Low-burden TP53 mutations in chronic phase of myeloproliferative neoplasms: association with age, hydroxyurea administration, disease type and JAK2 mutational status.

The multistep process of TP53 mutation expansion during myeloproliferative neoplasm (MPN) transformation into acute myeloid leukemia (AML) has been documented retrospectively. It is currently unknown how common TP53 mutations with low variant allele frequency (VAF) are, whether they are linked to hydroxyurea (HU) cytoreduction, and what disease progression risk they carry. Using ultra-deep next-generation sequencing, we examined 254 MPN patients treated with HU, interferon alpha-2a or anagrelide and 85 untreated patients. We found TP53 mutations in 50 cases (0.2-16.3% VAF), regardless of disease subtype, driver gene status and cytoreduction. Both therapy and TP53 mutations were strongly associated with older age. Over-time analysis showed that the mutations may be undetectable at diagnosis and slowly increase during disease course. Although three patients with TP53 mutations progressed to TP53-mutated or TP53-wild-type AML, we did not observe a significant age-independent impact on overall survival during the follow-up. Further, we showed that complete p53 inactivation alone led to neither blast transformation nor HU resistance. Altogether, we revealed patient's age as the strongest factor affecting low-burden TP53 mutation incidence in MPN and found no significant age-independent association between TP53 mutations and hydroxyurea. Mutations may persist at low levels for years without an immediate risk of progression.

Absence of polycythemia in a child with a unique erythropoietin receptor mutation in a family with autosomal dominant primary polycythemia.

Primary familial and congenital polycythemia (PFCP or familial erythrocytosis) is a rare proliferative disorder of erythroid progenitor cells, characterized by elevated erythrocyte mass and hemoglobin concentration, hypersensitivity of erythroid progenitors to erythropoietin (EPO), and autosomal dominant inheritance or sporadic occurrence. A number of EPO receptor (EPOR) mutations were found in subjects with PFCP; most of these mutations resulted in the truncation of the COOH-terminal of the EPOR protein. We studied a family with autosomal dominant inheritance of PFCP in which four subjects were affected in three generations. We screened the affected individuals for EPOR gene mutations using SSCP analysis and found a C5964G mutation in exon VIII that changes tyrosine codon 426 to a translation termination codon resulting in an EPOR protein truncated by 83 amino acids. The mutant C5964G-EPOR exhibited hypersensitive EPO-dependent proliferation compared to the wild-type EPOR when tested in a murine interleukin-3-dependent myeloid cell line (FDC-P1). We also examined the segregation of the mutation with PFCP in the family and found that a child in the third generation inherited the mutation without having laboratory evidence of polycythemia. Further in vitro analysis of the erythroid progenitor cells of this affected child revealed that the progenitor cells were hypersensitive to EPO (a hallmark of PFCP) suggesting the presence of the disease at the level of progenitor cells. Failure of this child to develop polycythemia suggests the existence of as yet unidentified environmental or genetic factors that may suppress disease development.

NOTCH1 activation in breast cancer confers sensitivity to inhibition of SUMOylation.

Breast cancer is genetically heterogeneous, and recent studies have underlined a prominent contribution of epigenetics to the development of this disease. To uncover new synthetic lethalities with known breast cancer oncogenes, we screened an epigenome-focused short hairpin RNA library on a panel of engineered breast epithelial cell lines. Here we report a selective interaction between the NOTCH1 signaling pathway and the SUMOylation cascade. Knockdown of the E2-conjugating enzyme UBC9 (UBE2I) as well as inhibition of the E1-activating complex SAE1/UBA2 using ginkgolic acid impairs the growth of NOTCH1-activated breast epithelial cells. We show that upon inhibition of SUMOylation NOTCH1-activated cells proceed slower through the cell cycle and ultimately enter apoptosis. Mechanistically, activation of NOTCH1 signaling depletes the pool of unconjugated small ubiquitin-like modifier 1 (SUMO1) and SUMO2/3 leading to increased sensitivity to perturbation of the SUMOylation cascade. Depletion of unconjugated SUMO correlates with sensitivity to inhibition of SUMOylation also in patient-derived breast cancer cell lines with constitutive NOTCH pathway activation. Our investigation suggests that SUMOylation cascade inhibitors should be further explored as targeted treatment for NOTCH-driven breast cancer.

Telomerase activity in plant cells.

