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N6-methyladenosine (m6A) is an abundant internal RNA modification1,2 that is catalysed predominantly by the METTL3–METTL14 methyltransferase complex3,4. The m6A methyltransferase METTL3 has been linked to the initiation and maintenance of acute myeloid leukaemia (AML), but the potential of therapeutic applications targeting this enzyme remains unknown5–7. Here we present the identification and characterization of STM2457, a highly potent and selective first-in-class catalytic inhibitor of METTL3, and a crystal structure of STM2457 in complex with METTL3–METTL14. Treatment of tumours with STM2457 leads to reduced AML growth and an increase in differentiation and apoptosis. These cellular effects are accompanied by selective reduction of m6A levels on known leukaemogenic mRNAs and a decrease in their expression consistent with a translational defect. We demonstrate that pharmacological inhibition of METTL3 in vivo leads to impaired engraftment and prolonged survival in various mouse models of AML, specifically targeting key stem cell subpopulations of AML. Collectively, these results reveal the inhibition of METTL3 as a potential therapeutic strategy against AML, and provide proof of concept that the targeting of RNA-modifying enzymes represents a promising avenue for anticancer therapy.Treatment with a specific inhibitor of the N6-methyladenosine methyltransferase METTL3 leads to reduced growth of cancer cells, indicating the potential of approaches targeting RNA-modifying enzymes for anticancer therapy. [ABSTRACT FROM AUTHOR]

N6-methyladenosine (m6A) is an abundant internal RNA modification in both coding and non-coding RNAs that is catalysed by the METTL3-METTL14 methyltransferase complex. However, the specific role of these enzymes in cancer is still largely unknown. Here we define a pathway that is specific for METTL3 and is implicated in the maintenance of a leukaemic state. We identify METTL3 as an essential gene for growth of acute myeloid leukaemia cells in two distinct genetic screens. Downregulation of METTL3 results in cell cycle arrest, differentiation of leukaemic cells and failure to establish leukaemia in immunodeficient mice. We show that METTL3, independently of METTL14, associates with chromatin and localizes to the transcriptional start sites of active genes. The vast majority of these genes have the CAATT-box binding protein CEBPZ present at the transcriptional start site, and this is required for recruitment of METTL3 to chromatin. Promoter-bound METTL3 induces m6A modification within the coding region of the associated mRNA transcript, and enhances its translation by relieving ribosome stalling. We show that genes regulated by METTL3 in this way are necessary for acute myeloid leukaemia. Together, these data define METTL3 as a regulator of a chromatin-based pathway that is necessary for maintenance of the leukaemic state and identify this enzyme as a potential therapeutic target for acute myeloid leukaemia. [ABSTRACT FROM AUTHOR]

Thep15gene (also known asCDKN2B,INK4B,p15INK4B), located in band 9p21, encodes a protein that induces a G1-phase cell cycle arrest through inhibition of CDK4/6 (cyclin-dependent kinase 4/6). It also plays an important role in the regulation of cellular commitment of hematopoietic progenitor cells and myeloid cell differentiation.p15can be silenced by several mechanisms, including deletion and hypermethylation of its promoter. Homozygousp15deletion is rare in acute myeloblastic leukemia (AML) and myelodysplastic syndromes (MDS) but frequent in acute lymphoblastic leukemia (ALL). On the contrary, methylation of thep15promoter is identified in some 50% of the patients with AML and MDS, but is less frequent in ALL. The analysis of the 28 studies available in the literature revealed conflicting results (unfavorable, favorable or no impact) that can be due, at least in part, to methodological and/or biological pitfalls. Among those, are the heterogeneity of the methylation patterns of thep15gene and the lack of a comprehensive analysis including transcriptional and translational inactivation that have major impact on its expression. Therefore, detection of thep15mRNA expression (quantitative or not) may represent a more appropriate method to determine the prognostic impact of thep15gene. [ABSTRACT FROM AUTHOR]

