Mutant Acute Myeloid Leukemia Cells Research Articles

Autophagy Promotes the Survival and Therapy Resistance of Human Acute Myeloid Leukemia Cells Under Hypoxia

Background: Acute myeloid leukemia (AML) is an aggressive hematological malignancy occurring primarily in older adults. Despite high remission rates following upfront therapy, the disease eventually recurs in most patients, and overall cure rates remain only 20-30%. Preclinical studies have recently demonstrated that the marrow microenvironment in acute leukemic hosts to be intrinsically hypoxic, with AML progression associated with further hypoxia. Moreover, human AML cells and primary AML colonies cultured under hypoxia are markedly less sensitive to cytarabine chemotherapy than normoxic cells. We hypothesized that AML cells may respond to hypoxic stress and mediate chemoresistance in part by invoking autophagy, a highly regulated catabolic process by which cells evade apoptosis by degrading damaged cellular components. To test our hypothesis, we investigated the effects of two known autophagy inhibitors (bafilomycin A1 (Baf) and chloroquine (CQ)) on the sensitivity of human AML cells to various therapeutic agents under differing oxygen levels.Methods: We treated HEL (FLT-3 wildtype) and MV4-11 (FLT-3 ITD mutant) AML cells with autophagy inhibitors (Baf and CQ) alone and in combination with a chemotherapeutic drug (cytarabine (AraC), doxorubicin (Dox), decitabine (Dac)) or a tyrosine kinase inhibitor (sorafenib, SFN) under normoxic (21% O2) or hypoxic (1% O2) conditions. Apoptosis /cell death and proliferation were measured by flow cytometry for Annexin-PI and MTT assays, respectively. Autophagy was assessed by flow cytometry using Cyto-ID Green Dye (Enzo Life Sciences), fluorescent microscropy for acridine orange dye accumulation, and western blot analysis.Results: Autophagy in human ALL and AML cell lines was significantly increased following 24-72 hours of hypoxia (1% O2) as compared with normoxia and was a relatively late response to prolonged low oxygen levels (> 24 hours). Treatment with cytotoxic agents (AraC or Dox) or hypomethylating agent (Dac) resulted in a dose-dependent increases in the number of autophagic vesicles in AML cells consistent with autophagy induction. Low-doses of Baf which selectively inhibits the vacuolar H+ ATPase to prevent lysosomal acidification, and CQ, which blocks lysosome-autophagosome fusion by raising the pH of lysosomes and endosomes, both resulted in buildup of autophagic vesicles by flow cytometry consistent with inhibition of autophagic flux in human AML cells. Combination treatment with an autophagy inhibitor (Baf, CQ) and cytotoxic chemotherapy (AraC, Dox) significantly enhanced apoptosis and cell death over single agent therapy. Treatment with Baf combined with hypomethylating therapy (Dac) synergistically improved the anti-leukemic effects as compared with monotherapy (CI 0.09-0.31)(see Figure). The addition of Baf also improved cell death induced by sorafenib (SFN) on FLT-3 ITD mutant human AML cells (MV4;11) (CI 0.36-0.9) (see Figure). Single agent Baf or CQ treatment resulted in significantly higher levels of apoptosis and cell death in AML cells under hypoxia. The anti-tumor activity of almost all combination regimens was consistently improved under hypoxic versus normoxic culture conditions. In vivo CQ treatment (25-50 mg/kg i.p. daily) in preclinical human AML xenograft models significantly inhibited systemic leukemia progression as a single agent. Further experiments investigating the in vivo effects of CQ combined with other chemotherapeutic agents in preclinical AML xenograft models are ongoing.Conclusions: Our data suggest that human AML cells preferentially induce autophagy to promote survival under chronic hypoxia and following cytotoxic, hypomethylating, and FLT-3 tyrosine kinase inhibitor therapy. Strategies targeting autophagy therefore may have the potential to improve therapeutic responses and overcome chemoresistance of AML cells within the hypoxic bone marrow microenvironment. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Genetic and Pharmacologic Notch4 Inhibition Synergizes with FLT3 Tyrosine Kinase Inhibition in Vitro to More Effectively Eliminate FLT3/ITD-Positive Leukemia Cells

