ALBERT

Description: Abstract 3472 Poster Board III-409 Background Tumor suppressor silencing via promoter CpG island hypermethylation is important in oncogenesis. DNA methyltransferase (DNMT) maintains promoter hypermethylation, and DNMT inhibitors (DNMTis) are of proven benefit for MDS and are in trials for AML and CML. Infant ALL is a rare, aggressive and distinctive subset of childhood ALL with 80% of cases harboring MLL gene rearrangements (MLL-r). Cooperating events leading to leukemia in these cases are largely unknown and recent studies suggest that oncogenesis is related to neither copy number alterations nor mutations in the tyrosine kinome. MLL has multiple domains with epigenetic activity, and silencing of several single tumor suppressor genes in MLL-r infant ALL cases has been described. We explored the role of genome-wide CpG island hypermethylation in MLL-r infant ALL, with a focus on its potential role as a novel molecular target for therapy. Methods HELP (HpaII-tiny fragment Enrichment by Ligation-mediated PCR), which accurately measures the abundance of DNA methylation at ∼30,000 CpG sites covering ∼13,000 promoter regions, was used to examine a cohorts of MLL-r infant ALL primary samples (n=5), other childhood ALL primary samples (TEL-AML1+, n=2 and hyperdiploid, n=3) and normal controls (CD34-selected- (n=2) and CD19-selected- (n=3) cord blood). To correlate HELP results with gene expression, we performed qRT-PCR on cDNA from the same samples for a set of 10 “genes of interest”, which were selected due to differential methylation in the HELP assay (n=6; DAPK1, DAXX, CASP9, LIFR, CCR6, HRK), or due to known biological significance (n=4; FLT3, HOXA9, MEIS1, FHIT). GAPDH and ABL were used as housekeeping genes. To explore the therapeutic potential of DNMTi in infant ALL, we treated MLL-r cell lines (n=3; SEMK2, KOPN8, HB1119) and an MLL-wt cell line (n=1; NALM6) with various concentrations (0, 0.5, 1, 2, 4 mM) of decitabine over multiple durations (8, 24, 48 and 72 hours), then performed MTT assays and qRT-PCR on the genes of interest for each concentration/time point combination. To validate the HELP assay findings and to determine whether the effects of decitabine on re-expression of silenced genes was due to DNMT activity, we also performed methylation specific PCR (MSP) for 3 of the genes of interest (DAPK1, CCR6, HRK) on the primary samples and the cell lines. Results Unsupervised analysis of the HELP assay showed tight clustering of samples into their known biological groups, indicating large differences in global methylation patterns, and the ability of these patterns to accurately sub-classify ALL samples. Global hypermethylation was seen in the MLL-r cohort compared to both the normals and the other ALLs, with ratios of significantly (p

Description: Cooperating leukemogenic events in MLL-rearranged (MLL-r) infant acute lymphoblastic leukemia (ALL) are largely unknown. We explored the role of promoter CpG island hypermethylation in the biology and therapeutic targeting of MLL-r infant ALL. The HELP (HpaII tiny fragment enrichment by ligation-mediated polymerase chain reaction [PCR]) assay was used to examine genome-wide methylation of a cohort of MLL-r infant leukemia samples (n = 5), other common childhood ALLs (n = 5), and normals (n = 5). Unsupervised analysis showed tight clustering of samples into their known biologic groups, indicating large differences in methylation patterns. Global hypermethylation was seen in the MLL-r cohort compared with both the normals and the others, with ratios of significantly (P 〈 .001) hypermethylated to hypomethylated CpGs of 1.7 and 2.9, respectively. A subset of 7 differentially hypermethylated genes was assayed by quantitative reverse-transcription (qRT)–PCR, confirming relative silencing in 5 of 7. In cell line treatment assays with the DNA methyltransferase inhibitor (DNMTi) decitabine, MLL-r (but not MLL wild-type cell lines) showed dose- and time-dependent cytotoxicity and re-expression of 4 of the 5 silenced genes. Methylation-specific PCR (MSP) confirmed promoter hypermethylation at baseline, and a relative decrease in methylation after treatment. DNMTi may represent a novel molecularly targeted therapy for MLL-r infant ALL.

