Search

The transcription factor lymphoid enhancer-binding factor 1 (LEF-1), which plays a definitive role in granulocyte colony-stimulating factor (G-CSF) receptor-triggered granulopoiesis, is downregulated in granulocytic progenitors of severe congenital neutropenia (CN) patients. However, the exact mechanism of LEF-1 downregulation is unclear. CN patients are responsive to therapeutically high doses of G-CSF and are at increased risk of developing acute myeloid leukemia. The normal expression of LEF-1 in monocytes and lymphocytes, whose differentiation is unaffected in CN, suggests the presence of a granulopoiesis-specific mechanism downstream of G-CSF receptor signaling that leads to LEF-1 downregulation. Signal transducer and activator of transcription 5 (STAT5) is activated by G-CSF and is hyperactivated in acute myeloid leukemia. Here, we investigated the effects of activated STAT5 on LEF-1 expression and functions in hematopoietic progenitor cells. We demonstrated that constitutively active STAT5a (caSTAT5a) inhibited LEF-1-dependent autoregulation of the LEF-1 gene promoter by binding to the LEF-1 protein, recruiting Nemo-like kinase and the E3 ubiquitin-ligase NARF to LEF-1, leading to LEF-1 ubiquitination and a reduction in LEF-1 protein levels. The proteasome inhibitor bortezomib reversed the defective G-CSF-triggered granulocytic differentiation of CD34(+) cells from CN patients in vitro, an effect that was accompanied by restoration of LEF-1 protein levels and LEF-1 messenger RNA autoregulation. Taken together, our data define a novel mechanism of LEF-1 downregulation in CN patients via enhanced ubiquitination and degradation of LEF-1 protein by hyperactivated STAT5.

Mutations in the metabolic enzymes isocitrate dehydrogenase 1 (IDH1) and 2 (IDH2) are frequently found in glioma, acute myeloid leukemia (AML), melanoma, thyroid cancer, and chondrosarcoma patients. Mutant IDH produces 2-hydroxyglutarate (2HG), which induces histone- and DNA-hypermethylation through inhibition of epigenetic regulators. We investigated the role of mutant IDH1 using the mouse transplantation assay. Mutant IDH1 alone did not transform hematopoietic cells during 5 months of observation. However, mutant IDH1 greatly accelerated onset of myeloproliferative disease-like myeloid leukemia in mice in cooperation with HoxA9 with a mean latency of 83 days compared with cells expressing HoxA9 and wild-type IDH1 or a control vector (167 and 210 days, respectively, P = .001). Mutant IDH1 accelerated cell-cycle transition through repression of cyclin-dependent kinase inhibitors Cdkn2a and Cdkn2b, and activated mitogen-activated protein kinase signaling. By computational screening, we identified an inhibitor of mutant IDH1, which inhibited mutant IDH1 cells and lowered 2HG levels in vitro, and efficiently blocked colony formation of AML cells from IDH1-mutated patients but not of normal CD34(+) bone marrow cells. These data demonstrate that mutant IDH1 has oncogenic activity in vivo and suggest that it is a promising therapeutic target in human AML cells.

Neurotrophins (NTs) and their receptors play a key role in neurogenesis and survival. The TRK (tropomyosin-related kinase) receptor protein tyrosine kinases (TRKA, TRKB, TRKC) are high-affinity NT receptors that are expressed in a variety of human tissues. Their role in normal and malignant hematopoiesis is poorly understood. In a prospective study involving 94 adult patients we demonstrate for the first time cell-surface expression of the 3 TRKs and constitutive activation in blasts from patients with de novo or secondary acute leukemia. At least one TRK was expressed in 55% of the analyzed cases. We establish a clear correlation between the TRK expression pattern and FAB classification. Although only few point mutations were found in TRK sequences by reverse-transcriptase–polymerase chain reaction (RT-PCR), we observed coexpression of BDNF (ligand for TRKB) in more than 50% of TRKB+ cases (16/30). Activation of TRKA or TRKB by NGF and BDNF, respectively, efficiently rescued murine myeloid cells from irradiation-induced apoptosis. Coexpression of TRKB/BDNF or TRKA/NGF in murine hematopoietic cells induced leukemia. Moreover, activation of TRKs was important for survival of both human and murine leukemic cells. Our findings suggest that TRKs play an important role in leukemogenesis and may serve as a new drug target.

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.

Hematopoiesis is tightly controlled by a family of cytokines that signal through a related set of receptors. The pleiotropic and overlapping response of a cell to different cytokines is reflected in the number and complex pattern of activated signal transducers. Of special interest is STAT5, which is stimulated by a large and diverse set of cytokines. In addition to the two highly homologous proteins, STAT5A and STAT5B, encoded by duplicated genes, expression and activation of a dominant-negative, carboxyl-truncated form has also been described in early hematopoietic progenitors. We show here that a protease expressed in early hematopoietic cells cleaves the α forms of STAT5A/5B (p96/p94) to generate carboxyl-truncated β forms (p80/p77). Inhibition studies assigned this protease to the serine class of endopeptidases. Cell fractionation experiments showed that the protease is associated with the nucleus in a constitutively activated form and does not require an activated STAT5 substrate. The ability of a protease to modulate the specificity of an activated transcription factor is unprecedented and underlines the importance of proteases in regulation of cell functions.

