Hi-C detects genomic structural variants in peripheral blood of pediatric leukemia patients.

B-cell acute lymphoblastic leukemia (B-ALL) is often driven by chromosome translocations that result in recurrent and well-studied gene fusions. Currently, fluorescent in situ hybridization probes are used to detect candidate translocations in bone marrow samples from B-ALL patients. Recently Hi-C, a sequencing-based technique originally designed to reconstruct the three-dimensional architecture of the nuclear genome, was shown to effectively recognize structural variants. Here, we demonstrate that Hi-C can be used as a genome-wide assay to detect translocations and other structural variants of potential clinical interest. Structural variants were identified in both bone marrow and peripheral blood samples, including an ETV6-RUNX1 translocation present in one pediatric B-ALL patient. Our report provides proof of principle that Hi-C could be an effective strategy to globally detect driver structural variants in B-ALL peripheral blood specimens, reducing the need for invasive bone marrow biopsies and candidate-based clinical tests.

Optimized serial expansion of human induced pluripotent stem cells using low-density inoculation to generate clinically relevant quantities in vertical-wheel bioreactors.

Human induced pluripotent stem cells (hiPSCs) have generated a great deal of attention owing to their capacity for self-renewal and differentiation into the three germ layers of the body. Their discovery has facilitated a new era in biomedicine for understanding human development, drug screening, disease modeling, and cell therapy while reducing ethical issues and risks of immune rejection associated with traditional embryonic stem cells. Bioreactor-based processes have been the method of choice for the efficient expansion and differentiation of stem cells in controlled environments. Current protocols for the expansion of hiPSCs use horizontal impeller, paddle, or rocking wave mixing method bioreactors which require large static cell culture starting populations and achieve only moderate cell fold increases. This study focused on optimizing inoculation, agitation, oxygen, and nutrient availability for the culture of hiPSCs as aggregates in single-use, low-shear, vertical-wheel bioreactors. Under optimized conditions, we achieved an expansion of more than 30-fold in 6 days using a small starting population of cells and minimal media resources throughout. Importantly, we showed that that this optimized bioreactor expansion protocol could be replicated over four serial passages resulting in a cumulative cell expansion of 1.06E6-fold in 28 days. Cells from the final day of the serial passage were of high quality, maintaining a normal karyotype, pluripotent marker staining, and the ability to form teratomas in vivo. These findings demonstrate that a vertical-wheel bioreactor-based bioprocess can provide optimal conditions for efficient, rapid generation of high-quality hiPSCs to meet the demands for clinical manufacturing of therapeutic cell products.

Molecular analysis distinguishes metastatic disease from second cancers in patients with retinoblastoma.

The pediatric ocular tumor retinoblastoma readily metastasizes, but these lesions can masquerade as histologically similar pediatric small round blue cell tumors. Since 98% of retinoblastomas have RB1 mutations and a characteristic genomic copy number "signature", genetic analysis is an appealing adjunct to histopathology to distinguish retinoblastoma metastasis from second primary cancer in retinoblastoma patients. Here, we describe such an approach in two retinoblastoma cases. In patient one, allele-specific (AS)-PCR for a somatic nonsense mutation confirmed that a temple mass was metastatic retinoblastoma. In a second patient, a rib mass shared somatic copy number gains and losses with the primary tumor. For definitive diagnosis, however, an RB1 mutation was needed, but heterozygous promoter→exon 11 deletion was the only RB1 mutation detected in the primary tumor. We used a novel application of inverse PCR to identify the deletion breakpoint. Subsequently, AS-PCR designed for the breakpoint confirmed that the rib mass was metastatic retinoblastoma. These cases demonstrate that personalized molecular testing can confirm retinoblastoma metastases and rule out a second primary cancer, thereby helping to direct the clinical management.

Five patients with a chromosome 1q21.1 triplication show macrocephaly, increased weight and facial similarities.