Description: FLT3 expression level in leukemia cells is associated with specific histone modifications that are associated with presence or absence of MLL fusion genes FLT3 is a class III receptor tyrosine kinase that is normally expressed in hematopoietic stem/progenitor cells. FLT3 expression is lost as hematopoietic cells differentiate. Based on murine knockout studies, FLT3 signaling is known to be important in the development of myeloid progenitors, B lymphoid progenitors, NK cells and dendritic cells. The FLT3 gene is mutated in about one third of acute myeloid leukemia. Apart from the activating mutations, it is also over-expressed in wide range of pre-B and myeloid leukemias. It is particularly highly expressed in leukemias harboring rearrangements of MLL at 11q23. The quantitative level of FLT3 expression in these leukemias is orders of magnitude higher than in germline (wild type) MLL leukemias of similar lineage. The MLL protein is known to have histone methyltransferase activity which resides in the C-terminal SET domain, which predominantly mediates H3K4 methylation. The MLL fusion proteins that result from MLL rearrangements lack the SET domain, but form complexes that interact with DOT1L, a H3K79 methyltransferase. These histone modifying properties of germline and rearranged MLL are central to the function of these proteins as master regulators of target gene expression. The mechanism(s) regulating the level of expression of FLT3 in hematopoietic cells have not been described. Given the association of high FLT3 expression levels with MLL rearrangements, and the recent elucidation of the role of the MLL gene and its fusion proteins in histone modification, we hypothesized that histone modifications may play an important role in the regulation of FLT3 expression. On histone H3, acetylation on lysines 9 and 14 and methylation on lysines 4, 36, and 79 are linked to active transcription, whereas tri-methylation on lysines 9 and 27 is linked to transcriptional repression. We analyzed the modification of histone H3 at lysine 9 (acetylation and tri-methylation) and 14 (acetylation) at the FLT3 promoter in different cell lines including pre-B ALL, monocytic AML, T-cell ALL and adenocarcinoma, with a range of quantitative FLT3 expression. For each of these cell lines, we performed ChIP using H3K9/14 acetyl and H3K9 tri-methyl antibodies followed by Real Time PCR with FLT3 promoter specific primers. The results are summarized in Table 1. Table 1 Cell line Lineage/origin Cytogenetics FLT3 expression mutational status ddC1 FLT3 promoter Acetyl/Methyl Ratio Acetyl H3K9 Methyl H3K9 MV4-11 monocylic AML MLL-AF4 High/TTD 11.1 5.55 2.00 Kopn 8 pre-BALL MLL-ENL High/wt 10.4 5.75 1.81 SEM pre-BALL MLL-AF4 High/wt 13.5 7.9 1.71 Nalm 6 pre-BALL ;(5;12) Low/wt 9 10.1 0.89 Jurkat TALL hypotetraploid Negative/wt 5.4 84 0.64 HeLa cervical CA aneuploid Negative/wt 4.05 9.5 0.43 We found that cell lines with robust FLT3 expression have higher acetylation at H3K9/14 than those with no or low FLT3 expression. Conversely, cell lines with no/low FLT3 expression have higher tri-methylation at H3K9 than those with high FLT3 expression. For individual cell lines, the ratio of acetyl H3K9/14 to tri-methyl H3K9 correlated with FLT3 expression. Furthermore, comparison of similar lineage cell lines with and without MLL rearrangements supports the hypothesis that the MLL mutational status (rearranged vs. germline) may dictate these differences. Kopn 8 (MLL rearranged, high FLT3) and Nalm6 (MLL germline, low FLT3) are both pre-B ALL leukemia cell lines that show converse H3K9/14 acetylation to H3K9 tri-methylation ratios. Together, these findings suggest that FLT3 expression may be controlled in part by histone modifications at its promoter, and that the mutational status of the MLL gene at 11q23 may be an important determinant of these modifications. In ongoing studies that will be reported at the meeting, we are expanding these studies to include additional H3 lysines (such as H3K4 and H3K79), additional cell lines, and primary patient leukemia samples. We are also performing ChIP assays on various sorted fractions of normal human bone marrow to ascertain whether histone modifications play a role in FLT3 expression during normal hematopoiesis.

Description: 2503 Epigenetic regulation of gene transcription is mediated both by methylation of DNA CpG islands and the local configuration of chromatin, which is dynamically regulated by post-translational modifications, or “marks”, of key lysines (K) of histones (especially H3). Some marks are associated with transcriptional repression [trimethylation (me3) of K9 and K27], and some with activation [me3 of K4, dimethylation (me2) of K79 and acetylation (Ac) of K9 and K14]. The MLL gene encodes a protein that functions as a master regulator of target gene expression by methylating H3K4 via its SET domain, and by interacting with other proteins with histone modifying properties. MLL is frequently rearranged (MLL-r) by translocations in acute leukemias, which exhibit a distinct global gene expression pattern. Many MLL-r partner genes form complexes that can methylate H3K79. Thus, histone modifications may be central to the function of both wild type (MLL-wt) and MLL-r. We hypothesized that aberrant histone coding of target genes contributes to MLL-r leukemogenesis. We characterized the histone code associated with the promoters of selected genes in n=5 MLL-r pre-B ALL samples (MLL-AF4 or MLL-ENL), n=4 MLL-wt pre-B ALL samples (TEL-AML1 or hyperdiploid) and normal control B-precursors (CD19+ cord blood cells). We selected 9 genes differentially overexpressed in MLL-r leukemia (HOXA7, HOXA9, MEIS1, FLT3, CCNA1, ZC3H12C, ATP8B4, C20orf103, and PROM1), and 3 control genes that are not MLL targets (HOXA1, HOXC8, LTF). We performed ChIP with antibodies specific for key H3 modifications (K4me3, K9me3, K9/14Ac, K27me3 and K79me2), followed by qPCR for the selected genes. Expression was measured by RT/qPCR. All 9 MLL target genes were significantly overexpressed in the MLL-r cohort, and this was associated with a specific “activating” histone code at the genes' promoters (fig 1 – MEIS1, e.g.). The opposite “repressive” code was found in the MLL-wt cohort, and in the MLL-r cohort at the promoters of the control genes. Compared to both sets of leukemias, normal B-precursors exhibited a paucity of histone modifications for all genes. For most genes, a specific developmental pattern of alterations in the histone code and corresponding relative change in expression could be traced from the normal B-precursors to the leukemia cells. This pattern was strikingly different in MLL-r leukemias than in MLL-wt leukemias, suggesting that the acquisition of MLL-r by normal B-precursors causes altered gene expression patterns via changes in the histone code. For most genes, normal B-precursors exhibit both the activating K4me3 mark and the repressive K27me3 mark, and express low but detectable levels of RNA. In MLL-r leukemias, upregulation of genes is associated with an increase in K4me3, loss of K27me3, and gain of K9/14Ac and/or K79me2. In MLL-wt leukemias, silencing of genes is associated with loss of K4me3 and gain of K9me3. To study the direct effects of MLL-wt and MLL-r on the histone code, we used 2 rounds of siRNA over 48 hours to knock down MLL-AF4 only, MLL-wt only or both in the RS4;11 cell line (MLL-AF4+ B-precursor ALL), then performed RT/qPCR and ChIP/qPCR. We achieved at least 60% knock down of MLL-AF4 and/or MLL-wt. Knock down of MLL-wt, with or without concomitant knock down of MLL-AF4, did not diminish the K4me3 mark for any genes, suggesting that MLL's SET domain is not required to maintain K4 methylation. While knock down of MLL-AF4 or MLL-wt alone did not diminish K79 methylation, knock down of both completely removed the K79me2 mark from all genes, suggesting that expression of either MLL-wt or MLL-AF4 is absolutely required for H3K79 methyltransferase activity. Two genes (HOXA7 and PROM1) demonstrated evidence of direct transcriptional regulation by MLL-AF4, since their expression decreased markedly after knock down of MLL-AF4 alone or with MLL-wt, but not with MLL-wt alone. In summary, primary MLL-r pre-B ALLs exhibit a distinct activating histone code at key overexpressed target genes when compared to MLL-wt pre-B ALLs and normal B-precursors. A causative role for MLL fusion proteins is suggested by the distinct pattern of histone code progression from normal B-precursors to MLL-r leukemias. Furthermore, knock down experiments provide direct evidence that some of the observed histone modifications in MLL-r leukemia, particularly H3K79 methylation, are directly downstream of wild type and mutant MLL. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 3506 Background: MLL gene rearrangements (MLL-r) are seen in all ages in both acute lymphocytic leukemia (ALL) and acute myeloid leukemia (AML). The most common fusion partners are AF4, AF9 and ENL/ELL. Murine models suggest that the expression of MLL fusion proteins is necessary but not sufficient for leukemogenesis, but true cooperating lesions have been difficult to identify. Our lab and others have previously shown that MLL-r infant ALL can be defined by a unique signature characterized by genome-wide hypermethylated CpG islands leading to tumor suppressor gene silencing. In this study we aimed to discover the driving mechanism of MLL-r infant ALL epigenetic signature and whether that signature is intrinsic to the MLL-r or possibly one of the elusive cooperating leukemogenic events. Methods: We measured DNA methylation levels at roughly 225,000 CpG sites covering over 19,000 genes using the HELP (HpaII-tiny fragment Enrichment by Ligation-mediated PCR) assay to evaluate 102 leukemia samples and 12 normal controls. Gene expression using Illumina HT-12 arrays were concurrently performed for future paired expression-methylation analyses. Leukemia samples were primary diagnostic patient samples representing ages infancy to adulthood and included 60 ALL samples (36 MLL-r and 24 MLL-wt) and 42 AML samples (29 MLL-r and 13 MLL-wt). We used unsupervised hierarchical clustering and principal component analysis (PCA) to evaluate whether the methylation patterns in the 102 samples discriminate between lineage (ALL vs. AML), MLL fusion status (MLL-r vs. MLL-wt), fusion partners, and age (infant, pediatric, adult). Results: Hierarchical clustering, using the top 50% of genes that show highest variance in methylation level across all 102 samples, discriminated leukemia samples into distinct lineage specific groups (ALL vs. AML). Within the ALL cluster, there were distinct sub-clusters separating T- and B- cell disease. In addition, In B- cell disease only, MLL-r and MLL-wt formed distinct sub-clusters while no MLL-r vs MLL-wt subclustering was noticed in AML. PCA recapitulated descriptive findings. Regression analysis of the first principal component of all 102 samples showed significant linear separation of AML vs. ALL (p