FMS-like tyrosine kinase-3 (FLT3) is a receptor tyrosine kinase expressed in hematopoietic stem/progenitor cells. Mutated in approximately 1/3 of patients, FLT3 is the most frequently mutated gene in acute myeloid leukemia (AML). The presence of FLT3 internal tandem duplication (ITD) mutations, which renders FLT3 constitutively active, confers a particular poor prognosis. While several FLT3 tyrosine kinase inhibitors (TKIs) have been developed to inhibit FLT3 signaling, the clinical success of these drugs is limited because they fail to achieve frequent complete durable responses, despite achieving high levels of FLT3 inhibition. These limited clinical responses suggest that monotherapy is unlikely to be curative. Current clinical trials of FLT3 inhibitors are administered in combination with chemotherapy to achieve success in inducing complete remissions. While there is hope that FLT3 inhibition in combination with chemotherapy will increase cure rates, it would be optimal to achieve cures without chemotherapy altogether and its inherent toxicities. Towards this goal it is necessary to uncover critical genes/pathways that collaborate with FLT3 mutations to transform cells. In recent years, the use of whole genome sequencing has greatly expanded the list of genes mutated in AML. However, many of these mutations do not represent a practical therapeutic target because they are present in a low frequency in the patient population, making molecular targeted therapy for each of these improbable to achieve. We hypothesize that these mutated genes likely funnel into a more limited number of signaling pathways, with some pathways being more important than others in contributing to FLT3/ITD AML. Identifying FLT3/ITD cooperative pathways whose inhibition might synergize with FLT3 inhibition could hold promise for greatly improving the cure rate in FLT3 mutant AML.We performed a loss of function RNAi screen in FLT3/ITD+ AML cell lines (Molm14 and MV411) to uncover genes and pathways whose inhibition combined with FLT3/ITD inhibition to more effectively kill FLT3 mutant AML cells. Notch4 signaling was identified as one such potential target. We found Notch4 to be overexpressed in FLT3/ITD+ AML cells lines as well as in the Lin-Sca+Kit+ fraction of bone marrow isolated from FLT3/ITD+ mice. Stable, tetracycline-inducible Notch4 knockdown cell lines were established in Molm14 and MV411 cell lines. While Notch4 knockdown alone slightly decreased cell growth, Notch4 knockdown in combination with FLT3 TKI demonstrated a significant decrease in proliferation compared to FLT3 TKI treated cells expressing a scrambled shRNA control. Additionally, colony forming unit assays revealed that Notch4 knockdown cells treated with FLT3 TKIs exhibited a decrease in clonogenicity compared to FLT3 TKI treated control cells. Pharmacologic inhibition of Notch4 by treatment with gamma secreatse inhibition (GSI) was also tested. Molm14, MV411, and THP-1 (FLT3 wild type AML) cells were treated with FLT3 TKIs (sorafenib, CEP-701, or AC220) alone, GSI alone, FLT3 TKI plus GSI, or vehicle control and assessed for anti-proliferative and apoptotic effects by MTT, and annexin V/7-AAD staining, respectively. In both assays, the combination of FLT3 TKI and GSI exhibited synergy with combination index (CI) values <1. Furthermore, Western blot analysis showed that while treatment of Molm14 and MV411 cells with GSI had little effect on FLT3 signaling (pSTAT5, pAKT, pMAPK activation), the combination of GSI plus FLT3 TKI inhibited FLT3 signaling significantly more than treatment with FLT3 TKI alone. The results of these experiments identify Notch4 signaling as a potential pathway whose inhibition can synergize with FLT3 inhibition to more effectively kill FLT3/ITD+ AML cells. Combining FLT3 inhibition with Notch4 inhibition may have potential for improving the cure rate of patients with FLT3 mutant AML. DisclosuresNo relevant conflicts of interest to declare.

