ALBERT

Description: The histone methyltransferase Enhancer of Zeste Homologue 2 (EZH2), a component of the polycomb group complex, is critical for normal hematopoietic stem cell development. EZH2 mediates transcriptional repression through histone tri-methylation (H3K27me3). The activity of EZH2 influences cell fate regulation, namely the balance between self-renewal and differentiation. The contribution of aberrant EZH2 expression to tumorigenesis is becoming increasingly recognized. Its role in hematological malignancies however, is complex. Both gain-of-function and loss-of-function mutations have been respectively reported in lymphoma and leukemia, suggesting that EZH2 may serve a dual purpose as an oncogene and tumor-suppressor gene. Impaired self-renewal via EZH2 inhibition has been observed and offers a potentially attractive therapeutic approach in acute myeloid leukemia. Indeed, overexpression of EZH2 has been reported in patients with AML, particularly in those with complex karyotypes. In the present study, we show that deletion of EZH2 compromises the growth potential of AML cells by promoting their differentiation. To understand the role of EZH2 in vitro, we first examined the cell growth and colony-forming ability of EZH2 knockdown vs WT HL-60 cells. We found that proliferation of HL-60 cells was severely compromised following deletion of EZH2. Additionally, EZH2 deletion resulted in retarded cell-cycle entry and resulted in increased apoptotic cell death Similarly, the number of total colonies generated by EZH2 deleted cells in the secondary and tertiary re-plating assays was considerably less than that of controls. EZH2 deleted cells tended to form dispersed colonies that were mainly composed of differentiated myeloid cells, whereas control cells mostly formed compact colonies composed of myeloblasts. The proportion of dispersed colonies in the EZH2deleted cell culture increased with serial replatings. Deletion of EZH2 affects the growth and replating capacity of AML cell in vitro. When EZH2 deleted HL-60 cells were treated with the retinoid all-trans-retinoic acid (ATRA), we observed a marked induction of differentiation (as measured by the myeloid maturation marker CD11b) compared to the effects of ATRA on differentiation in wild type (WT) cells. Similarly, impaired clonogenic survival was more pronounced following ATRA treatment in EZH2 deleted vs WT HL-60 cells (see figure). We then profiled a number of small molecule inhibitors of EZH2 alone (EPZ005687, EPZ-6438, GSK126, El1, DZNeP, UNC1999 and GSK343) and in combination with ATRA, confirming these phenotypic changes. To elucidate the mechanism for how EZH2 regulates the balance of self-renewal vs differentiation in AML, we examined the genome-wide distribution of H3K27me3 by ChIP-seq analysis. First, western blot analysis revealed a marked decrease in the levels of H3K27me3 in EZH2 deleted AML cells. Next, we examined the presence of H3K27me3 marks in leukemia cells purified by ChIP-seq analysis. We focused on the region from 5.0 kb upstream to 3.0 kb downstream of transcription start sites (TSSs) of reference sequence (RefSeq) genes (http://www.ncbi.nlm.nih.gov/RefSeq/) because H3K27me3 marks are usually enriched near TSSs or across the body of genes. As expected, the deletion of EZH2 caused a drastic reduction in these H3K27me3 marks. Targeting EZH2 presents and interesting dichotomy as a novel drug target since inhibition of this protein could potentially be beneficial or detrimental depending on the context of the disease. In the case of AML, EZH2 mutations likely impede differentiation and block retinoic acid led differentiation programs. Updated studies outlining the interaction between the retinoic acid signaling pathway and EZH2 will be presented. These studies justify clinical investigation of EZH2 inhibitors combined with ATRA for patients with AML. Figure 1. Knockdown of EZH2 (C) promotes differentiation of AML cells (A), impairs clonogenic survival and synergistically enhances the anti-leukemic effects of the retinoid all-trans-retinoic acid (ATRA) (B). Figure 1. Knockdown of EZH2 (C) promotes differentiation of AML cells (A), impairs clonogenic survival and synergistically enhances the anti-leukemic effects of the retinoid all-trans-retinoic acid (ATRA) (B). Disclosures No relevant conflicts of interest to declare.

