ALBERT

Description: Abstract 2382 Epigenetic modification process is required for the development of hematopoietic cells. DNA methyltransferase DNMT3A, responsible for de novo DNA methylation, was newly reported to have a high frequency of mutations in hematopoietic malignancies. Conditional knock-out of DNMT3A promoted self-renewal activity of murine hematopoietic stem cells (HSCs). However, the role of mutated DNMT3A in hematopoiesis and its regulative mechanism of epigenetic network mostly remain unknown. Here we showed that the Arg882His (R882H) hotspot locus on DNMT3A impaired the normal function of this enzyme and resulted in an abnormal increase of primitive hematopoietic cells. In both controlled in vivo and in vitro assays, we found that the cells transfected by R882H mutant promoted cell proliferation, while decreased the differentiation of myeloid lineage compared to those with wild type. Analysis of bone marrow (BM) cells from mice transduced by R882H reveals an expansion of Lin−Sca-1+C-kit+ populations and a reduction of mature myeloid cells. Meanwhile, a cluster of upregulated genes and downregulated lineage-specific differentiation genes associated with hematopoiesis were discovered in mice BM cells with R882H mutation. We further evaluated the association of mutated DNMT3A and HOXB4 which was previously detected to be highly expressed in clinical samples carrying R882 mutation. Compared with wildtype DNMT3A, R882H mutation disrupted the repression of HOXB4 by largely recruiting tri-methylated histone 3 lysine 4 (H3K4). Taken together, our results showed that R882H mutation disturbed HSC activity through H3K4 tri-methylation, and transcriptional activation of HSC-related genes. Disclosures: No relevant conflicts of interest to declare.

Description: Background. In previous study of adult and childhood B-cell acute lymphoblastic leukemia, through whole exome and transcriptome sequencing we identified three new categories of fusion genes with prognostic significance and much more mutations of epigenetic regulators in adult versus pediatric B-ALL cases. To address how these gene alterations contribute to leukemogenesis and affect prognosis, we performed detailed data analysis and functional study of the three new categories of B-ALL. In addition, the changes of epigenetic regulator were scrutinized in a systematic way. Methods. Differentially expressed genes analysis and gene set enrichment analysis (GSEA) were performed. The regulation of target genes by distinct fusions was further verified through lentivirus mediated overexpression, shRNA induced knockdown, quantitative real-time PCR as well as transcriptional reporter assays. The modification of histone acetylation was evaluated by Western bloting. To observe the effect of fusion genes in vivo, murine retroviral bone marrow transplantation models were established. Results. Through in-depth data analysis, differentially expressed genes and signaling pathways related to fusion genes were identified. Alternative splicing of transcription factor ERG was seen in most of the cases with DUX4 fusions. In some of the cases, both ERG deletion leading to frameshift of the open reading frame and an abnormal short transcript of ERG were also observed. This group can also be characterized by some outlier genes (AGAP1, STAP1, PTPRM1, PCDH17 and PLEKHA6). Overexpression of DUX4-IGH in human B-ALL cell line induced the appearance of the short form of ERG and upregulation of these outlier genes. In mouse models, both DUX4-IGH and the short ERG inhibited the development of B-cell lineage. NT5E, also known as CD73, which has been reported to promote anthracycline resistance, was significantly downregulated in this group, which could explain the favorable prognosis of this type of B-ALL. Pre-BCR signaling pathway maintaining the survival of leukemic cells at the pre-B stage was activated in patients with MEF2D fusions. Genes related to protein amimo acid phosphorylation and cell morphogenesis involved in differentiation were significantly upregulated in this group. Notably, this group exhibited rather poor survival rate, and the expression of CD73 in this group was as high as that in BCR-ABL1 and BCR-ABL1 like group. When juxtaposed to the upstream of ZNF384, EP300 and CREBBP lost the histone acetylation domain, which could reduce the histone acetylation level and be involved in the leukemogenesis. Indeed, EP300-ZNF384 induced the rapid emergence of acute leukemia in mouse with a median survival of 100 days. In addition, we checked the sequence variations in our whole exome/genome dataset containing 203 cases. When mutations of different gene categories were scrutinized, abnormalities of epigenetic regulators (ER) drew our attention. Using the ER database that St. Jude Children's Research Hospital established, we found a total of 140 somatic mutations in 80 ER classified into eight classes. Forty two percent of the 203 patients (56.5% of adult and 30.6% of children) harbored at least one gene mutation among the eight classes of ER. The fact that abnormalities of MLL2 and SETD2, the two ER genes with highest mutation rates, were mutually exclusive in most affected cases suggests that they should bear somehow functional resemblance. Conclusions. Detailed study of MEF2D, ZNF384 and DUX4-IGH gene fusions expands the extant knowledge about molecular subtypes of B-ALL, and provides more potential targets as well as promotes further personalized treatment in both childhood and adult B-ALL. The overview of ER alterations in adult and childhood B-ALL proves the importance of these genes in the pathogenesis of B-ALL, especially in adult cases. Disclosures No relevant conflicts of interest to declare.

