Aberrant BCAT1 expression augments MTOR activity and accelerates disease progression in chronic lymphocytic leukemia

Abstract

We performed gene expression profiling of mRNA/cDNA isolated from N = 117 flow sorted CLL. We detected aberrant expression of the metabolic enzyme branched chain amino acid transferase (BCAT1) in CLL with del17p/TP53mut. Through extensive validation, we confirmed the highly preferential expression of BCAT1 in CLL with del17p/TP53mut (66%) or trisomy 12 (77%). BCAT1 was not expressed in B cells isolated from normal human lymph nodes. The products of the bidirectional BCAT1 reaction, including leucine, acetyl-CoA, and alpha-ketoglutarate are known activators of MTOR. We measured an ~two-fold higher MTOR activity via normalized p-S6K levels in primary CLL with BCAT1 high versus absent expression before and after sIgM crosslinking. Through steady state metabolomics and heavy isotope metabolic tracing in primary CLL cells, we demonstrate that CLL cells are avid consumers of branched chain amino acids (BCAAs) and that BCAT1 in CLL engages in bidirectional substrate reactions. Of additional interest, CLL with aberrant BCAT1 expression were less sensitive to Venetoclax-induced apoptosis. Biologically, three CLL-derived cell lines with disruption of BCAT1 had substantially reduced growth ex vivo. Clinically, the expression of any detectable BCAT1 protein in CLL independently associated with shorter median survival (125 months versus 296 months; p < 0.0001), even after exclusion of del17p/TP53mut cases.

Fig. 1. Aberrant expression of BCAT1 in genomic CLL subgroups carrying del17p/TP53mut or trisomy 12.

A Displayed are immunoblot band density ratios of BCAT1: mean intrablot BCAT2 for detergent lysates from N = 205 primary CD19 + CLL purified via depletion of CD3+ and CD14+ cells categorized by indicated genomic status. B Representative BCAT1 immunoblot results for detergent lysates from primary CD19 + CLL. C Displayed are qRT-PCR based delta Ct values for BCAT1-PGK1 based on RNA/cDNA from N = 269 CD19+ flow sorted CLL categorized by indicated genomic status. A threshold delta CT value of 2.5 is highlighted above which no BCAT1 protein was detectable by immunoblotting. D Display of immunoblot band density ratios of BCAT1: mean intrablot BCAT2 (y-axis) and paired delta Ct values for BCAT1-PGK1 (x-axis) for individual CLL cases. A threshold delta CT value of 2.5 is highlighted above which no BCAT1 protein was detectable. BCAT1 branched chain amino acid transferase 1, BCAT2 branched chain amino acid transferase 2, CLL: Chronic Lymphocytic Leukemia, qRT-PCR quantitative Reverse Transcriptase-Polymerase Chain Reaction, Ct Cycle threshold, cDNA complementary DNA, PGK1 phosphoglycerate kinase 1.

Fig. 2. Elevated sIgM-crosslinking induced MTOR activation in CLL with aberrant BCAT1 expression.

A–E Primary CLL aberrantly expressing BCAT1 or BCAT1 null were purified via Miltenyi column-based depletion of CD3 (T-cells) and CD14 (macrophages) and subsequently incubated in serum supplemented RPMI medium in the presence of anti-IgM or not for 10’. Detergent cell lysates were fractionated by SDS-PAGE and protein prepared for immunoblotting with antibodies targeting the indicated epitopes. The CLL FISH and IGHV status is indicated. (1) diluted lysate from 293 T cells; (2) diluted lysate from 293 T cells transfected with a ATP6V1B2 cDNA [30]; (*) HG3 cell line lysate. F Displayed are immunoblot based band density ratios of normalized p-p70-S6K: normalized beta-actin for CLL with or without BCAT1 expression before and after anti-IgM treatment. Mann-Whitney test (unpaired, nonparametric), ***p < 0.001. sIgM surface immunoglobulin M, CLL Chronic Lymphocytic Leukemia, BCAT1 branched chain amino acid transferase 1, CLL FISH Chronic Lymphocytic Leukemia fluorescence in-situ hybridization, IGHV immunoglobulin heavy chain variable, S6K S6 protein kinase.

Fig. 3. BCAT1 expression is required for optimal growth of CLL-derived cell lines.

The BCAT1 or BCAT2 gene or the AAVS locus (control) were targeted in the CLL-derived cell lines C1, PGA1 and HG3, with a pool of four specific guides using pLENTI-CRISPRv2-Cas9 followed by puromycin selection and verification via immunoblotting that resulting cell pools lacked BCAT1 or BCAT2 expression expression E, J. The BCAT1 or BCAT2 targeted and AAVS control cells were grown in fully supplemented RPMI1640 media or RPMI1640 media with 25% of BCAA. A, C 12-day cell growth curves (y-axis log₂ scale), Black: AAVS (control) targeted cells; Red: BCAT1 knock-out cells. B, D cell counts on day 12 (y-axis log₂ scale). Two-Way ANOVA with the Holm-Šídák test, *p < 0.05; **p < 0.01; ***p < 0.001. F, H 12-day cell growth curves (y-axis log₂ scale), Black: AAVS (control) targeted cells; Red: BCAT2 knock-out cells. G, I cell counts on day 12 (y-axis log₂ scale). Two-Way ANOVA with the Holm-Šídák test, *p < 0.05; **p < 0.01. BCAT1 branched chain amino acid transferase 1, BCAT2 branched chain amino acid transferase 2, CLL: Chronic Lymphocytic Leukemia, AAVS adeno-associated virus site, CRISPR clustered regularly interspaced short palindromic repeats, BCAA branched chain amino acid.

