The chromosome 21 kinase DYRK1A: emerging roles in cancer biology and potential as a therapeutic target.

Dual-specificity tyrosine phosphorylation-regulated kinase 1 A (DYRK1A) is a serine/threonine kinase that belongs to the DYRK family of proteins, a subgroup of the evolutionarily conserved CMGC protein kinase superfamily. Due to its localization on chromosome 21, the biological significance of DYRK1A was initially characterized in the pathogenesis of Down syndrome (DS) and related neurodegenerative diseases. However, increasing evidence has demonstrated a prominent role in cancer through its ability to regulate biologic processes including cell cycle progression, DNA damage repair, transcription, ubiquitination, tyrosine kinase activity, and cancer stem cell maintenance. DYRK1A has been identified as both an oncogene and tumor suppressor in different models, underscoring the importance of cellular context in its function. Here, we review mechanistic contributions of DYRK1A to cancer biology and its role as a potential therapeutic target.

AML1-ETO-Related Fusion Circular RNAs Contribute to the Proliferation of Leukemia Cells.

The AML1-ETO (RUNX1-RUNX1T1) fusion gene created by the chromosome translocation t(8;21) (q21;q22) is one of the essential contributors to leukemogenesis. Only a few studies in the literature have focused on fusion gene-derived circular RNAs (f-circRNAs). Here, we report several AML1-ETO-related fusion circular RNAs (F-CircAEs) in AML1-ETO-positive cell lines and primary patient blasts. Functional studies demonstrate that the over-expression of F-CircAE in NIH3T3 cells promotes cell proliferation in vitro and in vivo. F-CircAE expression enhances the colony formation ability of c-Kit+ hematopoietic stem and progenitor cells (HSPCs). Meanwhile, the knockdown of endogenous F-CircAEs can inhibit the proliferation and colony formation ability of AML1-ETO-positive Kasumi-1 cells. Intriguingly, bioinformatic analysis revealed that the glycolysis pathway is down-regulated in F-CircAE-knockdown Kasumi-1 cells and up-regulated in F-CircAE over-expressed NIH3T3 cells. Further studies show that F-CircAE binds to the glycolytic protein ENO-1, up-regulates the expression level of glycolytic enzymes, and enhances lactate production. In summary, our study demonstrates that F-CircAE may exert biological activities on the growth of AML1-ETO leukemia cells by regulating the glycolysis pathway. Determining the role of F-CircAEs in AML1-ETO leukemia can lead to great strides in understanding its pathogenesis, thus providing new diagnostic markers and therapeutic targets.

Cumulus cells of euploid versus whole chromosome 21 aneuploid embryos reveal differentially expressed genes.

RESEARCH QUESTION: Can cumulus cells be used as a non-invasive target for the study of determinants of preimplantation embryo quality? DESIGN: Cumulus cells were collected from monosomy 21, trisomy 21 and euploid embryos and subjected to RNA sequencing analysis and real-time polymerase chain reaction assays. The differential gene expression was analysed for different comparisons. RESULTS: A total of 3122 genes in monosomy 21 cumulus cells and 19 genes in trisomy 21 cumulus cells were differentially expressed compared with euploid cumulus cells. Thirteen of these genes were differentially expressed in both monosomy and trisomy 21, compared with euploid, including disheveled segment polarity protein 2 (DVL2), cellular communication network factor 1 (CCN1/CYR61) and serum response factor (SRF), which have been previously implicated in embryo developmental competence. In addition, ingenuity pathway analysis revealed cell-cell contact function to be affected in both monosomy and trisomy 21 cumulus cells. CONCLUSIONS: These findings support the use of cumulus cell gene expression analysis for the development of biomarkers evaluating oocyte quality for patients undergoing fertility preservation of oocytes.

Mapping the cellular origin and early evolution of leukemia in Down syndrome.

