Li Chong Chan

Protein arginine-methyltransferase-dependent oncogenesis

Enzymes that mediate reversible epigenetic modifications have not only been recognized as key in regulating gene expression and oncogenesis, but also provide potential targets for molecular therapy. Although the methylation of arginine 3 of histone 4 (H4R3) by protein arginine methyltransferase 1 (PRMT1) is a critical modification for active chromatin and prevention of heterochromatin spread, there has been no direct evidence of any role of PRMTs in cancer. Here, we show that PRMT1 is an essential component of a novel Mixed Lineage Leukaemia (MLL) oncogenic transcriptional complex with both histone acetylation and H4R3 methylation activities, which also correlate with the expression of critical MLL downstream targets. Direct fusion of MLL with PRMT1 or Sam68, a bridging molecule in the complex for PRMT1 interaction, could enhance self-renewal of primary haematopoietic cells. Conversely, specific knockdown of PRMT1 or Sam68 expression suppressed MLL-mediated transformation. This study not only functionally dissects the oncogenic transcriptional machinery associated with an MLL fusion complex, but also uncovers — for the first time — an essential function of PRMTs in oncogenesis and reveals their potential as novel therapeutic targets in human cancer.

The interaction between EEN and Abi-1, two MLL fusion partners, and synaptojanin and dynamin : implications for leukaemogenesis

The mixed lineage leukaemia gene, MLL (also called HRX, ALL-1) in acute leukaemia is fused to at least 16 identified partner genes that display diverse structural and biochemical properties. Using GST pull down and the yeast two hybrid system, we show that two different MLL fusion partners with SH3 domains, EEN and Abi-1, interact with dynamin and synaptojanin, both of which are involved in endocytosis. Synaptojanin, a member of the inositol phosphatase family that has recently been shown to regulate cell proliferation and survival, is also known to bind to Eps15, the mouse homologue of AF1p, another fusion partner of MLL. Expression studies show that synaptojanin is strongly expressed in bone marrow and immature leukaemic cell lines, very weakly in peripheral blood leukocytes and absent in Raji, a mature B cell line. We found that the SH3 domains of EEN and Abi-1 interact with different proline-rich domains of synaptojanin while the EH domains of Eps15 interact with the NPF motifs of synaptojanin. In vitro competitive binding assays demonstrate that EEN displays stronger binding affinity than Abi-1 and may compete with it for synaptojanin. These findings suggest a potential link between MLLfusion-mediated leukaemogenesis and the inositol-signalling pathway.

The impact of differential binding of wild-type RARα, PML-, PLZF- and NPM-RARα fusion proteins towards transcriptional co-activator, RIP-140, on retinoic acid responses in acute promyelocytic leukemia

Retinoic acid receptor (RA) heterodimer (RAR/RXR) activities have been shown to be repressed by transcriptional co-repressor, SMRT/N-CoR, in the absence of the ligand while upon all-trans retionic acid (ATRA) treatment, SMRT/N-CoR is dissociated from RARα leading to gene expression by the recruitment of transcriptional co-activators to the transcriptional complex. The difference in response to ATRA therapy between acute promyelocytic leukemia (APL) patients with PML-RARα fusion and PLZF-RARα fusion has recently been found to be partially due to the strong association of the transcriptional co-repressor, SMRT/N-CoR, with PLZF domain. We demonstrate that SMRT association, as with PML-RARα, can be released from NPM-RARα at pharmacological concentration of ATRA (10−6 M). Moreover, we show for the first time that the interaction between the transcriptional co-activator, RIP-140, and PML-, PLZF- or NPM-RARα fusion proteins can be positively stimulated by ATRA although they are less sensitive as compared with the wild-type RARα. Our results suggest that the dissociation of transcriptional co-repressors, SMRT/N-CoR, and recruitment of co-activators, eg RIP-140, to APL-associated fusion proteins constitute a common molecular mechanism in APL and underlie the responsiveness of the disease to RA therapy.

