Bone Marrow Cells Of Healthy Donors Research Articles

Novel myeloperoxidase-derived HLA-A2-restricted peptides as therapeutic targets against myeloid leukemia

Background aimsHuman myeloperoxidase has been shown to be overexpressed in many types of leukemia, such as chronic myeloid leukemia, acute myeloid leukemia and myelodysplastic syndrome. The authors identified two myeloperoxidase-derived HLA-A2-restricted peptides, MY4 and MY8, as novel leukemia-associated antigens. MethodsEx vivo-elicited MY4- and MY8-specific cytotoxic T lymphocytes were generated, and tested for leukemia cell lysis in vitro and in NOD/SCID AML xenograft model. ResultsThese MY4- and MY8-specific cytotoxic T lymphocytes killed leukemic blasts while sparing healthy donor bone marrow cells. In addition, co-injection of MY4- and MY8-specific cytotoxic T lymphocytes into nonobese diabetic/severe combined immunodeficiency mice with acute myeloid leukemia drastically reduced tumor burden in vivo. The authors also found that MY4- and MY8-specific T cells could be detected in the peripheral blood mononuclear cells of allogeneic stem cell transplant recipients. ConclusionsThese antigen-specific T cells were significantly increased in blood samples from patients compared with healthy donors, suggesting that both MY4 and MY8 are immunogenic and that MY4- and MY8-specific cytotoxic T lymphocytes may play a role in reducing leukemia in vivo. Thus, the discovery of MY4 and MY8 as novel leukemia-associated antigens paves the way for targeting these antigens in immunotherapy against myeloid leukemia.

Integrative Study of EZH2 Mutational Status, Copy Number, Protein Expression and H3K27 Trimethylation in AML/MDS Patients

