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 Mll(Een) (/+) 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 Mll(Een) (/+) 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 Mll(Een) (/+) 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.

Homozygous L-SIGN (CLEC4M) plays a protective role in SARS coronavirus infection.

Severe acute respiratory syndrome (SARS) is caused by infection of a previously undescribed coronavirus (CoV). L-SIGN, encoded by CLEC4M (also known as CD209L), is a SARS-CoV binding receptor that has polymorphism in its extracellular neck region encoded by the tandem repeat domain in exon 4. Our genetic risk association study shows that individuals homozygous for CLEC4M tandem repeats are less susceptible to SARS infection. L-SIGN is expressed in both non-SARS and SARS-CoV-infected lung. Compared with cells heterozygous for L-SIGN, cells homozygous for L-SIGN show higher binding capacity for SARS-CoV, higher proteasome-dependent viral degradation and a lower capacity for trans infection. Thus, homozygosity for L-SIGN plays a protective role during SARS infection.

IDH2 mutations are frequent in angioimmunoblastic T-cell lymphoma.

Mutations in isocitrate dehydrogenase 1 (IDH1) and isocitrate dehydrogenase 2 (IDH2) occur in most grade 2 and 3 gliomas, secondary glioblastomas, and a subset of acute myelogenous leukemias but have not been detected in other tumor types. The mutations occur at specific arginine residues and result in the acquisition of a novel enzymatic activity that converts 2-oxoglutarate to D-2-hydroxyglutarate. This study reports IDH1 and IDH2 genotyping results from a set of lymphomas, which included a large set of peripheral T-cell lymphomas. IDH2 mutations were identified in approximately 20% of angioimmunoblastic T-cell lymphomas (AITLs), but not in other peripheral T-cell lymphoma entities. These results were confirmed in an independent set of AITL patients, where the IDH2 mutation rate was approximately 45%. This is the second common genetic lesion identified in AITL after TET2 and extends the number of neoplastic diseases where IDH1 and IDH2 mutations may play a role.

Cytokine combinations on the potential for ex vivo expansion of murine hematopoietic stem cells.

Hematopoietic stem cell (HSC) is a rare cell population, which is capable of self-renewal and differentiation to all blood lineages. The clinical potential of HSCs for treating hematological disorders has led to the use of cytokine stimulation for ex vivo expansion. However, little is known about the molecular features of the HSC populations expanded under different cytokine combinations. We studied the expansion of murine HSCs cultured with six different cytokine combinations under serum-containing or serum-free conditions for 14days. We found that all the cytokine combinations promoted expansion of murine HSCs. Although SCF/IL-3/IL-6 induced the highest expansion of the immunophenotypic Lineage(-)Sca-1(+)c-Kit(+) (LSK) cells at day 14, over 90% of them were FcεRIα(+) mast cells. In contrast, the serum-free medium with SCF/Flt3-L/IL-11 effectively promoted the expansion of LSK/FcεRIα(-) HSCs by over 50-fold. HSCs expanded by SCF/Flt3-L/IL-11 combination formed compact hematopoietic colonies and demonstrated a higher degree of multipotency compared to the HSCs cultured with other cytokine combinations. Surprisingly, despite the same LSK/FcεRIα(-) immunophenotype, HSCs cultured with different cytokine combinations demonstrated differential patterns of hematopoietic gene expression. HSCs cultured with SCF/Flt3-L/IL-11 maintained a transcription profile resembling that of freshly isolated HSCs. We propose that serum-free medium supplemented with SCF/Flt3-L/IL-11 is the optimal culture condition to maintain the stemness of ex vivo expanded HSCs. This study used molecular characterization of cytokine-expanded murine HSCs to facilitate the selection of cytokine combinations that could induce fully competent HSC for clinical applications.

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.

A 3-bp deletion in the HBS1L-MYB intergenic region on chromosome 6q23 is associated with HbF expression.

