CircRNAome of Childhood Acute Lymphoblastic Leukemia: Deciphering Subtype-Specific Expression Profiles and Involvement in TCF3::PBX1 ALL.

Childhood B-cell acute lymphoblastic leukemia (B-ALL) is a heterogeneous disease comprising multiple molecular subgroups with subtype-specific expression profiles. Recently, a new type of ncRNA, termed circular RNA (circRNA), has emerged as a promising biomarker in cancer, but little is known about their role in childhood B-ALL. Here, through RNA-seq analysis in 105 childhood B-ALL patients comprising six genetic subtypes and seven B-cell controls from two independent cohorts we demonstrated that circRNAs properly stratified B-ALL subtypes. By differential expression analysis of each subtype vs. controls, 156 overexpressed and 134 underexpressed circRNAs were identified consistently in at least one subtype, most of them with subtype-specific expression. TCF3::PBX1 subtype was the one with the highest number of unique and overexpressed circRNAs, and the circRNA signature could effectively discriminate new patients with TCF3::PBX1 subtype from others. Our results indicated that NUDT21, an RNA-binding protein (RBP) involved in circRNA biogenesis, may contribute to this circRNA enrichment in TCF3::PBX1 ALL. Further functional characterization using the CRISPR-Cas13d system demonstrated that circBARD1, overexpressed in TCF3::PBX1 patients and regulated by NUDT21, might be involved in leukemogenesis through the activation of p38 via hsa-miR-153-5p. Our results suggest that circRNAs could play a role in the pathogenesis of childhood B-ALL.

Characterisation of FLT3 alterations in childhood acute lymphoblastic leukaemia.

BACKGROUND: Alterations of FLT3 are among the most common driver events in acute leukaemia with important clinical implications, since it allows patient classification into prognostic groups and the possibility of personalising therapy thanks to the availability of FLT3 inhibitors. Most of the knowledge on FLT3 implications comes from the study of acute myeloid leukaemia and so far, few studies have been performed in other leukaemias. METHODS: A comprehensive genomic (DNA-seq in 267 patients) and transcriptomic (RNA-seq in 160 patients) analysis of FLT3 in 342 childhood acute lymphoblastic leukaemia (ALL) patients was performed. Mutations were functionally characterised by in vitro experiments. RESULTS: Point mutations (PM) and internal tandem duplications (ITD) were detected in 4.3% and 2.7% of the patients, respectively. A new activating mutation of the TKD, G846D, conferred oncogenic properties and sorafenib resistance. Moreover, a novel alteration involving the circularisation of read-through transcripts (rt-circRNAs) was observed in 10% of the cases. Patients presenting FLT3 alterations exhibited higher levels of the receptor. In addition, patients with ZNF384- and MLL/KMT2A-rearranged ALL, as well as hyperdiploid subtype, overexpressed FLT3. DISCUSSION: Our results suggest that specific ALL subgroups may also benefit from a deeper understanding of the biology of FLT3 alterations and their clinical implications.

Repurposing disulfiram, an alcohol-abuse drug, in neuroblastoma causes KAT2A downregulation and in vivo activity with a water/oil emulsion.

Neuroblastoma, the most common type of pediatric extracranial solid tumor, causes 10% of childhood cancer deaths. Despite intensive multimodal treatment, the outcomes of high-risk neuroblastoma remain poor. We urgently need to develop new therapies with safe long-term toxicity profiles for rapid testing in clinical trials. Drug repurposing is a promising approach to meet these needs. Here, we investigated disulfiram, a safe and successful chronic alcoholism treatment with known anticancer and epigenetic effects. Disulfiram efficiently induced cell cycle arrest and decreased the viability of six human neuroblastoma cell lines at half-maximal inhibitory concentrations up to 20 times lower than its peak clinical plasma level in patients treated for chronic alcoholism. Disulfiram shifted neuroblastoma transcriptome, decreasing MYCN levels and activating neuronal differentiation. Consistently, disulfiram significantly reduced the protein level of lysine acetyltransferase 2A (KAT2A), drastically reducing acetylation of its target residues on histone H3. To investigate disulfiram's anticancer effects in an in vivo model of high-risk neuroblastoma, we developed a disulfiram-loaded emulsion to deliver the highly liposoluble drug. Treatment with the emulsion significantly delayed neuroblastoma progression in mice. These results identify KAT2A as a novel target of disulfiram, which directly impacts neuroblastoma epigenetics and is a promising candidate for repurposing to treat pediatric neuroblastoma.

