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Invasive aspergillosis (IA) is a significant cause of morbidity and mortality following allogeneic hematopoietic cell transplantation (HCT). Previous studies have reported an association between IA development and single nucleotide polymorphisms (SNPs), but many SNPs have not been replicated in a separate cohort. The presence of a positive serum galactomannan assay (SGM+) has also been associated with a worse prognosis in patients with IA, and genetic determinants in this subset of patients have not been systematically studied. The study cohort included 2609 HCT recipients and their donor pairs: 483 with proven/probable IA (183 SGM+) and 2126 with no IA by standard criteria. Of 25 SNPs previously published, we analyzed 20 in 14 genes that passed quality control. Samples were genotyped via microarray, and SNPs that could not be genotyped were imputed. The primary aim was to replicate SNPs associated with proven/probable IA at 2 years; secondary goals were to explore the associations using an end point of SGM+ IA or proven/probable IA using a different genetic model or time to IA (3 months vs 2 years) compared with the original study. Two SNPs in 2 genes (PTX3, CLEC7a) were replicated. Thirteen SNPs in 9 genes had an association at P ≤ .05 using the secondary aims (PTX3, CLEC7a, CD209, CXCL10, TLR6, S100B, IFNG, PLG, TNFR1), with hazard ratios ranging from 1.2 to 3.29. Underlying genetic differences can influence development of IA following HCT. Identification of genetic predispositions to IA could have important implications in donor screening, risk stratification of recipients, monitoring, and prophylaxis.

B-cell adaptor for phosphatidylinositol 3-kinase (BCAP) is a signaling adaptor expressed in mature hematopoietic cells, including monocytes and neutrophils. Here we investigated the role of BCAP in the homeostasis and development of these myeloid lineages. BCAP−/− mice had more bone marrow (BM) monocytes than wild-type (WT) mice, and in mixed WT:BCAP−/− BM chimeras, monocytes and neutrophils skewed toward BCAP−/− origin, showing a competitive advantage for BCAP−/− myeloid cells. BCAP was expressed in BM hematopoietic progenitors, including lineage−Sca-1+c-kit+ (LSK), common myeloid progenitor, and granulocyte/macrophage progenitor (GMP) cells. At the steady state, BCAP−/− GMP cells expressed more IRF8 and less C/EBPα than did WT GMP cells, which correlated with an increase in monocyte progenitors and a decrease in granulocyte progenitors among GMP cells. Strikingly, BCAP−/− progenitors proliferated and produced more myeloid cells of both neutrophil and monocyte/macrophage lineages than did WT progenitors in myeloid colony-forming unit assays, supporting a cell-intrinsic role of BCAP in inhibiting myeloid proliferation and differentiation. Consistent with these findings, during cyclophosphamide-induced myeloablation or specific monocyte depletion, BCAP−/− mice replenished circulating monocytes and neutrophils earlier than WT mice. During myeloid replenishment after cyclophosphamide-induced myeloablation, BCAP−/− mice had increased LSK proliferation and increased numbers of LSK and GMP cells compared with WT mice. Furthermore, BCAP−/− mice accumulated more monocytes and neutrophils in the spleen than did WT mice during Listeria monocytogenes infection. Together, these data identify BCAP as a novel inhibitor of myelopoiesis in the steady state and of emergency myelopoiesis during demand conditions.

