ALBERT

Description: Leukemia caused by retroviral insertional mutagenesis after stem cell gene transfer has been reported in several experimental animals and in patients treated for X-linked severe combined immunodeficiency. Here, we analyzed whether gene transfer into mature T cells bears the same genotoxic risk. To address this issue in an experimental “worst case scenario,” we transduced mature T cells and hematopoietic progenitor cells from C57BL/6 (Ly5.1) donor mice with high copy numbers of gamma retroviral vectors encoding the potent T-cell oncogenes LMO2, TCL1, or ΔTrkA, a constitutively active mutant of TrkA. After transplantation into RAG-1–deficient recipients (Ly5.2), animals that received stem cell transplants developed T-cell lymphoma/leukemia for all investigated oncogenes with a characteristic phenotype and after characteristic latency periods. Ligation-mediated polymerase chain reaction analysis revealed monoclonality or oligoclonality of the malignancies. In striking contrast, none of the mice that received T-cell transplants transduced with the same vectors developed leukemia/lymphoma despite persistence of gene-modified cells. Thus, our data provide direct evidence that mature T cells are less prone to transformation than hematopoietic progenitor cells.

Description: After the report of two cases of leukaemia caused by insertional mutagenesis of a retroviral vector in children with SCID, it became clear that safety issues of therapeutic gene transfer must be addressed more thoroughly. We analysed whether gene transfer into mature T cells and haematopoietic stem cells bear the same risk of generating T cell leukaemia through activation of specific T cell oncogenes, such as LMO2, TCL1 and ΔTrkA. To address this issue, we used the Rag-1 mouse model, which allows long term analysis of transplanted T cells and haematopoietic stem cells. We were able to transduce mature T cells and haematopoietic stem cells of C57BL/6 (Ly5.1) donor mice with oncoretroviral vectors expressing LMO2, TCL1 and ΔTrkA. Transduction efficacies of up to 70% were achieved for mature T cells and approximately 90% for haematopoietic stem cells. After transplantation into Rag-1-deficient recipients, stem cell transplanted animals developed T cell lymphomas/leukemia for all investigated oncogenes after characteristic incubation times, mostly of a CD8+CD4+ double positive phenotype. T cell lymphomas were characterised by gross thymic mass, splenomegaly and heavily enlarged lymph nodes, although none of the control- vector- transduced mice developed lymphoma/leukaemia. LM PCR analysis revealed mono- or oligoclonality of the tumours. T cell transplanted animals showed no signs of leukaemia development so far. However, after several attempts, one immortalized T cell progenitor clone could be generated after transduction with LMO2. Our results so far indicate that mature T cells are less susceptible to transformation by known T cell proto-oncogenes, but the studies are still ongoing.

Description: Lymphoid enhancer-binding factor 1 (LEF-1) belongs to the canonical Wnt signaling pathway acting in transcriptional complexes with β-catenin. LEF-1 can also act independent of β-catenin (i.e. in the TGF-β or Notch pathway). Additionally, recent studies described LEF-1 dominant negative isoform (dnLEF-1), which lack the β-catenin binding domain and functions as either a transcriptional repressor or activator. To date, analysis of the role of LEF-1 in hematopoiesis has been restricted to the lymphoid compartment. Previously we described the crucial role of lymphoid enhancer-binding factor 1 (LEF-1) in granulopoiesis. We found that LEF-1 mediates proliferation, survival, and differentiation of granulocyte progenitor cells. Moreover, abrogated LEF-1 expression is one of the pathomechanism of severe congenital neutropenia CN (Skokowa et al., Nature Medicine, in press). Based on these findings, we aimed to characterize the molecular mechanisms of LEF-1 in the regulation of granulocytic differentiation. C/EBPα is well known as a key transcription factor in granulopoiesis and we found it to be a target gene directly regulated by LEF-1. A screen of the known 566 bp upstream promoter of C/EBPα gene revealed a putative LEF-1 binding site (− 559 bp to − 538 bp). We confirmed LEF-1 binding to C/EBPα promoter in nuclear extracts from CD34+ and CD33+ cells in the transcription factor binding NoShift and ChIP assays. Interestingly, LEF-1 binds to the C/EBPα promoter more efficiently in CD33+ myeloid progenitors than in CD34+ cells. The direct regulation of C/EBPα by LEF-1 was further confirmed in functional studies. We found that in line with down-regulation of LEF-1, expression of C/EBPα was also significantly reduced in CD33+ myeloid progenitors of CN patients. Moreover, LEF-1 rescue of these cells resulted in a marked up-regulation of C/EBPα mRNA expression and in vitro restoration of defective granulocytic differentiation. Remarkably, transduction of CN CD33+ cells with dnLEF-1 isoform, which lacks the ß-catenin-binding domain, resulted in up-regulation of C/EBPα to a similar degree as it was observed with full-length LEF-1. A direct regulatory link between LEF-1 and C/EBPα was additionally confirmed in LEF-1 inhibition experiments. C/EBPα expression was significantly down-regulated in CD34+ cells of healthy individuals, transduced with LEF-1 shRNA. Therefore, we propose that LEF-1 is a key regulator of myeloid differentiation acting in a β-catenin-independent manner, similar as it is known for LEF-1 regulation of T-lymphocyte development.

