CDK9 Inhibition Induces a Metabolic Switch that Renders Prostate Cancer Cells Dependent on Fatty Acid Oxidation

Abstract

Cyclin-dependent kinase 9 (CDK9), a key regulator of RNA-polymerase II, is a candidate drug target for cancers driven by transcriptional deregulation. Here we report a multi-omics-profiling of prostate cancer cell responses to CDK9 inhibition to identify synthetic lethal interactions. These interactions were validated using live-cell imaging, mitochondrial flux-, viability- and cell death activation assays. We show that CDK9 inhibition induces acute metabolic stress in prostate cancer cells. This is manifested by a drastic down-regulation of mitochondrial oxidative phosphorylation, ATP depletion and induction of a rapid and sustained phosphorylation of AMP-activated protein kinase (AMPK), the key sensor of cellular energy homeostasis. We used metabolomics to demonstrate that inhibition of CDK9 leads to accumulation of acyl-carnitines, metabolic intermediates in fatty acid oxidation (FAO). Acyl-carnitines are produced by carnitine palmitoyltransferase enzymes 1 and 2 (CPT), and we used both genetic and pharmacological tools to show that inhibition of CPT-activity is synthetically lethal with CDK9 inhibition. To our knowledge this is the first report to show that CDK9 inhibition dramatically alters cancer cell metabolism.

Figure 1

AT7519 treatment induces acute metabolic stress. A) AMPK phosphorylation in response to AT7519 in LNCaP cells. Data was recorded using the reverse-phase protein array (RPPA)-approach (all the data is provided in Suppl. Table 1). LNCaP cells were treated with 0.5 μM AT7519 for 4 and 24 hours and cell lysates were analyzed using RPPA profiling. Data shown is an average of four biological replicates with SEM and Student's t-test was used to evaluate statistical significance (*<.05, **<.01). B) LNCaP cells were treated with DMSO or increasing dose of AT7519 for 24 hours and samples were analyzed using western blotting. Densitometry was used to evaluate the abundance of each protein. This is a representative western blot of two replicates. C, D) AT7519 treatment decreases mitochondrial oxygen-consumption rate (OCR) in LNCaP cells. Cells were treated with either DMSO or AT7519 for 24 hours prior to start of the Seahorse XFe 96 analyzer OCR-measurements. Serial injections of oligomycin (Olig), Carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP), and a mix of rotenone and antimycin (Rot+AA) enabled measurements of ATP production, maximal respiration, and non-mitochondrial respiration, respectively. Proton leak and spare respiratory capacity were calculated using these parameters and basal respiration. Data shown is an average of 3–4 biological replicates with SEM and Student's t-test was used to assess the statistical significance (*<.05, **<.01, ***<.001). AT7519 treatment induces acute metabolic stress. A) AMPK phosphorylation in response to AT7519 in LNCaP cells. Data was recorded using the reverse-phase protein array (RPPA)-approach (all the data is provided in Suppl. Table 1). LNCaP cells were treated with 0.5 μM AT7519 for 4 and 24 hours and cell lysates were analyzed using RPPA profiling. Data shown is an average of four biological replicates with SEM and Student's t-test was used to evaluate statistical significance (*<.05, **<.01). B) LNCaP cells were treated with DMSO or increasing dose of AT7519 for 24 hours and samples were analyzed using western blotting. Densitometry was used to evaluate the abundance of each protein. This is a representative western blot of two replicates. C, D) AT7519 treatment decreases mitochondrial oxygen-consumption rate (OCR) in LNCaP cells. Cells were treated with either DMSO or AT7519 for 24 hours prior to start of the Seahorse XFe 96 analyzer OCR-measurements. Serial injections of oligomycin (Olig), Carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP), and a mix of rotenone and antimycin (Rot+AA) enabled measurements of ATP production, maximal respiration, and non-mitochondrial respiration, respectively. Proton leak and spare respiratory capacity were calculated using these parameters and basal respiration. Data shown is an average of 3–4 biological replicates with SEM and Student's t-test was used to assess the statistical significance (*<.05, **<.01, ***<.001).