Telomerase is a ribonucleoprotein enzyme which elongates the G-rich strand of telomeric DNA to compensate for the progressive reduction in its length due to incomplete replication of chromosome ends, which in human somatic cells leads to cell cycle arrest upon shortening of telomeres to a critical length. To examine the possible involvement of telomerase in metabolism of plant genetic material, we used cells of Nicotiana tabacum strain TBY-2, a stable long-term culture which has kept a constant pattern of restriction fragments from chromosome termini during its 6 month period of cultivation in our laboratory. In a direct assay for telomerase, a 5' end-labeled plant telomeric oligonucleotide 5' (TTTAGGG)(3')6 was elongated in a TBY-2 cell extract, showing a pausing pattern which is a characteristic feature of telomerases from other organisms. The elongation was inhibited by RNase A pretreatment of the extract. We conclude that plant cells possess telomerase which is used for maintenance of their telomeres.

The telomeric sequence is directly attached to the HRS60 subtelomeric tandem repeat in tobacco chromosomes.

PCR and primers derived from the telomeric repeat (CCCTAAA)n and from the tobacco subtelomeric tandemly repetitive sequence HRS60 (EMBL X12489) were used to amplify the region linking the two loci. A 131 bp PCR product was obtained both from total tobacco DNA and from the DNA fraction enriched for telomeres. Its sequence only consists of the telomeric primer and the attached region of the HRS60 repetitive unit up to the end of the sequence complementary to the HRS60 primer. The site of direct continuity between the two sequences is formed by a (dA)7 tract.

DNA curvature of the tobacco GRS repetitive sequence family and its relation to nucleosome positioning.

Recently, a highly repetitive DNA sequence family (GRS) from tobacco was described in our laboratory. These sequences were found to be localized predominantly in the pericentromeric heterochromatin of tobacco chromosomes. To test the hypothesis that these sequences play an important role in the formation of heterochromatin, we investigated the DNA curvature of the GRS sequences and its possible impact to the chromatin structure at these loci. Application of the nearest-neighbour wedge model of intrinsic DNA curvature for the GRS1 family member predicted two loci of curvature: a major bend at the 5' end of the sequence and a minor bend of opposite direction at the centre of the GRS1. The presence of the major and the minor loci of DNA curvature was studied experimentally using permutation analysis and site-directed mutagenesis. The experimental results were consistent with the computer predictions. We gave evidence that the described DNA curvature is also present in the entire GRS family. Genomic statistical sequencing showed the conservation of the major bend sequence determinants in the members of the GRS family. To investigate the chromatin structure at the GRS sequences, we determined the nucleosome positioning in vivo at these sequences using thermal cycle primer extension. A relation between the curvature pattern and the histone octamer position was observed: the major bend is excluded from the nucleosome surface to the linker region, while the minor bend is distributed along the core DNA. The suggestion is made that the sequences in the minor locus of curvature define the rotational setting of the nucleosome, and a possible role of the major bend as a factor, which defines the translational setting, is discussed.

MYBL2 haploinsufficiency increases susceptibility to age-related haematopoietic neoplasia.

The haematopoietic system is prone to age-related disorders ranging from deficits in functional blood cells to the development of neoplastic states. Such neoplasms often involve recurrent cytogenetic abnormalities, among which a deletion in the long arm of chromosome 20 (del20q) is common in myeloid malignancies. The del20q minimum deleted region contains nine genes, including MYBL2, which encodes a key protein involved in the maintenance of genome integrity. Here, we show that mice expressing half the normal levels of Mybl2 (Mybl2(+/Δ)) develop a variety of myeloid disorders upon ageing. These include myeloproliferative neoplasms, myelodysplasia (MDS) and myeloid leukaemia, mirroring the human conditions associated with del20q. Moreover, analysis of gene expression profiles from patients with MDS demonstrated reduced levels of MYBL2, regardless of del20q status and demonstrated a strong correlation between low levels of MYBL2 RNA and reduced expression of a subset of genes related to DNA replication and checkpoint control pathways. Paralleling the human data, we found that these pathways are also disturbed in our Mybl2(+/Δ) mice. This novel mouse model, therefore, represents a valuable tool for studying the initiation and progression of haematological malignancies during ageing, and may provide a platform for preclinical testing of therapeutic approaches.

Deletions of the transcription factor Ikaros in myeloproliferative neoplasms.

Transformation to acute leukemia is a major complication of myeloproliferative neoplasms (MPNs), however, the genetic changes leading to transformation remain largely unknown. We screened nine patients with post-MPN leukemia for chromosomal aberrations using microarray karyotyping. Deletions on the short arm of chromosome 7 (del7p) emerged as a recurrent defect. We mapped the common deleted region to the IKZF1 gene, which encodes the transcription factor Ikaros. We further examined the frequency of IKZF1 deletions in a total of 29 post-MPN leukemia and 526 MPN patients without transformation and observed a strong association of IKZF1 deletions with post-MPN leukemia in two independent cohorts. Patients with IKZF1 loss showed complex karyotypes, and del7p was a late event in the genetic evolution of the MPN clone. IKZF1 deletions were observed in both undifferentiated and differentiated myeloid cell types, indicating that IKZF1 loss does not cause differentiation arrest but rather renders progenitors susceptible to transformation, most likely through chromosomal instability. Induced Ikzf1 haploinsufficiency in primary murine progenitors resulted in elevated Stat5 phosphorylation and increased cytokine-dependent growth, suggesting that reduced expression of IKZF1 is sufficient to perturb growth regulation. Thus, IKZF1 loss is an important step in the leukemic transformation of a subpopulation of MPN patients.