Abstract 2678: All-trans-retinoic acid as a new therapeutic approach to target isocitrate dehydrogenase mutations in acute myeloid leukemia

Abstract Mutations of isocitrate dehydrogenases 1 and 2 (IDH1/2) occur in about 15% of acute myeloid leukemia (AML) patients, and lead to the production of an oncometabolite, the 2-hydroxyglutarate (2-HG). Importantly, many clinical studies demonstrated that IDH1/2 mutations are systematically conserved at relapse, suggesting that this mutated enzyme could contribute to early leukemogenesis. In an effort to identify therapy specifically targeting IDH1 R132H mutated blasts, we developed an AML cell line model carrying IDH1 R132H mutation. We first stably transduced HL60 cell line to establish clones carrying either empty vector, IDH1 WT gene or IDH1-R132H mutated gene. We then characterized these clones with different features including 2-HG production. Since demethylation of many genes involved in the retinol metabolism is impaired by IDH1-R132H mutation and because all-trans retinoic acid (ATRA), a retinol metabolite, is already used in clinic, we studied its effects on IDH1-R132H mutated AML cells. Here, we showed on our transduced models as well as on primary AML patient samples (n=6), that AML blasts harboring IDH1-R132H mutation were highly sensitive to low doses of ATRA, compared to the IDH1 WT. In fact, IDH1-R132H mutation sensitized to ATRA-induced differentiation, as shown by increased expression of the differentiation marker CD11b and morphological changes evidenced with May-Grünwald-Giemsa staining. This was followed by changes of CD14 and CD15 expression, as well as granulocytic enzymatic activity measured with the nitroblue tetrazolium reduction assay. This terminal granulocytic differentiation came with the reduction of proliferation and colony formation, and then, quickly led to apoptosis of IDH1 R132H mutated AML cells. Moreover, we showed that the level of differentiation of IDH1 R132H mutated models was correlated to the magnitude of IDH1-R132H protein expression and thus to the 2-HG production. We further demonstrated that treatment with a cell-permeable form of 2-HG sensitized empty construct to ATRA-induced differentiation, whereas the inhibition of IDH1-R132H activity reduced its ATRA-sensitivity in IDH1-R132H mutant. Altogether, our results show that IDH1-R132H mutation sensitizes to ATRA-induced granulocytic terminal differentiation in a 2-HG concentration-dependent manner and that it leads to their apoptosis. Since serum 2-HG concentration of IDH mutated patients is approximately 100 fold higher than for non mutated patient, we could expect a high and specific effect of ATRA treatment on IDH1 mutated AML patients. Furthermore, as this mutation could contribute to relapse, this might be a promising therapeutic strategy to achieve a long-term remission in AML with IDH1 mutations. Citation Format: Helena Boutzen, Fabienne De Toni, Estelle Saland, Eric Delabesse, Véronique De mas, Cécile Demur, Florence Castelli, Lara Gales, Virginie Penard-lacronique, Stéphane De Botton, Jean-Charles Portais, Christophe Junot, Stéphane Manenti, Christian Récher, Jean-emmanuel Sarry. All-trans-retinoic acid as a new therapeutic approach to target isocitrate dehydrogenase mutations in acute myeloid leukemia. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2678. doi:10.1158/1538-7445.AM2014-2678

Deguelin, a selective silencer of the NPM1 mutant, potentiates apoptosis and induces differentiation in AML cells carrying the NPM1 mutation.

Nucleophosmin (NPM1) is a multifunctional protein that functions as a molecular chaperone, shuttling between the nucleolus and the cytoplasm. In up to one third of patients with acute myeloid leukemia, mutation of NPM1 results in the aberrant cytoplasmic accumulation of mutant protein and is thought to be responsible for leukemogenesis. Deguelin, a rotenoid isolated from several plant species, has been shown to be a strong anti-tumor agent. Human leukemia cell lines were used for in vitro studies. Drug efficacy was evaluated by apoptosis and differentiation assays, and associated molecular events were assessed by Western blot. Gene silencing was performed using small interfering RNA (siRNA). Deguelin exhibited strong cytotoxic activity in the cell line of OCI-AML3 and selectively down-regulated the NPM1 mutant protein, which was accompanied by up-regulation of the activity of caspase-6 and caspase-8 in high concentrations. Deguelin induced differentiation of OCI-AML3 cells at a nontoxic concentration which was associated with a decrease in expression of activated caspase-8, p53, p21, and the 30-kD form of CCAAT/enhancer binding protein α (C/EBPα), whereas no effects were found in OCIM2 cells expressing NPM-wt. Moreover, treatment with siRNA in the NPM mutant cell line OCI-AML3 decreased expression of p53, p21, pro-caspase-8, and the 30-kD form of C/EBPα, and it inhibited proliferation and induced differentiation of the OCI-AML3 cells. In conclusion, deguelin is a potent in vitro inhibitor of the mutant form of NPM1, which provides the molecular basis for its anti-leukemia activities in NPM1 mutant acute myeloid leukemia cells.

