Search

Complete or partial deletions of chromosome 7 (-7/del7q) belong to the most frequent chromosomal abnormalities in myeloid neoplasm (MN) and are associated with a poor prognosis. The disease biology of -7/del7q and the genes responsible for the leukemogenic properties have not been completely elucidated. Chromosomal deletions may create clonal vulnerabilities due to haploinsufficient (HI) genes contained in the deleted regions. Therefore, HI genes are potential targets of synthetic lethal strategies. Through the most comprehensive multimodal analysis of more than 600 -7/del7q MN samples, we elucidated the disease biology and qualified a list of most consistently deleted and HI genes. Among them, 27 potentially synthetic lethal target genes were identified with the following properties: (i) unaffected genes by hemizygous/homozygous LOF mutations; (ii) prenatal lethality in knockout mice; and (iii) vulnerability of leukemia cells by CRISPR and shRNA knockout screens. In -7/del7q cells, we also identified 26 up or down-regulated genes mapping on other chromosomes as downstream pathways or compensation mechanisms. Our findings shed light on the pathogenesis of -7/del7q MNs, while 27 potential synthetic lethal target genes and 26 differential expressed genes allow for a therapeutic window of -7/del7q.

Human telomere biology disorders (TBD)/short telomere syndromes (STS) are heterogeneous disorders caused by inherited loss-of-function mutations in telomere-associated genes. Here, we identify 3 germline heterozygous missense variants in the RPA1 gene in 4 unrelated probands presenting with short telomeres and varying clinical features of TBD/STS, including bone marrow failure, myelodysplastic syndrome, T- and B-cell lymphopenia, pulmonary fibrosis, or skin manifestations. All variants cluster to DNA-binding domain A of RPA1 protein. RPA1 is a single-strand DNA-binding protein required for DNA replication and repair and involved in telomere maintenance. We showed that RPA1E240K and RPA1V227A proteins exhibit increased binding to single-strand and telomeric DNA, implying a gain in DNA-binding function, whereas RPA1T270A has binding properties similar to wild-type protein. To study the mutational effect in a cellular system, CRISPR/Cas9 was used to knock-in the RPA1E240K mutation into healthy inducible pluripotent stem cells. This resulted in severe telomere shortening and impaired hematopoietic differentiation. Furthermore, in patients with RPA1E240K, we discovered somatic genetic rescue in hematopoietic cells due to an acquired truncating cis RPA1 mutation or a uniparental isodisomy 17p with loss of mutant allele, coinciding with stabilized blood counts. Using single-cell sequencing, the 2 somatic genetic rescue events were proven to be independently acquired in hematopoietic stem cells. In summary, we describe the first human disease caused by germline RPA1 variants in individuals with TBD/STS.

Human telomere biology disorders (TBD)/short telomere syndromes (STS) are heterogeneous disorders caused by inherited loss-of-function mutations in telomere-associated genes. Here, we identify 3 germline heterozygous missense variants in the RPA1gene in 4 unrelated probands presenting with short telomeres and varying clinical features of TBD/STS, including bone marrow failure, myelodysplastic syndrome, T- and B-cell lymphopenia, pulmonary fibrosis, or skin manifestations. All variants cluster to DNA-binding domain A of RPA1 protein. RPA1 is a single-strand DNA-binding protein required for DNA replication and repair and involved in telomere maintenance. We showed that RPA1E240Kand RPA1V227Aproteins exhibit increased binding to single-strand and telomeric DNA, implying a gain in DNA-binding function, whereas RPA1T270Ahas binding properties similar to wild-type protein. To study the mutational effect in a cellular system, CRISPR/Cas9 was used to knock-in the RPA1E240Kmutation into healthy inducible pluripotent stem cells. This resulted in severe telomere shortening and impaired hematopoietic differentiation. Furthermore, in patients with RPA1E240K, we discovered somatic genetic rescue in hematopoietic cells due to an acquired truncating cis RPA1mutation or a uniparental isodisomy 17p with loss of mutant allele, coinciding with stabilized blood counts. Using single-cell sequencing, the 2 somatic genetic rescue events were proven to be independently acquired in hematopoietic stem cells. In summary, we describe the first human disease caused by germline RPA1variants in individuals with TBD/STS.

