FLT3 Expression Research Articles

Flow Cytometric FLT3 Expression in Acute Leukaemias Is of Diagnostic Value but Does Not Correlate with ITD / D835Y Mutation Status.

High level expression of FLT3 in acute myeloid (AML) and lymphoid (ALL) leukaemias has been demonstrated by PCR techniques. This suggests that FLT3 and its ligand may play a significant role in the proliferation of leukaemic blast cells. Mutations of FLT3 are the most common genetic lesion in AML, occurring in 20–30% of cases, and confer an adverse prognosis. Flow cytometry was used as an alternative method to investigate FLT3 expression in acute leukaemia. Surface expression of FLT3 was measured in 111 bone marrow or peripheral blood samples of patients with AML or ALL using a phycoerythrin-labelled monoclonal IgG1 antibody (Coulter-Immunotech). Exons 14 – 15 and 20 were amplified from DNA extracted from mononuclear cells of 38 of the 111 samples. Internal tandem duplication (ITD) and mutation of the D835 position of the activation loop of FLT3 were identified by SSCP and direct sequencing. 82% (47/57) of AML and 81% (34/42) of B-lineage ALL samples showed ≥ 20% (range, 20 – 97%) positivity for FLT3 using flow cytometry. The Mean Fluorescence Intensity (MFI) was similar between the two groups and no significant difference was found between adult (82%) and paediatric (80%) samples. However, FLT3 expression in all 9 T-ALL samples (pro, intermediate and late subtypes) was negative with markedly reduced MFIs. Of the 25 AML cases tested for ITD/D835Y, 5 (20%) were positive (4 ITDs, 1 D835Y). Surface FLT3 expression in these 5 cases (% and MFI) was the same as in those with wild-type FLT3. No mutations of FLT3 were found in the ALL cases tested (9 B-ALL, 4 T-ALL). Of three biphenotypic leukaemias, one case with myeloid/B lineage positivity was FLT3+ by flow cytometry, while another with myeloid/T lineage markers, was FLT3−. A diagnostic conundrum was a patient with AML morphologically (cytogenetics failed): CD33+, MPO−, CD13− CD117−; T-lineage markers CD2+, CD5+, CD7+, as well as TdT+. The T-lineage specific marker cytoplasmic CD3 was negative. Only FLT3 positivity allowed a definitive diagnosis of AML to be made. In conclusion, surface FLT3 expression was present in > 80% of AML and B-lineage ALL, but in 0% (0/9) of T-ALL cases. Detection of this antigen should be included in the diagnostic immunophenotyping of acute leukaemias and is important where T-ALL needs to be excluded. Flow cytometric FLT3 expression cannot differentiate cases with or without ITD or point mutations of FLT3.

High FLT3 mRNA Expression in Acute Myeloid Leukemia May Be Functionally an Alternative to Mutational Activation of the Receptor.

Mutations of the FLT3 gene are detectable in approximately 30% of all adult AML. These mutations lead to an autoactivation of the receptor inducing increased proliferation of the leukemic clone. An alternative mechanism of FLT3 activation might be mRNA overexpression. In the presented study the FLT3 expression level in 208 adult AML patients and 8 healthy donors was assessed by real time PCR and correlated to several parameters. In all patients cytomorphology, cytogenetics, and FLT3 mutation status was assessed. Significant differences of FLT3-expression levels were found in certain AML subgroups. The highest expression levels were found in FAB subtypes M5 and the lowest in M3. In total, increasing levels were shown in the following order: M3 <M3v <M6 <M2 <M4eo <M4 <M0 <M1 <M5a <M5b. Independent analysis of FLT3 expression in different cytogenetic AML subgroups showed the lowest median in the t(15;17) group, followed by t(8;21), inv(16), normal, complex karyotype and the highest median in the t(11q23) group. No difference was observed between the group of secondary AML following MDS, therapy related AML and the de novo AML (p=0.868, p=0.562, and p=0.570, respectively). Compared to clinical parameters, FLT3 expression correlated with high percentage of bone marrow blasts (p=0.0005) and high leukocyte count (p<0.001). In contrast to previous studies no difference in FLT3 expression levels was detected between AML with (n=74) and without (n=130) any FLT3 mutation. Assessment of FLT3 RNA by microarray analysis and FLT3 receptor surface expression (CD135) detected by flow cytometry correlated significantly with FLT3-expression as assessed by real time PCR (p<0.001, each). To analyze whether high FLT3 expression is a prognostic parameter 118 AML cases with normal karyotype were devided into two groups. Group 1 (n=75) was defined to have less and group 2 (n=43) more than the median of the FLT3 expression level found in the total group. No impact on OS and EFS could be shown (608 vs. 311 days, p=0.1283 and 398 vs. 208 day, p=0.3056). In conclusion, these data support the hypothesis that FLT3 activation through mRNA overexpression is an alternative mechanism to FLT3 mutations. Especially as it was found extremely high in 11q23 AML, that rarely reveal FLT3 mutations.

Homeodomain Expression in AML and T-ALL Cell Lines.

HOX genes are frequent targets of chromosomal translocations and retroviral integrations in human and murine acute leukemia, often involving genes at the 5′-end of the HOX clusters. We previously reported that HOX expression patterns in AML were related to prognostic cytogenetic subsets. We also identified a distinct subset of patients with intermediate cytogenetics based on high levels of HOX and FLT3 expression, frequent FLT3 mutations and a low incidence of C/EBPa mutations. Certain cases of T-ALL also have rearrangements of homeodomain genes and some T-ALLs express limited myeloid markers. To further explore the spectrum of homeodomain gene expression, we developed qRT-PCR assays for nearly all clustered HOXA-D genes, selected homeodomain genes on chromosomes often altered in AML, and selected polycomb (Pc) genes, FLT3 and MLL. Altogether, 52 genes were analyzed in 32 AML and T-ALL cell lines. FLT3 expression was confined to a subset of AMLs. HOX11, HOX11L2 and NKX2.5 were expressed only in cases involving rearrangements of these genes. The Pc and MLL genes were uniformly expressed. Among HOX clusters, the frequency of gene expression was HOXA>B>C>D. Genes more highly expressed in the HOXC and D clusters were those at the 5′-ends (e.g., D13, C10). Marked or selective overexpression of individual genes suggests their possible involvement in the disease process, immortalization or differentiation. Examples include EN1 (SUPT1), D13 (MEGA1, B9 (PEER). A hierarchical cluster analysis based on homeodomain genes successfully identified subsets of related cell lines. Thus, the analysis of quantitative HOX expression may provide an important new tool to better understand the biology of acute leukemia.

