The ribosomal protein S6 kinase alpha-1 (RPS6KA1) induces resistance to venetoclax/azacitidine in acute myeloid leukemia.

Venetoclax/azacitidine combination therapy is effective in acute myeloid leukemia (AML) and tolerable for older, multimorbid patients. Despite promising response rates, many patients do not achieve sustained remission or are upfront refractory. Identification of resistance mechanisms and additional therapeutic targets represent unmet clinical needs. By using a genome-wide CRISPR/Cas9 library screen targeting 18,053 protein- coding genes in a human AML cell line, various genes conferring resistance to combined venetoclax/azacitidine treatment were identified. The ribosomal protein S6 kinase A1 (RPS6KA1) was among the most significantly depleted sgRNA-genes in venetoclax/azacitidine- treated AML cells. Addition of the RPS6KA1 inhibitor BI-D1870 to venetoclax/azacitidine decreased proliferation and colony forming potential compared to venetoclax/azacitidine alone. Furthermore, BI-D1870 was able to completely restore the sensitivity of OCI-AML2 cells with acquired resistance to venetoclax/azacitidine. Analysis of cell surface markers revealed that RPS6KA1 inhibition efficiently targeted monocytic blast subclones as a potential source of relapse upon venetoclax/azacitidine treatment. Taken together, our results suggest RPS6KA1 as mediator of resistance towards venetoclax/azacitidine and additional RPS6KA1 inhibition as strategy to prevent or overcome resistance.

SAM-Competitive EZH2-Inhibitors Induce Platinum Resistance by EZH2-Independent Induction of ABC-Transporters.

T-cell lymphomas are heterogeneous and rare lymphatic malignancies with unfavorable prognosis. Consequently, new therapeutic strategies are needed. The enhancer of zeste homologue 2 (EZH2) is the catalytic subunit of the polycomb repressive complex 2 and responsible for lysine 27 trimethylation of histone 3. EZH2 is overexpressed in several tumor entities including T-cell neoplasms leading to epigenetic and consecutive oncogenic dysregulation. Thus, pharmacological EZH2 inhibition is a promising target and its clinical evaluation in T-cell lymphomas shows favorable results. We have investigated EZH2 expression in two cohorts of T-cell lymphomas by mRNA-profiling and immunohistochemistry, both revealing overexpression to have a negative impact on patients' prognosis. Furthermore, we have evaluated EZH2 inhibition in a panel of leukemia and lymphoma cell lines with a focus on T-cell lymphomas characterized for canonical EZH2 signaling components. The cell lines were treated with the inhibitors GSK126 or EPZ6438 that inhibit EZH2 specifically by competitive binding at the S-adenosylmethionine (SAM) binding site in combination with the common second-line chemotherapeutic oxaliplatin. The change in cytotoxic effects under pharmacological EZH2 inhibition was evaluated revealing a drastic increase in oxaliplatin resistance after 72 h and longer periods of combinational incubation. This outcome was independent of cell type but associated to reduced intracellular platinum. Pharmacological EZH2 inhibition revealed increased expression in SRE binding proteins, SREBP1/2 and ATP binding cassette subfamily G transporters ABCG1/2. The latter are associated with chemotherapy resistance due to increased platinum efflux. Knockdown experiments revealed that this was independent of the EZH2 functional state. The EZH2 inhibition effect on oxaliplatin resistance and efflux was reduced by additional inhibition of the regulated target proteins. In conclusion, pharmacological EZH2 inhibition is not suitable in combination with the common chemotherapeutic oxaliplatin in T-cell lymphomas revealing an EZH2-independent off-target effect.

Clonal hematopoiesis with DNMT3A and PPM1D mutations impairs regeneration in autologous stem cell transplant recipients.