Genetic complexity of myeloproliferative neoplasms.

Oncogenic mutations in JAK2 and MPL genes have recently been identified in myeloproliferative neoplasms (MPNs). In addition to these mutations, cytogenetic aberrations are frequently present at diagnosis but their role in the pathogenesis remains unclear. Two models of MPN pathogenesis have recently emerged based on either a single-hit or a multi-hit concept. The first model proposes that the acquisition of JAK2 mutations is the disease-initiating event, causing both the onset of disease phenotype and establishment of clonal hematopoiesis. The second model postulates the existence of 'pre-JAK2' mutations that establish clonal hematopoiesis before acquisition of JAK2 mutations and onset of disease phenotype. In this review, the two models have been critically evaluated in the context of the latest findings. At present, neither of the two models can be universally applied to all MPN patients due to their genetic heterogeneity. It is likely that the disease pathogenesis in some patients follows the first, and in other patients, the second model. Thus, the somatic mutations in MPN do not seem to be acquired in a predetermined order as seen in other malignancies, but occur randomly. Furthermore, the role of uniparental disomy in MPN and certain aspects of MPN therapy are discussed.

Congenital and inherited polycythemia.

Absolute polycythemia is a condition with increased red blood cell mass. There are a number of primary and secondary polycythemic disorders leading to absolute polycythemia. Primary polycythemias are caused by a defect intrinsic to the erythroid progenitor cells. The best characterized primary polycythemia is the autosomal dominant primary familial and congenital polycythemia (PFCP). Familial or childhood occurrence of the myeloproliferative disorder polycythemia vera are also discussed, emphasizing the importance of distinction between polycythemia vera and PFCP. Congenital or familial secondary polycythemic conditions are characterized by increased red cell mass, which is caused by circulating serum factors, typically erythropoietin.

Haematopoietic progenitors and signal transduction in polycythaemia vera and primary thrombocythaemia.

While significant progress has been made in understanding the cellular defect and molecular basis of polycythaemia vera (PV), elucidation of the primary mutation leading to PV remains elusive. While clinically useful, the PV diagnostic criteria put forward by the Polycythemia Vera Study Group are not based on the pathophysiology of this disorder and in some instances may lead to false diagnosis or may not be sufficient to diagnose an early PV. In diagnostically unclear situations, clinical and laboratory findings must take into account the acquired nature of PV, its clonality, and the presence of endogenous erythroid colony formation in serum-containing media. It is likely that other simpler assays may be developed based on the rapidly emerging knowledge of the cellular pathology of PV. Several intriguing observations of abnormalities pertaining to the erythroid signal transduction have been recently reported; these remain to be validated in other laboratories and to be proven specific for PV. The clinical concept of primary thrombocythaemia (PT) lags behind what we know about PV. While the diagnosis of PT is still based on the exclusion of other known causes of thrombocytosis, new knowledge is emerging. Recent clonality studies of a large number of PT females show that the majority are clonal. It is our belief that thrombocythaemic subjects who are not found to be clonal are those with secondary thrombocytosis. Multiple in vitro-based assays of megakaryocytic and erythroid progenitors have been developed and conflicting data published. It is likely that standardized assays of megakaryocytic progenitors will soon become available and a reproducible PT specific defect will be found. Such a specific test would be of immense diagnostic value in this most elusive of all myeloproliferative disorders.

Genetic heterogeneity of primary familial and congenital polycythemia.

Primary familial and congenital polycythemia (PFCP) is an inherited disorder of erythroid progenitor cells resulting in elevated erythrocyte mass. Several mutations of the erythropoietin receptor (EPOR) gene have been associated with PFCP, although in a few families the linkage between the EPOR gene and PFCP has been excluded. To examine the role of EPOR mutations in the pathogenesis of PFCP, we studied 43 unrelated PFCP subjects. Erythroid culture data were available in 26 subjects, and in all these subjects, we observed hypersensitivity of erythroid progenitors to erythropoietin (EPO). We screened all EPOR gene exons for mutations using ribonuclease cleavage assay and protein truncation test. We detected five mutations in exon VIII of the EPOR gene, four of which we reported earlier. A new EPOR gene mutation was found (G5959T) that changes codon 425 GAG to a termination codon, resulting in truncation of the EPOR by 84 amino acids. The G5959T mutation was found to segregate with the disease in the affected family and represents another example of a nonsense mutation associated with PFCP. We also report the first intronic mutation (A2706T) of the EPOR gene. The finding of only five disease-causing mutations in our PFCP patient pool of 43 subjects (12%) indicates that EPOR gene mutations are not the major genetic defect associated with PFCP. The hypersensitivity of erythroid progenitors to EPO seen in all examined PFCP subjects suggests a dominant lesion of an as yet unidentified gene either at the level of the EPOR signaling pathway or another erythropoiesis regulating pathway.