Reversal of Acquired Drug Resistance in FLT3-Mutated Acute Myeloid Leukemia Cells via Distinct Drug Combination Strategies

FMS-like tyrosine kinase-3 (FLT3) internal tandem duplication (FLT3-ITD) mutations are common in patients with acute myeloid leukemia (AML). These patients regularly develop resistance to FLT3 inhibitors suggesting that targeted combination drug strategies are needed to enhance AML therapy efficacy. Acquired point mutations of FLT3-ITD gene were screened using cDNA-based sequencing approach in vitro sorafenib-resistant cells, which were developed by long-term exposure of Ba/F3-ITD to increasing doses of sorafenib, and in FLT3-ITD mutated AML patients, who developed relapse following sorafenib therapy. Drug effects (e.g., proliferation inhibition, apoptosis induction, and changes in signal transduction protein expression) were assessed in AML cells harboring the point mutations in vitro and in FLT3-ITD-mutated AML patient samples. We identified several acquired point mutations in the tyrosine kinase domains (TKD) of the FLT3 gene in sorafenib-resistant murine leukemia cell line carrying human FLT3-ITD mutations, which were also detected in two of four sorafenib-resistant patient samples. Engineering these point mutations into Ba/F3-ITD cells generated sublines that demonstrated varying degrees of sorafenib [a type II tyrosine kinase inhibitor (TKI)] resistance. A similar pattern of resistance could be observed by exposing these sublines to the other type II TKIs AC220 and MLN518. However, these sublines retained sensitivity to the type I TKIs PKC412 or crenolanib. The combination of crenolanib with sorafenib demonstrated marked cytotoxic effects in all of the sorafenib-resistant sublines. These combination strategies could be clinically important in reversing acquired resistance to FLT3 inhibition in AML.

Study Of Activity Of E6201, a Dual FLT3 and MEK Inhibitor, In Acute Myelogenous Leukemia With FLT3 Or RAS Mutation

Internal tandem duplication (ITD) or point mutation of Fms-like tyrosine kinase 3 (FLT3) and N/KRAS mutations in patients with acute myeloid leukemia (AML) lead to aberrant activation of FLT3 and/or RAS–mitogen-activated protein kinase (MAPK) pathways and are associated with poor prognosis (Kottaridis et al, Leuk Lymphoma Vol. 44:905, 2003; Thiede et al, Blood Vol. 99;4326, 2002). Therapy with inhibitors targeting these pathways individually may at best result in short lasting responses in the appropriate mutational context (Borthakur et al, Haematologica Vol. 96:62, 2011; Cortes et al, Blood Vol. 114:636a, 2009). Persistent activation of MEK/ERK signaling pathway is seen in cells resistant to FLT3-ITD inhibitor sorafenib that harbor acquired point mutations of FLT3 in tyrosine kinase domains in addition to ITD mutation (Moore et al, Leukemia Vol. 26:1462, 2012).E6201 is a MEK1/FLT3 dual inhibitor with inhibitory activity in low nanomolar concentrations against both targets. We tested E6201 against AML cells including FLT3-inhibitor resistant cells, AML patient samples and investigated its efficacy in murine AML model. E6201 inhibited cell growth and induced apoptosis in AML cells with FLT3 ITD mutations (including sorafenib-resistant cells harboring ITD plus N676D/Y842C point mutations) at nanomolar levels, and showed 600 to 1000-fold more selective activity against cells with FLT3-ITD mutations than those with FLT3-WT (IC50s 0.003µM, 0.005 and 0.002µM, respectively, in Ba/F3-ITD and FLT3-ITD mutant MOLM13 and MV4-11cells compared to 3.18µM in Ba/F3-FLT3-WT cells). In addition, OCI/AML3 cells [FLT3 and RAS wild-type(WT)], which have high basal p-ERK level and are resistant to most of chemotherapeutic drugs, were sensitive to E6201 (IC50 = 0.037µM). Consistent with its MEK1 inhibitory activity, E6201 was more active against NRAS mutation carrying OCI/AML3 and MV4-11 cells than their NRAS-WT isogenic cells. E6201-induced apoptosis appears to be p53 dependent as p53-wild-type OCI/AML3 and MOLM13 cells were significantly more sensitive compared to their paired p53-knockdown cells. EC50 of E6201 was at sub-micromolar levels in all 5 FLT3-ITD mutant primary AML samples, which included one with FLT3-ITD/RAS dual mutation.NOG mice bearing xenografts of MOLM13-Luc-GFP (FLT3-ITD mutated) cells were treated with E6201 i.v. starting on day 5 after leukemia cell injection until day 21 on a twice-per-week schedule. Bioluminescence imaging revealed that the tumor burden (mean luminescence) was reduced (3.1 x 106 and 2.7 x 106vs. 5.6 x 106 Photons/sec, p< .01 in 20mg/kg and 40mg/kg groups compared with vehicle group)(Fig 1) and histologically leukemia cells infiltrations were profoundly reduced in the bone marrows, spleens, livers and lungs on Day 9 after first drug treatment. Additionally, the median survival was modestly extended from 16 days of vehicle-treated mice to 18 days of E6201-treated mice (P <0.01). [Display omitted] Mechanistically, E6201 significantly suppressed p-FLT3 and p-ERK in all tested FLT3 mutant AML cell lines and p53 wild-type OCI/AML3 cells. In addition, decrease of Bcl-xL and Mcl-1 levels and increase of cleaved-caspase-3 was observed in all FLT3 mutant cell lines after treatment with E6201 for 24 hours. In conclusion, E6201 is active against AML cells with FLT3 and/or RAS mutation. A clinical trial is in development in FLT3 and/or RAS mutant AML. Disclosures:Borthakur:Eisai, Inc.: Research Funding. Nomoto:Eisai, Inc.: Employment. Zhao:Eisai, Inc.: Employment.