Clonal hematopoiesis (CH) is an age-related condition driven by stem and progenitor cells harboring recurrent mutations linked to myeloid neoplasms. Currently, potential effects on hematopoiesis, stem cell function and regenerative potential under stress conditions are unknown. We performed targeted DNA sequencing of 457 hematopoietic stem cell grafts collected for autologous stem cell transplantation (ASCT) in myeloma patients and correlated our findings with high-dimensional longitudinal clinical and laboratory data (26,510 data points for blood cell counts/serum values in 25 days around transplantation). We detected CHrelated mutations in 152 patients (33.3%). Since many patients (n=54) harbored multiple CH mutations in one or more genes, we applied a non-negative matrix factorization (NMF) clustering algorithm to identify genes that are commonly co-mutated in an unbiased approach. Patients with CH were assigned to one of three clusters (C1-C3) and compared to patients without CH (C0) in a gene specific manner. To study the dynamics of blood cell regeneration following ASCT, we developed a time-dependent linear mixed effect model to validate differences in blood cell count trajectories amongst different clusters. The results demonstrated that C2, composed of patients with DNMT3A and PPM1D single and co-mutated CH, correlated with reduced stem cell yields and delayed platelet count recovery following ASCT. Also, the benefit of maintenance therapy was particularly strong in C2 patients. Taken together, these data indicate an impaired regenerative potential of hematopoietic stem cell grafts harboring CH with DNMT3A and PPM1D mutations.

Aspirin use and bleeding events during thrombocytopenia after autologous stem-cell transplantation for multiple myeloma.

Background: In patients with cardiovascular (CV) comorbidities that necessitate antiplatelet therapy (APT), its optimal management during chemotherapy-induced thrombocytopenia remains elusive, as the risk of bleeding has to be balanced against the risk of CV events. The purpose of this study was to assess the risk for bleeding with APT during thrombocytopenia in patients with multiple myeloma undergoing high-dose chemotherapy and subsequent autologous stem-cell transplantation (ASCT) with and without acetylsalicylic acid (ASA) as comedication. Methods: We assessed patients who underwent ASCT at the Heidelberg University Hospital between 2011 and 2020 for bleeding events, management strategies for ASA intake during thrombocytopenia, transfusion requirements, and the occurrence of CV events. Results: There were 57/1,113 patients who continued ASA until at least 1 day after ASCT; thus, a continuous platelet inhibition during thrombocytopenia was assumed. Most of the patients (41/57) continued ASA until they had a platelet count of 20-50/nl. This range reflects the kinetics of thrombocytopenia and nondaily measurements of platelets during ASCT. A tendency toward a higher risk for bleeding events in the ASA group was demonstrated (1.9% (control group) vs. 5.3% (ASA), p = 0.082). The risk factors for bleeding in multivariate analysis were the duration of thrombocytopenia < 50/nl, a history of gastrointestinal bleeding, and diarrhea. The factors predicting the duration of thrombocytopenia were age >60 years, a hematopoietic stem-cell transplantation comorbidity index ≥3, and an impaired bone marrow reserve at admission. CV events occurred in three patients; none of them took ASA or had an indication for APT. Conclusions: The intake of ASA until thrombocytopenia with a platelet count of 20-50/nl appears safe, although an elevated risk cannot be excluded. If ASA is indicated for the secondary prevention of CV events, the evaluation of risk factors for bleeding and a prolonged time of thrombocytopenia before conditioning is crucial to adapt the strategy for ASA intake during thrombocytopenia.

Clonally resolved single-cell multi-omics identifies routes of cellular differentiation in acute myeloid leukemia.

Inter-patient variability and the similarity of healthy and leukemic stem cells (LSCs) have impeded the characterization of LSCs in acute myeloid leukemia (AML) and their differentiation landscape. Here, we introduce CloneTracer, a novel method that adds clonal resolution to single-cell RNA-seq datasets. Applied to samples from 19 AML patients, CloneTracer revealed routes of leukemic differentiation. Although residual healthy and preleukemic cells dominated the dormant stem cell compartment, active LSCs resembled their healthy counterpart and retained erythroid capacity. By contrast, downstream myeloid progenitors constituted a highly aberrant, disease-defining compartment: their gene expression and differentiation state affected both the chemotherapy response and leukemia's ability to differentiate into transcriptomically normal monocytes. Finally, we demonstrated the potential of CloneTracer to identify surface markers misregulated specifically in leukemic cells. Taken together, CloneTracer reveals a differentiation landscape that mimics its healthy counterpart and may determine biology and therapy response in AML.

