Metabolic Changes in Venetoclax Resistance Are Determined By Differentiation State in T-Cell Acute Lymphoblastic Leukemia
Authors: Di Grande Alessandra , Roe Andrew , Lefeivre Thomas , Leon Theresa E. , Mansour Marc R. , Bond Jonathan , Van Vlierberghe Pieter , Ni Chonghaile Triona 
Published: November 2021

Abstract
T-cell acute lymphoblastic leukaemia (T-ALL) is an aggressive hematologic malignancy arising from the transformation of immune T-cell lymphocytes. Early T-cell progenitor (ETP-ALL) is a subgroup particularly associated with chemoresistance and a high risk for relapse. Recently, it was shown that ETP-ALL is dependent on the expression of the anti-apoptotic protein BCL-2, and is sensitive to inhibition with ABT-199, a BCL-2 specific BH3 mimetic 1,2. However, one issue with a targeted agent, such as ABT-199, is the development of acquired resistance. Interestingly, there have been numerous high impact papers connecting ABT-199 resistance to altered oxidative phosphorylation (OXPHOS) 3,4. While there are relatively few studies into T-ALL metabolism, there is evidence that aerobic glycolysis, the conversion of glucose to lactate, is greater in proliferating T-cells than in T-ALL and that NOTCH signalling can drive mitochondrial OXPHOS 5. A recent study showed that the transcription factor RUNX2 altered T-ALL metabolism, increasing both glycolysis and OXPHOS and enhancing leukemic cell migration 6. However, there has been relatively little research into the metabolic profile of T-ALL at distinct stages of differentiation. The aim of this study was to determine the role of ABT-199 resistance in altering metabolism and determine if that was due to the differentiation state of the T-ALL.
ABT-199 R LOUCY cells were generated by chronic exposure to increasing concentrations of ABT-199 administered every two days. The EZH2 KO Jurkat cell lines were previously generated through CRISPR-Cas9 engineering 7. In order to assess the metabolic profile, cells were attached to a 96 well plate using CellTak and the extracellular acidification rate (ECAR) and oxidative phosphorylation (OXPHOS) was measured on a Seahorse Bioscience XF96 Extracellular Flux Analyzer. Anti-apoptotic dependence was measured using BH3 profiling and cell death by Annexin V/propidium iodide staining. The mitochondrial structure was visualized using transmission electron microscopy.
Previously, we generated ABT-199 resistant ETP-ALL LOUCY cells (ABT-199 R) following continuous exposure to ABT-199 over a prolonged period of several months 8. The ABT-199 R cells showed dependence on BCL-XL for survival and sensitivity to the BCL-XL inhibitor WEHI 539. The ABT-199 R cells showed evidence of differentiation to a more mature T-cell. The ABT-199 R cells had increased surface CD3 (sCD3) expression and CD1A expression, along with increased expression of TAL1 and LMO2 genes compared to parental LOUCY cells. Interestingly, the ABT-199 R cells showed enhanced basal respiration, ATP production and max respiration compared to the parental cells. Indeed, analysis of the expression of OXPHOS complexes showed increased expression of complexes I-IV in the ABT-199 R cells, compared to the parental controls. Indeed, the parental LOUCY cells appeared to have reduced cristae number and length compared to the ABT-199R cells.
Next, we assessed if inhibiting OXPHOS with a series of inhibitors (oligomycin, rotenone, antimycin) could sensitize the ABT-199 R LOUCY cells to ABT-199. However, we did not detect any changes to sensitivity of ABT-199. This led us to hypothesize that perhaps the changes in OXPHOS were due differentiation state of the LOUCY cells. We confirmed that more typical T-ALL cell lines (JURKAT and CEM-CCRF) had higher OXPHOS than the ETP-ALL cell line LOUCY and had higher expression of the OXPHOS complexes I-IV by Western blotting.
To assess if de-differentiation of a more typical T-ALL cell line would cause a reduction in OXPHOS we turned to the EZH2 knockout (K/O) Jurkat cells 7. We found that EZH2 KO2 showed a reduction in the differentiation markers CD1A and CD3 on the cell surface and TAL1 gene expression, compared to WT control Jurkats. Next, we assessed the OXPHOS and found that the de-differentiated EZH2 cells had reduced OXPHOS compared to the parental controls, with altered mitochondrial structure. Suggesting, that de-differentiation of typical T-ALL cell line reduces OXPHOS.
In this study we show that metabolic phenotype is linked to the maturation stage of T-ALL. We believe that the altered metabolism identified in ABT-199 resistance is linked to the selection of a more mature cell type. Highlighting, that altered metabolism may not be a driver of resistance to ABT-199 but a consequence of the maturation stage of the resistant cell.

Disclosures
Di Grande: Novartis: Current Employment. Leon: BenevolentAI: Current Employment. Mansour: Astellas: Consultancy, Honoraria; Janssen: Consultancy. Bond: Haematology Association of Ireland Award funded by Novartis: Research Funding. Ni Chonghaile: AbbVie: Research Funding.