Thpo/Mpl signaling plays an important role in the maintenance of hematopoietic stem cells (HSCs) in addition to its role in megakaryopoiesis. Patients with inactivating mutations in Mpl develop thrombocytopenia and aplastic anemia because of progressive loss of HSCs. Yet, it is unknown whether this loss of HSCs is an irreversible process. In this study, we used the Mpl knockout (Mpl−/−) mouse model and expressed Mpl from newly developed lentiviral vectors specifically in the physiologic Mpl target populations, namely, HSCs and megakaryocytes. After validating lineage-specific expression in vivo using lentiviral eGFP reporter vectors, we performed bone marrow transplantation of transduced Mpl−/− bone marrow cells into Mpl−/− mice. We show that restoration of Mpl expression from transcriptionally targeted vectors prevents lethal adverse reactions of ectopic Mpl expression, replenishes the HSC pool, restores stem cell properties, and corrects platelet production. In some mice, megakaryocyte counts were atypically high, accompanied by bone neo-formation and marrow fibrosis. Gene-corrected Mpl−/− cells had increased long-term repopulating potential, with a marked increase in lineage−Sca1+cKit+ cells and early progenitor populations in reconstituted mice. Transcriptome analysis of lineage−Sca1+cKit+ cells in Mpl-corrected mice showed functional adjustment of genes involved in HSC self-renewal.

Previous studies have demonstrated leukemic complications in mice after high-copy retroviral gene transfer of the multidrug resistance 1 (MDR1) cDNA, encoding a membrane-located efflux pump expressed in hematopoietic stem cells. In contrast, no such complications or MDR1-associated alterations of hematopoiesis were observed in numerous other studies exploring MDR1 gene transfer into cell lines, mice, dogs, nonhuman primates, and human subjects. Here, we show that leukemias associated with retroviral expression of MDR1 depend on high vector dose, and involve the selection of clones with combinatorial insertional mutagenesis of proto-oncogenes or other signaling genes. Compared with insertion patterns in normal long-term repopulating hematopoietic cells, such hits were overrepresented in leukemic clones, pointing to a causal role. A similar constellation of insertion sites was also observed in a leukemia arising after high-copy retroviral gene transfer of a fluorescent protein. Spectral karyotyping demonstrated additional chromosomal translocations in a subset of cases, indicative of secondary genetic instability. We also show that insertional mutants can be amplified in vitro prior to transplantation. On the basis of these findings, we suggest the use of preclinical dose-escalation studies to define a therapeutic index for retroviral transgene delivery.

Recent conceptual and technical improvements have resulted in clinically meaningful levels of gene transfer into repopulating hematopoietic stem cells. At the same time, evidence is accumulating that gene therapy may induce several kinds of unexpected side effects, based on preclinical and clinical data. To assess the therapeutic potential of genetic interventions in hematopoietic cells, it will be important to derive a classification of side effects, to obtain insights into their underlying mechanisms, and to use rigorous statistical approaches in comparing data. We here review side effects related to target cell manipulation; vector production; transgene insertion and expression; selection procedures for transgenic cells; and immune surveillance. We also address some inherent differences between hematopoiesis in the most commonly used animal model, the laboratory mouse, and in humans. It is our intention to emphasize the need for a critical and hypothesis-driven analysis of “transgene toxicology,” in order to improve safety, efficiency, and prognosis for the yet small but expanding group of patients that could benefit from gene therapy.

