Targeting Cks1b in Multiple Myeloma Using Fluoxetine

Introduction

An initiating event in multiple myeloma development is malignant plasma cells re-entering the cell cycle. Cyclin kinase subunit 1b (Cks1b) is a cell cycle regulator and is over-expressed in a large proportion of myeloma. Over-expression is induced by various mechanisms including gains of chromosome 1q where Cks1b is located. Cks1b is known to form a complex with Skp2 to mediate p27 degradation and promote entry into S phase. Cks1b over-expression is associated with increased proliferation, increased risk of relapse, and drug resistance. Fluoxetine hydroxide (Prozac), a commonly prescribed antidepressant, has been identified as a potential Cks1b inhibitor and may have potential value in being repurposed as a therapy to target cycling multiple myeloma cells, including those patients with 1q gains who have a poor prognosis.

Materials and Methods

The human myeloma cell lines (HMCLs) used in this study were JJN3, U266 and KMS34, harbouring 6-8, 3 and 2 copies of 1q respectively. The levels of Cks1b protein correlated to the 1q copy number. Primary myeloma samples were also tested; all of these had normal 1q status. Cell viability following fluoxetine treatment was assessed using Alamar Blue (HMCLs) and CellTitre-Glo (primary samples). Cumulative growth was measured using Trypan blue exclusion counting. Colony forming unit assays were established using MethoCult H4100 (HMCLs) or H4534 (primary cells). The cell cycle was assessed using a combination of BrdU, 7AAD and PI flow cytometry; primary samples were CD138-selected. In situ proximity ligation assays (PLA) examined Cks1b-Skp2 interactions. Flow cytometry and Western blotting were used to measure p27. Cks1b knockdown was performed using siRNA introduced to cells via Nucleofection.

Results

Fluoxetine has a cytostatic and cytotoxic effect on HMCLs, regardless of 1q status, reducing cell viability and inhibiting colony growth. JJN3 cells were the most sensitive, with a dose of 10uM reducing viability in suspension cultures by 20% and precluding colony formation. 1uM and 5uM dosed daily over 5 days were highly effective at increasing cell doubling times in the cell lines (P<0.005). In contrast, stromal cell lines MS-5 and HS-5 were minimally affected by doses up to 50uM.

The mechanism by which fluoxetine exhibits an anti-myeloma effect was discerned in JJN3 cells. We have demonstrated that fluoxetine inhibits Cks1b-Skp2 complex assembly by binding selectively to the Skp2-binding region of Cks1b, preventing the interaction and resulting in significantly reduced Cks1b-Skp2 PLA foci (P<0.05). This leads to cell cycle arrest at the G1-S transition with accumulation of cells in G1 (P=0.005), and a subsequent 1.5-fold increase in p27. These results were replicated using siRNA-mediated knockdown of Cks1b, further confirming Cks1b as a target of fluoxetine.

Clinically relevant doses of fluoxetine (≤5uM), chosen to match a blood serum concentration equivalent to a patient taking a daily 80mg dose, were tested on CD138+ bone marrow derived primary cells. A concentration of 2uM reduced cell viability by 25-30%, increased cell death (P<0.0001), and reduced colony growth. Fluoxetine also induced a 1.4-fold decrease in S phase cells (P=0.0014), indicative of slowed cycling. Matched patient CD138- mononuclear cells were not sensitive (<1% cytotoxicity), suggesting that the cytotoxic effect of fluoxetine is myeloma cell-specific.

Conclusion

Fluoxetine has cytotoxic and cytostatic effects in myeloma cells, independent of 1q status. Importantly, it spares non-myeloma CD138- cells. It is a well-tolerated drug with a highly characterised safety profile. Fluoxetine is therefore worthy of further investigation and may have promising benefits for all patients, including those with 1q gains who have a poor prognosis. Our results support the concept of incorporating fluoxetine into current myeloma treatment regimens, with the aim of delaying disease progression and extending patient remission durations.

Smith:Abbvie, J&J, Takeda: Consultancy; BMS, Sanofi: Research Funding; J&J, Takeda, Menarini, BMS, Sanofi, Pfizer: Honoraria, Speakers Bureau.