Analysis of 3760 hematologic malignancies reveals rare transcriptomic aberrations of driver genes.

BACKGROUND: Rare oncogenic driver events, particularly affecting the expression or splicing of driver genes, are suspected to substantially contribute to the large heterogeneity of hematologic malignancies. However, their identification remains challenging. METHODS: To address this issue, we generated the largest dataset to date of matched whole genome sequencing and total RNA sequencing of hematologic malignancies from 3760 patients spanning 24 disease entities. Taking advantage of our dataset size, we focused on discovering rare regulatory aberrations. Therefore, we called expression and splicing outliers using an extension of the workflow DROP (Detection of RNA Outliers Pipeline) and AbSplice, a variant effect predictor that identifies genetic variants causing aberrant splicing. We next trained a machine learning model integrating these results to prioritize new candidate disease-specific driver genes. RESULTS: We found a median of seven expression outlier genes, two splicing outlier genes, and two rare splice-affecting variants per sample. Each category showed significant enrichment for already well-characterized driver genes, with odds ratios exceeding three among genes called in more than five samples. On held-out data, our integrative modeling significantly outperformed modeling based solely on genomic data and revealed promising novel candidate driver genes. Remarkably, we found a truncated form of the low density lipoprotein receptor LRP1B transcript to be aberrantly overexpressed in about half of hairy cell leukemia variant (HCL-V) samples and, to a lesser extent, in closely related B-cell neoplasms. This observation, which was confirmed in an independent cohort, suggests LRP1B as a novel marker for a HCL-V subclass and a yet unreported functional role of LRP1B within these rare entities. CONCLUSIONS: Altogether, our census of expression and splicing outliers for 24 hematologic malignancy entities and the companion computational workflow constitute unique resources to deepen our understanding of rare oncogenic events in hematologic cancers.

Comprehensive comparison between azacytidine and decitabine treatment in an acute myeloid leukemia cell line.

Azacytidine (AzaC) and decitabine (AzadC) are cytosine analogs that covalently trap DNA methyltransferases, which place the important epigenetic mark 5-methyl-2'-deoxycytidine by methylating 2'-deoxycytidine (dC) at the C5 position. AzaC and AzadC are used in the clinic as antimetabolites to treat myelodysplastic syndrome and acute myeloid leukemia and are explored against other types of cancer. Although their principal mechanism of action is known, the downstream effects of AzaC and AzadC treatment are not well understood and the cellular prerequisites that determine sensitivity toward AzaC and AzadC remain elusive. Here, we investigated the effects and phenotype of AzaC and AzadC exposure on the acute myeloid leukemia cell line MOLM-13. We found that while AzaC and AzadC share many effects on the cellular level, including decreased global DNA methylation, increased formation of DNA double-strand breaks, transcriptional downregulation of important oncogenes and similar changes on the proteome level, AzaC failed in contrast to AzadC to induce apoptosis efficiently in MOLM-13. The only cellular marker that correlated with this clear phenotypical outcome was the level of hydroxy-methyl-dC, an additional epigenetic mark that is placed by TET enzymes and repressed in cancer cells. Whereas AzadC increased hmdC substantially in MOLM-13, AzaC treatment did not result in any increase at all. This suggests that hmdC levels in cancer cells should be monitored as a response toward AzaC and AzadC and considered as a biomarker to judge whether AzaC or AzadC treatment leads to cell death in leukemic cells.

Suppression of SARS-CoV-2 Replication with Stabilized and Click-Chemistry Modified siRNAs.

The emergence of more transmissible or aggressive variants of SARS-CoV-2 requires the development of antiviral medication that is quickly adjustable to evolving viral escape mutations. Here we report the synthesis of chemically stabilized small interfering RNA (siRNA) against SARS-CoV-2. The siRNA can be further modified with receptor ligands such as peptides using CuI -catalysed click-chemistry. We demonstrate that optimized siRNAs can reduce viral loads and virus-induced cytotoxicity by up to five orders of magnitude in cell lines challenged with SARS-CoV-2. Furthermore, we show that an ACE2-binding peptide-conjugated siRNA is able to reduce virus replication and virus-induced apoptosis in 3D mucociliary lung microtissues. The adjustment of the siRNA sequence allows a rapid adaptation of their antiviral activity against different variants of concern. The ability to conjugate the siRNA via click-chemistry to receptor ligands facilitates the construction of targeted siRNAs for a flexible antiviral defence strategy.