Description: Abstract 1433 BACKGROUND High levels of 2-HG in serum were found among AML cases with IDH1/IDH2 mutations. However, the impact of 2-HG levels on biological characteristics and clinical outcomes has not been systematically evaluated in a large cohort of AML patients. METHODS Serum 2-HG levels in 699 patients with hematologic malignancies, including 405 AML patients, were measured by GC-TOFMS. Clinical prognostic power of 2-HG and genetic risk factors associated with 2-HG were also evaluated in AML patients. RESULTS Among all patients with hematopoietic malignancies investigated, high serum 2-HG levels were observed only in AML group. 64 out of 405 (15.8%) AML patients displayed aberrantly higher levels (7.14±2.11μg/ml) of 2-HG. Compared to cases with normal 2-HG (3.65±1.02μg/ml), these patients showed a higher prevalence in AML-M0/M1 subtypes, a closer correlation with mutated IDH1/2 genes, a distinct gene expression profile and an aberrant DNA methylation status in bone marrow blasts. Additionally, the patients with high 2-HG were associated with higher serum levels of α-ketoglutarate (α-KG) and glutamate, suggesting presence of impaired metabolic pathway involved in the biosynthesis of 2-HG. Univariate and multivariate analyses indicate that high level of 2-HG is among the most significant negative indicators for complete remission (CR) rate, overall survival (OS) and event-free survival (EFS) either in all AML cases or in cases with cytogenetically normal AML (CN-AML). CONCLUSIONS Serum 2-HG is an independent prognostic marker in AML. Patients with high 2-HG had significantly higher frequency of IDH1/IDH2 gene mutations and unfavorable prognosis compared to those with normal 2-HG. Disclosures: No relevant conflicts of interest to declare.

Description: The BCR-ABL fusion protein generated by t(9;22)(q34;q11) in chronic myeloid leukemia (CML) plays an essential role in the pathogenesis of the myeloproliferative disorder status at the chronic phase of the disease, but progression from the chronic phase to blast crisis (BC) is believed to require additional mutations. To explore the underlying mechanisms for BC, which is characterized by a blockage of blood cell differentiation, we screened several genes crucial to hematopoiesis and identified 10 types of mutations in RUNX1 among 11 of 85 (12.9%) patients with acute transformation of CML. Most of the mutations occurred in the runt homology domain, including H78Q, W79C, R139G, D171G, R174Q, L71fs-ter94, and V91fs-ter94. Further studies indicated that RUNX1 mutants not only exhibited decreased transactivation activity but also had an inhibitory effect on the WT RUNX1. To investigate the leukemogenic effect of mutated RUNX1, H78Q and V91fs-ter94 were transduced into 32D cells or BCR-ABL–harboring murine cells, respectively. Consistent with the myeloblastic features of advanced CML patients with RUNX1 mutations, H78Q and V91fs-ter94 disturbed myeloid differentiation and induced a BC or accelerated phase–like phenotype in mice. These results suggest that RUNX1 abnormalities may promote acute myeloid leukemic transformation in a subset of CML patients.

Description: BACKGROUND B-lineage acute lymphoblastic leukemia (B-ALL) represents the most common subtype in ALL. Genomic sequence information has been lacking in adult B-ALL, whose outcome remains dismal, while a comparison between genomic abnormalities in adult and pediatric groups is useful to further decipher disease mechanisms. METHODS We used whole exome sequencing (WES), copy number variation and molecular cytogenetics to catalog somatic mutations in 95 B-ALL patients (43 adults and 52 children).WES was conducted with appropriate depths for paired genomic DNA from bone marrow mononuclear cells at diagnosis and their matched peripheral blood samples during complete remission (CR) or saliva samples. Targeted deep sequencing (TDS) was performed in a validation cohort of 179 adult and 199 pediatric B-ALLs. RESULTS Eighty-four recurrent gene mutations were revealed by WES. Integrative analysis identified the involvement of 9 functional categories of genes: key fusions (KFs), epigenetic modifiers (EMs), signaling molecules (SMs), transcription factors (TFs), tumor suppressors (TSs), actin binding/cytoskeletons (ABCs), ion binding proteins (IBPs), trans-membrane proteins (TPs) and the others. Mutually cooperative or exclusive relationships were revealed among some of these categories. Genomic landscapes suggested two distinct mechanisms involved in the leukemogenesis: one is mainly driven by KFs together with mutations of TFs and TSs; the other results from the abnormalities of TFs and TSs, in cooperation with mutations of EMs, SMs, ABCs and IBPs recapitulating the role of KFs. A panel of histone/DNA methylation modifiers (HMMs) were revealed to bear potential value of relatively favorable prognosis in both adult and childhood patients. CONCLUSIONS We described the genome-wide abnormality patterns in adult B-ALL patients in comparison with those of pediatric cases, contributing to the understanding of leukemogenesis and the identification of potential new prognostic markers. Disclosures No relevant conflicts of interest to declare.