Introduction Enhancer of Zeste Homolog 2 (EZH2), the catalytic domain of Polycomb Repressive Complex 2 (PRC2), mediates the repressive mark of trimethylation of histone H3 lysine 27 (H3K27me3). The EZH2 gene is located on chromosome (chr.) 7q36.1 (frequently deleted in AML/MDS). Its role in tumorigenesis appears to be context-dependent since both EZH2 overexpression and loss of function are associated with different types of cancer. In patients (pts) with MDS or MDS/MPN, loss-of-function EZH2 mutations (mut) are recurrently found, associated with a poor prognosis, and EZH2 is considered a tumor suppressor gene (TSG). In AML, the incidence of EZH2-mut is lower and less well-studied. We wished to determine the effects of EZH2-mut and copy number (CN) on EZH2 expression, and the consequences for H3K27me3 levels in vivo. Methods Fifty-eight pts (51 AML, 3 MDS, 4 MDS/MPN), median age 63.5 years (yr, range 20-86, almost all diagnosed between 2015 and 2017), with available sequencing data, EZH2 CN status and EZH2 expression data were studied. Mutation status was determined by next-generation sequencing (NGS) including a panel of 54 genes (Illumina Myeloid NGS panel). Metaphase cytogenetics and/or fluorescence in situ hybridization and single nucleotide polymorphism arrays (selected pts) were conducted to determine EZH2 CN status. Protein expression of EZH2 and H3K27me3 was assessed semi-quantitatively by immunohistochemistry (IHC) on formalin-fixed EDTA-decalcified paraffin-embedded bone marrow (BM) core biopsies. The Kruskal-Wallis test and Dunn's multiple comparison test were used to address whether EZH2 protein expression was associated with EZH2-mut and CN. Results The EZH2 gene showed mutations (mostly missense or nonsense, median variant allele frequency (VAF) 45%, range 7-63%) in 13/58 pts and was unmutated in 45/58 pts. EZH2-mut pts had a median of 4 mutations. Additional mutations were most frequently found in ASXL1 (10/13; median VAF 35%, range 19-48%), less frequently in TET2, RUNX1, STAG2 (3/13 each), NRAS (2/13), and DNMT3A (1/13). In contrast, EZH2-wt pts had a median of 2 mutations, most frequently in DNMT3A (12/45), followed by NRAS (10/45), IDH1 (9/45), FLT3 (8/45), and NPM1 (7/45). Regarding chr. 7, 43 pts had no detectable deletion, 15 had 7q-/-7. Notably, the incidence of EZH2-mut was similar in pts with 7q-/-7 lesions (3/15, i.e. 20%) and pts with normal chr. 7 (10/43, 23%). EZH2 expression in neoplastic BM cells ranked from no (score 0) to strong expression (score 3). While the hematopoietic BM cells of healthy donors usually showed a moderate EZH2 expression (score 2), in our cohort the score was 0 in 3, 1 in 13, 2 in 23, and 3 in 19 pts, respectively. We next asked whether EZH2 protein expression differed between pts depending on EZH2-mut and chr. 7 status. In Figure 1, 4 subgroups are depicted, showing highest expression in pts with EZH2-wt and either no chr. 7 abnormalities (group A, n=33) or 7q-/-7 (group C, n=12). In comparison, expression was significantly reduced in pts with EZH2-mut and no chr. 7 abnormalities (group B, n=10), and lowest in pts with EZH2-mut and chr. 7 abnormalities (group D, n=3), p<0.05 (indicated by asterisks). Since functional EZH2 protein is necessary for the trimethylation of H3K27, the presence of this histone mark was also determined semi-quantitatively by IHC in 40 pts. H3K27me3 levels were variable, and a test for a possible association between EZH2 and H3K27me3 levels by linear regression analysis did not show an association (R²=0.13). Sixty-two % of EZH2-mut (median age 71 yr) and 51% of EZH2-wt pts (median age 61 yr) received allografting. Median overall survival in EZH2-mut pts was 16.1 months compared to 23.5 in EZH2-wt pts (p=0.16). Conclusions Inactivating EZH2 mutations are infrequent events in AML. In our study, they were strongly associated with mutations of ASXL1 (also involved in PRC2 function). We did not observe overrepresentation of EZH2-mut in 7q-/-7 pts, in line with Bejar et al. (N Engl J Med 2011); this is in contrast to the frequent cooperative events between TSG mutations and deletions, e.g. of TP53. Functionally, EZH2 mutations appeared to have a stronger effect on decreased EZH2 expression than hemizygosity. Global H3K27me3 levels were not altered by EZH2 reduction, which could be due to compensatory upregulation of EZH1, supporting the clinical development of EZH1/2 inhibition. Disclosures No relevant conflicts of interest to declare.

Abstract 935: Generation and preclinical characterization of CD123-CPI antibody-drug conjugate (ADC)