Epigenetic Anti-Cancer Treatment With a Stabilized Carbocyclic Decitabine Analogue.

5-Aza-2'-deoxycytidine (Decitabine, AzadC) is a nucleoside analogue, which is in clinical use to treat patients with myelodysplastic syndrome or acute myeloid leukemia. Its mode of action is unusual because the compound is one of the few drugs that act at the epigenetic level of the genetic code. AzadC is incorporated as an antimetabolite into the genome and creates covalent, inhibitory links to DNA methyltransferases (DNMTs) that methylate 2'-deoxycytidine (dC) to 5-methyl-dC (mdC). Consequently, AzadC treatment leads to a global loss of mdC, which presumably results in a reactivation of silenced genes, among them tumor suppressor and DNA damage response genes. Because AzadC suffers from severe instability, which limits its use in the clinic, a more sophisticated AzadC derivative would be highly valuable. Here, we report that a recently developed carbocyclic AzadC analogue (cAzadC) blocks DNMT1 in the AML cell line MOLM-13 as efficient as AzadC. Moreover, cAzadC has a surprisingly strong anti-proliferative effect and leads to a significantly higher number of double strand breaks compared to AzadC, while showing less off-target toxicity. These results show that cAzadC triggers more deleterious repair and apoptotic pathways in cancer cells than AzadC, which makes cAzadC a promising next generation epigenetic drug.

tRNA modification profiles in obligate and moderate thermophilic bacilli.

Transfer RNAs (tRNAs) are the most ancient RNA molecules in the cell, modification pattern of which is linked to phylogeny. The aim of this study was to determine the tRNA modification profiles of obligate (Anoxybacillus, Geobacillus, Paragebacillus) and moderate (Bacillus, Brevibacillus, Ureibacillus, Paenibacillus) thermophilic aerobic bacilli strains to find out its linkage to phylogenetic variations between species. LC-MS was applied for the quantification of modified nucleosides using both natural and isotopically labeled standards. The presence of m2A and m7G modifications at high levels was determined in all species. Relatively high level of i6A and m5C modification was observed for Paenibacillus and Ureibacillus, respectively. The lowest level of Cm modification was found in Bacillus. The modification ms2i6A and m1G were absent in Brevibacillus and Ureibacillus, respectively, while modifications Am and m22G were observed only for Ureibacillus. While both obligate and moderate thermophilic species contain Gm, m1G and ms2i6A modifications, large quantities of them (especially Gm and ms2i6A modification) were detected in obligate thermophilic ones (Geobacillus, Paragebacillus and Anoxybacillus). The collective set of modified tRNA bases is genus-specific and linked to the phylogeny of bacilli. In addition, the dataset could be applied to distinguish obligate thermophilic bacilli from moderate ones.

Redirected nuclear glutamate dehydrogenase supplies Tet3 with α-ketoglutarate in neurons.

Tet3 is the main α-ketoglutarate (αKG)-dependent dioxygenase in neurons that converts 5-methyl-dC into 5-hydroxymethyl-dC and further on to 5-formyl- and 5-carboxy-dC. Neurons possess high levels of 5-hydroxymethyl-dC that further increase during neural activity to establish transcriptional plasticity required for learning and memory functions. How αKG, which is mainly generated in mitochondria as an intermediate of the tricarboxylic acid cycle, is made available in the nucleus has remained an unresolved question in the connection between metabolism and epigenetics. We show that in neurons the mitochondrial enzyme glutamate dehydrogenase, which converts glutamate into αKG in an NAD+-dependent manner, is redirected to the nucleus by the αKG-consumer protein Tet3, suggesting on-site production of αKG. Further, glutamate dehydrogenase has a stimulatory effect on Tet3 demethylation activity in neurons, and neuronal activation increases the levels of αKG. Overall, the glutamate dehydrogenase-Tet3 interaction might have a role in epigenetic changes during neural plasticity.

Quantification of DNA Methylation and Its Oxidized Derivatives Using LC-MS.

