Global Gene Expression Analysis Reveals Genotoxic Effect of Nrf2 Pharmacological Inhibitor Brusatol in Myeloid Leukemia Cells- a Promising Novel Anticancer Agent

Combating drug resistance is a paramount task in treating acute myeloid leukemia (AML). NFE2 related factor 2 (NRF2) is a major player in inducing chemo resistance in both AML and solid tumors. We have previously reported that inhibition of NRF2 both by shRNA knockdown and pharmacological inhibition using brusatol sensitizes inherent resistant AML cell lines and patient samples to chemotherapy (Karathedath et al, Plos one, 2017). Recent studies have shown that brusatol acts as a potent protein translation inhibitor in A549 lung cancer cell line and this deleterious effect is due to the molar scale concentrations of the drug (Stokoe, et al., 2016, Harder et al, 2016).

As the available reports was in solid tumor lines and at higher concentrations, we undertook this study to elucidate the mechanism of action of brusatol in our experimental setup utilizing Nano molar concentration of brusatol in AML cell lines. We compared the global gene expression changes induced by brusatol in AML cell lines resistant to Ara-C, Dnr and ATO namely THP-1 and U937.

Total RNA was extracted from both cell lines with and without brusatol treatment (100nM for 6 hours) using Qiagen RNeasy mini kit, cDNA was synthesized and subjected to a One -color 8X60K Agilent micro-array analysis. Data normalization, and analysis was performed using Gene Spring GX (V 12.0) software using the 75th percentile shift. Using student t-Test, 3070 genes were identified to be differentially expressed between THP-1 control and brusatol treated while 6856 genes were identified to be differentially expressed between U937 control and brusatol treated samples. About 1242 differentially expressed genes were common between brusatol treated THP-1 and U937. Differentially expressed genes(DEGs) were clustered using hierarchical clustering based on Pearson coefficient correlation algorithm to identify significant gene expression patterns. Genes were classified based on functional category using biological analysis tool DAVID. The enriched genes in both THP-1 and U937 after brusatol treatment fell into the following; Molecular function: protein binding (1396 and 1701 genes), Biological function: regulation of transcription (350 and 502 genes), Cellular component: Mitochondrion (231 and 249 genes) and so on. KEGG analysis was performed to identify enriched pathways after brusatol treatment in DEGs. DEGs in THP1 and U937 were enriched in 33 and 57 pathways were respectively, combining the pathways revealed 20 common target pathways between THP1 and U937. Pathways involved in cellular stress response such as NFkB, TNF and MAPK pathways were selectively up-regulated in the common target pathways and genes involved in cell cycle pathway were down regulated. Select genes from these target pathways and few genes DOCK3, KLF10, NR4A3, GADD45B, KDM4B, GPR155, H2FAY, from the top 100 DEGs were validated by RQ-PCR (SyBr green) all the genes screened showed results consistent with the micro-array data.

Interestingly DNA damage response genes GADD45B, KDM4B, H2AFY were in the top up-regulated genes indicating that brusatol might also cause DNA damage in leukemic cells. NR4A3 the orphan nuclear receptor is an established tumor suppressor in AML and is generally silenced in both AML cell lines and primary samples. One of the key finding from our data is that brusatol strikingly up-regulated NRA4A3 expression in both THP1 and U937. This study has shed light into other modes of action of brusatol apart from being a modulator of the protein translational machinery. Mechanism of chemo sensitization by brusatol could be tissue, time and concentration specific. Our data reveals brusatol at nano-molar concentration acts as a potent DNA damaging agent and reactivates NR4A3 expression in AML apart from being an inhibitor of NRF2. We have also reported at nano-molar concentration brusatol has minimal toxicity towards normal Peripheral blood mono-nuclear cells. Brusatol could be an ideal compound to combat drug resistance in AML. Further experimental validation is warranted to substantiate these finding of the mode of action of brusatol in chemo sensitizing AML cells.