Targeting Aberrant Epigenetic Networks Mediated by PRMT1 and KDM4C in Acute Myeloid Leukemia

Summary Transcriptional deregulation plays a major role in acute myeloid leukemia, and therefore identification of epigenetic modifying enzymes essential for the maintenance of oncogenic transcription programs holds the key to better understanding of the biology and designing effective therapeutic strategies for the disease. Here we provide experimental evidence for the functional involvement and therapeutic potential of targeting PRMT1, an H4R3 methyltransferase, in various MLL and non-MLL leukemias. PRMT1 is necessary but not sufficient for leukemic transformation, which requires co-recruitment of KDM4C, an H3K9 demethylase, by chimeric transcription factors to mediate epigenetic reprogramming. Pharmacological inhibition of KDM4C/PRMT1 suppresses transcription and transformation ability of MLL fusions and MOZ-TIF2, revealing a tractable aberrant epigenetic circuitry mediated by KDM4C and PRMT1 in acute leukemia.

Expression and protein-binding studies of the EEN gene family, new interacting partners for dynamin, synaptojanin and huntingtin proteins.

EEN, identified initially as a fusion partner to the mixed-lineage leukaemia gene in human leukaemia, and its related members, EEN-B1 and EEN-B2, have recently been shown to interact with two endocytic molecules, dynamin and synaptojanin, as well as with the huntingtin protein. In the present study, we show that the expression of the EEN gene-family members is differentially regulated. Multiple-spliced variants were identified for EEN-B2. In the brain, EEN-B1 and EEN-B2 mRNA are preferentially expressed in the cerebellar Purkinje and granule cells, dentate gyrus cells, hippocampal pyramidal neurons and cerebral granule cells. The expression patterns of EEN-B1 and EEN-B2 mRNA in the brain overlap with those of dynamin-I/III, synaptojanin-I and huntingtin, whereas the ubiquitous expression of EEN is consistent with that of dynamin-II. In testes, members of the EEN family are co-expressed with testis-type dynamin and huntingtin in Sertoli cells and germ cells respectively. Our results on the overlapping expression patterns are consistent with the proposed interaction of EEN family members with dynamin, synaptojanin and huntingtin protein in vivo. Although all three EEN family members bind to dynamin and synaptojanin, EEN-B1 has the highest affinity for binding, followed by EEN and EEN-B2. We also demonstrate that amphiphysin, a major synaptojanin-binding protein in brain, can compete with the EEN family for binding to synaptojanin and dynamin. We propose that recruitment of the EEN family by dynamin/synaptojanin to clathrin-coated pits can be regulated by amphiphysin.

The Mll-Een knockin fusion gene enhances proliferation of myeloid progenitors derived from mouse embryonic stem cells and causes myeloid leukaemia in chimeric mice

Rearrangement of the mixed lineage leukaemia (MLL) gene with extra eleven nineteen (EEN) was previously identified in an infant with acute myeloid leukaemia. Using homologous recombination, we have created a mouse equivalent of the human MLL-EEN allele and showed that when MllEen/+ embryonic stem (ES) cells were induced to differentiate in vitro into haemopoietic cells, there was increased proliferation of myeloid progenitors with self-renewal property. We also generated MllEen/+ chimeric mice, which developed leukaemia displaying enlarged livers, spleens, thymuses and lymph nodes owing to infiltration of MllEen/+-expressing leukemic cells. Immunophenotyping of cells from enlarged organs and bone marrow (BM) of the MllEen/+ chimeras revealed an accumulation of Mac-1+/Gr-1− immature myeloid cells and a reduction in normal B- and T-cell populations. We observed differential regulation of Hox genes between myeloid cells derived from MllEen/+ ES cells and mouse BM leukemic cells which suggested different waves of Hox expression may be activated by MLL fusion proteins for initiation (in ES cells) and maintenance (in leukemic cells) of the disease. We believe studies of MLL fusion proteins in ES cells combined with in vivo animal models offer new approaches to the dissection of molecular events in multistep pathogenesis of leukaemia.