Children with Down syndrome have a 150-fold increased risk of developing myeloid leukemia, but the mechanism of predisposition is unclear. Because Down syndrome leukemogenesis initiates during fetal development, we characterized the cellular and developmental context of preleukemic initiation and leukemic progression using gene editing in human disomic and trisomic fetal hematopoietic cells and xenotransplantation. GATA binding protein 1 (GATA1) mutations caused transient preleukemia when introduced into trisomy 21 long-term hematopoietic stem cells, where a subset of chromosome 21 microRNAs affected predisposition to preleukemia. By contrast, progression to leukemia was independent of trisomy 21 and originated in various stem and progenitor cells through additional mutations in cohesin genes. CD117+/KIT proto-oncogene (KIT) cells mediated the propagation of preleukemia and leukemia, and KIT inhibition targeted preleukemic stem cells.

RUNX1/ETO and mutant KIT both contribute to programming the transcriptional and chromatin landscape in t(8;21) acute myeloid leukemia.

Acute myeloid leukemia development occurs in a stepwise fashion whereby an original driver mutation is followed by additional mutations. The first type of mutations tends to be in genes encoding members of the epigenetic/transcription regulatory machinery (i.e., RUNX1, DNMT3A, TET2), while the secondary mutations often involve genes encoding members of signaling pathways that cause uncontrolled growth of such cells such as the growth factor receptors c-KIT of FLT3. Patients usually present with both types of mutations, but it is currently unclear how both mutational events shape the epigenome in developing AML cells. To this end we generated an in vitro model of t(8;21) AML by expressing its driver oncoprotein RUNX1-ETO with or without a mutated (N822K) KIT protein. Expression of N822K-c-KIT strongly increases the self-renewal capacity of RUNX1-ETO-expressing cells. Global analysis of gene expression changes and alterations in the epigenome revealed that N822K-c-KIT expression profoundly influences the open chromatin landscape and transcription factor binding. However, our experiments also revealed that double mutant cells still differ from their patient-derived counterparts, highlighting the importance of studying patient cells to obtain a true picture of how gene regulatory networks have been reprogrammed during tumorigenesis.

Morphological characteristics, cytogenetic profile, and outcome of RUNX1-RUNX1T1-positive acute myeloid leukemia: Experience of an Indian tertiary care center.

INTRODUCTION: A prototype of good prognosis, t(8;21)-positive AML, has diverse clinical and genetic features which affect its outcome. This study aimed at evaluating the clinico-pathological spectrum of t(8;21)-positive AML and ascertaining prognostic factors influencing its outcome in the Indian subcontinent. METHODS: A retrospective analysis of 75 cases of t(8;21)-positive AML diagnosed over a period of six years (2013-2018) was carried out. Detailed clinical and laboratory data of the patients were collected from the electronic medical records and reviewed. RESULTS: Median age was 19.5 years (range 5-75 years) with a M:F of 1.7. Myeloid sarcoma was observed in 9.3% cases. There were 85% FAB AML-M2, 8% AML-M1, and 7% AML-M4 subtypes. Prominent morphological characteristics included dyspoiesis in maturing myeloid cells (83%), long thin tapered Auer rods (58%), cytoplasmic vacuoles (58%), eosinophilia (50%), and mast cells (22%). Auer rods in maturing granulocytes (4% cases) were highly suggestive of the translocation. Additional cytogenetic abnormalities were present in 53% cases. Seventy-one percent (25/35) achieved CR. The overall survival (OS) was 40%, with a median follow-up of 27 months (range 4-57 months). None of the hematological or cytogenetic factors correlated with OS, except for the presence of myeloid sarcoma which had a trend toward poor survival (P = .07). CONCLUSION: Outcome of t(8;21) AML is not influenced by any of the clinico-pathological parameters, except for a myeloid sarcoma, which may herald a poor prognosis. Recognition of this distinct subtype of AML would facilitate further molecular screening for risk stratification in resource-constrained settings.

RUNX1-ETO Depletion in t(8;21) AML Leads to C/EBPα- and AP-1-Mediated Alterations in Enhancer-Promoter Interaction.