Reduced proliferation of bone marrow MSC after allogeneic stem cell transplantation is associated with clinical outcome.

Engraftment and differentiation of donor hematopoietic stem cells is decisive for the clinical success of allogeneic stem cell transplantation (alloSCT) and depends on the recipient's bone marrow (BM) niche. A damaged niche contributes to poor graft function after alloSCT; however, the underlying mechanisms and the role of BM multipotent mesenchymal stromal cells (MSC) are ill-defined. Upon multivariate analysis in 732 individuals, we observed a reduced presence of proliferation-capable MSC in BM aspirates from patients (N = 196) who had undergone alloSCT. This was confirmed by paired analysis in 30 patients showing a higher frequency of samples with a lack of MSC presence post-alloSCT compared with pre-alloSCT. This reduced MSC presence was associated with reduced survival of patients after alloSCT and specifically with impaired graft function. Post-alloSCT MSC showed diminished in vitro proliferation along with a transcriptional antiproliferative signature, upregulation of epithelial-mesenchymal transition and extracellular matrix pathways, and altered impact on cytokine release upon contact with hematopoietic cells. To avoid in vitro culture bias, we isolated the CD146+/CD45-/HLA-DR- BM cell fraction, which comprised the entire MSC population. The post-alloSCT isolated native CD146+MSC showed a similar reduction in proliferation capacity and shared the same antiproliferative transcriptomic signature as for post-alloSCT colony-forming unit fibroblast-derived MSC. Taken together, our data show that alloSCT confers damage to the proliferative capacity of native MSC, which is associated with reduced patient survival after alloSCT and impaired engraftment of allogeneic hematopoiesis. These data represent the basis to elucidate mechanisms of BM niche reconstitution after alloSCT and its therapeutic manipulation.

A Dynamic rRNA Ribomethylome Drives Stemness in Acute Myeloid Leukemia.

The development and regulation of malignant self-renewal remain unresolved issues. Here, we provide biochemical, genetic, and functional evidence that dynamics in ribosomal RNA (rRNA) 2'-O-methylation regulate leukemia stem cell (LSC) activity in vivo. A comprehensive analysis of the rRNA 2'-O-methylation landscape of 94 patients with acute myeloid leukemia (AML) revealed dynamic 2'-O-methylation specifically at exterior sites of ribosomes. The rRNA 2'-O-methylation pattern is closely associated with AML development stage and LSC gene expression signature. Forced expression of the 2'-O-methyltransferase fibrillarin (FBL) induced an AML stem cell phenotype and enabled engraftment of non-LSC leukemia cells in NSG mice. Enhanced 2'-O-methylation redirected the ribosome translation program toward amino acid transporter mRNAs enriched in optimal codons and subsequently increased intracellular amino acid levels. Methylation at the single site 18S-guanosine 1447 was instrumental for LSC activity. Collectively, our work demonstrates that dynamic 2'-O-methylation at specific sites on rRNAs shifts translational preferences and controls AML LSC self-renewal. SIGNIFICANCE: We establish the complete rRNA 2'-O-methylation landscape in human AML. Plasticity of rRNA 2'-O-methylation shifts protein translation toward an LSC phenotype. This dynamic process constitutes a novel concept of how cancers reprogram cell fate and function. This article is highlighted in the In This Issue feature, p. 247.

Protein tyrosine kinase 2b inhibition reverts niche-associated resistance to tyrosine kinase inhibitors in AML.