Fig. 4. Results from untargeted metabolomics of primary human CLL with BCAT1 expression or lack of BCAT1 expression (N = 4 each).

Primary human CLL cells were purified via Miltenyi column-based depletion of CD3+ T cells and CD14+ macrophages, cultured in RPMI1640 media with 10% FCS and pellets extracted with ice cold Methanol. Organic cell extracts were analyzed via mass spectrometry as detailed in the methods section. Displayed is the mean and individual measurements of selected aminoacids, dipeptides and N-acetylaminoacids in BCAT1 expressing versus absent CLL cases. The mean for metabolite measurements in CLL with absent BCAT1 expression was indexed at 1. Y-axis linear, dimension less. Mann-Whitney test (unpaired, nonparametric), *p < 0.05. BCAT1 branched chain amino acid transferase 1, BCAT2 branched chain amino acid transferase 2, CLL: Chronic Lymphocytic Leukemia.

Fig. 5. Aberrant BCAT1 expression accelerates primary CLL cell leucine consumption but does not change leucine conversion to TCA cycle intermediates.

Primary CLL cells aberrantly expressing BCAT1 or BCAT1 null were purified via Miltenyi column-based depletion of CD3 (T-cells) and CD14 (macrophages) and subsequently incubated in serum supplemented RPMI medium in the presence of ¹³C-Leu added at equimolar amounts to unlabeled Leu for 4 h in three separate experiments. An aliquot of BCAT1 expressing CLL cells was pre-incubated with 100 µM of the BCAT1 inhibitor compound #2. Cell pellets were prepared for mass spectrometric tracing as described in the methods. A Simplified schema of the BCAT1 reaction, carbon tracing and link to downstream TCA cycle intermediates. B Residual M + 5 leucine (input) in CLL cells after 4 h of cell culture. Increased Leu consumption in BCAT1 high cells is evident and is reversed by pre-incubation with a BCAT1 inhibitor. C–G The fractional ¹³C labeling of each indicated metabolite. One-Way ANOVA with the Dunnett test, **p < 0.01; ***p < 0.001. BCAT1 branched chain amino acid transferase 1, BCAT2 branched chain amino acid transferase 2, CLL: Chronic Lymphocytic Leukemia, TCA tricarboxylic acid cycle, Leu Leucine, ANOVA Analysis of Variance.

Fig. 6. The BCAT1/2 reaction in primary CLL cells is rapidly bi-directional.

Primary CLL aberrantly expressing BCAT1 or BCAT1 null were purified via Miltenyi column-based depletion of CD3 (T-cells) and CD14 (macrophages) and subsequently incubated in serum supplemented RPMI medium in the presence of ¹⁵N-Leu added at equimolar amounts to unlabeled Leu for 1 h, 2 h or 4 h in three separate experiments. An aliquot of BCAT1 expressing CLL cells was pre-incubated with 100 µM of the BCAT1 inhibitor compound #2. Cell pellets were prepared for mass spectrometric tracing as described in the methods. A Simplified schema of the bi-directional BCAT1 reaction. B–H The fraction of each indicated metabolite labeled by ¹⁵N at various time points. Two-Way ANOVA with the Holm-Šídák test, *p < 0.05; **p < 0.01; ***p < 0.001. BCAT1 branched chain amino acid transferase 1, BCAT2 branched chain amino acid transferase 2, CLL: Chronic Lymphocytic Leukemia, Leu Leucine, ANOVA Analysis of Variance.

Fig. 7. Reduced overall survival of CLL patients aberrantly expressing BCAT1 (Kaplan-Meier plots).

Survival analysis of CLL patients untreated at the time of enrollment stratified by biomarkers. Total number of patients with available data and positive or negative marker measurements is indicated. A BCAT1:BCAT2 pooled immunoblot protein ratios (any detectable BCAT1 protein in orange). B Same as in (A) but exclusive of TP53 mutated CLL. C TP53 mutated versus WT. D IGHV mutation status MUT versus UM. E. CLL interphase FISH findings prioritized 17p > 11q > t12 > 13q14>normal. F SNP 6.0 array based genomic acquired copy number aberrations (complexity) per case. BCAT1 branched chain amino acid transferase 1, BCAT2 branched chain amino acid transferase 2, CLL: Chronic Lymphocytic Leukemia, CLL FISH Chronic Lymphocytic Leukemia fluorescence in-situ hybridization, IGHV immunoglobulin heavy chain variable, MUT mutated, UM unmutated SNP single nucleotide polymorphism.