Acute myeloid leukemia (AML) is associated with mutations in transcriptional and epigenetic regulator genes impairing myeloid differentiation. The t(8;21)(q22;q22) translocation generates the RUNX1-ETO fusion protein, which interferes with the hematopoietic master regulator RUNX1. We previously showed that the maintenance of t(8;21) AML is dependent on RUNX1-ETO expression. Its depletion causes extensive changes in transcription factor binding, as well as gene expression, and initiates myeloid differentiation. However, how these processes are connected within a gene regulatory network is unclear. To address this question, we performed Promoter-Capture Hi-C assays, with or without RUNX1-ETO depletion and assigned interacting cis-regulatory elements to their respective genes. To construct a RUNX1-ETO-dependent gene regulatory network maintaining AML, we integrated cis-regulatory element interactions with gene expression and transcription factor binding data. This analysis shows that RUNX1-ETO participates in cis-regulatory element interactions. However, differential interactions following RUNX1-ETO depletion are driven by alterations in the binding of RUNX1-ETO-regulated transcription factors.

ETV6-RUNX1 interacts with a region in SPIB intron 1 to regulate gene expression in pre-B-cell acute lymphoblastic leukemia.

The most frequently occurring genetic abnormality in pediatric B-lymphocyte-lineage acute lymphoblastic leukemia is the t(12;21) chromosomal translocation that results in a ETV6-RUNX1 (also known as TEL-AML1) fusion gene. Expression of ETV6-RUNX1 induces a preleukemic condition leading to acquisition of secondary driver mutations, but the mechanism is poorly understood. SPI-B (encoded by SPIB) is an important transcriptional activator of B-cell development and differentiation. We hypothesized that SPIB is directly transcriptionally repressed by ETV6-RUNX1. Using chromatin immunoprecipitation, we identified a regulatory region in the first intron of SPIB that interacts with ETV6-RUNX1. Mutation of the RUNX1 binding site in SPIB intron 1 prevented transcriptional repression in transient transfection assays. Next, we sought to determine to what extent gene expression in REH cells can be altered by ectopic SPI-B expression. SPI-B expression was forced using CRISPR-mediated gene activation and also using a retroviral vector. Forced expression of SPI-B resulted in altered gene expression and, at high levels, impaired cell proliferation and induced apoptosis. Finally, we identified CARD11 and CDKN1A (encoding p21) as transcriptional targets of SPI-B involved in regulation of proliferation and apoptosis. Taken together, this study identifies SPIB as an important target of ETV6-RUNX1 in regulation of B-cell gene expression in t(12;21) leukemia.

Activin A contributes to the definition of a pro-oncogenic bone marrow microenvironment in t(12;21) preleukemia.

The TEL-AML1 fusion gene, generated by the t(12;21) chromosome translocation, arises in a progenitor/stem cell and could induce clonal expansion of a persistent preleukemic B-cell clone which, on acquisition of secondary alterations, may turn into full-blown leukemia. During infections, deregulated cytokine signaling, including transforming growth factor ß (TGF-ß), can further accelerate this process by creating a protumoral bone marrow (BM) microenvironment. Here, we show that activin A, a member of the TGF-ß family induced under inflammatory conditions, inhibits the proliferation of normal progenitor B cells but not that of preleukemic TEL-AML1-positive clones, thereby providing a selective advantage to the latter. Finally, we find that activin A inhibits BM-derived mesenchymal stromal cell-mediated secretion of CXCL12, a major chemoattractant in the BM compartment, thereby contributing to shape a leukemia-promoting environment. Overall, our findings indicate that activin A, in concert with TGF-ß, could play an important role in the creation of a pro-oncogenic BM microenvironment and provide novel mechanistic insights into TEL-AML1-associated leukemogenesis.

Trisomy silencing by XIST normalizes Down syndrome cell pathogenesis demonstrated for hematopoietic defects in vitro.