Distinct transcriptomic profile of small arteries of hypertensive patients with chronic kidney disease identified miR-338-3p targeting GPX3 and PTPRS.

OBJECTIVE: Hypertension is associated with vascular injury, which contributes to end-organ damage. MicroRNAs regulating mRNAs have been shown to play a role in vascular injury in hypertensive mice. We aimed to identify differentially expressed microRNAs and their mRNA targets in small arteries of hypertensive patients with/without chronic kidney disease (CKD) to shed light on the pathophysiological molecular mechanisms of vascular remodeling. METHODS AND RESULTS: Normotensive individuals and hypertensive patients with/without CKD were recruited ( n  = 15-16 per group). Differentially expressed microRNAs and mRNAs were identified uniquely associated with hypertension (microRNAs: 10, mRNAs: 68) or CKD (microRNAs: 68, mRNAs: 395), and in both groups (microRNAs: 2, mRNAs: 32) with a P less than 0.05 and a fold change less than or greater than 1.3 in subcutaneous small arteries ( n  = 14-15). One of the top three differentially expressed microRNAs, miR-338-3p that was down-regulated in CKD, presented the best correlation between RNA sequencing and reverse transcription-quantitative PCR (RT-qPCR, R2  = 0.328, P  < 0.001). Profiling of human aortic vascular cells showed that miR-338-3p was mostly expressed in endothelial cells. Two of the selected top nine up-regulated miR-338-3p predicted targets, glutathione peroxidase 3 ( GPX3 ) and protein tyrosine phosphatase receptor type S ( PTPRS ), were validated with mimics by RT-qPCR in human aortic endothelial cells ( P  < 0.05) and by a luciferase assay in HEK293T cells ( P  < 0.05). CONCLUSION: A distinct transcriptomic profile was observed in gluteal subcutaneous small arteries of hypertensive patients with CKD. Down-regulated miR-338-3p could contribute to GPX3 and PTPRS up-regulation via the canonical microRNA targeting machinery in hypertensive patients with CKD.http://links.lww.com/HJH/C27.

Identification of new ETV6 modulators through a high-throughput functional screening.

ETV6 transcriptional activity is critical for proper blood cell development in the bone marrow. Despite the accumulating body of evidence linking ETV6 malfunction to hematological malignancies, its regulatory network remains unclear. To uncover genes that modulate ETV6 repressive transcriptional activity, we performed a specifically designed, unbiased genome-wide shRNA screen in pre-B acute lymphoblastic leukemia cells. Following an extensive validation process, we identified 13 shRNAs inducing overexpression of ETV6 transcriptional target genes. We showed that the silencing of AKIRIN1, COMMD9, DYRK4, JUNB, and SRP72 led to an abrogation of ETV6 repressive activity. We identified critical modulators of the ETV6 function which could participate in cellular transformation through the ETV6 transcriptional network.

Repurposing proscillaridin A in combination with decitabine against embryonal rhabdomyosarcoma RD cells.

