Xueqi Fu

Identification of an Acquired JAK2 Mutation in Polycythemia Vera

Polycythemia vera (PV) is a human clonal hematological disorder. The molecular etiology of the disease has not been identified. PV hematopoietic progenitor cells exhibit hypersensitivity to growth factors and cytokines, suggesting possible abnormalities in protein-tyrosine kinases and phosphatases. By sequencing the entire coding regions of cDNAs of candidate enzymes, we identified a G:C→ T:A point mutation of the JAK2 tyrosine kinase in 20 of 24 PV blood samples but none in 12 normal samples. The mutation has varying degrees of heterozygosity and is apparently acquired. It changes conserved Val617 to Phe in the pseudokinase domain of JAK2 that is known to have an inhibitory role. The mutant JAK2 has enhanced kinase activity, and when overexpressed together with the erythropoietin receptor in cells, it caused hyperactivation of erythropoietin-induced cell signaling. This gain-of-function mutation of JAK may explain the hypersensitivity of PV progenitor cells to growth factors and cytokines. Our study thus defines a molecular defect of PV.

Transgenic expression of JAK2V617F causes myeloproliferative disorders in mice

The JAK2(V617F) mutation was found in most patients with myeloproliferative disorders (MPDs), including polycythemia vera, essential thrombocythemia, and primary myelofibrosis. We have generated transgenic mice expressing the mutated enzyme in the hematopoietic system driven by a vav gene promoter. The mice are viable and fertile. One line of the transgenic mice, which expressed a lower level of JAK2(V617F), showed moderate elevations of blood cell counts, whereas another line with a higher level of JAK2(V617F) expression displayed marked increases in blood counts and developed phenotypes that closely resembled human essential thrombocythemia and polycythemia vera. The latter line of mice also developed primary myelofibrosis-like symptoms as they aged. The transgenic mice showed erythroid, megakaryocytic, and granulocytic hyperplasia in the bone marrow and spleen, displayed splenomegaly, and had reduced levels of plasma erythropoietin and thrombopoietin. They possessed an increased number of hematopoietic progenitor cells in peripheral blood, spleen, and bone marrow, and these cells formed autonomous colonies in the absence of growth factors and cytokines. The data show that JAK2(V617F) can cause MPDs in mice. Our study thus provides a mouse model to study the pathologic role of JAK2(V617F) and to develop treatment for MPDs.

Erlotinib effectively inhibits JAK2V617F activity and polycythemia vera cell growth.

JAK2V617F, a mutant of tyrosine kinase JAK2, is found in most patients with polycythemia vera (PV) and a substantial proportion of patients with idiopathic myelofibrosis or essential thrombocythemia. The JAK2 mutant displays a much increased kinase activity and generates a PV-like phenotype in mouse bone marrow transplant models. This study shows that the anti-cancer drug erlotinib (Tarceva™) is a potent inhibitor of JAK2V617F activity. In vitro colony culture assays revealed that erlotinib at micro-molar concentrations effectively suppresses the growth and expansion of PV hematopoietic progenitor cells while having little effect on normal cells. Furthermore, JAK2V617F-positive cells from PV patients show greater susceptibility to the inhibitor than their negative counterparts. Similar inhibitory effects were found with the JAK2V617F-positive human erythroleukemia HEL cell line. These data suggest that erlotinib may be used for treatment of JAK2V617F-positive PV and other myeloproliferative disorders.

LSD1-mediated epigenetic modification is required for TAL1 function and hematopoiesis

TAL1 is a critical transcription factor required for hematopoiesis. However, perturbation of its activity often leads to T cell leukemia. Whether and how its transcriptional activities are regulated during hematopoiesis remains to be addressed. Here, we show that TAL1 is associated with histone demethylase complexes containing lysine-specific demethylase 1 (LSD1), RE1 silencing transcription factor corepressor (CoREST), histone deacetylase 1 (HDAC1), and histone deacetylase 2 in erythroleukemia and T cell leukemia cells. The enzymatic domain of LSD1 plays an important role in repressing the TAL1-directed transcription of GAL4 reporter linked to a thymidine kniase minimal promoter. Furthermore, we demonstrate that the TAL1-associated LSD1, HDAC1, and their enzymatic activities are coordinately down-regulated during the early phases of erythroid differentiation. Consistent with the rapid changes of TAL1–corepressor complex during differentiation, TAL1 recruits LSD1 to the silenced p4.2 promoter in undifferentiated, but not in differentiated, murine erythroleukemia (MEL) cells. Finally, shRNA-mediated knockdown of LSD1 in MEL cells resulted in derepression of the TAL1 target gene accompanied by increasing dimeH3K4 at the promoter region. Thus, our data revealed that histone lysine demethylase LSD1 may negatively regulate TAL1-mediated transcription and suggest that the dynamic regulation of TAL1-associated LSD1/HDAC1 complex may determine the onset of erythroid differentiation programs.