Germline SAMD9and SAMD9Lmutations (SAMD9/9Lmut) predispose to myelodysplastic syndromes (MDS) with propensity for somatic rescue. In this study, we investigated a clinically annotated pediatric MDS cohort (n= 669) to define the prevalence, genetic landscape, phenotype, therapy outcome and clonal architecture of SAMD9/9L syndromes. In consecutively diagnosed MDS, germline SAMD9/9Lmutaccounted for 8% and were mutually exclusive with GATA2mutations present in 7% of the cohort. Among SAMD9/9Lmutcases, refractory cytopenia was the most prevalent MDS subtype (90%); acquired monosomy 7 was present in 38%; constitutional abnormalities were noted in 57%; and immune dysfunction was present in 28%. The clinical outcome was independent of germline mutations. In total, 67 patients had 58 distinct germline SAMD9/9Lmutclustering to protein middle regions. Despite inconclusive in silico prediction, 94% of SAMD9/9Lmutsuppressed HEK293 cell growth, and mutations expressed in CD34+cells induced overt cell death. Furthermore, we found that 61% of SAMD9/9Lmutpatients underwent somatic genetic rescue (SGR) resulting in clonal hematopoiesis, of which 95% was maladaptive (monosomy 7 ± cancer mutations), and 51% had adaptive nature (revertant UPD7q, somatic SAMD9/9Lmut). Finally, bone marrow single-cell DNA sequencing revealed multiple competing SGR events in individual patients. Our findings demonstrate that SGR is common in SAMD9/9LmutMDS and exemplify the exceptional plasticity of hematopoiesis in children.

Acquired aplastic anemia (AA) is a bone marrow failure resulting from an autoimmune T-cell attack against hematopoietic stem and progenitor cells (HSPCs). Several escape mechanisms within the HSPC compartment have been reported: i) adaptive alterations causing loss of high-risk HLA allele of AA development [deletion or uniparental disomy (UPD) of chromosome 6p including HLA cluster region, and loss-of-function (LOF) mutations in HLA gene], ii) clonal expansion of paroxysmal nocturnal hemoglobinuria (PNH) clone resulting from PIGAmutation, and iii) presence of clonal hematopoiesis of indeterminate potential (CHIP) mutations (i.e., BCOR/ BCORL1, DNMT3A, ASXL1). Multiple types of alterations often co-occur, however due to the inherent limitations of conventional methods, it is not known if i) these alterations are in the same or independent clones, ii) what is the mutational hierarchy, and iii) what is the cell of origin. We addressed these questions using single-cell and bulk genomic sequencing.

Chromosomal translocations are a genomic hallmark of many hematologic malignancies. Often as initiating events, these structural abnormalities result in fusion proteins involving transcription factors important for hematopoietic differentiation and/or signaling molecules regulating cell proliferation and cell cycle. In contrast, epigenetic regulator genes are more frequently targeted by somatic sequence mutations, possibly as secondary events to further potentiate leukemogenesis. Through comprehensive whole-transcriptome sequencing of 231 children with acute lymphoblastic leukemia (ALL), we identified 58 putative functional and predominant fusion genes in 54.1% of patients (n= 125), 31 of which have not been reported previously. In particular, we described a distinct ALL subtype with a characteristic gene expression signature predominantly driven by chromosomal rearrangements of the ZNF384gene with histone acetyltransferases EP300and CREBBP. ZNF384-rearranged ALL showed significant up-regulation of CLCF1and BTLAexpression, and ZNF384 fusion proteins consistently showed higher activity to promote transcription of these target genes relative to wild-type ZNF384 in vitro. Ectopic expression of EP300-ZNF384and CREBBP-ZNF384fusion altered differentiation of mouse hematopoietic stem and progenitor cells and also potentiated oncogenic transformation in vitro. EP300-and CREBBP-ZNF384fusions resulted in loss of histone lysine acetyltransferase activity in a dominant-negative fashion, with concomitant global reduction of histone acetylation and increased sensitivity of leukemia cells to histone deacetylase inhibitors. In conclusion, our results indicate that gene fusion is a common class of genomic abnormalities in childhood ALL and that recurrent translocations involving EP300and CREBBPmay cause epigenetic deregulation with potential for therapeutic targeting.