FLT3 Expression Is Increased by MEIS1 and Collaborates with NUP98-HOX Fusion Genes in the Induction of Acute Myeloid Leukemia.

The TALE family member and HOX cofactor MEIS1 is important in leukemic transformation. MEIS1 has, although non-leukemogenic on its own, been shown to strongly collaborate with several HOX genes and NUP98-HOX fusions to induce acute myeloid leukemia (AML). We have recently described a novel in vitro culture system of cell lines established from murine primary bone marrow cells transduced with the AML-associated fusion gene NUP98-HOXD13 or an engineered NUP98-HOXA10 fusion. These pre-leukemic NUP98-HOX cell lines are transplantable and can efficiently be converted into AML-inducing cells upon MEIS1 transduction. Conveniently, the MEIS1 transduced cells can be purified and preserve their leukemogenic potential even after extensive in vitro expansion. Thus, the availability of the NUP98-HOX cell lines system provided the opportunity to investigate and characterize the mechanism of MEIS1-mediated AML-conversion. Potentially interesting target or candidate genes were screened for expression changes between the parental pre-leukemic lines and AML-inducing MEIS1 transduced cell lines with quantitative RT-PCR and Western blotting. Aberrant expression or mutations of the receptor tyrosine kinase FLT3 gene is a common finding in human AML. Interestingly, Flt3 was found induced 5 to 10 fold in MEIS1 transduced cell lines compared to the parental cell lines. The observed increase in Flt3 expression provided the MEIS1 transduced cells with Flt3 ligand driven growth. This was not seen in the parental cell lines, which could not proliferate with Flt3 ligand as single cytokine or with a MEIS1-homeodomain mutant expressing cell line. Importantly, the Flt3 inhibitor AG1295 could block the proliferative effect of the Flt3 ligand in the MEIS1 transduced cell lines. To test whether Flt3 could substitute for MEIS1-mediated induction of AML in NUP98-HOX pre-leukemic cells, a NUP98-HOXA10 cell line was transduced with an MSCV-Flt3-IRES-YFP construct. The resulting Flt3-transduced cells were shown to express Flt3 at levels similar to that of MEIS1 transduced cells without any significant increase in endogenous Meis1 expression. Transplantation of these cells into mice led to lethal and transplantable AML with a median disease onset of 116 days (n=8) compared to 59 days for MEIS1 (n=4), whereas control transplants remained healthy (n=2). In conclusion, this study demonstrates that MEIS1 can induce Flt3 expression and that Flt3 can collaborate with NUP98-HOX fusion genes in the induction of acute myeloid leukemia. Furthermore, theses results suggest a model in which the leukemogenic synergism of MEIS1 on HOX-mediated leukemia might in part be mediated through FLT3-dependent pathways.

CEP701 and PKC412 Predictably and Reliably Inhibit FLT3 Phosphorylation in Primary AML Blasts but Their Induction of a Cytotoxic Response Appears to Be Much More Variable.

Activating mutations of the receptor tyrosine kinase FLT3 are present in approximately one-third of AML cases and are associated with an adverse prognosis. FLT3 is expressed in over 90% of cases of AML and many non-mutants show evidence of FLT3 activation, which may play a significant signalling role in leukaemogenesis, making FLT3 an attractive therapeutic target. CEP701 (Cephalon) and PKC412 (Novartis) are orally-bioavailable indolocarbazole derivatives that potently inhibit FLT3 phosphorylation. We studied the relationship between in vitro inhibition of FLT3 phosphorylation and induction of cytotoxicity in primary AML blasts from 12 patients. 7 of the cases were FLT3 mutants (6 ITDs and 1 D835 point mutant), the amount of mutant RNA varying between 7% and 84% of total FLT3 RNA expressed. The blasts were exposed for 1 hour to a range of concentrations of CEP701 and PKC412, lysed and immunoprecipitated with an anti-FLT3 antibody. After sequential immunoblotting with anti-phosphotyrosine and anti-FLT3 antibodies, inhibition of FLT3 phosphorylation was measured by densitometry. Both drugs inhibited FLT3 phosphorylation in all samples with lower concentrations required in FLT3 mutants. CEP701 inhibited FLT3 phosphorylation with median IC50s of 3.7nM and 11.9nM in mutant and wild type (WT) cases respectively (p=0.0006). IC50s for PKC412 were 7.7nM and 59.8nM in mutant and WT cases (p=0.0268). Induction of cytotoxicity was assessed by MTS assay following 72-hour exposure of blasts to a range of concentrations of CEP701 and PKC412. Cytotoxic responses to both drugs were greater in FLT3 mutants than WT cases at each dose studied and in terms of IC50 dose (median IC50s in mutant and WT cases: 95nM and 231nM with CEP701, 1.24 μM and 1.61μM with PKC412) although these differences did not reach statistical significance. Annexin V binding apoptosis assay produced similar dose response curves. Both agents showed greater inter-case variability in cytotoxic response than in sensitivity to inhibition of FLT3 phosphorylation. A lack of cytotoxic response to FLT3 inhibition with CEP701 was seen in the ITD mutant with the lowest ratio of mutant to WT FLT3 RNA (0.08) and several WT samples displayed resistance to in vitro induction of cytotoxicity despite almost complete inhibition of FLT3. Induction of cytotoxicity with PKC412 in both mutant and WT cases generally required doses well in excess of those required to fully inhibit FLT3 phosphorylation. Cases were further stratified by flow cytometric measurement of surface FLT3 expression, and by immunoblotting to measure STAT5 dephosphorylation in response to both drugs. No significant difference in overall FLT3 expression was seen between mutant and WT cases. Interestingly the highest FLT3 expression level was seen in a wild type case that was highly sensitive to CEP701. Inhibition of STAT5 phosphorylation appeared closely linked to FLT3 inhibition, although in some cases a good cytotoxic response was achieved despite failure to inhibit STAT5, suggesting involvment of other signalling pathways. In summary, although both CEP701 and PKC412 predictably and reliably inhibit FLT3 phosphorylation in primary AML blasts, their induction of cytotoxicity appears to be much more variable. A number of factors may influence this including variations in level of dependency on FLT3 signalling for blast survival, mutant to WT allele ratio and overall FLT3 expression level. Effects on targets other than FLT3 also need to be considered.