Description: Abstract 224 During hematopoiesis, all-trans-retinoic acid (ATRA), a natural derivative of vitamin A, has been shown to induce both myelomonocytic progenitor/stem cell differentiation and self-renewal. Although these opposing effects are likely to be partly due to developmental differences, it has been shown that pro- and anti-differentiation effects of ATRA are mediated by distinct retinoic acid receptor isotypes (RARα and RARγ, respectively). With the exception of acute promyelocytic leukemia (APL), ATRA treatment as a single agent has not been successful in other types of acute myeloid leukemia (AML). We have previously hypothesized that one of the underlying reasons for poor response of non-APL AML to ATRA (pan-RAR agonist) is aberrant expression and/or activities of RAR isotypes favoring RARγ and cell growth versus differentiation. Consistently, we have reported that expression of RARα isoforms, particularly ATRA-inducible RARα2, are down-regulated in AML (Blood. 2008; 111:2374). Epigenetic analysis of patient samples revealed that relative to normal CD33+ cells, the loss of RARα2 in AML is associated with a diminution in levels of histone histone H3 lysine 4 dimethylation (H3K4me2) on the ATRA-responsive RARA2 promoter (a modification associated with transcriptional activation). Interestingly, the H3K4me1/me2 demethylase LSD1/KDM1 (AOF2) is highly expressed in AML patients (www.proteinatlas.org). A number of small molecules that target this enzyme (LSD1i) are in development and, collectively, these data predict that the use of LSD1i will facilitate induction of expression of genes that are required for differentiation of AML cells. In this study we used tranylcypromine (TCP, a monoamine oxidase used as an antidepressant and anxiolytic agent in the clinical treatment of mood and anxiety disorders, respectively), which functions a time-dependent, mechanism-based inhibitor of LSD1. Here we show that TCP unlocked the ATRA-driven therapeutic differentiation response in non-APL AML cell lines including the TEX cell line, which is derived from primitive human cord blood cells immortalized by expression of the TLS-ERG oncogene. TEX cells are 〉90% CD34+, respond poorly to ATRA and mimic features of primary human AML and leukemia initiating cells (Leukemia. 2005; 19:1794). Consistent with this, ATRA/TCP treatment increased differentiation in primary patient samples. ATRA alone had in general only small effects in primary AML samples and TCP showed minimal activity in most cases. Furthermore, shRNA-mediated knockdown of LSD1 confirmed a critical role for this enzyme in blocking the ATRA response in AML cells. The effects of ATRA/TCP on AML cell maturation were paralleled by enhanced induction of genes associated with myelomonocytic differentiation, including direct ATRA targets. LSD1i treatment did not lead to an increase in genome-wide H3K4me2, but did increase H3K4 dimethylation of myelomonocytic differentiation-associated genes. Importantly, treatment with ATRA/TCP dramatically diminished the clonogenic capacity of AML cells in vitro and engraftment of cells derived from AML patients in vivo, suggesting that ATRA/TCP may also target leukemic stem cells. These data strongly suggest that LSD1 may, at least in part, contribute to AML pathogenesis by inhibiting the normal function of ATRA in myelomonocytic development and pave the way for effective differentiation therapy of AML. Disclosures: No relevant conflicts of interest to declare.