Summary Acute myeloid leukemia (AML) is an aggressive cancer with a poor prognosis, for which mainstream treatments have not changed for decades. To identify additional therapeutic targets in AML, we optimize a genome-wide clustered regularly interspaced short palindromic repeats (CRISPR) screening platform and use it to identify genetic vulnerabilities in AML cells. We identify 492 AML-specific cell-essential genes, including several established therapeutic targets such as DOT1L , BCL2 , and MEN1 , and many other genes including clinically actionable candidates. We validate selected genes using genetic and pharmacological inhibition, and chose KAT2A as a candidate for downstream study. KAT2A inhibition demonstrated anti-AML activity by inducing myeloid differentiation and apoptosis, and suppressed the growth of primary human AMLs of diverse genotypes while sparing normal hemopoietic stem-progenitor cells. Our results propose that KAT2A inhibition should be investigated as a therapeutic strategy in AML and provide a large number of genetic vulnerabilities of this leukemia that can be pursued in downstream studies. [ABSTRACT FROM AUTHOR]

In 1963, Jean Bernard, a French hematologist, opened a new chapter in hematology called geographic hematology (‘Hématologie Géographique’). He distinguished two research avenues. One dealt with the differences between the various populations (ethnic hematology), the other with various environmental factors (environmental hematology). In recent years, focus has been put on analyzing the genetic susceptibility in cancer and hematological malignancies, particularly in childhood acute lymphoblastic leukemia, using specific gene or (genome-wide association study) approach. However, almost 30 years ago, it was suggested by a few workers that chromosomal abnormalities observed in leukemia could have a geographic and/or ethnic distribution. In this review, we analyze the literature on chromosomal and molecular abnormalities in several types of leukemia. [ABSTRACT FROM PUBLISHER]

The EVI1 gene, located in chromosomal band 3q26, is a transcription factor that has stem cell-specific expression pattern and is essential for the regulation of self-renewal of hematopoietic stem cells. It is now recognized as one of the dominant oncogenes associated with myeloid leukemia. EVI1 overexpression is associated with minimal to no response to chemotherapy and poor clinical outcome. Several chromosomal rearrangements involving band 3q26 are known to induce EVI1 overexpression. They are mainly found in acute myeloid leukemia and blastic phase of Philadelphia chromosome-positive chronic myeloid leukemia, more rarely in myelodysplastic syndromes. They include inv(3)(q21q26), t(3;3)(q21;q26), t(3;21)(q26;q22), t(3;12)(q26;p13) and t(2;3)(p15-23;q26). However, many other chromosomal rearrangements involving 3q26/EVI1 have been identified. The precise molecular event has not been elucidated in the majority of these chromosomal abnormalities and most gene partners remain unknown. [ABSTRACT FROM AUTHOR]

The EVI1 gene, located in chromosomal band 3q26, is a transcription factor that has stem cell-specific expression pattern and is essential for the regulation of self-renewal of hematopoietic stem cells. It is now recognized as one of the dominant oncogenes associated with myeloid leukemia. EVI1 overexpression is associated with minimal to no response to chemotherapy and poor clinical outcome. Several chromosomal rearrangements involving band 3q26 are known to induce EVI1 overexpression. They are mainly found in acute myeloid leukemia and blastic phase of Philadelphia chromosome-positive chronic myeloid leukemia, more rarely in myelodysplastic syndromes. They include inv(3)(q21q26), t(3;3)(q21;q26), t(3;21)(q26;q22), t(3;12)(q26;p13) and t(2;3)(p15-23;q26). However, many other chromosomal rearrangements involving 3q26/ EVI1 have been identified. The precise molecular event has not been elucidated in the majority of these chromosomal abnormalities and most gene partners remain unknown. [ABSTRACT FROM AUTHOR]

