The mevalonate pathway is an actionable vulnerability of t(4;14)-positive multiple myeloma

Abstract

Multiple myeloma (MM) is a plasma cell malignancy that is often driven by chromosomal translocations. In particular, patients with t(4;14)-positive disease have worse prognosis compared to other MM subtypes. Herein, we demonstrated that t(4;14)-positive cells are highly dependent on the mevalonate (MVA) pathway for survival. Moreover, we showed that this metabolic vulnerability is immediately actionable, as inhibiting the MVA pathway with a statin preferentially induced apoptosis in t(4;14)-positive cells. In response to statin treatment, t(4;14)-positive cells activated the integrated stress response (ISR), which was augmented by co-treatment with bortezomib, a proteasome inhibitor. We identified that t(4;14)-positive cells depend on the MVA pathway for the synthesis of geranylgeranyl pyrophosphate (GGPP), as exogenous GGPP fully rescued statin-induced ISR activation and apoptosis. Inhibiting protein geranylgeranylation similarly induced the ISR in t(4;14)-positive cells, suggesting that this subtype of MM depends on GGPP, at least in part, for protein geranylgeranylation. Notably, fluvastatin treatment synergized with bortezomib to induce apoptosis in t(4;14)-positive cells and potentiated the anti-tumor activity of bortezomib in vivo. Our data implicate the t(4;14) translocation as a biomarker of statin sensitivity and warrant further clinical evaluation of a statin in combination with bortezomib for the treatment of t(4;14)-positive disease.

Fig. 1. The t(4;14) translocation is associated with statin sensitivity in MM.

a Schematic representation of the MVA pathway and its sterol-regulated feedback loop. Statins inhibit the rate-limiting enzyme of the MVA pathway, HMGCR. Statin-mediated cholesterol depletion activates the SREBP2 transcription factor, which induces genes involved in MVA metabolism, including HMGCR. b List of lovastatin-sensitive and insensitive MM cell lines previously characterized by Wong et al. [10]. c Differential gene expression analysis between statin-sensitive and insensitive MM cell lines listed in (b). The blue and red dots represent differentially expressed genes with a log₂(fold change) >2 or <−2. The genes in red further pass the adjusted p value cut-off when corrected for multiple testing (Bonferroni correction). Black dots represent genes with no significant difference in expression between statin-sensitive and insensitive MM cell lines. d Sensitivity to fluvastatin determined by MTT assays following 48 h of treatment. Fluvastatin IC₅₀ value and 95% confidence interval (CI) for each cell line are shown. The t(4;14) translocation status is indicated and t(4;14)-positive cell lines are shaded in gray. e Sensitivity to fluvastatin, lovastatin, and simvastatin in t(4;14)-positive or negative MM cell lines mined from the CTRPv2 database. Percent area above the drug dose-response curve (% AAC) values are plotted as a box plot with whiskers representing minimum and maximum values. A higher % AAC indicates greater drug sensitivity (unpaired, two-tailed Wilcoxon rank-sum test comparing t(4;14)-positive and negative MM cell lines). f Primary plasma cells from t(4;14)-positive or negative patients were cultured in the presence of 5 μM fluvastatin or ethanol as a solvent control. After 72 h, cells were labeled with PE-conjugated anti-CD138 and FITC-conjugated Annexin V, and then cells were analyzed by flow cytometry. The percent change in primary CD138+ cell viability is plotted. The data are represented as the mean ± SD, p value = 0.14 (unpaired, two-tailed Wilcoxon rank-sum test).

Fig. 2. Fluvastatin induces the integrated stress response (ISR) in t(4;14)-positive MM cells.

H929 and KMS11 cells were treated with 2 μM fluvastatin or ethanol as a solvent control for 24 h, after which RNA was isolated and RNA-seq was performed. a Gene set enrichment analysis of ATF4 target genes in a gene list of fluvastatin-perturbed genes, ranked by fold change between fluvastatin-treated and ethanol-treated cells. b H929 and KMS11 cells were treated with solvent controls, 4 μM fluvastatin or 0.5 μg/mL tunicamycin for 24 h, after which protein was isolated and immunoblotting was performed to evaluate phosphorylated eIF2α, ATF4, and ATF3 expression. c t(4;14)-positive cells (H929, KMS11, OPM2, and LP1; in blue) and t(4;14)-negative cells (JJN3, SKMM1, U266, and EJM; in orange) were treated with ethanol control (0 h fluvastatin) or 4 μM fluvastatin for 16 or 24 h, after which RNA was isolated for qRT-PCR. The ATF4 target genes CHOP and GADD34 were evaluated and expression was normalized to RPL13A. The data are represented as the mean + SD, n = 3, *p < 0.05 (one-way ANOVA with Bonferroni’s multiple comparisons test, where each group was compared to the ethanol control).

