Somatic mitochondrial DNA mutations are a source of heterogeneity among primary leukemic cells.

Somatic mitochondrial DNA (mtDNA) mutations are prevalent in tumors, yet defining their biological significance remains challenging due to the intricate interplay between selective pressure, heteroplasmy, and cell state. Utilizing bulk whole-genome sequencing data from matched tumor and normal samples from two cohorts of pediatric cancer patients, we uncover differences in the accumulation of synonymous and nonsynonymous mtDNA mutations in pediatric leukemias, indicating distinct selective pressures. By integrating single-cell sequencing (SCS) with mathematical modeling and network-based systems biology approaches, we identify a correlation between the extent of cell-state changes associated with tumor-enriched mtDNA mutations and the selective pressures shaping their distribution among individual leukemic cells. Our findings also reveal an association between specific heteroplasmic mtDNA mutations and cellular responses that may contribute to functional heterogeneity among leukemic cells and influence their fitness. This study highlights the potential of SCS strategies for distinguishing between pathogenic and passenger somatic mtDNA mutations in cancer.

Exploring GPCR signaling pathway networks as cancer therapeutic targets.

GPCR signaling can contribute to establishing the tumor microenvironment and influence the progression and metabolism of tumors. Arora et al.1 describe a systems-level approach to investigate the patterns of co-expression of GPCR signaling pathway networks across diverse tumors and identify network components that correlate with patient-survival data across different cancer types.

Peptide-scFv antigen recognition domains effectively confer CAR T cell multiantigen specificity.

The emergence of immune escape is a significant roadblock to developing effective chimeric antigen receptor (CAR) T cell therapies against hematological malignancies, including acute myeloid leukemia (AML). Here, we demonstrate feasibility of targeting two antigens simultaneously by combining a GRP78-specific peptide antigen recognition domain with a CD123-specific scFv to generate a peptide-scFv bispecific antigen recognition domain (78.123). To achieve this, we test linkers with varying length and flexibility and perform immunophenotypic and functional characterization. We demonstrate that bispecific CAR T cells successfully recognize and kill tumor cells that express GRP78, CD123, or both antigens and have improved antitumor activity compared to their monospecific counterparts when both antigens are expressed. Protein structure prediction suggests that linker length and compactness influence the functionality of the generated bispecific CARs. Thus, we present a bispecific CAR design strategy to prevent immune escape in AML that can be extended to other peptide-scFv combinations.

Modular chimeric cytokine receptors with leucine zippers enhance the antitumour activity of CAR T cells via JAK/STAT signalling.

The limited availability of cytokines in solid tumours hinders maintenance of the antitumour activity of chimeric antigen receptor (CAR) T cells. Cytokine receptor signalling pathways in CAR T cells can be activated by transgenic expression or injection of cytokines in the tumour, or by engineering the activation of cognate cytokine receptors. However, these strategies are constrained by toxicity arising from the activation of bystander cells, by the suboptimal biodistribution of the cytokines and by downregulation of the cognate receptor. Here we show that replacement of the extracellular domains of heterodimeric cytokine receptors in T cells with two leucine zipper motifs provides optimal Janus kinase/signal transducer and activator of transcription signalling. Such chimeric cytokine receptors, which can be generated for common γ-chain receptors, interleukin-10 and -12 receptors, enabled T cells to survive cytokine starvation without induction of autonomous cell growth, and augmented the effector function of CAR T cells in vitro in the setting of chronic antigen exposure and in human tumour xenografts in mice. As a modular design, leucine zippers can be used to generate constitutively active cytokine receptors in effector immune cells.

Defining the condensate landscape of fusion oncoproteins.

Fusion oncoproteins (FOs) arise from chromosomal translocations in ~17% of cancers and are often oncogenic drivers. Although some FOs can promote oncogenesis by undergoing liquid-liquid phase separation (LLPS) to form aberrant biomolecular condensates, the generality of this phenomenon is unknown. We explored this question by testing 166 FOs in HeLa cells and found that 58% formed condensates. The condensate-forming FOs displayed physicochemical features distinct from those of condensate-negative FOs and segregated into distinct feature-based groups that aligned with their sub-cellular localization and biological function. Using Machine Learning, we developed a predictor of FO condensation behavior, and discovered that 67% of ~3000 additional FOs likely form condensates, with 35% of those predicted to function by altering gene expression. 47% of the predicted condensate-negative FOs were associated with cell signaling functions, suggesting a functional dichotomy between condensate-positive and -negative FOs. Our Datasets and reagents are rich resources to interrogate FO condensation in the future.

Predicting evolutionary outcomes through the probability of accessing sequence variants.