Abstract 770 Mutations in the metabolic enzymes IDH1 and IDH2 are frequently found in glioma and AML patients. Mutant IDH produces R-2-hydroxyglutarate (2HG), which induces histone- and DNA-hypermethylation through inhibition of epigenetic regulators, thus linking metabolism to tumorigenesis. However, it is unknown whether R-2HG is required for transformation. To investigate the function of mutated IDH1 we established an in vivo mouse model, which allowed us to study R-2HG-dependent and –independent functions of mutated IDH1. We cloned wildtype IDH1 (IDH1wt) and mutated IDH1 (IDH1mut, R132C) from AML and MDS patients and identified a mutated splice variant that lacked exon 7 (IDH1mutΔ7) in several IDH1 mutated patients. To evaluate whether the mutated proteins produce the oncometabolite R-2HG, we expressed IDH1mut, IDH1mutΔ7, and IDH1wt in immortalized murine bone marrow cells and measured intracellular levels of 2HG by enantiomer-specific quantification. IDH1mut expressing cells produced high levels of R-2HG, but not IDH1 mutated cells that lacked exon 7 of IDH1, control-vector transduced cells (CTL), or IDH1 wildtype cells (P In vivo cell cycle analysis showed a significantly higher proportion of cells in S/G2/M phase in bone marrow cells transduced with IDH1mut or IDH1mutΔ7 when compared to cells transduced with IDH1wt or CTL. Also, cyclin-dependent kinase inhibitors (Cdkn) 1A (p21), 1B (p27), 2A (p16), and 2B (p15) were markedly downregulated in IDH1mut and IDH1mutΔ7 cells when compared to IDH1wt cells. We next investigated, whether the promoters of the repressed Cdk inhibitors were hypermethylated. The CpG island in the promoter of Cdkn2a and Cdkn2b showed low levels of DNA methylation in IDH1wt, IDH1mut, IDH1mutΔ7 and CTL cells (0.6% to 4.3% average methylation), suggesting that Cdk inhibitors are repressed in cells with mutated IDH1 independently of DNA methylation. Gene set enrichment analysis from microarray data of transduced bone marrow cells showed that genes related to MAP-kinase signalling were highly enriched in IDH1mut and IDHmutΔ7 cells compared to IDH1wt or CTL transduced cells. By Western blot we found that pERK was highly upregulated in IDH1mut and IDHmutΔ7 cells compared to IDH1wt or CTL cells. Pharmacologic inhibition of transduced cells in vitro showed that IDH1mut and IDH1mutΔ7 cells were resistant to inhibition with the MEK1/2 inhibitor UO126, suggesting that MAP-kinase signalling is a relevant target in IDH1 mutated AML. In summary, we show that an alternatively spliced isoform of mutated IDH1 promotes leukemogenesis independently of R-2HG in a mouse model of transformed hematopoietic cells with the same kinetics as mutated full-length IDH1 through transcriptional repression of cyclin-dependent-kinase inhibitors p15 and p16 and activation of MAP-kinase signalling. Our mouse model of mutated IDH1 represents a powerful tool to test the efficacy of newly developed drugs targeting mutated IDH1 in a 2-HG-dependent and –independent manner. Disclosures: No relevant conflicts of interest to declare.

Abstract 2415 Introduction. High activation of the PI3K-AKT-mTOR pathway is characteristic for T-cell acute lymphoblastic leukemia (T-ALL) and has been linked to c-myc independence, as well as resistance of T-ALL to Glucocorticoids and Notch inhibition. The master regulator of PI3K-AKT signaling is the lipid phosphatase PTEN. Despite impaired or lost PTEN activity in T-ALL experimental evidence suggests that input from receptor tyrosine kinases (RTKs) is needed to sustain high mTOR activation. Here we investigated how RTK signaling contributes to the pathogenesis of T-ALL. To mimic RTK signaling we expressed ΔTrkA, a constitutively active nerve growth factor RTK in primary murine hematopoietic cells. Injection of ΔTrkA-transduced cells into C57BL6 mice induced transplantable T-ALL with a latency of 120 days. Results. Signaling studies showed that ΔTRKA activates mTORC1 over the MAPK pathway, but not mTORC2. In contrast, ΔTRKA+ T-ALL showed a profound shift in the use of downstream signaling cascades, displaying a very high activation of mTORC1 and mTORC2 and absent MAPK signaling. To understand the rewired signaling network we first ruled out contribution of insertional mutagenesis by sequencing vector integration sites. Array-CGH revealed hetero- or homozygous loss of PTEN in 4 of 6 T-ALL lines. In the remaining 2 T-ALL lines, which showed normal PTEN protein levels, we detected two PTEN mutations in the phosphatase domain, D92G and G165E, the latter of which has not been described before. Re-expression of the mutants in PTEN−/− cells revealed complete loss of function for D92G and a partial loss for the G165E mutant. Restoration of wt PTEN expression in the T-ALLs resulted in reversion of the signaling pattern. Sequencing of Notch1 revealed both, PEST domain mutations and 5'-deletions in cis, in all but one investigated T-ALL. Northern and Western Blots confirmed the expression of truncated Notch1 transcripts and protein. Notch inhibition in PTEN−/− cell lines resulted in robust inhibition of mTORC2, showing that Notch contributes to mTORC2 activation independently of the Hes1-PTEN axis. Despite the loss of PTEN most T-ALLs remained addicted to the input of the leukemia initiating RTK, ΔTRKA. Importantly, the cooperation of three oncogenic pathways converging on mTOR culminated in an oncogenic addiction to mTOR. We screened several inhibitors of the mTOR pathway and found that ΔTRKA+ PTEN− Notch+ T-ALL are highly susceptible to an inhibition of cap dependent translation by 4EGI-1, whereas primary hematopoietic cells and freshly isolated thymocytes were far less susceptible. Polysome profiling indicated a profound decrease in ribosome occupancy upon treatment with 4EGI-1. Microarray analysis of polysome fractions revealed that mRNAs belonging to genes that were strongly upregulated between T-ALL and normal thymocytes were specifically shifted out of the actively translated transcriptome by 4EGI-1. These genes included members of the translational apparatus itself, mitochondrial matrix proteins, cell cycle regulators such as CyclinD1 and c-myc as well as Bcl-2. Western blots confirmed early loss of these proteins after cessation of cap dependent translation. Conclusions. Biochemical and genetic dissection of T-ALLs induced by constitutive RTK signaling uncovered activating Notch mutations and PTEN loss as cooperating events and revealed a strong, druggable addiction to cap-dependent translation downstream of oncogenic mTOR. The striking overlap of genes defining the molecular difference between T-ALL blasts and normal thymocytes and genes that preferentially lost ribosomal occupancy upon 4EGI-1 treatment suggests that this drug targets oncogenic core pathways in T-ALL and deserves further investigation. Disclosures: No relevant conflicts of interest to declare.

