Contributions
Abstract: EP316
Type: E-Poster Presentation
Session title: Acute lymphoblastic leukemia - Biology & Translational Research
Background
T-cell acute lymphoblastic leukemia (T-ALL) is a biologically and clinically heterogeneous disease mostly associated with NOTCH1 mutations that characterize over 60% of cases. Therapy-resistant or refractory T-ALL remains a major clinical challenge. Recently, the metabolism of Branched Chain Amino Acids (BCAAs) has been associated with tumor growth and survival. In-fact, aberrant expression of BCAA Transaminase 1 (BCAT1), a cytosolic aminotransferase, involved in the first step of BCAAs metabolism, has been demonstrated in different tumor models, including leukemia. However, its biological role in T-ALL remains to be elucidated.
Aims
The purpose of this study was to evaluate the role of BCAT1 in T-ALL leukemogenesis, with particular emphasis on a putative link between BCAT1 and NOTCH1 in promoting T-ALL initiation and progression.
Methods
We evaluated BCAT1 expression using RNAseq data from 264 T-ALL samples (TARGET cohort enrolled on Children's Oncology Group ALL0434 protocol). BCAT1 transcript (qRT-PCR) and protein levels (western blot) were determined in T-ALL cell lines (n=13) and patient derived xenografts (n=12). Five T-ALL samples were treated with a g secretase inhibitor (DBZ) to dampen NOTCH signaling. BCAT1 promoter occupancy by NOTCH1 was determined in two T-ALL cell lines using Chromatin Immunoprecipitation (ChIP) coupled with qPCR. Tandem affinity purification (TAP) and mass spectrometry (MS) analysis were used to identify BCAT1 interacting proteins from CUTLL1 T-ALL cells. Further, BCAT1 knock-down experiments were performed to address its role on T-ALL cell growth, proliferation and drug sensitivity.
Results
We found variable BCAT1 expression levels amongst the different T-ALL molecular subgroups, with higher expression levels in TLX1 (p<0.001), HOXA-TLX3 (p<0.05) and NKX2-1 (p<0.01) subtypes. Considering recurrent genetic alterations, we found BCAT1 expression to be significantly higher (p<0.01) in NOTCH1 mutated cases compared to un-mutated samples. Further, BCAT1 was more highly expressed in the phenotypically mature cortical T-ALL subgroup. Analysis of T-ALL cell lines and PDX samples also evidenced heterogeneous BCAT1 expression levels, with a trend toward higher expression in these cells compared to their normal cellular counterparts (thymocytes). In agreement with our previous analysis, NOTCH1 mutated PDX samples showed higher BCAT1 expression compared to un-mutated cases. Inhibition of NOTCH1 signaling with DBZ decreased BCAT1 expression in numerous T-ALL models. In addition, ChIP analysis demonstrated binding of NOTCH1 on BCAT1 promoter in T-ALL cells, suggesting that NOTCH1 may directly regulate its expression. Functionally, we found BCAT1 knock-down T-ALL cells to have a compromised capacity to form tumors in vivo. Further, TAP followed by MS analysis of BCAT1 interacting proteins disclosed that BCAT1 may be implicated in non-metabolic processes such as DNA replication and repair. Indeed, we found BCAT1 KD cells to be particularly sensitive to topoisomerase inhibitors such as etoposide.
Conclusion
BCAT1 is highly expressed in a subgroup of T-ALL cases, particularly in NOTCH1 mutated T-ALL. NOTCH1 may directly regulate BCAT1 expression. Genetic depletion of BCAT1 slows tumor growth in vivo and renders cells particularly sensitive to topoisomerase inhibitors. Our results identify BCAT1 as a novel therapeutic target and suggest that a combination of BCAT1 inhibitor and topoisomerase inhibitor could be particularly useful in BCAT1 overexpressing T-ALL cases.
