Adult Acute Myeloid Leukemia Cell Research Articles

Inhibition of NRF2 enhances the acute myeloid leukemia cell death induced by venetoclax via the ferroptosis pathway

Venetoclax, an inhibitor that selectively targets B cell lymphoma-2 (BCL-2) that has been approved for treating adult acute myeloid leukemia (AML) in combination with hypomethylating agents. However, its short duration of response and emergence of resistance are significant issues. In this study, we found that the sensitivity of AML cells to venetoclax was considerably enhanced by ML385, an inhibitor of the ferroptosis factor nuclear transcription factor erythroid 2-related factor 2 (NRF2). Using AML samples, we verified that NRF2 and its target gene ferritin heavy chain 1 (FTH1) were highly expressed in patients with AML and correlated with poor prognosis. Downregulation of NRF2 could inhibit FTH1 expression and significantly enhance the venetoclax-induced labile iron pool and lipid peroxidation. By contrast, NRF2 overexpression or administration of the reactive oxygen species inhibitor N-acetylcysteine and vitamin E could effectively suppress the anti-AML effects of ML385+venetoclax. Furthermore, the ferroptosis inducer erastin increased the anti-AML effects of venetoclax. Our study demonstrated that NRF2 inhibition could enhance the AML cell death induced by venetoclax via the ferroptosis pathway. Thus, the combination of ML385 with venetoclax may offer a favorable strategy for AML treatment.

Abstract LB035: The role of nucleophosmin1 mediated caspase2 activation in acute myeloid leukemia cell death and cell survival

Abstract Nucleophosmin 1 (NPM1) mutation is the primary genetic lesion in adult acute myeloid leukemia (AML). Typically, a nucleolar protein, NPM1 mutations cause cytoplasmic delocalization of NPM1(NPM1c+), triggering leukemogenesis. Interestingly, NPM1c+ mutation confers enhanced chemosensitivity. However, the mechanism underlying NPM1c+ mutation-induced oncogenesis or chemosensitivity remains unclear. We previously reported that, in response to genotoxic stimuli, NPM1 forms a platform for caspase-2 activation in the nucleolus. Caspase-2 is a pro-apoptotic protein and a known tumor suppressor. Our results indicate that nucleolar caspase-2 activation is specific to NPM1wt cells. In the NPM1c+ cells, caspase-2 activation is exclusively cytosolic, suggesting that caspase-2 is activated in the same sub-cellular compartment as NPM1 localization. Loss of caspase-2 rescued the increased sensitivity of NPM1c+ cells to apoptotic cell death. This is accompanied by reduced cleavage of the apoptotic substrate, caspase-3. Notably, caspase-2 deficient NPM1wt cells showed no significant difference in the percentage of apoptotic cell death or endogenous substrate cleavage compared to its caspase-2 wild-type counterpart. This suggests that caspase-2 may contribute to the chemosensitivity of NPM1c+ cells. Strikingly, in unstimulated cells, loss of caspase-2 leads to prolonged G1 arrest and progressive loss of cell viability. Immunophenotyping of NPM1c+ cells revealed a remarkable increase in the CD14+population representing monocyte/macrophage-like features, suggesting that loss of caspase-2 results in terminal differentiation. Exogenous expression of NPM1c+ in caspase-2 deficient NPM1wt cells impaired viability, confirming that NPM1c+ cells require caspase-2 for its growth and proliferation. Transcriptomic analysis revealed that loss of caspase-2 significantly downregulates pathways involved in stem cell pluripotency. In particular, FGF2, IGFR, WNT, LIF, and one of the core members of the self-renewal transcription factor, OCT 4, were downregulated in the absence of caspase-2 in NPM1c+ cells. This reveals a novel role for caspase-2 in maintaining NPM1c+ AML stemness. Taken together, our study shows that NPM1c+ mediated caspase-2 activation regulates AML cell death and survival cascades, a key determinant of chemosensitivity and leukemogenesis. Citation Format: Dharaniya Sakthivel, Alexandra Brown-Suedel, Francesca Keane, F. Kenneth McSherry, Chloe I. Charendoff, Kevin Dunne, Dexter Robichaux, Jonathan M. Flanagan, Lisa Bouchier-Hayes. The role of nucleophosmin1 mediated caspase2 activation in acute myeloid leukemia cell death and cell survival [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB035.

High trophinin-associated protein expression predicts good survival in acute myeloid leukemia with normal cytogenetics.

Nearly half of adult acute myeloid leukemia (AML) patients were classified into cytogenetic normal acute myeloid leukemia (CN-AML). The expression level of Trophinin associated protein (TROAP) was proven to be associated with the prognosis of several cancers, but it is still unclear in the prognosis of patients with CN-AML. We integrated CN-AML patients samples from 4 datasets to analyze the relationship between TROAP expression and the survival of CN-AML. In addition, we investigated 92 AML patients of The Cancer Genome Atlas (TCGA) database to analyze the relationship between TROAP expression and the survival of AML patients received chemotherapy. We investigated the relationship between the expression of TROAP and drug sensitivity in AML cell lines. CN-AML patients with high TROAP expression were related to good event-free survival (EFS) and overall survival (OS). In AML patients received chemotherapy, high TROAP expression was associated with good survival prognosis. Additionally, the expression of TROAP gene in leukemia stem cells (LSC) + group was lower. Among multiple drugs, the lower the expression of TROAP, the lower the IC50. TROAP could serve as an independent predictor of CN-AML patients and could act as a potential biomarker for the prognosis of CN-AML. TROAP expression levels were closely correlated with the drug sensitivity of multiple drugs.

Decrease of lncRNA MEG3 in Acute Myeloid Leukemia Affected the Expression of p53 and MDM2.

The aim was to determine the clinical role of long non coding maternally expressed gene 3 (lncRNA MEG3) in adult acute myeloid leukemia (AML). The expression levels of lncRNA MEG3 in bone marrow of AML patients and healthy donors were determined by qPCR. The correlation between expression levels of lncRNA MEG3 and clinical features was also analyzed. MTT assay and flow-cytometric assay were employed to determine the role of lncRNA MEG3 on the cell viability and apoptosis of AML cell lines. Expression levels of caspase-9, Bcl-2, MDM2, and p53 in those cells were determined by western blot. The expression levels of lncRNA MEG3 was significantly down-regulated in AML patients. Expression levels of lncRNA MEG3 were positively correlated with overall survival of patients. Up-regulation of lncRNA MEG3 could not only inhibit the cell growth and promoted apoptosis, but also increased the expression of caspase-9 and p53 and decreased the expression of Bcl-2 and MDM2. These results suggest that down-regulation of lncRNA MEG3 in AML patients might promote tumor progression and affect the p53-MDM2 pathway.