FLT3-Targeted Therapy Selectively Kills MLL-Rearranged Infant and Childhood ALL Blasts In Vitro and in Vivo.

The FLT3 receptor tyrosine kinase has been convincingly implicated in the pathogenesis of human leukemia. In childhood acute lymphoblastic leukemia (ALL), FLT3 is expressed in 94% of B-lineage disease and 32% of T-lineage disease. Co-expression of FLT3 ligand (FL) may frequently occur in these cases, as we have observed constitutive activation of the wild type receptor in cell lines and primary samples. A smaller percentage of ALL cases harbor activating mutations of FLT3. Cases of ALL with MLL gene rearrangements are common among infants less than 1 year of age, and have a distinctly poor prognosis, with overall survival rates of approximately 20%. Gene expression studies have revealed that these cases express the highest levels of FLT3, and activating mutations of FLT3 also occur in 18% of MLL-rearranged ALL. We hypothesized that inhibition of FLT3 signaling would be selectively cytotoxic to ALL blasts with high levels of FLT3 expression, particularly if MLL rearrangements were present. We determined the anti-leukemic activity of CEP-701, a potent (IC50=3 nM) and selective small molecule FLT3 inhibitor, in 36 bone marrow samples obtained at diagnosis from infants and children with various subtypes of ALL. FLT3 expression level was determined by RNA microarray analysis or by FLT3 immunoprecipitation and immunoblotting, and FLT3 mutation status was determined by PCR analysis. MTT cytotoxicity assays and annexin V binding apoptosis assays were performed on all samples. CEP-701 induced more pronounced cytotoxicity at all six dose levels (5–100 nM) in samples that expressed high levels of FLT3 (N=23) compared to samples with low levels of expression (N=13). At 50 nM, for example, the MTT mean optical density was 45% that of untreated control in the FLT3 high group vs. 84% in the FLT3 low group (P<0.0001). Cytotoxicity was particularly pronounced in samples with MLL gene rearrangements (N=11, P=0.0006). Seven samples (five with MLL rearrangements) that were sensitive to CEP-701, and six samples that were resistant (none with MLL rearrangements), were examined by FLT3 immunoprecipitation and immunoblotting. All seven sensitive samples demonstrated constitutively phosphorylated FLT3 that was potently inhibited by CEP-701. Conversely, 0 of 6 resistant samples expressed constitutively phosphorylated FLT3. To assess the in vivo activity of FLT3-targeted therapy for MLL-rearranged ALL, six week old NOD/SCID mice were injected with MLL-rearranged primary infant leukemia blasts. Mice were treated with vehicle control, CEP-701 or EB10 (a fully humanized anti-FLT3 monoclonal antibody) for 14 weeks. Bone marrow was then harvested and assessed for engraftment of human cells. Inhibition of engraftment was achieved in the CEP-701-treated mice (mean engraftment 45%, N=5) and EB10-treated mice (mean engraftment 28%, N=4) compared to vehicle controls (mean engraftment 96%, N=9). Finally, in cytotoxicity and apoptosis assays utilizing MLL-rearranged cell lines and primary blast samples, schedule-dependent synergy between CEP-701 and several chemotherapy agents active in ALL was demonstrable, including doxorubicin, dexamethasone, l-asparaginase, etoposide, vincristine and cytarabine. We conclude that FLT3-targeted therapy is a promising novel approach to the treatment of MLL-rearranged ALL, a disease with dismal prognosis with current treatment approaches. Clinical testing is warranted.

Genetic Evidence of a Novel Blood Differentiation Pathway from Lympho-Myeloid Hematopoietic Stem/Progenitor Cells, Independent of Common Myeloid Progenitors.

We have recently identified three novel subsets of multipotent hematopoietic stem/progenitor cells (HSCs) in the Lin−Sca-1+c-Kithi (LSK) compartment of adult murine bone marrow based on differential expression of CD34 and the cytokine tyrosine kinase receptor Flt3. Long-term HSCs (LT-HSCs) lack CD34 and Flt3 expression (LSKCD34-flt3-), whereas short-term HSCs (ST-HSCs) are LSKCD34+flt3−. A third LSK population is characterized by co-expression of CD34 and Flt3 (LSKCD34+flt3+) and possess a combined myeloid (granulocyte and monocyte) and lymphoid (B and T cell) differentiation potential, but surprisingly lack megakaryocytic (Mk) and erythroid (E) potential in vitro and in vivo. These findings implicate an alternative road map for blood lineage development distinct from the classical model in which the first lineage commitment step of HSCs is thought to result in a strict separation into myelopoiesis and lymphopoiesis. In the current study we sought genetic evidence in further support of this new model through genetic profiling of these three HSC subpopulations, using affymetrix chips, quantitative (Q)-PCR and single cell PCR. In contrast to the pluripotent LSKCD34−Flt3− LT-HSCs and LSKCD34+Flt3− ST-HSCs, LSKCD34+Flt3+ cells downregulated or turned of genes critically involved in promoting Mk and E lineage development, such as the Epo and Tpo receptors as well as the transcription factor Gata-1. In contrast, the gene for Il-7rα, critically involved in early B and T cell development was upregulated in LSKFlt3+ cells, but absent in LT-HSCs and ST-HSCs. However, in agreement with their sustained ability to produce granulocytes and monocytes, G-CSFR and Pu.1 expression was sustained from LT-HSC through the LSKCD34+flt3+ stage Particularly noteworthy, single cell PCR demonstrated that a fraction of single LSKCD34+Flt3+ cells upregulating Il-7rα gene expression, sustained and co-expressed G-CSFR expression. Thus, genetic profiling at the single cell level provide further and compelling evidence for a novel road map for blood lineage development, independent of the common myeloid progenitor (CMP). Our biological and gene expression data rather supports the existence of a pathway in which HSCs lose their lineage potentials one by one, starting with the Mk and E lineages.