Activating mutations in the juxtamembrane domain (FLT3-length mutations, FLT3-LM) and in the protein tyrosine kinase domain (TKD) of FLT3 (FLT3-TKD) represent the most frequent genetic alterations in acute myeloid leukemia (AML) and define a molecular target for therapeutic interventions by protein tyrosine kinase (PTK) inhibitors. We could show that distinct activating FLT3-TKD mutations at position D835 mediate primary resistance to FLT3 PTK inhibitors in FLT3-transformed cell lines. In the presence of increasing concentrations of the FLT3 PTK inhibitor SU5614, we generated inhibitor resistant Ba/F3 FLT3-internal tandem duplication (ITD) cell lines (Ba/F3 FLT3-ITD-R1-R4) that were characterized by a 7- to 26-fold higher IC50 (concentration that inhibits 50%) to SU5614 compared with the parental ITD cells. The molecular characterization of ITD-R1-4 cells demonstrated that specific TKD mutations (D835N and Y842H) on the ITD background were acquired during selection with SU5614. Introduction of these dual ITD-TKD, but not single D835N or Y842H FLT3 mutants, in Ba/F3 cells restored the FLT3 inhibitor resistant phenotype. Our data show that preexisting or acquired mutations in the PTK domain of FLT3 can induce drug resistance to FLT3 PTK inhibitors in vitro. These findings provide a molecular basis for the evaluation of clinical resistance to FLT3 PTK inhibitors in patients with AML.

Introduction: The combination of bendamustine (B) and rituximab (R) is an effective and relatively well tolerated treatment for B-cell malignancies. However, there is increased concern regarding infectious complications since FDA approval, in particular due to reports of prolonged and profound lymphopenia associated with BR. [Saito, Blood Cancer J 2015; Garcia-Munoz, Ann Hematol 2014] There have been numerous case reports of opportunistic infection (OI) with BR, such as viral reactivation (HBV, VZV, HSV, CMV, EBV) and Pneumocystis jiroveci. [Abkur, Clinical Case Reports 2015; Carter, Leuk Res 2011; Tsutsumi, Int J Hematol 2012; Lim, Ann Hematol 2011] A retrospective analysis conducted in Israel showed that B ± R therapy was associated with 47% incidence of infectious complications (ICs) with 2 out of 183 pts received antimicrobial prophylaxis (ppx) and 65% G-CSF use.[Gafter-Gvili, Blood 2014, abs 3077]Another study concluded that a prolonged period of VZV ppx may be advisable with BR.[Allen, Blood 2015, abs 4167] Brugger et al published a practice guide for B-based therapy with a section devoted to discussing potential ICs and considerations for antimicrobial ppx.[Brugger, Oncologist 2013] In prospective trials (StiL and Bright), Rummel et al reported a 37% incidence of unspecified infectious episodes and Flinn et al reported 55% incidence of all infections with 10% OI despite 30% G-CSF use.[Rummel, Lancet 2013, Flinn, Blood 2014] Antimicrobial ppx and G-CSF use were not mandated in those trials. With these reports and OI episodes in a few of our patients, we performed a retrospective analysis at MSKCC to evaluate the incidence of ICs and potential risk factors in patients treated with B and anti-CD20 antibody ± R maintenance. Methods: Pts ≥18 year old with CD20+ NHL and received ≥2 cycles of B and anti-CD20 antibody (rituximab or ofatumumab) ± R maintenance from 2008 through 2015 were included. Pts were excluded if they received B monotherapy, switched treatment before completion of planned course, or underwent stem cell transplantation right after completion of bendamustine combination. Infection data were collected for up to 1 yr post B-based treatment with a cutoff date of 5/1/2016. Adverse drug events (ADEs) including neutropenia, neutropenic fever (NF), lymphopenia, time to lymphocyte recovery, and liver function abnormalities were graded according to CTCAE v4.0. Univariate analysis with Fisher’s exact test was used to evaluate the potential risk factors (degree of lymphopenia and neutropenia, R maintenance, and line of therapy) for ICs. Results: 416 pts were included in this retrospective analysis (Table 1). Initial bendamustine dose ranged from 50mg/m2 to 120mg/m2, with 11.5% of pts requiring dose attenuation. 