Oncometabolite-Independent Anti-Apoptotic Feature Via Phospholipid Metabolic Adaptation in IDH2 Mutant AML Cells

Isocitrate dehydrogenase (IDH) gene mutation is found in approximately 20% of adult acute myeloid leukemia (AML) patients. IDH gene mutations give a pathological enzymatic activity producing oncometabolite, which impair hematopoietic differentiation while promoting leukemogenesis via epigenetic changes. Targeting mutant (mut) form of IDH enzymes, small molecule inhibitors have been developed. Among these inhibitors, AG-221 (enasidenib), which is the first-in-class, selective and orally available mut IDH2 inhibitor (Yen et al., Cancer Discov. 2017), has been tested in clinical trial. The clinical trial showed overall response rate was approximately 40% and more than half of the patient didn't respond to the treatment. This clinical study also showed clear correlation between clearance of IDH2 mut clones and complete clinical response rate (Stein et al., Blood 2019). Relapse cases under the AG-221 treatment, in which additional mutation in IDH2 or IDH1 gene occurred in the residual IDH2 mut clone have been reported (Intlekofer et al., Nature 2018, Harding et al., Cancer Discov. 2018). These reports suggested that IDH2 mut cells have an oncometabolite-independent survival advantage which contributes to refractoriness and relapse. However, most of the studies on IDH mut AML have been focused on oncometabolite-related mechanisms and oncometabolite-independent mechanism has not been investigated in detail so far. IDH gene mutations frequently coexist with other oncogenic mutations in AML patient cells, which make it difficult to analyze the precise role of IDH gene mutation in leukemogenesis. To address this issue, we utilized the cytokine-dependent AML cell line, TF-1 cells and its isogenic cell line with IDH2 gene mutation, which showed cytokine independent growth. Though AG-221 suppressed the cytokine independent growth in IDH2 mut cells, the growth of AG-221 treated IDH2 mut cells remained significantly faster than IDH2 wild type (WT) cells. These data indicated that IDH2 mut cells have a growth advantage machinery in an oncometabolite-independent manner. Metabolome analysis clearly separated AG-221 treated IDH2 mut cells from IDH2 WT cells and identified that inositol phosphate metabolism pathway showed significant difference between these two groups. High throughput drug screening using metabolic inhibitors also revealed that inositol phosphate related enzyme, phospholipase C (PLC) specific inhibitor promoted IDH2 mut cell proliferation. These data suggested that suppressed PLC activity induced oncometabolite-independent growth advantage in IDH2 mut AML cells. Since PLC indirectly release free arachidonic acid (AA) from phospholipid bilayer of cellular membrane and intracellular AA level has positive correlation to apoptosis (Cao et al. PNAS 2000), we analyzed intracellular AA and apoptosis in IDH2 mut cells. As expected, these analyses showed significantly lower intracellular AA and reduced Annexin V+ apoptotic cells in IDH2 mut cells irrespective of AG-221 treatment. Supporting these data, PLC activity related gene sets were negatively enriched and anti-apoptosis related genes were positively enriched in IDH2 mut cells in RNA seq analysis. Based on these novel mechanisms, we tested FDA-approved anti-inflammatory drugs, COX-2 and 5-LOX specific inhibitors (celecoxib and zileuton) to upregulate intracellular AA and induce apoptosis of TF-1 cells in vitro and in vivo xenograft. These drugs in combination with AG-221 successfully suppressed IDH2 mut cell proliferation and induced apoptosis both in vitro and in vivo. The mice treated with these triple drugs showed no sign of cytopenia nor therapy-associated complication, securing safety of this combination therapy. In this study, we identified unappreciated oncometabolite-independent anti-apoptotic feature acquisition via phospholipid metabolic adaptation in IDH2 mut AML cells. Moreover, targeting this metabolic adaptation, we identified that FDA-approved anti-inflammatory drugs in combination with mut IDH2 inhibitor could eradicate IDH2 mut AML cells by efficiently inducing apoptosis. Since these anti-inflammatory drugs have a track record of safety in clinical practice, the triple drug combination therapy could be an alternative therapeutic option for the IDH2 mut AML patients who couldn't tolerate existing therapy.

Inducible MyD88 Signaling Enhances the Anti-AML Activity of BiTE-Secreting ENG T-Cells

Background: Pediatric and adult acute myeloid Leukemia (AML) is a disease with poor prognosis due to its high relapse rate and treatment related mortality. Adoptive immunotherapy has the potential to improve outcomes and CD123 present a promising immunotherapy target for AML. CD123-specific engager (ENG) T-cells are cells genetically modified to secrete bispecific T-cell engagers (BiTEs) that recognize CD3+ on T-cells and CD123+ on AML. We had previously shown that inclusion of an inducible MyD88/CD40 costimulatory molecule, which is activated by a chemical inducer of dimerization (CID), improved anti-tumor effect against CD123+ targets in vitro. We now wanted to determine, which component of the costimulatory molecule is critical for the improved effector function of CD123-ENG T cells and perform in vivo studies. Methods: We generated a panel of retroviral vectors encoding CD20 as a safety switch, CD123-ENG, and an inducible costimulatory molecule consisting of a myristoylation sequence, FKBP dimerizer domains and one of three signaling costimulatory domains (MyD88 (iM) ,CD40(iC) or MyD88/CD40(iMC)). A vector specific for CD19 (CD20.CD19-ENG.iMC) and non-transduced (NT) T-cells served as controls. We genetically modified T-cells using a retroviral transduction protocol and evaluated their effector function +/- CID. We assessed the effector function of transduced T-cells using flow cytometry, ELISA and luciferase based cytotoxicity assays. We evaluated antitumor activity and persistence in vivo in a xenograft AML model. Results: We successfully generated CD20.CD123-ENG.iM, CD20.CD123-ENG.iC and CD20.CD123-ENG.iMC T-cells. Transduction efficiency ranged from 45 to 70% (±5). The immunophenotype of CD123-ENG T-cells was predominandly CD8+ effector memory. CD20.CD123-ENG T-cells expressing inducible costimulatory molecules secreted higher levels of CD123-specific BiTEs, IL2 and IFNγ in comparison to CD20.CD123-ENG T-cells when cocultured with of MOLM-13 (CD123+) and CID (p<0.005, N=3). In the presence of CID, CD20.CD123-ENG.iM or CD20.CD123-ENG.iMC T-cells were able to sequentially kill AML cells for up to 5 stimulations, as opposed to CD20.CD123 ENG, CD20.CD123 ENG.iC and NT T-cells (n=3, E:T=1:1). We evaluated the in vivo anti-AML activity of ENG T cells using an AML cell line expressing firefly luciferase (MOLM-13.ffluc) to allow for serial bioluminescence imaging. MOLM-13.ffluc bearing mice were treated with one i.v. dose of effector cells (CD20.CD123-ENG, CD20.CD123-ENG iM, CD20.CD123-ENG.iC, CD20.CD123-ENG.iMC or NT T-cells ) and 2 doses/week of CID for 2 weeks (100mg/mice, i.p.). CD20.CD123-ENG.iMC and iM T-cells had potent anti-leukemic activity, resulting in a significant survival advantage in comparison to CD20.CD123-ENG T-cells group (P<0.005, n=10 animals per group). Conclusion: We demonstrate here that inducible MyD88/CD40 costimulation significantly enhances the anti-AML activity of CD123-ENG T cells in vitro and in xenograft models. Mechanistic studies revealed that MyD88 costimulation is critical for the observed benefit. Thus, genetically modifying T-cells to express BiTEs and an inducible MyD88 costimulatory molecule may present a promising cell product for the immunotherapy of CD123+ hematological malignancies. Disclosures Bonifant: Patents filed in the field of engineered cellular therapies: Patents & Royalties: Patents filed in the field of engineered cellular therapies. Gottschalk:TESSA Therapeutics: Other: Research Collaboration; Tidal: Membership on an entity's Board of Directors or advisory committees; Sanofi: Honoraria; Inmatics: Membership on an entity's Board of Directors or advisory committees; Merck: Consultancy; MBIO: Other: St. Jude Children's Research Hospital has an existing exclusive license and ongoing partnership with Mustang Bio for the further clinical development and commercialization of this XSCID gene therapy; EMD Serono: Honoraria; ViraCyte: Consultancy; NHLBI: Research Funding; ASSISI fundation of Memphis: Research Funding; California Institute for Regenerative Medicine: Research Funding; Patents and patent applications in the fields of T-cell & Gene therapy for cancer: Patents & Royalties; America Lebanese Syrian Associated Charities: Research Funding. Velasquez:Rally! Foundation: Membership on an entity's Board of Directors or advisory committees; St. Jude: Patents & Royalties: Patent Applications in the Fields of Cell and Gene Therapy .