Flt3 Receptor Expression Rescues Dendritic Cell Developmental Potential in Megakaryocyte/Erythrocyte Progenitor Cells.

Flt3/Flt3-ligand has a non-redundant role in dendritic cell (DC) and interferon producing cell (IPC) development from hematopoietic progenitor cells. Flt3-ligand−/− mice display a severe reduction of DCs and IPCs, and both, Flt3-ligand injection and conditional over- expression of Flt3-ligand in mice leads to an increased pool size of both cellular subsets. Furthermore, DC and IPC developmental precursor activity segregates with Flt3+ progenitor populations in mouse bone marrow. We have reported that common myeloid progenitors (CMPs) and their progeny granulocyte/macrophage progenitors (GMPs) as well as common lymphoid progenitors (CLPs) and pro-T cells can give rise to DCs in vitro and in vivo. DC developmental potential was lost, however, once definitive megakaryocyte/erythrocyte or B cell commitment occurs, both steps that are accompanied by loss flt3 receptor expression. To test if DC/IPC developmental potential can be regained, we evaluated the effect of ecotropic expression of human Flt3 in lin−c-kit+Flt3−Thy1.1−IL-7Ra− cells (flt3− progenitors) and in megakaryocyte/erythrocyte progenitors (MEPs).hFlt3 was transduced into purified flt3− progenitors, MEPs, and GMPs using a retrovirus vector carrying both hFlt3 and GFP. The transduction efficiencies were 20~25%. hFlt3-transduced flt3− progenitors gave rise to functional DCs and IPCs, but not control-GFP-transduced flt3− progenitors in vitro. Similarly, hFlt3-transduced MEPs differentiated into functional DCs in Flt3L, SCF, GM-CSF, IL-4, and TNF-a supplemented cultures but not into TER119+ erythroid cells. DC developmental potential of hFlt3-transduced MEPs was comparable to GFP-transduced GMPs. Over expression of hFlt3 in GMPs resulted in augmentation of its DC developmental potential. Interestingly, both hFlt3-tarnsduced MEPs and GMPs preferentially differentiated into IPCs in Flt3L supplemented medium. RT-PCR analysis revealed that enforced Flt3 signaling in MEPs lead to up-regulation of GM- and/or DC-development affiliated genes such as G-CSF receptor, M-CSF receptor, and GM-CSF receptor, PU.1, C/EBPa and STAT3. Furthermore, hFlt3-transduced MEPs gave rise to myeloid colonies (CFU-GM/G/M) but not CFU-Mix colonies in GM-CSF, IL-3, SCF, Flt3L, Tpo, and Epo supplemented methylcellulose medium. These results show that artificial Flt3 signaling in otherwise Flt3− progenitors is sufficient to reestablish DC/IPC differentiation and GM-differentiation programs.

Intrathymic and Extrathymic Notch-Dependent T Lineage Checkpoints during Normal Development and after Bone Marrow Transplantation.

The essential role of Notch for T lineage commitment is well established, but it remains unclear where and on which progenitor subset(s) Notch signals act. Addressing these questions is critical to understand the regulation of lymphopoiesis in normal and lymphopenic settings, such as after bone marrow transplantation (BMT).In normal mice, a small number of progenitors settle in the thymus from the blood and expand to generate a pool of early T lineage progenitors (ETPs). At the population level, ETPs can give rise to T cells, NK cells, B cells, DCs and myeloid cells. We found that ETPs could be subdivided based on expression of the cytokine receptor Flt3, which is expressed on about 10% of ETPs and is inversely correlated with expression of Notch target genes. B lineage potential was restricted to ETPFlt3 positive cells. The Notch targets Hes-1, Hes-5, and Deltex1 were present at low levels in ETPFlt3 positive and high levels in ETPFlt3 negative cells. Induction of Notch signaling resulted in the rapid downregulation of surface Flt3 expression. In contrast, culture of ETPFlt3 negative cells in the absence of Notch ligands resulted in upregulation of Flt3. Although both ETP subsets were efficient T lineage progenitors, ETPFlt3 negative cells had a more rapid differentiation kinetics resembling DN2 thymocytes, consistent with a more advanced state of T lineage commitment relative to the Flt3 positive subset. In mice reconstituted with HSCs transduced with the pan-Notch inhibitor DNMAML1 (Maillard et al., Blood 2004), no ETPFlt3 negative and very few ETPFlt3 positive cells were observed, indicating that the generation of ETPs is Notch-dependent. These observations position the physiological Notch checkpoint either very early after thymic seeding and/or in a prethymic location.To further investigate if Notch-dependent prethymic T lineage commitment occurs in adult mice, we studied lymphoid reconstitution early after BMT. ETPs were absent in the thymus of recipients until ≥ 6 weeks post-BMT, despite the presence of donor-derived pre-T cells and double positive thymocytes as early as 2–3 weeks post-BMT. Instead, cells with pre-T cell characteristics were present in the spleen of BMT recipients in the first month post-BMT. These cells were absent when DNMAML1-transduced bone marrow was used as the source of HSCs, indicating that generation of extrathymic pre-T cells is Notch-dependent. Thus, extrathymic sites may be important for efficient lymphoid reconstitution after BMT. Altogether, our results indicate that both intrathymic and extrathymic Notch-dependent checkpoints regulate T lineage commitment during normal development and in the post-BMT setting.

