FLT3 Expression Research Articles

Disease Characteristics and Prognostic Implications of Cell-Surface FLT3 Receptor (CD135) Expression in Pediatric Acute Myeloid Leukemia: A Report from the Children's Oncology Group.

Purpose: The FLT3 cell-surface receptor tyrosine kinase (CD135) is expressed in a majority of both acute lymphoid leukemia (ALL) and myeloid leukemia (AML). However, the prognostic significance of CD135 expression in AML remains unclear. We therefore evaluated the association between FLT3 surface expression and disease characteristics and outcomes in pediatric patients with AML.Experimental Design: We analyzed FLT3 receptor expression on AML blasts by multi-dimensional flow cytometry and its association with disease characteristics, clinical outcomes, and FLT3 transcript level in 367 children with AML treated on the Children's Oncology Group trial AAML0531.Results: There was high variability in blast CD135 cell-surface expression across specimens. CD135 expression measured by flow cytometry was not correlated with FLT3 transcript expression determined by quantitative RT-PCR. Overall, CD135 expression was not significantly different for patients with FLT3/WT, FLT3/ITD, or FLT3/ALM (P = 0.25). High cell-surface CD135 expression was associated with FAB M5 subtype (P < 0.001), KMT2A rearrangements (P = 0.009), and inversely associated with inv(16)/t(16;16) (P < 0.001). Complete remission rate, overall survival, disease-free survival, and relapse rates were not significantly different between patients with low and high CD135 expression.Conclusions: FLT3 cell-surface expression did not vary by FLT3 mutational status, but high FLT3 expression was strongly associated with KMT2A rearrangements. Our study found that there was no prognostic significance of FLT3 cell surface expression in pediatric AML. Clin Cancer Res; 23(14); 3649-56. ©2017 AACR.

Niche-mediated depletion of the normal hematopoietic stem cell reservoir by Flt3-ITD-induced myeloproliferation.

Although previous studies suggested that the expression of FMS-like tyrosine kinase 3 (Flt3) initiates downstream of mouse hematopoietic stem cells (HSCs), FLT3 internal tandem duplications (FLT3 ITDs) have recently been suggested to intrinsically suppress HSCs. Herein, single-cell interrogation found Flt3 mRNA expression to be absent in the large majority of phenotypic HSCs, with a strong negative correlation between Flt3 and HSC-associated gene expression. Flt3-ITD knock-in mice showed reduced numbers of phenotypic HSCs, with an even more severe loss of long-term repopulating HSCs, likely reflecting the presence of non-HSCs within the phenotypic HSC compartment. Competitive transplantation experiments established that Flt3-ITD compromises HSCs through an extrinsically mediated mechanism of disrupting HSC-supporting bone marrow stromal cells, with reduced numbers of endothelial and mesenchymal stromal cells showing increased inflammation-associated gene expression. Tumor necrosis factor (TNF), a cell-extrinsic potent negative regulator of HSCs, was overexpressed in bone marrow niche cells from FLT3-ITD mice, and anti-TNF treatment partially rescued the HSC phenotype. These findings, which establish that Flt3-ITD-driven myeloproliferation results in cell-extrinsic suppression of the normal HSC reservoir, are of relevance for several aspects of acute myeloid leukemia biology.

The Cytokine Flt3-Ligand in Normal and Malignant Hematopoiesis.

The cytokine Fms-like tyrosine kinase 3 ligand (FL) is an important regulator of hematopoiesis. Its receptor, Flt3, is expressed on myeloid, lymphoid and dendritic cell progenitors and is considered an important growth and differentiation factor for several hematopoietic lineages. Activating mutations of Flt3 are frequently found in acute myeloid leukemia (AML) patients and associated with a poor clinical prognosis. In the present review we provide an overview of our current knowledge on the role of FL in the generation of blood cell lineages. We examine recent studies on Flt3 expression by hematopoietic stem cells and its potential instructive action at early stages of hematopoiesis. In addition, we review current findings on the role of mutated FLT3 in leukemia and the development of FLT3 inhibitors for therapeutic use to treat AML. The importance of mouse models in elucidating the role of Flt3-ligand in normal and malignant hematopoiesis is discussed.

Selective Expression of Flt3 within the Mouse Hematopoietic Stem Cell Compartment.