Description: Acute Promyelocytic Leukemia (APL) accounts for 5% of all cases of acute myeloid leukemia (AML). This disease is highly curable with all-trans-retinoic acid (ATRA) based therapy. In non-APL AML, ATRA has limited activity, and little is known about mechanisms of ATRA resistance. The apparent selective efficacy of ATRA in PML/RARα-associated APL poses an important question as to whether the presence of this fusion protein renders APL uniquely susceptible. Two compelling arguments can be made to counter this view. First, experiments in vitro show that ATRA effectively differentiates HL-60 cell lines, which lack the PML/RARα fusion protein. Second, clinical studies with ATRA in previously untreated older AML patients (excluding APL) have reported clinical activity. These observations confirm the therapeutic potential of ATRA beyond APL. In this context, our group has previously identified the lysine demethylase LSD-1, as a therapeutic target to re-sensitize leukemic blasts to ATRA. A clinical investigation of ATRA combined with LSD-1 inhibition is currently underway (NCT02273102). It is likely that other defects leading to ATRA resistance will be similarly amenable to pharmacologic manipulation. Defects in the proto-oncogene c-Myc have been widely implicated in the initiation and maintenance of AML. Over-expression of c-Myc in leukemic blasts enhances clonogenic survival and blocks ATRA induced differentiation. We hypothesized that down-regulation of c-Myc might increase the anti-leukemic effects of ATRA in AML. To date, c-Myc has been an evasive target for direct pharmacologic inhibition however, inhibitors of the PI3K/AKT/mTOR pathway have been shown to indirectly lower levels of c-Myc in leukemic blasts. In the current study, we show that the pro-differentiation effects of ATRA are markedly potentiated when combined with agents that target PI3K/AKT/mTOR signalling. In AML cell lines and primary patient samples, we observed additive pro-differentiation effects when ATRA was combined with inhibitors of PI3K (ZSTK474) and mTOR complex proteins (Torin-1, WYE-125132). However, when combined with the bromodomain inhibitor NVP-BEZ235, a dual inhibitor of PI3K and mTOR, we observed synergistic induction of CD11b by FACS analysis. Combination studies revealed loss of cell viability, cell cycle arrest in G1 phase, and impaired clonogenic survival, which was more prominent for ATRA combination treatments than with any agent used alone (Figure 1). To assess the role of c-Myc in mediating these effects, we measured c-Myc protein levels and PI3K/AKt/mTOR pathway markers at different time-points following treatment with ATRA alone and in combination with the inhibitors described above (Figure 2). Our findings suggest that ATRA alone quickly down-regulates c-Myc (within 6 hours) through transcriptional repression. Disruption of the PI3K/AKT/mTOR pathway further down-regulates c-Myc (within 3 hours) through destabilization and enhanced degradation. ATRA combined with NVP-BEZ235 produced maximal c-Myc suppression, and led to more cell kill than any other combination tested. Detailed analysis of changes in the transcriptome in MV-411 cells following treatment with ATRA and NVP-BEZ235 revealed that both agents act jointly on the regulation of the same biological pathways and processes, but regulate different sets of genes within these pathways. Updated mechanism based studies will be presented. In conclusion, suppression of c-Myc levels through disruption of PI3K/AKT/mTOR signalling augments the anti-leukemic effects of ATRA. These data support the clinical investigation of ATRA combined with rapalogs or bromodomain inhibitors. Figure 1. Combination treatment with PI3K/mTORC inhibitors and ATRA decreases cell viability in AML cells. MV4-11 cells were treated as indicated with combinations of BEZ (1µM), WYE (1µM) or ZSTK (2.5µM) and ATRA (0.1 µM). Number of cells was determined by CellTiter-Glo¨ luminescent cell viability assay (Promega). Data were analyzed by one-way ANOVA (P 〈 0.0001) followed by TukeyÕs post-hoc test. * P〈 0.05, ** P 〈 0.01, *** P 〈 0.001,**** P 〈 0.0001. Figure 1. Combination treatment with PI3K/mTORC inhibitors and ATRA decreases cell viability in AML cells. MV4-11 cells were treated as indicated with combinations of BEZ (1µM), WYE (1µM) or ZSTK (2.5µM) and ATRA (0.1 µM). Number of cells was determined by CellTiter-Glo¨ luminescent cell viability assay (Promega). Data were analyzed by one-way ANOVA (P 〈 0.0001) followed by TukeyÕs post-hoc test. * P〈 0.05, ** P 〈 0.01, *** P 〈 0.001,**** P 〈 0.0001. Figure 2. Reduced expression of MYC protein by inhibition of the PI3K/AKT/mTORC pathways. Immunoblotting/quantification of MYC protein levels in MV4-11 cells following treatment with combinations of WYE (1µM), BEZ (1µM), ZSTK (2.5µM) and ATRA (0.1 µM). Figure 2. Reduced expression of MYC protein by inhibition of the PI3K/AKT/mTORC pathways. Immunoblotting/quantification of MYC protein levels in MV4-11 cells following treatment with combinations of WYE (1µM), BEZ (1µM), ZSTK (2.5µM) and ATRA (0.1 µM). Disclosures No relevant conflicts of interest to declare.