A Novel Road Map for Blood Lineage Commitment and Development: Identification of a Lympho-Myeloid Stem/Progenitor Cell Lacking Erythro-Megakaryocytic Potential.

The recent identification of common myeloid and lymphoid progenitors (CMPs and CLPs, respectively) lends support to the classical and currently prevailing model for hematopoietic commitment and blood lineage development, suggesting that the first and decisive lineage commitment step of adult hematopoietic stem cells (HSCs) results in an immediate and complete separation of myelopoiesis and lymphopoiesis. Virtually all of multipotent (lympho-myeloid) stem and progenitor cells in adult mouse bone marrow (BM) reside in the small Lin−Sca1+kithi (LSK) compartment. We now present data demonstrating that the LSK compartment in adult BM, can be separated into three phenotypically and functionally distinct HSC subsets, based on differential expression of CD34 and flt3. Long-term HSCs (with extensive self-renewing potential) reside in the LSKCD34−flt3− fraction. The LSKCD34+ short-term HSC compartment consists of LSKCD34+flt3− cells fulfilling all criteria of short-term HSCs, being highly enriched in CFU-S activity, and capable of rapidly reconstituting myelo-erythropoiesis and thereby rescuing myeloablated mice. The short-term LSKCD34+flt3− HSCs give upon transplantation rise to LSKCD34+flt3+ cells, that although sustaining a combined lymphoid (B and T cell) and myeloid (granulocyte and monocyte) developmental potential at the single cell level, loose their ability to adapt megakaryocytic and erythroid fates in vitro as well as in vivo. These evidence for the existence of LSKCD34+flt3+ cells with granulocyte, monocyte, B and T cell, but not megakaryocyte and erythroid development potentials, are not compatible with the first lineage commitment step of HSCs resulting in strict separation of common lymphoid and common myeloid differentiation pathways. Based on the present findings we rather propose an alternative road map for blood lineage development in which LSKCD34+flt3− short-term HSCs genereate megakaryocyte-erythroid progenitors and LSKCD34+flt3+ cells upon asymmetric cell divisions.

Molecular Epidemiological Study in Pediatric Acute Myeloid Leukemia.

It is hypothesized that AML arises from two cooperative types of mutations: type I mutations mainly induce proliferation and type II mutations involved in the maturation arrest. AML is a rare disease in children and few molecular data are available on pediatric AML. We therefore studied N-RAS, K-RAS, FLT3-ITD, FLT3 , C-KIT mutations (type I), and CEBPA mutations (type II) as well as FLT3, EVI-1 and WT1 gene expression in 77 de novo AML.Patients and methods: All the patients (aged 1 month-17 years, median age: 6.9 years, male/female ratio 1.26) were treated for de novo AML between 1995 and 2003 in two French institutions and prospectively enrolled in LAM91, LAM01 and APLs French collaborative protocols. According to the FAB classification the repartition was: M0:6.5%, M1: 5.2%, M2: 22%, M3: 13%, M4 :14.3%, M5 :30%, M7: 6.5% and unclassified :2.5%. Cytogenetics features according to the MRC classification were favorable, intermediate or poor in 25% (t(8;21) n=5; t(15,17) n=8, inv(16) n=5), 65% (normal n=20 and 11q23 abnormalities n=15) and 10% (−7, n=4) respectively. With a median follow-up of 26 months (range 2–98 months), Complete Remission was obtained in 92% (71/77) of patients, OS was 71% and EFS 61%. CEBPA, N-RAS, K-RAS, C-KIT and FLT3 mutations detection was performed by direct sequencing. FLT3, EVI-1 and WT1 transcripts were quantified by RQ-PCR.Results: (1) Frequency of N-RAS and K-RAS mutations were 11% (8/75) and 16% (12/75) respectively. RAS-mutated patients belonged to favorable (30%), intermediate (60%) and poor (10%) cytogenetic subgroups. In univariate analysis only N-RAS mutations is associated with adverse outcome (OS 37% vs 79%, p<0.05). (2) CEBPA mutations were found in 8% (6/75), mostly belonged to the intermediate cytogenetic risk subgroup (66%). (3) C-KIT mutations were observed in 4% (4/75) always associated with fusion CBFb/MYH11 transcript and excellent outcome. (4) FLT3-ITD and FLT3 Asp835 mutations were obtained in 12% (9/74) and 4% (3/74) of patients respectively. Cytogenetic subgroups were favorable (33%) and intermediate (67%). (5) At diagnosis FLT3 overexpression was detected in 34% (24/70) and 11q23 abnormalities were associated in 7/24 patients. (6) EVI-1 overexpression was found in 21% (16/76), belonged to intermediate (85%) and poor (15%) cytogenetic subgroups with a significant frequency in monosomy 7 (p<0.025). The EVI-1 expression was specifically expressed (p<0.001) in M5 and M7-FAB subtypes. (7) WT1 overexpression was detected in 81% (62/76).Conclusion: In total, 48% of de novo AML in children had a mutation in N-RAS, K-RAS, FLT3-ITD, FLT3 Asp835, C-KIT or CEBPA with a high frequency of RAS mutations (27%) compared to adult AML and a significantly bad survival. Additional gene expression quantification of EVI-1, FLT3 and WT1 allows MDR detection in 95% of patients.

Gene Expression Profiles, Differentiation and Lineage Commitment of Human Myeloid Progenitors.