The fms-like tyrosine kinase 3 (Flt3) is a cell surface receptor that is expressed by various hematopoietic progenitor cells (HPC) and Flt3-activating mutations are commonly present in acute myeloid and lymphoid leukemias. These findings underscore the importance of Flt3 to steady-state and malignant hematopoiesis. In this study, the expression of Flt3 protein and Flt3 mRNA by single cells within the hematopoietic stem cell (HSC) and HPC bone marrow compartments of C57/BL6 mice was investigated using flow cytometry and the quantitative reverse transcription polymerase chain reaction. Flt3 was heterogeneously expressed by almost all of the populations studied, including long-term reconstituting HSC and short-term reconstituting HSC. The erythropoietin receptor (EpoR) and macrophage colony-stimulating factor receptor (M-CSFR) were also found to be heterogeneously expressed within the multipotent cell compartments. Co-expression of the mRNAs encoding Flt3 and EpoR rarely occurred within these compartments. Expression of both Flt3 and M-CSFR protein at the surface of single cells was more commonly observed. These results emphasize the heterogeneous nature of HSC and HPC and the new sub-populations identified are important to understanding the origin and heterogeneity of the acute myeloid leukemias.

Human Bronchial Epithelial Cells Induce CD141/CD123/DC-SIGN/FLT3 Monocytes That Promote Allogeneic Th17 Differentiation.

Little is known about monocyte differentiation in the lung mucosal environment and about how the epithelium shapes monocyte function. We studied the role of the soluble component of bronchial epithelial cells (BECs) obtained under basal culture conditions in innate and adaptive monocyte responses. Monocytes cultured in bronchial epithelial cell-conditioned media (BEC-CM) specifically upregulate CD141, CD123, and DC-SIGN surface levels and FLT3 expression, as well as the release of IL-1β, IL-6, and IL-10. BEC-conditioned monocytes stimulate naive T cells to produce IL-17 through IL-1β mechanism and also trigger IL-10 production by memory T cells. Furthermore, monocytes cultured in an inflammatory environment induced by the cytokines IL-6, IL-8, IL-1β, IL-15, TNF-α, and GM-CSF also upregulate CD123 and DC-SIGN expression. However, only inflammatory cytokines in the epithelial environment boost the expression of CD141. Interestingly, we identified a CD141/CD123/DC-SIGN triple positive population in the bronchoalveolar lavage fluid (BALF) from patients with different inflammatory conditions, demonstrating that this monocyte population exists in vivo. The frequency of this monocyte population was significantly increased in patients with sarcoidosis, suggesting a role in inflammatory mechanisms. Overall, these data highlight the specific role that the epithelium plays in shaping monocyte responses. Therefore, the unraveling of these mechanisms contributes to the understanding of the function that the epithelium may play in vivo.

RNA binding protein MSI2 positively regulates FLT3 expression in myeloid leukemia

FLT3 is frequently mutated and overexpressed in acute myelogenous leukemia (AML) and other hematologic malignancies. Although signaling events downstream of FLT3 receptor tyrosine kinase have been studied in depth, molecular mechanisms of how FLT3 expression is regulated at the post-transcriptional level in particular remain elusive. In this study, we investigated the roles of an RNA binding protein MSI2 as a regulator of FLT3 expression. MSI2 and FLT3 are significantly co-regulated in human AML and chronic myelogenous leukemia in blast crisis (BC-CML). Genetic loss of MSI2 leads to down-regulation of the FLT3 receptor in both AML and BC-CML cells and concomitant impairment of clonogenic growth potential. Furthermore, we demonstrate that MSI2 protein is physically bound to FLT3 mRNA transcripts, suggesting post-transcriptional control of FLT3 expression. Collectively, these results reveal a novel mode of FLT3 regulation essential for leukemia growth, which may aid in designing a targeted therapy to treat human myeloid leukemia.

Arsenic Trioxide Synergizes with FLT3 Tyrosine Kinase Inhibitors to Kill FLT3-ITD+ Leukemic Cells through Depressed Production and Degradation of FLT3 Protein

