Loss of the redox mitochondrial protein mitoNEET leads to mitochondrial dysfunction in B-cell acute lymphoblastic leukemia

Abstract

B-cell acute lymphoblastic leukemia (ALL) affects both pediatric and adult patients. Chemotherapy resistant tumor cells that contribute to minimal residual disease (MRD) underlie relapse and poor clinical outcomes in a sub-set of patients. Targeting mitochondrial oxidative phosphorylation (OXPHOS) in the treatment of refractory leukemic cells is a potential novel approach to sensitizing tumor cells to existing standard of care therapeutic agents. In the current study, we have expanded our previous investigation of the mitoNEET ligand NL-1 in the treatment of ALL to interrogate the functional role of the mitochondrial outer membrane protein mitoNEET in B-cell ALL. Knockout (KO) of mitoNEET (gene: CISD1) in REH leukemic cells led to changes in mitochondrial ultra-structure and function. REH cells have significantly reduced OXPHOS capacity in the KO cells coincident with reduction in electron flow and increased reactive oxygen species. In addition, we found a decrease in lipid content in KO cells, as compared to the vector control cells was observed. Lastly, the KO of mitoNEET was associated with decreased proliferation as compared to control cells when exposed to the standard of care agent cytarabine (Ara-C). Taken together, these observations suggest that mitoNEET is essential for optimal function of mitochondria in B-cell ALL and may represent a novel anti-leukemic drug target for treatment of minimal residual disease.

Keywords: Chemoresistance; Glitazones; Mitochondrial dysfunction; cisd2.

Fig. 1.

A) The mitochondrial protein mitoNEET was knocked out (KO) of REH cells using CRISPR/Cas9 for comparison to vector control cells (VC) and the protein expression determined with Western Blot. Beta-actin was used as the loading control (N = 3). B) Cellular localization of mitoNEET in REH cells. MitoNEET (red) is located on mitochondria, which are stained with MitoTracker Deep Red (green). Co-localization is observed from the yellow/orange coloring in the overlay in the VC cells. MitoNEET KO cells do not show localized staining in the mitochondria. Scale bar = 2 μm. C) TEM micrographs of REH cells. The KO cells are characterized by structural changes in the size and shape of the cristae in mitochondria, which are indicated by arrows, compared to the VC. Scale bar = 500 nm.

Fig. 2.

Mitochondrial function in REH leukemia cells is disrupted. A mitochondrial stress test kit with the Seahorse bioanalyzer shows reduction in several key aspects of mitochondrial function including: A) non-mitochondrial oxygen consumption, B) basal respiration, C) maximal respiration, D) proton leak, E) ATP production, F) spare respiratory capacity; G) oxygen consumption (OCR) graphs of mitochondrial function. H) The extrasellar acidification rate (ECAR) graph, and I) Glycolysis stress test (no significant difference). J) ATP content (μM) is reduced in the mitoNEET KO cells. Data are shown average ± SD, where N = 8. *P<0.05.

Fig. 3.

Mitochondrial electron transport chain (ETC) function is diminished in mitoNEET KO REH cells. A) OCR graph showing substrate and inhibitor addition, B) averaged OCR response, C) Complex I Activity from the ETC data and D) Complex II/III activity. Data are shown as the average ± SD, where N = 8. *P<0.05. Abbreviations: rotenone (rot), succinate (sue), antimycin A (AA), ascorbate (asc), and N,N,N′,N′-tetramethyl-p-phenylenediamine (TMPD).

Fig. 4.

MitoNEET KO leads to mitochondrial dysfunction in REH cells. A) hydrogen peroxide ROS levels are increased in KO cells compared to VC; B) mitochondrial membrane potential is decreased in KO cells compared to VC; C) DHE detection of superoxide indicates increased levels in the KO cells compared to VC. The positive control antimycin A increased superoxide in both the VC and KO cells. The antioxidant N-acetyl cysteine (NAC) decreased both endogenous as well as antimycin A-induced superoxide production. Data are shown as the average ± SD, where N = 3-4. *P<0.05. D) Thiol content in REH cells with levels of reduced (GSH) and E) oxidized (GSSH) glutathione were measured in the KO cells compared to the VC. The loss of mitoNEET was correlated with a decrease in thiol content. F) Western Blot analysis of glutathione peroxidase (GPX) 1 and 4 as well as superoxide dismutase 2 (SOD2) showed a decrease in protein content in the mitoNEET KO cells compared to the VC. Equal protein loading per lane verified with β-actin staining. Data are shown average ± SD, where N = 8. *P<0.05. G) Metabolites NADP and NADPH in REH cells show significant increase in mitoNEET KO cells compared to the controls (VC). RLU average ± SD, where N = 8. *P<0.05.

Fig. 5.

mitoNEET KO leads to a decrease in cellular lipid levels. A) Nile Red staining shows lipid levels and DAPI nuclear staining, scale bar = 20 μm; B) free fatty acid (FFA) levels are unchanged with mitoNEET KO, C) β-oxidation assay with the Seahorse BioFlux Analyzer show that OCR is reduced in mitoNEET KO cells, average ± SD, where N = 8.

Fig. 6.

MitoNEET KO results in decreased proliferation of REH cells compared to control cells (VC). Data are shown average ± SD, where N = 4. *P<0.05.

Fig. 7.

MitoNEET KO in REH cells increases sensitivity to the ALL anti-cancer drug Ara-C. Proliferation was significantly decreased in the KO cells compared to the VC. Data are shown average ± SD, where N = 4. *P<0.05.