Abstract 371: Synthetic lethal and resistance pathways in midostaurin-treated AML

Abstract Introduction: Mutations in fms-like tyrosine kinase 3 (FLT3) are common genomic abnormalities in adult acute myeloid leukemia (AML). When FLT3 is present as a dominant internal tandem duplication (ITD) AML clone, it is associated with poor treatment outcome and short overall survival (OS). A phase 3 study with the multi-kinase (including FLT3) inhibitor midostaurin improved OS of FLT3+ AML prompting widespread use, yet little is understood about resistance pathways and synergistic pharmacologic targets. We conducted a genome-wide CRISPR-KO (loss-of-function) screen in a FLT3+ AML cell line treated with midostaurin, validated results in vitro and in vivo, and identified distinct findings relative to limited CRISPR screening previously reported (Cancer Res 77:4402, 2017) with a very selective FLT3-ITD inhibitor (quizartinib). Methods: CRISPR pooled screens (GeCKOv2 library, >500 cells/sgRNA) of midostaurin versus vehicle were analyzed via MAGeCK. Gene essentiality was derived from guide abundance after 7-day selection of transduced cells compared to plasmid levels. In vitro validation included shRNA knockdown and/or inhibition followed by proliferation and clonogenic assays in cell lines and primary AML blasts. NSG mice (n=7/group) engrafted with MOLM-13-luciferase cells were treated with vehicle, single agents, or combination and evaluated for disease burden (IVIS) and survival (Kaplan-Meier). Results: We first confirmed our screening approach correlated well with previously reported essential genes (Cell Rep 17:1193, 2016; Cancer Cell 33:386, 2017;Genome Biol 16:281, 2015). We then identified >500 genes (FDR<2%) with significant depletion (synergistic) or enrichment (resistance) after midostaurin treatment. Top synergistic genes included BCL2, MEF2D, CCNG1, and RPS14, with no overlap to reported quizartinib results. We selected BCL2 for immediate validation based on demonstrated efficacy targeting this gene in AML. Indeed, shRNA knockdown of BCL2 sensitized FLT3+ AML cell lines to midostaurin. Similarly, combination treatment of FLT3+ cell lines and AML patient tumor samples with midostaurin and venetoclax displayed synergy at clinically relevant doses. In a xenograft model, combination therapy decreased disease burden and improved survival over single agents supporting synergy of these agents in vivo. Conclusion: FLT3 inhibitors represent the first targeted therapeutic to impact AML survival and herein we use genome-wide CRIPSR screening to demonstrate that resistance mechanisms of broad-spectrum kinase inhibitors are distinct from those previously reported with specific inhibitors (quizartinib). Furthermore, we validate the select target BCL2 genetically and with the therapeutic venetoclax demonstrating in vitro and in vivo synergy, thereby justifying pursuit of this combination in future clinical trials. Ongoing efforts include validation of other targets that will be presented. Citation Format: Lindsey T. Brinton, Katie Williams, Shelley Orwick, Larry Beaver, Daniel Canfield, Casey Cempre, Jordan Skinner, Ronni Wasmuth, Quais Hassan, John C. Byrd, Rosa Lapalombella, James S. Blachly. Synthetic lethal and resistance pathways in midostaurin-treated AML [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 371.

Members of the microRNA-106a-363 Cluster Associate with Unfavorable Outcome in Adult Acute Myeloid Leukemia Patients and Promote Leukemogenesis invivo through Increased Metabolic Activity