A number of selective FLT3 tyrosine kinase inhibitors (TKIs) have been tested for treatment of FLT3-ITD+ AML. However, monotherapy with FLT3 TKIs alone has achieved only transient and limited clinical responses due to several resistance mechanisms. Arsenic trioxide (ATO) has demonstrated significant efficacy in treating acute promyelocytic leukemia (APL). ATO has also shown some activity in treating non-APL myeloid leukemias. Recent studies have demonstrated that ATO can affect the degradation of oncogenic mutant proteins including mutant p53 and NPM through the ubiquitin-proteasome pathway (UPP). Here we investigated the feasibility of combining FLT3 TKIs with ATO in the treatment of FLT3-ITD+leukemia.Anti-leukemic effects against FLT3-ITD+ patient AML samples and leukemia cell lines (Molm14, MV4;11) by FLT3 TKIs (Sorafenib, quizartinib), ATO and the combination were examined by MTT, apoptosis, cell viability and colony forming assays. Our data revealed that the combination showed synergistic growth inhibition of the FLT3-ITD+ cell lines Molm14 and MV4;11, with combinatorial index (CI) values at ED50 below 1.0 for both cell lines (CI values were 0.46 and 0.56 for ATO + sorafenib, 0.65 and 0.57 for ATO + quizartinib in Molm14 and MV4;11 cells, respectively). In contrast, there was no synergy observed for the combination in treating leukemia cell lines that do not express mutant FLT3. Synergistic effects for the combination in inducing apoptosis and inhibiting colony formation were also observed for the FLT3-ITD+ cell lines. Furthermore, when the combination was used to treat primary FLT3-ITD+ patient samples, there was also significant reduction of viability and clonogenicity. In contrast, normal BM MNCs showed very limited responses to the combination. Western blot (WB) analysis of Molm14 and FLT3-ITD+ patient samples revealed the combination of ATO and sorefenib potently reduced phosphorylation of FLT3 and its downstream targets (STAT5, MAPK, and AKT). In vivoexperiments using the combination to treat NSG mice engrafted with Molm14 cells demonstrated a significant reduction in the level of leukemic cells.We further investigated the mechanism by which ATO contributes to an anti-leukemic effect on FLT3-ITD+ cells. Morphologic and flow cytometric analysis showed that ATO promoted the differentiation of Molm14 cells. The expression of C/EBPα and PU.1, two key regulators for myeloid differentiation, was increased in ATO-treated Molm14 cells at both the mRNA and protein levels. These data suggest ATO is capable of inducing the differentiation of leukemic cells. We also found that, in FLT3-ITD+ cells, ATO decreased expression of FLT3 protein. This could result from reduced FLT3 production and/or increased protein degradation. Further quantitative PCR analysis revealed ATO decreased expression of FLT3 and its upstream regulators HoxA9 and meis1. Co-immunoprecipitation assay showed that ATO facilitated poly-Ubiquitination and degradation of FLT3 in a dose- and time-dependent fashion. These results indicate that ATO exerts its anti-leukemic effects in FLT3-ITD+AML cell lines and primary samples at least partly through reducing the level of FLT3 protein.These studies together demonstrate that ATO has a unique activity towards FLT3-ITD+ leukemia cells. Based on these findings, ATO is a potential candidate to work in combination with FLT3 TKIs to improve the outcome of FLT3-ITD+ AML patients. DisclosuresLevis:Millennium: Consultancy, Research Funding; Daiichi-Sankyo: Consultancy, Honoraria; Astellas: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Honoraria, Research Funding.

Targeting Chromatin Regulators Inhibits Leukemogenic Gene Expression in NPM1 Mutant Leukemia.

MLL1 and DOT1L are chromatin regulators that control HOX, MEIS1, and FLT3 expression and are therapeutic targets in NPM1mut AML. Combinatorial small-molecule inhibition has synergistic on-target activity and constitutes a novel therapeutic concept for this common AML subtype. Cancer Discov; 6(10); 1166-81. ©2016 AACR.See related commentary by Hourigan and Aplan, p. 1087This article is highlighted in the In This Issue feature, p. 1069.

Silencing of GATA3 defines a novel stem cell-like subgroup of ETP-ALL.

BackgroundGATA3 is pivotal for the development of T lymphocytes. While its effects in later stages of T cell differentiation are well recognized, the role of GATA3 in the generation of early T cell precursors (ETP) has only recently been explored. As aberrant GATA3 mRNA expression has been linked to cancerogenesis, we investigated the role of GATA3 in early T cell precursor acute lymphoblastic leukemia (ETP-ALL).MethodsWe analyzed GATA3 mRNA expression by RT-PCR (n = 182) in adult patients with T-ALL. Of these, we identified 70 of 182 patients with ETP-ALL by immunophenotyping. DNA methylation was assessed genome wide (Illumina Infinium® HumanMethylation450 BeadChip platform) in 12 patients and GATA3-specifically by pyrosequencing in 70 patients with ETP-ALL. The mutational landscape of ETP-ALL with respect to GATA3 expression was investigated in 18 patients and validated by Sanger sequencing in 65 patients with ETP-ALL. Gene expression profiles (Affymetrix Human genome U133 Plus 2.0) of an independent cohort of adult T-ALL (n = 83) were used to identify ETP-ALL and investigate GATA3low and GATA3high expressing T-ALL patients. In addition, the ETP-ALL cell line PER-117 was investigated for cytotoxicity, apoptosis, GATA3 mRNA expression, DNA methylation, and global gene expression before and after treatment with decitabine.ResultsIn our cohort of 70 ETP-ALL patients, 33 % (23/70) lacked GATA3 expression and were thus defined as GATA3low. DNA methylation analysis revealed a high degree of GATA3 CpG island methylation in GATA3low compared with GATA3high ETP-ALL patients (mean 46 vs. 21 %, p < 0.0001). Genome-wide expression profiling of GATA3low ETP-ALL exhibited enrichment of myeloid/lymphoid progenitor (MLP) and granulocyte/monocyte progenitor (GMP) genes, while T cell-specific signatures were downregulated compared to GATA3high ETP-ALL. Among others, FLT3 expression was upregulated and mutational analyses demonstrated a high rate (79 %) of FLT3 mutations. Hypomethylating agents induced reversal of GATA3 silencing, and gene expression profiling revealed downregulation of hematopoietic stem cell genes and upregulation of T cell differentiation.ConclusionsWe propose GATA3low ETP-ALL as a novel stem cell-like leukemia with implications for the use of myeloid-derived therapies.Electronic supplementary materialThe online version of this article (doi:10.1186/s13045-016-0324-8) contains supplementary material, which is available to authorized users.