Description: Ectopic delivery of HOXB4 elicits the in vitro and in vivo expansion of hematopoietic stem cells (HSC) although the mechanism is still unknown. We have previously shown that overexpression of HOXB4 attenuates the TNFα signaling pathway at the transcriptional level (Schiedlmeier et al., 2007, PNAS). TNFα is expression is induced at high levels in vivo after irradiation used in bone marrow (BM) transplantation preparative regimens. Since HSC and progenitors (P) derived from Fanconi anemia (FA) knockout mice are hypersensitive to the action of inhibitory cytokines such as TNFα, we chose the Fancc−/− mouse as a model to study the physiologic effects of HOXB4 on TNFα sensitivity of HSC/P and the relationship of these effects to the engraftment defect of FA HSC. Competitive repopulating assays were used to evaluate the effect of HOXB4 overexpression upon engraftment of Fancc−/− BM. Control (CON, expressing eGFP only) transduced Fancc−/− BM demonstrated 80-fold lower engraftment compared with wild type (WT) eGFP+ BM at 26 weeks post-transplant (Table 1). In marked contrast, HOXB4 transduced (HOXB4+) Fancc−/− BM showed a 26-fold higher level of engraftment compared with CON transduced Fancc−/− BM and a 2-fold higher level of engraftment compared with Fancc−/− cells corrected with a FANCC-expressing vector. Fancc−/− BM co-transduced with both vectors (expressing HOXB4 and FANCC) further increased the level of engraftment to that of WT eGFP+ BM suggesting a synergistic correction of the FA HSC engraftment defect. To determine the potential role of TNFα signaling in these effects, we directly assessed the impact of HOXB4 expression on the response of Fancc−/− BM to treatment with TNFα. Fancc−/− BM cells transduced with the CON eGFP vector demonstrated 〉70% reduction in colony formation upon treatment with 10ng/ml TNFα (Table 2), while Fancc−/− BM overexpressing HOXB4 showed no significant inhibition of CFU at 10ng/ml TNFα compared to either untreated cells or to treated WT BM transduced with eGFP CON vector. Additionally, in vitro treatment of eGFP CON transduced Fancc−/− BM with 100ng/ml TNFα resulted in a 21±5% decrease in lineage-, Sca-1+, c-Kit+ (LSK) cells within 24 hrs. In contrast, similarly treated HOXB4+Fancc−/− demonstrated a 6±9% increase in LSK cells (p25% reduction in the number of cells that stained positive for either TNFR1 or TNFR2. In addition, there was a 〉35% decrease in the MFI of staining for TNFR1 in HOXB4+Fancc−/− LSK compared to other groups. In summary, ectopic HOXB4 protects Fancc−/− and WT HSC/P from the inhibitory effects of TNFα. Since HOXB4 expression also protects WT cells from TNFα treatment (data not shown), we propose that HOXB4 enhances engraftment by protecting HSC from the elevated TNFα levels, which is a result of conditioning regimens applied to transplant recipients. We suggest that this mechanism reveals a novel target for the pharmacologic manipulation of HSC during engraftment, thus avoiding the potential adverse effects of constitutive HOXB4 overexpression. Table 1: % peripheral blood chimerism at 26 weeks post-transplant Fancc−/− + eGFP 0.5±0.2 Fancc−/− + FANCC 7.8±6.5 Fancc−/− + HOXB4 18.1±6.0** Fancc−/− + FANCC + HOXB4 34.1±7.1** WT + eGFP 44.3±5.3** Table 2: CFU as % non-treated control ± SEM TNFα dose 1ng/ml 10ng/ml Fancc−/− + eGFP 47±7 20±7 Fancc−/− + FANCC 81±7** 57±7** Fancc−/− + HOXB4 99±5** 92±5** WT + eGFP 90±7** 77±7** Table 3: % (and MFI) of BM positive for TNFR1 and TNFR2 expression ± SEM TNFR1 TNFR2 **p