Natural selection can only operate on available genetic variation. Thus, determining the probability of accessing different sequence variants from a starting sequence can help predict evolutionary trajectories and outcomes. We define the concept of "variant accessibility" as the probability that a set of genotypes encoding a particular protein function will arise through mutations before subject to natural selection. This probability is shaped by the mutational biases of nucleotides and the structure of the genetic code. Using the influenza A virus as a model, we discuss how a more accessible but less fit variant can emerge as an adaptation rather than a more fit variant. We describe a genotype-accessibility landscape, complementary to the genotype-fitness landscape, that informs the likelihood of a starting sequence reaching different parts of genotype space. The proposed framework lays the foundation for predicting the emergence of adaptive genotypes in evolving systems such as viruses and tumors.

CCL22 mutations drive natural killer cell lymphoproliferative disease by deregulating microenvironmental crosstalk.

Chronic lymphoproliferative disorder of natural killer cells (CLPD-NK) is characterized by clonal expansion of natural killer (NK) cells where the underlying genetic mechanisms are incompletely understood. In the present study, we report somatic mutations in the chemokine gene CCL22 as the hallmark of a distinct subset of CLPD-NK. CCL22 mutations were enriched at highly conserved residues, mutually exclusive of STAT3 mutations and associated with gene expression programs that resembled normal CD16dim/CD56bright NK cells. Mechanistically, the mutations resulted in ligand-biased chemokine receptor signaling, with decreased internalization of the G-protein-coupled receptor (GPCR) for CCL22, CCR4, via impaired ß-arrestin recruitment. This resulted in increased cell chemotaxis in vitro, bidirectional crosstalk with the hematopoietic microenvironment and enhanced NK cell proliferation in vivo in transgenic human IL-15 mice. Somatic CCL22 mutations illustrate a unique mechanism of tumor formation in which gain-of-function chemokine mutations promote tumorigenesis by biased GPCR signaling and dysregulation of microenvironmental crosstalk.

Integrated Genomic Analysis Identifies UBTF Tandem Duplications as a Recurrent Lesion in Pediatric Acute Myeloid Leukemia.

The genetics of relapsed pediatric acute myeloid leukemia (AML) has yet to be comprehensively defined. Here, we present the spectrum of genomic alterations in 136 relapsed pediatric AMLs. We identified recurrent exon 13 tandem duplications (TD) in upstream binding transcription factor (UBTF) in 9% of relapsed AML cases. UBTF-TD AMLs commonly have normal karyotype or trisomy 8 with cooccurring WT1 mutations or FLT3-ITD but not other known oncogenic fusions. These UBTF-TD events are stable during disease progression and are present in the founding clone. In addition, we observed that UBTF-TD AMLs account for approximately 4% of all de novo pediatric AMLs, are less common in adults, and are associated with poor outcomes and MRD positivity. Expression of UBTF-TD in primary hematopoietic cells is sufficient to enhance serial clonogenic activity and to drive a similar transcriptional program to UBTF-TD AMLs. Collectively, these clinical, genomic, and functional data establish UBTF-TD as a new recurrent mutation in AML. SIGNIFICANCE: We defined the spectrum of mutations in relapsed pediatric AML and identified UBTF-TDs as a new recurrent genetic alteration. These duplications are more common in children and define a group of AMLs with intermediate-risk cytogenetic abnormalities, FLT3-ITD and WT1 alterations, and are associated with poor outcomes. See related commentary by Hasserjian and Nardi, p. 173. This article is highlighted in the In This Issue feature, p. 171.

The chemotherapeutic CX-5461 primarily targets TOP2B and exhibits selective activity in high-risk neuroblastoma.

Survival in high-risk pediatric neuroblastoma has remained around 50% for the last 20 years, with immunotherapies and targeted therapies having had minimal impact. Here, we identify the small molecule CX-5461 as selectively cytotoxic to high-risk neuroblastoma and synergistic with low picomolar concentrations of topoisomerase I inhibitors in improving survival in vivo in orthotopic patient-derived xenograft neuroblastoma mouse models. CX-5461 recently progressed through phase I clinical trial as a first-in-human inhibitor of RNA-POL I. However, we also use a comprehensive panel of in vitro and in vivo assays to demonstrate that CX-5461 has been mischaracterized and that its primary target at pharmacologically relevant concentrations, is in fact topoisomerase II beta (TOP2B), not RNA-POL I. This is important because existing clinically approved chemotherapeutics have well-documented off-target interactions with TOP2B, which have previously been shown to cause both therapy-induced leukemia and cardiotoxicity-often-fatal adverse events, which can emerge several years after treatment. Thus, while we show that combination therapies involving CX-5461 have promising anti-tumor activity in vivo in neuroblastoma, our identification of TOP2B as the primary target of CX-5461 indicates unexpected safety concerns that should be examined in ongoing phase II clinical trials in adult patients before pursuing clinical studies in children.

