(P 301) REDUCING THE INVASIVE POTENTIAL OF DEDIFFERENTIATED LIPOSARCOMA BY TARGETING ALTERNATIVE RNA SPLICING MACHINERY

Objective: Surgical resection is the only curative treatment option for patients with dedifferentiated liposarcoma (ddLPS). However, surgery is often precluded by local invasion of adjacent vital structures or organs. While cytotoxic chemotherapy has been the mainstay of treatment for patients with advanced ddLPS for more than 30 years, response rates are only 25%, and the overall survival at 5 years is 20-30%.
IWS1 is a transcription elongation and RNA processing factor that is activated by phosphorylation of AKT. The mechanism underlying how IWS1 promotes tumor progression is not well understood. Our recent findings demonstrate that IWS1 expression is associated with worse survival and a shorter time to relapse in patients with ddLPS. Moreso, knocking down IWS1 significantly reduced anchorage-independent growth, migration, and invasion in vitro, as well as reduced the number of tumor metastases in preclinical models of human ddLPS. In this study, we present novel IWS1 inhibitors and provide a rationale for targeting IWS1 pharmacologically as a potential treatment for preventing tumor invasion and metastases in patients with ddLPS.

Methods: To develop an IWS1 inhibitor, we focused on the IWS1/Spt6 interface, as this interaction has been well-characterized and shown to be biologically relevant in literature. Utilizing computer-aided, structure-based virtual screening approaches, we identified a class of compounds that exhibited inhibitory potential. A subset of FDA-approved drugs was evaluated by molecular docking calculations, and compounds with the highest calculated theoretical potency were selected for further analysis.
Lipo863 and Lipo815 cell lines were used as in vitro ddLPS models. To assess whether specific compounds could disrupt IWS1/Spt6 binding, Co-Immunoprecipitation (Co-IP) experiments were conducted. Fraction analysis was performed to examine the effect of a drug candidate on the cellular distribution of IWS1. Finally, to evaluate the impact of IWS1 inhibition on spheroid formation, a matrigel extracellular matrix scaffold was employed.

Results: Through virtual screening, we identified a class of compounds characterized by a specific chemical pattern featuring a tricyclic fused-ring structure (Fig 1). Co-IP of IWS1/Spt6 complex after 48-hour treatment with these drugs showed a decrease of over 50% of complex formation (Fig 2).
Migration of the ddLPS cell line, Lipo863, was reduced by 29% and 18% when treated with Desloratadine or Ketotifen respectively with no effect on cell viability. Moreover, this effect was dose-dependent: 1-10 uM solutions of Ketotifen decreased migration ability by 18-44%, 0.4-4 uM Desloratadine by 34-53% (Fig 3). In addition, the ratio between nuclear and cytosolic fractions of IWS1 increased by 1.1 for 20 uM Ketotifen and by 3.3 for 5 uM Desloratadine compared to DMSO with a ratio of 0.75. In a 3D model, a 14-day treatment with drugs dramatically reduced spheroid formation, confirming the impact on tumor growth and cell proliferation (Fig 4).

Conclusion: From the identified class of potential IWS1 inhibitors, two drugs effectively disrupted the IWS1/Spt6 complex, leading to a compensatory increase in IWS1 expression. Inhibition of IWS1 reduced the migratory potential in vitro and tumor growth in a 3D model. The dose-dependent effect observed for these compounds suggests that there may be an optimal in vivo concentration to reduce cells from invading nearby structures and/or metastasizing. Targeting IWS1 holds potential benefits for preventing recurrence and/or metastases of ddLPS, but in vivo experiments are needed to establish the efficacy of an IWS1 inhibitor. Based on these outcomes, we are currently conducting a follow-up virtual screening to identify additional compounds with greater potency.