With the objective to further elucidate the mechanism behind commitment to erythroid and neutrophil lineages, we isolated by cell sorting common myeloid progenitors (CMPs), granulocyte/monocyte progenitors (GMPs) and megakaryocyte/erythrocyte progenitors (MEPs), based on the surface expression of CD123 (IL3R-α) and CD45RA on human CD34+ bone marrow cells (first described by Manz et al. PNAS , 2002; 99:11872). Methylcellulose cultures supporting the growth of myeloid and erythroid progenitors, and real-time RT-PCR mapping the gene expression of Flt3, c-kit, TpoR, GATA-2, GATA-1, SCL, NF-E2, EpoR, ABO, β-globin, GPA, PU.1, C/EBPα, C/EBPε, G-CSFR, proteinase 3 (PR3) and lactoferrin, were used to validate and characterize the progenitors and their progeny. Cell sorted progenitors were labeled with CFDA, SE to track cell division and cultured in suspension to induce neutrophil or erythroid differentiation (SCF+G-CSF for neutrophil and Epo+IL–3+GM–CSF for erythroid culture). After 3–5 days, cells that had gone through 1–8 divisions were sorted and changes in clonogenicity and gene expression were studied. The CMP-population retained some clonogenicity after as many divisions as were tested (at the most six divisions in erythroid and eight in neutrophil culture) and the CMPs differentiated along the lineage defined by the culture system, as evidenced both by the methylcellulose cultures and an increasing expression of GATA-1 and EpoR in erythroid and PU.1, G-CSFR and PR3 in neutrophil cultures, respectively. On the other hand, the GMP-population displayed granulocyte/monocyte (G/M)-differentiation irrespective of the culture system used, although it divided fewer times and lost its clonogenic capacity faster in erythroid culture. Little or no clonogenicity remained after 4–5 divisions in erythroid culture, while some colony-forming capacity remained even after seven divisions in neutrophil culture (maximum number tested). The increased expression of the granulopoiesis-associated genes was also less pronounced in the erythroid culture. The MEP-population dominated by erythroid differentiation capacity retained colony-forming capacity for at least 6–7 divisions in both erythroid and neutrophil cultures, although with a higher overall clonogenicity in erythroid culture. Unexpectedly, however, MEPs were restricted to G/M-differentiation when cultured in neutrophil culture. In cells from erythroid culture the expression of GATA-1, EpoR and β-globin increased, while a corresponding pattern was seen for PU.1, G-CSFR and PR3 in neutrophil culture. Overall, our data support the progenitor classification, based on the surface expression of CD123 and CD45RA, with regard to CMP and GMP populations but question it with regard to the MEP-population. The change in differentiation course for the MEPs in neutrophil culture could be a result of an initially present G/M-potential or a less strict commitment susceptible to cytokine-induced redifferentiation.

Over-Expression of Flt3 Induces the Expression of NFkB Target Genes, cIAP-1 and IL-6.

Nuclear factor kappa B (NFkB) regulates transcription of many genes involved in the immune response, including cytokines (e.g. IL-6) and growth factors. NFkB suppresses apoptosis as well, through various mechanisms including activation of cellular inhibitor of apoptosis-1 (cIAP-1). NFkB can be activated by the activation of Ras/phosphatidylinositol 3′-kinase (PI3K)/protein kinase B pathway in acute myeloblastic leukemia (AML). It was reported that in large proportion of primary AML cells, NFkB is constitutively activated, and the target genes are upregulated. Flt3 is a member of receptor tyrosine kinases. Flt3 receptor phosphorylation/activation results in the activation of downstream kinase pathways, like Ras, PI3K, leading to abnormal cell growth and aberrant gene regulation. Increased levels of Flt3 transcript are observed in a large number of AML specimens, and the over-expression of Flt3 contributes to the phosphorylation of Flt3 and activation of this pathway. In this study, we addressed the role of Flt3 over-expression in the activation of NFkB, which is a target molecule of various kinase pathways. We first tested the effect of Flt3 expression to NFkB responsive reporter by transient transfection and revealed the significant induction of the reporter. Next, we generated Flt3 transgenic BaF3 (BaF3-Flt3) cells that stably over-expressing Flt3 to confirm this effect. The introduction of NFkB-responsive reporter, as well as serum response element (SRE)-luciferase fused reporter into these cells resulted in the activation of the reporters. These results suggest that Flt3 over-expression mediated signal transduction activates NFkB pathways and affects growth factor pathways as well. Real time quantitative PCR demonstrated that in BaF3-Flt3 cells, mRNA expression of IL-6 and cIAP-1, both known target genes of NFkB, were increased. AG1296 or PDTC, potent inhibitors of Flt3 and NFkB respectively, completely abrogated the induction of these genes, suggesting the importance of Flt3-NFkB pathway to these effects. Moreover, we measured the expression level of Flt3 and NFkB target genes in 24 primary AML samples. As a result, there was a tendency of moderate to weak positive correlation between Flt3 and NFkB target genes. (Flt3 vs IL-6 [Spearman's rank correlation coefficient: r= 0.35], Flt3 vs cIAP-1 [r=0.16]). These facts may also indicate the positive influence of Flt3 over-expression to NFkB target genes. Overall, these suggest a role of Flt3 over-expression in the activation of NFkB pathway in AML.

A Phase II Trial of the FLT3 Inhibitor CEP701 as First Line Treatment for Older Patients with Acute Myeloid Leukaemia Not Considered Fit for Intensive Chemotherapy.

