Research ArticleCancer

Metabolic perturbations sensitize triple-negative breast cancers to apoptosis induced by BH3 mimetics

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Science Signaling  08 Jun 2021:
Vol. 14, Issue 686, eabc7405
DOI: 10.1126/scisignal.abc7405
  • Fig. 1 HT-DBP screening identifies NAMPT inhibitors to increase mitochondrial apoptotic priming of TNBC cells but not normal cells.

    (A) HT-DBP was used to screen a library of 192 metabolism-perturbing small molecules (MPSMs) on the SUM149 cells 48 hours after treatment. MPSMs were assayed at 10-point dilutions (20 to 0.003 μM). Percent delta priming of each concentration of each MSPM was calculated by comparing cytochrome c abundance in treated cells to that of vehicle control cells. MPSM concentrations that induced priming that was more than 3× SD of the DMSO-treated wells (horizontal dotted line) were considered to increase the mitochondrial apoptotic priming of the cancer cells and are indicated in red. MPSM concentrations that decreased the cell count to less than 80% of the average cell count of DMSO-treated cells (vertical dotted line) were considered to have an effect on cell count. MPSM concentrations that show an increased % delta priming, but no reduction in cell count was considered hits and are indicated in bright red. Control (DMSO-treated) cells are indicated in blue. Data are average of technical replicates. (B) Thirty-two MPSMs that increased priming of SUM149 cells after 48 hours in RPMI medium were counter screened on the SUM149 cells cultured in human plasma-like medium (HPLM). Compounds that showed priming in RPMI but not in HPLM (indicated in brown) were excluded from further follow-up screenings. Control (DMSO-treated) cells are indicated in blue. ABT-263–treated cells (positive control) are indicated in red. Data are average of technical duplicates. (C) Metabolic compounds identified as hits in the initial screen on SUM149 were screened on MDA-MB-231, MDA-MB-468, HCC1143, and HCC1937 TNBC cells and counter screened on MCF10A, HMEC, and HK-2 cells. Data are represented as average of normalized AUC measurements of the % delta priming dilution curves of the metabolic agents tested in three independent screenings. Compounds that increase apoptotic priming in both TNBC and nonmalignant cells are grouped in group A, and compounds that at a given concentration only increase priming of TNBC cells are grouped in group B. ABT-263 is a positive control for increasing apoptotic priming; DMSO is negative control.

  • Fig. 2 NAMPT inhibition increases overall mitochondrial apoptotic priming, antiapoptotic dependencies, and sensitivity of TNBC cell lines to BH3 mimetics and apoptosis inducers.

    (A) Percent delta priming of TNBC cell lines and nonmalignant cells (MCF10A and HMEC) after 48 hours of exposure to NAMPT inhibitors (FK866 and GPP78) at the indicated concentrations. Priming was determined by DBP using BIM peptide. Delta priming is calculated relative to DMSO-treated samples. Data are mean and SD of two or three independent experiments. (B) AUC and SD of the % delta priming dilution curves in (A). (C) Binding properties of BH3 peptides to antiapoptotic BCL-2 family members as described previously (6). Green squares indicate binding, and white squares indicate no binding or an EC50 (median effective concentration) binding value >1 μM. (D) Heatmap of the % delta priming of TNBC cell lines and MCF10A cells exposed for 48 hours to 0.01 μM FK866 or 0.1 μM GPP78. Percent delta priming is calculated by comparing cytochrome c abundance in drug-treated versus vehicle-treated cells. Responses to the BIM and PUMA peptide indicates overall mitochondrial priming. Responses to the BAD, HRK, MS1, and FS1 peptides, and BH3 mimetics indicate specific antiapoptotic dependencies. BAD and ABT-263 indicate BCL-2, BCL-XL, or Bcl-w dependency; HRK, A-133, and A-115 indicate BCL-XL dependency; ABT-199 indicates BCL-2 dependency; MS1 and S63845 indicate MCL-1 dependency. Data are average of three independent experiments. Data are also presented as bar graphs ± SD in the Supplementary Materials (fig. S3). (E) Cell death measurements using microscopy-based AV-Hoechst cell death assay. TNBC cells and MCF10A cells were treated for 72 hours with 0.01 μM FK866 and 0.1 μM GPP78 in the presence or absence of 0.1 μM BH3 mimetics. Values are means of three independent experiments. Data are also presented as dot plots ± SD in the Supplementary Materials (fig. S4).

  • Fig. 3 NAMPT inhibition increases overall mitochondrial apoptotic priming, antiapoptotic dependencies, and sensitivity to S63845 of TNBC PDX2 but not PDX1.

