions and point mutations of canonical oncogenes were often acquired in the early decades of life. During a long latency, cancer-related genes were disrupted or amplified by complex rearrangements. The genomic landscape was different between subgroups-EGFR-mutant LADCs had frequent whole-genome duplications with p53 mutations, whereas fusion-oncogene-driven LADCs had frequent SETD2 mutations. Our study highlights LADC oncogenesis driven by endogenous mutational processes.

A Tumor-Agnostic NTRK (TRK) Inhibitor.

Larotrectinib is a small-molecule kinase inhibitor that targets NTRK fusions that occur in multiple types of cancer. Its FDA approval represents the first instance of a treatment indication being designated "tumor-agnostic" from the outset, being based on actionable genomic insights. To view this Bench to Bedside, open or download the PDF.

CBFß-SMMHC Inhibition Triggers Apoptosis by Disrupting MYC Chromatin Dynamics in Acute Myeloid Leukemia.

The fusion oncoprotein CBFß-SMMHC, expressed in leukemia cases with chromosome 16 inversion, drives leukemia development and maintenance by altering the activity of the transcription factor RUNX1. Here, we demonstrate that CBFß-SMMHC maintains cell viability by neutralizing RUNX1-mediated repression of MYC expression. Upon pharmacologic inhibition of the CBFß-SMMHC/RUNX1 interaction, RUNX1 shows increased binding at three MYC distal enhancers, where it represses MYC expression by mediating the replacement of the SWI/SNF complex component BRG1 with the polycomb-repressive complex component RING1B, leading to apoptosis. Combining the CBFß-SMMHC inhibitor with the BET inhibitor JQ1 eliminates inv(16) leukemia in human cells and a mouse model. Enhancer-interaction analysis indicated that the three enhancers are physically connected with the MYC promoter, and genome-editing analysis demonstrated that they are functionally implicated in deregulation of MYC expression. This study reveals a mechanism whereby CBFß-SMMHC drives leukemia maintenance and suggests that inhibitors targeting chromatin activity may prove effective in inv(16) leukemia therapy.

Small Molecules Co-targeting CKIα and the Transcriptional Kinases CDK7/9 Control AML in Preclinical Models.

CKIα ablation induces p53 activation, and CKIα degradation underlies the therapeutic effect of lenalidomide in a pre-leukemia syndrome. Here we describe the development of CKIα inhibitors, which co-target the transcriptional kinases CDK7 and CDK9, thereby augmenting CKIα-induced p53 activation and its anti-leukemic activity. Oncogene-driving super-enhancers (SEs) are highly sensitive to CDK7/9 inhibition. We identified multiple newly gained SEs in primary mouse acute myeloid leukemia (AML) cells and demonstrate that the inhibitors abolish many SEs and preferentially suppress the transcription elongation of SE-driven oncogenes. We show that blocking CKIα together with CDK7 and/or CDK9 synergistically stabilize p53, deprive leukemia cells of survival and proliferation-maintaining SE-driven oncogenes, and induce apoptosis. Leukemia progenitors are selectively eliminated by the inhibitors, explaining their therapeutic efficacy with preserved hematopoiesis and leukemia cure potential; they eradicate leukemia in MLL-AF9 and Tet2-/-;Flt3ITD AML mouse models and in several patient-derived AML xenograft models, supporting their potential efficacy in curing human leukemia.

Prion-like Domains Program Ewing's Sarcoma.

Prion-like domains have emerged as important drivers of neurodegenerative disease. Now, Boulay et al. establish that the translocated prion-like domain of the oncogenic EWS-FLI1 fusion protein enables phase-separation events, which inappropriately recruit chromatin-remodeling factors to elicit the aberrant transcriptional programs underlying Ewing's sarcoma.

Cancer-Specific Retargeting of BAF Complexes by a Prion-like Domain.

Alterations in transcriptional regulators can orchestrate oncogenic gene expression programs in cancer. Here, we show that the BRG1/BRM-associated factor (BAF) chromatin remodeling complex, which is mutated in over 20% of human tumors, interacts with EWSR1, a member of a family of proteins with prion-like domains (PrLD) that are frequent partners in oncogenic fusions with transcription factors. In Ewing sarcoma, we find that the BAF complex is recruited by the EWS-FLI1 fusion protein to tumor-specific enhancers and contributes to target gene activation. This process is a neomorphic property of EWS-FLI1 compared to wild-type FLI1 and depends on tyrosine residues that are necessary for phase transitions of the EWSR1 prion-like domain. Furthermore, fusion of short fragments of EWSR1 to FLI1 is sufficient to recapitulate BAF complex retargeting and EWS-FLI1 activities. Our studies thus demonstrate that the physical properties of prion-like domains can retarget critical chromatin regulatory complexes to establish and maintain oncogenic gene expression programs.

Oncogenic Role of Fusion-circRNAs Derived from Cancer-Associated Chromosomal Translocations.

