Research ArticleCancer

Metabolic reprogramming toward oxidative phosphorylation identifies a therapeutic target for mantle cell lymphoma

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Science Translational Medicine  08 May 2019:
Vol. 11, Issue 491, eaau1167
DOI: 10.1126/scitranslmed.aau1167
  • Fig. 1 Landscape of somatic mutations and DNA copy number alterations in 14 patients with MCL treated with ibrutinib.

    (A) Somatic mutations, where each column represents a patient tumor sample, and the clinical and pathological characteristics are annotated at the top. Genes with nonsynonymous mutations or copy number alterations in two or more patients are listed. The numbers on the left and right sides represent the percentages of MCL tumors carrying a mutation or copy number alteration of each specific gene in the ibrutinib-sensitive and ibrutinib-resistant groups, respectively. Mantle Cell Lymphoma International Prognostic Index (MIPI) score: 0 to 3, low risk; 4 to 5, intermediate risk; 6 to 11, high risk. CR, complete response; PR, partial response; PD, progressive disease; LOH, loss of heterozygosity. The asterisk at CDKN2A and MTAP denotes a statistically significant difference in the deletion of CDKN2A and MTAP in the ibrutinib-resistant cohort compared with the ibrutinib-sensitive cohort (P = 0.010). (B) Somatic copy number alterations in ibrutinib-resistant (top) and ibrutinib-sensitive (bottom) tumors. The chromosome numbers are labeled at the top, and the sample IDs are shown on the left. Blue indicates copy number loss, and red indicates copy number gain, where the intensity corresponds to the log2 ratio of each segment. (C) Copy number gains and losses in ibrutinib-sensitive (blue) and ibrutinib-resistant (pink) groups. The boxes in the box plot represent the interquartile range (IQR), where the centerline depicts the median. The upper whisker indicates the maximum value or 75th percentile +1.5 IQR, whichever is smaller; the lower whisker indicates the minimum value or 25th percentile −1.5 IQR, whichever is greater.

  • Fig. 2 DEGs between the ibrutinib-sensitive and ibrutinib-resistant tumors.

    (A) Unsupervised clustering showing the most DEGs between the ibrutinib-sensitive and ibrutinib-resistant tumors. (B) A cutoff fold change of ≥2 or ≤−2 and an FDR q value of ≤0.01 were applied, and only genes that met these criteria were selected for unsupervised clustering in (A) and labeled in the volcano plot. (C) Box plots showing two representative DEGs of metabolite transporters SLC16A1 and SLC1A5. (D) Immunoblotting showing the differential expression of the metabolite transporters SLC16A1 and SLC1A5 in ibrutinib-resistant MCL cell lines Maver-1, Z-138, Granta-519, and three MCL patient samples (PT4 to PT6) compared with the ibrutinib-sensitive MCL cell lines JeKo-1, Mino, SP-49, and three MCL patient samples (PT1 to PT3). To quantitate the proteins, all bands were first normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) by area using ImageJ software. A ratio was obtained for each band. The band in the first lane was treated as 1, and a fold change for the other bands compared with the first value was calculated to represent the relative protein expression. Ibrutinib-S/R, ibrutinib-sensitive/resistant.

  • Fig. 3 Signaling pathways driving ibrutinib resistance in patients with MCL.

    (A) The top significantly enriched signaling pathways in ibrutinib-resistant (versus ibrutinib-sensitive) tumors by GSEA pathway enrichment analysis. The normalized enrichment score (NES) reflects the extent of enrichment and allows comparison across gene sets. Listed pathways are ranked by their NES and colored by their type classification. The FDR q values are labeled on the right. (B) Representative enrichment plots for the hallmark OXPHOS pathway, mTORC1 signaling, and MYC and E2F targets. The top portion of each plot shows the running enrichment score (ES) for the gene set as the analysis walks down the ranked list of genes. ES reflects the degree to which a gene set is overrepresented at the top (positive ES) or bottom (negative ES) of a ranked list of genes. The score at the peak of the plot (the score furthest from 0.0) is the ES for the gene set. The black vertical line at the bottom shows where the members of the gene set appear in the ranked list of genes. The graded red to blue bars on the x axis represent the DESeq2 statistical values (resistant group versus sensitive group), with red and blue denoting up-regulation and down-regulation, respectively.

  • Fig. 4 The role of OXPHOS in the energy production of ibrutinib-resistant MCL cells.