Description: Histone modifications play a crucial role in the regulation of gene expression by activating or inactivating transcription. The Polycomb Group Protein Enhancer of Zeste Homologue 2 (EZH2) mediates trimethylation of histone H3K27, thereby inducing gene silencing. Overexpression of EZH2 has been reported to be associated with metastases and cancer progression in solid tumors like breast cancer or prostate cancer. However, loss of function mutations or deletions of EZH2 occur in myeloid malignancies and T-ALL. These mutations result in a poor prognosis. The aim of this study was to analyze the relevance of histone modifications for therapy resistance in AML. FLT3-ITD positive MV4-11 leukemic cells that were continuously cultured in media containing the kinase inhibitor PKC412 became resistant not only to PKC412 but also to standard chemotherapeutics Cytarabin (AraC) and Daunorubicin. Western blot analysis identified an almost complete loss of H3K27me3 in resistant MV4-11 cells (MV4-11R). This was accompanied by loss of EZH2 protein in the MV4-11R compared to the sensitive MV4-11. To test for acquisition of drug resistance due to reduced H3K27me3 levels, lentiviral knock-down (KD) of EZH2 was performed in the sensitive MV4-11 leading to diminished H3K27me3 levels. Knock-down cells showed resistance to the apoptosis-inducing effects of PKC412 compared to scrambled controls. Furthermore, resistance to standard chemotherapeutics AraC and Daunorubicin could be also observed in MV4-11 KD cells compared to control. To verify whether diminished levels of H3K27me3 can cause a more general, FLT3-ITD-independent drug resistance, knock-down of EZH2 was performed in FLT3-WT AML cell lines HL60, Kasumi-1 and ML-1. Again, this led to resistance to the standard chemotherapeutics AraC and Daunorubicin. In order to investigate the regulation of EZH2 in MV4-11R, promoter methylation and microRNA expression analysis was performed revealing no regulation of EZH2 expression via both mechanisms. Instead, the reduction of EZH2 protein expression was depended on posttranslational mechanisms that could be counteracted by CDK1-inhibitors. CDK1-inhibitors restored EZH2 protein and H3K27 trimethylation levels as well as drug sensitivity. By analyzing EZH2 mRNA expression of 220 primary diagnosed AML patients, a trend towards low EZH2 mRNA expression and poor overall as well as relapse free survival could be demonstrated. Thus, EZH2- and H3K27me3 protein expression were also analyzed by immunohistochemistry in bone marrow biopsies from AML patients (N=126). H3K27me3 and EZH2 protein expression correlated closely (r= 0.9, p

Description: Abstract 1046 Poster Board I-68 During hematopoiesis, all-trans-retinoic acid (ATRA), a natural derivative of vitamin A, has been shown to induce both myelomonocytic progenitor/stem cell differentiation and self-renewal. Although these opposing effects are likely to be partly due to developmental differences, it has been shown that pro- and anti-differentiation effects of ATRA are mediated by distinct retinoic acid receptor isotypes (RARαa and RARγ, respectively). With the exception of acute promyelocytic leukemia (APL) ATRA treatment as a single agent has not been successful in other types of acute myeloid leukemia (AML). We have hypothesized that one of the underlying reasons for poor response of non-APL AML to ATRA (pan-RAR agonist) is aberrant expression and/or activities of RAR isotypes favoring RARγ and cell growth versus differentiation. Consistently, we have reported that expression of RARαa isoforms, particularly ATRA-inducible RARαa2, are down-regulated in AML (Blood 2008; 