Abstract CD123 is expressed on cancer cells in a variety of hematologic malignancies including acute myeloid leukemia (AML). CD123 is frequently expressed on leukemic blasts and leukemic stem cells (LSCs), a cell population associated with relapse in patients, but there is minimal or no expression on most normal hematopoietic cells and solid tissues. Thus, CD123 is a promising target for AML. We have generated an anti-CD123 ADC that carries cyclopropylpyrroloindoline (CPI) payload that crosslinks DNA. A drug loading of 2 molecules of CPI per molecule of antibody was achieved by site-specific conjugation using our transglutaminase methodology. Upon binding to the CD123 antigen, CD123-ADC is internalized and delivered to the endosomal-lysosomal pathway whereupon the CPI payload is released from the antibody by proteolysis of the linker. The released CPI alkylates DNA, which activates ATR/ATM, CHK1, CHK2 and FANCD2, ultimately resulting in cell death. In vitro, CD123–CPI elicited cytotoxicity in a dose-dependent manner against several CD123-positive, but not against CD123-negative cell lines. Cell lines with higher CD123 expression level were more sensitive to the ADC. Long-Term Culture-Initiating Cells in vitro assay showed that AML patient bone marrow samples that naturally have high percentage of LSCs yielded substantially fewer colonies in CD123-ADC treated cells compared to control ADC. Importantly, CD123-ADC had no adverse effects in healthy donor bone marrow cells experimentally enriched in CD34+ primitive stem cells. This result suggests that CD123-ADC specifically inhibits the growth potential of leukemic blasts and progenitor cells. In vivo, robust antileukemic activity was observed in CD123-positive AML cell line-derived xenograft models. Low doses of CD123-ADC effectively regressed tumors whereas the tumor progressed in mice that received control ADC. Efficacy was also evaluated in disseminated AML PDX models (n≥7) established with patient samples of various cytogenetics and molecular abnormalities, and relapse/refractory. Flow cytometry analyses of leukemic load in peripheral blood and in bone marrow samples of mice showed that CD123-ADC was efficacious in reducing tumor burden even at the suboptimal doses. In summary, our data demonstrate that our CD123-ADC is highly active in a broad panel of primary AML samples. Currently, CD123-ADC in combination with other AML therapies is being tested in vivo. All these attributes of CD123-ADC make it an attractive agent to evaluate in clinical trials. Citation Format: Yoon-Chi Han, Fan Jiang, Nicole Piche-Nicholas, Madan Katragadda, Nadira Prashad, Manoj Charati, Wendy Hu, Mauricio Leal, Nathan Tumey, Andreas Maderna, Russell Dushin, Kenny Kim, LuAnna Lemon, Marc Damelin, HP Gerber, Lioudmila Tchistiakova, Frank Loganza, Chris O'Donnell, Puja Sapra. Generation and preclinical characterization of CD123-CPI antibody-drug conjugate (ADC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 935.

Abstract 17412: Paracrine Role of B Lymphocytes in Successful Bone Marrow Cell Therapy for Myocardial Infarction

Introduction: Treatment of myocardial infarction (MI) with bone marrow cells (BMCs) or cell-free BMC lysate substantially improves post-MI cardiac function in rodent experiments. However, clinical trials of BMC therapy have been less successful. While most mouse experiments use young healthy BMC donors, MI patients undergoing autologous cell therapy (i.e., receiving their own BMCs) are older and are post-MI. We have previously demonstrated that BMCs from aged and post-MI donor mice are therapeutically impaired, that donor MI induces inflammation leading to changes in BMC composition, and that BMCs from aged or post-MI mice have reduced levels of B cells, which reportedly can be a therapeutic BMC subpopulation. We recently observed that partial depletion of B cells from young healthy donor BMCs reduces the BMCs’ therapeutic effect, mimicking the effects of donor age or MI. Hypothesis: B cells play a crucial role in BMC therapy for MI, and B cell lysate is also therapeutic. Methods: We induced permanent coronary artery ligation MI, and implanted purified CD19+ bone marrow B cells from young healthy donors into the recipient infarct border zone 3 days post-MI via ultrasound guidance. CD3ε+ T cells and vehicle (HBSS) were injected as negative controls. A similar experiment was carried out with B cell lysate and T cell lysate. Results: In all groups, recipient left ventricular ejection fraction (EF) declined uniformly from baseline to day 2 post-MI, before cell injection. Injection of B cells significantly improved day 28 EF (p<.0005 adjusted for multiple comparisons). In contrast, T cells provided little or no benefit relative to HBSS. Notably, B cell lysate, but not T cell lysate, increased EF comparably to intact B cells. *p<.05 in figure. Conclusions: Bone marrow B cells play a crucial role in BMC therapy for MI via a paracrine mechanism. Reduction of bone marrow B cells by age or MI may explain why autologous BMC therapy in patients has not matched the success of rodent experiments.