Abstract: Chromosomal rearrangements involving the MLL gene have been associated with many different types of hematological malignancies. Fluorescent in situ hybridization with a panel of probes coupled with long distance inverse-PCR was used to identify chromosomal rearrangements involving the MLL gene. Between 1995 and 2010, 27 patients with an acute leukemia were found to have a fusion gene involving MLL. All seven ALL patients with B cell acute lymphoblastic leukemia were characterized by the MLL/AFF1 fusion gene resulting from a translocation (5 patients) or an insertion (2 patients). In the 19 AML patients with acute myeloblastic leukemia, 31.6% of all characterized MLL fusion genes were MLL/MLLT3, 21.1% MLL/ELL, 10.5% MLL/MLLT6 and 10.5% MLL/EPS15. Two patients had rare or undescribed fusion genes, MLL/KIAA0284 and MLL/FLNA. Seven patients (26%) had a complex chromosomal rearrangement (three-way translocations, insertions, deletions) involving the MLL gene. Splicing fusion genes were found in three patients, leading to a MLL/EPS15 fusion in two and a MLL/ELL fusion in a third patient. This study showed that fusion involving the MLL gene can be generated through various chromosomal rearrangements such as translocations, insertions and deletions, some being complex or cryptic. A systematic approach should be used in all cases of acute leukemia starting with FISH analyses using a commercially available MLL split signal probe. Then, the analysis has to be completed, if necessary, by further molecular cytogenetic and genomic PCR methods. [Copyright &y& Elsevier]

Chromosomal rearrangements involving the ABL1 gene, leading to a BCR-ABL1 fusion gene, have been mainly associated with chronic myeloid leukemia and B-cell acute lymphoblastic leukemia (ALL). At present, six other genes have been shown to fuse to ABL1. The kinase domain of ABL1 is retained in all chimeric proteins that are also composed of the N-terminal part of the partner protein that often includes a coiled-coil or a helix-loop-helix domain. These latter domains allow oligomerization of the protein that is required for tyrosine kinase activation, cytoskeletal localization, and neoplastic transformation. Fusion genes that have a break in intron 1 or 2 ( BCR-ABL1, ETV6-ABL1, ZMIZ1-ABL1, EML1-ABL1, and NUP214-ABL1) have transforming activity, although NUP214-ABL1 requires amplification to be efficient. The NUP214-ABL1 gene is the second most prevalent fusion gene involving ABL1 in malignant hemopathies, with a frequency of 5% in T-cell ALL. Both fusion genes ( SFPQ-ABL1 and RCSD1-ABL1) characterized by a break in intron 4 of ABL1 are associated with B-cell ALL, as the chimeric proteins lacked the SH2 domain of ABL1. Screening for ABL1 chimeric genes could be performed in patients with ALL, more particularly in those with T-cell ALL because ABL1 modulates T-cell development and plays a role in cytoskeletal remodeling processes in T cells. [ABSTRACT FROM AUTHOR]

The development of the bacterial artificial chromosome (BAC) systemwas driven in part by the human genome project in order to construct genomic DNA libraries and physicalmaps for genomic sequencing. The availability of BAC clones has become a valuable tool for identifying cancer genes. We report here our experience in identifying genes located at breakpoints of chromosomal rearrangements and in defining the size and boundaries of deletions in hematological diseases. The methodology used in our laboratory consists of a three-step approach using conventional cytogenetics followed by FISH with commercial probes, then BAC clones. One limitation to the BAC system is that it can only accommodate inserts of up to 300 kb. As a consequence, analyzing the extent of deletions requires a large amount of material. Array comparative genomic hybridization (array-CGH) using a BAC/PAC system can be an alternative. However, this technique has limitations also, and it cannot be used to identify candidate genes at breakpoints of chromosomal rearrangements such as translocations, insertions, and inversions. [ABSTRACT FROM AUTHOR]

A letter to the editor pertaining to a case study related to T-cell acute lymphoblastic leukemia in children is presented.

A letter to the editor on RUNX1 gene amplification in B-cell acute lymphoblastic leukemia is presented.

