ALBERT

Description: Journal of the American Chemical Society DOI: 10.1021/jacs.7b04418

Description: Background: Acquired aplastic anemia (AA), the prototypical bone marrow failure syndrome, is inferred to result from immune-mediated destruction of hematopoietic progenitors, as most patients respond to immunosuppressive therapies. Clonal hematopoiesis in AA is evident in the presence of paroxysmal nocturnal hemoglobinuria (PNH) cells in as many as half of patients and by identification of uniparental disomies involving 6p (6pUPD) chromosome in 13% of cases. In addition, "clonal transformation", as defined by the development of myelodysplastic syndromes (MDS) or acute myelogenous leukemia (AML) is a serious long-term complication in 10-15% AA patients. Methods: We performed targeted deep sequencing and SNP array-based copy number (CN) analysis of peripheral blood- or granulocyte-derived DNA from 439 patients with AA (280 from US and 159 from Japanese cohorts) for a panel of 103 candidate genes, chosen because they are known to be frequently mutated in myeloid neoplasms. Germline DNA was available for 288 out of 439 patients and was used to confirm the somatic origin of mutations. Whole exome sequencing (WES) was performed in 52 cases. Where serial samples were available, the chronology of detected mutations was also investigated. Results: Targeted deep sequencing provided highly concordant results between the US and Japanese cohorts; approximately one third of AA patients had mutations in genes commonly affected in myeloid neoplasms, and about one third of patients in whom mutations were identified had multiple mutations. Multi-lineage involvement of mutations was confirmed in 6 cases using flow-sorted bone marrow samples. However, compared to myeloid neoplasms, mutations in AA were at much lower variant allele frequencies (VAFs) (

Description: Abstract 2697 Adult T cell leukemia (ATL) is an aggressive peripheral T-cell neoplasm highly resistant to conventional therapies. The development of ATL is thought to be initiated by immortalization of T cells by human T cell leukemia virus type I (HTLV-I) infection in early childhood, followed by accumulating genetic hits during a long latency period, which eventually cause neoplastic transformation of T-cells. However, little has been known about those genetic hits that are involved in the pathogenesis of ATL, except for the role of tax protein in T-cell immortalization and alterations of other genes, including TP53, p16 and TCF8. So, in order to understand the genetic basis of ATL, we performed whole genome sequencing of paired-normal DNA from an ATL patient. In total 77 non-silent somatic mutations were detected, among which a TET2 mutation (R1261C) immediately drew our attention. TET2 mutations are found in a wide variety of myeloid malignancies in high frequency and implicated in their pathogenesis. The TET families of proteins are thought to be involved in the epigenetic regulation of gene expression through catalyzing conversion of 5'-methyl cytosine to 5'-hydroxymethyl cytosine, which are supposed to be further converted to unmethylated cytosine. One of the recent interests in TET2 mutations was the recent report of frequent TET2 mutations in peripheral T cell neoplasms, including angioimmunoblastic T-cell lymphomas, peripheral T-cell lymphomas not otherwise specified, as well as other B cell neoplasms, which is in agreement with the observation in TET2-deficient mice, which showed an expansion of bone marrow progenitor pools involving immature lymphoid populations, indicating that deregulated epigenetic machineries could be also involved in the development of mature lymphoid neoplasms. To explore this hypothesis, 144 samples of ATL were screened for mutations in TET2 and other epigenetic regulators commonly mutated in myeloid malignancies, including DNMT3A, IDH1/2 and spliceosome genes, using target deep sequencing. We found 16 TET2 mutations in 13 out of the 144 ATL cases (9%) together with other mutations, which was similar to the frequencies reported in other peripheral T cell neoplasms. Deep sequencing allowed for accurate estimation of allelic burden of these mutations. In most cases, TET2 mutations were found in the major tumor populations, indicating their early origin during clonal evolution. In addition, DNMT3A (2%), IDH1/2 (1%), SF3B1 (2%), ZRSR2 (1%) were also mutated, although at lower frequencies. In conclusion, we found TET2 mutation through whole genome sequencing. TET2 mutations were relatively common in ATL. Together with other mutations frequently found in myeloid malignancies, our finding provided an intriguing insight into the role of deregulated DNA methylation in the pathogenesis of ATL and also suggested that a common mechanism may underlie between the two very different blood cancers. Disclosures: No relevant conflicts of interest to declare.

