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Given the plasticity of hematopoietic stem and progenitor cells, multiple routes of differentiation must be blocked in the the pathogenesis of acute myeloid leukemia, the molecular basis of which is incompletely understood. We report that posttranscriptional repression of the transcription factor ARID3A by miR-125b is a key event in the pathogenesis of acute megakaryoblastic leukemia (AMKL). AMKL is frequently associated with trisomy 21 and GATA1 mutations (GATA1s), and children with Down syndrome are at a high risk of developing the disease. The results of our study showed that chromosome 21–encoded miR-125b synergizes with Gata1s to drive leukemogenesis in this context. Leveraging forward and reverse genetics, we uncovered Arid3a as the main miR-125b target behind this synergy. We demonstrated that, during normal hematopoiesis, this transcription factor promotes megakaryocytic differentiation in concert with GATA1 and mediates TGFβ-induced apoptosis and cell cycle arrest in complex with SMAD2/3. Although Gata1s mutations perturb erythroid differentiation and induce hyperproliferation of megakaryocytic progenitors, intact ARID3A expression assures their megakaryocytic differentiation and growth restriction. Upon knockdown, these tumor suppressive functions are revoked, causing a blockade of dual megakaryocytic/erythroid differentiation and subsequently of AMKL. Inversely, restoring ARID3A expression relieves the arrest of megakaryocytic differentiation in AMKL patient-derived xenografts. This work illustrates how mutations in lineage-determining transcription factors and perturbation of posttranscriptional gene regulation can interact to block multiple routes of hematopoietic differentiation and cause leukemia. In AMKL, surmounting this differentiation blockade through restoration of the tumor suppressor ARID3A represents a promising strategy for treating this lethal pediatric disease.

Given the plasticity of hematopoietic stem/progenitor cells, multiple routes of differentiation must be blocked during acute myeloid leukemia pathogenesis – the molecular basis of which is incompletely understood. Here we report that post-transcriptional repression of transcription factor ARID3A by miR-125b is a key event in megakaryoblastic leukemia (AMKL) pathogenesis. AMKL is frequently associated with trisomy 21 and GATA1 mutations (GATA1s), and children with Down syndrome are at a high risk of developing this disease. We show that chromosome 21-encoded miR-125b synergizes with Gata1s to drive leukemogenesis in this context. Leveraging forward and reverse genetics, we uncover Arid3a as the main miR-125b target underlying this synergy. We demonstrate that during normal hematopoiesis this transcription factor promotes megakaryocytic differentiation in concert with GATA1 and mediates TGFβ-induced apoptosis and cell cycle arrest in complex with SMAD2/3. While Gata1s mutations perturb erythroid differentiation and induce hyperproliferation of megakaryocytic progenitors, intact ARID3A expression assures their megakaryocytic differentiation and growth restriction. Upon knockdown, these tumor suppressive functions are revoked, causing a dual megakaryocytic/erythroid differentiation blockade and subsequently AMKL. Inversely, restoring ARID3A expression relieves the megakaryocytic differentiation arrest in AMKL patient-derived xenografts. This work illustrates how mutations in lineage-determining transcription factors and perturbation of post-transcriptional gene regulation interplay to block multiple routes of hematopoietic differentiation and cause leukemia. Surmounting this differentiation blockade in megakaryoblastic leukemia by restoring the tumor suppressor ARID3A represents a promising strategy for treating this lethal pediatric disease.Key pointsRepression of megakaryocytic transcription factor ARID3A by miR-125b synergizes with GATA1s to induce leukemiaRestoring ARID3A expression relieves megakaryocytic differentiation arrest in megakaryoblastic leukemiaGraphical abstract

Chromosomal translocations that generate oncogenic fusion proteins are causative for most pediatric leukemias and frequently affect the MLL/ KMT2A gene. In vivo modeling of bona fide chromosomal translocations in human hematopoietic stem and progenitor cells is challenging but essential to determine their actual leukemogenic potential. We therefore developed an advanced lentiviral CRISPR-Cas9 vector that efficiently transduced human CD34(+) hematopoietic stem and progenitor cells and induced the t(11; 19)/MLL-ENL translocation. Leveraging this system, we could demonstrate that hematopoietic stem and progenitor cells harboring the translocation showed only a transient clonal growth advantage in vitro. In contrast, t(11; 19)/MLL-ENL-harboring CD34(+) hematopoietic stem and progenitor cells not only showed longterm engraftment in primary immunodeficient recipients, but t(11; 19)/ MLL-ENL also served as a first hit to initiate a monocytic leukemia-like disease. Interestingly, secondary recipients developed acute lymphoblastic leukemia with incomplete penetrance. These findings indicate that environmental cues not only contribute to the disease phenotype, but also to t(11; 19)/ MLL-ENL-mediated oncogenic transformation itself. Thus, by investigating the true chromosomal t(11; 19) rearrangement in its natural genomic context, our study emphasizes the importance of environmental cues for the pathogenesis of pediatric leukemias, opening an avenue for novel treatment options.

