Alterations in Genome Organization in Lymphoma Cell Nuclei due to the Presence of the t(14;18) Translocation.

Chromosomal rearrangements have been shown to alter genome organization, consequently having an impact on gene expression. Studies on certain types of leukemia have shown that gene expression can be exacerbated by the altered nuclear positioning of fusion genes arising from chromosomal translocations. However, studies on lymphoma have been, so far, very limited. The scope of this study was to explore genome organization in lymphoma cells carrying the t(14;18)(q32;q21) rearrangement known to results in over-expression of the BCL2 gene. In order to achieve this aim, we used fluorescence in situ hybridization to carefully map the positioning of whole chromosome territories and individual genes involved in translocation in the lymphoma-derived cell line Pfeiffer. Our data show that, although there is no obvious alteration in the positioning of the whole chromosome territories, the translocated genes may take the nuclear positioning of either of the wild-type genes. Furthermore, the BCL2 gene was looping out in a proportion of nuclei with the t(14;18) translocation but not in control nuclei without the translocation, indicating that chromosome looping may be an essential mechanism for BCL2 expression in lymphoma cells.

From FISH to Hi-C: The Chromatin Architecture of the Chromosomal Region 7q36.3, Frequently Rearranged in Leukemic Cells, Is Evolutionary Conserved.

Fluorescence in situ hybridization (FISH) and Hi-C methods are largely used to investigate the three-dimensional organization of the genome in the cell nucleus and are applied here to study the organization of genes (LMBR1, NOM1, MNX1, UBE3C, PTPRN2) localized in the human 7q36.3 band. This region contains the MNX1 gene, which is normally not expressed in human lymphocytes beyond embryonic development. However, this homeobox gene is frequently activated in leukemic cells and its expression is associated with an altered gene positioning in the leukemia cell nuclei. In this study, we used FISH on 3D-preserved nuclei to investigate the nuclear positioning of MNX1 in the leukemia-derived cell line K562. Of the five copies of the MNX1 gene present in K562, four alleles were positioned in the nuclear periphery and only one in the nuclear interior. Using the Juicebox's Hi-C dataset, we identified five chromatin loops in the 7q36.3 band, with different extensions related to the size and orientation of the genes located here, and independent from their expression levels. We identified similar loops in 11 human and three mouse cell lines, showing that these loops are highly conserved in different human cell lines and during evolution. Moreover, the chromatin loop organization is well conserved also during neuronal cell differentiation, showing consistency in genomic organization of this region in development. In this report, we show that FISH and Hi-C are two different approaches that complement one another and together give complete information on the nuclear organization of specific chromosomal regions in different conditions, including cellular differentiation and genetic diseases.

Expression of the checkpoint kinase BUB1 is a predictor of response to cancer therapies.

The identification of clinically-relevant biomarkers is of upmost importance for the management of cancer, from diagnosis to treatment choices. We performed a pan-cancer analysis of the mitotic checkpoint budding uninhibited by benzimidazole 1 gene BUB1, in the attempt to ascertain its diagnostic and prognostic values, specifically in the context of drug response. BUB1 was found to be overexpressed in the majority of cancers, and particularly elevated in clinically aggressive molecular subtypes. Its expression was correlated with clinico-phenotypic features, notably tumour staging, size, invasion, hypoxia, and stemness. In terms of prognostic value, the expression of BUB1 bore differential clinical outcomes depending on the treatment administered in TCGA cancer cohorts, suggesting sensitivity or resistance, depending on the expression levels. We also integrated in vitro drug sensitivity data from public projects based on correlation between drug efficacy and BUB1 expression to produce a list of candidate compounds with differential responses according to BUB1 levels. Gene Ontology enrichment analyses revealed that BUB1 overexpression in cancer is associated with biological processes related to mitosis and chromosome segregation machinery, reflecting the mechanisms of action of drugs with a differential effect based on BUB1 expression.

The mitotic checkpoint kinase BUB1 is a direct and actionable target of MYB in adenoid cystic carcinoma.

Adenoid cystic carcinoma (ACC) is a head and neck cancer that frequently originates in salivary glands, but can also strike other exocrine glands such as the breast. A key molecular alteration found in the majority of ACC cases is MYB gene rearrangements, leading to activation of the oncogenic transcription factor MYB. In this study, we used immortalised breast epithelial cells and an inducible MYB transgene as a model of ACC. Molecular profiling confirmed that MYB-driven gene expression causes a transition into an ACC-like state. Using this new cell model, we identified BUB1 as a targetable kinase directly controlled by MYB, whose pharmacological inhibition caused MYB-dependent synthetic lethality, growth arrest and apoptosis of patient-derived cells and organoids.

Contribution of histone variants to aneuploidy: a cancer perspective.