Fig. 3. Fluvastatin-induced apoptosis and ISR activation can be rescued by exogenous GGPP.

a t(4;14)-positive H929 or KMS11 cells were treated with 4 μM fluvastatin ± 2 μM GGPP or 10 μM FPP for 48 h, and apoptosis was determined by Annexin V staining. The data are represented as the mean + SD, n = 3, *p < 0.05 (one-way ANOVA with Bonferroni’s multiple comparisons test, where each group was compared to the solvent controls group). b H929 or KMS11 cells were treated with 4 μM fluvastatin ± 2 μM GGPP or 10 μM FPP for 24 h, after which b protein was isolated and immunoblotting was performed to evaluate ATF4 expression or c RNA was isolated for qRT-PCR. The ATF4 target genes CHOP and GADD34 were evaluated and expression was normalized to RPL13A. The data are represented as the mean + SD, n = 3, *p < 0.05 (one-way ANOVA with Bonferroni’s multiple comparisons test, where each group was compared to the solvent controls group). d H929, KMS11, U266, and EJM cells were treated with solvent controls, 4 μM fluvastatin, 10 μM GGTI-298, or 20 μM FTI-277 for 24 h, after which RNA was isolated for qRT-PCR. The ATF4 target genes CHOP and GADD34 were evaluated and expression was normalized to RPL13A. The data are represented as the mean + SD, n = 3, *p < 0.05 (one-way ANOVA with Bonferroni’s multiple comparisons test, where each group was compared to the solvent controls group).

Fig. 4. Fluvastatin and bortezomib synergize to induce t(4;14)-positive MM cell death.

a H929 and b EJM cells were treated with fluvastatin (ranging from 0 to 4 μM for H929 and 0 to 8 μM for EJM) or bortezomib (ranging from 0 to 5 nM for both cell lines) as single agents or in combination for 48 h. The dose-response matrices, as determined by live-cell imaging and quantification of % dead cells, are representative of three independent experiments and depict the mean % dead cells ± SD. The mean % dead cell values for each dose combination were used to compute Bliss synergy scores for each cell line. Synergy plots for c H929 and d EJM cells are shown, where red represents synergy and green represents antagonism. e The Bliss synergy scores from the three independent experiments are plotted for H929 and EJM cells. The data are represented as the mean ± SD. The mean Bliss synergy score and 95% confidence interval (CI) of the mean for each cell line are reported in the table.

Fig. 5. Fluvastatin and bortezomib cooperate to induce the ISR and cell death in t(4;14)-positive MM cells.

a H929, b LP1, or c EJM cells were treated with solvent controls, fluvastatin or bortezomib (BTZ) at the indicated concentrations for 48 h, and apoptosis was determined by Annexin V staining. The data are represented as the mean + SD, n = 3–4, *p < 0.05 (one-way ANOVA with Tukey’s multiple comparisons test, or Kruskal–Wallis test with Dunn’s multiple comparisons test (a), where each group was compared to the solvent controls group), ^#p < 0.05 (one-way ANOVA with Tukey’s multiple comparisons test, comparing the two indicated groups). d H929, e LP1, or f EJM cells treated with solvent controls, fluvastatin, or BTZ at the indicated concentrations for 24 h, and RNA was isolated for qRT-PCR. The ATF4 target genes CHOP and GADD34 were evaluated and expression was normalized to RPL13A. The data are represented as the mean + SD, n = 3, *p < 0.05 (one-way ANOVA with Tukey’s multiple comparisons test, where each group was compared to the solvent controls group), ^#p < 0.05 (one-way ANOVA with Tukey’s multiple comparisons test, comparing the two indicated groups).

Fig. 6. Fluvastatin potentiates bortezomib activity in a t(4;14)-positive tumor model.

a NOD/SCID mice were injected with 5 million H929 cells subcutaneously (s.c.) in the flank. Once tumor volumes reached ~500 mm³, the mice were randomized to receive either bortezomib in combination with phosphate-buffered saline (PBS; vehicle control) or 50 mg/kg fluvastatin. Bortezomib was delivered twice per week at 1 mg/kg via intraperitoneal (i.p.) injection, up to a total of three doses. Fluvastatin was resuspended in PBS and delivered daily via oral gavage (per os; p.o.). Tumor measurement assessments were not blinded. b Percent change in tumor growth over time. The data are represented as the mean + SD, n = 5 mice per treatment group. *p < 0.05 (unpaired, two-tailed Wilcoxon rank-sum test). c Percent change in mouse body weight over the course of treatment. d Schematic diagram detailing the potential for statins to be used in combination with bortezomib for the personalized treatment of t(4;14)-positive MM.