# FL496, an FL118-derived small molecule, induces growth inhibition, senescence, and apoptosis of malignant pleural mesothelioma (MPM) cells, and exhibits anti-MPM tumor efficacy strikingly superior to the pemetrexed-cisplatin combination

## TL;DR

FL496, a new drug derived from FL118, shows strong anti-cancer effects in treating malignant pleural mesothelioma (MPM) and is more effective than current standard treatments.

## Contribution

FL496 is a novel FL118-derived small molecule that demonstrates superior anti-MPM tumor efficacy compared to the pemetrexed-cisplatin combination.

## Key findings

- FL496 induces growth inhibition, senescence, and apoptosis in MPM cells.
- FL496 is more effective in inhibiting MPM tumor growth than the pemetrexed-cisplatin combination.
- FL496 reduces the expression of anti-apoptotic proteins and increases pro-apoptotic markers in MPM cells.

## Abstract

Malignant pleural mesothelioma (MPM) responds poorly to chemotherapy and is a highly progressive malignancy with a median survival time of only 6–9 months. Therefore, the development of anti-MPM tumor agents with high efficacy and low toxicity is urgent and addresses an unmet need for MPM patients.

Medicinal chemistry synthesis of small molecules based on the FL118 drug platform were further comparatively investigated using multiple MPM and osteosarcoma cell/tumor in vitro and/or in vivo models. The method includes cell viability assay, Western blot analysis, colony formation assay, immunocytochemical staining, β-galactosidase senescence staining, flow cytometry, DNA fragmentation cell death detection, vector-free CRISPR-Cas9-mediated gene knockout, bioinformatic analysis, FL496 efficacy determination using severe combined immunodeficiency (SCID) mice with human MPM tumor, and immunohistochemistry (IHC) analysis of MPM tumors.

Here, we report that we identified a novel FL118-derived small molecule (FL496). FL496 appears to be strikingly more effective in inhibiting MPM tumor growth in MPM tumor animal models than the currently most prevalent pemetrexed-cisplatin combination in the clinic. The treatment of MPM cells with FL496 rapidly induced p53 and p21 accumulation, and Rb and p-Rb inhibition, which were associated with MPM cell senescence and G1/G0 arrest and apoptosis. Knockout (KO) of the TP53/p53 gene decreased the ability of FL496 to inhibit MPM cell growth (i.e., increase FL496 IC50 values) and colony formation. FL496-treated MPM cells resulted in strong inhibition of the expression of survivin, Mcl-1, Bcl-2, Bcl-XL, and the induction of active caspase-3, cleaved PARP, and PUMA, which were further confirmed using MPM tumor tissues via IHC analysis. High survivin in MPM patients’ tumors is associated with poor patient survival. Similar to FL118, FL496 treatment reduces DDX5 expression in MPM cells, but FL496 is more potent than FL118 in inhibiting MPM cell growth. Therefore, the mechanism of action (MOA) of FL496 overlaps with, but is likely beyond the scope of FL118 MOA, which needs further investigation.

Together, these results indicate that FL496 is a promising anti-MPM small molecule, and its high anti-MPM potential is worthy of being further explored as a monotherapeutic agent to treat MPM patients in clinical trials.

The online version contains supplementary material available at 10.1186/s13046-025-03547-9.