Determination of complex subclonal structures of hematological malignancies by multiplexed genotyping of blood progenitor colonies.

Current next-generation sequencing (NGS) technologies allow unprecedented insights into the mutational profiles of tumors. Recent studies in myeloproliferative neoplasms have further demonstrated that, not only the mutational profile, but also the order in which these mutations are acquired is relevant for our understanding of the disease. Our ability to assign mutation order from NGS data alone is, however, limited. Here, we present a strategy of highly multiplexed genotyping of burst forming unit-erythroid colonies based on NGS results to assess subclonal tumor structure. This allowed for the generation of complex clonal hierarchies and determination of order of mutation acquisition far more accurately than was possible from NGS data alone.Work in ARG lab has been supported by the Leukemia and Lymphoma Society (grant 7001-12), the National Institute of Health Research (grant NF-SI-0512-10079) and core support grants by the MRC and Wellcome Trust to the Cambridge Institute for Medical Research (100140/Z/12/Z) and Wellcome Trust-MRC Cambridge Stem Cell Institute (097922/Z/11/Z). Work in ARG's laboratory has in addition been supported by Cancer Research UK (grants C1163/A12765 and C1163/A21762), Bloodwise (grant 13003) and the Wellcome Trust (grant 104710/Z/14/Z

An RNA-Seq Strategy to Detect the Complete Coding and Non-Coding Transcriptome Including Full-Length Imprinted Macro ncRNAs

Imprinted macro non-protein-coding (nc) RNAs are cis-repressor transcripts that silence multiple genes in at least three imprinted gene clusters in the mouse genome. Similar macro or long ncRNAs are abundant in the mammalian genome. Here we present the full coding and non-coding transcriptome of two mouse tissues: differentiated ES cells and fetal head using an optimized RNA-Seq strategy. The data produced is highly reproducible in different sequencing locations and is able to detect the full length of imprinted macro ncRNAs such as Airn and Kcnq1ot1, whose length ranges between 80–118 kb. Transcripts show a more uniform read coverage when RNA is fragmented with RNA hydrolysis compared with cDNA fragmentation by shearing. Irrespective of the fragmentation method, all coding and non-coding transcripts longer than 8 kb show a gradual loss of sequencing tags towards the 3′ end. Comparisons to published RNA-Seq datasets show that the strategy presented here is more efficient in detecting known functional imprinted macro ncRNAs and also indicate that standardization of RNA preparation protocols would increase the comparability of the transcriptome between different RNA-Seq datasets

Frequent deletions of JARID2 in leukemic transformation of chronic myeloid malignancies

Chronic myeloproliferative neoplasms (MPN) and myelodysplastic syndromes (MDS) have an inherent tendency to progress to acute myeloid leukemia (AML). Using high-resolution SNP microarrays, we studied a total of 517 MPN and MDS patients in different disease stages, including 77 AML cases with previous history of MPN (N = 46) or MDS (N = 31). Frequent chromosomal deletions of variable sizes were detected, allowing the mapping of putative tumor suppressor genes involved in the leukemic transformation process. We detected frequent deletions on the short arm of chromosome 6 (del6p). The common deleted region on 6p mapped to a 1.1-Mb region and contained only the JARID2 genemember of the polycomb repressive complex 2 (PRC2). When we compared the frequency of del6p between chronic and leukemic phase, we observed a strong association of del6p with leukemic transformation (P = 0.0033). Subsequently, analysis of deletion profiles of other PRC2 members revealed frequent losses of genes such as EZH2, AEBP2, and SUZ12; however, the deletions targeting these genes were large. We also identified two patients with homozygous losses of JARID2 and AEBP2. We observed frequent codeletion of AEBP2 and ETV6, and similarly, SUZ12 and NF1. Using next generation exome sequencing of 40 patients, we identified only one somatic mutation in the PRC2 complex member SUZ12. As the frequency of point mutations in PRC2 members was found to be low, deletions were the main type of lesions targeting PRC2 complex members. Our study suggests an essential role of the PRC2 complex in the leukemic transformation of chronic myeloid disorders. Am. J. Hematol. 2012

Mutant calreticulin knockin mice develop thrombocytosis and myelofibrosis without a stem cell self-renewal advantage.