Histone variants, which generally differ in few amino acid residues, can replace core histones (H1, H2A, H2B, and H3) to confer specific structural and functional features to regulate cellular functions. In addition to their role in DNA packaging, histones modulate key processes such as gene expression regulation and chromosome segregation, which are frequently dysregulated in cancer cells. During the years, histones variants have gained significant attention as gatekeepers of chromosome stability, raising interest in understanding how structural and functional alterations can contribute to tumourigenesis. Beside the well-established role of the histone H3 variant CENP-A in centromere specification and maintenance, a growing body of literature has described mutations, aberrant expression patterns and post-translational modifications of a variety of histone variants in several cancers, also coining the term "oncohistones." At the molecular level, mechanistic studies have been dissecting the biological mechanisms behind histones and missegregation events, with the potential to uncover novel clinically-relevant targets. In this review, we focus on the current understanding and highlight knowledge gaps of the contribution of histone variants to aneuploidy, and we have compiled a database (HistoPloidyDB) of histone gene alterations linked to aneuploidy in cancers of the The Cancer Genome Atlas project.

Mechanisms associated with t(7;12) acute myeloid leukaemia: from genetics to potential treatment targets.

Acute myeloid leukaemia (AML), typically a disease of elderly adults, affects 8 children per million each year, with the highest paediatric incidence in infants aged 0-2 of 18 per million. Recurrent cytogenetic abnormalities contribute to leukaemia pathogenesis and are an important determinant of leukaemia classification. The t(7;12)(q36;p13) translocation is a high-risk AML subtype exclusively associated with infants and represents the second most common abnormality in this age group. Mechanisms of t(7;12) leukaemogenesis remain poorly understood. The translocation relocates the entire MNX1 gene within the ETV6 locus, but a fusion transcript is present in only half of the patients and its significance is unclear. Instead, research has focused on ectopic MNX1 expression, a defining feature of t(7;12) leukaemia, which has nevertheless failed to produce transformation in conventional disease models. Recently, advances in genome editing technologies have made it possible to recreate the t(7;12) rearrangement at the chromosomal level. Together with recent studies of MNX1 involvement using murine in vivo, in vitro, and organoid-based leukaemia models, specific investigation on the biology of t(7;12) can provide new insights into this AML subtype. In this review, we provide a comprehensive up-to-date analysis of the biological features of t(7;12), and discuss recent advances in mechanistic understanding of the disease which may deliver much-needed therapeutic opportunities to a leukaemia of notoriously poor prognosis.

Haematopoietic development and HSC formation in vitro: promise and limitations of gastruloid models.

Haematopoietic stem cells (HSCs) are the most extensively studied adult stem cells. Yet, six decades after their first description, reproducible and translatable generation of HSC in vitro remains an unmet challenge. HSC production in vitro is confounded by the multi-stage nature of blood production during development. Specification of HSC is a late event in embryonic blood production and depends on physical and chemical cues which remain incompletely characterised. The precise molecular composition of the HSC themselves is incompletely understood, limiting approaches to track their origin in situ in the appropriate cellular, chemical and mechanical context. Embryonic material at the point of HSC emergence is limiting, highlighting the need for an in vitro model of embryonic haematopoietic development in which current knowledge gaps can be addressed and exploited to enable HSC production. Gastruloids are pluripotent stem cell-derived 3-dimensional (3D) cellular aggregates which recapitulate developmental events in gastrulation and early organogenesis with spatial and temporal precision. Gastruloids self-organise multi-tissue structures upon minimal and controlled external cues, and are amenable to live imaging, screening, scaling and physicochemical manipulation to understand and translate tissue formation. In this review, we consider the haematopoietic potential of gastruloids and review early strategies to enhance blood progenitor and HSC production. We highlight possible strategies to achieve HSC production from gastruloids, and discuss the potential of gastruloid systems in illuminating current knowledge gaps in HSC specification.

Pan-Cancer Analysis Identifies MNX1 and Associated Antisense Transcripts as Biomarkers for Cancer.

The identification of diagnostic and prognostic biomarkers is a major objective in improving clinical outcomes in cancer, which has been facilitated by the availability of high-throughput gene expression data. A growing interest in non-coding genomic regions has identified dysregulation of long non-coding RNAs (lncRNAs) in several malignancies, suggesting a potential use as biomarkers. In this study, we leveraged data from large-scale sequencing projects to uncover the expression patterns of the MNX1 gene and its associated lncRNAs MNX1-AS1 and MNX1-AS2 in solid tumours. Despite many reports describing MNX1 overexpression in several cancers, limited studies exist on MNX1-AS1 and MNX1-AS2 and their potential as biomarkers. By employing clustering methods to visualise multi-gene relationships, we identified a discriminative power of the three genes in distinguishing tumour vs. normal samples in several cancers of the gastrointestinal tract and reproductive systems, as well as in discerning oesophageal and testicular cancer histological subtypes. Notably, the expressions of MNX1 and its antisenses also correlated with clinical features and endpoints, uncovering previously unreported associations. This work highlights the advantages of using combinatory expression patterns of non-coding transcripts of differentially expressed genes as clinical evaluators and identifies MNX1, MNX1-AS1, and MNX1-AS2 expressions as robust candidate biomarkers for clinical applications.