55.8% received B and anti CD-20 antibody as ≥ 2nd line therapy. The incidence of ICs was 20% (n = 83; 95% CI: 16 to 24%) and 6% (n = 25; 95% CI 3.7-8.5%) of which was OI in this cohort (Table 2). The 25 OI cases consisted of viral (n = 19), fungal (n = 1), and PJP (n = 5). Nine cases occurred during B-based treatment and 16 cases occurred up to a year post (one was on R maintenance). All 25 cases were associated with either no ppx (n = 21), early ppx cessation (≤ 1 month post) (n = 2), or non-compliance (n = 2). One pt died of disseminated histoplasmosis 1.5 years after completed rituximab maintenance without additional treatment. Antimicrobial ppx, mainly anti-viral and anti-PJP, was employed in 36.1% of pts and primary or secondary G-CSF ppx in 64.7%. ICs were not associated with SLL/CLL histology (p = 0.471), R maintenance (p = 0.843), line of therapy (p = 0.804), and grade of lymphopenia (p = 0.554) or grade of neutropenia (p = 0.839) (Table 2). However, OI was associated with lack of antimicrobial ppx (p = 0.048). Other ADEs included grade 3/4 neutropenia (65.8%), NF (2.9%), grade 3/4 lymphopenia (76%), and elevated liver function tests (91.5% grade 1). The median absolute lymphocyte counts nadired after cycle 3 and persisted for at least 6 months following completion of bendamustine combination (Figure 1). Conclusions: The 20% incidence of infectious complication and 6% of opportunistic infection with bendamustine and anti-CD20 antibody combination at MSKCC are somewhat lower than that reported in prospective trials and retrospective analysis by Gafter-Gvili et al, possibly due to antimicrobial ppx and G-CSF use. We have implemented a prophylaxis guideline at MSKCC. Download : Download high-res image (277KB) Download : Download full-size image Download : Download high-res image (265KB) Download : Download full-size image Download : Download high-res image (121KB) Download : Download full-size image Disclosures Hamlin: Celgene: Membership on an entity's Board of Directors or advisory committees; Xencor: Membership on an entity's Board of Directors or advisory committees; Molecular Templates: Research Funding; Seattle Genetics: Research Funding; Gilead: Membership on an entity's Board of Directors or advisory committees; Portola: Research Funding; Novartis: Research Funding. Kumar: Celgene: Honoraria, Other: Scientific Advisory Board; Celgene: Research Funding; Adaptive Biotechnologies: Research Funding; Seattle Genetics: Research Funding; Pharmacyclics: Research Funding. Moskowitz: Seattle Genetics: Honoraria, Research Funding; Merck: Honoraria; Bristol Myers Squibb: Honoraria. Moskowitz: Merck: Membership on an entity's Board of Directors or advisory committees, Research Funding; Bristol-Myers Squibb: Membership on an entity's Board of Directors or advisory committees, Research Funding; Seattle Genetics: Membership on an entity's Board of Directors or advisory committees, Research Funding. Noy: Pharmacyclics, LLC, an AbbVie Company: Other: travel, accommodations, expenses, Research Funding. Zelenetz: Gilead Sciences: Research Funding.

