(T1330-04-22) Exploring the Synergistic Potential of Fatty Acid Synthase Inhibitors and Geranylgeranyl Diphosphate Synthase Inhibitors in Myeloma Therapy

Purpose: Multiple myeloma (MM) remains an incurable cancer despite significant therapeutic advances. It ranks as the second most prevalent hematological malignancy, constituting roughly 13% of all blood cancers. It is characterized by the accumulation and proliferation of plasma cells in the bone marrow. Recognizing the potential of combination therapy in MM, targeting the isoprenoid biosynthetic pathway and fatty acid synthesis pathway could be beneficial, given their impact on MM proliferation. Geranylgeranyl diphosphate synthase (GGDPS) plays a pivotal role in the isoprenoid biosynthetic pathway that helps in Rab-mediated protein trafficking. Inhibiting GGDPS by geranylgeranyl diphosphate synthase inhibitor (GGSI) disrupts protein geranylgeranylation, hindering intracellular protein trafficking and ultimately triggering apoptosis. Similarly, fatty acid synthase (FASN) is a key enzyme involved in neoplastic lipogenesis responsible for tumor growth and survival. Several FASN inhibitors have been investigated in different types of cancers for their anti-cancer properties. Moreover, both FASN and GGDPS are overexpressed in MM cells making them excellent targets for combination therapy for MM treatment. Therefore, the primary objective for this study is to improve therapeutic anti-cancer benefits by targeting two crucial pathways simultaneously. Previously, GGSI (RAM2061) had successfully disrupted Rab proteins leading to apoptosis of MM cells [1]. Here, we hypothesize that combining FASN inhibitors with RAM 2061 will allow synergistic or additive effects between the two promising pathways and increase anti-MM efficacy.

Methods: First, an immunofluorescence assay was conducted to visualize the localization of FASN expression within the MM cells (RPMI-8226 and MM.1S cells). The cellular toxicity of FASN inhibitors (orlistat, TVB 3166, TVB 2640 and cerulenin) was evaluated in MM cell lines (RPMI 8226 and MM1.S) at 24, 48, and 72 hours timepoints by CCK-8 assay. Further, combination study assays were conducted sequentially and in concurrent incubation of RAM 2061 with FASN inhibitors on MM cells for 48 hours. Then, cell viability was assessed, synergy/antagonism effect were analyzed, and combination indices (CI) were calculated by Chou-Talalay method [2]. In all cellular assays, we compared single-drug treatments (MM cells treated with either RAM 2061 or the FASN inhibitor) with combination (sequential and concurrent) treatments. In the sequential treatment groups, the FASN inhibitor was added to MM cells 24 hours after the initial treatment with RAM 2061 and left for an additional 24 hours before conducting the assays. Conversely, in the concurrent treatment group, the FASN inhibitor was added to MM cells simultaneously with RAM2061 and left for 48 hours before conducting the assays. To investigate the influence of timing of drug administration on induction of cell death, we assessed induction of apoptosis by flow cytometry using Annexin V and propidium iodide staining. Then, we further examined the effects of the combination treatment on apoptosis markers (cleaved PARP, cleaved caspases 3, 8, and 9) via immunoblot analysis. Lastly, to understand the effects of synergic effects on the combination treatment, we explored the UPR markers (eIF2α, p-eIF2α, IRE1α and ATF-4) by immunoblot analysis.

Results: To investigate the potential of targeting FASN for the treatment of MM, we determined whether FASN is expressed in MM cells. Both cell lines (RPMI 8226 and MM.1S) showed expression of FASN as demonstrated by double immunofluorescent staining for FASN (green) and nuclear staining, DAPI (blue) (Fig. 1). Furthermore, FASN inhibitors - orlistat, TVB 3166, TVB 2640, and cerulenin showed comparable cytotoxicity profiles for RPMI 8226 and MM.1S cell lines at 24, 48, and 72 hours with IC₅₀ in micromolar range (Fig. 2B). Combination of RAM 2061 and FASN inhibitors with higher potencies (orlistat and cerulenin) exhibited either synergistic or additive effect at 48 hours in both cell lines which was unaffected by the timing of drug exposure (Fig. 2C). However, when accessing the effects on the apoptosis markers, MM cells treated with RAM2061 followed by sequential treatment of FASN inhibitors displayed enhanced expression of apoptotic markers relative to single treatment group and concurrent treatment group (Fig. 3). Previously, RAM 2061 exhibited increased activation of UPR markers in MM cells [3]. Immunoblot analysis was performed to evaluate the impact of combination treatment on UPR markers. Interestingly, no enhancement of UPR marker expression was observed in the combination groups compared to those treated with a single drug, prompting further investigation into the underlying reasons for the observed synergy.

Conclusion: The combination of FASN inhibitor and RAM2061 shows potential to become a new therapeutic approach for the treatment of MM due to the synergistic or additive effects. Furthermore, a variety of key metabolic parameters and functions will be investigated to fully define the underlying mechanism of observed synergy in MM cells.

References: [1] R.A. Matthiesen, M.L. Varney, P.C. Xu, A.S. Rier, D.F. Wiemer, S.A. Holstein, α-Methylation enhances the potency of isoprenoid triazole bisphosphonates as geranylgeranyl diphosphate synthase inhibitors, Bioorg Med Chem, 26 (2018) 376-385.
[2] T.C. Chou, Drug combination studies and their synergy quantification using the Chou-Talalay method, Cancer Res, 70 (2010) 440-446.
[3] S.L. Haney, Y.S. Chhonker, M.L. Varney, G. Talmon, L.M. Smith, D.J. Murry, S.A. Holstein, In Vivo Evaluation of Isoprenoid Triazole Bisphosphonate Inhibitors of Geranylgeranyl Diphosphate Synthase: Impact of Olefin Stereochemistry on Toxicity and Biodistribution, Journal of Pharmacology and Experimental Therapeutics, 371 (2019) 327-338.


Figure 1 Immunofluorescent staining of MM cells for FASN (green), and 4′,6-diamidino-2-phenylindole (DAPI) (blue).


Figure 2 (A) Chemical structures of FASN inhibitors used for this study. (B) IC50 of FASN inhibitors on MM cell lines (RPMI 8226 and MM1.S for 24, 48, and 72 hrs. (C) Combination indices of combination treatment on RPMI 8226 and MM.1S cells. Combination indices < 1 are considered synergistic in nature.


Figure 3 Effects of combination treatment on the apoptosis markers (cleaved PARP, caspases 3, 8, and 9) via immunoblot analysis. (A) RPMI 8226 cells. (B) MM.1S cells