Upregulation of Flt3 is a passive event in Hoxa9/Meis1-induced acute myeloid leukemia in mice.

HOXA9, MEIS1 and FLT3 are genes frequently upregulated in human acute myeloid leukemia. Hoxa9 and Meis1 also cooperate to induce aggressive AML with high Flt3 expression in mice, suggesting an important role for Flt3 in Hoxa9/Meis1-induced leukemogenesis. To define the role of Flt3 in AML with high Hoxa9/Meis1, we treated mice with Hoxa9/Meis1-induced AML with the Flt3 inhibitor AC220, used an Flt3-ligand (FL-/-) knockout model, and investigated whether overexpression of Flt3 could induce leukemia together with overexpression of Hoxa9. Flt3 inhibition by AC220 did not delay AML development in mice transplanted with bone marrow cells overexpressing Hoxa9 and Meis1. In addition, Hoxa9/Meis1 cells induced AML in FL-/- mice as rapid as in wild-type mice. However, FL-/- mice had reduced organ infiltration compared with wild-type mice, suggesting some Flt3-dependent effect on leukemic invasiveness. Interestingly, leukemic Hoxa9/Meis1 cells from sick mice expressed high levels of Flt3 regardless of presence of its ligand, showing that Flt3 is a passive marker on these cells. In line with this, combined engineered overexpression of Flt3 and Hoxa9 did not accelerate the progression to AML. We conclude that the Hoxa9- and Meis1-associated upregulation of Flt3 is not a requirement for leukemic progression induced by Hoxa9 and Meis1.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

FLT3 is implicated in cytarabine transport by human equilibrative nucleoside transporter 1 in pediatric acute leukemia.

FLT3 abnormalities are negative prognostic markers in acute leukemia. Infant leukemias are a subgroup with frequent MLL (KMT2A) rearrangements, FLT3 overexpression and high sensitivity to cytarabine, but dismal prognosis. Cytarabine is transported into cells by Human Equilibrative Nucleoside Transporter-1 (hENT1, SLC29A1), but the mechanisms that regulate hENT1 in acute leukemia have been scarcely studied.We explored the expression and functional link between FLT3 and main cytarabine transporters in 50 pediatric patients diagnosed with acute lymphoblastic leukemia and MLL rearrangement (ALL-MLL+) and other subtypes of leukemia, and in leukemia cell lines.A significant positive correlation was found between FLT3 and hENT1 expression in patients. Cytarabine uptake into cells was mediated mainly by hENT1, hENT2 and hCNT1. hENT1-mediated uptake of cytarabine was transiently abolished by the FLT3 inhibitor PKC412, and this effect was associated with decreased hENT1 mRNA and protein levels. Noticeably, the cytotoxicity of cytarabine was lower when cells were first exposed to FLT3 inhibitors (PKC412 or AC220), probably due to decreased hENT1 activity, but we observed a higher cytotoxic effect if FLT3 inhibitors were administered after cytarabine.FLT3 regulates hENT1 activity and thereby affects cytarabine cytotoxicity. The sequence of administration of cytarabine and FLT3 inhibitors is important to maintain their efficacy.

Hematopoietic stem cell-specific GFP-expressing transgenic mice generated by genetic excision of a pan-hematopoietic reporter gene.

Selective labeling of specific cell types by expression of green fluorescent protein (GFP) within the hematopoietic system would have great utility in identifying, localizing, and tracking different cell populations in flow cytometry, microscopy, lineage tracing, and transplantation assays. In this report, we describe the generation and characterization of a new transgenic mouse line with specific GFP labeling of all nucleated hematopoietic cells and platelets. This new "Vav-GFP" mouse line labels the vast majority of hematopoietic cells with GFP during both embryonic development and adulthood, with particularly high expression in hematopoietic stem and progenitor cells (HSPCs). With the exception of transient labeling of fetal endothelial cells, GFP expression is highly selective for hematopoietic cells and persists in donor-derived progeny after transplantation of HSPCs. Finally, we also demonstrate that the loxP-flanked reporter allows for specific GFP labeling of different hematopoietic cell subsets when crossed to various Cre reporter lines. By crossing Vav-GFP mice to Flk2-Cre mice, we obtained robust and highly selective GFP expression in hematopoietic stem cells (HSCs). These data describe a new mouse model capable of directing GFP labeling exclusively of hematopoietic cells or exclusively of HSCs.