KIT exon 8 mutations are located in the extracellular portion of the receptor and are strongly associated with core-binding factor (CBF)-acute myeloid leukemia (AML). To characterize the functional role of these mutants, we analyzed the proproliferative and antiapoptotic potential of 3 KIT exon 8 mutations in interleukin 3 (IL-3)-dependent Ba/F3 cells. All KIT exon 8 mutants induced receptor hyperactivation in response to stem cell factor (SCF) stimulation in terms of proliferation and resistance toward apoptotic cell death. A representative KIT exon 8 mutant showed spontaneous receptor dimerization, phosphorylation of mitogen-activated protein kinase (MAPK), and conferred IL-3-independent growth to Ba/F3 cells. MAPK and phosphatidylinositol 3-kinase (PI3-kinase) activation was essential for the phenotype of this mutant. Additionally, imatinib inhibited proliferation of KIT exon 8 mutant-expressing Ba/F3 cells. Our data show that KIT exon 8 mutations represent gain-of-function mutations and might represent a new molecular target for treatment of CBF leukemias. (Blood. 2005;105:3319-3321)

Safety and pharmacokinetics (PK) of recombinant von Willebrand factor (rVWF) combined at a fixed ratio with recombinant factor VIII (rFVIII) were investigated in 32 subjects with type 3 or severe type 1 von Willebrand disease (VWD) in a prospective phase 1, multicenter, randomized clinical trial. rVWF was well tolerated and no thrombotic events, inhibitors, or serious adverse events were observed. The PK of rVWF ristocetin cofactor activity, VWF antigen, and collagen-binding activity were similar to those of the comparator plasma-derived (pd) VWF-pdFVIII. In vivo cleavage of ultra-large molecular-weight rVWF multimers by ADAMTS13 (a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13; the endogenous VWF protease) and generation of characteristic satellite bands were demonstrated. In 2 subjects with specific nonneutralizing anti-VWF-binding antibodies already detectable before rVWF infusion, a reduction in VWF multimers and VWF activity was observed. Stabilization of endogenous FVIII was enhanced following post-rVWF-rFVIII infusion as shown by the difference in area under the plasma concentration curve compared with pdVWF-pdFVIII (AUC0-∞) (P < .01). These data support the concept of administering rVWF alone once a therapeutic level of endogenous FVIII is achieved.

Mutations in codon D816 of the KIT gene represent a recurrent genetic alteration in acute myeloid leukemia (AML). To clarify the biologic implication of activation loop mutations of the KIT gene, 1940 randomly selected AML patients were analyzed. In total, 33 (1.7%) of 1940 patients were positive for D816 mutations. Of these 33 patients, 8 (24.2%) had a t(8;21), which was significantly higher compared with the subgroup without D816 mutations. Analyses of genetic subgroups showed that KIT-D816 mutations were associated with t(8;21)/AML1-ETO and other rare AML1 translocations. In contrast, other activating mutations like FLT3 and NRAS mutations were very rarely detected in AML1-rearranged leukemia. KIT mutations had an independent negative impact on overall (median 304 vs 1836 days; P = .006) and event-free survival (median 244 vs 744 days; P = .003) in patients with t(8;21) but not in patients with a normal karyotype. The KIT-D816V receptor expressed in Ba/F3 cells was resistant to growth inhibition by the selective PTK inhibitors imatinib and SU5614 but fully sensitive to PKC412. Our findings clearly indicate that activating mutations of receptor tyrosine kinases are associated with distinct genetic subtypes in AML. The KIT-D816 mutations confer a poor prognosis to AML1-ETO-positive AML and should therefore be included in the diagnostic workup. Patients with KIT-D816-positive/AML1-ETO-positive AML might benefit from early intensification of treatment or combination of conventional chemotherapy with KIT PTK inhibitors.

Abstract 2171 Myeloid derived suppressor cells (MDSC) are a heterogeneous population of cells that accumulate during tumor progression in a process driven by soluble factors such as granulocyte-macrophage colony stimulating factor (GM-CSF). These cells contribute to the suppressive nature of the tumor microenvironment and interfere with the functions of cytotoxic anti-tumor T effector cells. To date, MDSC heterogeneity has presented a barrier to studying the properties of individual MDSC constituents in vivo. Herein, we find that GM-CSF, a cytokine that promotes the numeric and functional development of monocytes, granulocytes and dendritic cells, and is frequently used as a vaccine adjuvant, is also critical for the expansion of a monocyte-derived MDSC population characterized by the expression of CD11b and the chemokine receptor CCR2. We demonstrate that these cells mediate T cell suppression in a contact dependent fashion and via the function of Arginase and inducible nitric oxide synthase, consistent with known MDSC functions. CD11b+CCR2 negative cells do not have suppressive capability despite also being expanded numerically by the actions of GM-CSF. Utilizing a toxin-mediated ablation strategy that targets CCR2-expressing cells, we demonstrate that monocytic MDSCs regulate activated CD8 T cell entry into the tumor site in vivo, thereby limiting the efficacy of immunotherapy. Our results extend observations on the dual role of GM-CSF in both stimulation and suppression of tumor immunity and suggest therapeutic targeting of monocytic MDSC could enhance the outcomes of immunotherapy. Disclosures: No relevant conflicts of interest to declare.