Abstract 1381 T-cell acute lymphoblastic leukemia (T-ALL) remains a therapeutic challenge. T-ALLs are characterized by recurring chromosomal rearrangements causing aberrant expression of transcription factors (Myb; TAL/SCL; HOX) dividing patients into different subgroups. Activating mutations in NOTCH, the master regulator of T-cell development, are found in more than 60% of T-ALLs independently of subtype. Most T-ALLs display a hyperactivation of the PI3K-AKT-mTOR pathway, a potential target for therapeutic intervention. The master regulator of PI3K-AKT signalling is PTEN, which is frequently inactivated in cancer. Recent data suggests that complete PTEN loss due to mutation is rare in primary human T-ALL, whereas PTEN-inhibiting posttranslational modifications are more common (Barata et al., J. Clin. Invest. 2008, 118). As these modifications decrease, but do not abolish the phosphatase activity of PTEN, we hypothesized that further input from tyrosine kinases, particularly receptor tyrosine kinases (RTK), may be needed to sustain PI3K-AKT-mTOR activation. In order to investigate how RTK-signaling may contribute to the pathogenesis of T-ALL we used an established murine bone marrow transplantation model (Li et al. Blood 2009, 113). To mimic tyrosine-kinase signaling we expressed δTrkA, a constitutively active TRKA receptor tyrosine kinase (TRK =tropomyosin-related kinase) from gammaretroviral or lentiviral vectors in c-kit+ Sca-1+ Lin− (KSL) cells. Intravenous injection of δTrkA-transduced hematopoietic cells in C57BL6 mice (n=10) induced transplantable T-ALL with a latency of about 120 days. The resulting T-ALLs could be propagated in culture as clonal cell lines. Signaling studies showed that δTRKA activates predominantly ERK upon expression in murine hematopoietic cell lines. However, the obtained δTRKA+ T-ALL lines (n=7) showed a profound shift in the use of downstream signaling cascades, displaying a very high activation of AKT-mTOR and absent ERK phosphorylation, resembling human T-ALL. High AKT activation was uniformly detected regardless of PTEN protein expression in all but one T-ALL (#003). To understand the rewired signaling network we looked for a potential contribution of insertional mutagenesis and chromosomal aberrations. Array-CGH showed homozygous deletions on chr14c2 involving the T-cell receptor alpha and delta genes in 3/3 cell lines and heterozygous deletions in Ikzf1 in 2/3 cell lines. Viral integration sites showed no common insertion pattern and no insertion in genes implicated in RTK-signaling. The expression of genes in proximity to viral integrations (±500 kb) appeared unaltered as determined by cDNA-microarray analysis of the T-ALL cell line #483 against wild type CD4+CD8+ thymocytes. Microarray analysis revealed enrichment of Notch1 target genes in the T-ALL cell line #483. Sequencing of Notch1 revealed both, PEST domain mutations and the recently described (Aster et al, Blood 2010, 116) RAG mediated 5'-deletions in cis, in all but one investigated T-ALL. Northern and Western Blots confirmed the expression of truncated Notch1 transcripts and protein, respectively. The one cell line (#003) which retained the original δTrkA signaling pattern had no Notch mutation and could only be cultured on OP9-Delta-like-1 stroma cells, highlighting the importance of Notch signaling. As this cell line was established from a mouse displaying an enlarged thymus, but no full manifestation of T-ALL, our data suggests that acquisition of Notch mutations is a late, but necessary step required for overt leukemia, whereas the initiating events may arise in kinase signaling pathways of prethymic progenitors. All T-ALL cell lines were sensitive to mTOR or Notch inhibition with Rapamycin or Compound E, respectively. Finally, we used phosphoprotein-arrays to monitor the phosphorylation of 42 RTK in childhood T-ALL samples with different activating NOTCH mutations (n=5) and detected several activated RTK (e.g. MSPR, FGFR, ErbB4, VEGFR) in the patient samples. Taken together, our findings suggest a cooperation of RTK and activating NOTCH mutations in mTOR activation seen in T-ALL and encourage further investigation of 1) aberrant RTK-signaling in T-ALL 2) the role of RTK activation in creating a preleukemic cell clone, 3) evaluation of combined therapy targeting RTKs and NOTCH, and 4) the role of activated NOTCH on mTORC2-AKT activation independently of PTEN. Disclosures: Baum: Patent office: Patents & Royalties.