Engineered model of t(7;12)(q36;p13) AML recapitulates patient-specific features and gene expression profiles.

Acute myeloid leukaemia carrying the translocation t(7;12)(q36;p13) is an adverse-risk leukaemia uniquely observed in infants. Despite constituting up to 30% of cases in under 2-year-olds, it remains poorly understood. Known molecular features are ectopic overexpression of the MNX1 gene and generation of a fusion transcript in 50% of patients. Lack of research models has hindered understanding of t(7;12) biology, which has historically focused on MNX1 overexpression rather than the cytogenetic entity itself. Here, we employed CRISPR/Cas9 to generate t(7;12) in the human K562 cell line, and in healthy CD34+ haematopoietic progenitors where the translocation was not sustained in long-term cultures or through serial replating. In contrast, in K562 cells, t(7;12) was propagated in self-renewing clonogenic assays, with sustained myeloid bias in colony formation and baseline depletion of erythroid signatures. Nuclear localisation analysis revealed repositioning of the translocated MNX1 locus to the interior of t(7;12)-harbouring K562 nuclei - a known phenomenon in t(7;12) patients which associates with ectopic overexpression of MNX1. Crucially, the K562-t(7;12) model successfully recapitulated the transcriptional landscape of t(7;12) patient leukaemia. In summary, we engineered a clinically-relevant model of t(7;12) acute myeloid leukaemia with the potential to unravel targetable molecular mechanisms of disease.

Chromosomal Rearrangements and Altered Nuclear Organization: Recent Mechanistic Models in Cancer.

The last decade has seen significant progress in understanding how the genome is organized spatially within interphase nuclei. Recent analyses have confirmed earlier molecular cytogenetic studies on chromosome positioning within interphase nuclei and provided new information about the topologically associated domains (TADs). Examining the nuances of how genomes are organized within interphase nuclei will provide information fundamental to understanding gene regulation and expression in health and disease. Indeed, the radial spatial positioning of individual gene loci within nuclei has been associated with up- and down-regulation of specific genes, and disruption of normal genome organization within nuclei will result in compromised cellular health. In cancer cells, where reorganization of the nuclear architecture may occur in the presence of chromosomal rearrangements such as translocations, inversions, or deletions, gene repositioning can change their expression. To date, very few studies have focused on radial gene positioning and the correlation to gene expression in cancers. Further investigations would improve our understanding of the biological mechanisms at the basis of cancer and, in particular, in leukemia initiation and progression, especially in those cases where the molecular consequences of chromosomal rearrangements are still unclear. In this review, we summarize the main milestones in the field of genome organization in the nucleus and the alterations to this organization that can lead to cancer diseases.

MLL-Rearranged Acute Leukemia with t(4;11)(q21;q23)-Current Treatment Options. Is There a Role for CAR-T Cell Therapy?

The MLL (mixed-lineage leukemia) gene, located on chromosome 11q23, is involved in chromosomal translocations in a subtype of acute leukemia, which represents approximately 10% of acute lymphoblastic leukemia and 2.8% of acute myeloid leukemia cases. These translocations form fusions with various genes, of which more than 80 partner genes for MLL have been identified. The most recurrent fusion partner in MLL rearrangements (MLL-r) is AF4, mapping at chromosome 4q21, accounting for approximately 36% of MLL-r leukemia and particularly prevalent in MLL-r acute lymphoblastic leukemia (ALL) cases (57%). MLL-r leukemia is associated with a sudden onset, aggressive progression, and notoriously poor prognosis in comparison to non-MLL-r leukemias. Despite modern chemotherapeutic interventions and the use of hematopoietic stem cell transplantations, infants, children, and adults with MLL-r leukemia generally have poor prognosis and response to these treatments. Based on the frequency of patients who relapse, do not achieve complete remission, or have brief event-free survival, there is a clear clinical need for a new effective therapy. In this review, we outline the current therapy options for MLL-r patients and the potential application of CAR-T therapy.

The RS4;11 cell line as a model for leukaemia with t(4;11)(q21;q23): Revised characterisation of cytogenetic features.

