Inhibition of the aryl hydrocarbon receptor/polyamine biosynthesis axis suppresses multiple myeloma

Abstract

Polyamine inhibition for cancer therapy is, conceptually, an attractive approach but has yet to meet success in the clinical setting. The aryl hydrocarbon receptor (AHR) is the central transcriptional regulator of the xenobiotic response. Our study revealed that AHR also positively regulates intracellular polyamine production via direct transcriptional activation of 2 genes, ODC1 and AZIN1, which are involved in polyamine biosynthesis and control, respectively. In patients with multiple myeloma (MM), AHR levels were inversely correlated with survival, suggesting that AHR inhibition may be beneficial for the treatment of this disease. We identified clofazimine (CLF), an FDA-approved anti-leprosy drug, as a potent AHR antagonist and a suppressor of polyamine biosynthesis. Experiments in a transgenic model of MM (Vk*Myc mice) and in immunocompromised mice bearing MM cell xenografts revealed high efficacy of CLF comparable to that of bortezomib, a first-in-class proteasome inhibitor used for the treatment of MM. This study identifies a previously unrecognized regulatory axis between AHR and polyamine metabolism and reveals CLF as an inhibitor of AHR and a potentially clinically relevant anti-MM agent.

Keywords: Cancer; Cell Biology; Oncology; Polyamines.

Figure 1. AHR controls ODC1 and AZIN1 transcription.

(A) Predicted transcription factors binding AZIN1 and ODC1 promoters. (B) Extracts from WI-38 cells expressing empty vector control (Ctrl) or CA-AHR probed by immunoblotting for AZIN1 and ODC1. (C) RNA from cells as in B probed in qRT-PCR with the indicated primers and probes. Data represent the average ± SEM of 3 independent experiments performed in triplicate. (D) Schematic of conserved (black circles) or partially conserved (shaded circle) AHR binding sites in the indicated promoters. Hs, Homo sapiens; Mm, Mus musculus. (E) WI-38 DNA was immunoprecipitated with control (IgG) or AHR-specific antibodies and probed in qRT-PCR with primers for the CYP1a1 promoter (positive control), regions in AZIN1 and ODC1 promoters described in D, or GMPR (negative control). Luciferase activity for the AZIN1 and ODC1 promoter regions described in D with increasing amounts of CA-AHR (F) or BaP (G). The XRE-luc plasmid was used as a control. Data represent the average ± SEM of 2 independent experiments performed in duplicate. (H) Cell extracts of WI-38 cells expressing control shRNA (Ctrl-sh) or 2 independent shRNAs against AHR (sh1 and sh2) probed by immunoblotting with the indicated antibodies. (I) RNA from cells as in H probed in qRT-PCR with the indicated primers and probes. Data represent the average ± SEM of 4 independent experiments performed in triplicate. (J) Polyamine content in cells as in H. Data represent the average ± SEM of 4 independent experiments. (K) Extracts of WI-38 cells treated for 2 hours with DMSO or 20 μM CH223191 probed by immunoblotting for AZIN1 and ODC1. (L) RNA from cells as in K probed in qRT-PCR with the indicated primers and probes. Data represent the average ± SEM of 3 independent experiments performed in triplicate. (M) Polyamine content in WI-38 cells treated with CH223191 for 48 hours. Data represent the average ± SEM of 3 independent experiments. *P < 0.05 and **P < 0.001, by 2-tailed Student’s t test. AZI, AZIN1 ; CYP, CYP1a1; ODC, ODC1; TiP, TiPARP; Spd, spermidine; Put, putrescine; Spm, spermine.

Figure 2. CLF is an AHR antagonist.

(A) Extracts of WI38 cells treated for 2 hours with increasing concentrations of CLF, Har, or FF probed in the immunoblot for AZIN1 and ODC1. (B) Cytoplasmic and nuclear fractions of WI38 cells treated for 2 hours with DMSO, 4 μM CLF, or 20 μM CH223191 resolved in the immunoblot and probed for AHR. Tubulin and TBP were used as positive controls. Quantification of band intensity was performed with ImageJ (NIH). (C) WI38 cells treated as in B and immunostained for AHR (red), DNA (Hoechst-33342, blue), and actin (phalloidin, green). Images are representative of 2 independent experiments. Scale bars: 20 μm. (D) Luciferase activity assay of HEK293FT cells transduced with XRE-luc, with increasing concentrations of BaP and DMSO, 4 μM CLF, or 20 μM CH223191. Data represent the average ± SEM of 2 independent experiments performed in duplicate. (E) Cytosolic fractions of Hepa 1c1c7 cells preincubated with vehicle or 16 nM TCDD and increasing concentrations of CLF or CH223191, and then incubated with XRE-containing biotinylated oligonucleotides and resolved on native gels. Protein-DNA complexes were visualized with a chemiluminescence system. (F) RNA from WI-38 cells treated for 2 hours with DMSO or 4 μM CLF probed in qRT-PCR with the indicated primers and probes. Data represent the average ± SEM of 3 independent experiments performed in triplicate. (G) Polyamine content in WI38 cells treated with 4 μM CLF for 48 hours. Data represent the average ± SEM of 5 independent experiments. *P < 0.05 and **P < 0.001, by 2-tailed Student’s t test. (H) Proliferation of WI38 cells expressing empty vector (Ctrl) or CA-AHR, with increasing concentrations of CLF over time. Control cells were supplemented with 10 μM spermidine and 1 mM aminoguanidine. IC₅₀ values were determined using GraphPad Prism. Data represent the average of 2 experiments performed in quadruplicate. **P < 0.001, by extra sum-of-squares F test using GraphPad Prism.