FLT3 tyrosine kinase inhibitor (TKI) therapy evolved into a standard therapy in FLT3-mutated AML. TKI resistance, however, develops frequently with poor outcomes. We analyzed acquired TKI resistance in AML cell lines by multilayered proteome analyses. Leupaxin (LPXN), a regulator of cell migration and adhesion, was induced during early resistance development, alongside the tyrosine kinase PTK2B which phosphorylated LPXN. Resistant cells differed in cell adhesion and migration, indicating altered niche interactions. PTK2B and LPXN were highly expressed in leukemic stem cells in FLT3-ITD patients. PTK2B/FAK inhibition abrogated resistance-associated phenotypes, such as enhanced cell migration. Altered pathways in resistant cells, assessed by nascent proteomics, were largely reverted upon PTK2B/FAK inhibition. PTK2B/FAK inhibitors PF-431396 and defactinib synergized with different TKIs or daunorubicin in FLT3-mutated AML. Midostaurin-resistant and AML cells co-cultured with mesenchymal stroma cells responded particularly well to PTK2B/FAK inhibitor addition. Xenograft mouse models showed significant longer time to leukemia symptom-related endpoint upon gilteritinib/defactinib combination treatment in comparison to treatment with either drug alone. Our data suggest that the leupaxin-PTK2B axis plays an important role in acquired TKI resistance in AML. PTK2B/FAK inhibitors act synergistically with currently used therapeutics and may overcome emerging TKI resistance in FLT3-mutated AML at an early timepoint.

Venetoclax synergizes with gilteritinib in FLT3 wild-type high-risk acute myeloid leukemia by suppressing MCL-1.

BCL-2 inhibition has been shown to be effective in acute myeloid leukemia (AML) in combination with hypomethylating agents or low-dose cytarabine. However, resistance and relapse represent major clinical challenges. Therefore, there is an unmet need to overcome resistance to current venetoclax-based strategies. We performed high-throughput drug screening to identify effective combination partners for venetoclax in AML. Overall, 64 antileukemic drugs were screened in 31 primary high-risk AML samples with or without venetoclax. Gilteritinib exhibited the highest synergy with venetoclax in FLT3 wild-type AML. The combination of gilteritinib and venetoclax increased apoptosis, reduced viability, and was active in venetoclax-azacitidine-resistant cell lines and primary patient samples. Proteomics revealed increased FLT3 wild-type signaling in specimens with low in vitro response to the currently used venetoclax-azacitidine combination. Mechanistically, venetoclax with gilteritinib decreased phosphorylation of ERK and GSK3B via combined AXL and FLT3 inhibition with subsequent suppression of the antiapoptotic protein MCL-1. MCL-1 downregulation was associated with increased MCL-1 phosphorylation of serine 159, decreased phosphorylation of threonine 161, and proteasomal degradation. Gilteritinib and venetoclax were active in an FLT3 wild-type AML patient-derived xenograft model with TP53 mutation and reduced leukemic burden in 4 patients with FLT3 wild-type AML receiving venetoclax-gilteritinib off label after developing refractory disease under venetoclax-azacitidine. In summary, our results suggest that combined inhibition of FLT3/AXL potentiates venetoclax response in FLT3 wild-type AML by inducing MCL-1 degradation. Therefore, the venetoclax-gilteritinib combination merits testing as a potentially active regimen in patients with high-risk FLT3 wild-type AML.

CDK7/12/13 inhibition targets an oscillating leukemia stem cell network and synergizes with venetoclax in acute myeloid leukemia.