Abstract 3123 In gene therapy targeting hematopoietic cells, a quantitative assessment of the risk factors underlying insertional mutagenesis is required to assess the practical value of preventive actions. Emanating from an observation of the Copeland lab (Du et al., 2005) we developed an in vitro immortalization (IVIM) assay which determines the risk of transformation of murine bone marrow cells as a consequence of insertional upregulation of Evi1 or Prdm16. These functionally related genes encode master regulators of hematopoiesis which are involved in the pathogenesis of human leukemia and insertional transformation in human gene therapy. Using our standardized conditions, the assay can detect mutants arising with a low frequency (down to 1 in a million cells), based on their rescue and expansion upon replating. The genetic lesion associated with clonal transformation is easily identified, and we can quantify not only the incidence of mutants (number of cells required to form a mutant) but also their fitness (number of subclones obtained by replating). Using the IVIM assay, our published work has revealed the following: (1) relocating gammaretroviral enhancer-promoter sequences from the LTR to an internal position of a “self-inactivating” (SIN) vector reduces the fitness of mutants, as do mutations in transcription factor binding sites or insulators that reduce the enhancer activity; (2) cellular promoters located in SIN vectors, depending on their enhancer activity, may reduce the risk of transformation below the detection limit (>3 logs compared to standard gammaretroviral vectors); (3) the post-transcriptional regulatory element of the woodchuck hepatitis virus does not affect insertional transformation; and (4) the lentiviral integration pattern reduces the risk of insertional transformation by a factor of ∼3 compared to gammaretroviral vectors. In the meantime, the assay has been used to assess the transforming potential of new vectors developed to treat a variety of hematopoietic disorders, most notably X-SCID, X-CGD, WAS and globinopathies. Reproducibly we found that vectors containing cellular promoters reduced the risk of insertional transformation when compared to retroviral promoters, although not all cellular promoters appeared to be free of risk. The assay has also revealed major functional differences of various insulator elements, including synthetic ones designed to block enhancer-crosstalk. Testing a battery of 8 insulators that we obtained from collaborators or designed ourselves, we found that only a subset was potent enough to significantly reduce the transforming potential of a strong retroviral enhancer-promoter. Furthermore, we assessed the transforming potential of our new alpharetroviral SIN vectors (Suerth et al., JV 2010), modified to remove a residual TATA box of the LTR. When containing a retroviral internal promoter, alpharetroviral SIN vectors were ∼9-times and 3-times, respectively, less likely than the corresponding gammaretroviral and lentiviral constructs to induce strongly replicating clones. Mutants obtained with alpharetroviral SIN vector insertions in Evi1 were not only less frequent but also had a greatly reduced fitness compared to those induced by similarly designed gammaretroviral vectors. Alpharetroviral SIN vectors containing the human elongation factor 1 alpha promoter did not immortalize cells in this assay, as previously shown for gammaretroviral SIN vectors. Finally, we performed experiments to explore the mechanistic basis of the IVIM assay. Our data suggest that its principle is the selection of mutants that resist the differentiation-inducing effect of a myeloid growth factor cocktail. Therefore, variations of the cell culture conditions have a significant impact on the sensitivity of the assay, and potentially also on the spectrum of mutants that can be isolated. The established conditions typically select for upregulation of Evi1, Prdm16, or, more rarely observed, Ras -related genes. In summary, the IVIM assay quantifies the risk of insertional mutagenesis in gene therapy, related to vector sequences and integration pattern. It is specifically useful to assess the risk of insertional upregulation of Evi1 and Prdm16 via enhancer-mediated mechanisms, in myeloid progenitor cells. It thus serves as an animal replacement assay to screen for safety-enhancing vector modifications. Disclosures: Off Label Use: CliniMACS for selection of CD34+ hematopoietic cells.