Activating mutations of the receptor tyrosine kinase FLT3 are present in approximately one-third of cases of AML and are associated with an adverse prognosis. Activated FLT3 is also frequently expressed in wild type (WT) patients. The indolocarbazole derivative CEP701 (Cephalon Inc) has been shown to potently inhibit phosphorylation of both mutant and WT FLT3 in vitro causing cytotoxicity in primary AML blasts and prolonging survival in mouse AML models. We present results from the first phase II trial of CEP701 given as first line treatment in AML in patients aged over 70 years (or aged 60–69 with a poor performance score or cardiac toxicity) and not considered fit for intensive chemotherapy, irrespective of FLT3 mutation status. Patients with presenting white blood cell (WBC) count of over 30x109/l received hydroxycarbamide for up to 21 days to reduce the WBC to less than 10x109/l before starting CEP701. CEP701 was administered orally for 56 days, initially at a dose of 60mg bd with dose increase to 80mg bd from day 28. Response was assessed by BM and peripheral blood examination on days 14, 28 and 56. 24 patients entered the study: 12 male and 12 female with a median age of 73 (range 67–84). 5 are too early in treatment to assess response. 1 patient died from intracerebral haemorrhage and 2 were withdrawn due to toxicity (emesis) before response could be assessed. Of the sixteen fully evaluable patients 4 were FLT3 mutants (2 ITD, 2 point mutations) and the remaining 12 had WT FLT3. Bone marrow response (BMR) was defined as a greater than 50% reduction in bone marrow blasts without haematological recovery. Haematological response (HR) was defined as disappearance of blasts from the peripheral blood not attributable to hydroxycarbamide. 3 of the 4 FLT3 mutant cases responded to CEP701. A 72 year-old male point mutant (deletion 836) achieved a late HR with normalisation of counts lasting 6 months. A 74 year-old male point mutant (D835) continues CEP701 after achieving a HR on 60mg bd associated with transfusion independence and normalisation of counts. A 74 year-old female ITD mutant achieved a HR on 60mg bd followed by rapid disease progression after the drug was withdrawn due to profound fatigue. 3 of the 12 wild type cases responded including 1 BMR and 1 HR (both transient) on 80mg bd. A third wild type case had a transient BMR on 60mg bd and a further transient BMR following dose increase to 80mg bd. No patient achieved a conventional complete remission. 11 of the first 24 patients (46%) experienced grade 1/2 gastrointestinal toxicity. Cases have been further categorised by Affymetrix gene expression analysis to identify gene signatures associated with drug responsiveness. 2 clinical CEP701 responders among the first 10 cases analysed fit a previously-defined gene expression signature associated with high in vitro CEP701 responsiveness (Knapper et al. Blood 102:11). Flow cytometric surface FLT3 expression assay, MTS cytotoxicity assay and ex vivo surrogate bioassay of plasma FLT3 inhibitory activity show limited correlation with clinical response. The preliminary trial results suggest that CEP701 has haematological activity in both mutant and WT patients. In view of the modest response of WT cases it is proposed to amend the trial to assess sequential low dose cytarabine and CEP701 as a combined approach.

Effects of SU5416, a small molecule tyrosine kinase receptor inhibitor, on FLT3 expression and phosphorylation in patients with refractory acute myeloid leukemia

Acute myeloid leukemia (AML) is associated with dysregulated hematopoietic cell proliferation and increased bone marrow angiogenesis, each regulated by signaling through receptor tyrosine kinases (RTKs). SU5416 is a small molecule inhibitor of VEGF receptors, c-kit and FLT3 and therefore provides a novel opportunity to target both angiogenesis and proliferation in AML. SU5416 was assessed in a phase II hematological malignancy trial in the US, where partial responses were observed in two of 33 patients. Since AML provides a unique platform to evaluate mechanism of action of small molecule inhibitors, investigation of the effect of SU5416 on FLT3 expression and phosphorylation in blood and bone marrow was an additional focus of this trial. Phosphorylated FLT3 was detected by immunoprecipitation/Western analysis in peripheral blood samples from 17 of 22 patients, and seven exhibited strong inhibition of phosphorylation immediately following a 1 h SU5416 infusion, demonstrating that SU5416 can modulate RTK phosphorylation in humans. Although no clear correlation with clinical response was observed, analysis of patient plasma drug levels suggested that a threshold SU5416 concentration of 15 μM was associated with FLT3 inhibition. This observation was supported by data from an ex vivo model where AML cells were spiked into human blood, established to mimic the clinical setting and enable more rigorous analysis of effect of SU5416. In addition, FLT3 protein levels were downregulated in patient bone marrow samples, analyzed by an RIA assay. To identify putative predictors of response, patient plasma was analyzed for levels of secreted ligands of SU5416 targets; SCF and FLT3 ligand. Baseline levels of SCF in patients with stable or progressive disease were significantly higher than those in normal donors, whereas FLT3 ligand levels in patients who exhibited progressive disease were significantly lower than those in normal donors. The translational and clinical analyses described in this report provide some insights into the mechanism and duration of action of SU5416.

CD34+CD38- hematopoietic precursors derived from human embryonic stem cells exhibit an embryonic gene expression pattern

AbstractGene expression patterns of CD34+CD38- cells derived from human embryonic stem cells (ESCs) were compared with those of cells isolated from adult human bone marrow (BM) using microarrays; 1692 and 1494 genes were expressed at levels at least 3-fold above background in cells from BM and ESCs, respectively. Of these, 494 showed similar levels of expression in cells from both sources, 791 genes were overexpressed in cells from BM (BM versus ESCs, at least 2-fold), and 803 genes were preferentially expressed in cells from ESCs (ESCs versus BM, at least 2-fold). The message of the flt-3 gene was markedly decreased in cells from ESCs, whereas there was substantial flt-3 expression in cells from BM. High levels of embryonic ϵ-globin expression were observed—but no adult β-globin message—in CD34+CD38- cells from ESCs, whereas high levels of β-globin expression—but no embryonic ϵ-globin message—could be detected in cells from BM. Furthermore, high levels of major histocompatibility complex (MHC) gene expression were demonstrated in cells from BM but very low levels of MHC message in corresponding cells from ESCs. These observations demonstrate that CD34+CD38- cells derived from ESCs correspond consistently to an early developmental stage at which the yolk sac and fetal liver are the primary sites of hematopoiesis.

