Geranylgeranyl Diphosphate Synthase Inhibitor and Proteasome Inhibitor Combination Therapy in Multiple Myeloma

Introduction: Multiple myeloma (MM) is characterized by proliferation of malignant plasma cells that produce monoclonal protein (MP). MM cells, compared with non-secretory cells, have a lower threshold for induction of the pro-apoptotic components of the unfolded protein response pathway (UPR) as a consequence of near-maximal expression of the protective UPR elements. Proteasome inhibitors (PIs) are widely used in the treatment of MM and have been shown to disrupt the UPR. As an alternative strategy by which to target protein homeostasis in MM, we have focused on the development of geranylgeranyl diphosphate synthase (GGDPS) inhibitors. Inhibition of GGDPS results in disruption of Rab protein geranylgeranylation, which in turn results in disruption of MP trafficking, leading to accumulation of intracellular MP, induction of the UPR and apoptosis. We have previously reported preclinical studies with our lead GGDPS inhibitor (GGDPSi), RAM2061, which demonstrated the agent's metabolic stability, prolonged half-life (plasma elimination half-life of 29.2 (+6.0) hours (hrs)), systemic distribution, in vivo disruption of geranylgeranylation and anti-tumor efficacy in a mouse MM xenograft model (Haney et al., ASH 2018, abstract 215). We hypothesized that the combination of GGDPSi and PI therapy would result in enhanced disruption of the UPR and increase anti-MM efficacy.

Methods: MTT assays were conducted to evaluate the cytotoxic effects of combining RAM2061 with the PI bortezomib (Bor) in human MM cell lines (RPMI-8226, MM.1S) and isobologram analysis was performed. The effects of RAM2061 and/or Bor on markers of the UPR were evaluated via qRT-PCR (ATF4, ATF6, CHOP, PERK, IRE1) and immunoblot analysis (ATF4, IRE1, phosphorylated eIF2α). Apoptotic markers (cleaved caspases 3, 8 and 9 and cleaved PARP) were evaluated by immunoblot analysis. Cell assays were performed using both concurrent (both drugs added at the start of the 48 hr incubation period) and RAM2061 pre-treatment (RAM2061 added at the start, Bor added after 24 hrs) approaches. To evaluate the in vivo activity of the novel combination therapy, a flank xenograft model was used. NOD/SCID mice were subcutaneously inoculated in the flank with MM.1S cells. Once tumors became palpable, mice were randomly divided into four treatment groups (n=8 per group): control (PBS), RAM2061 (0.08 mg/kg IV two times/week (wk)), Bor (0.3 mg/kg SQ two times/wk) and RAM2061/Bor combination. Tumor volume (TV) and animal body weight were recorded three times/wk. Mice were euthanized when tumors reached 2000 mm³. Blood samples from day 9 post-initiation of treatment were analyzed via ELISA for human λ light chain. Blood samples collected at time of euthanasia were analyzed for hematologic, renal and hepatic parameters.

Results: Isobologram analysis revealed a primarily additive effect with concurrent GGDPSi/PI incubations and primarily synergistic effect with the RAM2061 pre-treatment approach. The effect of Bor on augmenting RAM2061-induced upregulation of UPR and apoptotic markers was both concentration- and time-dependent. While there was in general an enhanced induction of apoptosis with the combination, Bor partially abrogated the RAM2061-induced upregulation of UPR markers at the tested time points. Studies are ongoing to determine whether this effect is associated with an accelerated time course of UPR activation in the presence of Bor. Consistent with our previous in vivo studies, the primary toxicity associated with RAM2061 treatment was hepatic transaminase elevation. No additional toxicity was observed when Bor was combined with RAM2061 based on animal weight, hematological, renal or hepatic parameters. Tumor growth over time was significantly decreased in the RAM2061 (p=0.0009) and RAM2061/Bor (p=0.0002) treatment groups compared to control animals and this corresponded to prolonged time to sacrifice (p=0.09 for RAM2061 group and p=0.003 for RAM2061/Bor group). Consistent with TV, mean plasma human λ light chain levels were decreased in the RAM2061 and RAM2061/Bor treatment groups compared to the control mice (p<0.05).

Conclusion: These studies support further investigation into the therapeutic potential of GGDPSi/PI-based combination therapy for MM. Further studies are needed to dissect the mechanisms underlying the interactions between the two classes of drugs on the UPR and apoptotic pathways.