Arsenic Trioxide Upregulates Cereblon mRNA Expression and Potentiates Sensitivity of SKM-1 and MDS-L Cells to Lenalidomide

INTRODUCTION

Arsenic trioxide (ATO) was approved in the United States and Europe for the treatment of acute promyelocytic leukemia (APL). ATO was also studied in multiple myeloma (MM) and myelodysplastic syndrome (MDS), where it induced growth inhibition and apoptosis. ATO produced trilineage responses in MDS by several mechanisms of action. These mechanisms included induction of cell cycle arrest, apoptosis and autophagy, generation of reactive oxygen species (ROS), release of cytochrome c and activation of caspases, induction of differentiation and anti-angiogenesis. We showed that the high level of full-length cereblon (CRBN) mRNA and CRBN protein is important for the efficacy of lenalidomide (LEN) in lower-risk MDS patients (Jonasova et al. Eur J Haematol 2015; 95: 27-34; Fuchs et al. Leuk Res 2017; 55 (S1): S132, abstr. 227). ATO potentiated CRBN expression levels in MM cell lines (Jian et al. Oncol Lett 2017; 14:3243-3248).

AIMS

The aim of our study was to evaluate the levels of CRBN mRNA and Nrf2 (nuclear factor, erythroid-derived 2-like 2 or NF-E2-related factor 2) mRNA in SKM-1 (leukemic cell line established from a patient with progression of MDS to myelomonocytic leukemia) and MDS-L (a human myeloblastic cell line established from the bone marrow of an MDS patient with del(5q); Matsuoka et al. Leukemia 2010; 24: 748-755) cells treated with various concentrations of ATO as a single agent or with combinations of ATO with LEN, erythropoietin (EPO) and prednisone for 24 hours. We tested our hypothesis that ATO induced the transcription factor Nrf2 mRNA levels and increased CRBN mRNA expression as a single agent and also in combinations with LEN, EPO and prednisone.

METHODS

Full-length CRBN mRNA and Nrf2 mRNA levels were quantified in SKM-1 and MDS-L cells incubated without (controls) or with 0.05; 0.1; 0,5 and 1.0 microM ATO as a single agent for 24 hours using the reverse transcription-quantitative TaqMan PCR assay. Total RNA was extracted from cells, reverse transcribed and TaqMan PCR assay was provided for CRBN mRNA, Nrf2 mRNA and glyceraldehyde-3- phosphate dehydrogenase (GAPDH), a housekeeping gene. The effect of ATO (0.1 microM) in combinations with LEN (10 microM), EPO (2U/ml) and prednisone (100 microM) on full-length CRBN mRNA and Nrf2 mRNA levels was also studied in SKM-1 and MDS-L cells.

RESULTS

The levels of full-length CRBN mRNA and Nrf2 mRNA corresponded in both cell lines, SKM-1 and MDS-L, treated with ATO as a single agent or in combinations with LEN, EPO and prednisone. The most prominent effect of ATO as a single agent on the levels of both mRNAs was gained in 0.1 microM concentration in SKM-1 cells and 0.5 microM in MDS-L cells. Positive effects of combinations of ATO and prednisone; ATO, prednisone and LEN; or ATO, prednisone, LEN and EPO in both cell lines on the levels of full-length CRBN mRNA and Nrf2 mRNA were also detected.

CONCLUSIONS

Our findings showed that ATO induced CRBN mRNA and Nrf2 mRNA levels in SKM-1 and MDS-L cell lines and potentiated sensitivity of these cells to LEN and to combinations of LEN with EPO and prednisone. We speculate that one of the mechanism of ATO action is a generation of reactive oxygen species (ROS) and oxidative stress responses which induce expression of transcription factor Nrf2. This transcription factor stimulates CRBN mRNA expression. LEN is an immunomodulatory drug and therefore in vitro studies are not sufficient. Further in vivo studies are required to study the effect of ATO and its combinations with LEN, EPO and prednisone.

This work was supported by the research project for conceptual development of research organization (00023736; IHBT, Prague, Czech Republic) and Grant Agency of Charles University, project number 924616.