Description: The drug resistance gene MGMT(P140K) mutant has been extensively studied as a means to protect/select hematopoietic stem cells (HSC) following treatment with O6-alkylating agents such as temozolomide (TMZ) in tandem with the inhibitor of endogenous MGMT, O6-benzylguanine (6BG). Since the repair of O6-alkyl adducts by MGMT is stoichiometric, it has been suggested that higher levels of MGMT(P140K) will confer better protection upon HSC. To test this hypothesis, we developed a panel of SIN γ-retroviral vectors which express low to very high levels of MGMT(P140K) via different internal promoters. The MGMT activity in bone marrow cells (BMC) transduced with a viral LTR-derived promoter, SF(MGMT), was approximately 7-fold higher than in cells transduced with either cellular promoters PGK(MGMT) or EFS(MGMT) which were in turn 〉300-fold higher than in control transduced cells (CON). Mice transplanted with transduced BMC were treated with 6BG (30mg/kg) and TMZ (80mg/kg) on 3 consecutive days starting 7 weeks post-transplant. Surprisingly, we found that selection/protection was not evident in mice engrafted with SF(MGMT) transduced BMC, while robust long term in vivo selection/protection was observed in mice transduced with PGK(MGMT) and EFS(MGMT) BMC (Table 1). We next sought to explore the mechanism of these findings. 2 hours after 6BG/TMZ treatment, BMC harvested from SF-, PGK- and EFS(MGMT) groups showed equivalent repair of the O6-alkylguanine lesion. Thus, lower MGMT expression is adequate for O6-alkylguanine lesion repair, and the lack of selection by SF(MGMT) is not related to defective repair. In the absence of chemoselection, we found a modest but statistically significant decrease in the in vivo reconstitution of SF(MGMT)-transduced BMC compared to EFS- or PGK- transduced BMC in a competitive repopulation assay, while secondary recipients showed a more pronounced engraftment defect (18- and 32-fold lower engraftment of SF(MGMT) vs EFS(MGMT), (p

Description: Ectopic delivery of HOXB4 elicits the expansion of engrafting hematopoietic stem cells (HSCs). We hypothesized that inhibition of tumor necrosis factor-α (TNF-α) signaling may be central to the self-renewal signature of HOXB4. Because HSCs derived from Fanconi anemia (FA) knockout mice are hypersensitive to TNF-α, we studied Fancc−/− HSCs to determine the physiologic effects of HOXB4 on TNF-α sensitivity and the relationship of these effects to the engraftment defect of FA HSCs. Overexpression of HOXB4 reversed the in vitro hypersensitivity to TNF-α of Fancc−/− HSCs and progenitors (P) and partially rescued the engraftment defect of these cells. Coexpression of HOXB4 and the correcting FA-C protein resulted in full correction compared with wild-type (WT) HSCs. Ectopic expression of HOXB4 resulted in a reduction in both apoptosis and reactive oxygen species in Fancc−/− but not WT HSC/P. HOXB4 overexpression was also associated with a significant reduction in surface expression of TNF-α receptors on Fancc−/− HSC/P. Finally, enhanced engraftment was seen even when HOXB4 was expressed in a time-limited fashion during in vivo reconstitution. Thus, the HOXB4 engraftment signature may be related to its effects on TNF-α signaling, and this pathway may be a molecular target for timed pharmacologic manipulation of HSC during reconstitution.