    (A) Percent delta priming of freshly isolated PDX1 and PDX2 tumor cells in vitro exposed to 0.01 μM FK866 and 0.1 μM GPP78 for 48 hours. Percent delta priming is calculated by comparing cytochrome c abundance in drug-treated versus vehicle-treated cells. ABT-263 is used as a positive control for induction of apoptotic priming; DMSO (vehicle) is used as a negative control. Data are average and SD of two or three independent experiments. (B) AUC and SD of % delta priming data shown in (A). Statistical analysis as done using two-way analysis of variance (ANOVA) with Sidak’s post hoc test. (C) DBP of freshly isolated PDX1 and PDX2 tumor cells exposed in vitro to 0.01 μM FK866 and 0.1 μM GPP78 for 48 hours. Responses to the BIM and PUMA peptide indicate overall mitochondrial priming. Responses to the BAD, HRK, MS1, FS1 peptides, and BH3 mimetics indicate specific antiapoptotic dependencies. BAD and ABT-263 indicate BCL-2, BCL-XL, or BCL-W dependency; HRK, A-133, and A-115 indicate BCL-XL dependency; ABT-199 indicates BCL-2 dependency; MS1 and S63845 indicate MCL-1 dependency. Data are average of two individual experiments. (D) Treatment schedule and volume of PDX1 and PDX2 tumors measured during the short-term efficacy study. Treatment was started when tumors reached 150 to 200 mm3, indicated as day 0 on the x axis. Red arrowheads indicate FK866 (15 mg/kg) or vehicle (1% hydroxypropyl-β-cyclodextrin and 12% 1,2-propylenglycol in saline) treatment via intraperitoneal (IP) injection twice a day (BID). Blue arrowheads indicate S63845 (25 mg/kg) or vehicle (2% vitamin E/TPGS) administration via intravenous (IV) injections twice a week (BIW). Mice were treated for 21 days and sacrificed on day 22. Tumor volume graphs show average and SD, n = 4 mice per treatment group. The trend in tumor volume changes by treatment across time was modeled using a linear mixed model with a random effect per subject and a compound symmetry correlation structure using R 3.6.3. Family-wise error was adjusted via Tukey’s multiple correction method: *P < 0.05, **P < 0.01, and ***P < 0.001; no statistical labeling indicates that no statistical difference was observed. Combination treatment versus S63845, P = 0.007; combination treatment versus vehicle, P < 0.001; S63845 versus vehicle, P = 0.021; and FK866 versus vehicle, P < 0.001. (E) Percent delta priming of PDX1 and PDX2 tumor cells at the day 22 end point of the short-term in vivo efficacy study in (D). Tumors were dissociated and BH3 profiling was performed on the freshly isolated tumor cells. Percent delta priming refers to the difference in apoptotic priming of tumor cells derived from FK866-treated versus vehicle-treated animals. Data shown are average and SD, n = 3 or 4 tumors per treatment group. (F) Treatment schedule and PDX2 tumor volumes of mice in long-term efficacy study. Treatment was initiated when tumors reached 150 to 200 mm3, indicated as day 0 on the x axis. Red arrowheads indicate FK866 (15 mg/kg) or vehicle (1% hydroxypropyl-β-cyclodextrin and 12% 1,2-propylenglycol in saline) treatment by intraperitoneal injection twice a day (BID). Blue arrowheads indicate S63845 (25 mg/kg) or vehicle (2% vitamin E/TPGS) by intravenous injections twice a week (BIW). Mice were treated for 19 days. Animals were sacrificed when their tumors reached 1200 mm3. Study was ended when all vehicle-treated animals reached a tumor size of 1200 mm3 and were sacrificed (53 days posttreatment). Data are average and SD; n = 6 mice per treatment group. Statistical analysis was done using Tukey’s multiple testing correction on a linear mixed model in R 3.6.3: **P < 0.01; no statistical labeling means no statistical difference was observed. (G) Kaplan-Meier analysis of PDX2 data represented in (F). A log-rank test was used to estimate a difference in survival effects across time between all treatments; multiple testing–induced family-wise error was adjusted using Benjamini and Hochberg with R 3.6.3: *P < 0.05.

  • Fig. 4 NAD+ levels need to drop below a critical level for apoptotic priming and antiapoptotic dependencies to be increased in TNBC cells after NAMPT inhibition.