Human MC4R variants affect endocytosis, trafficking and dimerization revealing multiple cellular mechanisms involved in weight regulation.

The Melanocortin-4 Receptor (MC4R) plays a pivotal role in energy homeostasis. We used human MC4R mutations associated with an increased or decreased risk of obesity to dissect mechanisms that regulate MC4R function. Most obesity-associated mutations impair trafficking to the plasma membrane (PM), whereas obesity-protecting mutations either accelerate recycling to the PM or decrease internalization, resulting in enhanced signaling. MC4R mutations that do not affect canonical Gαs protein-mediated signaling, previously considered to be non-pathogenic, nonetheless disrupt agonist-induced internalization, ß-arrestin recruitment, and/or coupling to Gαs, establishing their causal role in severe obesity. Structural mapping reveals ligand-accessible sites by which MC4R couples to effectors and residues involved in the homodimerization of MC4R, which is disrupted by multiple obesity-associated mutations. Human genetic studies reveal that endocytosis, intracellular trafficking, and homodimerization regulate MC4R function to a level that is physiologically relevant, supporting the development of chaperones, agonists, and allosteric modulators of MC4R for weight loss therapy.

Biological impact of mutually exclusive exon switching.

Alternative splicing can expand the diversity of proteomes. Homologous mutually exclusive exons (MXEs) originate from the same ancestral exon and result in polypeptides with similar structural properties but altered sequence. Why would some genes switch homologous exons and what are their biological impact? Here, we analyse the extent of sequence, structural and functional variability in MXEs and report the first large scale, structure-based analysis of the biological impact of MXE events from different genomes. MXE-specific residues tend to map to single domains, are highly enriched in surface exposed residues and cluster at or near protein functional sites. Thus, MXE events are likely to maintain the protein fold, but alter specificity and selectivity of protein function. This comprehensive resource of MXE events and their annotations is available at: http://gene3d.biochem.ucl.ac.uk/mxemod/. These findings highlight how small, but significant changes at critical positions on a protein surface are exploited in evolution to alter function.

Amino acid homorepeats in proteins.

Amino acid homorepeats, or homorepeats, are polypeptide segments found in proteins that contain stretches of identical amino acid residues. Although abnormal homorepeat expansions are linked to pathologies such as neurodegenerative diseases, homorepeats are prevalent in eukaryotic proteomes, suggesting that they are important for normal physiology. In this Review, we discuss recent advances in our understanding of the biological functions of homorepeats, which range from facilitating subcellular protein localization to mediating interactions between proteins across diverse cellular pathways. We explore how the functional diversity of homorepeat-containing proteins could be linked to the ability of homorepeats to adopt different structural conformations, an ability influenced by repeat composition, repeat length and the nature of flanking sequences. We conclude by highlighting how an understanding of homorepeats will help us better characterize and develop therapeutics against the human diseases to which they contribute.

A conserved molecular switch in Class F receptors regulates receptor activation and pathway selection.

Class F receptors are considered valuable therapeutic targets due to their role in human disease, but structural changes accompanying receptor activation remain unexplored. Employing population and cancer genomics data, structural analyses, molecular dynamics simulations, resonance energy transfer-based approaches and mutagenesis, we identify a conserved basic amino acid in TM6 in Class F receptors that acts as a molecular switch to mediate receptor activation. Across all tested Class F receptors (FZD4,5,6,7, SMO), mutation of the molecular switch confers an increased potency of agonists by stabilizing an active conformation as assessed by engineered mini G proteins as conformational sensors. Disruption of the switch abrogates the functional interaction between FZDs and the phosphoprotein Dishevelled, supporting conformational selection as a prerequisite for functional selectivity. Our studies reveal the molecular basis of a common activation mechanism conserved in all Class F receptors, which facilitates assay development and future discovery of Class F receptor-targeting drugs.

Molecular Signatures of Fusion Proteins in Cancer.

Although gene fusions are recognized as driver mutations in a wide variety of cancers, the general molecular mechanisms underlying oncogenic fusion proteins are insufficiently understood. Here, we employ large-scale data integration and machine learning and (1) identify three functionally distinct subgroups of gene fusions and their molecular signatures; (2) characterize the cellular pathways rewired by fusion events across different cancers; and (3) analyze the relative importance of over 100 structural, functional, and regulatory features of ∼2200 gene fusions. We report subgroups of fusions that likely act as driver mutations and find that gene fusions disproportionately affect pathways regulating cellular shape and movement. Although fusion proteins are similar across different cancer types, they affect cancer type-specific pathways. Key indicators of fusion-forming proteins include high and nontissue specific expression, numerous splice sites, and higher centrality in protein-interaction networks. Together, these findings provide unifying and cancer type-specific trends across diverse oncogenic fusion proteins.

Human Diseases from Gain-of-Function Mutations in Disordered Protein Regions.