Expression of GADS enhances FLT3-induced mitogenic signaling.

GADS is a member of a family of SH2 and SH3 domain-containing adaptors that functions in tyrosine kinase-mediated signaling cascades. Its expression is largely restricted to hematopoietic tissues and cell lines. Therefore, GADS is mainly involved in leukocyte-specific protein tyrosine kinase signaling. GADS is known to interact with tyrosine-phosphorylated SHC, BCR-ABL and KIT. The SH2 domain of GADS has a similar binding specificity to that of GRB2 but its SH3 domain displays a different binding specificity, and thus it is involved in other downstream signaling pathways than GRB2. In the present study, we examined the role of GADS in FLT3 signaling. FLT3 is a type III receptor tyrosine kinase, which is mutated in more than 30% of acute myeloid leukemia (AML) and the most common mutations is the internal tandem duplication (ITD) mutations. We observed that expression of GADS enhanced oncogenic FLT3-ITD-induced cell proliferation and colony formation in vitro. In a mouse xenograft model, GADS accelerated FLT3-ITD-dependent tumor formation. Furthermore, expression of GADS induced a transcriptional program leading to upregulation of MYC and mTORC1 target genes. GADS localizes to the cell membrane and strongly binds to ligand-stimulated wild-type FLT3 or is constitutively associated with the oncogenic mutant FLT3-ITD. We mapped the binding sites in FLT3 to pY955 and pY969 which overlaps with the GRB2 binding sites. Expression of GADS enhanced FLT3-mediated phosphorylation of AKT, ERK1/2, p38 and STAT5. Taken together, our data suggests that GADS is an important downstream component of FLT3 signaling and expression of GADS potentiates FLT3-mediated mitogenic signaling.

Role of FLT3 in the proliferation and aggressiveness of hepatocellular carcinoma

Previously we showed that Fms-like tyrosine kinase (FLT3) changes its cellular localization upon partial hepatectomy, suggesting a role in liver regeneration. FLT3 was also shown to play an important function in cellular proliferation and activation of PI3K and Ras. Thus, we aimed to investigate the role of FLT3 in hepatocellular tumorigenesis utilizing in vitro and in vivo models. We used Snu398 cells that express FLT3. We investigated these cells' in vitro proliferation and invasion abilities by treatment with the FLT3 inhibitor K-252a or by knocking-down with FLT3 shRNA,. Furthermore, the effect of blocking FLT3 activity and expression during in vivo tumorigenesis was assessed with xenograft models. After K-252a treatment or stable knock-down, these cells' proliferation and migration abilities were highly diminished in vitro. In addition, significant diminution in tumorigenicity of Snu398 cells was also obtained in vivo. When FLT3 knocked-down Snu398 cells were injected into nude mice, we did not detect αSMA expression in these tumors, suggesting a role for FLT3 in in vivo invasiveness. Our data provided evidence that FLT3 has a crucial role both in hepatocarcinogenesis and its invasiveness. Therefore, targeting FLT3 and/or its activity may be a promising tool for combating hepatocellular carcinomas.

FLT3 Internal Tandem Duplication Mutation, c MPL and CD34 Expressions Predict Low Survival in Acute Myeloid Leukemia Patients

To detect FMS-like tyrosine kinase-3 internal tandem duplicate (FLT3 ITD) mutation, Myeloproliferative leukemia virus oncogene (cMPL) and Ephrin A 4 receptor (EphA4) expressions in Acute myeloid leukemia (AML) and their correlation to patient's clinicopathological characteristics and survival.RNA was extracted from blood samples of 58 AML patients (39 adults and 19 children) and 20 age and sex matched controls. FLT3 ITD mutation, cMPL and EphA4 expression was studied using RT-PCR and correlated to the clinical and survival data of the patients.FLT3 ITD mutation, cMPL and EphA4 expression was positive in 35.9%, 76.9% and 56.4% of adult AML patients respectively and in 15.8%, 47.4% and 36.8% of pediatric AML patients respectively. 76.9% of adult and 89.5% of pediatric patients expressed CD33. 64.1 % of adults and 42.1% of children expressed CD34. CD34 expression was significantly associated with both FLT3 ITD mutation and cMPL expression. CD34, FLT3 and cMPL negative cases have significantly higher overall survival than positive cases.CD34 expression is significantly associated with both FLT3 ITD mutation and cMPL expression which could be used as a marker for low survival. Normal FLT3 and negative expression of CD34 and cMPL may predict a longer overall survival. Further studies are needed to investigate the mechanism that may correlate CD34 to both markers.