Recurrent rearrangements of chromosome 1q21.1 that occur as a consequence of non-allelic homologous recombination (NAHR) show considerable variability in phenotypic expression and penetrance. Chromosome 1q21.1 deletions (OMIM 612474) have been associated with microcephaly, intellectual disability, autism, schizophrenia, cardiac abnormalities and cataracts. Phenotypic features in individuals with 1q21.1 duplications (OMIM 612475) include macrocephaly, learning difficulties, developmental delay, intellectual disability and mild dysmorphic features. Half of these patients show autistic behavior. For the first time, we describe five patients, including monozygotic twins, with a triplication of the 1q21.1 chromosomal segment. Facial features common to all patients include a high, broad forehead; a flat and broad nasal bridge; long, downslanted palpebral fissures and dysplastic, low-set ears. Likely associated features include macrocephaly and increased weight. We observed that the triplications arose through different mechanisms in the patients: it was de novo in one patient, inherited from a triplication carrier in two cases, while the father of the twins is a 1q21.1 duplication carrier. The de novo triplication contained copies of both maternal alleles, suggesting it was generated by a combination of inter- and intrachromosomal recombination.

MicroRNA-223 dose levels fine tune proliferation and differentiation in human cord blood progenitors and acute myeloid leukemia.

A precise understanding of the role of miR-223 in human hematopoiesis and in the pathogenesis of acute myeloid leukemia (AML) is still lacking. By measuring miR-223 expression in blasts from 115 AML patients, we found significantly higher miR-223 levels in patients with favorable prognosis, whereas patients with low miR-223 expression levels were associated with worse outcome. Furthermore, miR-223 was hierarchically expressed in AML subpopulations, with lower expression in leukemic stem cell-containing fractions. Genetic depletion of miR-223 decreased the leukemia initiating cell (LIC) frequency in a myelomonocytic AML mouse model, but it was not mandatory for rapid-onset AML. To relate these observations to physiologic myeloid differentiation, we knocked down or ectopically expressed miR-223 in cord-blood CD34⁺ cells using lentiviral vectors. Although miR-223 knockdown delayed myeloerythroid precursor differentiation in vitro, it increased myeloid progenitors in vivo following serial xenotransplantation. Ectopic miR-223 expression increased erythropoiesis, T lymphopoiesis, and early B lymphopoiesis in vivo. These findings broaden the role of miR-223 as a regulator of the expansion/differentiation equilibrium in hematopoietic stem and progenitor cells where its impact is dose- and differentiation-stage-dependent. This also explains the complex yet minor role of miR-223 in AML, a heterogeneous disease with variable degree of myeloid differentiation.

Cell fate decisions in malignant hematopoiesis: leukemia phenotype is determined by distinct functional domains of the MN1 oncogene.

Extensive molecular profiling of leukemias and preleukemic diseases has revealed that distinct clinical entities, like acute myeloid (AML) and T-lymphoblastic leukemia (T-ALL), share similar pathogenetic mutations. It is not well understood how the cell of origin, accompanying mutations, extracellular signals or structural differences in a mutated gene determine the phenotypic identity of leukemias. We dissected the functional aspects of different protein regions of the MN1 oncogene and their effect on the leukemic phenotype, building on the ability of MN1 to induce leukemia without accompanying mutations. We found that the most C-terminal region of MN1 was required to block myeloid differentiation at an early stage, and deletion of an extended C-terminal region resulted in loss of myeloid identity and cell differentiation along the T-cell lineage in vivo. Megakaryocytic/erythroid lineage differentiation was blocked by the N-terminal region. In addition, the N-terminus was required for proliferation and leukemogenesis in vitro and in vivo through upregulation of HoxA9, HoxA10 and Meis2. Our results provide evidence that a single oncogene can modulate cellular identity of leukemic cells based on its active gene regions. It is therefore likely that different mutations in the same oncogene may impact cell fate decisions and phenotypic appearance of malignant diseases.

CHD2 haploinsufficiency is associated with developmental delay, intellectual disability, epilepsy and neurobehavioural problems.