The heterogeneous response of acute myeloid leukemia (AML) to current anti-leukemic therapies is only partially explained by mutational heterogeneity. We previously identified GPR56 as a surface marker associated with poor outcome across genetic groups, which characterizes two leukemia stem cell (LSC)-enriched compartments with different self-renewal capacities. How these compartments self-renew remained unclear. Here, we show that GPR56+ LSC compartments are promoted in a complex network involving epithelial-to-mesenchymal transition (EMT) regulators besides Rho, Wnt, and Hedgehog (Hh) signaling. Unexpectedly, Wnt pathway inhibition increased the more immature, slowly cycling GPR56+ CD34+ fraction and Hh/EMT gene expression, while Wnt activation caused opposite effects. Our data suggest that the crucial role of GPR56 lies in its ability to co-activate these opposing signals, thus ensuring the constant supply of both LSC subsets. We show that CDK7 inhibitors suppress both LSC-enriched subsets in vivo and synergize with the Bcl-2 inhibitor venetoclax. Our data establish reciprocal transition between LSC compartments as a novel concept underlying the poor outcome in GPR56high AML and propose combined CDK7 and Bcl-2 inhibition as LSC-directed therapy in this disease.

Divergent Effects of EZH1 and EZH2 Protein Expression on the Prognosis of Patients with T-Cell Lymphomas.

T-cell lymphomas are highly heterogeneous and their prognosis is poor under the currently available therapies. Enhancers of zeste homologue 1 and 2 (EZH1/2) are histone H3 lysine-27 trimethyltransferases (H3K27me3). Despite the rapid development of new drugs inhibiting EZH2 and/or EZH1, the molecular interplay of these proteins and the impact on disease progression and prognosis of patients with T-cell lymphomas remains insufficiently understood. In this study, EZH1/2 mutation status was evaluated in 33 monomorphic epitheliotropic intestinal T-cell lymphomas by next generation sequencing and EZH1/2 and H3K27me3 protein expression levels were detected by immunohistochemistry in 46 T-cell lymphomas. Correlations with clinicopathologic features were analyzed and survival curves generated. No EZH1 mutations and one (3%) EZH2 missense mutation were identified. In univariable analysis, high EZH1 expression was associated with an improved overall survival (OS) and progression-free survival (PFS) whereas high EZH2 and H3K27me3 expression were associated with poorer OS and PFS. Multivariable analysis revealed EZH1 (hazard ratio (HR) = 0.183; 95% confidence interval (CI): 0.044-0.767; p = 0.020;) and EZH2 (HR = 8.245; 95% CI: 1.898-35.826; p = 0.005) to be independent, divergent prognostic markers for OS. In conclusion, EZH1/2 protein expression had opposing effects on the prognosis of T-cell lymphoma patients.

Integrated RNAi screening identifies the NEDDylation pathway as a synergistic partner of azacytidine in acute myeloid leukemia.

Treatment of acute myeloid leukemia (AML) remains challenging and novel targets and synergistic therapies still need to be discovered. We performed a high-throughput RNAi screen in three different AML cell lines and primary human leukemic blasts to identify genes that synergize with common antileukemic therapies. We used a pooled shRNA library that covered 5043 different genes and combined transfection with exposure to either azacytidine or cytarabine analog to the concept of synthetic lethality. Suppression of the chemokine CXCL12 ranked highly among the candidates of the cytarabine group. Azacytidine in combination with suppression of genes within the neddylation pathway led to synergistic results. NEDD8 and RBX1 inhibition by the small molecule inhibitor pevonedistat inhibited leukemia cell growth. These findings establish an in vitro synergism between NEDD8 inhibition and azacytidine in AML. Taken together, neddylation constitutes a suitable target pathway for azacytidine combination strategies.

Hotspot DNMT3A mutations in clonal hematopoiesis and acute myeloid leukemia sensitize cells to azacytidine via viral mimicry response.

Somatic mutations in DNA methyltransferase 3A (DNMT3A) are among the most frequent alterations in clonal hematopoiesis (CH) and acute myeloid leukemia (AML), with a hotspot in exon 23 at arginine 882 (DNMT3AR882). Here, we demonstrate that DNMT3AR882H-dependent CH and AML cells are specifically susceptible to the hypomethylating agent azacytidine (AZA). Addition of AZA to chemotherapy prolonged AML survival solely in individuals with DNMT3AR882 mutations, suggesting its potential as a predictive marker for AZA response. AML and CH mouse models confirmed AZA susceptibility specifically in DNMT3AR882H-expressing cells. Hematopoietic stem cells (HSCs) and progenitor cells expressing DNMT3AR882H exhibited cell autonomous viral mimicry response as a result of focal DNA hypomethylation at retrotransposon sequences. Administration of AZA boosted hypomethylation of retrotransposons specifically in DNMT3AR882H-expressing cells and maintained elevated levels of canonical interferon-stimulated genes (ISGs), thus leading to suppressed protein translation and increased apoptosis.