Cdx2 Cooperates with Flt3-ITD to Induce Acute Myeloid Leukaemia in Mice

The caudal-related homeobox gene CDX2 is ectopically expressed in 90% of human acute myeloid leukaemia (AML), but not normal haematopoietic stem and progenitor cells (HSPC). Retroviral expression of Cdx2 causes a highly penetrant, lethal AML in mouse models, while short hairpin RNA-mediated targeted knockdown of CDX2 in vitro impairs growth and clonogenic potential of AML cell lines (Scholl et al. 2007). These findings implicate Cdx2 overexpression as a clinically relevant model of myeloid leukaemogenesis, however existing studies have been limited by the requirement for ex vivo manipulation, retroviral overexpression and transplantation.To understand the role of Cdx2 expression in de novo leukaemic transformation of HSC, we generated an inducible transgenic mouse model of Cdx2 expression linked to the mCherry fluorescent reporter. Cdx2 was specifically activated in adult HSC using the tamoxifen-inducible SclCreERT:Cdx2 model and compared with LysMCre:Cdx2 mice that selectively activated Cdx2 in the myeloid lineage, commencing at committed myeloid progenitors. SclCreERT:Cdx2 mice developed a lethal myelodysplastic phenotype with a long latency (median survival, 184 days) characterised by leukopenia, anaemia, thrombocytopenia and reduced marrow cellularity. Bone marrow histology revealed prominent megakaryocytic dysplasia. Competitive transplantation assays showed dramatic loss of long-term HSC self-renewal after tamoxifen induction of Cdx2 in the secondary recipients. Conversely, LysMCre:Cdx2 mice developed a myeloproliferative phenotype with leukocytosis, splenomegaly and extramedullary haematopoiesis. Interestingly, both cohorts exhibited marked neutrophil hypersegmentation compared to non-Cdx2 controls, a characteristic that is compatible with myelodysplasia, and suggests that Cdx2 not only controls HSC development and differentiation, but is required for granulocyte maturation. In contrast to retroviral models, AML was never observed with Cdx2 expression alone.FLT3-internal tandem duplication (ITD) represents a tyrosine kinase mutation that is present in 35% of AML patients, making it one of the most common genetic alterations found in AML. Transgenic Flt3ITD mice develop myelodysplastic syndrome (MDS)/myeloproliferative neoplasm (MPN) with long latency (approx. 10-15 months) but do not develop AML (Lee et al. 2007), suggesting that additional mutations are required for complete leukaemogenesis. Preliminary data has shown a correlation between CDX2 and FLT3 expression in AML. We therefore crossed SclCreERT:Cdx2 with Flt3ITD/+ and Flt3ITD/ITD mice. SclCreERT:Cdx2xFlt3ITD/+ mice developed a rapidly progressive lethal MDS with marked anaemia and thrombocytopenia, splenomegaly, morphological dysplasia and a shorter latency (median survival, 45 days) with a low incidence of transformation to AML. Strikingly, SclCreERT:Cdx2xFlt3ITD/ITD mice developed a fully penetrant AML characterised by marked leukocytosis, splenomegaly and hepatomegaly, and >20% circulating blast cells, demonstrating that Cdx2 cooperates with Flt3ITD to induce AML in a Flt3ITD gene dosage-dependent manner (Fig 1). Transplantation experiments of these samples into lethally irradiated, syngeneic wildtype (WT) recipients revealed that cells from SclCreERT:Cdx2xFlt3ITD/+ or SclCreERT:Cdx2xFlt3ITD/ITD but not WT, Flt3ITD/+ or Flt3ITD/ITD resulted in direct phenocopy of the primary disease with a short latency lethality, demonstrating the disease is intrinsic to transformed HSPC populations.Mechanistically, SclCreERT:Cdx2xFlt3ITD/+ and SclCreERT:Cdx2xFlt3ITD/ITD samples showed marked depletion of long-term HSC, abnormal cell cycle regulation and widespread gene expression changes leading to the activation of a leukaemia stem cell (LSC) program, including the aberrant expression of several Hox genes.Altogether, this work demonstrates that conditional Cdx2 expression has a critical role in the transformation of HSPC populations to AML LSC. This is a novel, inducible model of de novo leukaemic transformation and reflects common genetic aberrations seen in human AML. Thus, this model may help to identify tractable susceptibilities to target LSC populations and improve clinical outcomes. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Up-Regulation of Flt3 Is a Passive Event in Hoxa9/Meis1-Induced Acute Myeloid Leukemia

