Abstract 3977: Pharmacological inhibition of Nrf2 overcomes acquired resistance to arsenic trioxide in FLT3 mutated acute myeloid leukemia
Authors: Jebanesan Daniel Zechariah , Illangeswaran Raveen Stephen , Das Saswati , Vidhyadharan Rakhi Thalayattu , Bijukumar Nayanthara Karpillymoola , Rajamani Bharathi M. , Balakrishnan Balaji , Mathews Vikram , Velayudhan Shaji Ramachandran , Balasubramanian Poonkuzhali 
Published: June 2022

Abstract
De novo acute myeloid leukemia (AML) patients with fms-like tyrosine kinase 3 internal tandem duplications (FLT3-ITD) have worse treatment outcomes. Arsenic trioxide (ATO) used in the treatment of acute promyelocytic leukemia (APL) had been reported to be effective in degrading the FLT3 protein in AML cell lines and sensitizing non-APL AML patient samples in-vitro (Liu et al. 2020). We have previously reported that primary cells from FLT3-ITD mutated AML patients were sensitive to ATO in-vitro compared to other non-M3 AML (Abraham et al, 2014) and molecular/pharmacological inhibition of NF-E2 related factor 2 (Nrf2), a master regulator of antioxidant response mechanism improved the chemosensitivity to ATO and daunorubicin (Karathedath et al. 2017). Here we assessed the role of pharmacological inhibition of Nrf2 in modulating acquired resistance to ATO in an FLT3-ITD mutated AML cell line (MV4-11 ATO-R).MV4-11 cell line containing FLT3-ITD was made resistant to ATO (MV4-11 ATO-R) using incremental method and showed 2-fold increase in IC50 to ATO. The basal expression of Nrf2 transcript and protein levels were compared between MV4-11 (naïve) and MV4-11 ATO-R (resistant) cells using quantitative RTPCR, immunoblotting, and immunofluorescence respectively. Mutations in the Keap1 gene were screened in naive and resistant cells by Sanger sequencing. The effect of known pharmacological inhibitor of Nrf2 (Digoxin (DIG)) in overcoming acquired resistance to ATO was tested by in-vitro cytotoxicity and apoptosis assays. Further, to identify the upstream activator of Nrf2, expression of Akt protein with and without DIG treatment was assessed by immunoblotting. Finally, the efficacy of Nrf2 inhibition by DIG in resistant cells were tested in a transplantable AML mice model.There was no significant increase in NRF2 RNA expression (&lt;2-fold) while there was a higher nuclear Nrf2 expression and localization as well as increased Akt protein expression in the resistant cells compared to naïve cells. DIG treatment reduced expression of Nrf2 resulting in improved sensitivity (IC50 ATO-3.5µM vs ATO+DIG-2.05µM) and increased apoptosis (≈2 fold in apoptotic rates) to ATO in resistant cells. There was a reduction in Akt protein expression in DIG treated cells confirming that Nrf2 activation in the resistant cells is mediated by Akt. Treating AML mice with either DIG alone or in combination with ATO showed reduction in leukemic load and significant increase in overall median survival (26 & 23 days, p&lt;0.001) when compared to untreated and ATO alone (20 & 16 days).
Our study suggests that Nrf2 mediates acquired resistance to ATO in a FLT3-ITD mutated AML cell line. Inhibition of Nrf2 using a drug routinely used in the clinic was able to re-sensitize the resistant cells to ATO in-vitro and reduced leukemic burden in-vivo.
Citation Format: Daniel Zechariah Jebanesan, Raveen Stephen Illangeswaran, Saswati Das, Rakhi Thalayattu Vidhyadharan, Nayanthara Karpillymoola Bijukumar, Bharathi M. Rajamani, Balaji Balakrishnan, Vikram Mathews, Shaji Ramachandran Velayudhan, Poonkuzhali Balasubramanian. Pharmacological inhibition of Nrf2 overcomes acquired resistance to arsenic trioxide in FLT3 mutated acute myeloid leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3977.