NOP10 predicts lung cancer prognosis and its associated small nucleolar RNAs drive proliferation and migration.

Non-small cell lung cancer (NSCLC) is the leading cause of cancer death worldwide underlining the urgent need for new biomarkers and therapeutic targets for this disease. Long noncoding RNAs are critical players in NSCLC but the role of small RNA species is not well understood. In the present study, we investigated the role of H/ACA box small nucleolar RNAs (snoRNAs) and snoRNA-bound ribonucleoproteins (snoRNPs) in the tumorigenesis of NSCLC. H/ACA box snoRNPs including the NOP10 core protein were highly expressed in NSCLC. High levels of either NOP10 mRNA or protein were associated with poor prognosis in NSCLC patients. Loss of NOP10 and subsequent reduction of H/ACA box snoRNAs and rRNA pseudouridylation inhibited lung cancer cell growth, colony formation, migration, and invasion. A focused CRISPR/Cas9 snoRNA knockout screen revealed that genomic deletion of SNORA65, SNORA7A, and SNORA7B reduced proliferation of lung cancer cells. In line, high levels of SNORA65, SNORA7A, and SNORA7B were observed in primary lung cancer specimens with associated changes in rRNA pseudouridylation. Knockdown of either SNORA65 or SNORA7A/B inhibited growth and colony formation of NSCLC cell lines. Our data indicate that specific H/ACA box snoRNAs and snoRNA-associated proteins such as NOP10 have an oncogenic role in NSCLC providing new potential biomarkers and therapeutic targets for the disease.

Site-specific methylation of 18S ribosomal RNA by SNORD42A is required for acute myeloid leukemia cell proliferation.

Noncoding RNAs, including small nucleolar RNAs (snoRNAs), play important roles in leukemogenesis, but the relevant mechanisms remain incompletely understood. We performed snoRNA-focused CRISPR-Cas9 knockout library screenings that targeted the entire snoRNAnome and corresponding host genes. The C/D box containing SNORD42A was identified as an essential modulator for acute myeloid leukemia (AML) cell survival and proliferation in multiple human leukemia cell lines. In line, SNORD42A was consistently expressed at higher levels in primary AML patient samples than in CD34+ progenitors, monocytes, and granulocytes. Functionally, knockout of SNORD42A reduced colony formation capability and inhibited proliferation. The SNORD42A acts as a C/D box snoRNA and directs 2'-O-methylation at uridine 116 of 18S ribosomal RNA (rRNA). Deletion of SNORD42A decreased 18S-U116 2'-O-methylation, which was associated with a specific decrease in the translation of ribosomal proteins. In line, the cell size of SNORD42A deletion carrying leukemia cells was decreased. Taken together, these findings establish that high-level expression of SNORD42A with concomitant U116 18S rRNA 2'-O-methylation is essential for leukemia cell growth and survival.

Hepatic leukemia factor is a novel leukemic stem cell regulator in DNMT3A, NPM1, and FLT3-ITD triple-mutated AML.