    (A) The basal, ATP-coupled, and reserve OCR in ibrutinib-sensitive (JeKo-1 and Mino) and ibrutinib-resistant (Z-138 and Maver-1) MCL cell lines (ibrutinib-sensitive comparison versus ibrutinib-resistant comparison: basal OCR, P < 0.0001; ATP-coupled OCR, P = 0.0059; reserve OCR, P = 0.0001, linear regression model; n = 3 biological replicates; means ± SD). (B) OCR:ECAR ratios calculated for MCL cell lines [from left to right: (ibrutinib sensitive) JeKo-1, Mino, and Rec-1 and (ibrutinib resistant) JVM-13, Z-138, and Maver-1; P < 0.0001, linear regression model] and MCL primary clinical specimens (n = 3 sensitive and 3 resistant; P < 0.0001, linear regression model). Each color represents a cell line (JeKo-1, blue; Mino, green; Rec-1, maroon; JVM-13, brown; Z-138, purple; and Maver-1, red) or clinical sample from a distinct patient (each color represents a different patient). (C) Relative abundance of metabolites extracted from ibrutinib-sensitive and ibrutinib-resistant MCL cell lines. Each row represents a single metabolite, and each column represents the indicated MCL cell line. (D) Reverse phase protein array (RPPA) analysis of ibrutinib-resistant MCL cell lines (Z-138 and Maver-1 in triplicate) versus ibrutinib-sensitive MCL cell lines (Rec-1 and Mino in triplicate) was used to generate the depicted volcano plot. A cutoff fold change of ≥2 or ≤−2 and an FDR q value of ≤0.01 were applied, and only proteins that met these criteria were selected for unsupervised clustering. (E) Immunoblotting showing GLS protein amounts in the indicated ibrutinib-sensitive and ibrutinib-resistant MCL cell lines and primary MCL cells freshly isolated from patients. (F) ROS detected by flow cytometry in MCL cell lines as indicated in the absence or presence of 2 mM glutamine (Q). DCF-DA, 2,7-dichlorofluorescein diacetate.

  • Fig. 5 Targeting the OXPHOS pathway to overcome ibrutinib resistance.

    (A) Cell growth inhibition of ibrutinib-resistant (Z-138 and Maver-1) and ibrutinib-sensitive (Mino and JeKo-1) MCL cell lines after a 72-hour incubation with IACS-010759 at the indicated doses (β regression model; P < 0.0001; n = 3 biological replicates, means ± SEM). (B and C) The mitochondria OCR quantitated in ibrutinib-resistant (Z-138 and Maver-1) and ibrutinib-sensitive (Mino and JeKo-1) MCL cell lines (B) and ibrutinib-sensitive and ibrutinib-resistant primary MCL cells (C) treated with 20 nM IACS-010759 for 1 hour (mixed effects regression model; P < 0.0001 between the resistant and the sensitive cell lines and primary MCL cells freshly isolated from patients with MCL; n = 3 biological replicates, means ± SEM). IACS-010759 produced an average reduction of 358.9406 (95% confidence interval, 321.1402-396.741) OCR compared with the control; mixed effects regression model; P < 0.0001. (D) Mitochondrial membrane potential (Δψm) depicted as histograms in the indicated MCL cell lines treated with 20 nM IACS-010759 and/or 5 μM BPTES for 24 hours before staining with tetramethylrhodamine, ethyl ester (TMRE) dye. (E) Quantitative analysis of apoptosis in ibrutinib-resistant Z-138 and Maver-1 compared with ibrutinib-sensitive Mino and JeKo-1 MCL cell lines (n = 3 biological replicates, means ± SD). Single-agent IACS-010759 treatment comparisons between the ibrutinib-sensitive and ibrutinib-resistant MCL cell lines calculated by linear regression P < 0.0001; IACS-010759 + BPTES versus single-agent IACS-010759 treatment in the ibrutinib-resistant MCL cell lines (P = 0.0059).

  • Fig. 6 The anti-MCL activity of IACS-010759 in an ibrutinib-resistant MCL PDX model.

    Vehicle control, ibrutinib (50 mg/kg oral gavage, daily), or IACS-010759 (10 mg/kg oral gavage, five consecutive days per week) was administered to the mice beginning 5 days after engraftment until the endpoint. (A) Tumor volume calculated to reflect tumor burden [n = 5; P = 0.6397 (ibrutinib versus vehicle) and P < 0.0001 (vehicle versus IACS-010759), mixed effects regression model after logarithmic transformation] as indicated. (B) Human β2M concentrations used to monitor tumor burden [P < 0.0001 (vehicle versus IACS-010759), mixed effects regression model after logarithmic transformation] on days 0, 10, and 20 of treatment. (C) Mouse body weight calculated during drug treatment [P = 0.3304, means ± SEM (vehicle versus IACS-010759), mixed effects regression model after logarithmic transformation]. (D) H&E and CD20 staining of representative mouse tumors dissected at the end of treatment. Scale bars, 100 μM.