Mutated FLT3 defines a promising target for the treatment of acute myeloid leukemia (AML) with specific protein tyrosine kinase (PTK) inhibitors. The clinical efficacy of this approach, however, is limited due to molecular mechanisms that remain to be elucidated. As we demonstrated previously, overexpression of antiapoptotic proteins of the BCL2 family lead to resistance against PTK inhibitors in cell lines with activating FLT3 mutations (Bagrintseva, Blood, 2005). In primary AML samples tested so far in our study, we found a correlation of the expression level of BCL-XL, a known downstream target of FLT3, and the presence of activating FLT3 mutations whereas MCL1 protein, another antiapoptotic member of the BCL2 family, showed very low expression. In contrast, we found a high expression level of BCL2 protein in all AML samples whether or not FLT3 mutations were present and this level remained unchanged after dephosphorylation of mutated FLT3. Thus, we speculated that an overexpression of BCL2 independent from FLT3 activation might at least in part explain the limited clinical efficacy of PTK inhibitors in the treatment of FLT3 positive AML. To test this hypothesis, we stably expressed mock, BCL2 or BCL-XL, respectively, in Ba/F3 cell lines carrying constitutively activated FLT3 with internal tandem duplication. The cells overexpressing BCL2 or BCL-XL, respectively, did not respond to treatment with the FLT3 specific inhibitor SU5614 up to high doses. To overcome the observed resistance, we tested the small molecule inhibitor ABT-737 (kindly provided by Abbott Laboratories) that has been described to efficiently disrupt intracellular BCL2 family interactions by binding to the hydrophobic BH3 groove of these proteins (Oltersdorf, Nature, 2005). Surprisingly, treatment of our generated cell lines with ABT-737 alone did not result in increased levels of apoptotic cell death. This finding is in line with previous reports showing that mono-treatment with ABT-737 does not directly activate proapoptotic proteins, but needs activator BH3-only proteins such as BID or BIM. Co-treatment of the cell lines with SU5614 and ABT-737, however, rendered them again susceptible to the PTK inhibitor in a concentration-dependent manner. SU5614 and ABT-737 showed synergism as confirmed by immunoblotting against cleaved and full-length caspase-3. As a negative control to all our experiments, we used the functionally inactive enantiomer of ABT-737 (ABT control) that caused significant cytotoxicity neither alone nor in combination with SU5614 up to high doses. To underline the clinical relevance of these findings, a panel of AML patient samples is currently tested for response to ABT-737 alone or in combination with PTK inhibitors. Two AML samples tested so far showed an IC50 of 10 and 25nM (vs. 300 and 1000 nM, respectively, for ABT control) after 24h of mono-treatment with ABT-737, whereas peripheral blood mononuclear cells of a healthy donor showed an IC50 of 80 nM for ABT-737 and 400nM for ABT control. This might be explained by recent findings indicating that native tumor and leukemia cells are addicted to the expression of antiapoptotic proteins and tonically exposed to proapoptotic stimuli (Certo, Cancer Cell, 2006). Since BCL2 has been reported to be involved in cell cycle regulation by facilitating G0/G1 arrest, we are also going to study the effects of ABT-737 on non-proliferating CD34+ progenitor AML cells that cannot be eliminated by conventional chemotherapy.