Trans-regulation of Histone Deacetylase Activities through Acetylation

HDAC1 and -2 are highly conserved enzymes and often coexist in the same coregulator complexes. Understanding the regulation of histone deacetylase activities is extremely important because these enzymes play key roles in epigenetic regulation in normal and cancer cells. We previously showed that HDAC1 is required for glucocorticoid receptor-mediated transcription activation and that its activity is regulated through acetylation by p300 during the induction cycle. Here, we showed that HDAC2 is also required for glucocorticoid receptor-mediated gene activation. HDAC2, however, is regulated through a different mechanism from that of HDAC1. HDAC2 is not acetylated by p300, although 5 of 6 acetylated lysine residues in HDAC1 are also present in HDAC2. More importantly, the activity of HDAC2 is inhibited by acetylated HDAC1. Additionally, we showed that acetylated HDAC1 can trans-regulate HDAC2 through heterodimerization. Thus, this study uncovered fundamental differences between HDAC1 and HDAC2. It also unveiled a new mechanism of collaborative regulation by HDAC1/2 containing coregulator complexes.

Death Receptor 6 Induces Apoptosis Not through Type I or Type II Pathways, but via a Unique Mitochondria-dependent Pathway by Interacting with Bax Protein

Background: DR6-induced apoptosis mechanism is unknown. Results: DR6-induced apoptosis is dependent on cytochrome c release and Bax translocation, but is independent of caspase-8 and Bid. Conclusion: DR6-induced apoptosis is mediated by a unique pathway, different from type I and type II pathways. Significance: This study will lead to a better understanding of the mechanism by which DR6 induces apoptosis. Cells undergo apoptosis through two major pathways, the extrinsic pathway (death receptor pathway) and the intrinsic pathway (the mitochondrial pathway). These two pathways can be linked by caspase-8-activated truncated Bid formation. Very recently, death receptor 6 (DR6) was shown to be involved in the neurodegeneration observed in Alzheimer disease. DR6, also known as TNFRSF21, is a relatively new member of the death receptor family, and it was found that DR6 induces apoptosis when it is overexpressed. However, how the death signal mediated by DR6 is transduced intracellularly is not known. To this end, we have examined the roles of caspases, apoptogenic mitochondrial factor cytochrome c, and the Bcl-2 family proteins in DR6-induced apoptosis. Our data demonstrated that Bax translocation is absolutely required for DR6-induced apoptosis. On the other hand, inhibition of caspase-8 and knockdown of Bid have no effect on DR6-induced apoptosis. Our results strongly suggest that DR6-induced apoptosis occurs through a new pathway that is different from the type I and type II pathways through interacting with Bax.

Dynamic interaction between TAL1 oncoprotein and LSD1 regulates TAL1 function in hematopoiesis and leukemogenesis

TAL1/SCL is a hematopoietic-specific oncogene and its activity is regulated by associated transcriptional co-activators and corepressors. Dysregulation of TAL1 activity has been associated with T-cell leukemogenesis. However, it remains unclear how the interactions between TAL1 and corepressors versus co-activators are properly regulated. Here, we reported that protein kinase A (PKA)-mediated phosphorylation regulates TAL1 interaction with the lysine-specific demethylase (LSD1) that removes methyl group from methylated Lys 4 on histone H3 tails. Phosphorylation of serine 172 in TAL1 specifically destabilizes the TAL1–LSD1 interaction leading to promoter H3K4 hypermethylation and activation of target genes that have been suppressed in normal and malignant hematopoiesis. Knockdown of TAL1 or LSD1 led to a derepression of the TAL1 target genes in T-cell acute lymphoblast leukemia (T-ALL) Jurkat cells, which is accompanied by elevating promoter H3K4 methylation. Similarly, treatment of PKA activator forskolin resulted in derepression of target genes by reducing its interaction with LSD1 while PKA inhibitor H89 represses them by suppressing H3K4 methylation levels. Consistent with the dual roles of TAL1 in transcription, TAL1-associated LSD1 is decreased while recruitment of hSET1 is increased at the TAL1 targets during erythroid differentiation. This process is accompanied by a dramatic increase in H3K4 methylation. Thus, our data revealed a novel interplay between PKA phosphorylation and TAL1-mediated epigenetic regulation that regulates hematopoietic transcription and differentiation programs during hematopoiesis and leukemogenesis.

