Ibrutinib Resistance Is Reduced by an Inhibitor of Fatty Acid Oxidation in Primary CLL Lymphocytes

Abstract

Chronic Lymphocytic Leukemia (CLL) is an incurable disease, characterized by the accumulation of malignant B-lymphocytes in the blood stream (quiescent state) and homing tissues (where they can proliferate). In CLL, the targeting of B-cell receptor signaling through a Burton's tyrosine kinase inhibitor (ibrutinib) has rendered outstanding clinical results. However, complete remission is not guaranteed due to drug resistance or relapse, revealing the need for novel approaches for CLL treatment. The characterization of metabolic rewiring in proliferative cancer cells is already being applied for diagnostic and therapeutic purposes, but our knowledge of quiescent cell metabolism-relevant for CLL cells-is still fragmentary. Recently, we reported that glutamine metabolism in primary CLL cells bearing the del11q deletion is different from their del11q negative counterparts, making del11q cells especially sensitive to glutaminase and glycolysis inhibitors. In this work, we used our primary CLL lymphocyte bank and compounds interfering with central carbon metabolism to define metabolic traits associated with ibrutinib resistance. We observe a differential basal metabolite uptake linked to ibrutinib resistance, favoring glutamine uptake and catabolism. Upon ibrutinib treatment, the redox balance in ibrutinib resistant cells is shifted toward NADPH accumulation, without an increase in glutamine uptake, suggesting alternative metabolic rewiring such as the activation of fatty acid oxidation. In accordance to this idea, the curtailing of fatty acid oxidation by CPT1 inhibition (etomoxir) re-sensitized resistant cells to ibrutinib. Our results suggest that fatty acid oxidation could be explored as a target to overcome ibrutinib resistance.

Keywords: CLL; del17p; drug resistance; fatty acid oxidation; ibrutinib; metabolism.

Figure 1

CLL lymphocytes display metabolic differences associated with ibrutinib sensitivity. (A–C) Survival fraction (relative to non-treated control [NT]) of CLL lymphocytes treated with metabolic inhibitors for 48 h, (A) (n = 26), (B) (n = 15–26), (C) (n = 11), (mean ± SEM). Basal metabolite uptake differences between ibrutinib sensitive and resistant CLL lymphocytes, (D) Glucose (n = 20), (E) Ammonia (n = 22), (F) Glutamine (n = 16), (G) Glutamate (n = 23). Negative values indicate metabolite excretion to the media. (H) Basal raw (Resazurin) signal of mitochondrial reductive capacity of ibrutinib sensitive and resistant CLL lymphocytes (n = 25). (I) Basal raw ROS (CellRox) signal of ibrutinib sensitive and resistant CLL lymphocytes (n = 25). Basal protein expression of ibrutinib sensitive and resistant CLL lymphocytes (relative to actin signal) (J) GLUT1 (n = 22) and (K) GLUT4 (n = 10), *p < 0.05.

Figure 2

Metabolic and Redox effects of ibrutinib on CLL lymphocytes. (A) Quantification of total glutathione after 24 h treatment with ibrutinib (n = 15), (mean ± SEM). (B) Reduced/Oxidized glutathione ratio after 24 h treatment with ibrutinib (n = 14). (C) NADPH/NADP ratio after 24 h of ibrutinib treatment (n = 8) (mean ± SEM). (D) NADH/NAD ratio after 24 h of ibrutinib treatment (n = 11) (mean ± SEM). Metabolite uptake after 24 h of ibrutinib treatment. (E) Glutamine (n = 21) and (F) Glutamate (n = 20) (mean ± SEM), *p < 0.05.

Figure 3

Inhibition of FAO decreases ibrutinib-induced cytotoxicity in CLL primary lymphocytes. (A) Survival fraction (relative to non-treated control) after 48 h treatment with ibrutinib alone or in combination with Fatty acids (FA), (n = 10). (B) Survival fraction (relative to non-treated control) after 48 h treatment with etomoxir alone or in combination with ibrutinib, (n = 12). (C) Total glutathione levels after 24 h treatment with etomoxir alone or in combination with ibrutinib (n = 11). Protein expression levels (relative to actin) after 24 h treatment with etomoxir alone or in combination with ibrutinib (D) GS (n = 11), (E) GAC (n = 10), (I) GLUT1 (n=10). (F) NADPH/NADP ratio (n = 8), and (G) NADH/NAD ratio (n = 11) after 24 h treatment with etomoxir alone or in combination with ibrutinib. (H) Relative ROS levels after 48 h treatment with etomoxir alone or in combination with ibrutinib (n = 12), (mean ± SEM). (J) Glucose uptake after 24 h treatment with ibrutinib and etomoxir (n = 8). *p < 0.05, **p < 0.001.

Figure 4

Effect of FAO inhibition in ibrutinib treated CLL lymphocytes positive for del17p. (A) Survival fraction (relative to non-treated control [NT]) after 48 h treatment with the indicated metabolic inhibitors (n = 20–30) (mean ± SEM). (B) Basal NADH/NAD ratio (n = 11), and (C) Basal NADPH/NADP ratio (n = 14) of CLL lymphocytes negative and positive to del17p. (D) Reduced/Oxidized glutathione ratio (n = 18), and (E) Basal total glutathione levels (n = 19) of CLL lymphocytes negative and positive to del17p. (F) Basal raw ROS (CellRox) signal on CLL lymphocytes negative and positive to del17p (n = 29). (G) Survival fraction (relative to non-treated control) after 48 h treatment with combinations of ibrutinib and etomoxir (n = 4). wt -del17p negative CLL lymphocytes.

Figure 5

Model for metabolic rewiring associated to ibrutinib resistance in CLL lymphocytes. Upon ibrutinib treatment, ibrutinib resistant cells initiate a compensation mechanism increasing fatty acid oxidation metabolism as well as α-KG production from glutamate, to maintain mitochondrial homeostasis. In line with this, we propose that α-KG follows preferentially an oxidation process in the TCA. Transporters are colored in purple, enzymes in green, and inhibitors in red. Enzymes overexpressed in CLL lymphocytes are in bold green characters. Blue arrows indicate reactions increased in basal ibrutinib resistant cells compared to the sensitive subset. Purple arrows represent the processes that are increased in resistant cells upon ibrutinib treatment. Dashed red arrows represent the processes inhibited upon ibrutinib treatment. Brown arrows indicate the proposed alternative metabolic rewiring to increase fatty acid and Acetyl-CoA availability.