Hhexencodes a homeodomain transcription factor that is widely expressed in hematopoietic stem and progenitor cell populations. Its enforced expression induces T‐cell leukemia and we have implicated it as an important oncogene in early T‐cell precursor leukemias where it is immediately downstream of an LMO2‐associated protein complex. Conventional Hhexknockouts cause embryonic lethality precluding analysis of adult hematopoiesis. Thus, we induced highly efficient conditional knockout (cKO) using vav‐Cre transgenic mice. Hhex cKOmice were viable and born at normal litter sizes. At steady state, we observed a defect in B‐cell development that we localized to the earliest B‐cell precursor, the pro‐B‐cell stage. Most remarkably, bone marrow transplantation using Hhex cKOdonor cells revealed a more profound defect in all hematopoietic lineages. In contrast, sublethal irradiation resulted in normal myeloid cell repopulation of the bone marrow but markedly impaired repopulation of T‐ and B‐cell compartments. We noted that Hhex cKOstem and progenitor cell populations were skewed in their distribution and showed enhanced proliferation compared to WT cells. Our results implicate Hhexin the maintenance of LT‐HSCs and in lineage allocation from multipotent progenitors especially in stress hematopoiesis. StemCells2015;33:2628—2641 The conditional deletion of Hhexaffects the function of hematopoietic stem and progenitor cells, especially perturbing the differentiation of lymphoid lineages.

LIM domain only 2(Lmo2) is frequently deregulated in sporadic and gene therapy‐induced acute T‐cell lymphoblastic leukemia (T‐ALL) where its overexpression is an important initiating mutational event. In transgenic and retroviral mouse models, Lmo2expression can be enforced in multiple hematopoietic lineages but leukemia only arises from T cells. These data suggest that Lmo2confers clonal growth advantage in T‐cell progenitors. We analyzed proliferation, differentiation, and cell death in CD2‐Lmo2transgenic thymic progenitor cells to understand the cellular effects of enforced Lmo2expression. Most impressively, Lmo2transgenic T‐cell progenitor cells were blocked in differentiation, quiescent, and immortalized in vitro on OP9‐DL1 stromal cells. These cellular effects were concordant with a transcriptional signature in Lmo2transgenic T‐cell progenitor cells that is also present in hematopoietic stem cells (HSCs) and early T‐cell precursor ALL. These results are significant in light of the crucial role of Lmo2in the maintenance of the HSC. The cellular effects and transcriptional effects have implications for LMO2‐dependent leukemogenesis and the treatment of LMO2‐induced T‐ALL. STEMCELLS2013;31:882–894

No relevant conflicts of interest to declare.

Hematopoietically expressed homeobox(Hhex) is a T-cell oncogene. It is frequently deregulated in murine retroviral insertional mutagenesis screens and its enforced expression induces T-cell leukemia in bone marrow transduction and transplantation experiments. We discovered that HHEXis a direct transcriptional target of an LIM domain Only-2 (LMO2)-associated protein complex. HHEXclusters with LMO2-overexpressing T-ALLs and is especially overexpressed in Early T-cell Precursor (ETP) – ALL where it is a direct transcriptional target of LMO2. To further understand Hhex's function, we induced a conditional knockout in floxed Hhexmice with the Vav-iCretransgene. Mice were viable and showed normal blood cell counts with highly efficient deletion of Hhexin all hematopoietic tissues. Thymocytes from conditional knockouts showed a normal pattern of development. Most impressively, Hhexconditional knockout markedly prolonged the latency of T-ALL onset in CD2-Lmo2transgenic mice (figure 1). Hhexconditional knockouts (HhexcKOs) also had a significant decrease in mature B cells in the spleen and bone marrow. Interestingly, hematopoietic stem and progenitor cells plated on OP9-GFP or OP9-DL1 stromal cells showed proliferative defects and incomplete differentiation towards both B and T lineage. Also under stress conditions such as sublethal irradiation and competitive bone marrow transplants, Hhex conditional knockouts show a marked defect in both B and T lineages but an increase in early progenitor populations. Our experiments show that Hhexis a critical transcription factor in lymphoid development and in LMO2-induced T-ALL.Figure 1Hhexconditional knockout markedly prolonged the latency of T-ALL onset in CD2-Lmo2transgenic miceFigure 1. Hhex conditional knockout markedly prolonged the latency of T-ALL onset in CD2-Lmo2 transgenic mice

No relevant conflicts of interest to declare.

Abstract 4637