BACKGROUND: The chromodomain helicase DNA binding domain (CHD) proteins modulate gene expression via their ability to remodel chromatin structure and influence histone acetylation. Recent studies have shown that CHD2 protein plays a critical role in embryonic development, tumor suppression and survival. Like other genes encoding members of the CHD family, pathogenic mutations in the CHD2 gene are expected to be implicated in human disease. In fact, there is emerging evidence suggesting that CHD2 might contribute to a broad spectrum of neurodevelopmental disorders. Despite growing evidence, a description of the full phenotypic spectrum of this condition is lacking. METHODS: We conducted a multicentre study to identify and characterise the clinical features associated with haploinsufficiency of CHD2. Patients with deletions of this gene were identified from among broadly ascertained clinical cohorts undergoing genomic microarray analysis for developmental delay, congenital anomalies and/or autism spectrum disorder. RESULTS: Detailed clinical assessments by clinical geneticists showed recurrent clinical symptoms, including developmental delay, intellectual disability, epilepsy, behavioural problems and autism-like features without characteristic facial gestalt or brain malformations observed on magnetic resonance imaging scans. Parental analysis showed that the deletions affecting CHD2 were de novo in all four patients, and analysis of high-resolution microarray data derived from 26,826 unaffected controls showed no deletions of this gene. CONCLUSIONS: The results of this study, in addition to our review of the literature, support a causative role of CHD2 haploinsufficiency in developmental delay, intellectual disability, epilepsy and behavioural problems, with phenotypic variability between individuals.

Cell of origin in AML: susceptibility to MN1-induced transformation is regulated by the MEIS1/AbdB-like HOX protein complex.

Pathways defining susceptibility of normal cells to oncogenic transformation may be valuable therapeutic targets. We characterized the cell of origin and its critical pathways in MN1-induced leukemias. Common myeloid (CMP) but not granulocyte-macrophage progenitors (GMP) could be transformed by MN1. Complementation studies of CMP-signature genes in GMPs demonstrated that MN1-leukemogenicity required the MEIS1/AbdB-like HOX-protein complex. ChIP-sequencing identified common target genes of MN1 and MEIS1 and demonstrated identical binding sites for a large proportion of their chromatin targets. Transcriptional repression of MEIS1 targets in established MN1 leukemias demonstrated antileukemic activity. As MN1 relies on but cannot activate expression of MEIS1/AbdB-like HOX proteins, transcriptional activity of these genes determines cellular susceptibility to MN1-induced transformation and may represent a promising therapeutic target.

HLA-DR(negative), CD34(negative) hypergranular acute myeloid leukemia with trisomy 6 and del(5)(q22q33): case report and review of the literature.

We report a unique pediatric case of hypergranular acute myeloid leukemia with myelodysplasia-related changes. The patient presented with moderate leukocytosis with neutrophilia with left-shift maturation and dysplasia, anemia, and multiple sclerotic bone lesions. The bone marrow was hypercellular with a predominance of myeloblast cells and/or abnormal promyelocytes with hypergranular cytoplasm. Flow cytometric immunophenotyping showed that the leukemic cells were positive for CD13, CD33, and myeloperoxidase, and negative for HLA-DR and CD34. Morphology and immunophenotyping were highly suggestive of acute promyelocytic leukemia. The classic t(15;17) or other RARα rearrangements were not detected by cytogenetic or molecular assays, ruling out acute promyelocytic leukemia. Standard cytogenetic analysis showed that the karyotype of the predominant clone was 47,XY,+6 with evidence of clonal evolution to 47,XY,+6,del(5)(q22q33). A literature and database review showed that trisomy 6 is a rare occurrence in hematological malignancies and, to our knowledge, has never been reported in association with del(5)(q22q33) in a child presenting with hypergranular acute myeloid leukemia with myelodysplasia-related changes. We present a current review of the literature and summarize the clinical features of 57 cases of trisomy 6 as the primary chromosomal abnormality in hematological disease.

Comprehensive analysis of mammalian miRNA* species and their role in myeloid cells.

Processing of pre-miRNA through Dicer1 generates an miRNA duplex that consists of an miRNA and miRNA* strand. Despite the general view that miRNA*s have no functional role, we further investigated miRNA* species in 10 deep-sequencing libraries from mouse and human tissue. Comparisons of miRNA/miRNA* ratios across the miRNA sequence libraries revealed that 50% of the investigated miRNA duplexes exhibited a highly dominant strand. Conversely, 10% of miRNA duplexes showed a comparable expression of both strands, whereas the remaining 40% exhibited variable ratios across the examined libraries, as exemplified by miR-223/miR-223* in murine and human cell lines. Functional analyses revealed a regulatory role for miR-223* in myeloid progenitor cells, which implies an active role for both arms of the miR-223 duplex. This was further underscored by the demonstration that miR-223 and miR-223* targeted the insulin-like growth factor 1 receptor/phosphatidylinositol 3-kinase axis and that high miR-223* levels were associated with increased overall survival in patients with acute myeloid leukemia. Thus, we found a supporting role for miR-223* in differentiating myeloid cells in normal and leukemic cell states. The fact that the miR-223 duplex acts through both arms extends the complexity of miRNA-directed gene regulation of this myeloid key miRNA.