Background: The HOXA9, MEIS1, and FLT3 genes are frequently up-regulated and co-expressed in human acute myeloid leukemia (AML). Over-expression of Hoxa9 and Meis1 also cooperate strongly to induce aggressive AML with high Flt3 expression in mice, suggesting an important role for Flt3 in Hoxa9/Meis1 leukemogenesis. However, a previous study using an Flt3-/- knock-out mouse model indicated that FLT3 is dispensable for HOXA9/MEIS1-induced AML (Morgado et al., Blood 2007). However, the lack of Flt3 dependency seen using this germline Flt3-/- knock-out model may be the result from an adaptation by the leukemia initiating cells to Flt3-deficiency or that Flt3's functions have been compensated for. Several FLT3-inhibitors have been tested in clinical trials with varying efficacy on AML harboring the FLT3 -ITD mutation but the potential of using wild-type FLT3 as a drug target in AML remains unclear.Aim and methods: To further define the role of expression of wild-type FLT3 in HOXA9/MEIS1-induced AML we used the murine AML model based on concurrent Hoxa9 and Meis1 overexpression. We inhibited Flt3-signaling by two means; with the FLT3 inhibitor AC220 and with the use of an Flt3 -ligand (Fl-/-) knock-out mouse model to minimize the risk that the leukemia initiating cells get adapted to Flt3 deficiency beforehand. In addition, we investigated whether Flt3 can substitute for some of Meis1's functions and accelerate leukemia onset together with Hoxa9 in vivo.Results: Hematopoietic cells with concurrent over-expression of both Hoxa9 and Meis1 showed a robust upregulation of Flt3 transcripts compared to cells expressing only Hoxa9 (p < 0.001). In addition, the Hoxa9/Meis1 cells could grow in media supplemented with the Flt3-ligand (Fl) as the only growth factor while cells expressing only Hoxa9 could not. Treatment with the FLT3-inhibitor AC220 effectively inhibited this Flt3-dependent proliferative advantage in vitro. However, the aggressive AML developed in mice transplanted with hematopoietic cells over-expressing Hoxa9 and Meis1 could not be ameliorated with AC220 treatment compared to placebo controls.Mice without expression of the Flt3-ligand (Fl-/-) transplanted with bone marrow cells expressing Hoxa9 and Meis1 showed a slower engraftment of leukemic cells (p < 0.001), lower white blood cell counts (p < 0.01), and less organ infiltration (p < 0.001) compared to wild-type recipient mice. However, the rapid development of anemia, leukemia and death were not different in Fl-/- mice compared to wild-type mice. Also, leukemic cells from Fl-/- mice expressed similar levels of Flt3 compared to leukemic cells from wild-type mice. In addition, overexpression of Flt3 together with Hoxa9 in hematopoietic cells could not accelerate the progression to AML in mice.Conclusions: We conclude that FLT3-signaling likely contributes to proliferation and engraftment of leukemic cells with high Hoxa9 and Meis1 expression when its ligand is present. However, bone marrow cells over-expressing Hoxa9 and Meis1 upregulate Flt3 expression also in the absence of Flt3-ligand and inhibition of the signaling through Flt3 does not prevent or delay leukemia development induced by Hoxa9 and Meis1. Thus, the Hoxa9- and Meis1-associated upregulation of Flt3 seems to be a passive event with regard to leukemia development, with limited relevance to the AML pathology. Treating AML with high HOXA9 and MEIS1 expression using FLT3 inhibitors will therefore most likely be unsuccessful. DisclosuresNo relevant conflicts of interest to declare.

RNA-Sequencing of Cytogenetically Normal Acute Myeloid Leukemia to Extend Routine Molecular Diagnostics

