Lars Palmqvist

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.

Candidate Genes for Expansion and Transformation of Hematopoietic Stem Cells by NUP98-HOX Fusion Genes

Background Hox genes are implicated in hematopoietic stem cell (HSC) regulation as well as in leukemia development through translocation with the nucleoporin gene NUP98. Interestingly, an engineered NUP98-HOXA10 (NA10) fusion can induce a several hundred-fold expansion of HSCs in vitro and NA10 and the AML-associated fusion gene NUP98-HOXD13 (ND13) have a virtually indistinguishable ability to transform myeloid progenitor cells in vitro and to induce leukemia in collaboration with MEIS1 in vivo. Methodology/Principal Findings These findings provided a potentially powerful approach to identify key pathways mediating Hox-induced expansion and transformation of HSCs by identifying gene expression changes commonly induced by ND13 and NA10 but not by a NUP98-Hox fusion with a non-DNA binding homedomain mutation (N51S). The gene expression repertoire of purified murine bone marrow Sca-1+Lin- cells transduced with retroviral vectors encoding for these genes was established using the Affymetrix GeneChip MOE430A. Approximately seventy genes were differentially expressed in ND13 and NA10 cells that were significantly changed by both compared to the ND13(N51S) mutant. Intriguingly, several of these potential Hox target genes have been implicated in HSC expansion and self-renewal, including the tyrosine kinase receptor Flt3, the prion protein, Prnp, hepatic leukemia factor, Hlf and Jagged-2, Jag2. Consistent with these results, FLT3, HLF and JAG2 expression correlated with HOX A cluster gene expression in human leukemia samples. Conclusions In conclusion this study has identified several novel Hox downstream target genes and provides important new leads to key regulators of the expansion and transformation of hematopoietic stem cells by Hox.

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.

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

OBJECTIVEnMEIS1, 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.nnnMATERIALS AND METHODSnTo further resolve these mechanisms, we conducted a structure-function analysis of MEIS1 and gene-expression profiling, in the context of NUP98-HOXD13 (ND13) leukemogenesis.nnnRESULTSnWe 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.nnnCONCLUSIONnThis 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.

Influence of the apolipoprotein E ε4 allele on human embryonic development

Human apolipoprotein E (apoE) exists in three major isoforms encoded by distinct alleles (APOE epsilon2, epsilon3 and epsilon4) and has important functions in nerve development and repair. Inheritance of the 4 allele is a major risk factor for the development of Alzheimers disease. To investigate the role of APOE polymorphisms in embryonic development, we analyzed the APOE genotypes of 81 spontaneously aborted embryos and 110 adult controls using a solid-phase minisequencing technique. The epsilon4 allele was significantly less frequent in the spontaneous abortion group than in the control group (P=0.009), while the frequency of epsilon3 was significantly increased (P=0.005), suggesting that epsilon4 may have protective effects during embryogenesis. These protective effects might counterbalance the deleterious age-related effects of the epsilon4 allele in natural selection.

Promoter DNA methylation pattern identifies prognostic subgroups in childhood T-cell acute lymphoblastic leukemia

Background Treatment of pediatric T-cell acute lymphoblastic leukemia (T-ALL) has improved, but there is a considerable fraction of patients experiencing a poor outcome. There is a need for better prognostic markers and aberrant DNA methylation is a candidate in other malignancies, but its potential prognostic significance in T-ALL is hitherto undecided. Design and Methods Genome wide promoter DNA methylation analysis was performed in pediatric T-ALL samples (nu200a=u200a43) using arrays covering >27000 CpG sites. Clinical outcome was evaluated in relation to methylation status and compared with a contemporary T-ALL group not tested for methylation (nu200a=u200a32). Results Based on CpG island methylator phenotype (CIMP), T-ALL samples were subgrouped as CIMP+ (high methylation) and CIMP− (low methylation). CIMP− T-ALL patients had significantly worse overall and event free survival (pu200a=u200a0.02 and pu200a=u200a0.001, respectively) compared to CIMP+ cases. CIMP status was an independent factor for survival in multivariate analysis including age, gender and white blood cell count. Analysis of differently methylated genes in the CIMP subgroups showed an overrepresentation of transcription factors, ligands and polycomb target genes. Conclusions We identified global promoter methylation profiling as being of relevance for subgrouping and prognostication of pediatric T-ALL.