Since the first specific chromosomal abnormality was identified in leukemia more than 50 years ago, technology has much evolved, now allowing the deciphering of cancer genomes in ever-greater detail. However, much has still to be learned as we have not yet completely dissected all the genomic aberrations driving the genesis and the evolution of malignant hemopathies. The first techniques that have been developed allowed 'gross' chromosomal abnormalities to be identified. They include conventional and molecular cytogenetics and microarray-based techniques. However, these techniques can only reveal part of the problem, as genes can be altered in a number of ways (mutations, methylation and so on). This led to the development of what is now known as next-generation sequencing (NGS). Each method has advantages and limits. At present, no single method can decipher all the mechanisms involved in leukemogenesis. Therefore, in our view, it is unlikely that a particular technique will become the 'gold standard'. [ABSTRACT FROM AUTHOR]

A letter to the editor is presented which the multiple mixed lineage leukemia (MLL)-ELL fusion gene in the cases of two infants with acute monoblastic leukemia and myeloid sarcoma (MS).

A letter to the editor is presented regarding myeloid hemopathy as a heterogenous clinical identity of myelodysplastic syndromes (MDS).

The article presents a case study of a 5 month-old boy who was hospitalized for left hemiplegia and right hemianopsia. In scan imaging it was revealed that he had a thrombosis in the right sylvian artery. Also, a diagnosis of myelomonoblastic leukaemia with severe diffuse intravascular coagulopathy was made. The boy was given palliative care thereafter. He died 2 months after the admission.

A letter to the editor is presented in response to the article "A complex 1;19;11 translocation involving the MLL gene in a patient with congenital acute monoblastic leukemia identified by molecular and cytogenetic techniques," by Etienne De Braekeleer and Claus Meyer in the December 28, 2004 issue.

Precise genome editing using CRISPR-Cas9 is a promising therapeutic avenue for genetic diseases, although off-target editing remains a significant safety concern. Guide RNAs shorter than 16 nucleotides in length effectively recruit Cas9 to complementary sites in the genome but do not permit Cas9 nuclease activity. Here we describe CRISPR Guide RNA Assisted Reduction of Damage (CRISPR GUARD) as a method for protecting off-targets sites by co-delivery of short guide RNAs directed against off-target loci by competition with the on-target guide RNA. CRISPR GUARD reduces off-target mutagenesis while retaining on-target editing efficiencies with Cas9 and base editor. However, we discover that short guide RNAs can also support base editing if they contain cytosines within the deaminase activity window. We explore design rules and the universality of this method through in vitro studies and high-throughput screening, revealing CRISPR GUARD as a rapidly implementable strategy to improve the specificity of genome editing for most genomic loci. Finally, we create an online tool for CRISPR GUARD design. Off-target editing remains a concern for therapeutic applications of CRISPR-Cas9. Here the authors present CRISPR GUARD, which uses very short non-cleaving gRNAs to prevent editing at off-target sites. [ABSTRACT FROM AUTHOR]

We recently identified the splicing kinase gene SRPK1 as a genetic vulnerability of acute myeloid leukemia (AML). Here, we show that genetic or pharmacological inhibition of SRPK1 leads to cell cycle arrest, leukemic cell differentiation and prolonged survival of mice transplanted with MLL-rearranged AML. RNA-seq analysis demonstrates that SRPK1 inhibition leads to altered isoform levels of many genes including several with established roles in leukemogenesis such as MYB, BRD4 and MED24. We focus on BRD4 as its main isoforms have distinct molecular properties and find that SRPK1 inhibition produces a significant switch from the short to the long isoform at the mRNA and protein levels. This was associated with BRD4 eviction from genomic loci involved in leukemogenesis including BCL2 and MYC. We go on to show that this switch mediates at least part of the anti-leukemic effects of SRPK1 inhibition. Our findings reveal that SRPK1 represents a plausible new therapeutic target against AML. SRPK1, a kinase involved in splicing regulation, is a potential therapeutic target for AML patients. Here, the authors show that SRPK1 inhibition changes isoform levels of key epigenetic regulators, including BRD4, and it has anti-tumor effects specifically against MLL-rearranged AML cells. [ABSTRACT FROM AUTHOR]