Fetal hemoglobin (HbF) is regulated as a multigenic trait. By genome-wide association study, we confirmed that HBS1L-MYB intergenic polymorphisms (HMIP) and BCL11A polymorphisms are highly associated with HbF in Chinese ß-thalassemia heterozygotes. In this population, the variance in HbF resulting from the HMIP is 13.5%; that resulting from the BCL11A polymorphism is 6.4%. To identify the functional variant in HMIP, we used 1000 Genomes Project data, single nucleotide polymorphism imputation, comparisons of association results across populations, potential transcription factor binding sites, and analysis of phylogenetic conservation. Based on these studies, a hitherto unreported association between HbF expression and a 3-bp deletion, between 135 460 326 and 135 460 328 bp on chromosome 6q23 was found. This 3-bp deletion is in complete linkage disequilibrium with rs9399137, which is the single nucleotide polymorphism in HMIP most significantly associated with HbF among Chinese, Europeans, and Africans. Chromatin immunoprecipitation assays confirmed erythropoiesis-related transcription factors binding to this region in K562 cells. Based on transient expression of a luciferase reporter plasmid, the DNA fragment encompassing the 3-bp deletion polymorphism has enhancer-like activity that is further augmented by the introduction of the 3-bp deletion. This 3-bp deletion polymorphism is probably the most significant functional motif accounting for HMIP modulation of HbF in all 3 populations.

CD44 activation in mature B-cell malignancies by a novel recurrent IGH translocation.

Using inverse polymerase chain reaction, we identified CD44, located on chromosome 11p13, as a novel translocation partner of IGH in 9 of 114 cases of gastric, nongastric extranodal, follicular, and nodal diffuse large B-cell lymphoma (DLBCL). Notably, these translocations involving IGHSmu were detected in follicular lymphomas and exclusively in germinal center B cell-ike (GCB)-DLBCLs. CD44 is not expressed in reactive GC B cells. The IGHSmu/CD44 translocations substitute Smu for the CD44 promoter and remove exon 1 of CD44, resulting in the overexpression of Imu-CD44 hybrid mRNA transcripts activated from derivative 11 that encode a new CD44 variant lacking the leader peptide and with a unique C-terminus (CD44DeltaEx1). When overexpressed in vitro in the CD44(-) GCB-DLBCL cell line BJAB, CD44DeltaEx1-green fluorescent protein localized to the cytoplasm and nucleus, whereas CD44s-green fluorescent protein (standard form) localized to the plasma membrane. The ectopic expression of CD44DeltaEx1 in BJAB cells enhanced their proliferation rate and clonogenic ability, indicating a possible pathogenic role of the translocation.

Rethinking the globalisation of problem-based learning: how culture challenges self-directed learning.

CONTEXT: Medical schools worldwide are increasingly switching to student-centred methods such as problem-based learning (PBL) to foster lifelong self-directed learning (SDL). The cross-cultural applicability of these methods has been questioned because of their Western origins and because education contexts and learning approaches differ across cultures. OBJECTIVES: This study evaluated PBL's cross-cultural applicability by investigating how it is applied in three medical schools in regions with different cultures in, respectively, East Asia, the Middle East and Western Europe. Specifically, it investigated how students' cultural backgrounds impact on SDL in PBL and how this impact affects students. METHODS: A qualitative, cross-cultural, comparative case study was conducted in three medical schools. Data were collected through 88 semi-structured, in-depth interviews with Year 1 and 3 students, tutors and key persons involved in PBL, 32 observations of Year 1 and 3 PBL tutorials, document analysis, and contextual information. The data were thematically analysed using the template analysis method. Comparisons were made among the three medical schools and between Year 1 and 3 students across and within the schools. RESULTS: The cultural factors of uncertainty and tradition posed a challenge to Middle Eastern students' SDL. Hierarchy posed a challenge to Asian students and achievement impacted on both sets of non-Western students. These factors were less applicable to European students, although the latter did experience some challenges. Several contextual factors inhibited or enhanced SDL across the cases. As students grew used to PBL, SDL skills increased across the cases, albeit to different degrees. CONCLUSIONS: Although cultural factors can pose a challenge to the application of PBL in non-Western settings, it appears that PBL can be applied in different cultural contexts. However, its globalisation does not postulate uniform processes and outcomes, and culturally sensitive alternatives might be developed.

Hb A2 Hong Kong - A novel δ-globin variant in a Chinese family masks the diagnosis of ß-thalassemia trait.