We previously demonstrated that an integrated XIST transgene can broadly repress one chromosome 21 in Down syndrome (DS) pluripotent cells. Here we address whether trisomy-silencing can normalize cell function and development sufficiently to correct cell pathogenesis, tested in an in vitro model of human fetal hematopoiesis, for which DS cellular phenotypes are best known. XIST induction in four transgenic clones reproducibly corrected over-production of megakaryocytes and erythrocytes, key to DS myeloproliferative disorder and leukemia. A contrasting increase in neural stem and iPS cells shows cell-type specificity, supporting this approach successfully rebalances the hematopoietic developmental program. Given this, we next used this system to extend knowledge of hematopoietic pathogenesis on multiple points. Results demonstrate trisomy 21 expression promotes over-production of CD43+ but not earlier CD34+/CD43-progenitors and indicates this is associated with increased IGF signaling. This study demonstrates proof-of-principle for this epigenetic-based strategy to investigate, and potentially mitigate, DS developmental pathologies.

Updates in the Pathology of Precursor Lymphoid Neoplasms in the Revised Fourth Edition of the WHO Classification of Tumors of Hematopoietic and Lymphoid Tissues.

PURPOSE OF REVIEW: Acute lymphoblastic leukemias (ALL) are malignant disorders of immature B or T cells that occur characteristically in children, usually under the age of 6 (75%). Approximately 6000 new cases of ALL are diagnosed each year in the USA, 80-85% of which represent B-ALL forms. Most presentations of B-ALL are leukemic, whereas T-ALL presents with a mediastinal mass, with or without leukemic involvement. The revised fourth edition of the World Health Organization (WHO) classification (2017) has introduced some changes in both B and T-ALL. Here, we summarize the categories of lymphoblastic leukemia/lymphomas as defined by the WHO and recent developments in the understanding of this group of hematologic malignancy. RECENT FINDINGS: Two provisional categories of B-ALL have now been identified including B-ALL, BCR-ABL1-like, and B-ALL with iAMP21. The Philadelphia chromosome-like B-ALL includes forms of the disease that shares the expression profiling of B-ALL with t(9;22) but lack such rearrangement. The second one shows amplification of part of the chromosome 21. Both entities are associated with worse prognosis. Within the T-ALL group, an early precursor T cell form has now been introduced as a provisional category. Such group demonstrates expression of stem cell and myeloid markers in conjunction with the T cell antigens. The current review summarizes the recent updates to the WHO classification.

Methylation-associated silencing of BASP1 contributes to leukemogenesis in t(8;21) acute myeloid leukemia.

The AML1-ETO fusion protein (A/E), which results from the t(8;21) translocation, is considered to be a leukemia-initiating event. Identifying the mechanisms underlying the oncogenic activity of A/E remains a major challenge. In this study, we identified a specific down-regulation of brain acid-soluble protein 1 (BASP1) in t(8;21) acute myeloid leukemia (AML). A/E recognized AML1-binding sites and recruited DNA methyltransferase 3a (DNMT3a) to the BASP1 promoter sequence, which triggered DNA methylation-mediated silencing of BASP1. Ectopic expression of BASP1 inhibited proliferation and the colony-forming ability of A/E-positive AML cell lines and led to apoptosis and cell cycle arrest. The DNMT inhibitor decitabine up-regulated the expression of BASP1 in A/E-positive AML cell lines. In conclusion, our data suggest that BASP1 silencing via promoter methylation may be involved in A/E-mediated leukemogenesis and that BASP1 targeting may be an actionable therapeutic strategy in t(8;21) AML.

Elucidating Pathogenic Mechanisms of Early-onset Alzheimer's Disease in Down Syndrome Patients.