Ageing can be defined as the time-dependent decline of physiological functions of an organism. The molecular causes for the ageing process are multiple, involving both genetic and environmental factors. It has been proposed that antioxidants may positively influence the ageing process, protecting the organism against free radical-induced damage. Here we show that the antioxidant N-acetylcysteine (NAC) has a life-extending effect on Drosophila melanogaster. Dietary uptake of NAC results in a dose-dependent increase in median and maximum life span. Flies fed on 1 mg/ml NAC food live 16.6% longer; at 10 mg/ml, life span increases by 26.6%. We have examined the effect of NAC treatment on protein and RNA levels: we observe an NAC-dependent increase in absolute amounts of total RNA and ribosomal RNA, but no differences in protein levels. The NAC effect on longevity may involve differential expression of specific mRNA genes, as suggested by RNA finger-printing experiments.

Reciprocal chromosomal translocations are the causative genetic aberration in almost 60% of the pediatric acute myeloid leukemia cases. Amongst these, rearrangements of the MLL1/KMT2A gene are most frequent. Retroviral overexpression of MLL fusion genes has been shown to be sufficient to transform human hematopoietic stem and progenitor cells (HSPCs). Whether endogenous MLL-rearrangements have a similarly potent transformation capacity remains an open question. As an emerging technology, the clustered regularly interspaced short palindromic repeats (CRISPR) - CRISPR-associated-9 (Cas9) system now offers the opportunity to engineer chromosomal rearrangements, allowing the investigation of fusion oncogenes in the endogenous context. The successful transfer to the target cell type represents the only limitation. With the aim to elucidate the transformative nature of endogenous MLL-rearrangements in primary human HSPCs, we developed and advanced an all-in-one lentiviral CRISPR-Cas9 system with two sgRNA expression cassettes (LentiCRISPR-CT2.0). The improved lentiviral architecture with additional viral enhancer elements yielded a vector capable of producing higher-titer virus (2.5-fold; p=080%) to generate the t(11;19)/MLL-ENL translocation. T7 endonuclease assays for the top five off-target sites and the on-target sites of our pre-selected sgRNAs verified high on-target and no detectable off-target activity at the endogenous loci. Dual sgRNA expression from a H1 promoter in combination with a U6 promoter was incorporated thereby establishing an efficient, recombination- and off-target-free all-in-one lentiviral CRISPR-Cas9 system for induction of chromosomal rearrangements. Based on these results, we tested generation of chromosomal rearrangements in hematopoietic cell lines. MLL-ENL transcript and the genomic breakpoint were robustly detectable in the transduced bulk population (K562 cells). To determine the impact of endogenous MLL-ENL on HSPCs, we transduced cord blood derived CD34+ HSPCs. In three independent experiments using methylcellulose-based colony-forming assays, MLL-ENL expression was detectable, resulting in a rearrangement efficacy of at least 1.58x10-3 (detection/total colony number). MLL-ENL containing cells, verified on DNA and RNA level, had an extended -but not unlimited- replating capacity. Our experiments thus provide strong evidence that endogenous MLL-ENL translocations provide a growth advantage and limited self-renewal to human HSPCs. These findings were further supported by clonal outgrowth in one out of two experiments performed in liquid culture. Transformation by MLL-rearrangements is guided by up-regulation of HOXA genes and their co-factors MEIS1 and PBX3. In line with these findings, MLL-ENL harboring cells showed robust up-regulation of HOXA9, HOXA10, MEIS1, and PBX3. Interestingly, genes associated with leukemic stem cell activity (CBX5, HMGB3, MYBL2) after retroviral MLL fusion gene expression in mice, were found down-regulated in our study. This finding highlights crucial differences to the previous, retrovirus-based studies in mice and the need to study chromosomal rearrangements at their endogenous locus in the primary human cell context. With the results of our in vitro studies, we next aimed to interrogate the transforming capacity of endogenous MLL-rearrangements in vivo. CD34+ HSPCs, freshly transduced with the LentiCRISPR-CT2.0, were transplanted into immunodeficient mice. Detection of MLL-ENL genomic breakpoints in the mice (8 weeks post transplant) strongly supports our in vitro findings of successful HSPC modification and underlines the power of our approach. Further follow up of our in vivo studies will yield new insights on clonal behavior and downstream events of endogenous MLL-rearrangements in human HSPCs. In aggregate, our study uncovers the oncogenic potency and limitations of endogenous MLL translocations in human HSPCs and highlights the power of the CRISPR-Cas9 system to generate precise cancer models, which will allow us to test the efficacy of targeted therapies, and to investigate the mechanisms of drug resistance in vitro and in vivo. Disclosures Charpentier: CRISPR Therapeutics AG: Other: Co-founder of CRISPR Therapeutics AG and a member of the scientific advisory board of CRISPR Therapeutics AG and Horizon Discovery Group..