The miR-106a-363 cluster, encoding six miRNAs (miR-106a, miR-18b, miR-20b, miR-19b, miR-92a and miR-363), is a paralogue of the oncogenic miR-17-92a polycistron and its role in leukemia is at present largely unknown. We aimed to investigate the putative oncogenic role of the miR-106a-363 cluster in adult acute myeloid leukemia (AML) and to dissect the contributions of its individual members to disease formation and progression.First, we analyzed the expression of each miRNA in AML patient samples as well as their clinical relevance. To determine the association of the miR-106a-363 cluster in AML with active disease, we quantified all six miRNAs individually in AML patient samples at initial diagnosis (n=33) and in AML patients in complete remission after chemotherapy (n=6). Hereof, miR-106a-5p, miR-19b-3p and miR-92a-3p levels were significantly lower in remission samples (p=0.0015, p=0.0013 and p=0.0004, respectively), confirming that these miRNAs are upregulated in AML. Stratifying AML patients within the LAML miRNA-Seq dataset of The Cancer Genome Atlas (TCGA) Research Network (n=187) (Ley et al., NEJM, 2013) according to their cytogenetic risk group demonstrated that all members of the cluster, except for miR-18b-5p, significantly associated with adverse cytogenetics. In addition, with the exception of miR-18b-5p, all members associated with an inferior overall survival (OS) in AML patients within the TCGA-LAML dataset, further supporting a pro-leukemogenic role for the cluster. Of note, miR-106a-5p was the most abundantly expressed unique miRNA of the polycistron, both in the TCGA patient cohort and in 11 myeloid leukemia cell lines quantified by quantitative real-time PCR (qRT-PCR).Since the miR-106a-363 cluster is associated with high risk AML, we hypothesized that increased levels of the entire cluster as well as individual members would significantly shorten the survival time in a murine transplantation model mimicking aggressive AML. Therefore, we engineered transplantable, primary murine AML cell lines based on retroviral overexpression of Hoxa9 and Meis1 exhibiting a median disease latency of 39 days (n=14) after syngeneic transplantation in mice. Enforced lentiviral expression of miR-106a-363 (n=13, p<0.0001), miR-106a (n=15, p=0.0003), miR-18b (n=8, p<0.0001), miR-20b (n=13, p<0.0001) and miR-363 (n=13, p<0.0001) in Hoxa9/Meis1 cells significantly accelerated leukemogenesis compared to the control arm. The most pronounced anemia (p=0.03) and the most immature phenotype, based on a significantly higher proportion of c-kit+ (p=0.0147) and a concurrent lower percentage of Mac-1+ and Gr-1+ (p=0.0051) cells, were observed in mice transplanted with Hoxa9/Meis1/miR-106a cells.Based on these results, we focused on the mechanism by which miR-106a contributed to the pathogenesis of AML and performed a proteomics screen comparing Hoxa9/Meis1/miR-106a and Hoxa9/Meis1/control cells. In particular, mitochondrial respiration processes, such as oxidative phosphorylation and electron transport chain components were induced by miR-106a as shown by Gene Set Enrichment Analysis. Preliminary results using high-resolution respirometry further indicated an increased number of mitochondria in Hoxa9/Meis1/miR-106a cells, supporting these findings.In conclusion, we highlight the previously unrecognized oncogenic contribution of the miR-106a-363 polycistron in adult AML. Functional dissection of this cluster, in particular miR-106a, revealed a new therapeutic angle for high risk AML. DisclosuresDöhner:Astellas: Consultancy, Honoraria; AbbVie: Consultancy, Honoraria; Bristol Myers Squibb: Research Funding; Astex Pharmaceuticals: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Bristol Myers Squibb: Research Funding; Celator: Consultancy, Honoraria; Jazz: Consultancy, Honoraria; Astex Pharmaceuticals: Consultancy, Honoraria; Jazz: Consultancy, Honoraria; Celator: Consultancy, Honoraria; Pfizer: Research Funding; Sunesis: Consultancy, Honoraria, Research Funding; AROG Pharmaceuticals: Research Funding; Agios: Consultancy, Honoraria; Celgene: Consultancy, Honoraria, Research Funding; Amgen: Consultancy, Honoraria; Astellas: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; AROG Pharmaceuticals: Research Funding; Seattle Genetics: Consultancy, Honoraria; Sunesis: Consultancy, Honoraria, Research Funding; Pfizer: Research Funding; Seattle Genetics: Consultancy, Honoraria; Novartis: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Research Funding; Agios: Consultancy, Honoraria; Novartis: Consultancy, Honoraria, Research Funding; AbbVie: Consultancy, Honoraria; Janssen: Consultancy, Honoraria.

Combinatorial Genetics Uncovers Novel Targets for the Treatment of Npm1/Cohesin Mutated AML

Current precision medicine approaches typically target a single genetic mutation. However, adult acute myeloid leukemia (AML) is difficult to treat due to its genetic complexity. Approximately 30 somatic mutations have been found to be recurrent, with an average of 5-15 mutations present per patient (Cancer Genome Atlas 2013). Therefore, combinatorial genetic approaches have the power to uncover novel gene targets that can be used to tailor therapies to an individual's unique mutational spectrum. Mutations in NPM1 commonly occur in in AML. Approximately 25-30% of patients harbor a specific variant of the NPM1 mutation referred to as NPM1cA, which results in mislocalization of the Npm1 protein from the nucleus to the cytoplasm (Cancer Genome Atlas 2013; Falini 2005). While NPM1cA is considered to be a driver of AML development, mice with this mutation develop AML with a long latency (18 months) and with incomplete penetrance (Vassiliou 2011). In addition, those mice that do develop AML acquire additional genetic mutations, suggesting that NPM1cA cooperates with serially acquired mutations to drive AML development (Dovey 2017).It was recently discovered that mutations in the cohesin complex (consisting of the genes STAG2, SMC1A, SMC3, and RAD21) are also common in AML, with mutation in any of the four components resulting in haploinsufficiency (Cancer Genome Atlas 2013). Cohesin haploinsufficiency is enriched in patients with NPM1 mutations. Although cohesin mutations alone are insufficient to generate AML in mice (Viny 2015), they do result in increased hematopoietic stem and progenitor cell (HSPC) self-renewal (Mazumdar 2015; Fisher 2017). As the cohesin complex has known roles in chromosomal organization and the regulation of gene expression, we hypothesized that cohesin mutations would cooperate with NPM1cA to uniquely alter gene expression, resulting in AML. We crossed inducible NPM1cAflox/+ and SMC3flox/+ mouse models to examine this genetic interaction.Our current data show that the double mutant mice develop AML with increased penetrance compared with the Npm1cA/+ mice alone, with a trend toward decreased latency. The double mutant HSPCs also exhibit increased self-renewal in vitro compared to the Npm1cA/+ or SmcΔ/+ single mutants. To examine changes in gene expression, we performed RNA sequencing on lineage-depleted bone marrow in 3 mice from each genotype (WT, Npm1cA/+, Smc3Δ/+, and double) 4 weeks post excision. Consistent with our hypothesis, additive changes in gene expression were not observed. Instead, a unique set of genes were found to be deregulated in Npm1cA/+; Smc3Δ/+ marrow.In an effort to specifically target Npm1cA/+; Smc3Δ/+ mutant AML, we screened our list of uniquely-affected genes for those associated with AML. We found DOCK1 to be overexpressed in the double, but not Npm1cA/+ single, cells. High expression of this gene has been correlated with decreased overall and disease-free survival in AML patients (Lee 2017). To determine if DOCK1 contributes to the enhanced cell growth observed in vitro in our leukemic lines, we used an inhibitor that targets Dock1. This inhibitor induced apoptosis in our double leukemic cell lines but was less effective in our NPM1 single mutant or WT cells. Additionally, no effect was observed with a genetically unrelated AML line, MLL-AF9. Similarly, shRNA-mediated knockdown of Dock1 resulted in decreased cell viability in Npm1cA/+; Smc3Δ/+ leukemic lines but not in Npm1cA/+ only lines. We thus hypothesize that Dock1 represents a unique target for the treatment of patients harboring the Npm1/Cohesin mutational combination. Our results provide validity to the concept that combinatorial genetics can be used to target the unique genetic landscape of an individual patient. Future studies will focus on the impact of Dock1 inhibition in human Npm1/Cohesion mutated AML cell lines. DisclosuresVassiliou:KYMAB: Consultancy, Equity Ownership; Celgene: Research Funding. Levine:Isoplexis: Equity Ownership; Epizyme: Patents & Royalties; Imago: Equity Ownership; Loxo: Consultancy, Equity Ownership; Janssen: Consultancy, Honoraria; Gilead: Honoraria; Prelude: Research Funding; Novartis: Consultancy; Celgene: Consultancy, Research Funding; Roche: Consultancy, Research Funding; C4 Therapeutics: Equity Ownership; Qiagen: Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