A 42-year-old Chinese woman (FP) was the mother of a patient with ß-thalassemia major (ß-TM) due to a compound heterozygosity for ß(0)-thalassemia (ß(0)-thal) mutations. She was also found to have a low Hb A(2) level of 1.6% by high performance liquid chromatography (HPLC) despite being a heterozygous carrier of the codons 41/42 (-TCTT) (HBB:c.126_129delCTTT) ß(0)-thal mutation. Doubling the amount of hemolysate loaded for chromatography revealed a widened Hb A(2) peak and raised the level to 4.1%, consistent with ß-thal trait. Direct nucleotide sequencing detected a novel δ-globin gene mutation at codon 29 (HBD:c.89G>A), which leads to a glycine to aspartic acid substitution. A homologous mutation at codon 29 in the ß-globin gene [Hb Lufkin or ß29(B11)Gly→Asp] has been reported in Black families. This report highlights the importance of genotype-phenotype correlation and the potential pitfall of relying on Hb A(2) level for phenotypic diagnosis of ß(0)-thal trait.

Histone lysine demethylase KDM5B maintains chronic myeloid leukemia via multiple epigenetic actions.

The histone lysine demethylase KDM5 family is implicated in normal development and stem cell maintenance by epigenetic modulation of histone methylation status. Deregulation of the KDM5 family has been reported in various types of cancers, including hematological malignancies. However, their transcriptional regulatory roles in the context of leukemia remain unclear. Here, we find that KDM5B is strongly expressed in normal CD34+ hematopoietic stem/progenitor cells and chronic myeloid leukemia (CML) cells. Knockdown of KDM5B in K562 CML cells reduced leukemia colony-forming potential. Transcriptome profiling of KDM5B knockdown K562 cells revealed the deregulation of genes involved in myeloid differentiation and Toll-like receptor signaling. Through the integration of transcriptome and ChIP-seq profiling data, we show that KDM5B is enriched at the binding sites of the GATA and AP-1 transcription factor families, suggesting their collaborations in the regulation of transcription. Even though the binding of KDM5B substantially overlapped with H3K4me1 or H3K4me3 mark at gene promoters, only a small subset of the KDM5B targets showed differential expression in association with the histone demethylation activity. By characterizing the interacting proteins in K562 cells, we discovered that KDM5B recruits protein complexes involved in the mRNA processing machinery, implying an alternative epigenetic action mediated by KDM5B in gene regulation. Our study highlights the oncogenic functions of KDM5B in CML cells and suggests that KDM5B is vital to the transcriptional regulation via multiple epigenetic mechanisms.

BCL11A is a major HbF quantitative trait locus in three different populations with beta-hemoglobinopathies.

Increased HbF levels or F-cell (HbF containing erythrocyte) numbers can ameliorate the disease severity of beta-thalassemia major and sickle cell anemia. Recent genome-wide association studies reported that single nucleotide polymorphisms (SNPs) in BCL11A gene on chromosome 2p16.1 were correlated with F-cells among healthy northern Europeans, and HbF among Sardinians with beta-thalassemias. In this study, we showed that SNPs in BCL11A were associated with F-cell numbers in Chinese with beta-thalassemia trait, and with HbF levels in Thais with either beta-thalassemia or HbE trait and in African Americans with sickle cell anemia. Taken together, the data suggest that the functional motifs responsible for modulating F-cells and HbF levels reside within a 3 kb region in the second intron of BCL11A.

Variation and heritability of Hb F and F-cells among beta-thalassemia heterozygotes in Hong Kong.

Enhanced fetal hemoglobin (Hb F) production can partially compensate for the lack of adult hemoglobin (Hb A) in patients with beta-thalassemia major or intermedia, and ameliorate the clinical severity of these diseases. To further elucidate factors governing Hb F levels, we evaluated demographic, clinical, laboratory, and genetic characteristics in 241 unrelated adult beta-thalassemia carriers in Hong Kong. They had wide variations in Hb F and F-cell numbers skewing toward higher levels. Individuals who coinherited the Xmn IT-allele in the (G)gamma-globin gene promoter had higher Hb F and more F-cells compared with those lacking the Xmn I T-allele. However, both groups exhibited a similarly wide spread of Hb F and F-cells. The correlation of Hb F and F-cells corresponded well to both linear and exponential models, suggesting multiple mechanisms for Hb F augmentation. The heritabilities of Hb F and F-cells were calculated in 66 families (111 parents who were beta-thalassemia carriers and 82 asymptomatic offspring) to be 0.7 to 0.9. The Xmn I polymorphism accounted for 9% of the Hb F and 13% of the F-cell heritabilities. These results suggest that these family members are well suited for genome wide association studies that will identify genetic loci regulating Hb F production, and likely novel pharmacological targets for reactivating Hb F production in adults.