Von Willebrand factor (VWF) is a multimeric protein that mediates adhesion of platelets to sites of vascular injury and is therefore essential for primary haemostasis. The modulation of VWF multimers size is critical to the control of the hemostatic activity, where the large multimeric forms are most hemostatically active. It has been shown that various proteases, such as Thrombospondin-1 (facilitates reduction of the disufide bonds), are critical in the degradation of VWF multimers. This might be an important aspect for VWF coagulation factor concentrates used in the treatment of von Willebrands disease, best treated with high molecular weight VWF multimers and least content of human TSP-1 (huTSP-1). Determination of huTSP-1 (ChemiKine EIA Kit der Fa. Chemicon International, USA) were done in 11 commercially available VWF/FVIII-concentrates (3 different batches each) in duplicates. HuTSP-1 content in the VWF/FVIII concentrates varied from 0,08 ug/mL to 12,7 ug/mL (variance in all duplicates less than 15%), as shown in the figure. The results show a significant difference in huTSP-1 content. VWD patients might receive as much as 250 ug huTSP-1 or even higher during a single treatment course. If the huTSP-1 is still active, requires further investigations. Figure Figure

Experimental data have shown that two of the most frequent genetic alterations in AML, the AML1-ETO (A/E) fusion gene and the FLT3 length mutation (FLT3-LM) are both mostly insufficient on their own to induce leukemia. These findings support the model that collaboration of two classes of genetic alterations, altering proliferation or differentiation, is necessary for leukemogenesis. When we first analyzed 135 patients with A/E positive AML, additional mutations affecting signal transduction were found in 38 % of all cases (FLT3-LM 10.3 %, KIT 8.1 % and NRAS 9.6 %). In contrast, none of the patient with A/E positive leukemia had alterations associated with transcriptional regulation such as MLL-PTD. To test the hypothesis that A/E collaborates with FLT3-LM in inducing acute leukemia, we transplanted mice with bone marrow (BM) cells retrovirally expressing A/E, FLT3-LM or both alterations. Mice transplanted with BM cells expressing A/E or FLT3-LM alone did not develop any disease. In contrast, mice (n=11) transplanted with BM cells expressing both alterations succumbed to an aggressive acute leukemia. Intriguingly, developing leukemias differed with regard to their phenotype with 7 animals developing AML and 4 animals developing ALL. Furthermore, the majority of AML cases showed simultaneous expression of lymphoid antigens as described in patients with A/E positive AML. The collaboration of A/E with FLT3-LM was depending on DNA binding activity of the fusion gene as the L148D point mutation in the Runx1 domain of the construct abrogated collaboration of A/E with the FLT3-LM in the CFU-S assay. Furthermore, inactivation of the kinase activity of the FLT3-LM (FLT3-LM K672R mutant) resulted in the complete loss of collaboration with the A/E fusion. Treatment of cells co-infected with A/E and FLT3-LM with the kinase inhibitor PKC412 resulted in a 62 % reduction of the CFU-S frequency. To further explore a possible contribution of retroviral insertional mutagenesis to the transformation process in this model, 10 retroviral integration sites were subcloned and sequenced from 4 leukemic mice: all 10 sites were unique with no indication of a common integration site associated with the leukemic transformation. Moreover, 5 sites were intergenic or not linked to known genes. The remaining sites were in introns in a 5′ to 3′ orientation most likely to lead to gene knockdown rather than activation. These data provide direct functional evidence for the oncogenic collaboration between A/E with a class of activating mutations, recurrently found in patients with t(8;21), and add experimental data to the clinical observation which demonstrated a significant inferior treatment outcome in patients with AML1-ETO and additional mutations of receptor tyrosine kinases.