Abstract 671 Gene therapy with gammaretroviral and lentiviral vectors has shown its potential for the treatment of inherited hematopoietic disorders. However, the occurrence of severe adverse events, namely the induction of leukemias in clinical trails for the treatment of severe combined immunodeficiency (SCID)-X1, chronic granulomatous disease and Wiskott Aldrich Syndrome due to insertional activation of proto-oncogenes by the integrated vector raised safety concerns. Lentiviral vectors are assumed to be safer due to their integration bias to transcription units in comparison to gammaretroviral vectors that have an integration preference to promoter regions and CpG islands. However, the recent report on the development of clonal dominance in a clinical trial for b-thalassemia mediated by deregulated gene expression due to alternative splicing after lentiviral insertion into an intron of HMGA2 highlights potential risks of lentiviral gene therapy. Here we report a case of B-cell lymphoblastic leukemia in a murine bone marrow (BM) transplantation model 199 days post transplantation of cells transduced by a lentiviral vector expressing eGFP from the human glycoprotein-I-b-alpha (GPIba) promoter (-286 to +309 relative to the transcription start site). The mouse developed leukocytosis (179×103/μl white blood cells counts) and leukemic infiltrations were found in the BM, spleen, liver and lung. The leukemia was clonal as determined by Southern blot analysis, derived from the donor cells as determined by the CD45.1 chimerism marker and reinitiated disease in secondary animals (n=7). Leukemic cells had the morphology of lymphoid blasts/progenitors, stained positive for the markers B220, CD43, and partially CD19 and were negative for IgM. We therefore concluded that the cells were arrested and the PrePro-B to Pro-B cell stage. Two lentiviral integrations were identified in the leukemic clone of which one was mapped to intron 8 of the Early B-cell factor 1 (Ebf1) gene, a major regulator of B-cell development. The other insertion site was detected in the first intron of the Nance-Horan-Syndrome-gene that has no known function in hematopoiesis and we could not detect altered expression of this gene. No further genomic alterations were identified by spectral karyotyping and Array CGH analysis. Because the vector contained various splice sites in its backbone and the internal GPIba promoter, we next analyzed potential splice products and detected mRNAs formed from splice events between Ebf1 exons 8 and 9 to the integrated vector. Some of these could result in early termination of the mRNA which would generate a truncated Ebf1 protein containing only the DNA-binding domain. However, the overall contribution of this early stop mRNAs was low (5% of total Ebf1 message) and we could not detect a truncated Ebf1 protein by Western blot. Overexpression of the truncated Ebf1 protein in a B-cell differentiation assay on OP9 cells in vitro did not interfere with B-cell development. In contrast, we found the Ebf1 full length mRNA to be ∼4-fold downregulated and the EBf1 protein in leukemic cells to be reduced compared to stage matched BM B-cell progenitors (CD19+CD43+). Genome wide expression analysis of leukemic cells compared to B-cell progenitors revealed downregulation of Ebf1 target genes and definite signs of transdifferentiation to the myeloid lineage as it was described upon loss of Ebf1 or its downstream target Pax5 in other studies. In addition, Gene Set Enrichment and Gene Ontology analyses suggested upregulation of genes implicated in JAK-STAT signalling. Indeed, we observed strong upregulation of Flt3 and high levels of STAT5 activation, in line with recent reports of B-cell leukemia development in Ebf1+/− mice overexpressing constitutively active STAT5 (Heltemes-Harris et al., J. Exp. Med 2011). STAT5 activation is also found in a large subset of human B-ALL and the occurrence of mono- or bi-allelic EBF1 deletions in human B-ALL is known (Mullighan et al., Nature, 2007). As the lentiviral integration was an early event in the leukemia development in our experiments, haploinsufficiency of Ebf1 will have been most likely the leukemia initiating event. Taken together our results highlight the risk of intragenic lentiviral vector integration of vectors containing splice sites that can induce alternative splicing, which, as shown here, may lead to inactivation of haploinsufficient tumour suppressor genes. Disclosures: Off Label Use: CliniMACS for selection of CD34+ hematopoietic cells.

Abstract 388 LEF-1 is a decisive transcription factor involved in G-CSFR triggered granulopoiesis and is downregulated in severe congenital neutropenia (CN). However the exact mechanism of LEF-1 downregulation is still unclear. CN patients show response to G-CSF therapy, however therapeutically high doses of G-CSF are required to increase the neutrophil counts. Also CN patients are found to be at increased risk of developing AML (cumulative incidence ∼ 20%). Since LEF-1 is normally expressed in monocytes and lymphocytes whose differentiation is not affected in CN, it must be a granulopoiesis-specific mechanism downstream of G-CSFR signalling leading to LEF-1 down-regulation. STAT5 is one of the factors activated by G-CSF signalling pathway and has also been found to be hyper-activated in AML blasts. Also STAT5 is known to suppress LEF-1 target gene C/EBPα. Therefore we investigated the effects of activated STAT5 on LEF-1 gene expression and functions. We performed chromatin immunoprecipitation (ChIP) assay from CD34+ cell nuclear lysates and found two putative STAT5 binding sites. Reporter gene assays performed using LEF-1 gene promoter, which contains these STAT5 binding sites co-transfected with LEF-1 and STAT5a cDNA showed significant down-regulation of LEF-1 dependent activation in presence of constitutive active STAT5a (caSTAT5a). However, when we introduced deletion mutations in both STAT5 binding regions and performed the reporter gene assays we found that STAT5 dependent down regulation was independent of its direct binding to the LEF-1 gene promoter. Therefore we further checked the effects of activated STAT5 on LEF-1 protein levels. We found that co-transfection of caSTAT5a cDNA with LEF-1 significantly reduces the LEF-1 protein levels. This reduction in LEF-1 protein levels could be normalised by treating the cells with proteosome inhibitors, bortezomib and MG132. Moreover addition of bortezomib or MG132 to the reporter assays could rescue the effect of STAT5 dependent downregulation on LEF-1 gene autoregulation. We indentified a novel endogenous interaction of STAT5 with LEF-1 protein in the co-immunoprecipites from jurkat cell lysates and this interaction was confirmed insitu using Duolink proximity ligation assay. Furthermore we analysed G-CSF dependent granulocytic differentiation of CD34+ from CN patients treated additionally with bortezomib. Granulocytic differentiation was assessed by FACS using myeloid-granulocytic surface markers CD15, CD16 and CD11b. Interestingly, we found that addition of bortezomib to G-CSF resulted in granulocytic differentiation of CD34+ cells of CN patients in vitro, which was abolished in CN cells treated with G-CSF alone. This granulocytic differentiation was accompanied by restoration of LEF-1 protein levels by bortezomib. Taken together we identified a novel mechanism of LEF-1 downregulation in CN patients, which is via elevated degradation of LEF-1 protein mediated by hyper-activated STAT5. This in turn leads to abrogated autoregulation of LEF-1 mRNA expression. Along with, this study opens gates to a new therapeutic approach using bortezomib in combination with G-CSF for CN patients who do not respond or need high doses of G-CSF. Disclosures: No relevant conflicts of interest to declare.

