The Role of NRAS G12D Mutations in the Response to Conventional Chemotherapy and 5-Azacitidine in Secondary AML
Authors: Garitano-Trojaola Andoni , Teufel Eva , Da Via' Matteo Claudio , Sancho Ana , Rodhes Nadine , Stuehmer Thorsten , Barrio Garcia Santiago , Groll Jürgen , Haferlach Torsten , Tibes Raoul , Rosenwald Andreas , Einsele Hermann , Kortüm Martin 
Published: October 2019

Abstract
Secondary Acute Myeloid Leukemia (sAML) accounts for 10-30% of all AML. It arises from a preexisting clonal disorder of hematopoiesis, such as myelodysplastic syndromes (MDS) or chronic myeloproliferative neoplasia (cMPN) in most cases (60-70%) or from exposure to a leukemogenic agent e.g. chemotherapy. sAML is generally considered to be of unfavorable prognosis, as treatment sensitivity is reduced, compared to de novo AML (dnAML) and overall survival is shortened. The incidence of AML associated NRAS are similar between sAML and dnAML (10 to 15%, Jelena D. Milosevic et al.). Prognostic impact of such mutations have been controversially discussed, but have been linked to favorable response to high dose cytarabine treatment in dnAML patients (Andreas Neubauer et al.), thus providing the first example of an oncogenic mutation impacting drug response in dnAML. This effect, however, has not yet been shown in sAML, therefore the aim of this work is to study the role of mutated NRAS in the response to chemotherapy and the hypomethylating agent (HMA) 5-azacitidine in sAML.
We utilized two sAML cell lines SET-2 and HEL (both NRAS wildtype) in which we stably introduced the NRAS WT and the known activating hotspot mutation NRAS G12D using the sleeping Beauty technology. The dose-response assays of conventional chemotherapy and 5-azacitidine were carried out in the parental cell lines (SET-2/HEL) compared to NRAS WT (SET-2 NRAS WT/HEL NRAS WT) and NRAS G12D (SET-2 NRAS G12D/HEL NRAS G12D). In contrast to our expectations, both NRAS G12D mutation harboring cell lines, SET-2 and HEL developed resistance to cytarabine, idarubicin and 5-azacytidine, whereas the ones with wildtype NRAS remained susceptible to the drugs. To reverse the resistance we tested the MEK inhibitors Binimetinib and Trametinib in our SET-2 NRAS G12D cell line model according to recent reports about preclinical efficacy of MEK inhibition in NRAS mutant dnAML cells (Michael R. Burgess et al.). And in fact, single agent Binetimib and Trametinib treatments reduced cell viability by 20% at 48 hours. Strikingly, in combination with 5-azacitidine, Binimetinib and Trametinib treatments led to a viability reduction by 90%. Next we induced necroptosis in our NRAS mutant cell line models through the combination of Birinapant (SMAC mimetics) and Emricasan (Inhibitor of Caspase 8), as recently described by Brumatti et al. and were, in addition, able to reduce the cell viability by 60 %.
In summary, we provide first evidence, that in contrast to dnAML, activating NRAS mutations may promote resistance to conventional chemotherapy and 5-azacitidine in sAML cell lines. Furthermore we were able to demonstrate, that the combination of MEK inhibitors (Binimetinib and Trametinib) and 5-azacitidine as well as the induction of necroptosis such as the combination birinapant and emricasan, may provide a potential strategy to overcome the resistance.

Disclosures
Haferlach: MLL Munich Leukemia Laboratory: Employment, Equity Ownership.