t cell mediated rejection of human cd34 cells is prevented by costimulatory blockade in a xenograft model

Abstract A xenograft model of stem cell rejection was developed by co-transplantating human CD34 + and allogeneic CD3 + T cells into NOD-scid ɣ-chain null mice. T cells caused graft failure when transplanted at any CD34/CD3 ratio between 1:50 and 1:.1. Kinetics experiments showed that 2 weeks after transplantation CD34 + cells engrafted the marrow and T cells expanded in the spleen. Then, at 4 weeks only memory T cells populated both sites and rejected CD34 + cells. Blockade of T cell costimulation was tested by injecting the mice with abatacept (CTLA4-IgG1) from day –1 to +27 (group A), from day –1 to +13 (group B), or from day +14 to +28 (group C). On day +56 groups B and C had rejected the graft, whereas in group A graft failure was completely prevented, although with lower stem cell engraftment than in controls ( P  = .03). Retransplantation of group A mice with same CD34 + cells obtained a complete reconstitution of human myeloid and B cell lineages and excluded latent alloreactivity. In this first xenograft model of stem cell rejection we showed that transplantation of HLA mismatched CD34 + cells may be facilitated by treatment with abatacept and late stem cell boost

Synergistic Cytotoxic Effect of Busulfan and the PARP Inhibitor Veliparib in Myeloproliferative Neoplasms

ABSTRACT Patients with high-risk myeloproliferative neoplasms (MPNs), and in particular myelofibrosis (MF), can be cured only with allogeneic hematopoietic stem cell transplantation (HSCT). Because MPNs and JAK2V617F-mutated cells show genomic instability, stalled replication forks, and baseline DNA double-strand breaks, DNA repair inhibition with poly(ADP-ribose) polymerase-1 (PARP-1) inhibitors represents a potential novel therapy. Because the alkylating agent busulfan is integral in conditioning regimens for HSCT and leads to stalled replication forks through DNA strand cross-linking, we hypothesized that PARP inhibition with veliparib in combination with busulfan may lead to synergistic cytotoxicity in MPN cells. We first treated 2 MPN cell lines harboring the JAK2V617F mutation (SET2 and HEL) with veliparib at increasing concentrations and measured cell proliferation. SET2 and HEL cells were relatively sensitive to veliparib (IC50 of 11.3 μM and 74.2 μM, respectively). We next treated cells with increasing doses of busulfan in combination with 4 μM veliparib and found that the busulfan IC50 decreased from 27 μM to 4 μM in SET2 cells and from 45.1 μM to 28.1 μM in HEL cells. The mean combination index was .55 for SET2 cells and .40 for HEL cells. Combination treatment of SET2 cells caused G2M arrest in 53% of cells, compared with 30% with veliparib alone and 35% with busulfan alone. G2M arrest was associated with activation of the ATR-Chk1 pathway, as shown by an immunofluorescence assay for phosphorylated Chk1 (p-Chk1). We then tested in vivo the effect of combined low doses of busulfan and veliparib in a JAK2V617F MPN-AML xenotransplant model. Vehicle- and veliparib-treated mice had similar median survival of 39 and 40 days, respectively. Combination treatment increased median survival from 47 days (busulfan alone) to 50 days (P = .02). Finally, we tested the combined effect of busulfan and veliparib on CD34+ cells obtained from the bone marrow or peripheral blood of 5 patients with JAK2V617F-mutated and 2 patients with CALR-mutated MF. MF cells treated with the combination of veliparib and busulfan showed reduced colony formation compared with busulfan alone (87% versus 68%; P = .001). In contrast, treatment of normal CD34+ cells with veliparib did not affect colony growth. Here we show that in vivo confirmation that treatment with the PARP-1 inhibitor veliparib and busulfan results in synergistic cytotoxicity in MPN cells. Our data provide the rationale for testing novel pretransplantation conditioning regimens with combinations of PARP-1 inhibition and reduced doses of alkylators, such as busulfan and melphalan, for high-risk MPNs or MPN-derived acute myelogenous leukemia

The Oncoprotein EVI1 and the DNA Methyltransferase Dnmt3 Co-Operate in Binding and De Novo Methylation of Target DNA