The amount of 5-methyl-2'-deoxycytidine (m5dC) and its oxidized derivatives 5-hydroxymethyl-dC (hm5dC), 5-formyl-dC (f5dC), and 5-carboxy-dC (ca5dC) inside mammalian cells provides valuable information concerning cellular state and fate. LC-MS methods enable reliable quantification of these noncanonical DNA modifications in the low femtomolar range. Here, we describe a broadly applicable protocol to quantify m5dC, hm5dC, f5dC, and ca5dC in vertebrate-derived cells using ultra-HPLC triple quadrupole MS (UHPLC-QQQ-MS).

5-Hydroxymethyl-, 5-Formyl- and 5-Carboxydeoxycytidines as Oxidative Lesions and Epigenetic Marks.

The four non-canonical nucleotides in the human genome 5-methyl-, 5-hydroxymethyl-, 5-formyl- and 5-carboxydeoxycytidine (mdC, hmdC, fdC and cadC) form a second layer of epigenetic information that contributes to the regulation of gene expression. Formation of the oxidized nucleotides hmdC, fdC and cadC requires oxidation of mdC by ten-eleven translocation (Tet) enzymes that require oxygen, Fe(II) and α-ketoglutarate as cosubstrates. Although these oxidized forms of mdC are widespread in mammalian genomes, experimental evidence for their presence in fungi and plants is ambiguous. This vagueness is caused by the fact that these oxidized mdC derivatives are also formed as oxidative lesions, resulting in unclear basal levels that are likely to have no epigenetic function. Here, we report the xdC levels in the fungus Amanita muscaria in comparison to murine embryonic stem cells (mESCs), HEK cells and induced pluripotent stem cells (iPSCs), to obtain information about the basal levels of hmdC, fdC and cadC as DNA lesions in the genome.

The cGMP-Dependent Protein Kinase 2 Contributes to Cone Photoreceptor Degeneration in the Cnga3-Deficient Mouse Model of Achromatopsia.

Mutations in the CNGA3 gene, which encodes the A subunit of the cyclic guanosine monophosphate (cGMP)-gated cation channel in cone photoreceptor outer segments, cause total colour blindness, also referred to as achromatopsia. Cones lacking this channel protein are non-functional, accumulate high levels of the second messenger cGMP and degenerate over time after induction of ER stress. The cell death mechanisms that lead to loss of affected cones are only partially understood. Here, we explored the disease mechanisms in the Cnga3 knockout (KO) mouse model of achromatopsia. We found that another important effector of cGMP, the cGMP-dependent protein kinase 2 (Prkg2) is crucially involved in cGMP cytotoxicity of cones in Cnga3 KO mice. Virus-mediated knockdown or genetic ablation of Prkg2 in Cnga3 KO mice counteracted degeneration and preserved the number of cones. Analysis of markers of endoplasmic reticulum stress and unfolded protein response confirmed that induction of these processes in Cnga3 KO cones also depends on Prkg2. In conclusion, we identified Prkg2 as a novel key mediator of cone photoreceptor degeneration in achromatopsia. Our data suggest that this cGMP mediator could be a novel pharmacological target for future neuroprotective therapies.

Recent evolution of a TET-controlled and DPPA3/STELLA-driven pathway of passive DNA demethylation in mammals.

Genome-wide DNA demethylation is a unique feature of mammalian development and naïve pluripotent stem cells. Here, we describe a recently evolved pathway in which global hypomethylation is achieved by the coupling of active and passive demethylation. TET activity is required, albeit indirectly, for global demethylation, which mostly occurs at sites devoid of TET binding. Instead, TET-mediated active demethylation is locus-specific and necessary for activating a subset of genes, including the naïve pluripotency and germline marker Dppa3 (Stella, Pgc7). DPPA3 in turn drives large-scale passive demethylation by directly binding and displacing UHRF1 from chromatin, thereby inhibiting maintenance DNA methylation. Although unique to mammals, we show that DPPA3 alone is capable of inducing global DNA demethylation in non-mammalian species (Xenopus and medaka) despite their evolutionary divergence from mammals more than 300 million years ago. Our findings suggest that the evolution of Dppa3 facilitated the emergence of global DNA demethylation in mammals.

Distinct and stage-specific contributions of TET1 and TET2 to stepwise cytosine oxidation in the transition from naive to primed pluripotency.