PURPOSE: Embryonal rhabdomyosarcoma (eRMS) is the most common type of rhabdomyosarcoma in children. eRMS is characterized by malignant skeletal muscle cells driven by hyperactivation of several oncogenic pathways including the MYC pathway. Targeting MYC in cancer has been extremely challenging. Recently, we have demonstrated that the heart failure drug, proscillaridin A, produced anticancer effects with specificity toward MYC expressing leukemia cells. We also reported that decitabine, a hypomethylating drug, synergizes with proscillaridin A in colon cancer cells. Here, we investigated whether proscillaridin A exhibits epigenetic and anticancer activity against eRMS RD cells, overexpressing MYC oncogene, and its combination with decitabine. METHODS: We investigated the anticancer effects of proscillaridin A in eRMS RD cells in vitro. In response to drug treatment, we measured growth inhibition, cell cycle arrest, loss of clonogenicity and self-renewal capacity. We further evaluated the impact of proscillaridin A on MYC expression and its downstream transcriptomic effects by RNA sequencing. Then, we measured protein expression of epigenetic regulators and their associated chromatin post-translational modifications in response to drug treatment. Chromatin immunoprecipitation sequencing data sets were coupled with transcriptomic results to pinpoint the impact of proscillaridin A on gene pathways associated with specific chromatin modifications. Lastly, we evaluated the effect of the combination of proscillaridin A and the DNA demethylating drug decitabine on eRMS RD cell growth and clonogenic potential. RESULTS: Clinically relevant concentration of proscillaridin A (5 nM) produced growth inhibition, cell cycle arrest and loss of clonogenicity in eRMS RD cells. Proscillaridin A produced a significant downregulation of MYC protein expression and inhibition of oncogenic transcriptional programs controlled by MYC, involved in cell replication. Interestingly, significant reduction in total histone 3 acetylation and on specific lysine residues (lysine 9, 14, 18, and 27 on histone 3) was associated with significant protein downregulation of a series of lysine acetyltransferases (KAT3A, KAT3B, KAT2A, KAT2B, and KAT5). In addition, proscillaridin A produced synergistic growth inhibition and loss of clonogenicity when combined with the approved DNA demethylating drug decitabine. CONCLUSION: Proscillaridin A produces anticancer and epigenetic effects in the low nanomolar range and its combination with decitabine warrants further investigation for the treatment of eRMS.

Chromosome 2 Fragment Substitutions in Dahl Salt-Sensitive Rats and RNA Sequencing Identified Enpep and Hs2st1 as Vascular Inflammatory Modulators.

Chromosome 2 introgression from normotensive Brown Norway (BN) rats into hypertensive Dahl salt-sensitive (SS) background (SS-chromosome 2BN/Mcwi; consomic S2B) reduced blood pressure and vascular inflammation under a normal-salt diet (NSD). We hypothesized that BN chromosome 2 contains anti-inflammatory genes that could reduce blood pressure and vascular inflammation in rats fed NSD or high-salt diet (HSD). Four- to 6-week old male SS and congenic rats containing the BN chromosome 2 distal portion (SS.BN-[rs13453786-rs66377062]/Aek; S2Ba) and middle segment (SS.BN-[rs106982173-rs65057186]/Aek; S2Bb) were fed NSD or HSD (4% NaCl) up to age 12 to 13 weeks. Systolic blood pressure determined by telemetry was higher in SS rats fed HSD versus NSD. Systolic blood pressure was lower in both congenic rats than in SS under NSD, but similar under HSD versus SS. Reactive oxygen species generation using dihydroethidium staining, expression of vascular cell adhesion molecule-1 and monocyte chemoattractant protein-1, and immune cell infiltration by immunofluorescence demonstrated that S2Ba rats present less inflammation under NSD and more under HSD versus SS rats. RNA sequencing and reverse transcription-quantitative PCR identified 2 differentially expressed genes encoded within BN chromosome 2 distal portion that could act as regulators of vascular inflammation. These were downregulated glutamyl aminopeptidase (Enpep) that was anti-inflammatory under NSD and upregulated heparan sulfate 2-O-sulfotransferase 1 (Hs2st1) that was proinflammatory under HSD. In conclusion, 2 differentially expressed genes encoded within introgressed BN chromosome 2 distal fragment were identified: Enpep associated with reduced vascular inflammation under NSD, and Hs2st1, associated with increased vascular inflammation under HSD.

Role of rs10406069 in miR-5196 in hyperdiploid childhood acute lymphoblastic leukemia.

Aim: To determine the role of single nucleotide polymorphisms (SNPs) in noncoding RNAs in childhood acute lymphoblastic leukemia (ALL) subtypes. Materials & methods: We screened all SNPs in 130 pre-miRNA genes to assess their role in the susceptibility of the most common subtypes of ALL: hyperdiploid and ETV6-RUNX1. Results: In two independent cohorts, we found a significant association between rs10406069 in miR-5196 and the risk of developing hyperdiploid ALL. This observation could be explained by the impact of the SNP on miR-5196 expression and in turn, in its target genes. Indeed, rs10406069 was associated with expression changes in SMC1A, a gene involved in sister chromatin cohesion. Conclusion: rs10406069 in miR-5196 may have a relevant role in hyperdiploid ALL risk.