Somatic mutations in the endoplasmic reticulum chaperone calreticulin (CALR) are detected in approximately 40% of patients with essential thrombocythemia (ET) and primary myelofibrosis (PMF). Multiple different mutations have been reported, but all result in a +1-bp frameshift and generate a novel protein C terminus. In this study, we generated a conditional mouse knockin model of the most common CALR mutation, a 52-bp deletion. The mutant novel human C-terminal sequence is integrated into the otherwise intact mouse CALR gene and results in mutant CALR expression under the control of the endogenous mouse locus. CALRdel/+ mice develop a transplantable ET-like disease with marked thrombocytosis, which is associated with increased and morphologically abnormal megakaryocytes and increased numbers of phenotypically defined hematopoietic stem cells (HSCs). Homozygous CALRdel/del mice developed extreme thrombocytosis accompanied by features of MF, including leukocytosis, reduced hematocrit, splenomegaly, and increased bone marrow reticulin. CALRdel/+ HSCs were more proliferative in vitro, but neither CALRdel/+ nor CALRdel/del displayed a competitive transplantation advantage in primary or secondary recipient mice. These results demonstrate the consequences of heterozygous and homozygous CALR mutations and provide a powerful model for dissecting the pathogenesis of CALR-mutant ET and PMF

Complex Patterns of Chromosome 11 Aberrations in Myeloid Malignancies Target CBL, MLL, DDB1 and LMO2

Exome sequencing of primary tumors identifies complex somatic mutation patterns. Assignment of relevance of individual somatic mutations is difficult and poses the next challenge for interpretation of next generation sequencing data. Here we present an approach how exome sequencing in combination with SNP microarray data may identify targets of chromosomal aberrations in myeloid malignancies. The rationale of this approach is that hotspots of chromosomal aberrations might also harbor point mutations in the target genes of deletions, gains or uniparental disomies (UPDs). Chromosome 11 is a frequent target of lesions in myeloid malignancies. Therefore, we studied chromosome 11 in a total of 813 samples from 773 individual patients with different myeloid malignancies by SNP microarrays and complemented the data with exome sequencing in selected cases exhibiting chromosome 11 defects. We found gains, losses and UPDs of chromosome 11 in 52 of the 813 samples (6.4%). Chromosome 11q UPDs frequently associated with mutations of CBL. In one patient the 11qUPD amplified somatic mutations in both CBL and the DNA repair gene DDB1. A duplication within MLL exon 3 was detected in another patient with 11qUPD. We identified several common deleted regions (CDR) on chromosome 11. One of the CDRs associated with de novo acute myeloid leukemia (P=0.013). One patient with a deletion at the LMO2 locus harbored an additional point mutation on the other allele indicating that LMO2 might be a tumor suppressor frequently targeted by 11p deletions. Our chromosome-centered analysis indicates that chromosome 11 contains a number of tumor suppressor genes and that the role of this chromosome in myeloid malignancies is more complex than previously recognized

Somatic Mutations of Calreticulin in Myeloproliferative Neoplasms

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Oncogene and tumor suppressor network in the myeloproliferative neoplasms