Delineating domains and functions of NUP98 contributing to the leukemogenic activity of NUP98-HOX fusions.

To determine the contribution of the common N-terminal truncation of NUP98 in NUP98-translocations resulting in acute myeloid leukemia, we have conducted a structure-function analysis of NUP98 in the context of NUP98-HOXA10HD, a novel, canonical NUP98-Hox fusion that significantly enhances the self-renewal capacity of hematopoietic stem cells and collaborates with Meis1 to induce AML in our mouse models. Our results identify that NUP98 functions by transcriptional activation likely by recruitment of CBP/p300 via its FG/GLFG repeats. In contrast, the functional interaction of NUP98 with Rae1 or the anaphase promoting complex appears non-essential for its role in NUP98-leukemogenic fusions.

Identification of E74-like factor 1 (ELF1) as a transcriptional regulator of the Hox cofactor MEIS1.

OBJECTIVE: Myeloid ectropic viral integration site 1 (MEIS1) is a Hox cofactor known for its role in development and is strongly linked to normal and leukemic hematopoiesis. Although previous studies have focused on identifying protein partners of MEIS1 and its transcriptionally regulated targets, little is known about the upstream transcriptional regulators of this tightly regulated gene. Understanding the regulation of MEIS1 is important to understanding normal hematopoiesis and leukemogenesis. MATERIALS AND METHODS: Here we describe our studies focusing on the evolutionary conserved putative MEIS1 promoter region. Phylogenetic sequence analysis and reporter assays in MEIS1-expressing (K562) and nonexpressing (HL60) leukemic cell line models were used to identify key regulatory regions and potential transcription factor binding sites within the candidate promoter region followed by functional and expression studies of one identified regulator in both cell lines and primary human cord blood and leukemia samples. RESULTS: Chromatin status of MEIS1 promoter region is associated with MEIS1 expression. Truncation and mutation studies coupled with reporter assays revealed that a conserved ETS family member binding site located 289 bp upstream of the annotated human MEIS1 transcription start site is required for promoter activity. Of the three ETS family members tested, only ELF1 was enriched on the MEIS1 promoter as assessed by both electrophoretic mobility shift assay and chromatin immunoprecipitation experiments in K562. This finding was confirmed in MEIS1-expressing primary human samples. Moreover, small interfering RNA-mediated knockdown of ELF1 in K562 cells was associated with a decreased MEIS1 expression. CONCLUSIONS: We conclude that the ETS transcription factor ELF1 is an important positive regulator of MEIS1 expression.

Linkage of the potent leukemogenic activity of Meis1 to cell-cycle entry and transcriptional regulation of cyclin D3.

MEIS1 is a three-amino acid loop extension class homeodomain-containing homeobox (HOX) cofactor that plays key roles in normal hematopoiesis and leukemogenesis. Expression of Meis1 is rate-limiting in MLL-associated leukemias and potently interacts with Hox and NUP98-HOX genes in leukemic transformation to promote self-renewal and proliferation of hematopoietic progenitors. The oncogenicity of MEIS1 has been linked to its transcriptional activation properties. To further reveal the pathways triggered by Meis1, we assessed the function of a novel engineered fusion form of Meis1, M33-MEIS1, designed to confer transcriptional repression to Meis1 target genes that are otherwise up-regulated in normal and malignant hematopoiesis. Retroviral overexpression of M33-Meis1 resulted in the rapid and complete eradication of M33-Meis1-transduced normal and leukemic cells in vivo. Cell-cycle analysis showed that M33-Meis1 impeded the progression of cells from G(1)-to-S phase, which correlated with significant reduction of cyclin D3 levels and the inhibition of retinoblastoma (pRb) hyperphosphorylation. We identified cyclin D3 as a direct downstream target of MEIS1 and M33-MEIS1 and showed that the G(1)-phase accumulation and growth suppression induced by M33-Meis1 was partially relieved by overexpression of cyclin D3. This study provides strong evidence linking the growth-promoting activities of Meis1 to the cyclin D-pRb cell-cycle control pathway.