Chromosomal translocations encode oncogenic fusion proteins that have been proven to be causally involved in tumorigenesis. Our understanding of whether such genomic alterations also affect non-coding RNAs is limited, and their impact on circular RNAs (circRNAs) has not been explored. Here, we show that well-established cancer-associated chromosomal translocations give rise to fusion circRNAs (f-circRNA) that are produced from transcribed exons of distinct genes affected by the translocations. F-circRNAs contribute to cellular transformation, promote cell viability and resistance upon therapy, and have tumor-promoting properties in in vivo models. Our work expands the current knowledge regarding molecular mechanisms involved in cancer onset and progression, with potential diagnostic and therapeutic implications.

Tuning levels of low-complexity domain interactions to modulate endogenous oncogenic transcription.

Gene activation by mammalian transcription factors (TFs) requires multivalent interactions of their low-complexity domains (LCDs), but how such interactions regulate transcription remains unclear. It has been proposed that extensive LCD-LCD interactions culminating in liquid-liquid phase separation (LLPS) of TFs is the dominant mechanism underlying transactivation. Here, we investigated how tuning the amount and localization of LCD-LCD interactions in vivo affects transcription of endogenous human genes. Quantitative single-cell and single-molecule imaging reveals that the oncogenic TF EWS::FLI1 requires a narrow optimum of LCD-LCD interactions to activate its target genes associated with GGAA microsatellites. Increasing LCD-LCD interactions toward putative LLPS represses transcription of these genes in patient-derived cells. Likewise, ectopically creating LCD-LCD interactions to sequester EWS::FLI1 into a well-documented LLPS compartment, the nucleolus, inhibits EWS::FLI1-driven transcription and oncogenic transformation. Our findings show how altering the balance of LCD-LCD interactions can influence transcriptional regulation and suggest a potential therapeutic strategy for targeting disease-causing TFs.

The FUS::DDIT3 fusion oncoprotein inhibits BAF complex targeting and activity in myxoid liposarcoma.

Mammalian SWI/SNF (mSWI/SNF or BAF) ATP-dependent chromatin remodeling complexes play critical roles in governing genomic architecture and gene expression and are frequently perturbed in human cancers. Transcription factors (TFs), including fusion oncoproteins, can bind to BAF complex surfaces to direct chromatin targeting and accessibility, often activating oncogenic gene loci. Here, we demonstrate that the FUS::DDIT3 fusion oncoprotein hallmark to myxoid liposarcoma (MLPS) inhibits BAF complex-mediated remodeling of adipogenic enhancer sites via sequestration of the adipogenic TF, CEBPB, from the genome. In mesenchymal stem cells, small-molecule inhibition of BAF complex ATPase activity attenuates adipogenesis via failure of BAF-mediated DNA accessibility and gene activation at CEBPB target sites. BAF chromatin occupancy and gene expression profiles of FUS::DDIT3-expressing cell lines and primary tumors exhibit similarity to SMARCB1-deficient tumor types. These data present a mechanism by which a fusion oncoprotein generates a BAF complex loss-of-function phenotype, independent of deleterious subunit mutations.

Oncogenic chimeric transcription factors drive tumor-specific transcription, processing, and translation of silent genomic regions.

Many cancers are characterized by gene fusions encoding oncogenic chimeric transcription factors (TFs) such as EWS::FLI1 in Ewing sarcoma (EwS). Here, we find that EWS::FLI1 induces the robust expression of a specific set of novel spliced and polyadenylated transcripts within otherwise transcriptionally silent regions of the genome. These neogenes (NGs) are virtually undetectable in large collections of normal tissues or non-EwS tumors and can be silenced by CRISPR interference at regulatory EWS::FLI1-bound microsatellites. Ribosome profiling and proteomics further show that some NGs are translated into highly EwS-specific peptides. More generally, we show that hundreds of NGs can be detected in diverse cancers characterized by chimeric TFs. Altogether, this study identifies the transcription, processing, and translation of novel, specific, highly expressed multi-exonic transcripts from otherwise silent regions of the genome as a new activity of aberrant TFs in cancer.

MLL::AF9 degradation induces rapid changes in transcriptional elongation and subsequent loss of an active chromatin landscape.

MLL rearrangements produce fusion oncoproteins that drive leukemia development, but the direct effects of MLL-fusion inactivation remain poorly defined. We designed models with degradable MLL::AF9 where treatment with small molecules induces rapid degradation. We leveraged the kinetics of this system to identify a core subset of MLL::AF9 target genes where MLL::AF9 degradation induces changes in transcriptional elongation within 15 minutes. MLL::AF9 degradation subsequently causes loss of a transcriptionally active chromatin landscape. We used this insight to assess the effectiveness of small molecules that target members of the MLL::AF9 multiprotein complex, specifically DOT1L and MENIN. Combined DOT1L/MENIN inhibition resembles MLL::AF9 degradation, whereas single-agent treatment has more modest effects on MLL::AF9 occupancy and gene expression. Our data show that MLL::AF9 degradation leads to decreases in transcriptional elongation prior to changes in chromatin landscape at select loci and that combined inhibition of chromatin complexes releases the MLL::AF9 oncoprotein from chromatin globally.

Oncogenic transcription factors and neogenes: New opportunities for cancer immunotherapy?

