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.

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.

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.

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.

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.

A childhood acute lymphoblastic leukemia-specific lncRNA implicated in prednisolone resistance, cell proliferation, and migration.

Childhood acute lymphoblastic leukemia (cALL) is the most common pediatric cancer and, despite an 85% cure rate, still represents a major cause of disease-related death in children. Recent studies have implicated long non-coding RNAs (lncRNAs) in cALL etiology, progression, and treatment response. However, barring some exceptions little is known about the functional impact of lncRNAs on cancer biology, which limits their potential as potential therapeutic targets. We wanted to investigate the functional role of lncRNAs identified as specifically overexpressed in pre-B cALL by whole-transcriptome sequencing. Here we report five lncRNAs specifically upregulated in pre-B cALL that had significant impacts on cancer hallmark traits such as cell proliferation, migration, apoptosis, and treatment response. In particular, silencing of the RP11-137H2.4 lncRNA effectively restored normal glucocorticoid (GC) response in a GC-resistant pre-B cALL cell line and specifically modulated expression of members of both the NRAS/BRAF/NF-κB MAPK cascade and cell cycle pathways. Since GC form the cornerstone of cALL chemotherapy and resistance in cALL confers a dismal prognosis, characterizing RP11-137H2.4'sexact role and function in this process will be critical to the development of new therapeutic approaches to overcome GC resistance in children treated for cALL.

SNooPer: a machine learning-based method for somatic variant identification from low-pass next-generation sequencing.

BACKGROUND: Next-generation sequencing (NGS) allows unbiased, in-depth interrogation of cancer genomes. Many somatic variant callers have been developed yet accurate ascertainment of somatic variants remains a considerable challenge as evidenced by the varying mutation call rates and low concordance among callers. Statistical model-based algorithms that are currently available perform well under ideal scenarios, such as high sequencing depth, homogeneous tumor samples, high somatic variant allele frequency (VAF), but show limited performance with sub-optimal data such as low-pass whole-exome/genome sequencing data. While the goal of any cancer sequencing project is to identify a relevant, and limited, set of somatic variants for further sequence/functional validation, the inherently complex nature of cancer genomes combined with technical issues directly related to sequencing and alignment can affect either the specificity and/or sensitivity of most callers. RESULTS: For these reasons, we developed SNooPer, a versatile machine learning approach that uses Random Forest classification models to accurately call somatic variants in low-depth sequencing data. SNooPer uses a subset of variant positions from the sequencing output for which the class, true variation or sequencing error, is known to train the data-specific model. Here, using a real dataset of 40 childhood acute lymphoblastic leukemia patients, we show how the SNooPer algorithm is not affected by low coverage or low VAFs, and can be used to reduce overall sequencing costs while maintaining high specificity and sensitivity to somatic variant calling. When compared to three benchmarked somatic callers, SNooPer demonstrated the best overall performance. CONCLUSIONS: While the goal of any cancer sequencing project is to identify a relevant, and limited, set of somatic variants for further sequence/functional validation, the inherently complex nature of cancer genomes combined with technical issues directly related to sequencing and alignment can affect either the specificity and/or sensitivity of most callers. The flexibility of SNooPer's random forest protects against technical bias and systematic errors, and is appealing in that it does not rely on user-defined parameters. The code and user guide can be downloaded at https://sourceforge.net/projects/snooper/ .

Genomic characterization of pediatric T-cell acute lymphoblastic leukemia reveals novel recurrent driver mutations.

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematologic malignancy with variable prognosis. It represents 15% of diagnosed pediatric ALL cases and has a threefold higher incidence among males. Many recurrent alterations have been identified and help define molecular subgroups of T-ALL, however the full range of events involved in driving transformation remain to be defined. Using an integrative approach combining genomic and transcriptomic data, we molecularly characterized 30 pediatric T-ALLs and identified common recurrent T-ALL targets such as FBXW7, JAK1, JAK3, PHF6, KDM6A and NOTCH1 as well as novel candidate T-ALL driver mutations including the p.R35L missense mutation in splicesome factor U2AF1 found in 3 patients and loss of function mutations in the X-linked tumor suppressor genes MED12 (frameshit mutation p.V167fs, splice site mutation g.chrX:70339329T>C, missense mutation p.R1989H) and USP9X (nonsense mutation p.Q117*). In vitro functional studies further supported the putative role of these novel T-ALL genes in driving transformation. U2AF1 p.R35L was shown to induce aberrant splicing of downstream target genes, and shRNA knockdown of MED12 and USP9X was shown to confer resistance to apoptosis following T-ALL relevant chemotherapy drug treatment in Jurkat leukemia cells. Interestingly, nearly 60% of novel candidate driver events were identified among immature T-ALL cases, highlighting the underlying genomic complexity of pediatric T-ALL, and the need for larger integrative studies to decipher the mechanisms that contribute to its various subtypes and provide opportunities to refine patient stratification and treatment.

CLIC5: a novel ETV6 target gene in childhood acute lymphoblastic leukemia.

The most common rearrangement in childhood precursor B-cell acute lymphoblastic leukemia is the t(12;21)(p13;q22) translocation resulting in the ETV6-AML1 fusion gene. A frequent concomitant event is the loss of the residual ETV6 allele suggesting a critical role for the ETV6 transcriptional repressor in the etiology of this cancer. However, the precise mechanism through which loss of functional ETV6 contributes to disease pathogenesis is still unclear. To investigate the impact of ETV6 loss on the transcriptional network and to identify new transcriptional targets of ETV6, we used whole transcriptome analysis of both pre-B leukemic cell lines and patients combined with chromatin immunoprecipitation. Using this integrative approach, we identified 4 novel direct ETV6 target genes: CLIC5, BIRC7, ANGPTL2 and WBP1L To further evaluate the role of chloride intracellular channel protein CLIC5 in leukemogenesis, we generated cell lines overexpressing CLIC5 and demonstrated an increased resistance to hydrogen peroxide-induced apoptosis. We further described the implications of CLIC5's ion channel activity in lysosomal-mediated cell death, possibly by modulating the function of the transferrin receptor with which it colocalizes intracellularly. For the first time, we showed that loss of ETV6 leads to significant overexpression of CLIC5, which in turn leads to decreased lysosome-mediated apoptosis. Our data suggest that heightened CLIC5 activity could promote a permissive environment for oxidative stress-induced DNA damage accumulation, and thereby contribute to leukemogenesis.