Cytosine DNA bases can be methylated by DNA methyltransferases and subsequently oxidized by TET proteins. The resulting 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC) are considered demethylation intermediates as well as stable epigenetic marks. To dissect the contributions of these cytosine modifying enzymes, we generated combinations of Tet knockout (KO) embryonic stem cells (ESCs) and systematically measured protein and DNA modification levels at the transition from naive to primed pluripotency. Whereas the increase of genomic 5-methylcytosine (5mC) levels during exit from pluripotency correlated with an upregulation of the de novo DNA methyltransferases DNMT3A and DNMT3B, the subsequent oxidation steps turned out to be far more complex. The strong increase of oxidized cytosine bases (5hmC, 5fC, and 5caC) was accompanied by a drop in TET2 levels, yet the analysis of KO cells suggested that TET2 is responsible for most 5fC formation. The comparison of modified cytosine and enzyme levels in Tet KO cells revealed distinct and differentiation-dependent contributions of TET1 and TET2 to 5hmC and 5fC formation arguing against a processive mechanism of 5mC oxidation. The apparent independent steps of 5hmC and 5fC formation suggest yet to be identified mechanisms regulating TET activity that may constitute another layer of epigenetic regulation.

Influencing Epigenetic Information with a Hydrolytically Stable Carbocyclic 5-Aza-2'-deoxycytidine.

5-Aza-2'-deoxycytidine (AzadC) is an antimetabolite in clinical use, which reduces the level of the epigenetic modification 5-methyl-2'-deoxycytidine (mdC). AzadC is incorporated into the genome of proliferating cells, where it inhibits DNA methyltransferases (DNMTs), leading to a reduction of mdC. The loss of mdC, which is a transcriptional silencer in the promoter region found upstream of genes, leads to the reactivation of the corresponding gene, including tumor-suppressor genes, which elicits a beneficial effect. The problem associated with AzadC is that the compound is hydrolytically unstable. It decomposes during treatment to a variety of poorly characterized hydrolysis products. After its incorporation into the genome, this hydrolytic instability generates abasic sites. It is consequently difficult to dissect whether the activity of the compound is caused by DNMT inhibition or more generally by DNA lesion formation. We now discovered that a disarmed version of AzadC, in which the ribose oxygen was replaced by a CH2 group, is surprisingly stable under a variety of pH values while keeping activity against the DNMTs.

Isotope-dilution mass spectrometry for exact quantification of noncanonical DNA nucleosides.

DNA contains not only canonical nucleotides but also a variety of modifications of the bases. In particular, cytosine and adenine are frequently modified. Determination of the exact quantity of these noncanonical bases can contribute to the characterization of the state of a biological system, e.g., determination of disease or developmental processes, and is therefore extremely important. Here, we present a workflow that includes detailed description of critical sample preparation steps and important aspects of mass spectrometry analysis and validation. In this protocol, extraction and digestion of DNA by an optimized spin-column and enzyme-based method are described. Isotopically labeled standards are added in the course of DNA digestion, which allows exact quantification by isotope dilution mass spectrometry. To overcome the major bottleneck of such analyses, we developed a short (~14-min-per-sample) ultra-HPLC (UHPLC) and triple quadrupole mass spectrometric (QQQ-MS) method. Easy calculation of the modification abundance in the genome is possible with the provided evaluation sheets. Compared to alternative methods, the quantification procedure presented here allows rapid, ultrasensitive (low femtomole range) and highly reproducible quantification of different nucleosides in parallel. Including sample preparation and evaluation, quantification of DNA modifications can be achieved in less than a week.

The chemistries and consequences of DNA and RNA methylation and demethylation.

Chemical modification of nucleobases plays an important role for the control of gene expression on different levels. That includes the modulation of translation by modified tRNA-bases or silencing and reactivation of genes by methylation and demethylation of cytosine in promoter regions. Especially dynamic methylation of adenine and cytosine is essential for cells to adapt to their environment or for the development of complex organisms from a single cell. Errors in the cytosine methylation pattern are associated with most types of cancer and bacteria use methylated nucleobases to resist antibiotics. This Point of View wants to shed light on the known and potential chemistry of DNA and RNA methylation and demethylation. Understanding the chemistry of these processes on a molecular level is the first step towards a deeper knowledge about their regulation and function and will help us to find ways how nucleobase methylation can be manipulated to treat diseases.