Growth Inhibitory Function Of a Novel T-LAK Cell-Originated Protein Kinase Inhibitor On FLT3-ITD Positive Acute Myeloid Leukemia (AML) Cells

Abstract T-LAK cell-originated protein kinase (TOPK/PBK), a serine-threonine mitogen-activated protein kinase kinase is highly expressed and correlated with more aggressive disease in several types of cancer, but is undetectable in normal tissues except the testis and fetal tissues. TOPK is up-regulated in a variety of hematologic malignancies including acute myeloid leukemia (AML) and may be involved in disease pathogenesis. To investigate the role of TOPK in AML and to develop the rationale for therapeutic targeting, we initially examined the expression level of TOPK protein in AML cell lines by western blot analysis. TOPK expression was detected at high levels in 8 out of 11 AML cell lines. Importantly, TOPK was not detected in mobilized, peripheral blood-derived stem cells from healthy donors. To determine the role of TOPK in AML, we utilized a loss of function approach in two AML cell lines (MV4-11 and U937 cells) and assessed cell viability and apoptosis by MTS and Annexin/PI staining assays, respectively. Cells transfected with TOPK-siRNA showed significant decrease in cell viability (∼70%, P&lt;0.001, both cell lines) and significant increase in apoptosis 48 hours following transfection compared to cells transfected with control-siRNA. A small molecule compound that inhibits TOPK kinase is currently undergoing pharmaceutical development for cancer treatment. We treated nine AML cell lines with the compound for 48 hours and assessed cells viability by MTS assay. Decrease in cell viability following treatment with increasing doses of the compound was noted with variable sensitivities among AML cell lines. In addition, the compound showed enhancement of cell differentiation assessed by CD11b staining in U937 cells. Cell lines with FLT3 mutations (MV4-11, MOLM13 and KOCL-48) were significantly more sensitive to the treatment compared to cell lines with unmutated FLT3. We next treated cells with increasing doses of the compound and assessed cells viability and apoptosis by MTS and flow cytometry assays. Following the treatment with the compound, IC50 was &lt;10nM for MV4-11 and MOLM13 cells (FLT3-ITD positive cell lines), and &gt;20nM for U937 and KG1 (FLT3-ITD negative cell lines). 40 nM of the compound induced apoptosis by ∼80% in MV4-11 and MOLM13 cells compared to 49% in U937 and 9% in KG1 cells, at 48 hours following treatment. We also validated the anti-leukemia activity in primary blasts from patients with AML (n=2). FLT3-ITD positive blasts had an IC50 of ∼15nM and showed 40% increase in apoptosis following treatment with 20nM of the compound. To gain a mechanistic insight into why FLT3-ITD mutant cells are preferentially sensitive to this TOPK inhibitor, we performed a gene expression profile microarray analysis on MV4-11 cells treated with 20nM of the compound or transfected with TOPK siRNA in comparison with untreated cells and cells transfected with control-siRNA. We observed significant downregulation in genes involved in cell cycle control pathways in the signatures associated with the compound-treated and TOPK siRNA-transfected cells. Interestingly, FLT3 was among the significantly downregulated genes in the compound treated cells (∼80%) and in TOPK-siRNA transfected cells (∼30%). We then examined the activity and the expression levels of FLT3 protein following the treatment. Consistently, we found that both the phospho-FLT3 and the total FLT3 protein levels were completely depleted in cells treated with 10, 20 and 40 nM of the compound as early as 16 hours following treatment. In conclusion, TOPK is highly expressed in AML and may act as novel therapeutic target. A novel TOPK kinase inhibitor exhibits preferential cytotoxicity to FLT3-ITD mutated AML cells, possibly through inhibition of FLT3 protein expression. Although further research is needed to determine the mechanism by which the compound inhibits FLT3 protein expression, this novel compound may represent a new targeted therapy for this adverse risk subset of patients with AML. Disclosures: Matsuo: OncoTherapy Science, Inc.: Employment. Nakamura:Oncotherapy Science. Ltd.: share holder Other.