Although there is much focus on the impact of mutations on structured protein domains, less is known about their impact on unstructured regions. In this issue, Meyer et al. demonstrate that mutations resulting in the emergence of new short linear peptide motifs within intrinsically disordered protein regions can cause human genetic diseases by gain of function.

Cotranslational protein assembly imposes evolutionary constraints on homomeric proteins.

Cotranslational protein folding can facilitate rapid formation of functional structures. However, it can also cause premature assembly of protein complexes, if two interacting nascent chains are in close proximity. By analyzing known protein structures, we show that homomeric protein contacts are enriched toward the C termini of polypeptide chains across diverse proteomes. We hypothesize that this is the result of evolutionary constraints for folding to occur before assembly. Using high-throughput imaging of protein homomers in Escherichia coli and engineered protein constructs with N- and C-terminal oligomerization domains, we show that, indeed, proteins with C-terminal homomeric interface residues consistently assemble more efficiently than those with N-terminal interface residues. Using in vivo, in vitro and in silico experiments, we identify features that govern successful assembly of homomers, which have implications for protein design and expression optimization.

Revealing the Determinants of Widespread Alternative Splicing Perturbation in Cancer.

It is increasingly appreciated that alternative splicing plays a key role in generating functional specificity and diversity in cancer. However, the mechanisms by which cancer mutations perturb splicing remain unknown. Here, we developed a network-based strategy, DrAS-Net, to investigate more than 2.5 million variants across cancer types and link somatic mutations with cancer-specific splicing events. We identified more than 40,000 driver variant candidates and their 80,000 putative splicing targets deregulated in 33 cancer types and inferred their functional impact. Strikingly, tumors with splicing perturbations show reduced expression of immune system-related genes and increased expression of cell proliferation markers. Tumors harboring different mutations in the same gene often exhibit distinct splicing perturbations. Further stratification of 10,000 patients based on their mutation-splicing relationships identifies subtypes with distinct clinical features, including survival rates. Our work reveals how single-nucleotide changes can alter the repertoires of splicing isoforms, providing insights into oncogenic mechanisms for precision medicine.

The contribution of intrinsically disordered regions to protein function, cellular complexity, and human disease.

In the 1960s, Christian Anfinsen postulated that the unique three-dimensional structure of a protein is determined by its amino acid sequence. This work laid the foundation for the sequence-structure-function paradigm, which states that the sequence of a protein determines its structure, and structure determines function. However, a class of polypeptide segments called intrinsically disordered regions does not conform to this postulate. In this review, I will first describe established and emerging ideas about how disordered regions contribute to protein function. I will then discuss molecular principles by which regulatory mechanisms, such as alternative splicing and asymmetric localization of transcripts that encode disordered regions, can increase the functional versatility of proteins. Finally, I will discuss how disordered regions contribute to human disease and the emergence of cellular complexity during organismal evolution.

Molecular Principles of Gene Fusion Mediated Rewiring of Protein Interaction Networks in Cancer.

Gene fusions are common cancer-causing mutations, but the molecular principles by which fusion protein products affect interaction networks and cause disease are not well understood. Here, we perform an integrative analysis of the structural, interactomic, and regulatory properties of thousands of putative fusion proteins. We demonstrate that genes that form fusions (i.e., parent genes) tend to be highly connected hub genes, whose protein products are enriched in structured and disordered interaction-mediating features. Fusion often results in the loss of these parental features and the depletion of regulatory sites such as post-translational modifications. Fusion products disproportionately connect proteins that did not previously interact in the protein interaction network. In this manner, fusion products can escape cellular regulation and constitutively rewire protein interaction networks. We suggest that the deregulation of central, interaction-prone proteins may represent a widespread mechanism by which fusion proteins alter the topology of cellular signaling pathways and promote cancer.

Discovering and understanding oncogenic gene fusions through data intensive computational approaches.

Although gene fusions have been recognized as important drivers of cancer for decades, our understanding of the prevalence and function of gene fusions has been revolutionized by the rise of next-generation sequencing, advances in bioinformatics theory and an increasing capacity for large-scale computational biology. The computational work on gene fusions has been vastly diverse, and the present state of the literature is fragmented. It will be fruitful to merge three camps of gene fusion bioinformatics that appear to rarely cross over: (i) data-intensive computational work characterizing the molecular biology of gene fusions; (ii) development research on fusion detection tools, candidate fusion prioritization algorithms and dedicated fusion databases and (iii) clinical research that seeks to either therapeutically target fusion transcripts and proteins or leverages advances in detection tools to perform large-scale surveys of gene fusion landscapes in specific cancer types. In this review, we unify these different-yet highly complementary and symbiotic-approaches with the view that increased synergy will catalyze advancements in gene fusion identification, characterization and significance evaluation.