BCAT1 is a NOTCH1 target and sustains the oncogenic function of NOTCH1

Abstract

High levels of branched-chain amino acid (BCAA) transaminase 1 (BCAT1) have been associated with tumor aggressiveness and drug resistance in several cancer types. Nevertheless, the mechanistic role of BCAT1 in T-cell acute lymphoblastic leukemia (T-ALL) remains uncertain. We provide evidence that Bcat1 was over-expressed following NOTCH1-induced transformation of leukemic progenitors and that NOTCH1 directly controlled BCAT1 expression by binding to a BCAT1 promoter. Further, using a NOTCH1 gain-of-function retroviral model of T-ALL, mouse cells genetically deficient for Bcat1 showed defects in developing leukemia. In murine T-ALL cells, Bcat1 depletion or inhibition redirected leucine metabolism towards production of 3-hydroxy butyrate (3-HB), an endogenous histone deacetylase inhibitor. Consistently, BCAT1-depleted cells showed altered protein acetylation levels which correlated with a pronounced sensitivity to DNA damaging agents. In human NOTCH1-dependent leukemias, high expression levels of BCAT1 may predispose to worse prognosis. Therapeutically, BCAT1 inhibition specifically synergized with etoposide to eliminate tumors in patient-derived xenograft models suggesting that BCAT1 inhibitors may have a part to play in salvage protocols for refractory T-ALL.

Figure 1.

BCAT1 is upregulated during NOTCH1-dependent transformation. (A) Heat map showing the top 50 most downregulated and upregulated genes between normal double-positive (DP) cells and ICN1-induced DP leukemic cells (NIC Tumors). (B) Expression levels (quantitative polymerase chain reaction [qRT-PCR]; left) of Bcat1 in thymocytes obtained from 6-8-week-old C57/ Bl6 mice and leukemic cells from 6 AE-NOTCH1 T-cell acute lymphoblastic leu kemia (T-ALL) tumors (NOTCH1-T). Significance was calculated using an unpaired two-tailed t test. **P<0.01. Western blot (right) showing protein expression levels of ICN1 and Bcat1. (3-actin and tubulin are shown as loading controls. Graphical representation of Bcat1/|3-actin ratios (extreme right). Bars represent mean values. ICN1: intracellular NOTCH1. (C) Box plot showing the expression of BCAT1 mRNA in T-ALL patients (N=57) and thymocyte subsets (7 thymocyte and mature T-cell subsets derived from [N=3] independent donors; quantile-normalized microarray results downloaded from GSE33469 and GSE33470). CD3⁺ and CD3- DP cells were grouped together. CD1⁺ and CD1-CD34⁺ cells were grouped together. Boxes represent first and third quartiles and line represents the median. Statistical analysis between groups was performed using unpaired two-sided t test. (D) BCAT1 transcript (top) and protein levels (bottom) in total human thymus, NOTCH1 wild-type and NOTCH1-activated/mutated patient derived T-ALL patient-derived xenografts (PDX). Significance was calculated using a non-parametric t test (Mann-Whitney). **P<0.01. ICN1, MYC and PTEN protein levels are also shown. (3-actin is shown as loading control. (E) PDX samples were treated in vivo with DBZ (10 μg/kg every 8 hours [h] for a total of 3 injections) or vehicle (dimethyl suldoxide [DMSO]) for 24 h before analysis of BCAT1 transcript levels. For statistical analysis, an unpaired t test was used. **P<0.01, ***P<0.001. (F) NOTCH1 chromatin immunoprecipitation (ChIP)-sequencing binding (left) in the BCAT1 locus in HPB T-ALL cells. Inset shows the location of ChIP-quantitative polymerase chain reaction (qPCR) amplicons near NOTCH-1 peak region (P1-P2) and in a negative control region (NL). Chromatin from PF382 cells was subjected to ChIP using a NOTCH1 antibody (right). The indicated regions (P1, P2 and NL) were PCR amplified from the precipitated and input DNA. Fold enrichment was calculated as a ratio of amplification efficiency of ChIP sample over that of the immunoglobulin G (IgG) control. Shown are means ± standard deviation SD (N≥3). For statistical analysis, an unpaired t test was used. ***P<0.001. NS: not significant.

Figure 2.