    (A) Classification of TNBC cell lines according to their sensitivity for BH3 mimetics after NAMPT inhibition, on the basis of the “average viability” calculated from the data presented in Fig. 2E and fig. S5: Average viability=[%viability cells (FK866+ABT199)+%viable cells (FK866+ABT263)+%viable cells (FK866+A133)+%viable cells (FK866+S63845)%viable cells (FK866+Staurosporin)]5 Cell lines are indicated in shades of red for sensitive, shades of gray for intermediately sensitive, and shades of blue for insensitive lines. Statistical analysis as done using one-way ANOVA with Tukey’s post hoc test. (B) NAD+ levels in indicated cell lines after 48-hour treatment with 0.01 μM FK866 or 0.1 μM GPP78, measured using the NAD/NADH kit (Promega). Data are average of two independent experiments. (C) NAD+ levels in PDX tumors after in vivo treatment with single-agent FK866 as described in Fig. 3D, measured using untargeted metabolomics and expressed as peak areas. Data are average and SD of n = 3 or 4 animals per treatment arm. Statistical analysis as done using one-way ANOVA with Tukey’s post hoc test. (D) Schematics of different NAD+ synthesis pathways and expression levels of rate-limiting enzymes: de novo synthesis in yellow, Preiss-Handler pathway in red, and salvage pathway in blue. Abbreviations not yet defined: NMNAT, NMN adenylyl transferase; NRKs, nicotinamide riboside kinases; NR, nicotinamide riboside; NADSYN, NAD synthetase; NAAD, nicotinic acid adenine dinucleotide; NAMN, nicotinic acid mononucleotide; QART, quinolinic acid phosphoribosyltransferase; QA, quinolinic acid. Expression levels of NAD+ synthesis enzymes are determined at baseline. Data are mean and SE of three independent experiments. (E) NAD+ levels in indicated cell lines after 24-hour treatment with 0.01 μM FK866 in the presence or absence of 25 μM NMN, measured using the NAD/NADH kit (Promega). Data are mean and SD of three independent experiments. Statistical analysis was done using one-way ANOVA with Tukey’s post hoc test. (F) Viability data of indicated cell lines treated for 72 hours with 0.01 μM FK866, 0.1 μM BH3 mimetics, 0.03 μM staurosporine, or combination in the presence or absence of 25 μM nicotinamide mononucleotide (NMN) or 25 μM nicotinic acid (NA). Data are means of three independent experiments. Data are also presented as dot plots of average and SD in the Supplementary Materials (fig. S10). (G) Percent delta priming of sensitive cell lines MDA-MB-468 and MDA-MB-231 after 48 hours of exposure to 0.01 μM FK866 in the presence or absence of NA or NMN (25 μM). Data are means of three independent experiments. Data are also presented as bar graphs of average ± SD in the Supplementary Materials (fig. S11). *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

  • Fig. 5 NAD+ depletion induces apoptotic priming and antiapoptotic dependencies in TNBC by dysregulation of specific metabolic pathways rather than an overall metabolic collapse.

    (A) Metabolomics data of intermediates of the glycolysis, pentose phosphate pathway (PPP), tricarboxylic acid (TCA) cycle, nucleoside synthesis, and the oxidative stress pathways extracted from cell lines treated for 48 hours with 0.01 μM FK866 or 0.1 μM GPP78. Data are expressed as fold changes compared to vehicle-treated samples. Data are average of technical duplicates. Sensitive models are indicated in red, intermediately sensitive models are indicated in gray, and insensitive models are indicated in blue. (B) Metabolomics data of intermediates of the glycolysis, pentose phosphate pathway (PPP), tricarboxylic acid (TCA) cycle, nucleoside synthesis, and the oxidative stress pathways extracted from PDX tumors treated in vivo with FK866 (7.5 or 15 mg/kg) according to treatment schedule in Fig. 3D. Data are expressed as fold changes compared to vehicle-treated samples. Data are average of n = 2 to 4 mice per treatment arm. (C) Microscopy-based annexin V–Hoechst cell viability data of MDA-MB-468 and MDA-MB-231 cells cotreated for 72 hours with 0.01 μM FK866 and 0.1 μM BH3 mimetics in the presence or absence of different metabolites. Data are means of three independent experiments. (D) Percent delta priming of TNBC cells after 48 hours of exposure to 0.01 μM FK866 in the absence or presence of 25 μM adenine. Data are average of three independent experiments. (E) NAD+ levels after 48 hours NAMPT inhibition (0.01 μM FK866) in the presence or absence of 25 μM adenine. Data are average and SD of three independent experiments. Statistical analysis as done using one-way ANOVA with Tukey’s post hoc test: ****P < 0.0001; no statistical labeling indicates no statistical difference was observed. ns, not significant.

  • Fig. 6 Mechanism of action of NAMPT inhibitors plus BH3 mimetics.

    TNBC cells that are sensitive to the combination treatment of a NAMPT inhibitor and a BH3 mimetic are reliant on NAMPT to maintain critical levels of NAD+. In these cells, other NAD+ synthesis pathways, with NAPRT and TDO and IDO as rate-limiting enzymes, cannot compensate for NAMPT inhibition and NAD+ levels drop below a critical threshold. While several metabolic pathways are dysregulated, it is adenine depletion in particular that increases the mitochondrial apoptotic priming. TNBC cells that are insensitive to the combination treatment activate compensatory NAD+ synthesis pathways upon NAMPT inhibition. While NAD+ levels drop, they remain above the critical threshold and no increased mitochondrial apoptotic priming is observed.

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