Figure 2

AT7519 induced accumulation of acyl-carnitines promotes prostate cancer cell survival. A) Targeted metabolite profiling of LNCaP cells treated as indicated for 24 hours. Data shown is an average of four biological replicates with SEM. Only metabolites whose abundance changed at least 20% in comparison to DMSO are shown. Metabolite levels were quantitatively measured using mass spectrometry and normalized to cell count. Control sample was set to 100% and treatments were also normalized to this. Student's t-test was used to assess the statistical significance (*<.05, **<.01). B) Measurement of the absolute amount of carnitines. Data shown is an average of 4 biological replicates with SEM and t-test was used to evaluate the statistical significance (*<.05). C) Schematic presentation of acyl-carnitine transport across the mitochondrial membrane. D) Knockdown (KD) of CPT1 and CPT2 sensitizes cells to AT7519 treatment. KD was performed for 4 days as indicated, after which cells were treated with AT7519 for 3 days and viability was assessed using the CellTiter-Glo assay. Data shown is an average of at least 3 biological replicates with SEM and t-test was used to evaluate the statistical significance (*<.05, **<.01). E) The efficacy of CPT1 and CPT2 KD was evaluated using western blotting.

Figure 3

CDK9 inhibitors are synthetically lethal with inhibitors of fatty acid oxidation. A) AT7519 renders prostate cancer cells sensitive to Perhexiline as measured using oxygen-consumption rate (OCR). A Seahorse XFe 96 analyzer was used to measure OCR of cells upon acute injection of perhexiline (indicated with an arrow). LNCaP cells were pre-treated with AT7519 for 24 hours as indicated. Data shown is an average of 3 biological replicates with SEM; t-test was used to evaluate statistical significance (*<.05, **<.01). B) Combination of 0.5 μM AT7519 with 10 μM Perhexiline induces DNA damage and cell death in LNCaP cells. Cells were treated as indicated for 24 hours and western blotting was used to detect the proteins of interest. Densitometry was used to evaluate the abundance of each protein. C) Combination of AT7519 with Perhexiline increases the sub-G1 population of cells. Cell cycle distribution was assessed using propidium iodide staining and flow cytometry. Data shown is an average of two biological replicates with SEM. D) Activation of cell death in response to AT7519 and Perhexiline treatments. The cumulative activation of Caspases 3 and 7 was recorded using live-cell imaging until 36 hours and normalized to cell confluency. Data shown is an average four biological replicate experiments with SEM; a t-test was used to evaluate the statistical significance. E) Growth rate of cells was recorded using live-cell imaging. Data shown is an average of four biological replicates with SEM and t-test was used to assess the statistical significance between combination treatments against any single treatment. F) LNCaP cells were treated with NVP2 for 24 hours and the abundance of ATP was determined using the CellTiter-Glo-assay. Data shown is an average of three technical replicates with STDEV. G) LNCaP cells were treated with DMSO or increasing dose of NVP2 for 24 hours and samples were analyzed using western blotting. Densitometry was used to evaluate the abundance of each protein. H) LNCaP cells were treated with 20 nM NVP2 for 4 hours, mRNA isolated and used for RT-qPCR. Transcript abundance was normalized to luciferase RNA that was added to each sample in the cell lysis buffer. Data shown is an average of three biological replicates with SEM and t-test was used to assess the statistical significance. I) Growth rate of cells was recorded using live-cell imaging. Data shown is an average of four biological replicates with SEM and t-test was used to assess the statistical significance between combination treatments against any single treatment. J) Activation of cell death in response to AT7519 and Perhexiline treatments. The cumulative activation of Caspases 3 and 7 was recorded using live-cell imaging until 36 hours and normalized to cell confluency. Data shown is an average of four biological replicate experiments with SEM and t-test was used to evaluate the statistical significance (*<.05, **<.01).