Functional effects of BCAT1 depletion. (A) Kaplan-Meier survival curves of overall survival in lethally irradiated C57BL/6J hosts transplanted with bone marrow (BM) cells (wild-type [WT] or knockout [KO] for Bcat1) transduced with ΔE-NOTCH1 allele. Data from 2 independent transplantation experiments were pooled together. Log-rank Mantel-Cox test was performed to calculate P value. ^***P<0.001. Shaded area represents 95% confidence interval (CI). (B) Representative plots (left) and bar graph representation (S-phase fraction; right) of ex vivo EdU incorporation in ΔE-NOTCH1 leukemias WT and null for Bcat1. Data for bar graph is shown as mean ± standard deviation (SD). Significance was calculated using an unpaired two-tailed t test. ^***P<0.001. (C) Representative plots (left) and bar graph representation (S-phase fraction; right) of MOLT4 cells transduced with small hairpin control vector (shCTRL), shBCAT1 #1 or shBCAT1 #2 7 days post-puromycin selection and assessed for EdU incorporation by fluorescence-activated cell sorting (FACS) analysis. Data for bar graph is shown as mean ± standard deviation (SD). Significance was calculated using an unpaired two-tailed t test. ^***P<0.001. (D) T-ALL cells were transduced with control vector (shCTRL) or vector containing shRNA sequences against BCAT1 (shBCAT1 #1, shBCAT1 #2). Expression of BCAT1 and tubulin was analyzed by immunoblotting 7 days post transduction (left panels) in DND41 and MOLT4. Starting from 7 days post-puromycin selection, cell proliferation was evaluated by determining cell number (DND41) or ATP levels by bioluminescence (MOLT4). Significance was calculated using an unpaired two-tailed t test. ^*P<0.05, ^**P<0.01. (E) Representative images of bioluminescence (left) and quantitative analysis of tumor burden (right) in NSG mice xenografted with CCRF-CEM cells expressing luciferase and transduced with shCTRL or shBCAT1 (#1 and #2). Analysis after 15 days post-transplant is shown. Significance was calculated using an unpaired two-tailed t test. ^**P<0.01. (F) Representative images of bioluminescence (top) and quantitative analysis of tumor burden (bottom) in NSG mice xenografted with MOLT4 cells expressing luciferase and transduced with shCTRL or shBCAT1 (#1 and #2). Analysis after 15 days post-transplant is shown. Significance was calculated using an unpaired two-tailed t test. ^*P<0.05, ^**P<0.01.

Figure 3.

Canonical functions of Bcat1. (A) Heat map representation of the top down-and upregulated genes between AE-NOTCH1 tumors wild-type (WT) or knockout (KO) for Bcat1. Two independent WT and KO tumors were analyzed. Bcat1 gene is highlighted in red. (B) Gene set enrichment analysis (GSEA) identified 3 significantly enriched gene sets involved in cell cycle regulation downregulated in Bcat1 KO T-cell acute lymphoblastic leukemia (T-ALL) cells. The normalized enrichment score (NES) and the nominal P value are illustrated. (C) GSEA identified 3 significantly enriched gene sets involved in DNA damage response upregulated in Bcat1 KO T-ALL cells. The NES and the nominal P value are illustrated. (D) Immunoblots of yH2AX, p21 and Bcat1 in tumors WT or KO for Bcat1 (top). a-tubulin is shown as loading control. T-ALL cells (CCRF-CEM, DND41, MOLT4) transduced with shCTRL/ CAS9 or shBCAT1 (#1 and #2)/sgBCAT1 were analyzed by immunoblotting for yH2AX and BCAT1 (bottom). (3-actin is shown as loading control. (E) Heatmap representation (left) of the top 50 differentially expressed metabolites identified by capillary electrophoresis time-of-flight mass spectrometry (CE-TOFMS) between AE-NOTCH1 leukemias WT and KO for Bcat1. Metabolites that are significantly and consistently differentially expressed in multiple comparisons are highlighted in red. Volcano plot (right) showing differentially expressed metabolites (≥1.5 fold change, P<0.05; in red and blue) identified by CE-TOFMS between AENOTCH1 leukemias WT and KO for Bcat1. Metabolites that are significantly and consistently differentially expressed in multiple comparisons are encircled.

Figure 4.

ERG245, a BCAT1-specific inhibitor mimics the functional consequences of Bcat1 depletion. (A) Representative plots of apoptosis in AE-NOTCH1 leukemia wild-type for Bcat1 (WT#6) treated in vitro for 48 hours (h) with phosphate-buffered saline (PBS) (vehicle) or increasing doses of ERG245 (200 μM - 1 mM). (B) Representative plots of ΔE NOTCH1 leukemia WT for Bcat1 (WT#6) treated in vitro for 48 h with PBS (vehicle) or increasing doses of ERG245 (200-500 \iM). Cells were then assessed for EdU incorporation by fluorescence-activated cell sorting (FACS) analysis. (C) Representative cell viability analysis in AE-NOTCH1 tumors WT (WT#3) or knockout (KO) (KO#3, #1) for Bcat1. Murine T-cell acute lymphoblastic leukemia (T-ALL) cells were treated in vitro for 48 h with PBS (vehicle) or increasing doses of ERG245 (100 ^M - 1 mM). Data is shown as mean ± standard deviation (SD). (D) Representative plots of T-ALL cell lines (CCRF-CEM, DND41) treated in vitro for 72 h with PBS (vehicle) or ERG245 (300 μM). Cells were then assessed for EdU incorporation by FACS analysis. (E) Representative plots of PDX#27 treated in vitro for 72 h with PBS (vehicle) or increasing doses of ERG245 (300 μM - 1 mM). Cells were then assessed for EdU incorporation by FACS analysis.

Figure 5.