Identification of Novel Therapeutic Strategies for NUP98-NSD1-Positive AML By Drug Sensitivity Profiling

BackgroundThe t(5;11)(q35;p15.5) translocation resulting in fusion of the nucleoporin NUP98 and methyltransferase NSD1 (NUP98-NSD1) genes is a recurrent aberration observed in pediatric and adult AML. The NUP98-NSD1 fusion often co-occurs with the FLT3-ITD mutation and characterizes a group of cytogenetically normal AML patients with very poor prognosis. Despite advances in the understanding of the biology of NUP98-NSD1-positive AML, its therapeutic success rate has remained low. We aimed to identify novel candidate drugs for NUP98-NSD1-positive AML by testing primary patient cells and in vitro cell models with a high-throughput drug sensitivity platform.MethodsLeukemic blasts were Ficoll separated from bone marrow (BM) aspirates of an AML patient positive for t(5;11)(q35;p15.5) and FLT3-ITD. RNA extracted from primary cells was used for RNA sequencing and gene expression analysis. NUP98-NSD1 cDNA was amplified from primary cell RNA and expressed from a lentiviral vector (LeGO-iCer2) also encoding the cerulean fluorescent marker. The NUP98-NSD1/LeGo-iCer2 and empty LeGo-iCer2 viruses were used to establish stably expressing Ba/F3 cell lines. Primary murine (BALB/c) BM cells were transduced with NUP98-NSD1 and FLT3-ITD retroviruses alone or in combination (NNF) in vitro (“preleukemic”) or passaged in vivo (“leukemic”) as previously described (Thanasopoulou et al, 2014). For screening, 309 small molecule inhibitors including FDA/EMA-approved and investigational oncology drugs were plated on 384-well plates in a 10,000-fold concentration range. Cells were dispensed on the pre-drugged plates and incubated at 37°C for 72h, and then cell viability measured using the CellTiter-Glo® luminescent assay. Drug response curves were generated and a drug sensitivity score determined (Yadav et al, 2014). Select drug sensitivity was calculated for each drug by comparing results between primary leukemic and healthy donor BM cells or between the cell constructs and empty vector transduced controls cells.ResultsPrimary patient cells and murine BM cells expressing FLT3-ITD alone or in combination with NUP98-NSD1 were selectively sensitive to specific FLT3 inhibitors (e.g. quizartinib, sorafenib and lestaurtinib), and broad-spectrum receptor tyrosine kinase inhibitors targeting FLT3-ITD (e.g. cabozantinib, crenolanib, foretinib, midostaurin, MGCD-265 and ponatinib). Furthermore, these cells were highly sensitive to checkpoint kinase 1/2- inhibitor AZD7762. The primary murine cells expressing both NUP98-NSD1 and FLT3-ITD showed higher sensitivity to all of the above-mentioned drugs compared to cells expressing either of the events alone indicating functional synergy. A very distinct drug response pattern was observed in the leukemic NNF cells cultured in vivo compared to the same cells cultured in vitro suggesting that microenvironment may also affect the observed drug responses. Interestingly, the preleukemic murine cells expressing NUP98-NSD1 with or without FLT3-ITD as well as the primary patient cells showed extreme vulnerability to BCL2/BCL-xL inhibitor navitoclax. Furthermore, primary murine cells expressing NUP98-NSD1 alone showed high select sensitivity to JAK-inhibitors ruxolitinib, BMS-911543, AZD1480 and tofacitinib indicating the fusion may stimulate JAK/STAT-signaling. Similar sensitivity was also observed in the Ba/F3-cells expressing NUP98-NSD1. In support of these findings, gene expression analyses showed high expression of anti-apoptotic factors BCL2, BCL-xL and MCL1 in the patient cells. MCL1 is regulated by STAT3 while BCL-xL is regulated by STAT5, which were also highly expressed.ConclusionsIn summary, we have observed an enhanced response to specific and non-specific FLT3 inhibitors in cells expressing NUP98-NSD1 and FLT3-ITD together compared to cells expressing either of the two alone. This coincides with previous findings that functional co-operation between NUP98-NSD1 and FLT3-ITD is important in AML (Thanasopoulou et al, 2014). We have seen high in-vitro-in-vivo correlation between primary patient cells and murine cells expressing NUP98-NSD1 and FLT3-ITD. Moreover, we have identified potential candidate compounds targeting oncogenic signaling activated by these two events. These data form a basis for clinical evaluation of candidate compounds for NUP98-NSD1-positive AML. DisclosuresPorkka:Bristol-Myers Squibb: Honoraria, Research Funding; Novartis: Honoraria, Research Funding. Heckman:Celgene: Research Funding.

