Role of NF-E2 related factor 2 (Nrf2) on chemotherapy resistance in acute myeloid leukemia (AML) and the effect of pharmacological inhibition of Nrf2

<div><p>Cytarabine (Ara-C) and Daunorubicin (Dnr) forms the backbone of acute myeloid leukemia (AML) therapy. Drug resistance and toxic side effects pose a major threat to treatment success and hence alternate less toxic therapies are warranted. NF-E2 related factor-2 (Nrf2), a master regulator of antioxidant response is implicated in chemoresistance in solid tumors. However, little is known about the role of Nrf2 in AML chemoresistance and the effect of pharmacological inhibitor brusatol in modulating this resistance. Primary AML samples with high <i>ex-vivo</i> IC50 to Ara-C, ATO, Dnr had significantly high <i>NRF2</i> RNA expression. Gene-specific knockdown of <i>NRF2</i> improved sensitivity to these drugs in resistant AML cell lines by decreasing the expression of downstream antioxidant targets of Nrf2 by compromising the cell’s ability to scavenge the ROS. Treatment with brusatol, a pharmacological inhibitor of Nrf2, improved sensitivity to Ara-C, ATO, and Dnr and reduced colony formation capacity. AML cell lines stably overexpressing <i>NRF2</i> showed increased resistance to ATO, Dnr and Ara-C and increased expression of downstream targets. This study demonstrates that Nrf2 could be an ideal druggable target in AML, more so to the drugs that function through ROS, suggesting the possibility of using Nrf2 inhibitors in combination with chemotherapeutic agents to modulate drug resistance in AML.</p></div

<i>NRF2</i> overexpression in AML cell lines increases IC50 of ATO & Dnr by up-regulating downstream target expression.

<p>Two AML cell lines (HL-60 and MOLM-13) with relatively low expression of <i>NRF2</i> were made to stably over express <i>NRF2</i>. <b>(A)</b> Overexpression was further confirmed by quantitative real-time PCR (i) and western blot (ii) for Nrf2 <b>(B)</b> Nrf2 downstream target genes (<i>GCLC</i>, <i>GCLM</i>, <i>NQO1</i>, <i>and HMOX-1</i>) were analyzed by real time quantitative PCR. <b>(C)</b> <i>In-vitro</i> cytotoxicity of overexpressed cells to Dnr and ATO was compared with control cells. Overexpression increases resistance to Dnr and ATO.</p

Primary AML cells and cell lines resistant to cytarabine, daunorubicin and arsenic trioxide show increased Nrf2 expression.

<p><b>(A)</b> AML samples categorised based on median IC50 values to Ara-C (6μM), Dnr (0.4μM) and ATO (2.42μM) were analysed for the expression of <i>NRF2</i> by quantitative real time PCR. Y axis denotes relative expression (2^-dCT) of <i>NRF2</i> normalised to <i>GAPDH</i>. <b>(B)</b> <i>NRF2</i> levels in AML cell lines resistant (THP1, U937) or sensitive (HL-60, MOLM-13) to Ara-C, Dnr and ATO were analysed and represented as fold change (2^-ddCT) normalised to Molm13 (with low <i>NRF2</i> RNA)} (n = 5). *Statistical significance were calculated based on Kruskal–Wallis test and p<0.05 is indicated. <b>(C)</b> Flow cytometric analysis of total Nrf2 expression in AML cell lines (n = 4). <b>(D)</b> Immunofluorescence analysis of nuclear Nrf2 expression in AML cell lines {4′,6-diamidino-2-phenylindole (blue) and Nrf2 antibody (red) stained images overlay} (n = 2). <b>(E)</b> Immunoblotting of total Nrf2 in AML cell lines (β-actin was used as loading control) (n = 3). <b>(F)</b> Nrf2 downstream target expression {(<i>HO-1</i>, <i>NQO1</i>, <i>GCLC</i> and <i>GCLM</i>) normalized to <i>GAPDH</i> and expressed relative to that of MOLM-13 cell line (n = 4)}.*Statistical significance were calculated based on Kruskal–Wallis test and p<0.05 is indicated.</p

Brusatol treatment brings down Nrf2 expression and reduces colony forming capacity of THP1 cells.