The assessment of ERα, PgR and HER2 status is routinely performed today to determine the endocrine responsiveness of breast cancer samples. Such determination is usually accomplished by means of immunohistochemistry and in case of HER2 amplification by means of fluorescent in situ hybridization (FISH). The analysis of these markers can be improved by simultaneous measurements using quantitative real-time PCR (Qrt-PCR). In this study we compared Qrt-PCR results for the assessment of mRNA levels of ERα, PgR, and the members of the human epidermal growth factor receptor family, HER1, HER2, HER3 and HER4. The results were obtained in two independent laboratories using two different methods, SYBR Green I and TaqMan probes, and different primers. By linear regression we demonstrated a good concordance for all six markers. The quantitative mRNA expression levels of ERα, PgR and HER2 also strongly correlated with the respective quantitative protein expression levels prospectively detected by EIA in both laboratories. In addition, HER2 mRNA expression levels correlated well with gene amplification detected by FISH in the same biopsies. Our results indicate that both Qrt-PCR methods were robust and sensitive tools for routine diagnostics and consistent with standard methodologies. The developed simultaneous assessment of several biomarkers is fast and labor effective and allows optimization of the clinical decision-making process in breast cancer tissue and/or core biopsies.

Genome editing with the CRISPR–Cas9 system has enabled unprecedented efficacy for reverse genetics and gene correction approaches. While off-target effects have been successfully tackled, the effort to eliminate variability in sgRNA efficacies—which affect experimental sensitivity—is in its infancy. To address this issue, studies have analyzed the molecular features of highly active sgRNAs, but independent cross-validation is lacking. Utilizing fluorescent reporter knock-out assays with verification at selected endogenous loci, we experimentally quantified the target efficacies of 430 sgRNAs. Based on this dataset we tested the predictive value of five recently-established prediction algorithms. Our analysis revealed a moderate correlation (r = 0.04 to r = 0.20) between the predicted and measured activity of the sgRNAs, and modest concordance between the different algorithms. We uncovered a strong PAM-distal GC-content-dependent activity, which enabled the exclusion of inactive sgRNAs. By deriving nine additional predictive features we generated a linear model-based discrete system for the efficient selection (r = 0.4) of effective sgRNAs (CRISPRater). We proved our algorithms’ efficacy on small and large external datasets, and provide a versatile combined on- and off-target sgRNA scanning platform. Altogether, our study highlights current issues and efforts in sgRNA efficacy prediction, and provides an easily-applicable discrete system for selecting efficient sgRNAs.

Summary Myeloid leukemia in Down syndrome (ML-DS) clonally evolves from transient abnormal myelopoiesis (TAM), a preleukemic condition in DS newborns. To define mechanisms of leukemic transformation, we combined exome and targeted resequencing of 111 TAM and 141 ML-DS samples with functional analyses. TAM requires trisomy 21 and truncating mutations in GATA1; additional TAM variants are usually not pathogenic. By contrast, in ML-DS, clonal and subclonal variants are functionally required. We identified a recurrent and oncogenic hotspot gain-of-function mutation in myeloid cytokine receptor CSF2RB. By a multiplex CRISPR/Cas9 screen in an in vivo murine TAM model, we tested loss-of-function of 22 recurrently mutated ML-DS genes. Loss of 18 different genes produced leukemias that phenotypically, genetically, and transcriptionally mirrored ML-DS.