Neurotrophins (NTs) and their receptors play a key role in neurogenesis and survival. The TRK (tropomyosin-related kinase) receptor protein tyrosine kinases (TRKA, TRKB, TRKC) are high affinity NT-receptors that are expressed in a variety of human tissues. Their role in normal and malignant hematopoiesis is poorly understood. Recently, we and others have obtained evidence for potential involvement of this receptor system in leukemia. In a prospective study involving 94 adult patients, we demonstrated for the first time cell surface expression of the three TRKs and constitutive activation in blasts from patients with de novo or secondary acute leukemia. At least one TRK receptor was expressed in 55% of the analyzed cases (Li Z et al., ASH 2007). Altered TRK signaling efficiently transformed murine hematopoietic stem/progenitor cells (Meyer J et al., Leukemia 2007; Li Z et al., ASH 2007). We observed constitutive activation of mammalian target of rapamycin (mTOR) both in murine and human leukemic cells. Murine leukemic cells induced by altered TRK signaling were very sensitive to rapamycin treatment in vitro. We next tested the therapeutic effect of rapamycin on altered TRK-induced leukemia in a mouse model (C57Bl/6J). Leukemic cells isolated from #483 mouse transplanted with primary hematopoietic stem/progenitor cells modified with deltaTrkA, an active mutant of TRKA isolated from a patient with acute myeloid leukemia, grew factor-independently. Treatment of #483 cells with rapamycin (10–50nM) induced apoptosis and showed a dose-dependent growth inhibition (up to 100%) in colony forming assay. Consistently, mTOR was strongly dephosphorylated. In pilot studies, we found that transplantation of 106 #483 cells into recipients constituted with sublethal irradiation (7.5Gy) gave rise to leukemia development in all animals after a latency of 8 weeks; Daily i.p. injection of 2mg/kg rapamycin in healthy animals mediated a high level of rapamycin in whole blood (around 50ng/ml). Thus, after irradiation, 20 animals were transplanted with 106 #483 cells and randomized in two groups. One group was treated daily with rapamycin 2mg/kg i.p., the other group received only carrier (placebo). The treatment begun 3 weeks after transplantation and continued until the last animal succumbed to leukemia. Rapamycin treatment significantly prolonged the survival of animals compared with control group (mean survival 48.5 and 32 days, respectively, P=0.0087) (Figure 1). Concentration of rapamycin in whole blood at the time of end point analysis ranged 26–151ng/ml. Our findings suggest that mTOR plays an important role in leukemogenesis induced by altered TRK signaling. TRKs and mTOR might serve as therapeutic targets in acute leukemia. Figure 1 Kaplan-Meier estimate of survival of animals after treatment. Figure 1. Kaplan-Meier estimate of survival of animals after treatment.

The thrombopoietin receptor Mpl is required for regeneration of hematopoietic stem cells and governs megakaryocytic differentiation. Patients with inherited MPL deficiency suffer from severe thrombocytopenia which progresses to aplastic anemia, a disorder called congenital amegakaryocytic thrombocytopenia (CAMT). As a first step towards a potential gene therapy for MPL deficiency, we retrovirally expressed the receptor in a murine bone marrow transplantation model. An initial series of vectors used a strong enhancer-promoter derived from the spleen-focus forming virus (SFFV). Mice transplanted with hematopoietic cells modified by these constructs developed a profound yet transient elevation of multi-lineage hematopoiesis due to increased signaling of the Thpo receptor on target and non-target cells. Mice developed increased leukocyte, erythrocyte and platelet counts (2–3 times elevated) compared to GFP control animals. Histopathology revealed an elevated number of mature megakaryocytes with atypical features like numerous giant forms in the bone marrow and the spleen and atypical neoformation of bone in the spleen leading to the diagnosis of chronic myeloproliferative disorder (CMPD). A minority of mice (3/27) developed erythroleukemia with almost 100% CD71 and TER119 double positive cells as detected by flow cytometry. Histopathology presented infiltration of erythroblasts in all hematopoietic tissues like the BM, spleen and liver. Molecular analysis revealed retroviral vector insertions in sfpi1, fli1 and klf3 that seem to be the major driving force for the development of leukemia in these animals. Somewhat unexpectedly, in the majority of mice the CMPD converted into a progressive, potentially lethal pancytopenia. Animals had severely reduced blood cell counts with only 50% of leukocyte, 20% of erythrocyte and 10% of platelet counts compared to GFP control animals. This population crisis affected all major blood lineages and also involved co-existing unmodified hematopoiesis. Histopathology presented a dysmegakaryopoiesis with an increased number of atypical micro-megakaryocytes, histiocytes with erythrocytophagocytosis and atypical mast cell proliferation diagnosed as a myelodysplastic syndrome (MDS)-like disorder. In the bone marrow, pancytopenic mice had reduced cell numbers of the primitive cell fraction (LSK cells). To address the mechanism of pancytopenia, we expressed a dominant negative form of Mpl (dnMpl) consisting of the extracellular and transmembrane domain and lacking the intracellular signal transduction domain. Animals transplanted with dnMpl-modified cells failed to show the initial CMPD but developed the same pancytopenic, MDS-like end stage. A vector expressing Mpl under control of the PGK promoter or a fragment of the Mpl-promoter reduced or completely avoided the side effects (CMPD, MDS-like disorder) observed with vectors using stronger promoters. The induction of a hematopoietic population homeostasis thus depends upon Mpl expression levels, indicating the need for strictly regulated transgene expression in gene therapy for MPL deficiency. As ectopic expression of the extracellular domain is sufficient to cause MDS, sequestration of crucial niche factors like Thpo may contribute to the pathogenesis of this disorder. This study demonstrates that ectopic expression of a hematopoietic growth factor receptor may disturb organ homeostasis through interference with intra- and extracellular mechanisms of cell communication.