Single-cell analysis of childhood leukemia reveals a link between developmental states and ribosomal protein expression as a source of intra-individual heterogeneity.

Childhood acute lymphoblastic leukemia (cALL) is the most common pediatric cancer. It is characterized by bone marrow lymphoid precursors that acquire genetic alterations, resulting in disrupted maturation and uncontrollable proliferation. More than a dozen molecular subtypes of variable severity can be used to classify cALL cases. Modern therapy protocols currently cure 85-90% of cases, but other patients are refractory or will relapse and eventually succumb to their disease. To better understand intratumor heterogeneity in cALL patients, we investigated the nature and extent of transcriptional heterogeneity at the cellular level by sequencing the transcriptomes of 39,375 individual cells in eight patients (six B-ALL and two T-ALL) and three healthy pediatric controls. We observed intra-individual transcriptional clusters in five out of the eight patients. Using pseudotime maturation trajectories of healthy B and T cells, we obtained the predicted developmental state of each leukemia cell and observed distribution shifts within patients. We showed that the predicted developmental states of these cancer cells are inversely correlated with ribosomal protein expression levels, which could be a common contributor to intra-individual heterogeneity in cALL patients.

Circulating let-7g-5p and miR-191-5p Are Independent Predictors of Chronic Kidney Disease in Hypertensive Patients.

BACKGROUND: Hypertension (HTN) is associated with target organ damage such as cardiac, vascular, and kidney injury. Several studies have investigated circulating microRNAs (miRNAs) as biomarkers of cardiovascular disease, but few have examined them as biomarker of target organ damage in HTN. We aimed to identify circulating miRNAs that could serve as biomarkers of HTN-induced target organ damage using an unbiased approach. METHODS AND RESULTS: Fifteen normotensive subjects, 16 patients with HTN, 15 with HTN associated with other features of the metabolic syndrome (MetS), and 16 with HTN or chronic kidney disease (CKD) were studied. Circulating RNA extracted from platelet-poor plasma was used for small RNA sequencing. Differentially expressed (DE) genes were identified with a threshold of false discovery rate <0.1. DE miRNAs were identified uniquely associated with HTN, MetS, or CKD. However, only 2 downregulated DE miRNAs (let-7g-5p and miR-191-5p) could be validated by reverse transcription-quantitative PCR. Let-7g-5p was associated with large vessel stiffening, miR-191-5p with MetS, and both miRNAs with estimated glomerular filtration rate (eGFR) and neutrophil and lymphocyte fraction or number and neutrophil-to-lymphocyte ratio. Using the whole population, stepwise multiple linear regression generated a model showing that let-7g-5p, miR-191-5p, and urinary albumin/creatinine ratio predicted eGFR with an adjusted R2 of 0.46 (P = 8.5e-7). CONCLUSIONS: We identified decreased circulating let-7g-5p and miR-191-5p as independent biomarkers of CKD among patients with HTN, which could have pathophysiological and therapeutic implications.

Heart failure drug proscillaridin A targets MYC overexpressing leukemia through global loss of lysine acetylation.

BACKGROUND: Cardiac glycosides are approved for the treatment of heart failure as Na+/K+ pump inhibitors. Their repurposing in oncology is currently investigated in preclinical and clinical studies. However, the identification of a specific cancer type defined by a molecular signature to design targeted clinical trials with cardiac glycosides remains to be characterized. Here, we demonstrate that cardiac glycoside proscillaridin A specifically targets MYC overexpressing leukemia cells and leukemia stem cells by causing MYC degradation, epigenetic reprogramming and leukemia differentiation through loss of lysine acetylation. METHODS: Proscillaridin A anticancer activity was investigated against a panel of human leukemia and solid tumor cell lines with different MYC expression levels, overexpression in vitro systems and leukemia stem cells. RNA-sequencing and differentiation studies were used to characterize transcriptional and phenotypic changes. Drug-induced epigenetic changes were studied by chromatin post-translational modification analysis, expression of chromatin regulators, chromatin immunoprecipitation, and mass-spectrometry. RESULTS: At a clinically relevant dose, proscillaridin A rapidly altered MYC protein half-life causing MYC degradation and growth inhibition. Transcriptomic profile of leukemic cells after treatment showed a downregulation of genes involved in MYC pathways, cell replication and an upregulation of hematopoietic differentiation genes. Functional studies confirmed cell cycle inhibition and the onset of leukemia differentiation even after drug removal. Proscillaridin A induced a significant loss of lysine acetylation in histone H3 (at lysine 9, 14, 18 and 27) and in non-histone proteins such as MYC itself, MYC target proteins, and a series of histone acetylation regulators. Global loss of acetylation correlated with the rapid downregulation of histone acetyltransferases. Importantly, proscillaridin A demonstrated anticancer activity against lymphoid and myeloid stem cell populations characterized by MYC overexpression. CONCLUSION: Overall, these results strongly support the repurposing of proscillaridin A in MYC overexpressing leukemia.