Long non-coding RNA taurine-upregulated gene 1 correlates with poor prognosis, induces cell proliferation, and represses cell apoptosis via targeting aurora kinase A in adult acute myeloid leukemia.

This study aimed to investigate the correlation of long non-coding RNA (lncRNA) taurine-upregulated gene 1 (TUG1) with clinicopathological feature and prognosis, and to explore its effect on cell proliferation and apoptosis as well as the relevant target genes in adult acute myeloid leukemia (AML). LncRNA TUG1 expression was detected in bone marrow samples from 186 AML patients and 62 controls. Blank mimic, lncRNA TUG1 mimic, blank inhibitor, and lncRNA TUG1 inhibitor lentivirus vectors were transfected in KG-1 cells. Rescue experiment was performed by transfection of lncRNA TUG1 inhibitor and aurora kinase A (AURKA) mimic lentivirus vectors. Cell proliferation, apoptosis, RNA, and protein expressions were determined by CKK-8, annexin V-FITC-propidium iodide, quantitative polymerase chain reaction, and western blot assays. LncRNA TUG1 expression was higher in AML patients compared to controls and correlated with higher white blood cell counts, monosomal karyotype, FLT3-ITD mutation, poor-risk stratification, and poor prognosis, which independently predicted worse event-free survival and overall survival. In vitro, lncRNA TUG1 expression was higher in AML cell lines (KG-1, MOLM-14, HL-60, NB-4, and THP-1 cells) compared to controls. LncRNA TUG1 mimic promoted cell proliferation and decreased cell apoptosis rate, while lncRNA TUG1 inhibitor repressed cell proliferation and increased cell apoptosis rate. Rescue experiment showed that AURKA attenuated the influence of lncRNA TUG1 on AML cell proliferation and apoptosis. In conclusion, lncRNA TUG1 associates with advanced disease and worse prognosis in adult AML patients, and it induces AML cell proliferation and represses cell apoptosis via targeting AURKA.

Abstract 3766: Targeting promoter regions of c-Myc and Bcl-2 in AML cells using G-quadruplex interacting drugs

Abstract Cancer is a disease that can be characterized by overexpression of key oncogenic drivers that support tumor development and maintenance. In many instances, these oncogenic drivers are ‘undruggable’ because of structural challenges, the inability to effectively inhibit high concentrations of overexpressed proteins, and the development of drug resistance mutations. An alternative therapeutic approach is to directly inhibit gene transcription by targeting unique secondary DNA structures, called G-quadruplexes, that are associated with subsets of promoters. We investigated the activity of a class of compounds termed G-quadruplex Interacting Drugs (GQIDs) that are able to shut down the expression of specific oncogenic drivers, such as c-Myc and Bcl-2, used by tumor cells for growth and survival. We tested the activity of two GQIDs, GQC-05 and GSA-1103, on cell growth of a panel of eight pediatric and eight adult acute myeloid leukemia (AML) cell lines. Drug dose response analysis showed IC50 values ranging from approximately 10 nM to 1 μM for GQC-05 and from 40 nM to 2 μM for GSA-1103. The AML cell lines had different sensitivities to each GQID indicating a different mechanism of action for each compound. Three AML cell lines that were highly resistant to cytarabine were among the more sensitive to GQC-05. Four cell lines sensitive to GQC-05 had high expression of either c-Myc and/or Bcl-2, both of which are potential targets of these two GQIDs. Furthermore, treatment of the highly sensitive line MV-4-11 with GQC-05 showed a 3-4 fold decrease in expression of c-Myc and Bcl-2 mRNA. Furthermore, GQC-05 treatment resulted in decreased levels of both c-Myc and Bcl-2 proteins. These studies will help define a novel approach of repressing key drivers of leukemia by targeting selective promoter structures. Citation Format: Megan Turnidge, Justin J. Montoya, Apurvi Patel, David W. Lee, Eiman Aleem, Daniel H. Wai, Laurence H. Hurley, Vijay Gokhale, Robert J. Arceci, David O. Azorsa. Targeting promoter regions of c-Myc and Bcl-2 in AML cells using G-quadruplex interacting drugs. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3766.

The Induction of Inhibitory Pathways in Dendritic Cells May Hamper the Efficient Activation of Anti-Leukemia T Cells within Chemotherapy-Induced Immunogenic Cell Death