FLT3 mutations in childhood acute lymphoblastic leukemia

AbstractActivating mutations of the FLT3 receptor tyrosine kinase are common in acute myelogenous leukemia (AML) but are rare in adult acute lymphoblastic leukemia (ALL). We have recently shown that FLT3 is highly expressed and often mutated in ALLs with rearrangement of the mixed lineage leukemia (MLL) gene on chromosome 11q23. Because hyperdiploid ALL samples also show high-level expression of FLT3, we searched for the presence of FLT3 mutations in leukemic blasts from 71 patients with ALL. The data show that approximately 25% (6 of 25) of hyperdiploid ALL samples possess FLT3 mutations, whereas only 1 of 29 TEL/AML1-rearranged samples harbored mutations (P = .04, Fisher exact test). Three mutations are novel in-frame deletions within a 7-amino acid region of the receptor juxtamembrane domain. Finally, 3 samples from patients whose disease would relapse harbored FLT3 mutations. These data suggest that patients with hyperdiploid or relapsed ALL might be considered candidates for therapy with newly described small-molecule FLT3 inhibitors. (Blood. 2004;103: 3544-3546)

Hox expression in AML identifies a distinct subset of patients with intermediate cytogenetics.

We previously reported that favorable and poor prognostic chromosomal rearrangements in acute myeloid leukemia (AML) were associated with distinct levels of HOX expression. We have now analyzed HOX expression in 50 independent adult AML patients (median age=62 years), together with FLT3 and FLT3-ligand mRNA levels, and FLT3 mutation determination. By cluster analysis, we could divide AMLs into cases with low, intermediate and high HOX expression. Cases with high expression were uniquely restricted to a subset of AMLs with intermediate cytogenetics (P=0.0174). This subset has significantly higher levels of FLT3 expression and appears to have an increase of FLT3 mutations (44%), while CEBPalpha mutations were infrequent (6%). FLT3 mRNA levels were correlated with the expression of multiple HOX genes, whereas FLT3 mutations were correlated with HOXB3. In some cases, FLT3 was expressed at levels equivalent to GAPDH in the absence of genomic amplification. We propose that high HOX expression may be characteristically associated with a distinct biologic subset of AML. The apparent global upregulation of HOX expression could be due to growth-factor signaling or, alternatively, these patterns may reflect a particular stage of differentiation of the leukemic cells.

Effects of SU5416, a small molecule tyrosine kinase receptor inhibitor, on FLT3 expression and phosphorylation in patients with refractory acute myeloid leukemia

Acute myeloid leukemia (AML) is associated with dysregulated hematopoietic cell proliferation and increased bone marrow angiogenesis, each regulated by signaling through receptor tyrosine kinases (RTKs). SU5416 is a small molecule inhibitor of VEGF receptors, c-kit and FLT3 and therefore provides a novel opportunity to target both angiogenesis and proliferation in AML. SU5416 was assessed in a phase II hematological malignancy trial in the US, where partial responses were observed in two of 33 patients. Since AML provides a unique platform to evaluate mechanism of action of small molecule inhibitors, investigation of the effect of SU5416 on FLT3 expression and phosphorylation in blood and bone marrow was an additional focus of this trial. Phosphorylated FLT3 was detected by immunoprecipitation/Western analysis in peripheral blood samples from 17 of 22 patients, and seven exhibited strong inhibition of phosphorylation immediately following a 1 h SU5416 infusion, demonstrating that SU5416 can modulate RTK phosphorylation in humans. Although no clear correlation with clinical response was observed, analysis of patient plasma drug levels suggested that a threshold SU5416 concentration of 15 μM was associated with FLT3 inhibition. This observation was supported by data from an ex vivo model where AML cells were spiked into human blood, established to mimic the clinical setting and enable more rigorous analysis of effect of SU5416. In addition, FLT3 protein levels were downregulated in patient bone marrow samples, analyzed by an RIA assay. To identify putative predictors of response, patient plasma was analyzed for levels of secreted ligands of SU5416 targets; SCF and FLT3 ligand. Baseline levels of SCF in patients with stable or progressive disease were significantly higher than those in normal donors, whereas FLT3 ligand levels in patients who exhibited progressive disease were significantly lower than those in normal donors. The translational and clinical analyses described in this report provide some insights into the mechanism and duration of action of SU5416.

Expression change of Flk-2/Flt-3 on murine hematopoietic stem cells in an activating state

Objective The receptor tyrosine kinase Flk-2/Flt-3 (Flt-3) represents an important molecule involved in early hematopoiesis. Murine hematopoietic stem cells (HSCs) have been shown to be negative for the expression of Flt-3. We now present clear evidence for the expression change of Flt-3 − HSCs in an activating state, and the reversibility of Flt-3 expression by HSCs in vivo. Materials and methods Bone marrow cells isolated from Ly5.1 mice were sorted on the basis of Flt-3 expression and transplanted into lethally irradiated Ly5.2 recipients. After 24 weeks, peripheral blood was analyzed for donor contribution by flow cytometry. Results Although long-term engraftment was predominantly detected in Flt-3 − populations as previously described, a 6-day cultivation of Lin −/lowc-kit +Sca-1 + Flt-3 − bone marrow cells with stem cell factor and interleukin-11 resulted in the generation of Flt-3 + HSCs with long-term engraftment capabilities. However, the Flt-3 ligand had no significant effect on self-renewal of the Flt-3 + HSCs. Next, to examine reversible expression of this receptor molecule, Flt-3 + cells converted in vitro from Ly5.1 Lin −/lowc-kit +Sca-1 + Flt-3 − bone marrow cells were isolated and transplanted into Ly5.2 primary recipients. After 24 weeks, Ly5.1 Lin −/low bone marrow cells were again separated into Flt-3 − and Flt-3 + cells and retransplanted into Ly5.2 secondary recipients. The majority of donor HSCs with long-term engraftment capabilities were detected in the Flt-3 − populations, indicating the reversion of Flt-3 + to Flt-3 − HSCs. Conclusions These observations suggest that Flt-3 is a useful cell-surface marker of HSC activation and that this phenotypic change is reversible.