miR-431-5p Knockdown Protects Against Angiotensin II-Induced Hypertension and Vascular Injury.

Vascular injury is an early manifestation in hypertension and a cause of end-organ damage. MicroRNAs play an important role in cardiovascular disease, but their implication in vascular injury in hypertension remains unclear. This study revealed using an unbiased approach, microRNA and mRNA sequencing with molecular interaction analysis, a microRNA-transcription factor coregulatory network involved in vascular injury in mice made hypertensive by 14-day Ang II (angiotensin II) infusion. A candidate gene approach identified upregulated miR-431-5p encoded in the conserved 12qF1 (14q32 in humans) microRNA cluster, whose expression correlated with blood pressure, and which has been shown to be upregulated in human atherosclerosis, as a potential key regulator in Ang II-induced vascular injury. Gain- and loss-of-function in human vascular smooth muscle cells demonstrated that miR-431-5p regulates in part gene expression by targeting ETS homologous factor. In vivo miR-431-5p knockdown delayed Ang II-induced blood pressure elevation and reduced vascular injury in mice, which demonstrated its potential as a target for treatment of hypertension and vascular injury.

Recurrent somatic BRAF insertion (p.V504_R506dup): a tumor marker and a potential therapeutic target in pilocytic astrocytoma.

Pilocytic astrocytoma (PA) is emerging as a tumor entity with dysregulated RAS/RAF/MEK/ERK signaling. In this study, we report the identification of a novel recurrent BRAF insertion (p.V504_R506dup) in five PA cases harboring exclusively this somatic tandem duplication. This recurrent alteration leads to an addition of three amino acids in the kinase domain of BRAF and has functional impact on activating MAPK phosphorylation. Importantly, we show that this mutation confers resistance to RAF inhibitors without changing effectiveness while downstream MEK inhibitors remain effective. Our results further emphasize the importance of BRAF alterations in PA and the need to characterize them in a given tumor as this can affect therapeutic strategies and their potential use as tumor marker in molecular diagnostics.

Very long intergenic non-coding RNA transcripts and expression profiles are associated to specific childhood acute lymphoblastic leukemia subtypes.

Very long intergenic non-coding RNAs (vlincRNAs) are a novel class of long transcripts (~50 kb to 1 Mb) with cell type- or cancer-specific expression. We report the discovery and characterization of 256 vlincRNAs from a cohort of 64 primary childhood pre-B and pre-T acute lymphoblastic leukemia (cALL) samples, of which 61% are novel and specifically expressed in cALL. Validation was performed in 35 pre-B and pre-T cALL primary samples. We show that their expression is cALL immunophenotype and molecular subtype-specific and correlated with epigenetic modifications on their promoters, much like protein-coding genes. While the biological functions of these vlincRNAs are still unknown, our results suggest they could play a role in cALL etiology or progression.

Genome wide mapping of ETV6 binding sites in pre-B leukemic cells.

Genetic alterations in the transcriptional repressor ETV6 are associated with hematological malignancies. Notably, the t(12;21) translocation leading to an ETV6-AML1 fusion gene is the most common genetic alteration found in childhood acute lymphoblastic leukemia. Moreover, most of these patients also lack ETV6 expression, suggesting a tumor suppressor function. To gain insights on ETV6 DNA-binding specificity and genome wide transcriptional regulation capacities, we performed chromatin immunoprecipitation experiments coupled to deep sequencing in a t(12;21)-positive pre-B leukemic cell line. This strategy led to the identification of ETV6-bound regions that were further associated to gene expression. ETV6 binding is mostly cell type-specific as only few regions are shared with other blood cell subtypes. Peaks localization and motif enrichment analyses revealed that this unique binding profile could be associated with the ETV6-AML1 fusion protein specific to the t(12;21) background. This study underscores the complexity of ETV6 binding and uncovers ETV6 transcriptional network in pre-B leukemia cells bearing the recurrent t(12;21) translocation.

