Differential regulation of myeloid leukemias by the bone marrow microenvironment

Like their normal hematopoietic stem cell counterparts, leukemia stem cells (LSC) in chronic myelogenous leukemia (CML) and acute myeloid leukemia (AML) are presumed to reside in specific niches in the bone marrow microenvironment (BMM)1, and may be the cause of relapse following chemotherapy.2 Targeting the niche is a novel strategy to eliminate persistent and drug-resistant LSC. CD443,4 and IL-65 have been implicated previously in the LSC niche. Transforming growth factor (TGF)-β1 is released during bone remodeling6 and plays a role in maintenance of CML LSCs7, but a role for TGF-β1 from the BMM has not been defined. Here, we show that alteration of the BMM by osteoblastic cell-specific activation of the parathyroid hormone (PTH) receptor8,9 attenuates BCR-ABL1-induced CML-like myeloproliferative neoplasia (MPN)10 but enhances MLL-AF9-induced AML11 in mouse transplantation models, possibly through opposing effects of increased TGF-β1 on the respective LSC. PTH treatment caused a 15-fold decrease in LSCs in wildtype mice with CML-like MPN, and reduced engraftment of immune deficient mice with primary human CML cells. These results demonstrate that LSC niches in chronic and acute myeloid leukemias are distinct, and suggest that modulation of the BMM by PTH may be a feasible strategy to reduce LSC, a prerequisite for the cure of CML

ElaD, a Deubiquitinating protease expressed by E. coli.

BACKGROUND: Ubiquitin and ubiquitin-like proteins (Ubl) are designed to modify polypeptides in eukaryotes. Covalent binding of ubiquitin or Ubls to substrate proteins can be reversed by specific hydrolases. One particular set of cysteine proteases, the CE clan, which targets ubiquitin and Ubls, has homologs in eukaryotes, prokaryotes, and viruses. FINDINGS: We have cloned and analyzed the E. coli protein elaD, which is distantly related to eukaryotic CE clan members of the ULP/SENP protease family that are specific for SUMO and Nedd8. Previously misannotated as a putative sulfatase/phosphatase, elaD is an efficient and specific deubiquitinating enzyme in vitro. Interestingly, elaD is present in all intestinal pathogenic E. coli strains, but conspicuously absent from extraintestinal pathogenic strains (ExPECs). Further homologs of this protease can be found in Acanthamoeba Polyphaga Mimivirus, and in Alpha-, Beta-and Gammaproteobacteria. CONCLUSION: The expression of ULP/SENP-related hydrolases in bacteria therefore extends to plant pathogens and medically relevant strains of Escherichia coli, Legionella pneumophila, Rickettsiae, Chlamydiae, and Salmonellae, in which the elaD ortholog sseL has recently been identified as a virulence factor with deubiquitinating activity. As a counterpoint, our phylogenetic and functional examination reveals that ancient eukaryotic ULP/SENP proteases also have the potential of ubiquitin-specific hydrolysis, suggesting an early common origin of this peptidase clan

Biochemical assay for substrate specificity of elaD.

<p>³⁵S-methionine-labeled <i>in vitro</i> translated wildtype elaD forms covalent adducts with suicide inhibitors based on ubiquitin (ubiquitin-vinylmethylester, VME) and Nedd8 (Nedd8-vinylsulfone, VS), but not with probes based on SUMO1 and ISG15. All probes were tested for activity with <i>bona fide</i> substrates (not shown) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000381#pone.0000381-Hemelaar1" target="_blank">[14]</a>. Mutation of the active-site cysteine at position 313 to serine abolishes adduct formation of elaD to electrophilic probes. Samples were resolved by reducing SDS-PAGE and visualized by fluorography. Indicated at the right is the molecular mass in kDa.</p

Phylogram representation of CE clan proteases in viruses, bacteria, and eukaryotes.

<p>Eukaryotic peptidases (in blue) belong to the C48 subfamily and can be separated into three groups: ULP1 (including the mammalian proteases SENP1, 2, 3, and 5), ULP2 (including SENP6 and 7), and the SENP8 group with proposed specificity for SUMO (ULP1 and ULP2 group) and Nedd8 (SENP8 group), respectively. Bacterial proteins are indicated with a preceding “B”, viral proteins with a “V”. We have further divided microbial protease homologs by color: green for biochemically tested proteases, red indicating the absence of published data on the function of these putative proteases, and yellow for the group representing elaD and its orthologs. The C5 family contains Adenovirus proteases with deubiquitinating activity, C55 comprises the bacterial YopJ homologs, and C57 the Vacciniavirus I7 peptidases. Based on sequence similarity, two bacterial C48 family groups can be distinguished: a group of Proteobacteria (located at one o'clock) which appear to be closely related to fungal SENP8 homologs (common node indicated with a circle, bootstrap support>60%), and <i>Chlamydiae</i>, for which we had previously shown the presence of deubiquitinating and deneddylating activity. Three additional groups have not yet been assigned to specific CE clan subfamilies in the MEROPS database, including Mimivirus (“group I”), Gammaproteobacteria (“group II”), and <i>Rickettsiae</i> (“group III”). The African Swine Fever Virus protease and the I7 Vacciniavirus protease have not been tested for deubiquitinating or Ubl-specific activity, but they both require a glycine-based motif at the C-terminus of the substrate, as found in ubiquitin or Ubls. The unrelated CD clan peptidase Clostripain is used as outgroup in this phylogram. For clarity, this tree does not contain all orthologs and paralogs of the different groups or families. Sequence information is provided in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000381#pone-0000381-t002" target="_blank">Table 2</a>.</p

A. Biochemical assay for substrate specificity of GZ8, the SENP8 homolog of the fungus <i>G. zeae.</i>

<p>Like the chlamydial deubiquitinase/deneddylase CT868 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000381#pone.0000381-Misaghi1" target="_blank">[23]</a>, GZ8 reacts with the electrophilic traps ubiquitin-vinylmethylester and Nedd8-vinylsulfone, but not with SUMO1-vinylsulfone or ISG15-vinylsulfone. Shown is SDS-PAGE and fluorography of ³⁵S-Methionine-labeled <i>in vitro</i> translated GZ8. The cysteine-alkylating agent N-ethylmaleimide (NEM) abrogates activity of the cysteine protease GZ8. Molecular weight markers are indicated at the left (in kDa). B. Biochemical assay for hydrolytic activity of GZ8. Like CT868, but unlike human SENP8, GZ8 exerts hydrolytic activity on ubiquitin-and Nedd8-AMC conjugates. Fluorescence measurement was assayed 16 hours after incubation of human SENP8, GZ8, and chlamydial CT868 (values defined as 100%) with ubiquitin-AMC and Nedd8-AMC. GZ8 and CT868 hydrolyze both substrates, whereas human SENP8 only cleaves Nedd8-AMC. Hydrolysis is sensitive to NEM treatment (not shown). The y-axis depicts relative fluorescence. Shown are the mean values of a representative experiment with triplicate measurements+/−standard deviations.</p

Activity-based profiling of DUBs.

<p>IVT was used to generate DUBs for profiling activity toward ISG15VS, UbVME and SUMO1VME probes. After incubation with the probes, samples were analyzed by SDS-PAGE, as shown for USP18 (A), USP14 (B), USP5 (C), USP13 (D), USP2 (E), and CGI-77 (F). Binding was inhibited by preincubation of the proteases with NEM. The SUMO protease SENP2 interacts specifically with SUMO1VME (G). Molecular weight in kDa is indicated on the left.</p