A dual colour dual fusion fluorescence in situ hybridisation study on the genesis of complex variant translocations in chronic myelogenous leukaemia.

Complex variant 9;22 translocations occur in a significant minority of chronic myelogenous leukaemia (CML) patients. Different mechanisms of their formation have been described. We report dual colour dual fusion fluorescence in situ hybridisation data in 12 Chinese CML patients with complex translocations. Three previously reported breakpoint hotspots in a third partner chromosome (14q32, 17q25, 1q21) were observed. In 10/12 (83.3%) patients, the abnormality occurred as a single step 3-break event. Only a single abnormal clone harbouring the complex translocation was seen in this group. The remaining 2 cases in the chronic phase showed a 4-break mechanism (2/12, 16.7%). Deletion of 5' ABL at der(9) was not observed in any of the 12 patients, however, the loss of 3' BCR was observed in 1 patient (1/12, 8.3%). Together with previous findings, these data suggest that these variant translocations occur more often as a 3-break single-step process with no reciprocal ABL-BCR fusion. On the other hand, a 4-break event is also regularly seen during the initial stages of leukaemogenesis, which likely predisposes to der(9) deletion. The observed difference in rates of der(9) deletion reported in a series of CML patients with variant translocations may be related to a difference in rates of a 4-break event.

The role of a PBL tutor: a personal perspective.

Based on my experience as a PBL tutor in the Faculty of Medicine since 1997, it is clear that the role of a PBL tutor is one of a master of many trades. Whilst the primary role of a PBL tutor is to ensure, as a facilitator and a guide, that students engage in self-directed learning within the tutorial setting, he or she should be able to identify issues within and outside the tutorial setting that impact on learning. A PBL tutor should know the case well before the tutorial starts, establish ground rules and recognize that the quality of learning which takes place prior to and after the tutorial by students affect personal and group dynamics within the tutorial setting. The PBL tutor occupies a central and unique role in influencing students' learning and as a mentor to students' development.

Hoxb3 deficiency impairs B lymphopoiesis in mouse bone marrow.

OBJECTIVE: Hox genes are involved in hematopoietic lineage commitment and differentiation. In this study, we investigated the roles of Hoxb3 in hematopoiesis by examining the phenotypes of a Hoxb3 knockout mutant mouse line. RESULTS: Despite previous reports describing the apparently normal phenotype of these mutant mice, we found that by 6 months of age, Hoxb3(-/-) mice began to exhibit significantly impaired B lymphopoiesis in the bone marrow (BM). The cellularity was reduced by 30% in mutant BM compared to age- and sex-matched heterozygous and wild-type controls. The population size of B220(+)CD43(+) progenitor B cells showed a twofold reduction while that of B220(+)CD43(-)IgM(-) precursor B cells was decreased fivefold. Sorting-purified Hoxb3(-/-) progenitor B cells displayed significantly reduced proliferative response to IL-7 in culture, consistent with our findings of reduced IL-7 receptor expression in Hoxb3(-/-) progenitor B cells. However, the peripheral B cell pool in the spleen of Hoxb3(-/-) mice was maintained with a similar size as in wild-type littermates. CONCLUSION: Analysis of T-cell development in the thymus and B1 cell compartment in the peritoneal cavity showed no significant changes. Thus, our findings suggest that the Hoxb3 gene plays an essential role in regulating B lymphopoiesis in the BM of adult mice.

A laboratory strategy for genotyping haemoglobin H disease in the Chinese.