Identification of miR-145 and miR-146a as mediators of the 5q- syndrome phenotype.

5q- syndrome is a subtype of myelodysplastic syndrome characterized by severe anemia and variable neutropenia but normal or high platelet counts with dysplastic megakaryocytes. We examined expression of microRNAs (miRNAs) encoded on chromosome 5q as a possible cause of haploinsufficiency. We show that deletion of chromosome 5q correlates with loss of two miRNAs that are abundant in hematopoietic stem/progenitor cells (HSPCs), miR-145 and miR-146a, and we identify Toll-interleukin-1 receptor domain-containing adaptor protein (TIRAP) and tumor necrosis factor receptor-associated factor-6 (TRAF6) as respective targets of these miRNAs. TIRAP is known to lie upstream of TRAF6 in innate immune signaling. Knockdown of miR-145 and miR-146a together or enforced expression of TRAF6 in mouse HSPCs resulted in thrombocytosis, mild neutropenia and megakaryocytic dysplasia. A subset of mice transplanted with TRAF6-expressing marrow progressed either to marrow failure or acute myeloid leukemia. Thus, inappropriate activation of innate immune signals in HSPCs phenocopies several clinical features of 5q- syndrome.

Modeling the functional heterogeneity of leukemia stem cells: role of STAT5 in leukemia stem cell self-renewal.

Although the cancer stem cell (CSC) concept implies that CSCs are rare, recent reports suggest that CSCs may be frequent in some cancers. We hypothesized that the proportion of leukemia stem cells would vary as a function of the number of dysregulated pathways. Constitutive expression of MN1 served as a 1-oncogene model, and coexpression of MN1 and a HOX gene served as a 2-oncogene model. Leukemia-initiating cell (LIC) number and in vitro expansion potential of LICs were functionally assessed by limiting dilution analyses. LIC expansion potential was 132-fold increased in the 2- compared with the 1-oncogene model, although phenotypically, both leukemias were similar. The 2-oncogene model was characterized by granulocyte-macrophage colony-stimulating factor (GM-CSF) hypersensitivity and activated STAT/ERK signaling. GM-CSF hypersensitivity of the 2-oncogene model (MN1/HOXA9) was lost in Stat5b(-/-) cells, and the LIC expansion potential was reduced by 86- and 28-fold in Stat5b(-/-) and Stat1(-/-) cells, respectively. Interestingly, in 201 acute myeloid leukemia (AML) patients, coexpression of MN1 and HOXA9 was restricted to patients with the poorest prognosis and was associated with highly active STAT signaling. Our data demonstrate the functional heterogeneity of LICs and show that STAT signaling is critical for leukemia stem cell self-renewal in MN1- and HOXA9-expressing leukemias.

CBL exon 8/9 mutants activate the FLT3 pathway and cluster in core binding factor/11q deletion acute myeloid leukemia/myelodysplastic syndrome subtypes.

PURPOSE: CBL is a negative regulator of activated receptor tyrosine kinases (RTK). In this study, we determined the frequency of CBL mutations in acute leukemias and evaluated the oncogenic potential of mutant CBL. EXPERIMENTAL DESIGN: The cDNA of 300 acute myeloid leukemia (AML)/myelodysplastic syndrome (MDS) and acute lymphoblastic leukemia (ALL) patients and 82 human leukemic cell lines was screened for aberrations in the linker and RING finger domain of CBL. The oncogenic potential of identified mutants was evaluated in hematopoietic cells. RESULTS: We identified 3 of 279 AML/MDS patients expressing CBL exon 8/9 deletion mutants. Three of four cases at diagnosis expressed deleted transcripts missing exon 8 or exon 8/9. In remission samples a weak or no expression of mutant CBL was detected. No aberrations were found in normal hematopoietic tissues. One of 116 sequenced AML/MDS cases carried a R420G missense mutation. All AML/MDS patients with identified CBL mutants belonged to the core binding factor and 11q deletion AML subtypes. Functionally, CBL negatively regulated FMS-like tyrosine kinase 3 (FLT3) activity and interacted with human FLT3 via the autophosphorylation sites Y589 and Y599 and colocalized in vivo. Expression of CBLDeltaexon8 and CBLDeltaexon8+9 in FLT3-WT-Ba/F3 cells induced growth factor-independent proliferation associated with autophosphorylation of FLT3 and activated the downstream targets signal transducer and activator of transcription 5 (STAT5) and protein kinase B (AKT). FLT3 ligand-dependent hyperproliferation of CBL mutant cells could be abrogated by treatment with the FLT3 PTK inhibitor PKC412 (midostaurin). CONCLUSION: CBL exon8/9 mutants occur in genetically defined AML/MDS subtypes and transform hematopoietic cells by constitutively activating the FLT3 pathway. This phenotype resembles the one of mutated RTKs and suggests that CBL mutant AML patients might benefit from treatment with FLT3 PTK inhibitors.