Metastatic progression remains a major burden for cancer patients and is associated with eventual resistance to prevailing therapies such as chemotherapy. Here, we reveal how chemotherapy induces an extracellular matrix (ECM), wound healing, and stem cell network in cancer cells via the c‐Jun N‐terminal kinase (JNK) pathway, leading to reduced therapeutic efficacy. We find that elevated JNK activity in cancer cells is linked to poor clinical outcome in breast cancer patients and is critical for tumor initiation and metastasis in xenograft mouse models of breast cancer. We show that JNK signaling enhances expression of the ECM and stem cell niche components osteopontin, also called secreted phosphoprotein 1 (SPP1), and tenascin C (TNC), that promote lung metastasis. We demonstrate that both SPP1 and TNC are direct targets of the c‐Jun transcription factor. Exposure to multiple chemotherapies further exploits this JNK‐mediated axis to confer treatment resistance. Importantly, JNK inhibition or disruption of SPP1 or TNC expression sensitizes experimental mammary tumors and metastases to chemotherapy, thus providing insights to consider for future treatment strategies against metastatic breast cancer. Synopsis: JNK activity in breast cancer cells induces expression of the ECM and stem cell niche components osteopontin (SPP1) and tenascin C (TNC) to promote metastatic progression. This molecular axis is further induced upon exposure to chemotherapy and limits therapeutic efficacy. JNK activity in breast cancer cells is required for metastatic progression in vivo.JNK signaling drives a molecular network enriched in genes associated with wound healing and developmental processes, as well as mammary stem cell (MaSC) and ECM functions.Upon growth of metastatic nodules, the proportion of cancer cells with active JNK is reduced, but increases again in response to chemotherapy.Breast cancer cells can exploit chemotherapy‐induced JNK signaling to upregulate SPP1 and TNC and undermine the treatment. JNK activity in breast cancer cells induces expression of the ECM and stem cell niche components osteopontin (SPP1) and tenascin C (TNC) to promote metastatic progression. This molecular axis is further induced upon exposure to chemotherapy and limits therapeutic efficacy. [ABSTRACT FROM AUTHOR]

A letter is presented that discusses a probably LMBRD1-CHD6 fusion resulting from a novel translocation in acute myeloid leukemia.

The article reports on research which was conducted to investigate the candidate genes involved in translocation in a 13 year old male patient and a 40 year old female patient with acute myeloid leukemia. After conducting testing on both patients researchers found that recurrent translocation (10;17)(p15;q21) in acute poorly differentiated myeloid leukemia probably led to ZMYND11 – MBTD1 fusion.

The article describes the case of a 5-year-old patient with acute megakaryoblastic leukemia with ETV6 gene and nipped-B-like protein (NIPBL) fusion. Topics explored include the symptoms presented including bilateral cervical adenopathies, anemia, and splenomegaly, the results of the physical and laboratory examinations performed including white blood cell count and immunophenotyping, and the treatment course administered including a combination of cytarabine and mercaptopurine.

The development of the bacterial artificial chromosome (BAC) system was driven in part by the human genome project in order to construct genomic DNA libraries and physical maps for genomic sequencing. The availability of BAC clones has become a valuable tool for identifying cancer genes. We report here our experience in identifying genes located at breakpoints of chromosomal rearrangements and in defining the size and boundaries of deletions in hematological diseases. The methodology used in our laboratory consists of a three-step approach using conventional cytogenetics followed by FISH with commercial probes, then BAC clones. One limitation to the BAC system is that it can only accommodate inserts of up to 300 kb. As a consequence, analyzing the extent of deletions requires a large amount of material. Array comparative genomic hybridization (array-CGH) using a BAC/PAC system can be an alternative. However, this technique has limitations also, and it cannot be used to identify candidate genes at breakpoints of chromosomal rearrangements such as translocations, insertions, and inversions. [ABSTRACT FROM AUTHOR]