BACKGROUND: The thalassaemias are the commonest blood disorders worldwide, with South East Asia and southern China as areas of high prevalence. Accurate diagnosis of these disorders helps in clinical management with improved outcome. METHODS: The alpha-globin genotypes of 100 Chinese patients in Hong Kong with haemoglobin H (Hb H) disease were characterised. Single-tube multiplex gap-PCR was used to detect --(SEA), -alpha(3.7) and -alpha(4.2), while Hb CS, Hb QS and codon 30 (DeltaGAG) were identified by single-tube multiplex amplification refractory mutation system (ARMS). Automated direct nucleotide sequencing of the amplified alpha2- and alpha1-globin genes was performed to characterise other non-deletional alpha-thalassaemia determinants. RESULTS: In the 100 cases studied, 99 cases had --(SEA) in combination with deletional alpha(+)-thalassaemia or non-deletional alpha-globin gene mutation involving the alpha2-globin gene. In 70 cases of the deletional form, 43 cases showed the genotype of (--(SEA)/-alpha(3.7)) and 27 cases of (--(SEA)/-alpha(4.2)). Three of the 27 cases of (--(SEA)/-alpha(4.2)) were found to have Hb Q-Thailand linked in-cis with -alpha(4.2). The remaining 30 cases were of non-deletional form with the following genotypes: 11 cases of (--(SEA)/alpha(HbCS)alpha), 9 cases of (--(SEA)/alpha(HbQS)alpha), 3 cases of (--(SEA)/alpha(cd30 (DeltaGAG))alpha), 3 cases of (--(SEA)/alpha(cd31)alpha), 2 cases of (--(SEA)/alpha(poly-A)alpha), 1 case of (--(SEA)/alpha(HbWestmead)alpha) and 1 case of (--(non-SEA)/alpha(HbQS)alpha). CONCLUSIONS: Based on two rapid diagnostic tests, multiplex gap-PCR and multiplex ARMS, more than 90% of the cases were genetically characterised. This laboratory strategy should be widely applicable for genetic diagnosis of alpha-thalassaemia.

Diagnostic utility of dual fusion PML/RARalpha translocation DNA probe (D-FISH) in acute promyelocytic leukemia.

Translocation(15;17) leading to the formation of fusion gene PML/RARalpha is the diagnostic hallmark of acute promyelocytic leukemia (APL). Interphase fluorescence in situ hybridization (FISH) is one of the diagnostic tools employed for the detection of PML/RARalpha rearrangement. Using a dual color dual fusion (D-FISH) PML/RARalpha translocation DNA probe which hybridises both to PML/RARalpha and RARalpha/PML fusion genes, we characterised the FISH pattern of 52 APL patients at diagnosis and correlated the findings with conventional cytogenetics and RT-PCR analysis. The diagnostic sensitivity of the probe for PML/RARalpha was 100%. Seven patients had atypical D-FISH patterns; two had a masked PML/RARalpha fusion signal caused by the insertion of PML into RARalpha on 17q; 3 had an extra copy of PML/RARalpha in the form of isochromosome der(17)(q10)t(15;17) and one had duplication of the normal RARalpha gene with an ider(17q) masquerading as i(17)(q10). There was also one case of t(7;17;15) with a typical D-FISH pattern and in which metaphase FISH suggested an unusual 4-point break. In summary, PML/RARalpha D-FISH is a highly sensitive method for confirming diagnosis of APL. However D-FISH cannot be solely relied on for the diagnosis of APL owing to atypical patterns which are infrequently observed in cases with additional 17q structural abnormalities, gene insertion and gene duplication.

Histone deacetylase inhibitors induce leukemia gene expression in cord blood hematopoietic stem cells expanded ex vivo.

Umbilical cord blood is a valuable source of hematopoietic stem cells. While cytokine stimulation can induce ex vivo hematopoietic cell proliferation, attempts have been made to use epigenetic-modifying agents to facilitate stem cell expansion through the modulation of cellular epigenetic status. However, the potential global effect of these modifying agents on epigenome raises concerns about the functional normality of the expanded cells. We studied the ex vivo expansion of cord blood hematopoietic stem and progenitor cells (HSPCs) by histone deacetylase (HDAC) inhibitors, trichostatin A and valproic acid. Treatment with HDAC inhibitors resulted in mild expansion of the total hematopoietic cell number when compared with cytokine stimulated sample. Nevertheless, we observed 20-30-fold expansion of the CD34+ CD38- HSPC population. Strikingly, cord blood cells cultured with HDAC inhibitors exhibited aberrant expression of leukemia-associated genes, including CDKN1C, CEBPα, HOXA9, MN1, and DLK1. Our results thus suggest that the expansion of HSPCs by this approach may provoke a pre-leukemic cell state. We propose that the alteration of epigenome by HDAC inhibitors readily expands cord blood HSPC population through the re-activation of the leukemia gene transcription. The present study provides an assessment of the leukemogenic potential of HSCs expanded ex vivo using HDAC inhibitors for clinical applications.

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

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.