In-depth characterization of the microRNA transcriptome in a leukemia progression model.

MicroRNAs (miRNAs) have been shown to play important roles in physiological as well as multiple malignant processes, including acute myeloid leukemia (AML). In an effort to gain further insight into the role of miRNAs in AML, we have applied the Illumina massively parallel sequencing platform to carry out an in-depth analysis of the miRNA transcriptome in a murine leukemia progression model. This model simulates the stepwise conversion of a myeloid progenitor cell by an engineered overexpression of the nucleoporin 98 (NUP98)-homeobox HOXD13 fusion gene (ND13), to aggressive AML inducing cells upon transduction with the oncogenic collaborator Meis1. From this data set, we identified 307 miRNA/miRNA species in the ND13 cells and 306 miRNA/miRNA species in ND13+Meis1 cells, corresponding to 223 and 219 miRNA genes. Sequence counts varied between two and 136,558, indicating a remarkable expression range between the detected miRNA species. The large number of miRNAs expressed and the nature of differential expression suggest that leukemic progression as modeled here is dictated by the repertoire of shared, but differentially expressed miRNAs. Our finding of extensive sequence variations (isomiRs) for almost all miRNA and miRNA species adds additional complexity to the miRNA transcriptome. A stringent target prediction analysis coupled with in vitro target validation revealed the potential for miRNA-mediated release of oncogenes that facilitates leukemic progression from the preleukemic to leukemia inducing state. Finally, 55 novel miRNAs species were identified in our data set, adding further complexity to the emerging world of small RNAs.

Linkage of Meis1 leukemogenic activity to multiple downstream effectors including Trib2 and Ccl3.

OBJECTIVE: MEIS1, a HOX cofactor, collaborates with multiple HOX and NUP98-HOX fusion proteins to accelerate the onset of acute myeloid leukemia (AML) through largely unknown molecular mechanisms. MATERIALS AND METHODS: To further resolve these mechanisms, we conducted a structure-function analysis of MEIS1 and gene-expression profiling, in the context of NUP98-HOXD13 (ND13) leukemogenesis. RESULTS: We show, in a murine bone marrow transplantation model, that the PBX-interaction domain, the homeodomain, and the C-terminal domain of MEIS1, are all required for leukemogenic collaboration with ND13. In contrast, the N-terminal domain of MEIS1 is dispensable for collaboration with ND13, but is required for Flt3 upregulation, indicating additional roles for MEIS1 in induction of leukemia independent of alterations in Flt3 expression. Gene-expression profiling of a cloned ND13 preleukemic cell line transduced with wild-type or Meis1 mutant forms revealed deregulation of multiple genes, including a set not previously implicated as MEIS1 targets. Chromatin immunoprecipitation revealed the in vivo occupancy of MEIS1 on regulatory sequences of Trib2, Flt3, Dlk1, Ccl3, Ccl4, Pf4, and Rgs1. Furthermore, engineered overexpression of Trib2 complements ND13 to induce AML while Ccl3 potentiates the repopulating ability of ND13. CONCLUSION: This study shows that Meis1-induced leukemogenesis with ND13 can occur in the absence of Flt3 upregulation and reveals the existence of other pathways activated by MEIS1 to promote leukemia.