Keyword(s): Notch signaling, T cell acute lymphoblastic leukemia
Abstract: EP316
Type: E-Poster Presentation
Session title: Acute lymphoblastic leukemia - Biology & Translational Research
Background
T-cell acute lymphoblastic leukemia (T-ALL) is a biologically and clinically heterogeneous disease mostly associated with NOTCH1 mutations that characterize over 60% of cases. Therapy-resistant or refractory T-ALL remains a major clinical challenge. Recently, the metabolism of Branched Chain Amino Acids (BCAAs) has been associated with tumor growth and survival. In-fact, aberrant expression of BCAA Transaminase 1 (BCAT1), a cytosolic aminotransferase, involved in the first step of BCAAs metabolism, has been demonstrated in different tumor models, including leukemia. However, its biological role in T-ALL remains to be elucidated.
Aims
The purpose of this study was to evaluate the role of BCAT1 in T-ALL leukemogenesis, with particular emphasis on a putative link between BCAT1 and NOTCH1 in promoting T-ALL initiation and progression.
Methods
We evaluated BCAT1 expression using RNAseq data from 264 T-ALL samples (TARGET cohort enrolled on Children's Oncology Group ALL0434 protocol). BCAT1 transcript (qRT-PCR) and protein levels (western blot) were determined in T-ALL cell lines (n=13) and patient derived xenografts (n=12). Five T-ALL samples were treated with a g secretase inhibitor (DBZ) to dampen NOTCH signaling. BCAT1 promoter occupancy by NOTCH1 was determined in two T-ALL cell lines using Chromatin Immunoprecipitation (ChIP) coupled with qPCR. Tandem affinity purification (TAP) and mass spectrometry (MS) analysis were used to identify BCAT1 interacting proteins from CUTLL1 T-ALL cells. Further, BCAT1 knock-down experiments were performed to address its role on T-ALL cell growth, proliferation and drug sensitivity.
Results
We found variable BCAT1 expression levels amongst the different T-ALL molecular subgroups, with higher expression levels in TLX1 (p<0.001), HOXA-TLX3 (p<0.05) and NKX2-1 (p<0.01) subtypes. Considering recurrent genetic alterations, we found BCAT1 expression to be significantly higher (p<0.01) in NOTCH1 mutated cases compared to un-mutated samples. Further, BCAT1 was more highly expressed in the phenotypically mature cortical T-ALL subgroup. Analysis of T-ALL cell lines and PDX samples also evidenced heterogeneous BCAT1 expression levels, with a trend toward higher expression in these cells compared to their normal cellular counterparts (thymocytes). In agreement with our previous analysis, NOTCH1 mutated PDX samples showed higher BCAT1 expression compared to un-mutated cases. Inhibition of NOTCH1 signaling with DBZ decreased BCAT1 expression in numerous T-ALL models. In addition, ChIP analysis demonstrated binding of NOTCH1 on BCAT1 promoter in T-ALL cells, suggesting that NOTCH1 may directly regulate its expression. Functionally, we found BCAT1 knock-down T-ALL cells to have a compromised capacity to form tumors in vivo. Further, TAP followed by MS analysis of BCAT1 interacting proteins disclosed that BCAT1 may be implicated in non-metabolic processes such as DNA replication and repair. Indeed, we found BCAT1 KD cells to be particularly sensitive to topoisomerase inhibitors such as etoposide.
Conclusion
BCAT1 is highly expressed in a subgroup of T-ALL cases, particularly in NOTCH1 mutated T-ALL. NOTCH1 may directly regulate BCAT1 expression. Genetic depletion of BCAT1 slows tumor growth in vivo and renders cells particularly sensitive to topoisomerase inhibitors. Our results identify BCAT1 as a novel therapeutic target and suggest that a combination of BCAT1 inhibitor and topoisomerase inhibitor could be particularly useful in BCAT1 overexpressing T-ALL cases.
Keyword(s): Notch signaling, T cell acute lymphoblastic leukemia