Characterization Of The HLA Class II Ligandome In Acute Myeloid Leukemia (AML) Reveals Novel Candidates For Peptide-Based Immunotherapy

CD4+ T cells are crucial for the induction and maintenance of cytotoxic T cell responses, but can also mediate direct tumor rejection. The therapeutic efficacy of peptide-based cancer vaccines may thus be improved by including HLA class II epitopes to stimulate T helper cells. In contrast to HLA class I ligands, only a small number of class II ligands of TAA has been described so far. We recently reported on the overexpression of HLA class II in AML cells as compared to autologous monocytes and granulocytes as well as on the first HLA class I leukemia associated antigens identified directly on the cell surface of primary AML cells (Stickel et. al. abstract in Blood 2012). In this study we characterized the HLA class II ligandome in AML to identify additional ligands for a peptide-based immunotherapy approach.HLA class II ligands from primary AML cells as well as bone marrow and peripheral blood mononuclear cell (BMNCs/PBMCs) of healthy donors were analyzed using the approach of direct isolation and identification of naturally presented HLA peptides by affinity chromatography and mass spectrometry (LC-MS/MS). LC-MS/MS peptide analysis provided qualitative and semi-quantitative information regarding the composition of the respective ligandomes. Comparative analysis of malignant and benign samples served to identify ligandome-derived tumor associated antigens (LiTAAs) and to select peptide vaccine candidates. Most abundantly detected peptides were functionally characterized with regard to their ability to induce a specific CD4+ T-cell response in healthy donors and in tumor patients using ELISpot. Samples from 10 AML patients (5 FLT3-ITD mutated) and 18 healthy donors were analyzed. We identified more than 2,100 AML-derived HLA class II ligands representing >1,000 different source proteins, of which 315 were exclusively represented in AML, but not in healthy PBMC/BMNC. Data mining for broadly represented LiTAAs pinpointed 26 HLA class II ligands from 8 source proteins that were presented exclusively on more than 40% of all analyzed AML samples as most promising targets. Amongst them were already described TAAs (e.g., RAB5A) as well as several so far understated proteins (e.g. calsyntenin 1, glycophorin A, mannose-binding lectin 2). Subset analysis revealed 58 LiTAAs presented exclusively on FLT3-ITD mutated AML cells. Additional screening for HLA class II ligands from described leukemia associated antigens showed positive results for NPM1 (1 peptide sequence) and MPO (13 peptide sequences). Peptides from calsyntenin 1 and RAB5A were able to elicit CD4+-T-cell response in 25% of tested AML patients (n=16). Thus, our study identified, for the first time, HLA class II tumor associated antigens directly obtained from the HLA ligandomes of AML patients and thereby represents a further step to our goal of developing a multipeptide vaccine for immunotherapy of AML. Disclosures:No relevant conflicts of interest to declare.