BCAT1 loss induces a dysfunctional DNA damage response following etoposide treatment. (A) Representative cell viability analysis (top) in AE-NOTCH1 tumors wild-type (WT) (WT#1) or knockout (KO) (KO#6) for Bcat1. Murine T-cell acute lymphoblastic leukemia (T-ALL) cells were treated in vitro for 48 hours (h) with dimethyl sulfoxide (DMSO) (vehicle) or increasing concentrations of etoposide (25-100 nM). Data is shown as mean ± standard deviation (SD). Significance was calculated using an unpaired two-tailed t test. *P<0.05, **P<0.01. Quantification of apoptosis (bottom) in AE-NOTCH1 tumors WT (WT#1) or KO (KO#6) for Bcat1 treated in vitro for 48 h with DMSO (vehicle) or increasing concentrations of etoposide (25-100 nM). Data is shown as mean ± SD. Significance was calculated using an unpaired two-tailed t test. *P<0.05, **P<0.01. (B) Representative plots of apoptosis (left) in CCRF-CEM T-ALL cells transduced with small hairpin control vector (shCTRL) or shBCAT1 (#1 and #2) and treated in vitro for 48 h with DMSO (vehicle) or etoposide (100 nM or 1 μM). Quantification of apoptosis (right) in CCRF-CEM T-ALL cells transduced with shCTRL or shBCAT1 (#1 and #2) and treated in vitro for 48 h with DMSO (vehicle) or etoposide (100 nM - 2 μM). Significance was calculated using an unpaired two-tailed t test. **P< 0.01, ***P<0.001. (C) Expression of cleaved PARP-1 and phosphorylated yH2AX was analyzed by immunoblotting in CCRF-CEM T-ALL cells transduced with shCTRL or shBCAT1 (#1 and #2) and treated in vitro for 48 h with DMSO (vehicle) or etoposide (1 μM). [3-actin and GADPH are shown as loading controls. (D) Representative neutral comet images (left) performed in CCRF-CEM cells infected with a control shRNA (shCTRL) or BCAT1-targeting shRNA (shBCAT1#1, shBCAT1#2) either untreated or after etoposide treatment (1µM) for 2 or 6 hours. Dot plot (right) showing individual percentages of comet tail DNA. The median value of 50-80 nuclei per experimental condition is indicated. Statistical analysis was conducted by using the Mann-Whitney test. Data are representative of 2 independent experiments.

Figure 6.

BCAT1-specific inhibition increases response to DNA-damaging agents, especially etoposide, in vitro and in vivo. (A) Representative plots of apoptosis in MOLT4 T-cell acute lymphoblastic leukemia (T-ALL) cells treated with vehicle (dimethyl sulfoxide [DMSO]), BCAT1 inhibitor (ERG245), etoposide (Etop; 50-75 nM) or the combination (ERG245 + Etop) for 48 hours (h). (B) Representative plots of Annexin V staining (left panels) in DND41 T-ALL cells treated with vehicle (DMSO), BCAT1 inhibitor (ERG245), Etop or the combination (ERG245 + Etop) for 48 h. Western blot analysis (right panels) of PARP-1 (total or cleaved PARP-1), and phosphorylated yH2AX in DND41 cells treated for 48 h with DMSO (vehicle), ERG245 (200 µM), Etop (500 nM) or ERG245 + Etop. GADPH was used as protein loading control. (C) Representative plots (left) and bar graph representation (right) of apoptosis (Annexin V-positive) in ex vivo obtained PDX#39 cells treated with vehicle (DMSO), BCAT1 inhibitor (ERG245), Etop (50-250 nM) or the combination (ERG245 + Etop) for 48 h. Significance was calculated using an unpaired two-tailed t test. **P<0.01, ***P<0.001. (D) Representative images of bioluminescence (left) in NSG mice xenografted with PDX#27 cells expressing luciferase (PDX#27-luc) and treated with vehicle (DMSO), BCAT1 inhibitor (ERG245; 30 mg/kg 3 times a week), Etop (15 mg/kg twice a week) or the combination (ERG245 + Etop). Analysis before (day 13 post-transplantation) and 15 days after start of treatment (day 28 post-transplantation) is shown. Quantitative analysis of tumor load (right) via in vivo bioluminescence imaging of NSG mice xenografted with PDX#27-luc after treatment (day 28 post-transplantation) with vehicle (DMSO), BCAT1 inhibitor (ERG245), Etop or the combination (ERG245 + Etop). Significance was calculated using an unpaired two-tailed t test. **P<0.01, ***P<0.001. NS: not significant.

Figure 7.

Schematic illustration of the proposed role of BCAT1 in regulating T-cell acute lymphoblastic leukemia response to DNA-damaging agents in BCAT1-high and BCAT1-silenced/functionally inhibited T-cell acute lymphoblastic leukemia cells. BCAT1 inhibition induces a partial break in the tricarboxylic acid cycle cycle between citrate and succinate leading to citrate accumulation and directing leucine metabolism towards 3-HB synthesis. 3-HB is known to act as an energy source in the absence of sufficient glucose and as it builds up it inhibits class I histone deacetylases (HDAC), leading to increased acetylation of proteins such as histones and DNA damage response proteins (including Ku70 and Ku80) modifying their activity^, and possibly priming cells to the deleterious effects of DNA-damaging agents. Following the exposure to DNA-damaging agents (etoposide) this leads to accentuated DNA damage leading to cell death.