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

Severe congenital neutropenia (CN) is a hematological disorder characterized by an early stage maturation arrest of granulopoiesis in bone marrow at promyelocytic stage. Previously we found that LEF-1 and its target gene C/EBPα are severely abrogated in promyelocytes of CN patients, which plays a decisive role in the pathomechanism of defective granulopoiesis (Skokowa J., et al., Nat. Med.2006;12:1191). Recently it has been shown that constitutively activated STAT5a inhibits myeloid differentiation of human CD34+ cells by downregulation of C/EBPα (Moore MA, et al., Exp. Hematol.2007;35:105). Therefore, we investigated whether the presence of constitutive activated STAT5a in CD34+ cells of healthy individuals leads to downregulation of LEF-1 and whether STAT5a is activated in myeloid progenitors of CN patients. A screen of the 10kb upstream region of LEF-1 gene revealed two putative STAT5a binding sites (−3913 bp to −3894 bp and −3728 bp to −3709 bp) and specificity of the STAT5a binding to the LEF-1 promoter in nuclear extracts of CD34+ cells was confirmed in a chromatin immunoprecipitation (ChIP) assay. Additionally, our data demonstrated that introduction of retroviral constructs expressing IRES-GFP and cDNA of constitutively activated STAT5a (mutant of STAT5a, (STAT5A[1*6]) into CD34+ cells of healthy individuals resulted in 20-fold downregulation of LEF-1 mRNA expression. To compare G-CSF- dependent phosphorylation of STAT5 in CD33+ bone marrow myeloid progenitors of CN patients and healthy individuals, we treated these cells with G-CSF in vitro and measured phosphorylation of STAT5 by intracellular staining of cells with phosphoSTAT5 (Y694) antibody. We found that G-CSF stimulation resulted in a significantly higher phosphorylation of STAT5 in myeloid progenitors of four CN patients, as compared to four healthy volunteers (mean fluorescence intensity (MFI) 436 ± 57 in CN patients vs 212 ± 30 in healthy controls). Interestingly, augmented phosphorylation of STAT5 has been observed in both groups of CN patients carrying either ELA2 or HAX1 mutations. These data confirm the hypothesis that downregulation of LEF-1 and LEF-1 target gene C/EBPα is at least in part a consequence of increased activation of STAT5a in both CD34+ cells of healthy individuals and in patients with CN.

Various neurotrophins (NT) are expressed in the hematopoietic microenvironment to deliver their signals through TRK receptor tyrosine kinases. Although previous reports suggested a transforming potential of activated TRK signaling in hematopoiesis, the target cells and underlying mechanisms are largely unknown. In this study, we investigated in vivo leukemogenesis of ΔTrkA, a mutant of TRKA isolated from a patient with acute myeloid leukemia (AML). Retroviral expression of ΔTrkA in myeloid 32D cells induced AML at ~4 weeks after transplantation into syngeneic C3H/Hej mice (n=11). C57BL/6J mice (n=15) transplanted with ΔTrkA-transduced primary bone marrow cells developed myeloid and/or lymphoid leukemia with infiltration in multiple organs including bone marrow, spleen, liver, lung, and central nervous system. While activation of a tyrosine kinase generally is not sufficient to cause AML, surprisingly, 7 out of 15 C57BL/6J mice developed a polyclonal AML with a latency of 78 days. This suggests that ΔTrkA can also transform long-term repopulating cells with a lymphoid potential. Retroviral insertion site analyses and spectral karyotyping revealed that induction of ALL by ΔTrkA required additional genetic lesions: Leukemic cells showed retroviral insertions in proto-oncogenes Bcl11a and Bcl11b, among others. Characterization of signal transduction demonstrated that PI3K and mTOR-raptor were crucial components of the transforming pathway induced by ΔTrkA. Phospholipase D was an important contributing factor, whereas STAT and MAP kinase pathways were not involved. In summary, our findings reveal potent transforming properties of altered NT receptor signaling in leukemia induction, which are in many respects distinct from other oncogenic tyrosine kinases. We would suggest to further evaluate the role of NT receptor signaling in leukemia pathogenesis, prognosis and treatment.