Figure 3. CLF and AHR work in overlapping pathways.

Volcano plot of differentially expressed genes in CLF-treated and AHR-KD WI38 cells. A FC of 1.5 and a P < 0.05 threshold were applied. Black dots indicate no significance, blue dots indicate genes that changed significantly only with CLF treatment, and orange dots indicate genes that changed significantly with both CLF treatment and AHR KD.

Figure 4. CLF inhibits polyamines in MM cells.

(A) Survival distribution of patients with MM (GEO GSE4581, https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi), separated by AHR expression levels. The cohort consisted of 256 patients, 64 of whom were classified as “high AHR” (average expression value = 340.14) and 192 as “low AHR” (average expression value = 27.45). Statistical analysis was performed with a log-rank test. (B) Cell extracts of the human MM cells MM.1S, RPMI-8226, and U266 were probed by immunoblotting with the indicated antibodies. (C) Cell extracts as in B were treated for 2 hours with 2 μM CLF and probed by immunoblotting. Quantification of band intensity was performed with ImageJ. (D) Extracts of MM.1S and 8226 cells were transduced with control shRNA (Ctrl-sh) or 2 independent shRNA against AHR (sh3 and sh4) and probed by immunoblotting with the indicated antibodies. (E) Extracts of MM.1S and 8226 cells transduced with empty vector (Ctrl) or CA-AHR were probed by immunoblotting as in D. Quantification of band intensity was performed with ImageJ. (F) Polyamine content in MM.1S and 8226 cells transduced with control or CA-AHR. Data represent the average ± SEM of 4 (MM1.S) or 2 (RPMI-8226) independent experiments. Cell proliferation of MM.1S (G) or 8226 (H) cells transduced with control or CA-AHR and treated for 48 hours with increasing concentrations of CLF. Control cells were supplemented with 10 μM spermidine and 1 mM aminoguanidine. IC₅₀ values were determined using GraphPad Prism. Viability of MM.1S (I) or RPMI-8226 (J) cells (WT or resistant to BTZ-R) treated with the indicated drugs and doses for 24 hours. Data represent the average ± SEM of 2 experiments performed in duplicate. *P < 0.05 and **P < 0.001 compared with the untreated control, by 2-tailed Student’s t test (F, I, and J) and sum-of-squares F test with GraphPad Prism (G and H).

Figure 5. CLF inhibits MM xenograft growth.

(A) MM.1S cells were inoculated s.c. into both flanks of 4- to 6-week-old female SCID mice. The animals were randomized into 2 groups (6 animals/group) and treated with daily i.p. injections of vehicle in PBS or CLF (10 mg/kg). Tumor volumes were recorded every 5 days. *P < 0.05, by 2-tailed Student’s t test. (B) Protein extracts from 6 tumors/group from A were resolved by immunoblotting and probed for CYP1a1, AZIN1, or ODC1. Growth of MM.1S (C) or RPMI-8226 (D) cell xenografts was monitored as in A. Animals were randomized into 3 treatment groups: PBS, CLF, or BTZ (1 mg/kg, biweekly i.p. injections). Tumor measurement distributions were tested for normality by Shapiro-Wilk test, and the significance of individual comparisons (treated/untreated) were determined by Student’s t test with Bonferroni’s correction.

Figure 6. CLF suppresses MM burden.

(A) 12 Vk*Myc mice with similar disease burdens (day 0) were randomized into 2 groups and treated with vehicle in PBS or CLF (10 mg/kg). Mice were bled once a week, and total murine IgG levels were determined by ELISA. Statistical significance was determined by 2-tailed Student’s t test. (B) Nine Vk*Myc mice with similar disease burdens were randomized into 3 treatment groups: PBS, CLF, or BTZ (1 mg/kg, biweekly i.p. injections). Total murine IgG levels were determined as in A. Statistical significance was determined by 1-way ANOVA.