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•The prevalence of myeloid CHIP in patients with CLL was 12% in untreated and 24% in treated patients (85% with prior chemotherapy exposure).•The presence of ≥2 M-CHIP mutations was associated with survival, even accounting for prior treatment and age.

Richter syndrome (RS) arising from chronic lymphocytic leukemia (CLL) exemplifies an aggressive malignancy that develops from an indolent neoplasm. To decipher the genetics underlying this transformation, we computationally deconvoluted admixtures of CLL and RS cells from 52 patients with RS, evaluating paired CLL–RS whole-exome sequencing data. We discovered RS-specific somatic driver mutations (including IRF2BP2, SRSF1, B2M, DNMT3Aand CCND3), recurrent copy-number alterations beyond del(9p21)(CDKN2A/B), whole-genome duplication and chromothripsis, which were confirmed in 45 independent RS cases and in an external set of RS whole genomes. Through unsupervised clustering, clonally related RS was largely distinct from diffuse large B cell lymphoma. We distinguished pathways that were dysregulated in RS versus CLL, and detected clonal evolution of transformation at single-cell resolution, identifying intermediate cell states. Our study defines distinct molecular subtypes of RS and highlights cell-free DNA analysis as a potential tool for early diagnosis and monitoring.

Recent advances in cancer characterization have consistently revealed marked heterogeneity, impeding the completion of integrated molecular and clinical maps for each malignancy. Here, we focus on chronic lymphocytic leukemia (CLL), a B cell neoplasm with variable natural history that is conventionally categorized into two subtypes distinguished by extent of somatic mutations in the heavy-chain variable region of immunoglobulin genes (IGHV). To build the ‘CLL map,’ we integrated genomic, transcriptomic and epigenomic data from 1,148 patients. We identified 202 candidate genetic drivers of CLL (109 new) and refined the characterization of IGHV subtypes, which revealed distinct genomic landscapes and leukemogenic trajectories. Discovery of new gene expression subtypes further subcategorized this neoplasm and proved to be independent prognostic factors. Clinical outcomes were associated with a combination of genetic, epigenetic and gene expression features, further advancing our prognostic paradigm. Overall, this work reveals fresh insights into CLL oncogenesis and prognostication.

Neural progenitor cells undergo somatic retrotransposition events, mainly involving L1 elements, which can be potentially deleterious. Here, we analyze the whole genomes of 20 brain samples and 80 non-brain samples, and characterized the retrotransposition landscape of patients affected by a variety of neurodevelopmental disorders including Rett syndrome, tuberous sclerosis, ataxia-telangiectasia and autism. We report that the number of retrotranspositions in brain tissues is higher than that observed in non-brain samples and even higher in pathologic vs normal brains. The majority of somatic brain retrotransposons integrate into pre-existing repetitive elements, preferentially A/T rich L1 sequences, resulting in nested insertions. Our findings document the fingerprints of encoded endonuclease independent mechanisms in the majority of L1 brain insertion events. The insertions are “non-classical” in that they are truncated at both ends, integrate in the same orientation as the host element, and their target sequences are enriched with a CCATT motif in contrast to the classical endonuclease motif of most other retrotranspositions. We show that L1Hs elements integrate preferentially into genes associated with neural functions and diseases. We propose that pre-existing retrotransposons act as “lightning rods” for novel insertions, which may give fine modulation of gene expression while safeguarding from deleterious events. Overwhelmingly uncontrolled retrotransposition may breach this safeguard mechanism and increase the risk of harmful mutagenesis in neurodevelopmental disorders.

Adenosine to inosine (A-to-I) RNA editing is a post-transcriptional modification catalyzed by the ADAR (adenosine deaminase that acts on RNA) enzymes, which are ubiquitously expressed among metazoans. Technical requirements have limited systematic mapping of editing sites to a small number of organisms. Thus, the extent of editing across the metazoan lineage is largely unknown. Here, we apply a computational procedure to search for RNA-sequencing reads containing clusters of editing sites in 21 diverse organisms. Clusters of editing sites are abundant in repetitive genomic regions that putatively form double-stranded RNA (dsRNA) structures and are rarely seen in coding regions. The method reveals a considerable variation in hyper-editing levels across species, which is partly explained by differences in the potential of sequences to form dsRNA structures and the variability of ADAR proteins. Several commonly used model animals exhibit low editing levels and editing levels in primates is not exceptionally high, as previously suggested. Editing by ADARs is highly prevalent across the Metazoa, mostly targeting dsRNA structures formed by genomic repeats. The degree to which the transcriptome of a given species undergoes hyper-editing is governed by the repertoire of repeats in the underlying genome. The strong association of RNA editing with the long dsRNA regions originating from non-coding repetitive elements is contrasted by the almost non-existing signal seen in coding regions. Hyper-edited regions are rarely expressed in a non-edited form. These results support the notion that the main role of ADAR is to suppress the cellular response to endogenous dsRNA structures.