Description: Adult T-cell leukemia/lymphoma (ATL) is a distinct form of peripheral T-cell lymphoma (PTCL). ATL is initiated by immortalization of T-cells by human T-lymphotropic virus type I (HTLV-1) infection during early infancy, followed by an accumulation of multiple genetic hits to develop leukemia. However, little has been known about these genetic hits. Recently, frequent somatic mutations in RHOA, DNMT3A, IDH2 and TET2 have been reported in angioimmunoblastic T cell lymphoma (AITL) and PTCL not otherwise specified (PTCL-NOS), which are characterized by follicular helper T-cell phenotypes. Most of the RHOA mutations in AITL invariably cause an identical amino acid change (Gly17Val), which was shown to inhibit wild-type RHOA function in a dominant-negative manner. In addition, RHOA mutations have been also identified in gastric cancer and Burkitt lymphoma, with mutational hotspots including Tyr42 and Arg5. Interestingly, some of them were reported as working in a gain-of function manner. In the current study, we investigated the mutational status of RHOA, as well as TET2, IDH2, and DNMT3A in a total of 205 patients with ATL in order to reveal their role in ATL pathogenesis. In targeted deep sequencing, RHOA was mutated in 32 (15.6%) cases. In contrast to the exclusive G17V mutation found in AITL and PTCL-NOS, mutations in ATL was widely distributed within the regions responsible for GTP-binding with Cys16Arg being most frequently mutated (N = 11; 5.4%), although Gly17Val mutations were also found in 4 cases (1.9%). Being commonly located in the same amino acid with the GTP binding site, Ala161Pro and Ala161Glu mutations were exclusively found in ATL and AITL/PTCL-NOS, respectively. We found no significant differences in the RHOA mutational status among different ATL subtypes, and no significant prognostic impact of RHOA mutations on survival. TET2 mutations were very common and tightly associated with RHOA mutation in AITL, but much less common (N = 21; 10.2%) and may not associated with RHOA mutations in ATL. Mutations of IDH2 (N = 2; 1%) and DNMT3A (N = 2; 1%) were rare in ATL. RHOA encodes a ras-related GTP-binding protein that functions as a molecular switch in a variety of biological processes through cycling between an active (GTP-bound) state and an inactive (GDP-bound) state. Known biological functions of RHOA are related to actin organization, cell migration and transcriptional activation. Modeling of three-dimensional structures suggested that most of mutations identified in ATL located near nucleotide binding pocket, which affected the GTP-binding ability of RHOA protein. Indeed, we performed luciferase assay to measure transcriptional activities of wild-type and mutant RHOA. As previously reported, dominant-negative Gly17Val RHOA mutant suppressed transcriptional activity. In contrast, ATL specific RHOA mutant such as Cys16Arg or Ala161Pro enhanced transcriptional activity, similar to or even more than wild-type RHOA. In addition, when we assessed actin stress fiber formation in cells transduced with wild-type or mutant RHOA, Gly17Val or Ala161Glu RHOA attenuated actin stress fiber formations, whereas Cys16Arg or Ala161Pro RHOA induced actin stress fiber formation, similar to known gain-of-function Gly14Val RHOA mutant. These functional assays implicated Cys16Arg and Ala161Pro RHOA mutant showed different biological behaviors from Gly17Val and Ala161Glu RHOA mutant, indicating gain-of-function mechanisms of Cys16Arg or Ala161Pro mutants. In summary, RHOA mutations were also common in ATL as in AITL and PTCL-NOS. Nevertheless, mutational patterns and functional consequences of RHOA mutations identified in ATL were distinct from those in other PTCLs, suggesting differential driver roles of RHOA mutations in ATL compared to other PTCLs. Disclosures No relevant conflicts of interest to declare.

Description: Adult T-cell leukemia/lymphoma (ATL) is an aggressive form of peripheral T-cell lymphoma (PTCL), which is etiologically associated with human T-lymphotropic virus type I (HTLV-1) infection during early infancy. Although HTLV-1 can effectively immortalize T-cells, there is a long latency period of ∼50 years prior to the onset of ATL, suggesting that HTLV-1 infection alone may not be sufficient for the development of ATL, but additional acquired genetic hits that occur in immortalized T-cells during the later life are essential for its pathogenesis. However, little has been known about those genetic hits that are involved in the pathogenesis of ATL. The purpose of this study is to understand the genetic basis of ATL, 32 cases with different ATL subtypes, including acute (N=15), chronic (N=6), lymphoma (N=10), smoldering (N=1) types were analyzed by whole exome sequencing as well as copy number analysis. With a mean coverage of 119, 94% of the target sequences were analyzed at more than 20 depth on average. A total of 2,862 somatic changes were detected in 32 cases with a true positive rate of 99% (329 of the 334 tested were confirmed by PCR-based deep sequencing). These consisted of 2,512 missense mutations, 174 nonsense mutations, 65 splice-site mutations, and 111 indels. The mutation rate of 89 (44-227) per sample was significantly higher than that in acute myeloid leukemia (7.3–13), myelodysplastic syndromes (9.2) and chronic lymphocytic leukemia (11.5). Recurrent mutations were observed in 350 genes, of which 192 were considered to be significantly mutated (q 〈 0.05) compared to background mutation rates (1.79 mutations per megabase). To investigate significantly mutated pathways, each pathway registered in the Kyoto Encyclopedia of Genes and Genomes, BioCarta, Reactome, Sigma-Aldrich and Signaling Transduction KE was tested on the basis of the background mutation rate observed in whole-exome sequencing data, which revealed a number of significantly mutated functional pathways, including pathways involeved in T cell receptor signaling, leukocyte trans-endothelial migration, VEGF and WNT signalings and other signaling pathways. Genes for epigenetic regulations were also among the frequent targets of gene mutations. We performed targeted deep sequencing of TET2, IDH1/2 and DNMT3A in 182 ATL cases. In total, 19 TET2 mutations were identified in 16 cases (8.7%). Different subtypes of ATL were almost evenly affected with 9 out of 67 acute, 3 out of 42 chronic and 4 out of 56 lymphoma types having TET2 mutations. Less frequent mutations of IDH2 and DNMT3A (both 1%) were also identified. Our findings on genetic alterations provide a novel insight into the pathogenesis of ATL. Disclosures: No relevant conflicts of interest to declare.