Hoxa9/Meis1 Mediate Leukemic Programming through Microrna-155

Synergistic deregulation of HOXA9 and the HOX-gene cofactor MEIS1 is a commonly observed phenomenon in acute myeloid leukemia (AML). The leukemogenic potential of aberrant Hoxa9 and Meis1 expression has been shown in several AML models. However, the molecular mechanisms behind Hoxa9- and Meis1-induced leukemogenesis are still not well understood.In order to identify functionally relevant Meis1-induced microRNAs (miRNA), we profiled the global miRNA expression using a Hoxa9-Meis1 murine AML progression model. This two-step model allowed us to quantify miRNAs at a pre-leukemic stage through the overexpression of the proto-oncogene Hoxa9 (Hoxa9/ctrl), as well as after full leukemic transformation through co-overexpression of Hoxa9 and Meis1 (Hoxa9/Meis1). The pre-leukemic stage is characterized by in vitro immortalization without in vivo engraftment, whereas the transplanted leukemic cells induce full-blown AML in vivo. MiR-155 turned out to be one of the most significant differentially expressed miRNA species and its upregulation was independently validated in Hoxa9/Meis1 cells by qRT-PCR.Subsequent analysis of various AML subtypes (CN-AML, t(11q23), t(8;21), t(15;17), n=38) showed significantly elevated levels of miR-155 in CN-AML with NPM1mut (n=10, p<0.01) and AML with t(11q23) (n=8, p<0.05) compared to healthy donor bone marrow (MNC). These results are in line with overexpression of HOXA9 (CN-AML NPM1mut: p<0.05, t(11q23): p<0.05) and MEIS1 (CN-AML NPM1mut: p<0.01, t(11q23): p<0.05) in these AML samples compared to healthy donor bone marrow cells (MNC). Expression analysis of miR-155 in healthy murine bone marrow (mbm) cells revealed miR-155 enrichment in hematopoietic stem- and progenitor cells compared to mature myeloid cells (p<0.05), mirroring a similar expression pattern as observed for Meis1.Therefore, to dissect the leukemic potential of miR-155 to program mbm, 5-FU-stimulated mbm cells were retrovirally transduced with miR-155, leading to significantly increased proliferation in vitro (p<0.05). This finding suggests enhancement of self-renewal on the stem-/progenitor cell level by miR-155. Furthermore, mbm cells overexpressing Hoxa9 together with miR-155 (Hoxa9/miR-155) significantly increased colony formation (p<0.05) in a methylcellulose assay. In turn, absence of miR-155 (miR-155-/- mbm) significantly reduced colony formation in conjunction with Hoxa9 (p<0.05) and MLL-AF9 (p=0.05), a known positive regulator of Hoxa9 and Meis1. These findings suggest a role for miR-155 in both proliferation and self-renewal indicating that the oncogenic program of Hoxa9/Meis1 relies on the presence of miR-155.The leukemic potency of Hoxa9/miR-155 was further investigated in a murine transplantation model in vivo. Transplantation of mbm co-overexpressing Hoxa9/miR-155 led to significantly increased engraftment levels already after four weeks (wks) (57.8%±31.3, n=16) compared to Hoxa9/ctrl (11.7%±19.3%, p<0.0001, n=17), but less than with Hoxa9/Meis1 (74.5%±20.3%, p<0.01, n=14). In contrast to Hoxa9/ctrl (22±7 wks), mice that received Hoxa9/miR-155 mbm cells had a significantly accelerated onset of a myeloproliferative disease (MPD)-like leukemia within 11 wks (11±6 wks, p<0.0001), but still a less aggressive course of disease compared to mice transplanted with Hoxa9/Meis1 (5±1 wks, p<0.0001). This result is striking considering the aggressive nature of the Hoxa9/Meis1 AML model and given how little is known about its central mechanisms. It also highlights the relevant contribution of miR-155 to the leukemic programming induced by Hoxa9/Meis1 and provides a further rational to target miR-155 in AML.Considering the central role of the Hoxa9/Meis1 in both myeloid and lymphoid acute leukemias, we demonstrate for the first time the leukemogenic relevance of a miRNA within this transcriptional axis. DisclosuresNo relevant conflicts of interest to declare.

