(W1030-04-22) Combination Therapy of Geranylgeranyl Diphosphate Synthase Inhibitor and Fatty Acid Synthase Inhibitor in Multiple Myeloma

Purpose: Multiple myeloma (MM) is characterized by accumulation and proliferation of plasma cells in the bone marrow (BM). It is the second most common hematological malignancy and accounts for approximately 13% of all blood cancers. Geranylgeranyl diphosphate synthase (GGDPS) is a key enzyme in isoprenoid pathway that leads to Rab-mediated protein trafficking which is crucial for the proliferation of MM cells. Inhibiting the GGDPS by geranylgeranyl diphosphate synthase inhibitor (GGSI) causes the disruption of protein geranylgeranylation which hinders intracellular protein trafficking resulting in apoptosis. Similarly, fatty acid synthase (FASN) is a pivotal enzyme involved in neoplastic lipogenesis and has been regarded as an important factor for tumor growth and survival. Many FASN inhibitors have been studied in several types of cancers for their anticancer effects. Multiple myeloma remains an incurable malignancy despite significant therapeutic advances. Most new cancer therapies comprise of a combination of two or more anti-cancer agents. Moreover, both FASN and GGDPS are overexpressed in MM cells making them excellent targets for combination studies for MM treatment. The main rationale for this study is to improve therapeutic anti-cancer benefits by simultaneously targeting two crucial pathways. In the past, it has been demonstrated that a GGSI (RAM2061) 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 effect between the two promising pathways and increase anti-MM efficacy

Methods: The cellular toxicity of FASN inhibitors (orlistat, TVB 3166, TVB 2640 and cerulenin) was evaluated in multiple myeloma 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 and 72 hours (Figure 1). Then, cell viability was assessed, and synergy/antagonism effect were analyzed using the freely available software, Combenefit. Lastly, combination indices (CI) were calculated by CompuSyn software.

Results: 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 (Figure 2). Combination of RAM 2061 and FASN inhibitors exhibited either synergistic or additive effect at 48 hr in both cell lines, which was influenced by the timing of drug exposure. Based on the synergy score and CI, treatment of RAM 2061 prior to orlistat and cerulenin was preferred, while simultaneous treatment was more effective for TVB 3166 and TVB 2640 on RPMI 8226 cells (table 1). No such pattern was observed for MM.1S cell line.

Conclusion: The combination of fatty acid synthase inhibitor and RAM2061 show potential to become a new therapeutic approach for the treatment of MM due to the synergistic or additive effects. Moreover, apoptosis markers and mechanism of apoptosis induced by FASN inhibitors in combination with RAM 2061 will be investigated to fully define the underlying mechanism of FASN inhibitors 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.

Acknowledgements:Acknowledgement: Department of pharmaceutical sciences, University of Nebraska Medical Center

Funding: National Institute of Health: R01 CA258621

Figure 1: Experimental design of (A) sequential and (A) con-current treatment for determination of synergy score and combination index using CCK-8 assay .

Figure 2: IC50 values determined by CCK-8 assay on 24hr, 48hr and 72 hours. A) RPMI8226 cells B) MM.1S cells

Table 1: Representative Combination Index (CI) of RPMI 8226 cells treated with RAM 2061 and FASN inhibitors for 48 hours. A) sequential treatment B) concurrent treatment