BACKGROUND: Haematological malignancies harbouring rearrangements of the KMT2A gene represent a unique subtype of leukaemia, with biphenotypic clinical manifestations, a rapid and aggressive onset, and a generally poor prognosis. Chromosomal translocations involving KMT2A often cause the formation of oncogenic fusion genes, such as the most common translocation t(4;11)(q21;q23) producing the KMT2A-AFF1 chimera. AIM: The aim of this study was to confirm and review the cytogenetic and molecular features of the KMT2A-rearranged RS4;11 cell line and put those in context with other reports of cell lines also harbouring a t(4;11) rearrangement. METHODS AND RESULTS: The main chromosomal rearrangements t(4;11)(q21;q23) and i(7q), described when the cell line was first established, were confirmed by fluorescence in situ hybridisation (FISH) and 24-colour karyotyping by M-FISH. Additional cytogenetic abnormalities were investigated by further FISH experiments, including the presence of trisomy 18 as a clonal abnormality and the discovery of one chromosome 8 being an i(8q), which indicates a duplication of the oncogene MYC. A homozygous deletion of 9p21 containing the tumour-suppressor genes CDKN2A and CDKN2B was also revealed by FISH. The production of the fusion transcript KMT2A-AFF1 arising from the der(11)t(4;11) was confirmed by RT-PCR, but sequencing of the amplified fragment revealed the presence of multiple isoforms. Two transcript variants, resulting from alternative splicing, were identified differing in one glutamine residue in the translated protein. CONCLUSION: As karyotype evolution is a common issue in cell lines, we highlight the need to monitor cell lines in order to re-confirm their characteristics over time. We also reviewed the literature to provide a comparison of key features of several cell lines harbouring a t(4;11). This would guide scientists in selecting the most suitable research model for this particular type of KMT2A-leukaemia.

Deletions of Chromosome 7q Affect Nuclear Organization and HLXB9Gene Expression in Hematological Disorders.

The radial spatial positioning of individual gene loci within interphase nuclei has been associated with up- and downregulation of their expression. In cancer, the genome organization may become disturbed due to chromosomal abnormalities, such as translocations or deletions, resulting in the repositioning of genes and alteration of gene expression with oncogenic consequences. In this study, we analyzed the nuclear repositioning of HLXB9 (also called MNX1), mapping at 7q36.3, in patients with hematological disorders carrying interstitial deletions of 7q of various extents, with a distal breakpoint in 7q36. We observed that HLXB9 remains at the nuclear periphery, or is repositioned towards the nuclear interior, depending upon the compositional properties of the chromosomal regions involved in the rearrangement. For instance, a proximal breakpoint leading the guanine-cytosine (GC)-poor band 7q21 near 7q36 would bring HLXB9 to the nuclear periphery, whereas breakpoints that join the GC-rich band 7q22 to 7q36 would bring HLXB9 to the nuclear interior. This nuclear repositioning is associated with transcriptional changes, with HLXB9 in the nuclear interior becoming upregulated. Here we report an in cis rearrangement, involving one single chromosome altering gene behavior. Furthermore, we propose a mechanistic model for chromatin reorganization that affects gene expression via the influences of new chromatin neighborhoods.

Folate deficiency as predisposing factor for childhood leukaemia: a review of the literature.

BACKGROUND: Folic acid and its derivates, known as folates, are chemoprotective micronutrients of great interest because of their essential role in the maintenance of health and genomic integrity. The supplementation of folic acid during pregnancy has long been known to reduce the risk of neural tube defects (NTDs) in the foetus. Folate metabolism can be altered by many factors, including adequate intake through diet. Folate deficiency can compromise the synthesis, repair and methylation of DNA, with deleterious consequences on genomic stability and gene expression. These processes are known to be altered in chronic diseases, including cancer and cardiovascular diseases. MAIN BODY: This review focuses on the association between folate intake and the risk of childhood leukaemia. Having compiled and analysed studies from the literature, we show the documented effects of folates on the genome and their role in cancer prevention and progression with particular emphasis on DNA methylation modifications. These changes are of crucial importance during pregnancy, as maternal diet has a profound impact on the metabolic and physiological functions of the foetus and the susceptibility to disease in later life. Folate deficiency is capable of modifying the methylation status of certain genes at birth in both animals and humans, with potential pathogenic and tumorigenic effects on the progeny. Pre-existing genetic polymorphisms can modify the metabolic network of folates and influence the risk of cancer, including childhood leukaemias. The protective effects of folic acid might be dose dependent, as excessive folic acid could have the adverse effect of nourishing certain types of tumours. CONCLUSION: Overall, maternal folic acid supplementation before and during pregnancy seems to confer protection against the risk of childhood leukaemia in the offspring. The optimal folic acid requirements and supplementation doses need to be established, especially in conjunction with other vitamins in order to determine the most successful combinations of nutrients to maintain genomic health and wellbeing. Further research is therefore needed to uncover the role of maternal diet as a whole, as it represents a main factor capable of inducing permanent changes in the foetus.