BACKGROUND: Overall survival of adult acute myeloid leukemia (AML) is still poor due to the lack of novel and effective therapies. In different malignancies including AML, some chemotherapy agents, such as daunorubicin (DNR) but not cytarabine (Ara-C), activate the immune response via the cross-priming of anti-tumor T cells by dendritic cells (DCs). Such process, known as immunogenic cell death (ICD), is characterized by intracellular and pericellular modifications of tumor cells, such as the cell surface translocation of calreticulin (CRT) and heat shock proteins 70/90 (HSPs 70/90), the extracellular release of ATP and pro-inflammatory factor HMGB1. Alongside with ICD, chemotherapy is known to induce inflammatory modifications within the tumor microenvironment, which may also elicit immunosuppressive pathways. In particular, DCs may be driven to acquire tolerogenic features, which may ultimately affect anti-tumor T-cell responses. In this study, we characterize ICD in AML to evaluate the involvement of some DC-related inhibitory pathways, such as the expression of indoleamine-2,3-dioxygenase 1 (IDO1) and the activation of PD-L1/PD-1 axis.METHODS: AML patients were analyzed at diagnosis.Before and after DNR-based chemotherapy, patient-derived T cells were extensively characterized by FACS and analyzed for their capacity to produce IFN-γ in response to autologous blasts. The AML cell line HL-60 and primary AML cells were then exposed, in vitro, to different drugs, including DNR and, as control drug, Ara-C. Dying cells were tested for the surface expression of CRT and HSPs 70/90, the release of HMGB1 and ATP. Functionally, immature DCs generated from healthy donors were pulsed with DNR-treated AML cells. Then, loaded DCs were tested for the expression of maturation-associated markers and of inhibitory pathways, such as IDO1 and PD-L1 and used to stimulate autologous CD3+ T cells. After co-culture, autologous healthy donor T cells were analyzed for IFN-g production, PD-1 expression and Tregs induction. A mouse model was set up to investigate in vivo the mechanism(s) underlying ICD in AML. The murine myelomonocytic leukemia cell line WEHI was transfected with luciferase PmeLUC probe, inoculated subcutaneously into BALB/c mice and used to measure in vivo ATP release after chemotherapy. Tumor-infiltrating T cells and DCs were characterized and correlated with ATP release.RESULTS: DNR treatment induced ICD-related modifications in both AML cell lines and primary blasts, including CRT, HSP70 and HSP90 exposure on cell surface, HMGB1 release from nucleus to cytoplasm and supernatant increase of ATP. Ex vivo, T-cell monitoring of DNR-treated AML patients displayed an increase in leukemia-specific IFN-g-producing CD4+ and CD8+ T cells in 20/28 evaluated patients. However, FACS analysis of CD8+ effector T cells emerging after chemotherapy showed a significant up-regulation of exhaustion marker such as LAG3 and PD-1, which paralleled with their reduced ability to produce active effector molecules, such as perforin and granzyme. Moreover, an increase of circulating Tregs was observed after DNR-based chemotherapy. In vitro, loading of chemotherapy-treated AML cells into DCs resulted not only in the induction of a maturation phenotype, but also in over-expression of inhibitory pathways, such as IDO1 and PD-L1. The silencing of IDO1 increased the capacity of DCs loaded with DNR-treated AML cells to induce leukemia-specific IFN-γ production by CD4+ and CD8+ T cells. In vivo, DNR therapy of mice inoculated with established murine AML cell line resulted in increased ATP release. Similarly to ex vivo and in vitro results, tumor-infiltrating DCs showed an increase in maturation status. Moreover, CD4+ and CD8+ T cells had increased IFN-γ production, but showed an exhausted phenotype.CONCLUSIONS: Our data confirm that chemotherapy-induced ICD may be active in AML and results in increased leukemia-specific T-cell immune response. However, a deep, ex vivo, in vitro and in vivo characterization of chemotherapy-induced T cells demonstrated an exhausted phenotype, which may be the result of the inhibitory pathways induction in DCs, such as IDO and PD-L1. The present data suggest that combination of chemotherapy with inhibitors of IDO1 and PD-L1 may represent an interesting approach to potentiate the immunogenic effect of chemotherapy, thus resulting in increased anti-leukemia immune response. DisclosuresCavo:Janssen-Cilag, Celgene, Amgen, BMS: Honoraria.

Preclinical Activity of a New Proteasome Inhibitor, MLN9708, in Acute Myelogenous Leukemias Expressing NPM1 Mutated Protein

ABSTRACT: The nucleophosmin-1 (NPM1) mutation represents the most common genetic lesion (30-35%) in adult acute myeloid leukemia (AML) with the mutation resulting in the cytoplasmic delocalization of the NPM protein (NPMc+, most commonly mutation type A, c.860_863dupTCTG). Outside of conventional chemotherapy, treatment options for those with relapsed disease are extremely limited. We have previously discovered that leukemic cells expressing NPMc+ are preferentially sensitive to the cytotoxic effects of the first generation proteasome inhibitor, bortezomib, as result of the induction of superoxide in these cells (Huang M, et al., Leukemia, 2013). However, bortezomib has demonstrated only modest anti-leukemic activity as a single agent in early phase clinical trials that have included primarily heavily pretreated patients not stratified according to mutational genotype. In addition, the high incidence of clinically significant peripheral neuropathy in these studies led to early discontinuation of treatment. MLN9708 (Ixazomib) is a novel second generation proteasome inhibitor that is orally bioavailable and has shown promising activity and improved tolerability in early phase clinical trials in lymphoma and in multiple myeloma, where it is currently in a Phase III clinical trial. The efficacy of MLN9708 in relapsed patients with AML has not yet been examined. We have investigated the in vitro effects of MLN9708 on the cytotoxicity, generation of reactive oxygen species (ROS), and cellular glutathione in both cultured NPMc+ AML cell lines and primary leukemic samples. Cultured OCI-AML3 cells (human AML cell line featuring the NPMc+ mutation) were approximately 50-fold more sensitive to MLN9708 with a mean half inhibitory concentrations (IC50 ± SD) of 67 nM ± 1.83 nM at 24 hours than were an AML cell line (THP-1) expressing wild type NPM1. Knockdown of both NPM1/NPMc+ by inducible NPM1 shRNA expression significantly attenuated the MLN9708-mediated cytotoxicity, as determined by MTS colorimetric and flow cytometric apoptosis assays. As with bortezomib, MLN9708 treatment at 75 nM induced a 2.1-fold (p < 0.002) increase in superoxide-specific DHE (PE)-derived fluorescence as compared to controls. Pre-incubation of cells with 25 mM N-acetylcysteine, a ROS scavenger, dramatically reduced cytotoxicity, strongly implicating the generation of ROS in the mechanism of MLN9708-induced cytotoxicity. Knockdown of NPM1/ NPMc+ expression resulted in an increase of glutathione levels of 2.6-fold (p < 0.04) in untreated cells, and 2.3-fold (p < 0.005) and 2.9-fold (p < 0.004) after a 22 hour incubation with MLN9708 at pharmacologically achievable doses of 75 nM and 150 nM, respectively (Figure 1). In addition, a concomitant 4-fold reduction (p < 0.04) in the NADP+/NADPH ratio was observed in the NPM1/NPMc+ depleted OCI-AML3 cells. In vitro apoptosis assays of primary leukemic blasts isolated from five patients with relapsed NPMc+ AML showed an IC50 for MLN9708 ranging from 49.963-467.487nM (183.8 nM ± 176.8 nM). Based on these promising in vitro studies, a Phase 2 clinical trial of single-agent oral MLN9708 (clinicaltrials.gov identifier: NCT02030405) has been initiated for patients with relapsed or refractory NPMc+ AML.We conclude that the expression of NPMc+ renders AML cells more susceptible to MLN9708 toxicity and that this effect results at least in part from the generation of superoxide. Lower glutathione levels in NPMc+ expressing cells suggests an underlying defect in the antioxidant defense pathway. Demonstration of the sensitivity of NPMc+ AML cells to MLN9708 as a single agent may serve as a platform for the development of novel combination strategies with this drug.Acknowledgments: Research was facilitated by the ASH Clinical Research Training Institute and AACR/ASCO Clinical Cancer Research training program. J.S.G is funded by the Leukemia & Lymphoma Society Career Development Award and ASCO YIA award through a grant supported by Mr. Aaron Sasson. J.S.G. is further supported by the Stanford Hematology Fellowship Program. [Display omitted] DisclosuresOff Label Use: MLN9708 is an investigational compound by Millennium Pharmaceuticals, Inc. (MPI). Through an MTA we have secured the compound for in vitro analysis. This data has led to a clinical trial and drug for that study is supplied by MPI.. Medeiros:Agios: Consulting - Ad board Other.