Genomic mutations in key genes are known to drive tumorigenesis and have been the focus of much attention in recent years. However, genetic content also may change farther downstream. RNA editing alters the mRNA sequence from its genomic blueprint in a dynamic and flexible way. A few isolated cases of editing alterations in cancer have been reported previously. Here, we provide a transcriptome-wide characterization of RNA editing across hundreds of cancer samples from multiple cancer tissues, and we show that A-to-I editing and the enzymes mediating this modification are significantly altered, usually elevated, in most cancer types. Increased editing activity is found to be associated with patient survival. As is the case with somatic mutations in DNA, most of these newly introduced RNA mutations are likely passengers, but a few may serve as drivers that may be novel candidates for therapeutic and diagnostic purposes.

Neuberg: Pharmacyclics: Research Funding; Celgene: Research Funding; Madrigak Pharmaceuticals: Current equity holder in publicly-traded company. Getz:Broad Institute: Patents & Royalties: MuTect, ABSOLUTE, MutSig, MSMuTect, MSMutSig, POLYSOLVER and TensorQTL; Pharmacyclics: Research Funding; IBM: Research Funding; Scorpion Therapeutics: Consultancy, Current equity holder in publicly-traded company, Other: Founder. Wu:BionTech: Current equity holder in publicly-traded company; Pharmacyclics: Research Funding.

Amongst the novel putative drivers identified by large-scale sequencing studies of chronic lymphocytic leukemia (CLL) is the ribosomal protein RPS15. Mutated in 5.3% of CLL, it co-occurs with heterozygous TP53alterations in 36% of RPS15-mutated samples. Mutation of this mediator of ribosome maturation and translation is associated with poor disease prognosis and enriched in cohorts with del(17p) and relapsed CLL, suggesting a role in disease progression and therapeutic resistance. However, the impact of RPS15mutation on B cell function and CLL development, in the presence or absence of TP53mutation, has yet to be characterized. To this end, we developed overexpression HG3 CLL cell lines modeling four common RPS15mutations (G134R, H137Y, S138F, and S139F) and a conditional knock-in mouse model of the S138F mutation with and without heterozygous Trp53deletion (generated by crossing Rps15and Trp53mutant mice with Cd19-Cre mice).

Although chronic lymphocytic leukemia (CLL) has been consistently at the forefront of genomic discovery, complete characterization of its genomic landscape has been limited by sample size and cohort diversity. To address this challenge, we assembled and generated new genomic data from CLL samples from ~1100 patients (‘CLL-1100‘). In total, we analyzed 984 whole-exome sequences (WES), 177 whole-genome sequences (WGS), RNA-sequencing of 717 cases, and 758 methylome profiles.

Background: Mobile elements comprise a large fraction of metazoan genomes. Accumulation of mobile elements is bound to produce multiple putative double-stranded RNA (dsRNA) structures within the transcriptome. These endogenous dsRNA structures resemble viral RNA and may trigger false activation of the innate immune response, leading to severe damage to the host cell. Adenosine to inosine (A-to-I) RNA editing is a common post-transcriptional modification, abundant within repetitive elements of all metazoans. It was recently shown that a key function of A-to-I RNA editing by ADAR1 is to suppress the immunogenic response by endogenous dsRNAs. Results: Here, we analyze the transcriptomes of dozens of species across the Metazoa and identify a strong genomic selection against endogenous dsRNAs, resulting in their purification from the canonical transcriptome. This purifying selection is especially strong for long and nearly perfect dsRNAs. These are almost absent from mRNAs, but not pre-mRNAs, supporting the notion of selection due to cytoplasmic processes. The few long and nearly perfect structures found in human transcripts are weakly expressed and often heavily edited. Conclusion: Purifying selection of long dsRNA is an important defense mechanism against false activation of innate immunity. This newly identified principle governs the integration of mobile elements into the genome, a major driving force of genome evolution. Furthermore, we find that most ADAR1 activity is not required to prevent an immune response to endogenous dsRNAs. The critical targets of ADAR1 editing are, likely, to be found mostly in non-canonical transcripts.