Mutations within the FLT3-gene are of growing importance for classification, risk assessment and therapeutic targeting in acute myeloid leukemia (AML). An increasing number of activating mutations have been reported during the last few years. A D324N variant located in the extracellular region of the FLT3 protein has been described recently in 4/94 (4.3%) of AML patients (Ley TJ et al., PNAS 2003). We have analyzed 705 de novo AML for D324N using a LightCycler based screening assay and found a gac to aac change in codon 324 in 43 cases (6.1%). This is approximately the same frequency that has been described for tyrosine kinase domain mutations in FLT3. However, in contrast to other FLT3 mutations the D324N was associated with a low leucocyte count (6.700/μl) and had no association to any AML subtype nor a prognostic impact regarding overall survival and event free survival (235 D324N- cases vs. 13 D324+ cases with normal karyotype analyzed). To analyze whether this FLT3 variant might be a polymorphism we analyzed peripheral blood of 329 healthy donors with a similar ethnic background. In this population we could also detect the D324N variant, but only in 4 cases (1.2%). This difference between AML and healthy donors was statistically significant (p=.0001). Three of the cases were heterozygous and one was homozygous for the D324N variant. Of one of the heterozygous cases a buccal smear was evaluated and the same heterozygous pattern could be detected in this material. In addition, of three D324N positive AML at diagnosis a sample from any time point in CCR was available that was negative for the leukemia clone with a sensitivity of 10−4 to 10−6 as assessed by quantitative PML-RARA- (1 case) or CBFB-MYH11- (1 case) specific PCR or by immunophenotyping (1 case). In these remission samples again a 50% ratio of the normal and the D324N variant was detectable. To functionally characterize the FLT3-D324N in vitro, FLT3-WT, FLT3-D324N, and FLT3-ITD cDNA were retrovirally transduced into IL-3 dependent Ba/F3 cells. Stably expressing cell lines were grown for 72h in the absence of IL-3 with varying doses of human FLT3 ligand (FL) and the number of viable cells was assessed by trypan blue exclusion. In contrast to FLT3-ITD expressing cells, FLT3-D324N transduced cells were not able to grow in the absence of IL-3. The growth of FLT3-WT and FLT3-D324N, but not vector expressing cell lines could be stimulated by exogenous FL in a dose-dependent manner. No significant difference could be demonstrated between FLT3-WT and FLT3-D324N cells. In apoptosis assays using annexin-V-PE and 7-AAD staining FL stimulation protected both D324N mutant and FLT3-WT expressing Ba/F3 cells from apoptotic cell death to a similar degree. These results strongly support the hypothesis that the D324N variant in the FLT3 gene represents a functionally silent polymorphism. The fivefold higher frequency in patients with AML compared to healthy donors raises the question whether this FLT3 variant is associated with a higher risk for AML.

Mutations in the extracellular portion of the KIT receptor tyrosine kinase (exon 8 mutations) are strongly associated with core binding factor (CBF) - acute myeloid leukemia (AML), but the functional role of these mutations has not been elucidated. In 93% of cases, codon Asp419 is deleted and exon 8 mutations were reported to confer an impaired prognosis to patients with CBF-AML. In this study, we are the first to report pro-proliferative and antiapoptotic potential of representative KIT exon 8 mutations in a cell culture model and to show a significant difference to KIT wildtype (KIT-WT). Three representative exon 8 mutants including a single deletion of codon 419 were created by in vitro site-directed mutagenesis. The integrity of all constructs was assessed by complete nucleotide sequencing. After stable expression in IL-3 dependent Ba/F3 cells (confirmed by FACS analysis and immunoblotting), exon 8 KIT mutants were characterized by a hypersensitivity to stem cell factor (SCF) stimulation in terms of proliferation and resistance to apoptotic cell death. The differences to KIT-WT occurred in the physiological range of SCF from 1 to 10ng/ml. The proliferative response caused by stimulation with SCF was reversed in KIT-WT and exon 8 mutants in the presence of Imatinib® (Novartis) in contrast to the activation loop mutant D816V which could not be inhibited. These biological effects were confirmed by demonstrating increased phosphorylation of the KIT downstream targets mitogen-activated protein kinase (MAPK) and AKT after SCF stimulation compared to the KIT-WT receptor. Furthermore, the MEK inhibitor PD98059 and the PI3 kinase inhibitor LY294002 resulted in a dose dependent inhibition of SCF induced proliferation in exon 8 mutants. Our data show that KIT exon 8 mutations represent gain-of-function mutations by inducing receptor hypersensitivity to its ligand SCF by activation of MAPK and PI3K and might represent a new molecular target for treatment of CBF leukemias.