Abstract 5220: Identifying AML-specific key targeted drugs using high-throughput drug sensitivity and resistance testing profiles of AML cells.

Abstract Introduction: Conventional cytotoxic chemotherapy regimens for adult acute myeloid leukemia (AML) are effective in curing less than 50% of the patients, and there is a major need for targeted drugs with better anti-cancer selectivity. Here, our aim was to i) identify potential clinically used or emerging cancer drugs by quantitative drug sensitivity and resistance testing (DSRT) of 16 AML cell lines ii) compare the cell line data with results obtained from tested 24 ex vivo AML patient specimens iii) identify genomic correlations potentially explaining drug responsiveness. Methods: The cancer pharmacopeia-wide drug collection is composed of 119 FDA approved and 90 investigational chemical compounds including cytotoxic agents and cell signaling molecule inhibitors. Each drug was tested over a 10,000-fold concentration range and that has generated quantitative five point dose-response curves. AML cells were plated in 384 well plates (where the drugs were pre-printed using an acoustic nano-dispensing technology, Labcyte®) and incubated in standard cell culture conditions. Cell viability was measured by Cell Titer Glow® luminescence assays. Analysis of dose response curves using Dotmatics® software resulted in IC50 values. Moreover, the genomic profiles of the AML cell lines were determined by microarrays and/or next-gen sequencing data for further integration with drug responses. Results: Comprehensive data analysis of 16 AML cell lines indicated that specific targeted drugs were selectively killing AML cells. The data analysis revealed relatively strong responses for MEK inhibitors in most AML cell lines (e.g. refametinib 87%, trametinib 82%, selumetinib 75%) while 21% of ex vivo AML patient samples were sensitive to these MEK inhibitors. In case of rapalog sensitivity, 80% of AML cell lines (e.g. temsirolimus 82%, everolimus 71%, sirolimus 81%) and 25% of ex vivo AML patient cases were responsive to the mTOR inhibitors. The AML cell lines carrying FLT3-ITD mutations were extremely sensitive to FLT3 inhibitors (e.g. quizartinib, lestaurtinib, tandutinib, and sorafenib) but very few responses to FLT3 inhibitors were observed in AML patients carrying an ITD mutation in the FLT3 kinase. Summary: Systematic DSRT profiling of AML cell lines illustrates drug sensitivity patterns to classify the cell lines as sensitive or resistant to specific classes of drugs. mTOR and MEK inhibitors were among the most effective inhibitors for most cell lines and also in some ex vivo patient cases suggesting that these drugs may have potential as therapeutic agents in AML. Also, bioinformatics predictions can be used to identify key synergistic combinations of tested drugs for effective AML therapy. Further integration of molecular profiles and functional responses of AML cell lines will help provide better understanding of drug efficacy based on known genetic background of the disease. Citation Format: Disha Malani, Astrid Murumägi, Tea Pemovska, Bhagwan Yadav, Evgeny Kulesskiy, Jing Tang, John Patrick Mpindi, Maija Wolf, Riikka Karjalainen, Tero Aittokallio, Caroline Heckman, Kimmo Porkka, Krister Wennerberg, Olli Kallioniemi. Identifying AML-specific key targeted drugs using high-throughput drug sensitivity and resistance testing profiles of AML cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5220. doi:10.1158/1538-7445.AM2013-5220

Abstract 3188: Development of a cancer pharmacopeia-wide ex-vivo drug sensitivity and resistance testing (DSRT) platform for AML: Towards individually optimized therapy and improved understanding of drug resistance patterns

Abstract In order to discover unexpected anti-cancer efficacies of approved and emerging drugs, we established a diagnostic ex vivo drug sensitivity and resistance testing (DSRT) platform covering the entire cancer pharmacopeia as well as emerging anti-cancer compounds. Here, the platform was applied to analyze bone marrow (BM) mononuclear cells from 17 adult acute myeloid leukemia (AML) patients, 3 healthy donors as well as 7 AML cell lines. The DSRT panel covered FDA-approved small molecule oncology drugs (n=120), as well as emerging, investigational and pre-clinical oncology compounds (n=120), such as kinase (e.g. RTKs, checkpoint and mitotic kinases, Raf, MEK, JAKs, mTOR, PI3K), and non-kinase inhibitors (e.g. HSP, Bcl, activin, HDAC, PARP, Hh). To generate dose-response curves, each of the drugs was applied over a 10,000-fold concentration range. In addition, the samples underwent deep molecular profiling including exome- and transcriptome sequencing, as well as phosphoproteomic analysis. DSRT provided consistent and reliable data from ex vivo samples with a high correlation between data from individual healthy BM samples (r=0.93). Bioinformatic processing of the data from AML resulted in several key observations. First, overall drug response profiles of AML blast cells were distinctly different from healthy BM controls suggesting several potential leukemia-selective effects, such as multi-kinase (dasatinib), MEK, and mTOR inhibitors. Second, the overall drug responses from AML cell lines and the patient ex vivo samples showed differences, suggesting that ex vivo testing may reveal cancer-selective effects not previously seen in established cancer cell line panels. Third, the response data from patient samples clustered many drugs consistently into the expected functional classes (such as topoisomerase II inhibitors, MEK inhibitors and rapalogs), whereas other drug classes were more dispersed (such as FLT3 inhibitors with quizartinib clustering away from all other tyrosine kinase inhibitors), suggesting secondary targets playing a key role in drug efficacy. Fourth, analysis of serial samples from patients developing clinical resistance to targeted agents showed striking agreement between the ex-vivo DSRT profiles and clinical responses. In conclusion, comprehensive DSRT platform generated powerful novel insights on AML drug response and may enable individual optimization of therapies, particularly for recurrent leukemias. DSRT will also serve as a powerful hypothesis-generator for clinical trials, particularly for emerging drugs. The ability to correlate ex vivo response profiles for hundreds of drugs in clinical samples with deep molecular profiling data will yield exciting new translational and pharmacogenomic opportunities for cancer therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3188. doi:1538-7445.AM2012-3188

PLX3397 Is An Investigational Selective FLT3 Inhibitor That Retains Activity Against the Clinically-Relevant FLT3-ITD/F691L “Gatekeeper” Mutation in Vitro

