IntroductionChronic myeloid leukemia (CML) is a myeloproliferative disease in which leukemic cells exhibit the reciprocal translocation t(9;22) and the bcr/abl oncogene. [1][2][3] The resulting oncoprotein, BCR/ABL, displays constitutive tyrosine kinase activity and activates a number of signaling molecules including RAS/RAF/MAP kinases, the phosphoinositide 3-kinase (PI3-kinase), and signal transducer and activator of transcription 5 (STAT5). [4][5][6][7][8] In addition, BCR/ABL converts cytokine-dependent cell lines to growth factor independence and acts oncogenic in mice. 9 Several different mechanisms have been implicated in BCR/ ABL-dependent growth and accumulation of leukemic cells in CML. [10][11][12] One important mechanism may be inhibition of apoptosis. 10,[13][14][15][16] Thus, a number of antiapoptotic molecules are expressed in CML cells and may contribute to enhanced survival of leukemic cells. [10][11][12][13][14][15][16][17] Likewise, it has been shown that CML cells express several members of the BCL-2 family including BCL-2, BCL-x L , or A1. [13][14][15][16][17] However, the relative contribution of each of these molecules to inhibition of apoptosis in CML cells has not been defined yet. In addition, some of these molecules may only be expressed in leukemic cells in a subgroup of patients. 17 MCL-1 is a well-characterized member of the BCL-2 family that is considered to act antiapoptotic in various neoplastic cells including several leukemia-derived cell lines. [18][19][20][21] Originally, MCL-1 was described as a survival-enhancing molecule that is expressed during 12-O-tetra decanoyl phorbol 13-acetate (TPA) induced differentiation of leukemic ML-1 cells. 18 The CML-derived cell line K562 has also been described to express the MCL-1 protein. 19,[21][22][23] However, little is known so far about expression of MCL-1 in primary CML cells, the regulation of expression of MCL-1 in these cells, and the role that this antiapoptotic molecule may play in survival and accumulation of leukemic cells in patients with CML.In the present study, we have investigated the role of BCR/ABL in expression of MCL-1 in leukemic cells and analyzed underlying signaling pathways. The results of our study show that primary CML cells express MCL-1 in a constitutive manner and that BCR/ABL promotes the expression of MCL-1 through activation of the RAS/RAF/MAP kinase pathway. Moreover, our data show that down-regulation of MCL-1 by antisense oligonucleotides (ASOs) counteracts growth Patients, materials, and methods ConstructsPlasmids used in this study were pDCR-ras-G12V 24,25 (kindly provided by Yoel Kloog, Hadassah Medical Center, Jerusalem, Israel), pMT-Ha-ras-N17 26 (kindly provided by Mark Ewen, Dana Farber Cancer Institute, Boston, MA), and pBSK-mcl-1 27,28 (kindly provided by Stanley J. Korsmeyer, Dana Farber Cancer Institute). cDNAs were cloned into pcDNA3.1 ϩ vector (Invitrogen, Carlsbad, CA). The mcl-1 reporter gene construct (mcl-1-Luc) 29 was a kind gift from Steven W. Edwards (School of Biological Sciences...

We recently developed a murine protocol for the induction of allogeneic mixed chimerism and tolerance employing nonmyeloablative total body irradiation (TBI), standard-dose bone marrow transplantation (BMT), and costimulation blockade (cobl) with an anti-CD154 monoclonal antibody (mAb) plus CTLA4Ig.

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We thank the patients and their families for their trust in taking part in this study. The study was academically funded and supported by the Medical University Vienna, the General Hospital Vienna, and the Research Center for Molecular Medicine (CeMM) of the Austrian Academy of Sciences. We gratefully acknowledge funding from the Vienna Science and Technology Fund (LS16-034 to GSF and UJ), the Austrian Science Fund (F4704-B20 to PV, F4711-B20 to GSF, and P27132-B20 to PBS), and the European Molecular Biology Organization Long Term Fellowship (1543-2012 to GIV, 733-2016 to TP). BS acknowledges

Leukaemic stem cells in patients with CD33(+) AML express CD33. This observation is in favour of novel treatment concepts employing CD33-targeting antibodies in AML.

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Peripheral and central clonal deletion are important tolerance mechanisms in models using bone marrow transplantation (BMT) with costimulation blockade (CB). However, since tolerance can be found before peripheral deletion is complete and since elimination of recipient CD4+ cells at the time of BMT prevents tolerance induction, we investigated the potential roles of regulation and anergy in such a murine model. We found that transient elimination of CD25 + cells or neutralization of IL2 immediately after BMT and CB prevented the induction of skin graft tolerance. Cotransfer into SCID mice of CD4 + cells taken from chimeras early after BMT, together with naïve recipient-type CD4 + cells significantly prolonged donor skin graft survival. In contrast, cotransfer of CD4 + cells harvested from chimeras late after BMT did not prolong donor skin graft survival. Besides, depletion of CD25 + cells in established chimeras several months post-BMT did not break tolerance. In vivo administration of recombinant IL2 inhibited chimerism and tolerance neither early nor late post-BMT, arguing against a decisive role for classical anergy. Thus, CD4 cell-mediated regulation contributes significantly to tolerance induction early after BMT, but appears to have no critical role in the maintenance of tolerance.