Influence of Different Genetic Alterations On the Engraftment Capacity and Serial Transplantation Efficiency of AML Patient-Derived Bone Marrow and Peripheral Blood Cells in NSG Mice

AbstractAbstract 3511 Introduction:In order to allow a better understanding of acute myeloid leukemia (AML) and develop more promising therapeutic strategies the establishment of functional and reproducible in vivo models is widely pursued. Of available model systems, xenografts in immunodeficient mice reproduce the clinical situation best. Here, we performed extensive analysis of AML engraftment in NOD/SCID-IL2-receptor-gamma-chain−/− (NSG) mice comparing tail vein versus intratibial injection and growth behavior of AML patient-derived bone marrow versus peripheral blood cells. Furthermore, tumor growth characteristics in the murine host were correlated with the disease stage and the molecular risk factor profile of the individual donors. Methods:Bone marrow and peripheral blood cells from 17 AML patients were injected intratibially into NSG mice (n=4–8/patient, 82 mice in total). As controls, 14 mice received bone marrow from three different donors and 5 mice were mock-injected. Tumor growth was monitored via a) determination of overall survival, b) fluorescence-based in vivo imaging (IVI, Kodak FX, Alexa750 labeled anti-human CD45 or CD33 and c) confirmation of IVI data by histological and immunohistochemical examination of bone marrow and spleen. When highly positive IVI signals and/or the overall condition of individual mice indicated enlarged tumor burden, the respective animals were sacrificed and the human AML cells transferred to another animal. In parallel the engraftment pattern of AML cells 2–4 weeks posttransplant was correlated with clinical disease activity, application route and origin of the particular tumor cells. Results:Patients included in the present study represent multiple different French-American-British (FAB) subtypes, various karyotypes and molecular features in terms of the mutational status of NPM1 and FLT3. All patient-derived specimens were capable of recapitulating the disease in NSG mice at 4–6 weeks after transplantation. Over a period of 13 months 12 out of 17 xenografts could be passaged once and 9 at least twice. Up to six passages were performed for an individual AML xenograft. In contrast, engraftment of healthy donor bone marrow cells could be determined merely until day 56 after implantation. The human bone marrow cells of the healthy donors did not engraft in serial passages. Mean survival time of AML bearing animals ranged between 21 and 82 days for a respective xenograft. No differences could be determined between engraftment capacities of peripheral blood or bone marrow cells of one patient. Neither karyotype, FAB classification nor leucocyte count or the percentage of monomorphic blasts in the bone marrow seemed to have an impact on engraftment capacity in the murine organism. However, mice bearing AML xenografts with mutations in FLT3 as well as in NPM1 showed particular short overall survival times and high tumor cell engraftment determined by IVI. This phenomenon became more obvious along the different passages. The intratibial approach proved to be superior in comparison to the intravenous application as cells of an individual patient engrafted faster when injected directly into the bone marrow microenvironment. Determination or tumor load via IVI permits to closely monitor not only the growth behavior but also the homing characteristics of the human cells over time. The positive IVI signals in bone marrow and spleen could be confirmed by histological examination as well as by immunohistochemistry specific for human CD45 and CD33. Conclusions:Our xenografts show a close resemblance to the AML-disease regarding the level of dissemination and organ involvement. Collection of whole-body IVI data proved to be a time- and animal-saving analysis that allows to closely monitor AML growth. As AML is characterized by an increasing number of molecular subtypes with completely different therapeutic options it seems to be extremely worthwhile to develop patient derived xenograft models representing as many AML subtypes as possible. Our results suggest that this model reflects the heterogeneity and important clinical characteristics of the disease, and thus may serve as a tool for preclinical drug testing and investigation of the pathophysiology of AML. Disclosures:No relevant conflicts of interest to declare.