P53 activation of mesenchymal stromal cells partially abrogates microenvironment-mediated resistance to FLT3 inhibition in AML through HIF-1α–mediated down-regulation of CXCL12

AbstractFms-like tyrosine kinase-3 (FLT3) inhibitors have been used to overcome the dismal prognosis of acute myeloid leukemia (AML) with FLT3 mutations. Clinical results with FLT3 inhibitor monotherapy have shown that bone marrow responses are commonly less pronounced than peripheral blood responses. We investigated the role of p53 in bone marrow stromal cells in stromal cell-mediated resistance to FLT3 inhibition in FLT3 mutant AML. While the FLT3 inhibitor FI-700 induced apoptosis in FLT3 mutant AML cells, apoptosis induction was diminished under stromal coculture conditions. Protection appeared to be mediated, in part, by CXCL12 (SDF-1)/CXCR4 signaling. The protective effect of stromal cells was significantly reduced by pre-exposure to the HDM2 inhibitor Nutlin-3a. p53 activation by Nutlin-3a was not cytotoxic to stromal cells, but reduced CXCL12 mRNA levels and secretion of CXCL12 partially through p53-mediated HIF-1α down-regulation. Results show that p53 activation in stroma cells blunts stroma cell-mediated resistance to FLT3 inhibition, in part through down-regulation of CXCL12. This is the first report of Nutlin effect on the bone marrow environment. We suggest that combinations of HDM2 antagonists and FLT3 inhibitors may be effective in clinical trials targeting mutant FLT3 leukemias.

NPMc+ AML cell line shows differential protein expression and lower sensitivity to DNA-damaging and p53-inducing anticancer compounds

Nucleophosmin (NPM), an important regulator in p53 signaling pathway, is one of the most frequently mutated genes in acute myeloid leukemia (AML). In our previous study, we found that hexamethylene bisacetamide inducible protein 1 (HEXIM1) interacted with both wild-type NPM and cytoplasmic-misallocated NPMc+ mutant, leading to an increase in RNA polymerase II transcription. Here, we examine the protein expression in wild-type NPM (AML2) and NPMc+ mutant (AML3) AML cell lines. Significant lower levels of NPM, HEXIM1 and p53 proteins are detected in AML3 cells, and such differential protein expression is not regulated at transcriptional or post-translational stages. Effects of several anticancer compounds on cell viability of AML2 and AML3 cells are investigated. Compared to AML3 cells, AML2 cells are more sensitive to the treatment of the DNA-damaging compounds (doxorubicin and etoposide) and a specific p53-inducing compound (nutlin-3). However, no significant difference in cytotoxicity was observed when AML2 and AML3 cells were treated with cyclin-dependent kinase inhibitors, flavopiridol and CYC202. Our results provide a novel insight into the functional impact of the NPMc+ mutation on protein expression and the potential approaches for selective therapy of AML.

Long-Term Expansion and Self Renewal Is Contained in the CD34+, but Not the CD34- Subfraction of Nucleophosmin Mutated Acute Myeloid Leukemia Cells.

Mutations in the nucleophosmin (NPM) gene are found in about 30% of cases of acute myeloid leukemia (AML) and lead to a dislocation of the nucleophosmin protein from the nucleus to the cytoplasm (NPMc+ AML). NPMc+ AML shows distinctive biological and clinical features, including a unique gene expression profile, a distinct microRNA signature, low percentage of CD34+ cells, increased incidence of Flt3-ITD (about 40% of cases), good response to induction chemotherapy and (in the absence of Flt3-ITD) a favourable prognosis. Despite significant progress in the characterization of the NPMc+ AML subgroup, questions remain about the leukemia-initiating cell. Distinct features of NPMc+ AML, including multilineage involvement and overexpression of HOX-genes, may point to an early progenitor as the leukemia-initiating cell, but the characteristic low percentage of CD34+ cells may point to a more differentiated leukemic stem cell in NPMc+ AML. To gain more insight in the leukemia-initiating cell in AML with mutated NPM, NPMc+ AML cells were sorted based on the expression of CD34 (n=8, the percentage of CD34+ in the total AML fraction varied between 0.06 and 37%). Western blotting, using an antibody that specifically recognizes the nucleophosmin mutant protein revealed that the NPM mutant protein is expressed in both CD34+ and CD34− cells, proving that the CD34+ NPMc+ AML cells belong to the leukemic clone. This was verified by sequencing the NPM gene in CD34+ and CD34− AML cells. Importantly, culture of sorted CD34+ and CD34− NPMc+ AML cells on a stromal layer revealed that the CD34+ but not the CD34− cells of NPMc+ AML were capable of expanding and initiating long-term growth. In the first 5 weeks of culture an at least 16 fold (range 16–208) expansion of CD34+ AML cells was seen in 5 out of 6 NPMc+ AML cases. This expansion was associated with the formation of cobblestone areas (CAs) under the stromal layer within 3 weeks after plating. The NPMc+ AML cells which expanded in culture were able to expand further after replating in 4 out of 5 investigated cases (fold expansion range 1.6–2.5), indicative of the self renewal capacity of these CD34+ NPMc+ AML cells. Gene expression analysis of CD34+ and CD34− NPMc+ AML cells of 4 cases analyzed thus far revealed the presence of the characteristic HOX-overexpression profile in both CD34+ and CD34− NPMc+ AML cells. In summary, this study shows that the NPM mutation is not only present in CD34−, but also in CD34+ cells of NPMc+ AML and that the properties of long-term expansion and self renewal belong exclusively to the CD34+ subfraction of NPMc+ AML.