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.

Increased risk for vascular complications in PRV-1 positive patients with essential thrombocythaemia

Summary. Essential thrombocythaemia (ET) is a heterogeneous disorder with respect to plasma erythropoietin concentration at diagnosis and clonality of haematopoiesis. Polycythaemia rubra vera‐1 (PRV‐1) positivity, i.e. PRV‐1 mRNA overexpression, is known to be present in the vast majority of patients with polycythaemia vera and also in some patients with ET. In the present study, PRV‐1 expression was quantified by real‐time polymerase chain reaction in 70 ET patients; 17 of them (24%) were found to be PRV‐1 positive. Ten of the 17 PRV‐1 positive ET patients had experienced thromboembolic complications compared with 14 of 53 PRV‐1 negative patients, the difference between the two groups being statistically significant (Pu2003=u20030·02). In addition, the frequency of total vascular complications, thromboembolic events and major bleedings, was significantly higher in the group of PRV‐1 positive as compared with PRV‐1 negative ET patients (Pu2003=u20030·03). The time from diagnosis of ET to the requirement of platelet‐lowering therapy was significantly shorter in PRV‐1 positive compared with PRV‐1 negative ET patients (Pu2003=u20030·014). It can be concluded that PRV‐1 positive patients appear to suffer from a more aggressive disorder with increased risk for vascular complications and a greater need for platelet‐lowering therapy, compared with PRV‐1 negative ET patients.

A novel splicing mutation causes an undescribed type of analbuminemia.

Analbuminemia is a rare autosomal recessive disorder manifested by the absence or severe reduction of circulating serum albumin in homozygous subjects. In this report we describe a new molecular defect that caused the analbuminemic trait in a newborn of Iraqi origin. When the parents DNA was analyzed, both subjects were found to be heterozygous for the same mutation found in the infant. All the 14 exon and flanking intron sequences of the albumin gene were amplified via PCR and screened for mutations by SSCP and heteroduplex analysis. A mutation in the DNA region encoding exon 1 and its flanking intron was revealed by the presence of a heteroduplex. The fragment, which was directly DNA sequenced, contains a previously unreported single nucleotide change, consisting in a G to A substitution at nucleotide 118 in the structural gene of the human protein. This mutation, involving the first base of intron 1, destroys the GT dinucleotide consensus sequence found at the 5 end of most intervening sequences and causes the defective pre-mRNA splicing responsible for the analbuminemic trait.

The presence of a significant association between elevated PRV-1 mRNA expression and low plasma erythropoietin concentration in essential thrombocythaemia

Approximately 45% of newly diagnosed patients with essential thrombocythaemia (ET) demonstrate subnormal plasma erythropoietin (EPO) concentrations, which constitutes a risk factor for occlusive vascular events. In 58 ET patients, a possible association between polycythaemia rubra vera‐1 (PRV‐1) overexpression and subnormal plasma EPO was investigated, which was always measured prior to the institution of platelet lowering agents. At the time when PRV‐1 expression was measured, 28 of 58 (48%) ET patients had received platelet lowering treatment. PRV‐1 expression was measured by quantitative real‐time reverse transcription–polymerase chain reaction assay of mRNA extracted from purified peripheral blood buffy coat. The cycle threshold (CT) value of PRV‐1 was determined and was divided with the CT value for the housekeeping GAPDH gene transcript. A quotient <0.93 was defined as PRV‐1 positive. Of the ET patients 12 of 58 (21%) were PRV‐1 positive and 19 of 58 (33%) demonstrated subnormal plasma EPO. In the 58 ET patients there was a significant association between low plasma EPO and PRV‐1 positive results (Pu2003=u20030.001). The 30 ET patients who had not received any platelet lowering treatment showed a significant (Pu2003=u20030.005) relation between PRV‐1 positivity and subnormal plasma EPO. No such relationship was present in the 28 ET patients who had received prior treatment with the above drugs (Pu2003=u20030.147).