Split Renilla Luciferase Protein Fragment-assisted Complementation (SRL-PFAC) to Characterize Hsp90-Cdc37 Complex and Identify Critical Residues in Protein/Protein Interactions

Hsp90 requires cochaperone Cdc37 to load its clients to the Hsp90 superchaperone complex. The purpose of this study was to utilize split Renilla luciferase protein fragment-assisted complementation (SRL-PFAC) bioluminescence to study the full-length human Hsp90-Cdc37 complex and to identity critical residues and their contributions for Hsp90/Cdc37 interaction in living cells. SRL-PFAC showed that full-length human Hsp90/Cdc37 interaction restored dramatically high luciferase activity through Hsp90-Cdc37-assisted complementation of the N and C termini of luciferase (compared with the set of controls). Immunoprecipitation confirmed that the expressed fusion proteins (NRL-Hsp90 and Cdc37-CRL) preserved their ability to interact with each other and also with native Hsp90 or Cdc37. Molecular dynamic simulation revealed several critical residues in the two interaction patches (hydrophobic and polar) at the interface of Hsp90/Cdc37. Mutagenesis confirmed the critical residues for Hsp90-Cdc37 complex formation. SRL-PFAC bioluminescence evaluated the contributions of these critical residues in Hsp90/Cdc37 interaction. The results showed that mutations in Hsp90 (Q133A, F134A, and A121N) and mutations in Cdc37 (M164A, R167A, L205A, and Q208A) reduced the Hsp90/Cdc37 interaction by 70–95% as measured by the resorted luciferase activity through Hsp90-Cdc37-assisted complementation. In comparison, mutations in Hsp90 (E47A and S113A) and a mutation in Cdc37 (A204E) decreased the Hsp90/Cdc37 interaction by 50%. In contrast, mutations of Hsp90 (R46A, S50A, C481A, and C598A) and mutations in Cdc37 (C54S, C57S, and C64S) did not change Hsp90/Cdc37 interactions. The data suggest that single amino acid mutation in the interface of Hsp90/Cdc37 is sufficient to disrupt its interaction, although Hsp90/Cdc37 interactions are through large regions of hydrophobic and polar interactions. These findings provides a rationale to develop inhibitors for disruption of the Hsp90/Cdc37 interaction.

Acetylation of histone deacetylase 1 regulates NuRD corepressor complex activity

Background: HDAC1-containing NuRD complex is required for GATA-1-mediated repression and activation. Results: GATA-1 associated with acetylated HDAC1-containing NuRD complex, which has no deacetylase activity, for gene activation. Conclusion: Acetylated HDAC1 converts NuRD complex from a repressor to an activator during GATA-1-directed erythroid differentiation program. Significance: HDAC1 acetylation may function as a master regulator for the activity of HDAC1 containing complexes. Histone deacetylases (HDACs) play important roles in regulating cell proliferation and differentiation. The HDAC1-containing NuRD complex is generally considered as a corepressor complex and is required for GATA-1-mediated repression. However, recent studies also show that the NuRD complex is involved in GATA-1-mediated gene activation. We tested whether the GATA-1-associated NuRD complex loses its deacetylase activity and commits the GATA-1 complex to become an activator during erythropoiesis. We found that GATA-1-associated deacetylase activity gradually decreased upon induction of erythroid differentiation. GATA-1-associated HDAC1 is increasingly acetylated after differentiation. It has been demonstrated earlier that acetylated HDAC1 has no deacetylase activity. Indeed, overexpression of an HDAC1 mutant, which mimics acetylated HDAC1, promotes GATA-1-mediated transcription and erythroid differentiation. Furthermore, during erythroid differentiation, acetylated HDAC1 recruitment is increased at GATA-1-activated genes, whereas it is significantly decreased at GATA-1-repressed genes. Interestingly, deacetylase activity is not required for Mi2 remodeling activity, suggesting that remodeling activity may be required for both activation and repression. Thus, our data suggest that NuRD can function as a coactivator or repressor and that acetylated HDAC1 converts the NuRD complex from a repressor to an activator during GATA-1-directed erythroid differentiation.

Potent and selective inhibition of T-cell protein tyrosine phosphatase (TCPTP) by a dinuclear copper(II) complex

A dinuclear Cu(II) complex, [Cu(2)(μ-IDA)(phen)(3)(NO(3))]NO(3)·4H(2)O (phen = 1,10-phenanthroline, H(2)IDA = iminodiacetic acid), was found to potently and selectively inhibit T-cell protein tyrosine phosphatase, and lead to the anti-proliferation and apoptosis of C6 glioma cells.