Mutational dynamics of early and late relapsed childhood ALL: rapid clonal expansion and long-term dormancy.

Childhood acute lymphoblastic leukemia (cALL) is the most frequent pediatric cancer. Refractory/relapsed cALL presents a survival rate of ∼45% and is still one of the leading causes of death by disease among children. Mechanisms, such as clonal competition and evolutionary adaptation, govern treatment resistance. However, the underlying clonal dynamics leading to multiple relapses and differentiating early (<36 months postdiagnosis) from late relapse events remain elusive. Here, we use an integrative genome-based analysis combined with serial sampling of relapsed tumors (from primary tumor to ≤4 relapse events) from 19 pre-B-cell cALL patients (8 early and 11 late relapses) to assess the fitness of somatic mutations and infer their ancestral relationships. By quantifying both general clonal dynamics and newly acquired subclonal diversity, we show that 2 distinct evolutionary patterns govern early and late relapse: on one hand, a highly dynamic pattern, sustained by a putative defect of DNA repair processes, illustrating the quick emergence of fitter clones, and on the other hand, a quasi-inert evolution pattern, suggesting the escape from dormancy of leukemia stem cells likely spared from initial cytoreductive therapy. These results offer new insights into cALL relapse mechanisms and highlight the pressing need for adapted treatment strategies to circumvent resistance mechanisms.

LncRNAs downregulated in childhood acute lymphoblastic leukemia modulate apoptosis, cell migration, and DNA damage response.

Childhood acute lymphoblastic leukemia (cALL) accounts for 25% of pediatric cancers and is one of the leading causes of disease-related death in children. Although long non-coding RNAs (lncRNAs) have been implicated in cALL etiology, progression, and treatment response, little is known about their exact functional role. We had previously sequenced the whole transcriptome of 56 cALL patients and identified lncRNA transcripts specifically silenced in tumoral cells. Here we investigated the impact of restoring the expression of three of these (RP11-624C23.1, RP11-203E8, and RP11-446E9) in leukemic cell lines had dramatic impact on cancer hallmark cellular phenotypes such as apoptosis, cell proliferation and migration, and DNA damage response. Interestingly, both RP11-624C23.1 and RP11-203E8 had very similar impacts on DNA damage response, specifically displaying lower γ-H2A.X and higher apoptosis levels than control cells in response to genotoxic stress. These results indicate that silencing RP11-624C23.1 or RP11-203E8 could provide a selective advantage to leukemic cells by increasing resistance to genotoxic stress, possibly by modulating the DDR pathway. Since genotoxic agents are fundamental parts of antineoplastic treatment, further investigation of the mechanisms these lncRNAs impact would provide novel and interesting avenues for overcoming treatment resistance.

Specific expression of novel long non-coding RNAs in high-hyperdiploid childhood acute lymphoblastic leukemia.

Pre-B cell childhood acute lymphoblastic leukemia (pre-B cALL) is a heterogeneous disease involving many subtypes typically stratified using a combination of cytogenetic and molecular-based assays. These methods, although widely used, rely on the presence of known chromosomal translocations, which is a limiting factor. There is therefore a need for robust, sensitive, and specific molecular biomarkers unaffected by such limitations that would allow better risk stratification and consequently better clinical outcome. In this study we performed a transcriptome analysis of 56 pre-B cALL patients to identify expression signatures in different subtypes. In both protein-coding and long non-coding RNAs (lncRNA), we identified subtype-specific gene signatures distinguishing pre-B cALL subtypes, particularly in t(12;21) and hyperdiploid cases. The genes up-regulated in pre-B cALL subtypes were enriched in bivalent chromatin marks in their promoters. LncRNAs is a new and under-studied class of transcripts. The subtype-specific nature of lncRNAs suggests they may be suitable clinical biomarkers to guide risk stratification and targeted therapies in pre-B cALL patients.