IntroductionDespite the discovery of new genetic alterations the cytogenetically normal acute myeloid leukemia (CN-AML) subset is still insufficiently characterized. As mRNA-sequencing (RNA-seq) is becoming more accessible, sequencing of the transcriptome could reveal not only information regarding deregulated expression patterns in leukemias, but also enable mutational evaluation of expressed alleles. To address this issue we performed RNA-seq of 5 AML patients from diagnostic bone marrow aspirates to assess the validity of the following: 1) remission samples can be used as control, 2) concordance between standard clinical laboratory analysis and RNA-seq data, and 3) implementation of existing microarray data repository to infer expected mutational status of the sequenced samples based on expression patterns.MethodsDiagnostic samples were selected among patients with high leukemic blast fraction (mean of 75%) and paired remission samples with very low, or no detectable, molecular minimal residual disease. A panel of recurrent somatic mutations was assessed by means of quantitative PCR (qPCR) and fragment analysis for sequencing comparisons. Sequencing was performed on single HiSeq lane aimed at minimum 66 million reads per sample (AROS Applied Biotechnology, Aarhus, Denmark). Biomedical Genomics Workbench 2 was employed for alignment, mutation calling and analysis of differential expression (Robinson-Smyth exact test, Bonferroni corrected). R and Mathematica were used for comparison of expression patterns from the individual samples in conjunction with CN-AML (251) and control bone marrow (73) data from microarray repositories (Gene Expression Omnibus, GSE15434 and GSE13159).Results259 genes were differentially expressed as defined by the following thresholds: normalized fold-change > 2, expression range values > 10 RPKM and p<0.05. Of these, 41 genes were upregulated in AML samples (p<0.01 subset heatmap is shown in fig. 1A). Supervised clustering of the samples on the basis of differential expression patterns efficiently divided the data into 2 groups according to diagnosis and remission status (fig. 1A, dendrogram). A median of 61 mutations per sample was observed (35 to 675). Thirty-four mutations occurred twice or more (fig. 1B, size reflects transcript fraction of mutated allele).A close correlation between routine molecular diagnostics and sequencing data was found. As expected, routine minimal residual disease marker WT1 was in agreement and found to be clearly expressed at diagnosis, but not at time of remission.By comparing patient expression patterns with NPM1, FLT3 or CEPBA mutation specific microarray expression signatures we were largely able to deduce expected mutational status for each patient (fig. 1C, showing the individual matching fraction of mutation specific gene expression signature in terms of upregulated or downregulated) in favor of qPCR analysis shown in table 1.Table 1Comparison of mutational status from qPCR/RNA-seqFLT3mutNPM1mutIDH1mutWT1mutCEBPAmutKITmut#1+/++/++/+-/--/--/-#2+/++/+-/--/--/--/-#3+/++/--/--/--/--/-#4-/-+/-+/++/--/--/-#5+/+-/--/--/--/--/-ConclusionsThis approach serves as a proof of the concept that RNA-sequencing can be directly implemented in the routine laboratory. Moreover, transcriptome data such as these can extend the molecular survey in a dynamic manner by aiding in therapy-related decision-making for the application of targeted therapy and for delineating the reasons for treatment. While publicly available repositories of RNA-seq data are being generated for referencing, it is possible to include microarray data to support molecular classification of the individual patients, as is shown here. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Gadd45a Loss Enhances MLL Leukemogenesis By Promoting Beta-Catenin Mitochondrial Translocation and Inhibiting Apoptosis

Gadd45a is a mediator of growth arrest and its expression is suppressed in primary human MLL-rearranged AML (predominantly MLL-AF9) and AML1-ETO AML with increased FLT3 expression (Valk et al., N Engl J Med 2004; Perugini et al., Leukemia 2009). While Gadd45a is frequently silenced in several human cancers, including leukemia, little is know about how Gadd45a loss contributes to leukemogenesis. Our studies have shown that Gadd45a loss promotes MLL-AF9 mediated development in AML in mice and protects leukemic cells from apoptosis via novel β-catenin/mitochondria pathway.To explore the functional role of Gadd45a loss in MLL-rearranged AML, we retrovirally introduced MLL-AF9 into c-Kithigh progenitors isolated from Gadd45a knockout mouse bone marrow cells. Our results showed that Gadd45a knockout markedly reduced the colony forming capacity of pre-leukemic cells and significantly shortened the latency for MLL leukemia development in mice (p = 0.037, N = 6 mice). As Gadd45a could induce cell death through the mitochondria pathway (Tong et al., Mol Cell Biol 2005), we next examined the expression of proteins involved in the mitochondrial apoptosis in Gadd45a knockout pre-leukemic cells. Our western blot analysis showed that Gadd45a knockout dramatically decreased expression of pro-death proteins Txnip, Bim and Bax but interestingly, significantly increased active β-catenin expression in mitochondria. Given that Gadd45a has been reported to regulate β-catenin distribution (Ji et al., Oncogene 2007), we postulate that Gadd45a loss facilitates the translocation of β-catenin into mitochondria. We have previously demonstrated that β-catenin is highly expressed in MLL-AF9 transformed pre-leukemic cells and is lower in Hoxa9/Meis1 transformed pre-leukemic cells, while increased β-catenin expression is correlated with a poor survival rate in mice. Here, we retrovirally introduced a constitutively active form of β-catenin into Hoxa9/Meis1 pre-leukemic cells. As expected, activation of β-catenin reduced endogenous expression of Txnip and Bim in mitochondria, exhibiting an expression pattern similar to that regulated by Gadd45a knockout. Consistently, both Gadd45a knockout and β-catenin activation not only reduced the basal cellular reactive oxygen species (ROS) levels but also inhibited Antimycin A (an ROS generator)-induced mitochondrial ROS production.Taken together, our study discovers a previously unrecognized Gadd45a loss/β-catenin/mitochondria pathway that prevents mitochondrial ROS accumulation and protects leukemic cells from cell death in MLL leukemogenesis. DisclosuresNo relevant conflicts of interest to declare.