3,4-Diarylmaleimides Effectively Inhibit Proliferation of FLT3-ITD-Positive Leukemic Cells, Induce Apoptosis and Show Additive Effects in Combination with Chemotherapeutic Drugs.

Internal tandem duplication (ITD) mutations of FLT3 are present in leukemic blasts of approximately 30% of AML patients. ITD-mutations of FLT3 confer a worse prognosis and decreased overall survival. Therefore, FLT3-tyrosine kinase is considered an attractive drug target in AML and several FLT3-tyrosine kinase inhibitors (TKIs) are currently being tested in clinical trials (CEP701, MLN518, Sorafenib, PKC412). However, using these drugs as monotherapy, against the setting of remarkable efficacy has emerged the problem of short duration of remission indicating rapid development of secondary resistance. In addition, up to 30% of patients may show primary resistance to currently available FLT3-TKIs. Therefore, to overcome these limitations further clinical development requires novel approaches (e.g. combination with chemotherapy) and development of more efficacious FLT3-TKI. Here, we investigated two novel TKIs (3,4-Diarylmaleimides, compounds 53 and 80) previously described as angiogenesis inhibitors (Peifer, J. Med. Chem. 2006), to determine their mechanism of action and efficacy in FLT3-ITD transfected 32D cells as well as in primary ITD-positive AML blasts. Western Blotting experiments of 32D-FLT3-ITD cells confirmed dose dependent inhibition/dephosphorylation of downstream targets as STAT5 (pTyr694/699), ERK (pThr202/pTyr204) and AKT (pSer473) upon incubation with either compound using nanomolar concentrations. Both compounds (cpd.) showed induction of apoptosis with an IC50 of 350nM (cpd. 53) and 800nM (cpd. 80), respectively, as detected by sub G1-fraction in cell cycle analysis. Using colony assays we defined a possible therapeutic window of these inhibitors: Bone-marrow cells of healthy donors were incubated with increasing concentrations of both inhibitors revealing no significant inhibition of colony formation up to a dose of 5 μM. As clinical studies in AML will evaluate FLT3 inhibitors in combination with chemotherapy, we tested both cpds. in vitro in combination with cytosine arabinosid (Ara-C) and daunorubicine (DNR). Within a range of 1–50nM DNR and 0.3–10μM Ara-C, both cpds. showed additive effects in combination with chemotherapy in 32D-FLT3-ITD cells as revealed by apoptosis assays. Primary AML blasts harboring the FLT3-ITD length mutation were also investigated to determine the efficacy in patient material. These experiments revealed apoptosis rates of 10–30% upon incubation with 3,4-Diarylmaleimides for 72 hours. Cellular up-take of cpd. 53 in primary AML blasts could be demonstrated within 5 minutes of incubation using FACS analysis. Increased fluorescence (200–400% of baseline values) could be detected for more than 24 hours suggesting stable intracellular concentration of the inhibitor in AML blasts within this time period. To evaluate signaling targets of 3,4-Diarylmaleimides in detail, we applied a kinomics peptide chip containing a set of 960 kinase substrates (human sequences only) in triplicate on a slide. Phosphorylation of peptide substrates was detected by autoradiography. Results of evaluation will be presented. In conclusion, 3,4-Diarylmaleimides show efficacy in FLT3-ITD-positive cells alone as well as in combination with chemotherapy. Currently, experiments evaluating toxicity in murine models are planned to explore a possible clinical application.