Description: Frequent pathway mutation involving multiple components of the RNA splicing machinery is a cardinal feature of myeloid neoplasms showing myeloid dysplasia, in which the major mutational targets include U2AF35, ZRSR2, SRSF2 and SF3B1. Among these, SF3B1 mutations were strongly associated with MDS subtypes characterized by increased ring sideroblasts, such as refractory anemia and refractory cytopenia with multiple lineage dysplasia with ring sideroblasts, suggesting the critical role of SF3B1 mutations in these MDS subtypes. However, currently, the molecular mechanism of SF3B1mutation leading to the ring sideroblasts formation and MDS remains unknown. The SF3B1 is a core component of the U2-small nuclear ribonucleoprotein (U2 snRNP), which recognizes the 3′ splice site at intron–exon junctions. It was demonstrated that Sf3b1 null mice were shown to be embryonic lethal, while Sf3b1 +/- mice exhibited various skeletal alterations that could be attributed to deregulation of Hox gene expression due to haploinsufficiency of Sf3b1. However, no detailed analysis of the functional role of Sf3b1 in hematopoietic system in these mice has been performed. So, to clarify the role of SF3B1 in hematopoiesis, we investigated the hematological phenotype of Sf3b1 +/- mice. There was no significant difference in peripheral blood counts, peripheral blood lineage distribution, bone marrow total cellularity or bone marrow lineage composition between Sf3b1 +/+ and Sf3b1 +/- mice. Morphologic abnormalities of bone marrow and increased ring sideroblasts were not observed. However, quantitative analysis of bone marrow cells from Sf3b1 +/- mice revealed a reduction of the number of hematopoietic stem cells (CD34 neg/low, cKit positive, Sca-1 positive, lineage-marker negative: CD34-KSL cells) measured by flow cytometry analysis, compared to Sf3b1 +/+ mice. Whereas examination of hematopoietic progenitor cells revealed a small decrease in KSL cell populations and megakaryocyte - erythroid progenitors (MEP) in Sf3b1 +/- mice, and common myeloid progenitors (CMP), granulocyte - monocyte progenitors (GMP) and common lymphoid progenitors (CLP) remained unchanged between Sf3b1 +/+ and Sf3b1 +/- mice. In accordance with the reduced number of hematopoietic stem cells in Sf3b1 +/- mice, the total number of colony-forming unit generated from equal number of whole bone marrow cells showed lower colony number in Sf3b1 +/- mice in vitro. Competitive whole bone marrow transplantation assay, which irradiated recipient mice were transplanted with donor whole bone marrow cells from Sf3b1 +/+ or Sf3b1 +/- mice with an equal number of competitor bone marrow cells, revealed impaired competitive whole bone marrow reconstitution capacity of Sf3b1 +/- mice in vivo. These data demonstrated Sf3b1 was required for hematopoietic stem cells maintenance. To further examine the function of hematopoietic stem cells in Sf3b1 +/- mice, we performed competitive transplantation of purified hematopoietic stem cells from Sf3b1 +/+ or Sf3b1 +/- mice into lethally irradiated mice together with competitor bone marrow cells. Sf3b1 +/- progenitors showed reduced hematopoietic stem cells reconstitution capacity compared to those from Sf3b1 +/+ mice. In serial transplantation experiments, progenitors from Sf3b1 +/- mice showed reduced repopulation ability in the primary bone marrow transplantation, which was even more pronounced after the second bone marrow transplantation. Taken together, these data demonstrate that Sf3b1 plays an important role in normal hematopoiesis by maintaining hematopoietic stem cell pool size and regulating hematopoietic stem cell function. To determine the molecular mechanism underlying the observed defect in hematopoietic stem cells of Sf3b1 +/- mice, we performed RNA-seq analysis. We will present the results of our biological assay and discuss the relation of Sf3b1 and hematopoiesis. Disclosures: No relevant conflicts of interest to declare.