Mutations That Cooperate with Nf1 Inactivation in Leukemogenesis Influence Therapeutic Response to MEK Inhibition.

Hyperactive Ras is a common feature of many cancers, including myelodysplastic syndrome (MDS), myeloproliferative disease (MPD), and acute myeloid leukemia (AML). Conditional inactivation of the Nf1 tumor suppressor, which encodes a GTPase activating protein that negatively regulates Ras, in murine hematopoietic cells in Mx1-Cre, Nf1flox/flox mice induces MPD with 100% penetrance. This MPD does not evolve to AML, which implies that cooperating mutations are necessary for transformation to acute leukemia. Upon infection with MOL4070LTR, a retroviral insertional mutagen, ∼25% of Mx1-Cre, Nf1flox/flox mice develop AML. The Ras effectors MEK and ERK are hyper-phosphorylated in Nf1 mutant MPDs and AMLs with Nf1 inactivation. We investigated the effects of CI-1040, a highly selective inhibitor of the MEK kinase, on the growth of hematopoietic progenitor and blast colonies in methylcellulose cultures stimulated with GM-CSF. Blast colony formation from Nf1 mutant AML cells was abrogated at much lower CI-1040 concentrations (0.25 μM) than wild-type or Nf1 mutant MPD cells (50 μM) suggesting a therapeutic index. Nf1 mutant mice with MPD that were treated with CI-1040 showed no improvement in leukocyte counts or survival despite transient MEK inhibition in vivo. In contrast, mice transplanted with three unique Nf1 deficient AMLs responded to CI-1040 treatment with decreased leukocyte counts and markedly prolonged survival compared to vehicle treated controls (24 versus 7 days in the vehicle-treated cohort; OR 3.5 95% CI 3.0–3.8). Despite lower white blood cell counts and prolonged survival, all of the leukemic mice receiving CI-1040 eventually developed reemergence of peripheral blood blasts and died from AML. Leukemias that relapsed during CI-1040 treatment are remarkably less sensitive to CI-1040 in vitro than the vehicle treated AMLs, and do not respond to treatment in secondary recipients. This cellular resistance is not due to an acquired change in kinase sensitivity to CI-1040 as we observe equivalent inhibition of pERK in sensitive and resistant AMLs that are exposed to a range of CI-1040 concentrations. Importantly, when compared with untreated leukemias, CI-1040-resistant AMLs contain new retroviral integrations. Cloning the integration sites from sensitive and resistant Nf1 deficient leukemia pairs resulted in identification of several candidate resistance genes including members of the RasGRP family and Mapk14, which encodes p38. Inhibition of p38 with SB 202190, a relatively selective inhibitor, confers resistance to CI-1040 in sensitive leukemias. Our data support the idea that MEK is a relevant biochemical target in myeloid leukemia, and show that cooperating mutations strongly modulates response. CI-1040 and related MEK inhibitors merit further investigation in myeloid malignancies. This model provides a tractable system for identifying cooperating mutations that result in progression to acute leukemia and modulate drug sensitivity.