BCAT1 increases sensitivity to Cytarabine and confers CXXC motif derived resistance to pro-oxidant treatment in Acute Myeloid Leukaemiacells

Hillier, James, Heaselgrave, Wayne and Coles, Steven ORCID: https://orcid.org/0000-0002-1109-6971 (2020) BCAT1 increases sensitivity to Cytarabine and confers CXXC motif derived resistance to pro-oxidant treatment in Acute Myeloid Leukaemiacells. In: British Society for Haematology Annual Scientific Meeting, 26-29th April 2020, Birmingham. ISSN 1365-2141 (Unpublished)

Abstract

Recently increased branched‐chain amino transferase (BCAT1) expression has been associated with poor prognosis in AML making it an attractive potential target for the development of novel therapies. BCAT1 features a CXXC motif characterised by redox active cysteine thiols, a feature common to antioxidant enzymes which utilise thiol groups as reducing agents. We previously demonstrated BCAT1 can metabolise H202 in vitro and overexpression in U937 cells results in a decrease in cellular reactive oxygen species (ROS). Given excessive ROS can induce apoptosis we hypothesised BCAT1 CXXC antioxidant properties may protect against pro‐oxidant treatment. Here we demonstrate for the first time BCAT1 decreases ROS and increases survival when challenged with pro‐oxidant Rotenone.

To evaluate the BCAT1 CXXC motif we utilised site directed mutagenesis to modify the C‐terminal Cysteine of the CXXC motif creating a Cysteine → Serine mutant (BCAT1‐CXXS) as previously described. This mutation eliminated the C‐terminal thiol significantly reducing the ability to metabolise H202 in vitro compared to BCAT1‐WT. Following lentiviral transduction stable expression in U937 cells was confirmed by western‐blot and qPCR; BCAT1‐WT (5·57 ± 1·31 fold increase) and BCAT1‐CXXS (5·93 ± 2·6 fold increase).

Transgenic U937 cells were challenged with Rotenone and cell density was measured after 72 h. Dose response analysis revealed the LD50 was significantly higher for BCAT1‐WT (40·9 ± 1·14 nmol/l) compared to either BCAT1‐CXXS(20·8 ± 1·14 nmol/l) and EV control (20·7 ± 1·18 nmol/l) (n = 3, P < 0·001). This finding suggests that the BCAT1 CXXC provides protection against ROS mediated cell death. To support this we measured intracellular ROS following Rotenone treatment using dichlorofluorescin diacetate. Treatment with 60 nmol/l Rotenone resulted in a significantly decreased ROS signal in BCAT1‐WT (491 ± 39 nmol/l) compared to either BCAT1‐CXXS(707 ± 29 nmol/l) and EV control (659 ± 22 nmol/l) (n = 3, P < 0·05).

Previously BCAT1 has been shown to promote cellular proliferation in a variety of cancer cell lines. First line AML therapy relies on Cytarabine (AraC) a nucleoside analogue that interrupts cell division however to date there has been no examination how BCAT1 expression affects treatment with AraC. We theorised the proliferative advantage of BCAT1 overexpressing cells may render them vulnerable to AraC. Firstly we confirmed BCAT1 provides a proliferative advantage in a U937 model, we seeded a T75 flask with 2 × 05 cells/ml and monitored cell density at 24 h intervals. BCAT1 cells reached a higher peak cell density after 96 h (1·57 × 106 ± 8·53 × 104 cells/ml) compared to EV control (1·1 × 106 ± 7·71 × 104) cells/ml) a relative increase of 42%. (n = 9, P < 0·01). Moreover at mid‐exponential phase (48 h) BCAT‐WT cells displayed a consistently higher percentage of cells in G2M phase (23·2 ± 1·4%) compared to EV control (16·4 ± 1·4%) (n = 4, P < 0·05). Next cell lines were incubated with AraC and cell density was measured after 72 h. LD50 values were significantly lower for BCAT1‐WT(0·442 ± 1·34 µmol/l) compared to EV control (2·36 ± 1·18 µmol/l) (n = 3 P < 0·0001), confirming increased AraC sensitivity in BCAT1‐WT over‐expressing cells.

In summary here we have demonstrated for the first time that BCAT1 expression protects against pro‐oxidant treatment whilst increasing vulnerability standard therapeutic treatment. This data opens up a new therapeutic avenue for clinicians seeking novel strategies in the stratified treatment of BCAT1 high and BCAT1 low expressing patients.

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