Subcellular localization of EEN/endophilin A2, a fusion partner gene in leukaemia

EEN (extra eleven nineteen), also known as EA2 (endophilin A2), a fusion partner of the MLL (mixed-lineage leukaemia) gene in human acute leukaemia, is a member of the endophilin A family, involved in the formation of endocytic vesicles. We present evidence to show that EEN/EA2 is localized predominantly in nuclei of various cell lines of haemopoietic, fibroblast and epithelial origin, in contrast with its reported cytoplasmic localization in neurons and osteoclasts, and that EEN/EA2 exhibits nucleocytoplasmic shuttling. During the cell cycle, EEN/EA2 shows dynamic localization: it is perichromosomal in prometaphase, co-localizes with the bipolar spindle in metaphase and anaphase and redistributes to the midzone and midbody in telophase. This pattern of distribution coincides with changes in protein levels of EEN/EA2, with the highest levels being observed in G2/M-phase. Our results suggest that distinct subcellular localization of the endophilin A family members probably underpins their diverse cellular functions and indicates a role for EEN/EA2 in the cell cycle.

Epigenetic dysregulation of leukaemic HOX code in MLL-rearranged leukaemia mouse model

HOX genes are frequently dysregulated in human leukaemia with the gene rearrangement between mixed lineage leukaemia (MLL) and partner genes. The resultant MLL fusion proteins are known to mediate leukaemia through disruption of the normal epigenetic regulation at the target gene loci. To elucidate the pathogenic role of MLL fusion proteins in HOX dysregulation in leukaemia, we generated a novel haematopoietic lineage‐specific Mll–Een knock‐in mouse model using a Cre‐mediated inversion strategy. The MllEen/+ invertor mice developed acute myeloid leukaemia, with organomegaly of the spleen, liver and mesenteric lymph nodes caused by infiltration of blast cells. Using Mll–Een‐expressing leukaemic cell lines derived from bone marrow of MllEen/+ mutant mice, we showed that induction of Hox genes in leukaemic cells was associated with hypomethylated promoter regions and an aberrant active chromatin state at the Hox loci. Knock‐down of Prmt1 was insufficient to reverse the active chromatin status and the hypomethylated Hox loci, suggesting that Prmt1‐mediated histone arginine methylation was only partially involved in the maintenance of Hox expression in leukaemic cells. Furthermore, in vivo analysis of bone marrow cells of MllEen/+ mice revealed a Hox expression profile similar to that of wild‐type haematopoietic stem cells. The leukaemic Hox profile was highly correlated with aberrant hypomethylation of Hox promoters in the mutant mice, which highlights the importance of DNA methylation in leukaemogenic mechanisms induced by MLL fusion proteins. Our results point to the involvement of dynamic epigenetic regulations in the maintenance of the stem cell‐like HOX code that initiates leukaemic stem cells in MLL‐rearranged leukaemia. This provides insights for the development of alternative strategies for leukaemia treatment. Copyright

Etiological Mechanisms in Childhood Acute Lymphoblastic Leukemia

Childhood acute lymphoblastic leukemia (ALL) is a biologically diverse malignancy. The major subsets correspond to B or T precursor cells, although some ALL may originate in lympho—myeloid stem cells. Multiple molecular alterations contribute to the pathogenesis of ALT., including some that are subset specific. In those cases (~5%) with a Philadelphia chromosome, the genetic basis of karyotypic alteration and the resultant activated ABL kinase have been determined and offer new strategies for molecular diagnosis and monitoring. Although these changes at the DNA level are likely to be functionally relevant to the underlying mechanisms of leukemogenesis, the etiology of childhood ALL remains an enigma. A hypothesis is proposed that takes into account geographic and time trend variations in incidence rates, unusual properties of lymphocyte precursors, and promoting effects of immune responses in infancy.

Involvement of the D segment (DQ52) nearest to the JH region in immunoglobulin gene rearrangements of lymphoid-cell precursors.

The specific antigen recognition molecules expressed by B and T lymphocytes, the immunoglobulins (Ig) and the T-cell receptor for antigen (TCR) are coded for by genes which are assembled in an ordered series of somatic DNA recombination events during lymphocyte differentiation. In B lymphocytes the heavy chain gene of the Ig molecule (IgH) is the first to be assembled and this occurs by two successive DNA rearrangements in which first a diversity segment (D) and then a variable gene segment (V) are joined, usually by a process of intrachromosomal deletion, to a joining segment (J) to form a complete variable region sequence (for a review, see Alt et al. 1986). A similar sequence of DNA recombinations leads to the assembly of variable region sequences from V, D and J gene segments in the TCR genes (for a recent review, see Kronenberg et al. 1986).