EVI1 has pleiotropic functions during murine embryogenesis and its targeted disruption leads to prenatal death by severely affecting the development of virtually all embryonic organs. However, its functions in adult tissues are still unclear. When inappropriately expressed, EVI1 becomes one of the most aggressive oncogenes associated with human hematopoietic and solid cancers. The mechanisms by which EVI1 transforms normal cells are unknown, but we showed recently that EVI1 indirectly upregulates self-renewal and cell-cycling genes by inappropriate methylation of CpG dinucleotides in the regulatory regions of microRNA-124-3 (miR-124-3), leading to the repression of this small gene that controls normal differentiation and cell cycling of somatic cells. We used the regulatory regions of miR-124-3 as a read-out system to investigate how EVI1 induces de novo methylation of DNA. Here we show that EVI1 physically interacts with DNA methyltransferases 3a and 3b (Dnmt3a/b), which are the only de novo DNA methyltransferases identified to date in mouse and man, and that it forms an enzymatically active protein complex that induces de novo DNA methylation in vitro. This protein complex targets and binds to a precise region of miR-124-3 that is necessary for repression of a reporter gene by EVI1. Based on our findings, we propose that in cooperation with Dnmt3a/b EVI1 regulates the methylation of DNA as a sequence-specific mediator of de novo DNA methylation and that inappropriate EVI1 expression contributes to carcinogenesis through improper DNA methylation

Repression of RUNX1 activity by EVI1: a new role of EVI1 in leukemogenesis

Recurring chromosomal translocations observed in human leukemia often result in the expression of fusion proteins that are DNA-binding transcription factors. These altered proteins acquire new dimerization properties that result in the assembly of inappropriate multimeric transcription complexes that deregulate hematopoietic programs and induce leukemogenesis. Recently, we reported that the fusion protein AML1/MDS1/EVI1 (AME), a product of a t(3;21)(q26;q22) associated with chronic myelogenous leukemia and acute myelogenous leukemia, displays a complex pattern of self-interaction. Here, we show that the 8th zinc finger motif of MDS1/EVI1 is an oligomerization domain involved not only in interaction of AME with itself but also in interactions with the parental proteins, RUNX1 and MDS1/EVI1, from which AME is generated. Because the 8th zinc finger motif is also present in the oncoprotein EVI1, we have evaluated the effects of the interaction between RUNX1 and EVI1 in vitro and in vivo. We found that in vitro, this interaction alters the ability of RUNX1 to bind to DNA and to regulate a reporter gene, whereas in vivo, the expression of the isolated 8th zinc finger motif of EVI1 is sufficient to block the granulocyte colony-stimulating factor-induced differentiation of 32Dcl3 cells, leading to cell death. As EVI1 is not detected in normal bone marrow cells, these data suggest that its inappropriate expression could contribute to hematopoietic transformation in part by a new mechanism that involves EVI1 association with key hematopoietic regulators, leading to their functional impairment

The zinc finger motifs 1 and 6 of EVI1 are required for interaction with the catalytic domain of <i>de novo</i> DNA methyltransferases.

<p>A. Schematic diagram of EVI1 and Dnmt3a shows the relevant domains analyzed in this study. B. EVI1 interacts with the catalytic domain of Dnmt3a. 293T cells were transiently co-transfected with full-length EVI1 and each one of the Flag-tagged separate domains of Dnmt3a as indicated, and analyzed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0020793#pone-0020793-g002" target="_blank">Figure 2</a>. Lanes 1 to 5 show the expression of EVI1 (bottom panel) and Dnmt3a domains (top panel) in the transfected cells. Lanes 6 to 10 show the proteins after IP with anti-EVI1 antibody. C. The proximal zinc finger domain of EVI1 interacts with the catalytic domain of Dnmt3a. 293T cells were transiently co-transfected with the HA-tagged EVI1 proximal domain (7ZnF) and each one of the Flag-tagged domains of Dnmt3a. The cells were processed and analyzed as described above. Lanes 1 to 5 show the expression of 7ZnF domain (bottom panel) and Dnmt3a domains (top panel) in the transfected cells. Lanes 6 to 10 show the proteins after IP with anti-EVI1 antibody. The proximal domain, 7ZnF, interacts only with Dnmt3a catalytic domain (lane 10). D. Zinc finger motifs 1 and 6 must be intact for interaction with Dnmt3a. 293T cells were transiently co-transfected with the Flag-tagged catalytic domain of Dnmt3a alone (lanes 2 and 7) or in combination with the HA-tagged 7ZnF domain (lanes 3 and 8) or with the mutant 7ZnF-(1+6Mut) domain (lanes 4 and 9) or with EVI1-Δ7ZnF (lanes 5 and 10). The proteins in the cell extracts were analyzed by Western blot after co-IP with anti-HA antibody. Interaction is observed only when the intact proximal domain is expressed (lane 8).</p

EVI1 interacts with <i>de novo</i> DNA methyltransferases.