111:2374). Epigenetic analysis of patient samples revealed that relative to normal CD33+ cells, the loss of RARαa2 in AML is associated with a diminution in histone H3K4me2 and an increase H3K27me3 on the RARA2 promoter (modifications associated with transcriptional activation and silencing, respectively). Interestingly, H3K4 demethylase LSD1 (AOF2) and the polycomb represive complex 2 (PCR2)-associated H3K27 methyltransferase EZH2 are highly expressed in AML (www.proteinatlas.org). Small molecules that target these enzymes are in development and, given the above results, we predict that the use of such agents in combination with ATRA will enhance the effects of ATRA-mediated induction of gene expression and differentiation of AML cells. To test this hypothesis, we used ATRA-responsive HL-60 AML cells and the TEX cell line. TEX cells are derived from primitive human cord blood cells immortalized by expression of the TLS-ERG oncogene. These cells, the ATRA-responsiveness of which is not known, mimic features of primary human AML and leukemia initiating cells (Leukemia. 2005; 19:1794). LSD1 activity was inhibited using monoaminoxidase inhibitor (MAOI) trans-2-phenylcyclopropylamine (Parnate, 1μM) in combination with pharmacological (1μM) and sub-optimal (0.1μM) concentrations of ATRA. Co-treatment with Parnate potentiated the HL-60 response to sub-optimal ATRA concentration. While ATRA appeared to be a less potent inducer of TEX cell differentiation, Parnate nevertheless enhanced their maturation at pharmacological ATRA concentrations and sensitized these cells to differentiation induction under sub-optimal ATRA levels. Additionally, we investigated the biguanide polyamine analogue 1,15-bis[N5-[3,3-(diphenyl) propyl]-N1-biguanido]-4,12-diazapentadecane (2d), which is structurally unrelated to Parnate, obtaining similar results. Biguanide polyamine analogue inhibitors of LSD1 may have several benefits over MAOIs, including DNA targeting due their cationic nature. We also tested 3-deazaneplanocin A (DZNep), which diminishes levels of H3K27 trimethylation via depletion of the EZH2 catalytic subunit of the PCR2. Consistent with our hypothesis and the above data, co-treatment of HL-60 and TEX cells with DZNep (0.05μM) and ATRA (0.1μM and/or 1μM) led to more robust differentiation response than when ATRA was used as a single agent. The use of ATRA in combination with DZNep and LSD1 inhibitors at the same time led to a better differentiation response, as measured by CD11b/CD11c expression, morphology and superoxide production (NBT assay), than when either drug alone was used with ATRA. The effects of these drug combinations on AML cell maturation were paralleled by synergistic induction of endogenous ATRA target genes and expected changes in the levels of H3K4/K27 methylation. At the concentrations used with ATRA neither Parnate, 2d nor DZNep induced differentiation when used as single agents, however, when used at higher concentrations both singly and in combination with ATRA, these drugs exerted cytotoxic effects. Importantly, the above described combination treatments were specific for AML blasts as they had no cytotoxic effects on normal CD33+/CD34+ cell populations. These data demonstrate existence of therapeutically relevant crosstalks between the ATRA-induced differentiation pathway and histone H3K4 and K27 methylation and that targeting LSD1 and/or EZH2 in combination with ATRA may represent a promising treatment for AML. Disclosures: Marton: Progen Pharmaceuticals: Employment. Woster:Progen Pharmaceuticals: Consultancy, Research Funding. Casero:Progen Pharmaceuticals: Consultancy, Research Funding.