Description: Pre-B-cell colony-enhancing factor (PBEF) was first isolated from blood lymphocytes and was found to be involved in the maturation of B-cell precursors. Recently, PBEF was described as cytosolic enzyme nicotinamide phosphoribosyltranserase (NAmPRTase), promoted NAD+ biosynthesis. In this study we tested the ability of PBEF to induce myelopoiesis. Primary CD34+ cells treated with 50 ng/ml of PBEF in the serum-free RPMI medium without any additional cytokines, led to differentiation into mature granulocytes and macrophages, as assessed by FACS analysis (expression of CD11b, CD14, CD16), and by morphology. The same results were observed after lentiviral transduction of CD34+ progenitors with PBEF-GFP reporter. PBEF-dependent maturation was accompanied by increased mRNA expression of hematopoietic transcription factors such as C/EBPα , C/EBPε , PU.1, and ELA2. Moreover, PBEF had synergistic effect on G-CSF-triggered myeloid differentiation of CD34+ cells. Interestingly, G-CSF-treated differentiated CD34+ cells produced increased amounts of PBEF mRNA, as well as of intracellular and secreted PBEF protein in a time-dependent manner, as measured by real-time RT-PCR, ELISA, FACS analysis, and confocal microscopy. Increased synthesis of PBEF was in line with high NAD+ levels in G-CSF-treated cells. Expression of PBEF mRNA during myeloid differentiation was measured by laser-assisted single-cell picking and real-time quantitative RT-PCR analysis of different myeloid precursor cells (myeloblasts, promyelocytes, myelocytes, metamyelocytes, mature granulocytes and monocytes) from bone marrow smears of healthy individuals. PBEF mRNA expression was continually increased during myeloid differentiation with highest levels in mature granulocytes and monocytes. In addition, we compared expression patterns of PBEF mRNA/protein in CD33+ progenitors and PMNs of healthy individuals after in vivo and in vitro treatment with G-CSF. We found PBEF mRNA/protein expression to be markedly increased in both cell types after G-CSF simulation (12 times for granulocytes, and 10 times for monocytes). Intriguingly, PBEF mRNA/protein expression in CD33+ cells and PMNs from G-CSF-treated patients with severe congenital neutropenia (CN) was also up-regulated and was significantly higher, as in G-CSF-treated healthy donors, suggesting that myeloid cells from CN patients try to overcome the differentiation arrest by upregulating PBEF. This was also supported by the fact that CN patients had at least 20 times higher PBEF plasma levels, in comparison to control group. G-CSF-dependent increase of PBEF expression was in line with increased NAD+ levels in G-CSF-treated cells. Indeed, in vitro PBEF treatment as well as lentiviral transduction of CD34+ cells from one CN patient with PBEF-GFP reporter gene resulted in partial restoration of myelopoiesis in vitro. Taken together, PBEF promotes maturation of myeloid cells by NAD+ dependent pathway. Moreover, increased expression of PBEF with subsequent up-regulation of NAD+ synthesis in CD33+ cells and neutrophils from CN patients could reveal mechanisms of G-CSF-dependent restoration of defective myelopoiesis in these patients. rPBEF protein and anti-PBEF antibody were provided by Amgen, USA

Description: CD37 is a lineage-specific B-cell antigen that represents an attractive target for immunotherapy in B-cell malignancies, especially in chronic lymphocytic leukemia (CLL) and non-Hodgkin’s lymphoma (NHL). CD37-specific small modular immuno pharmaceutical (CD37-SMIP™) drug is an engineered protein therapeutic directed to the CD37 antigen using a single chain variable region (scFv) linked to a modified human IgG1 hinge, CH2, and CH3 domains. We have previously presented that CD37-SMIP™ drug induces both ADCC and apoptosis against primary CLL cells and B-cell lymphoma cells and therapeutic efficacy was observed in a Raji cell disseminated leukemia xenograft model. Herein, we sought to determine the effector cell type(s) mediating ADCC and explore if agents that deplete NK cell inhibitory T-regulatory cells influence CD37-SMIP™ efficacy. Immunostaining of human peripheral blood mononuclear cells (PBMCs) from CLL patients demonstrated no expression of CD37 on CD3+ T cells, CD16+ or CD56+ NK cells, or CD64+ monocytes whereas CD37 was highly expressed on CD19+ B cells. Using purified human PBMCs as effector cells and Cr-51 labelled CLL B cells as targets, we found the CD37-SMIP™ dependent ADCC was predominantly mediated by NK cells, but not naïve or activated monocytes. Consistent with these in-vitro results, the in-vivo therapeutic efficacy of CD37-SMIP™ drug was significantly compromised by depletion of NK cells in the Raji cell disseminated leukemia xenograft SCID mouse model. The median survival time of CD37-SMIP™ treated mice decreased from 51 days (95% CI: 38, 78) to 27 days (95% CI: 25, 37) (p=0.017) with the depletion of NK cells. Consistent with previous studies, ADCC is not diminished by fludarabine, an agent that may deplete T-regulatory cells, suggesting that the anti-CD37 protein and fludarabine might combine for increased efficacy in-vivo. We therefore examined the effect of fludarabine on CD37-SMIP™ in-vitro apoptotic activity. These data demonstrate that direct cell death mediated by CD37-SMIP™ drug (5 ug/mL) synergizes with fludarabine-induced caspase-dependent apoptosis, as measured by both the MTT assay and annexin V/PI staining of CLL cells (combination index, CI=0.44). Overall, these data suggest that the CD37-SMIP™ is a promising therapeutic agent against CD37+ B-cell malignancies as either monotherapy or in combination with fludarabine. Further clinical development is warranted to investigate the effect of CD37-SMIP™ drug in CLL and NHL. (SMIP trademark is owned by Trubion Pharmaceuticals).