Unter dem Begriff "myeloproliferative Neoplasien" (MPN) werden verschiedene Erkrankungen der Blutbildung zusammengefasst, die eine Überproduktion eines oder mehrerer ausgereifter Zelltypen im periphären Blut zeigen. Ziel dieser Arbeit war es zu einem besseren Verständnis der genetischen Basis dieser Erkrankungen beizutragen. Wir untersuchten über 400 MPN Patienten mittels DNA Mikroarrays und fanden chromosomale Veränderungen in 62,5% aller Fälle. Eine höhere Anzahl genetischer Veränderungen assoziierte signifikant mit dem Alter der Patienten und mit dem Fortschreiten der Erkrankung. Ein Bruchteil der Patienten entwickelt eine post-MPN akute myeloische Leukämie (AML). Wir beschrieben Veränderungen an den Chromosomen 1, 3, 5, 6, 7, 19 und 22, die signifikant mit der Transformation zur AML assoziiert sind. Für häufig vorkommende chromosomale Veränderungen konnten wir folgende Zielgene identifizieren: FOXP1 (Chromosom 3p), TET2 (4q), IKZF1 (7p), CUX1 (7q), ETV6 (12p) und RUNX1 (21q). In einer zweiten Studie kombinierten wir Daten von DNA Mikroarrays mit "whole-exome" Sequenzierdaten um eine detaillierte Analyse von Veränderungen auf Chromosom 11 durchzuführen. Dazu analysierten wir Mikroarraydaten von über 800 Patienten die MPN oder andere myeloide Erkrankungen hatten. Uniparentale Disomien von Chromosom 11 waren mit Mutationen in den CBL, MLL und DDB1 Genen assoziiert. Das Zielgen von Deletionen auf Chromosom 11p war LMO2. Der Verlust eines anderen Abschnitts auf Chromosom 11p war signifikant mit de novo AML assoziiert. Die genetischen Ursachen von MPN erscheinen komplex. Im Blickwinkel des systembiologischen Kontextes lässt sich zeigen, dass verschiedene genetische Veränderungen zu gemeinsamen Mechanismen beitragen. Ein solcher systembiologischer Ansatz erscheint vielversprechend für ein besseres Verständnis der Pathogenese von MPN.Myeloproliferative neoplasms (MPN) are a group of clonal, stem cell derived disorders of hematopoiesis associated with overproduction of one or more cell types in peripheral blood. We analyzed over 400 MPN patients for chromosomal aberrations using microarray technology. We found that 62.5% of the patients harbored at least one chromosomal aberration. Older patients had significantly more lesions than younger patients. The most significant association was observed between the number of genetic lesions in a patient and disease progression from chronic MPN to acute myeloid leukemia (AML). We found specific aberrations of chromosomes 1, 3, 5, 6, 7, 19 and 22 to be significantly associated with the transformation to AML. We were able to map target genes of recurrent chromosomal lesions including FOXP1, TET2, IKZF1, CUX1, ETV6 and RUNX1 on chromosomes 3p, 4q, 7p, 7q, 12p, and 21q, respectively. In a second project we complemented microarray technology with whole-exome sequencing. We performed a detailed analysis of chromosome 11 in MPN and other myeloid malignancies by analyzing over 800 patient samples. Uniparental disomies of chromosome 11 were associated with mutations in the CBL, MLL and DDB1 genes. A common deletion of chromosome 11p targeted the LMO2 transcription factor. Another frequently deleted region on chromosome 11p was significantly associated with de novo AML. The genetic basis of MPN appears to be complex. Interpretation of the data in a systems biology context has led to the identification of common mechanisms downstream of different lesions - a promising approach towards a better understanding of MPN pathogenesis.submitted by Thorsten KlampflAbweichender Titel laut Übersetzung der Verfasserin/des VerfassersZsfassung in dt. SpracheWien, Med. Univ., Diss., 2013OeBB(VLID)171442

Mutant Calreticulin in the Myeloproliferative Neoplasms.

AbstractMutations in the gene for calreticulin (CALR) were identified in the myeloproliferative neoplasms (MPNs) essential thrombocythaemia (ET) and primary myelofibrosis (MF) in 2013; in combination with previously described mutations in JAK2 and MPL, driver mutations have now been described for the majority of MPN patients. In subsequent years, researchers have begun to unravel the mechanisms by which mutant CALR drives transformation and to understand their clinical implications. Mutant CALR activates the thrombopoietin receptor (MPL), causing constitutive activation of Janus kinase 2 (JAK2) signaling and cytokine independent growth in vitro. Mouse models show increased numbers of hematopoietic stem cells (HSCs) and overproduction of megakaryocytic lineage cells with associated thrombocytosis. In the clinic, detection of CALR mutations has been embedded in World Health Organization and other international diagnostic guidelines. Distinct clinical and laboratory associations of CALR mutations have been identified together with their prognostic significance, with CALR mutant patients showing increased overall survival. The discovery and subsequent study of CALR mutations have illuminated novel aspects of megakaryopoiesis and raised the possibility of new therapeutic approaches

Kinase-inactivated CDK6 preserves the long-term functionality of adult hematopoietic stem cells

Inactivated CDK6; Hematopoietic stem cellsCDK6 inactivat; Células madre hematopoyéticas adultasCDK6 inactivat; Cèl·lules mare hematopoètiques adultesHematopoietic stem cells (HSCs) are characterized by the ability to self-renew and to replenish the hematopoietic system. The cell-cycle kinase cyclin-dependent kinase 6 (CDK6) regulates transcription, whereby it has both kinase-dependent and kinase-independent functions. Herein, we describe the complex role of CDK6, balancing quiescence, proliferation, self-renewal, and differentiation in activated HSCs. Mouse HSCs expressing kinase-inactivated CDK6 show enhanced long-term repopulation and homing, whereas HSCs lacking CDK6 have impaired functionality. The transcriptomes of basal and serially transplanted HSCs expressing kinase-inactivated CDK6 exhibit an expression pattern dominated by HSC quiescence and self-renewal, supporting a concept, in which myc-associated zinc finger protein (MAZ) and nuclear transcription factor Y subunit alpha (NFY-A) are critical CDK6 interactors. Pharmacologic kinase inhibition with a clinically used CDK4/6 inhibitor in murine and human HSCs validated our findings and resulted in increased repopulation capability and enhanced stemness. Our findings highlight a kinase-independent role of CDK6 in long-term HSC functionality. CDK6 kinase inhibition represents a possible strategy to improve HSC fitness