Description: Abstract 3468 The development of drug resistance is a common feature in AML that occurs towards classical cytotoxic drugs as well as novel kinase inhibitors. Mutations in primary drug targets explain a significant fraction of acquired drug resistance but the mechanisms of resistance still remain unknown in most patients. About 20–30% of all AML patients possess the mutant FLT3-ITD leading to a constitutive activation of the FLT3 kinase. Flt3-mutations can be targeted by treatment with tyrosine kinase inhibitors. Resistance to these kinase inhibitors is an increasing phenomenon whose mechanisms are not entirely understood today. As epigenetic mechanisms are shown to play an important role in leukemia pathogenesis they are also likely to influence drug resistance. To elucidate the potential role of epigenetic mechanisms in the development of drug resistance towards kinase inhibitors we used a PKC412 partially resistant clone (MV4-11R) of the AML cell line MV4-11, which harbors a homozygous FLT3 internal tandem duplication (ITD) mutation. An initial screening for histone modifying enzymes revealed a downregulation of EZH2 on mRNA as well as protein level compared with the parental cell line. The reduction of EZH2, a H3K27 methyltransferase, in MV4-11R is furthermore correlating with globally diminished H3K27me3 levels. ChIP-Seq experiments using H3K27me3 antibody revealed differences in histone 3 K27 methylation at specific promoter sites between the parental and resistant MV4-11. To test for an increased drug resistance due to reduced EZH2 protein levels lentiviral knock-down of EZH2 was performed in the MV4-11 parental cell line and three individual knock-down cell clones were investigated for their drug resistance potential. These knock downs all showed elevated IC50 values as well as resistance towards the apoptosis-inducing effects of PKC412 compared with the scrambled shRNA cells. Furthermore, EZH2 protein levels of 5 FLT3-ITD-positive AML patient samples were determined by Western Blot, samples were treated with PKC412 for 3 days and cell survival was assayed. Using this approach higher EZH2 levels in patients could also be associated with a higher sensitivity to PKC412 pointing to a putative role of EZH2 in the development of PKC412 resistance in vivo. As EZH2 has been shown to interact with DNMTs in the context of the Polycomb Repressive Complex 2 and 3 (Viré et al., Nature 2006), we analyzed whether parental and resistant MV4-11 differ in their DNA methylation pattern. To identify hyper-/hypomethylated genes in MV4-11R, we applied the Illumina 27k Methylation BeadChip approach as well as Reduced Representation Bisulfite Sequencing (RRBS) for a genome wide CpG methylation analysis. In particular genes associated with apoptosis pathways and signal transduction were hypermethylated in MV4-11R cells compared to the parental cell line. Based on the observation of DNA methylation changes between the parental cell line and MV4-11R, treatment with the demethylating agent 2-deoxy-5-azacytidine (Aza dC) was conducted to investigate recovery of drug sensitivity. Incubation with 250 nM Aza dC for 5 days could restore the sensitivity of MV4-11R towards PKC412 as shown in proliferation and apoptosis assays. Using the Affymetrix Human Gene 1.0 ST Array platform we identified 110 genes whose expression was reactivated after treatment of MV4-11R with Aza dC, predominantly genes playing a role in signal transduction, apoptosis pathways and cell cycle. A cell cycle analysis of the MV4-11 and MV4-11R cells indeed showed that the resistant cell line is cycling less than the parental one, which possibly also favors the resistance development towards PKC412. Summarized, our data show that a loss of EZH2 accompanied by DNA methylation changes lead to PKC412 resistance in a FLT3-ITD AML cell line model possibly reflecting a way of acquisition of drug resistance in patients. Disclosures: Thiede: Novartis: Lectures, Research Funding. Müller-Tidow:Novartis: Research Funding.