Down syndrome (DS) patients demonstrate the neuropathology of Alzheimer's disease (AD) characterized by the formation of senile plaques and neurofibrillary tangles by age 40-50 years. It has been considered for a number of years that 1.5-fold expression of the gene for the amyloid precursor protein (APP) located on chromosome 21 leading to overproduction of amyloid-ß peptide (Aß) results in the early onset of AD in adults with DS. However, the mean age of onset of familial AD with the Swedish mutation on APP which has high affinity for ß-secretase associated with a dramatic increase in Aß production is about 55 years. This paradox indicates that there is a poor correlation between average ages of AD onset and the theoretical amount of Aß production and that there are factors exacerbating AD on chromosome 21. We therefore focused on dual-specificity tyrosine phosphorylation-regulated kinase 1A (DYRK1A), since overexpressing transgenic mice show AD-like brain pathology. The overexpression of DYRK1A caused suppression of the activity of neprilysin (NEP), which is a major Aß-degrading enzyme in the brain, and phosphorylation at the NEP cytoplasmic domain. NEP activity was markedly reduced in fibroblasts derived from DS patients compared with that in fibroblasts derived from healthy controls. This impaired activity of NEP was rescued by DYRK1A inhibition. These results show that DYRK1A overexpression causes suppression of NEP activity through its phosphorylation in DS patients. Our results suggest that DYRK1A inhibitors could be effective against AD not only in adults with DS but also in sporadic AD patients.

Chromosome-wise Protein Interaction Patterns and Their Impact on Functional Implications of Large-Scale Genomic Aberrations.

Gene copy-number changes influence phenotypes through gene-dosage alteration and subsequent changes of protein complex stoichiometry. Human trisomies where gene copy numbers are increased uniformly over entire chromosomes provide generic cases for studying these relationships. In most trisomies, gene and protein level alterations have fatal consequences. We used genome-wide protein-protein interaction data to identify chromosome-specific patterns of protein interactions. We found that some chromosomes encode proteins that interact infrequently with each other, chromosome 21 in particular. We combined the protein interaction data with transcriptome data from human brain tissue to investigate how this pattern of global interactions may affect cellular function. We identified highly connected proteins that also had coordinated gene expression. These proteins were associated with important neurological functions affecting the characteristic phenotypes for Down syndrome and have previously been validated in mouse knockout experiments. Our approach is general and applicable to other gene-dosage changes, such as arm-level amplifications in cancer.

Prognostic Value of miRNA-155 Expression in B-Cell Non-Hodgkin Lymphoma.

OBJECTIVE: MicroRNA-155 (miRNA-155) resides within the B-cell integration cluster gene on chromosome 21. It can act either as an oncogene or as a tumor-suppressor gene, depending on the cell background in which miRNA-155 is performing its specific target gene controlling function. Therefore, the aim of this study was to investigate miRNA-155 expression in patients with B-cell non-Hodgkin lymphoma (NHL) and its relation to disease prognosis in diffuse large B-cell lymphoma (DLBCL) patients. MATERIALS AND METHODS: Reverse transcription-polymerase chain reaction assay was performed to evaluate the expression levels of miRNA-155 in 84 patients with newly diagnosed B-cell NHL and 15 normal controls. RESULTS: Compared with normal controls, miRNA-155 expression was significantly upregulated in patients. Moreover, higher levels of miRNA-155 were associated with the presence of B symptoms, involvement of extranodal sites, and high Eastern Cooperative Oncology Group (ECOG) score. Higher levels of miRNA-155 in DLBCL were associated with non-germinal B-cell-like type, the presence of B symptoms, involvement of extranodal sites, and higher International Prognostic Index (IPI) and ECOG scores. Only the high IPI score and high miRNA-155 expression indicated a higher risk of lower event-free survival using multivariate Cox regression analysis. Our data demonstrated that the expression of miRNA-155 was upregulated in newly diagnosed B-cell NHL patients. miRNA-155 is expressed at a lower level in GCB-subtype DLBCL. Low IPI score and miRNA-155 expression were predictors of longer event-free survival. CONCLUSION: Despite contradicting literature reports, the current findings suggest the potential value of miRNA-155 as a biomarker of prognosis and monitoring in B-cell NHL, and especially that of the DLBCL type.

Expression patterns of the chromosome 21 MicroRNA cluster (miR-99a, miR-125b and let-7c) in chorioamniotic membranes.