<p>THP1 cells were treated with 1μM of brusatol reconstituted in DMSO (final concentration of 0.01%) for 6hrs. The expression of Nrf2 after treatment with brusatol was assessed by <b>(A)</b> flow cytometry (n = 2) (i), western blot with β—actin as the loading control (n = 3) (ii) and subcellular expression (iii). <b>(B)</b> THP1 cells were treated with 100nM of Brusatol for 6h and downstream targets of Nrf2 <i>GCLC</i>, <i>GCLM</i>, <i>HO-1</i>, <i>and NQO1</i> was evaluated by quantitative real time PCR (n = 4). RNA expression of all target genes was normalized to <i>GAPDH</i>. *Statistical significance were calculated based on unpaired t test and p<0.05 is indicated. <b>(C)</b> Apoptosis upon incubation with two different concentrations 100nM and 1000nM of Brusatol was assessed by Annexin V-7AAD.</p

Pharmacological inhibition of Nrf2 with brusatol brings down the IC50 of Ara-C, Dnr & ATO in resistant AML cell lines.

<p>THP1 cells were incubated with Nrf2 inhibitor Brusatol 100nM for 6hrs, followed by increasing concentration of <b>(A)</b> Ara-C (0.1–80μM), <b>(B)</b> Dnr (0.0025–1μM) and <b>(C)</b> ATO (0.1–6μM) for 48hrs. <i>In-vitro</i> cytotoxicity was measured by MTT assay (n = 9). <b>(D)</b> IC50 of THP1 to Dnr and ATO was compared with untreated control cells. <b>(E)</b> THP1 cells were incubate with 100nM of Brusatol followed by treatment with 5μM of Ara-C, 0.25μM of Dnr or 6μM of ATO for 24hrs. Control and treated (5*10³) cells were seeded in methyl cellulose medium (n = 2) and enumerated on day 14. Light microscopic images of colonies were taken on day 14 (10X magnification) (i). Control or treated (5*10³) cells seeded in methyl cellulose medium were stained with methylene blue (ii) and colonies were enumerated (n = 2)(iii). Brusatol alone minimally reduces the colony forming capacity of THP1 cells and considerably reduces in combination with Ara-C, Dnr and ATO.</p

Prognostic plasma biomarkers of early complications and graft‐versus‐host disease in patients undergoing allogeneic hematopoietic stem cell transplantation

Abstract Early complications post hematopoietic stem cell transplantation (HSCT) such as sinusoidal obstruction syndrome (SOS) and graft versus host disease (GVHD) can be life threatening. Although several biomarkers have been identified to correlate with these complications and their response to treatment, these are yet to be used in clinical practice. Here, we evaluated circulating endothelial cells (CECs) (n = 26) and plasma biomarkers (ST2, REG3α, VCAM1, ICAM1, TIM3) (N = 210) at early time points, to determine their association with early complications post‐HSCT. Elevated CEC counts at the end of conditioning was associated with GVHD, indicating endothelial damage during HSCT. Plasma levels of REG3α, VCAM1, ICAM1, and TIM3 on day 14 (D14) and D14 ICAM1 and D28 ST2 were significantly higher in patients with SOS and aGVHD, respectively. Upon sub‐group analysis, D28 ST2, D14/D28 REG3α, and D14 ICAM1 levels were significantly higher in patients with gastrointestinal GVHD, while D28 ST2 was higher in those with skin/liver GVHD. High ST2 levels on D28 was significantly associated with non‐relapse mortality (NRM) and overall survival. Our results suggest that elevated ST2 levels on D28 could predict the likelihood of developing aGVHD and could influence NRM and OS