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Targeting FLT3-ITD in AML
dem duplications (FLT3-ITD) are the most common muta- tions in the FLT3 receptor gene. FLT3-mutated AML account for 25-35% of all AML, and their prognosis is poor, particularly in unfit, refractory or relapsed patients.
Targeting the mutated FLT3 receptor is a promising approach to treat this specific AML subset. Midostaurin (PKC412) is a first generation type III receptor tyrosine kinase inhibitor that has been extensively studied in vitro and in clinical trials as a treatment for AML patients with mutated FLT3.2,3 After successful phase II clinical trials, midostaurin was found to significantly prolong survival of FLT3-mutated AML patients when combined with con- ventional induction and consolidation therapies in a ran- domized phase III clinical trial leading to the first new drug approval in AML in over 40 years.4 Midostaurin is a multi-targeted kinase inhibitor able to block FLT3 auto- phosphorylation and to induce growth arrest and apopto- sis in FLT3-dependent leukemia.5 Midostaurin is orally administered and generally well tolerated as a single agent. Quizartinib (ACC220) and gilteritinib (ASP2215) are second and third generation FLT3 inhibitors currently in evaluation for the treatment of FLT3-mutated AML.6-8
Targeting the p53 antagonist MDM2 is a novel approach to restore the crucial p53 tumor suppressor function in AML cells.9 Idasanutlin (RG7833) is a second generation MDM2 inhibitor that has been studied in vitro and in vivo as a treatment for AML patients with wild type TP53.10 NVP-CGM09711 and NVP-HDM20112 are second genera- tion MDM2 inhibitors that are currently evaluated in sin- gle-agent phase I studies in patients with advanced tumors with wild type TP53 (clinicaltrials.gov identifiers 01760525 and 02143635). Like midostaurin, NVP-HDM201 is orally administered and expected to be well tolerated as single agent.
In this study, we investigated the combined treatment with MDM2- and FLT3- inhibitors, in particular NVP- HDM201 and midostaurin, on AML cells in order to iden- tify a potentially effective treatment specifically for FLT3-ITD AML refractory to or unfit for intensive chemotherapy. The study might provide the rationale for initiating a clinical study in FLT3-ITD AML evaluating this combination.
Methods
Patient samples
Mononuclear cells of AML patients diagnosed and treated at the University Hospital, Bern, Switzerland between 2005 and 2015 were included in this study. Informed consent from all patients was obtained according to the Declaration of Helsinki, and the studies were approved by decisions of the local ethics committee of Bern, Switzerland. Mutational screening for FLT3, NPM1, TP53 and conventional karyotype analysis of at least 20 metaphases were performed for each patient. Peripheral blood mononuclear cells (PBMCs) and bone marrow mononuclear cells (BMMCs) were collected at the time of diagnosis before initiation of treat- ment.
AML cell lines
OCI-AML2 (AML-M4, FLT3wt, DNMT3A R882C, NPM1wt, TP53wt); OCI-AML3 (AML-M4, FLT3wt, DNMT3A R882C, NPM1mut, TP53wt), MOLM-13 (AML-M5, t(9;11), FLT3-ITD, TP53wt), MOLM-16 (AML-M0, FLT3wt, TP53mut), MV4-11 (AML-M5, t(4;11), FLT3-ITD, TP53wt), ML-2 (AML-M4, t(6;11), FLT3wt, TP53mut), PL-21 (AML-M3, FLT3wt, TP53hemi) and HL-60 (AML-M2, FLT3wt, TP53null) cells were supplied by the Leibniz Institute DSMZ, German Collection of Microorganisms and Cell Cultures. AML cells were grown in RPMI 1640 (SIGMA-ALDRICH, St. Louis, MO, USA) supplemented with 20% fetal bovine serum (FBS, Biochrom GmbH, Germany).
Cytotoxicity assays
AML cells were treated with the MDM2 inhibitors NVP- HDM201, NVP-CGM097, idasanutlin (RG7388), the FLT3 inhibitors midostaurin (PKC412), quizartinib (ACC220), gilteri- tinib (ASP2215) or with genotoxic compounds cytarabine and idarubicin in equimolar concentrations. NVP-HDM201 and NVP-CGM097 investigational compounds were supplied by Novartis, Switzerland, whereas RG7833, PKC412, ACC220 and ASP2215 were purchased at MCE (MedChemExpress, Monmouth Junction, NJ, USA). Cytarabine and idarubicin were purchased at Sigma-Aldrich (St.Louis, MO, USA) and SelleckChem (Houston, TX, USA). Cell viability was deter- mined using the MTT-based in vitro toxicology assay (TOX1, Sigma-Aldrich) with four repeat measurements per dosage. Data
Table 1. Genetic variants in AML cell lines.
ID FLT3
HL-60 wt
MOLM-13 ITD MOLM-16 wt
MV4-11 ITD OCI-AML2 wt
A680V
OCI-AML3 wt
TP53
del
wt V173M C238S
wt wt
wt
NPM1 mutated genes
wt NRAS Q61L CDKN2A R80X
wt MLL-AF9 (t9;11) wt MLL V1368L MTOR T571K
wt MLL-ENL (t4;11) wt DNMT3A R635W
MLL K1751*
L287fs DNMT3A R882C
NRAS Q61L PL-21 wt wt wt KRASA146V
P336L P36fs
wt: wild type; ITD: internal tandem duplication; del: deletion
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