  • Fig. 7 The anti-lymphoma effects of IACS-010759 in an ibrutinib-resistant B cell lymphoma PDX model.

    Vehicle control, ibrutinib (50 mg/kg oral gavage, daily), or IACS-010759 (10 mg/kg oral gavage, five consecutive days per week) was administered to the mice beginning 5 days after engraftment until the endpoint. (A) Tumor volume calculated to reflect tumor burden [n = 5; P = 0.7227 (ibrutinib versus vehicle) and P < 0.0001 (vehicle versus IACS-010759), mixed effects regression model after logarithmic transformation]. (B) Human β2M concentrations used to monitor tumor burden on days 0, 7, and 15 of treatment [n = 5; P = 0.6911 (ibrutinib versus vehicle) and P = 0.0003 (IACS-010759 versus vehicle), mixed effects regression model after logarithmic transformation]. (C) Mouse body weight calculated during drug treatment [P = 0.1964 (IACS-010759 versus vehicle), mixed effects regression model after logarithmic transformation]. (D) Survival curve of the ibrutinib-resistant PDX model [n = 5; P = 0.0035 (IACS-010759 versus vehicle), log-rank test].

Supplementary Materials

  • stm.sciencemag.org/cgi/content/full/11/491/eaau1167/DC1

    Materials and Methods

    Fig. S1. Survival outcomes of patients with MCL relative to ibrutinib response.

    Fig. S2. Tumor cellularity comparison between the ibrutinib-sensitive and ibrutinib-resistant clinical specimens.

    Fig. S3. DEGs between ibrutinib-sensitive and ibrutinib-resistant tumors using the nanoString nCounter system 63-gene ibrutinib resistance panel.

    Fig. S4. DEGs between the ibrutinib-sensitive and ibrutinib-resistant tumors using the nanoString nCounter system panCancer panel.

    Fig. S5. Relative abundance of metabolites in ibrutinib-sensitive and ibrutinib-resistant MCL cell lines.

    Fig. S6. OXPHOS pathway activation in ibrutinib-resistant MCL cells.

    Fig. S7. OXPHOS pathway member up-regulation in BTK KD ibrutinib-resistant MCL cell lines.

    Fig. S8. The growth inhibition effects of rotenone treatment on ibrutinib-resistant MCL cells.

    Fig. S9. Reductions in mitochondrial activity and ATP production and induction of ROS production in ibrutinib-resistant MCL cells.

    Fig. S10. Induction of apoptosis in ibrutinib-resistant MCL cells.

    Fig. S11. The anti-cancer effects of IACS-010759 in an ibrutinib-resistant MCL PDX model.

    Table S1. Information on the samples used for the nanoString nCounter system assay using the 63-gene ibrutinib resistance panel.

    Table S2. The IC50 values of ibrutinib across MCL cell lines.

    Data file S1. The clinical and pathological data of patients with MCL.

    References (82, 83)

  • The PDF file includes:

    • Materials and Methods
    • Fig. S1. Survival outcomes of patients with MCL relative to ibrutinib response.
    • Fig. S2. Tumor cellularity comparison between the ibrutinib-sensitive and ibrutinib-resistant clinical specimens.
    • Fig. S3. DEGs between ibrutinib-sensitive and ibrutinib-resistant tumors using the nanoString nCounter system 63-gene ibrutinib resistance panel.
    • Fig. S4. DEGs between the ibrutinib-sensitive and ibrutinib-resistant tumors using the nanoString nCounter system panCancer panel.
    • Fig. S5. Relative abundance of metabolites in ibrutinib-sensitive and ibrutinib-resistant MCL cell lines.
    • Fig. S6. OXPHOS pathway activation in ibrutinib-resistant MCL cells.
    • Fig. S7. OXPHOS pathway member up-regulation in BTK KD ibrutinib-resistant MCL cell lines.
    • Fig. S8. The growth inhibition effects of rotenone treatment on ibrutinib-resistant MCL cells.
    • Fig. S9. Reductions in mitochondrial activity and ATP production and induction of ROS production in ibrutinib-resistant MCL cells.
    • Fig. S10. Induction of apoptosis in ibrutinib-resistant MCL cells.
    • Fig. S11. The anti-cancer effects of IACS-010759 in an ibrutinib-resistant MCL PDX model.
    • Table S1. Information on the samples used for the nanoString nCounter system assay using the 63-gene ibrutinib resistance panel.
    • Table S2. The IC50 values of ibrutinib across MCL cell lines.
    • References (82, 83)

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

    • Data file S1 (Microsoft Excel format). The clinical and pathological data of patients with MCL.

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