Vibert et al. (2022) demonstrate that oncogenic transcription factor fusion proteins bind otherwise silent genomic regions, producing RNAs that can be spliced, exported, and translated. These "neogenes" represent possible targets for immunotherapy and may even be universal byproducts of altered transcription in cancer.

In vivo temporal resolution of acute promyelocytic leukemia progression reveals a role of Klf4 in suppressing early leukemic transformation.

Genome organization plays a pivotal role in transcription, but how transcription factors (TFs) rewire the structure of the genome to initiate and maintain the programs that lead to oncogenic transformation remains poorly understood. Acute promyelocytic leukemia (APL) is a fatal subtype of leukemia driven by a chromosomal translocation between the promyelocytic leukemia (PML) and retinoic acid receptor α (RARα) genes. We used primary hematopoietic stem and progenitor cells (HSPCs) and leukemic blasts that express the fusion protein PML-RARα as a paradigm to temporally dissect the dynamic changes in the epigenome, transcriptome, and genome architecture induced during oncogenic transformation. We found that PML-RARα initiates a continuum of topologic alterations, including switches from A to B compartments, transcriptional repression, loss of active histone marks, and gain of repressive histone marks. Our multiomics-integrated analysis identifies Klf4 as an early down-regulated gene in PML-RARα-driven leukemogenesis. Furthermore, we characterized the dynamic alterations in the Klf4 cis-regulatory network during APL progression and demonstrated that ectopic Klf4 overexpression can suppress self-renewal and reverse the differentiation block induced by PML-RARα. Our study provides a comprehensive in vivo temporal dissection of the epigenomic and topological reprogramming induced by an oncogenic TF and illustrates how topological architecture can be used to identify new drivers of malignant transformation.

Functions of bromodomain-containing proteins and their roles in homeostasis and cancer.

Bromodomains (BRDs) are evolutionarily conserved protein-protein interaction modules that are found in a wide range of proteins with diverse catalytic and scaffolding functions and are present in most tissues. BRDs selectively recognize and bind to acetylated Lys residues - particularly in histones - and thereby have important roles in the regulation of gene expression. BRD-containing proteins are frequently dysregulated in cancer, they participate in gene fusions that generate diverse, frequently oncogenic proteins, and many cancer-causing mutations have been mapped to the BRDs themselves. Importantly, BRDs can be targeted by small-molecule inhibitors, which has stimulated many translational research projects that seek to attenuate the aberrant functions of BRD-containing proteins in disease.

Redox modification of nuclear actin by MICAL-2 regulates SRF signaling.

The serum response factor (SRF) binds to coactivators, such as myocardin-related transcription factor-A (MRTF-A), and mediates gene transcription elicited by diverse signaling pathways. SRF/MRTF-A-dependent gene transcription is activated when nuclear MRTF-A levels increase, enabling the formation of transcriptionally active SRF/MRTF-A complexes. The level of nuclear MRTF-A is regulated by nuclear G-actin, which binds to MRTF-A and promotes its nuclear export. However, pathways that regulate nuclear actin levels are poorly understood. Here, we show that MICAL-2, an atypical actin-regulatory protein, mediates SRF/MRTF-A-dependent gene transcription elicited by nerve growth factor and serum. MICAL-2 induces redox-dependent depolymerization of nuclear actin, which decreases nuclear G-actin and increases MRTF-A in the nucleus. Furthermore, we show that MICAL-2 is a target of CCG-1423, a small molecule inhibitor of SRF/MRTF-A-dependent transcription that exhibits efficacy in various preclinical disease models. These data identify redox modification of nuclear actin as a regulatory switch that mediates SRF/MRTF-A-dependent gene transcription.

Decoding the function of an oncogenic transcription factor: finding the first responders.

Transcription factors (TFs) are frequently altered in human diseases. Identifying the direct and immediate target genes of TFs is critical to understanding their role in pathophysiology. Stengel et al. (2020) applied chemogenetic and nascent transcriptome mapping technologies to define the core gene set regulated by AML1-ETO-an oncogenic TF fusion protein frequently found in acute myeloid leukemia (AML).

Definition of a small core transcriptional circuit regulated by AML1-ETO.

Transcription factors regulate gene networks controlling normal hematopoiesis and are frequently deregulated in acute myeloid leukemia (AML). Critical to our understanding of the mechanism of cellular transformation by oncogenic transcription factors is the ability to define their direct gene targets. However, gene network cascades can change within minutes to hours, making it difficult to distinguish direct from secondary or compensatory transcriptional changes by traditional methodologies. To overcome this limitation, we devised cell models in which the AML1-ETO protein could be quickly degraded upon addition of a small molecule. The rapid kinetics of AML1-ETO removal, when combined with analysis of transcriptional output by nascent transcript analysis and genome-wide AML1-ETO binding by CUT&RUN, enabled the identification of direct gene targets that constitute a core AML1-ETO regulatory network. Moreover, derepression of this gene network was associated with RUNX1 DNA binding and triggered a transcription cascade ultimately resulting in myeloid differentiation.