Trisomy 21 (T21) is the most common chromosome abnormality in humans and is associated with a spectrum of phenotypes, including cognitive impairment, congenital heart defects and immune system defects. In addition, T21 is also associated with abnormalities of fetal membranes including chorioamniotic separation, delayed fusion of the chorioamniotic membranes, defects in syncytiotrophoblast formation, as well as amniocyte senescence. There is evidence indicating miRNAs encoded by sequences on chromosome 21 (Chr-21) are involved in several of the cognitive and neurological phenotypes of T21, but the role of Chr-21 derived miRNAs in fetal membrane abnormalities associated with T21 has not been investigated. In the current study, we determined the expression patterns of three miRNAs derived from a cluster on Chr-21 - hsa-miR-99a, hsa-miR-125b and hsa-let-7c in chorioamniotic membranes obtained from term pregnancies with spontaneous rupture (n = 20). Tissue and location specific expression patterns within the chorioamniotic membranes were identified. The rupture zone in the choriodecidua had distinct expression patterns compared to other fetal membrane locations. Despite the increased gene dosage associated with T21, the expression of all three miRNAs was reduced in cultured T21 amniocytes as compared to cultured euploid amniocytes. In silico analysis of experimentally validated targets of the three miRNAs suggest these Chr-21 derived miRNAs play a potential role in fetal membrane rupture and the fetal membrane defects associated with T21.

ZBTB7A mutations in acute myeloid leukaemia with t(8;21) translocation.

The t(8;21) translocation is one of the most frequent cytogenetic abnormalities in acute myeloid leukaemia (AML) and results in the RUNX1/RUNX1T1 rearrangement. Despite the causative role of the RUNX1/RUNX1T1 fusion gene in leukaemia initiation, additional genetic lesions are required for disease development. Here we identify recurring ZBTB7A mutations in 23% (13/56) of AML t(8;21) patients, including missense and truncating mutations resulting in alteration or loss of the C-terminal zinc-finger domain of ZBTB7A. The transcription factor ZBTB7A is important for haematopoietic lineage fate decisions and for regulation of glycolysis. On a functional level, we show that ZBTB7A mutations disrupt the transcriptional repressor potential and the anti-proliferative effect of ZBTB7A. The specific association of ZBTB7A mutations with t(8;21) rearranged AML points towards leukaemogenic cooperativity between mutant ZBTB7A and the RUNX1/RUNX1T1 fusion.

A Study of Single Nucleotide Polymorphisms of the SLC19A1/RFC1 Gene in Subjects with Autism Spectrum Disorder.

Autism Spectrum Disorder (ASD) is a group of neurodevelopmental disorders with complex genetic etiology. Recent studies have indicated that children with ASD may have altered folate or methionine metabolism, suggesting that the folate-methionine cycle may play a key role in the etiology of ASD. SLC19A1, also referred to as reduced folate carrier 1 (RFC1), is a member of the solute carrier group of transporters and is one of the key enzymes in the folate metabolism pathway. Findings from multiple genomic screens suggest the presence of an autism susceptibility locus on chromosome 21q22.3, which includes SLC19A1. Therefore, we performed a case-control study in a Japanese population. In this study, DNA samples obtained from 147 ASD patients at the Kanazawa University Hospital in Japan and 150 unrelated healthy Japanese volunteers were examined by the sequence-specific primer-polymerase chain reaction method pooled with fluorescence correlation spectroscopy. p < 0.05 was considered to represent a statistically significant outcome. Of 13 single nucleotide polymorphisms (SNPs) examined, a significant p-value was obtained for AA genotype of one SNP (rs1023159, OR = 0.39, 95% CI = 0.16-0.91, p = 0.0394; Fisher's exact test). Despite some conflicting results, our findings supported a role for the polymorphism rs1023159 of the SLC19A1 gene, alone or in combination, as a risk factor for ASD. However, the findings were not consistent after multiple testing corrections. In conclusion, although our results supported a role of the SLC19A1 gene in the etiology of ASD, it was not a significant risk factor for the ASD samples analyzed in this study.