Organization of telomeric and subtelomeric chromatin in the higher plant Nicotiana tabacum.

We have examined the structure and chromatin organization of telomeres in Nicotiana tabacum. In tobacco the blocks of simple telomeric repeats (TT-TAGGG)n are many times larger than in other plants, e.g., Arabidopsis thaliana or tomato. They are resolved as multiple fragments 60-160 kb in size (in most cases 90-130 kb) on pulsed-field gel electrophoresis (PFGE) of restriction endonuclease-digested DNA. The major subtelomeric repeat of the HRS60 family forms large homogeneous blocks of a basic 180 bp motif having comparable lengths. Micrococcal nuclease (MNase) cleaves tobacco telomeric chromatin into subunits with a short repeat length of 157 +/- 5 bp; the subtelomeric heterochromatin characterized by tandemly repeated sequences of the HRS60 family is cut by MNase with a 180 bp periodicity. The monomeric and dimeric particles of telomeric and subtelomeric chromatin differ in sensitivity to MNase treatment: the telomeric particles are readily digested, producing ladders with a periodicity of 7 bp, while the subtelomeric particles appear to be rather resistant to intranucleosomal cleavage. The results presented show apparent similarities in the organization of telomeric chromatin in higher plants and mammals.

A polymorphism of the X-linked gene IDS increases the number of females informative for transcriptional clonality assays.

Studies of clonality have been essential for understanding the hierarchy of hematopoiesis and the biology of malignancies. Most clonality assays are based on the X chromosome inactivation phenomenon in females; these assays detect protein polymorphisms, differences in DNA methylation, or transcripts of the active X chromosome. Assays based on protein polymorphisms or DNA methylation have significant shortcomings. The major disadvantage of transcriptional assays is their limited applicability since only approximately half of females are informative for these studies. We have developed a new transcriptional assay based on an exonic polymorphism of the X-chromosome gene IDS. This gene is located in the same X-chromosome region (Xq28) as G6PD and p55, two genes with exonic polymorphisms for which we previously developed transcriptional assays. We developed non-radioactive PCR-based assays for rapid screening of genotype and determination of clonality. We also report reaction conditions for a quantitative ligase detection assay of IDS allelic transcripts. The frequency of the IDS polymorphism is 46% in Caucasian females and 39% in African-American females; in combination with G6PD and p55, 76% of Caucasian females and 62% of African-American females are informative for these assays. While this gene is highly polymorphic in Caucasian and African-American females, it is not informative in Oriental females. We established that the IDS gene is in linkage equilibrium with G6PD and p55. Unlike methylation-based assays, this assay is suitable for studying clonality in non-nucleated cells such as platelets and reticulocytes. With the discovery of exonic polymorphisms of other X-chromosome genes, all females should eventually be suitable for X-chromosome transcriptional clonality analysis.

The erythropoietin receptor gene is not linked with the polycythemia phenotype in a family with autosomal dominant primary polycythemia.

Primary familial and congenital polycythemia (PFCP or familial erythrocytosis) is a rare hematological disorder with either autosomal-dominant inheritance or sporadic occurrence. It is characterized by an increased proliferation of erythroid precursors that results in an elevated red blood cell mass. In some of the PFCP families, the disease phenotype is associated with mutations of the erythropoietin receptor (EPOR). Mutations in other genes are likely to cause PFCP as well, but no evidence so far has been provided to support this contention. In this study, we present a family in which 6 of 15 family members were affected in three generations. We screened exon VIII of the EPOR gene for mutations and found a C-->T substitution (C6148T) in the maternal grandmother of the propositus. The mutated allele of the affected grandmother was not passed to either of her two affected children or to her one healthy child; thus, the disease phenotype was not linked to the C6148T mutation in this family. Further examination of the inheritance of the EPOR gene alleles and sequence analysis ruled out linkage between the disease phenotype and the EPOR gene; therefore, an abnormality in another gene must be the cause of PFCP in this particular family. In three affected family members tested, erythroid progenitors were hypersensitive to EPO. This in vitro behavior of the progenitors confirms the diagnosis of PFCP in these subjects. Moreover, it suggests a dominant lesion of an as-yet unidentified gene, either at the level of the EPOR-signaling pathway or another erythropoiesis-regulating pathway that may be responsible for enhanced proliferation of the erythroid progenitors.