Abstract 764 Background:Activating mutations in FLT3 are detected in approximately 30% of adult acute myeloid leukemia (AML) cases, most commonly involving internal tandem duplication (ITD) events. The clinically-active FLT3 inhibitor AC220 (quizartinib) was recently reported to be associated with a composite complete remission (CR) rate of 43% in relapsed/refractory FLT3-ITD+ AML patients in an interim analysis of a phase II study (Cortes, et al, EHA 2011, abstract #1019). AC220 is vulnerable in vitro to a limited number of resistance-conferring mutations in the FLT3 kinase domain (Smith et al, AACR 2011, abstract #4737). Mutations at two of these residues, F691 and D835, have been detected in 9/9 patients with AML with loss of response to AC220 analyzed to date (Smith et al, ASH 2011, submitted). Mutations at “gatekeeper” residues such as F691 have repeatedly surfaced as important mediators of clinical resistance to inhibitors of BCR-ABL, EGFR, ALK and KIT kinases. Identifying tyrosine kinase inhibitors that retain activity against these substitutions has proven challenging. PLX3397 is a potent and selective inhibitor of FMS, KIT and oncogenic FLT3 with an elimination half-life of 20 hours in humans. Steady-state plasma concentrations of 10–20 uM have been achieved in solid tumor patients enrolled in a phase I study, where minimal myelosuppression has been observed (Anthony et al, J Clin Oncol 29: 2011, suppl abstr 3093). Results:PLX3397 selectively inhibited the proliferation of the human FLT3-ITD+ AML cell lines MV4-11 and MOLM-14 with a 50% inhibitory concentration (IC50) in the submicromolar range (0.09–0.2 uM). PLX3397 inhibited phosphorylation of FLT3-ITD in cells with a dose response similar to the growth inhibition range. We next evaluated the ability of PLX3397 to inhibit the proliferation of Ba/F3 cells transformed with FLT3-ITD and AC220-resistant FLT3-ITD mutant isoforms F691L, D835V/Y, and Y842C/H. PLX3397 inhibited the proliferation of Ba/F3/FLT3-ITD cells (IC50 0.07 uM) at a concentration comparable to that needed to inhibit the growth of MV4-11. Encouragingly, PLX3397 retained activity against Ba/F3 cells expressing the clinically-relevant F691L gatekeeper mutation at a similar concentration (IC50 0.37 uM). Other AC220-resistant mutations evaluated conferred substantial cross-resistance to PLX3397 (IC50 >200x IC50 of FLT3-ITD alone for all mutations; range 14.3–130,000 uM). Western blot analysis of FLT3 autophosphorylation demonstrated dose responsiveness in the same concentration range as observed in cellular proliferation results for each FLT3-ITD mutant isoform. Based upon molecular docking studies, PLX3397 forms non-specific hydrophobic interactions with the gatekeeper side-chain, and thus is less sensitive to the F691L mutation than AC220, for which the combined effects of steric hindrance and polarity mismatch impede its binding to the FLT3-ITD/F691L. To further assess if PLX3397 warrants clinical evaluation as a FLT3 inhibitor in AML, we performed a modified plasma inhibitory assay by incubating MOLM-14 cells in either normal donor or AML patient plasma spiked with increasing concentrations of PLX3397 as well as unmanipulated, steady-state plasma samples from the solid tumor PLX3397 phase I trial. Using phospho-specific flow cytometry to evaluate FLT3 signaling through the downstream protein ribosomal S6, we observed near-maximal reductions in phospho-S6 in both normal and AML patient plasma containing >10 uM PLX3397 as well as plasma samples obtained from the solid tumor trial. Conclusions:PLX3397 harbors substantial promise for the treatment of FLT3-ITD+ AML. Our data demonstrate that potent FLT3 inhibitory concentrations are achieved in humans. Moreover, while mutations in the FLT3-ITD kinase domain represent an important cause of loss of response to clinically-active FLT3 inhibitors, PLX3397 retains activity against the FLT3-ITD/F691L “gatekeeper” mutation, which we have found to be a common cause of preclinical and acquired clinical resistance to AC220. A multi-site phase I/II study of PLX3397 in FLT3-ITD+ AML has been initiated. Disclosures:Off Label Use: Investigational agent PLX3397 will be discussed. Zhang:Plexxikon Inc.: Employment. Carroll:Agios Pharmaceuticals: Research Funding; TetraLogic Pharmaceuticals: Research Funding; Sanofi Aventis Corporation: Research Funding; Glaxo Smith Kline, Inc.: Research Funding. Shah:Novartis: Consultancy; Bristol-Myers Squibb: Consultancy, Research Funding; Ariad: Consultancy, Research Funding.

Expression of topoisomerase (topo) II in adult acute myeloid leukemia (AML): Relationships to immunophenotype and treatment outcomes.

6564 Background: Anthracyclines used in the treatment of AML target topo II. Topo II α and β are genetically distinct with different expression (expr) patterns and cellular distribution. While topo IIα has been associated with increased proliferation and an adverse prognosis in AML, there is no information regarding topo IIβ. This study examined the relationships of topo IIα and β expr to immunophenotype (IP), multidrug resistant (MDR) phenotype and outcomes in adult AML. Methods: 230 pts with non-M3 AML by FAB criteria who received frontline ara‐C/daunomycin (AD) therapy during the years 1992-98 on studies SWOG-9031, ‐9126, ‐9333 or ‐9500 were included (median age 63 years [18-88], WBC 29x109/L [0.8-274]). RT-PCR of RNA from marrow blasts was run with topo IIα and β specific primers (ABI PRISM 7900HT). Topo IIα and β expr in pts, normalized to internal standard β2‐microglobulin, was measured by log (fold-change) relative to the Kasumi AML cell line. Treatment outcomes - complete response (CR), resistant disease (RD), and relapse-free and overall survival (RFS, OS) - were analyzed by logistic or Cox regression. Results: Topo IIα and β data were available for 211 pts. Topo IIα expr in pts ranged from 298-fold less to 91-fold more than in the Kasumi AML cell line, but was not significantly correlated with any pt characteristic, IP or treatment outcome in uni- or multivariate analysis. In contrast, topo IIβ ranged from only 9‐fold less to 10-fold more than the cell line, but was associated with higher blast percentage in marrow (P=0.0001) or blood (P=0.0055); higher CD7 (P=0.0071); and lower CD14 (P<0.0001) and CD54 (P<0.0001) in multivariate regression analysis. In 105 pts with MDR data, topo IIβ was also inversely correlated with Rhodamine efflux (P<0.0001) and CD11c (P=0.0010). However after adjusting for prognostic factors, topo IIβ was not significantly related to any outcome, nor did topo IIβ interact significantly with any prognostic factors. Conclusions: Topo IIβ expr reflects aspects of disease biology, such as highly proliferative disease (higher blasts), IP and MDR, and thus may not be an independent variable influencing outcome in adult AML pts treated with anthracycline-based therapy.