Description: CD37 is a lineage-specific B-cell antigen highly expressed on both normal and transformed B-cells. The significant B-cell selective CD37 expression makes it a valuable target for therapy against B cell malignancies including chronic lymphocytic leukemia (CLL), hairy cell leukemia (HCL), and non-Hodgkins lymphoma (B-NHL). A novel CD37-specific small modular immunopharmaceutical (CD37SMIP) was engineered to contain a single chain variable region (scFv) linked to a human IgG1 hinge, CH2 and CH3 domains. CD37SMIP induces potent apoptosis in the presence of a crosslinker and antibody dependent cellular cytotoxicity (ADCC) against primary CLL cells. Significant in vivo therapeutic efficacy was demonstrated in a SCID mouse xenograft leukemia/lymphoma model that is dependent upon NK cell function. The induced cytotoxicity in primary CLL cells was independent of activation of caspase cascade, and consistent with this, the pan-caspase inhibitor z-VAD-fmk failed to rescue CD37 SMIP drug induced cytotoxicity. In an attempt to define the CD37 mediated early signaling events and their role in cytotoxicity, we investigated protein tyrosine phosphorylation as a potential early activation event responsible for CD37 SMIP drug mediated cytotoxicity. Primary B CLL cells were stimulated with increasing concentration of CD37 SMIP with or without crosslinker. Western blot analysis of cellular lysates with anti-phosphotyrosine antibody revealed several tyrosine phosphorylated proteins including a predominant ∼65KDa protein in response to the cross-linking of CD37 SMIP within 10 minutes. Further, pre-treatment with the tyrosine kinase inhibitor herbimycin prevented tyrosine phosphorylation of the predominant 65KDa protein and inhibited CD37 SMIP induced apoptosis in a dose dependent manner. Attempts to identify the tyrosine phosphorylated proteins, using MALDI-TOF mass spectrometry, and to define the potential role of these target proteins in the apoptotic pathway are ongoing. To further examine the molecular mechanism whereby CD37 SMIP triggers cell death, we investigated the involvement of mitochondrial pathways including changes in mitochondrial potential (Δψm) and generation of reactive oxygen species. Treatment with CD37 SMIP induces a time dependent mitochondrial membrane depolarization and increased production of ROS. Further, primary B CLL cells cultured with CD37SMIP induced cleavage and mitochondrial translocation of Bax and upregulation of Bim in a time dependent manner indicating a potential role for Bim and Bax pro apoptotic proteins in CD37 induced apoptosis. These findings provide strong justification for CD37 as a therapeutic target aimed at modulation of pro apoptotic proteins and introduce small modular immunopharmaceuticals as a novel class of targeted therapies for B-cell malignancies. (SMIP trademark is owned by Trubion Pharmaceuticals).

Description: The neurotrophins (NTs), which include nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), NT-3, NT-4, and NT-6, play a major role in neuronal survival. NTs are unique in that they utilize two different classes of receptors: the TRK (tropomyosin-related kinase) receptor protein tyrosine kinase (TRKA, TRKB, TRKC) and the low affinity NGF receptor (LNGFR=p75NTR). Recently, we and others have obtained evidence for potential involvement of this receptor system in leukemia. In the present study, we demonstrate for the first time expression of the three TRKs on the protein level in blasts from patients with newly diagnosed de novo or secondary acute leukemia. TRK expression was detected by flow cytometry using monoclonal antibodies previously validated on cell lines expressing retrovirally encoded TRK and considered positive if 〉20% of leukemic blasts expressed the respective receptor. 93 adult patients (41 female, 52 male) with a mean age of 53.8 years and diagnosis of AML (87%), ALL (12%), or AUL (1%) were enrolled after informed consent. Positive expression for at least one TRK was found in 55% of the analyzed cases without statistically meaningful differences in expression rate between AML (42/81) and ALL (8/11). Interestingly, while TRKB was expressed alone in blasts, TRKA or TRKC expression always occurred concomitantly with TRKB. In contrast to a previous study, we established a clear correlation of TRK expression pattern and FAB classification. In particular, TRKA expression occurred in 19 of 32 myelo-monocytic/monocytic leukemias (59%) whereas only 5 of 39 non-myelo-monocytic/monocytic leukemias (13%) were positive (p