Description: Abstract 479 The identification of activating mutations in the FLT3 gene and their impact on prognosis has been crucial to the rationale behind the development of FLT3 inhibitors. While it has been shown that some leukemic cells with high tumorigenic potential exist mostly in a dormant state, it is unclear if this quiescent/non-cycling leukemia-initiating fraction carries the FLT3-ITD mutation and if it is successfully targeted by FLT3 inhibitors. As a paradigm, quiescent Ph+ stem cells in CML have been shown to exhibit resistance to bcr-abl targeted inhibitors. Additionally, results from clinical trials suggest that FLT3 inhibitors reduce the leukemic blast count in peripheral blood but are less successful in the bone marrow where factors regulating hematopoietic stem cell quiescence are active. In order to investigate the non-cycling and cycling human leukemic cell boundary, we devised a biological model that allowed us to distinguish non-cycling AML cells from cycling AML progenitors in human FLT3-ITD positive AML samples. CD34+ cells were isolated from AML samples using magnetic cell sorting, labeled with the cell membrane dye PKH26 to enable tracking of cell division, and cultured on murine stroma for 12 days. Non-cycling AML cells were then separated from cycling cells by FACS sorting and were found to retain a CD34+ primitive phenotype in contrast to expanding leukemic blasts. Fluorescence in situ hybridization analyses revealed that non-cycling cells carried leukemic gene rearrangements (trisomy 8, trisomy 13, t[3;21]and t[16;16] in our cases), and were therefore part of the original leukemic clone. PCR for the FLT3-ITD region showed that in four out of five cases, the FLT3-ITD mutation was present in the non-cycling fraction. To examine the distribution of FLT3-ITD to FLT3 wild type (WT) bearing cells, non-cycling AML cells were FACS sorted, DNA extracted and the PCR products subsequently cloned. Bacterial colonies were sequenced and colony-PCR used to determine the ratio of FLT3-ITD to WT bearing colonies for each patient. These data indicated that at least 25% of non-cycling cells (range 25%-100%) harbored the FLT3-ITD mutation. We then assessed the impact of a potent FLT3-directed inhibitor, TKI258 (Novartis), on leukemic cell expansion and the viability of non-cycling cells. TKI258 has been found to induce apoptosis of FLT3-ITD bearing cells of the human acute monocytic leukemia MV4;11 cell line. In our present study, CD34+ AML blasts from the same FLT3-ITD positive patient samples were grown in vitro in the presence of 0 μM, 0.3 μM (IC50 dose) and 1.25 μM TKI258. In stromal cultures, TKI258 significantly reduced leukemic cell expansion (range 2.13 to 20 fold for untreated cultures and 0.07 to 2.27 fold for 1.25 μM TKI258 treated cultures at day 12, p ≤ 0.05). In methylcellulose colony assays, TKI258 exposure resulted in dose-dependent suppression of colony formation of CD34+ FLT3-ITD positive leukemia cells (60% to 81% reduction in the mean plating efficiency of CD34+ AML cells at 0.3 μM TKI258). Despite this striking anti-proliferative effect, the majority of non-cycling cells from AML patients showed resistance to TKI258 (five out of six cases). In these samples, FLT3-ITD positive non-cycling cells could still be detected after treatment with the equivalent highest clinical dose (1.25 μM) of TKI258. Moreover, at a functional level, limiting-dilution experiments on non-cycling AML cells pre-treated with TKI258 showed no impairment in a modified leukemic cobblestone assay at four weeks compared to untreated non-cycling cells. These results suggest that the majority of non-cycling AML cells that harbor FLT3-ITD are unaffected by a FLT3 inhibitor and may constitute an as yet untargeted disease reservoir. Only one FLT3-ITD AML case showed exquisite sensitivity to TKI258 with elimination of the non-cycling fraction observed from 0.3 μM of TKI258 upwards. Possible explanations for this may include specific mutant receptor sensitivities or generic multi-drug resistance mechanisms operating in dormant cells. Interestingly, TKI258 selectively eradicated an ‘intermediate' dividing progenitor population in two of the insensitive cases, an indication that leukemic progenitors may be rendered sensitive to FLT3 inhibition on transiting the dormancy-cycling boundary. Further studies are needed to determine if these findings are representative of a generation of FLT3 inhibitors or specific for TKI258. Disclosures: No relevant conflicts of interest to declare.