<p>A. EVI1 (lane 7) but not EVI1-(1+6Mut) (lane 8) interacts with Dnmt3a. 293T cells were co-transfected with Myc-tagged Dnmt3a alone (lanes 2 and 6) or in combination with HA-tagged EVI1 or EVI1-(1+6Mut) (lanes 3 and 7, or lanes 4 and 8, respectively). Two days after transfection cell lysates were collected and incubated with anti-HA beads (lanes 5 to 8) followed by IP. The immunoprecipitated proteins (lanes 5 to 8) and proteins from the cell lysates (lanes 1 to 4) were separated by electrophoresis, transferred to a PVDF membrane and probed as marked in the Figure. Lanes 1 and 5 represent the results with mock transfected cells. B, C. 293T cells were transfected with a plasmid encoding EVI1 (lanes 2 and 5) or EVI1-(1+6Mut) (lanes 3 and 6). Lanes 1 and 4 represent mock-transfected cells. Two days after transfection cell lysates were collected and incubated with anti-Dnmt3a (B) or anti-Dnmt3b (C) antibody (lanes 4 to 6) followed by IP. The immunoprecipitated proteins (lanes 4 to 6) and proteins from cell lysates (lanes 1 to 3) were separated by electrophoresis, transferred to a PVDF membrane and probed as marked in the Figure.</p

EVI1 and <i>de novo</i> DNA methyltransferases occupy the regulatory region of miR-124-3.

<p>A. ChIP assay was performed with chromatin fragments obtained from 293T cells transiently transfected with empty vector (lanes 1, 4, and 7), EVI1 (lanes 2, 5, and 8) and EVI1-(1+6Mut) (lanes 3, 6, and 9). For the ChIP, we used IgG (lanes 1 to 3) or anti-HA (lanes 4 to 6) or anti-Dnmt3b (lanes 7 to 9) antibodies. Transfected EVI1 (lane 5, lower panel) and endogenous Dnmt3b (lane 8, lower panel) are present together on the putative miR-124-3 promoter. In contrast, when EVI1 is not expressed (lanes 4 and 7) or is mutated (lanes 6 and 9) Dnmt3b is less capable of binding to chromatin. Lanes 1–3 represent negative control with unspecific IgG. B. Dnmt3a is also enriched at the putative miR-124-3 promoter in EVI1-transfected cells (lane 4). ChIP assay was performed with chromatin fragments derived from 293T cells transiently transfected with EVI1. C. Cooperation between EVI1 and Dnmt3a in promoter occupancy. 293T cells were transiently transfected with the empty vector (lane 1), EVI1 and EVI1-(1+6Mut) alone (lanes 2 and 3) or in combination with Dnmt3a (lanes 4 and 5). The cells were used for chromatin fragments isolation/ChIP with anti-HA antibody. Normal EVI1 and Dnmt3a are more efficient in ChIP (compare lanes 2 and 4). D. ChIP quantification. The signal for cells transfected with the empty vector was arbitrarily taken as 1. Lanes numbering in C and D is the same.</p

EVI1 and <i>de novo</i> DNA methyltransferases form an enzymatically active complex.

<p>A. ES cells stably transfected with the empty vector (lane 1), EVI1 (lane 2) or EVI1-(1+6Mut) (lane 3) were used for <i>in vitro</i> DMTase assay as described in Material and Methods. 293T cells were transiently transfected with plasmids encoding EVI1 (lane 4), Dnmt3b (lane 5), or EVI1 and Dnmt3b (lane 6), and the IP proteins were used for <i>in vitro</i> DMTase assay. A strong band evident in lane 2 represents the DNA methyltransferase activity of EVI1 and endogenous dnDMTs from ES cells. A strong band evident in lane 6 results from the activity of co-transfected Dnmt3b and EVI1. B. Quantitative RT-PCR shows that the expression of EVI1 (lane 2) and EVI1-(1+6Mut) (lane 3) in ES cells is comparable. Western blot analysis shows the expression of EVI1 (lane 4), Dnmt3b (lane 5) and EVI1+Dnmt3b (lane 6) in 293T cells used for <i>in vitro</i> DMTase assay.</p