FLT3, DNMT3A, and NPM1 are the most frequently mutated genes in cytogenetically normal acute myeloid leukemia (AML), but little is known about how these mutations synergize upon cooccurrence. Here we show that triple-mutated AML is characterized by high leukemia stem cell (LSC) frequency, an aberrant leukemia-specific GPR56 highCD34low immunophenotype, and synergistic upregulation of Hepatic Leukemia Factor (HLF). Cell sorting based on the LSC marker GPR56 allowed isolation of triple-mutated from DNMT3A/NPM1 double-mutated subclones. Moreover, in DNMT3A R882-mutated patients, CpG hypomethylation at the HLF transcription start site correlated with high HLF mRNA expression, which was itself associated with poor survival. Loss of HLF via CRISPR/Cas9 significantly reduced the CD34+GPR56+ LSC compartment of primary human triple-mutated AML cells in serial xenotransplantation assays. HLF knockout cells were more actively cycling when freshly harvested from mice, but rapidly exhausted when reintroduced in culture. RNA sequencing of primary human triple-mutated AML cells after shRNA-mediated HLF knockdown revealed the NOTCH target Hairy and Enhancer of Split 1 (HES1) and the cyclin-dependent kinase inhibitor CDKN1C/p57 as novel targets of HLF, potentially mediating these effects. Overall, our data establish HLF as a novel LSC regulator in this genetically defined high-risk AML subgroup.

appreci8: a pipeline for precise variant calling integrating 8 tools.

Motivation: The application of next-generation sequencing in research and particularly in clinical routine requires valid variant calling results. However, evaluation of several commonly used tools has pointed out that not a single tool meets this requirement. False positive as well as false negative calls necessitate additional experiments and extensive manual work. Intelligent combination and output filtration of different tools could significantly improve the current situation. Results: We developed appreci8, an automatic variant calling pipeline for calling single nucleotide variants and short indels by combining and filtering the output of eight open-source variant calling tools, based on a novel artifact- and polymorphism score. Appreci8 was trained on two data sets from patients with myelodysplastic syndrome, covering 165 Illumina samples. Subsequently, appreci8's performance was tested on five independent data sets, covering 513 samples. Variation in sequencing platform, target region and disease entity was considered. All calls were validated by re-sequencing on the same platform, a different platform or expert-based review. Sensitivity of appreci8 ranged between 0.93 and 1.00, while positive predictive value ranged between 0.65 and 1.00. In all cases, appreci8 showed superior performance compared to any evaluated alternative approach. Availability and implementation: Appreci8 is freely available at https://hub.docker.com/r/wwuimi/appreci8/. Sequencing data (BAM files) of the 678 patients analyzed with appreci8 have been deposited into the NCBI Sequence Read Archive (BioProjectID: 388411; https://www.ncbi.nlm.nih.gov/bioproject/PRJNA388411). Supplementary information: Supplementary data are available at Bioinformatics online.

Correction: Patients with Acute Myeloid Leukemia Admitted to Intensive Care Units: Outcome Analysis and Risk Prediction.

[This corrects the article DOI: 10.1371/journal.pone.0160871.].

AML1-ETO requires enhanced C/D box snoRNA/RNP formation to induce self-renewal and leukaemia.

Leukaemogenesis requires enhanced self-renewal, which is induced by oncogenes. The underlying molecular mechanisms remain incompletely understood. Here, we identified C/D box snoRNAs and rRNA 2'-O-methylation as critical determinants of leukaemic stem cell activity. Leukaemogenesis by AML1-ETO required expression of the groucho-related amino-terminal enhancer of split (AES). AES functioned by inducing snoRNA/RNP formation via interaction with the RNA helicase DDX21. Similarly, global loss of C/D box snoRNAs with concomitant loss of rRNA 2'-O-methylation resulted in decreased leukaemia self-renewal potential. Genomic deletion of either C/D box snoRNA SNORD14D or SNORD35A suppressed clonogenic potential of leukaemia cells in vitro and delayed leukaemogenesis in vivo. We further showed that AML1-ETO9a, MYC and MLL-AF9 all enhanced snoRNA formation. Expression levels of C/D box snoRNAs in AML patients correlated closely with in vivo frequency of leukaemic stem cells. Collectively, these findings indicate that induction of C/D box snoRNA/RNP function constitutes an important pathway in leukaemogenesis.