Small-molecule PIK-93 modulates the tumor microenvironment to improve immune checkpoint blockade response.

Immune checkpoint inhibitors (ICIs) targeting PD-L1 immunotherapy are state-of-the-art treatments for advanced non-small cell lung cancer (NSCLC). However, the treatment response of certain patients with NSCLC is unsatisfactory because of an unfavorable tumor microenvironment (TME) and poor permeability of antibody-based ICIs. In this study, we aimed to discover small-molecule drugs that can modulate the TME to enhance ICI treatment efficacy in NSCLC in vitro and in vivo. We identified a PD-L1 protein-modulating small molecule, PIK-93, using a cell-based global protein stability (GPS) screening system. PIK-93 mediated PD-L1 ubiquitination by enhancing the PD-L1-Cullin-4A interaction. PIK-93 reduced PD-L1 levels on M1 macrophages and enhanced M1 antitumor cytotoxicity. Combined PIK-93 and anti-PD-L1 antibody treatment enhanced T cell activation, inhibited tumor growth, and increased tumor-infiltrating lymphocyte (TIL) recruitment in syngeneic and human peripheral blood mononuclear cell (PBMC) line-derived xenograft mouse models. PIK-93 facilitates a treatment-favorable TME when combined with anti-PD-L1 antibodies, thereby enhancing PD-1/PD-L1 blockade cancer immunotherapy.

Vorinostat combined with brigatinib overcomes acquired resistance in EGFR-C797S-mutated lung cancer.

The development of a new generation of tyrosine kinase inhibitors (TKIs) has improved the treatment response in lung adenocarcinomas. However, acquired resistance often occurs due to new epidermal growth factor receptor (EGFR) mutations. In particular, the C797S mutation confers drug resistance to T790M-targeting EGFR TKIs. To address C797S resistance, a promising therapeutic avenue is combination therapy that targets both total EGFR and acquired mutations to increase drug efficacy. We showed that combining vorinostat, a histone deacetylase inhibitor (HDACi), with brigatinib, a TKI, enhanced antitumor effects in primary culture and cell lines of lung adenocarcinomas harboring EGFR L858R/T790M/C797S mutations (EGFR-3M). While EGFR phosphorylation was decreased by brigatinib, vorinostat reduced total EGFR-3M (L858R/T790M/C797S) proteins through STUB1-mediated ubiquitination and degradation. STUB1 preferably ubiquitinated other EGFR mutants and facilitated protein turnover compared to EGFR-WT. The association between EGFR and STUB1 required the functional chaperone-binding domain of STUB1 and was further enhanced by vorinostat. Finally, STUB1 levels modulated EGFR downstream functions. Low STUB1 expression was associated with significantly poorer overall survival than high STUB1 expression in patients harboring mutant EGFR. Vorinostat combined with brigatinib significantly improved EGFR-TKI sensitivity to EGFR C797S by inducing EGFR-dependent cell death and may be a promising therapy in treating C797S-resistant lung adenocarcinomas.

Antimetabolite pemetrexed primes a favorable tumor microenvironment for immune checkpoint blockade therapy.

BACKGROUND: The immune checkpoint blockade (ICB) targeting programmed cell death-1 (PD-1) and its ligand (PD-L1) has been proved beneficial for numerous types of cancers, including non-small-cell lung cancer (NSCLC). However, a significant number of patients with NSCLC still fail to respond to ICB due to unfavorable tumor microenvironment. To improve the efficacy, the immune-chemotherapy combination with pemetrexed, cis/carboplatin and pembrolizumab (anti-PD-1) has been recently approved as first-line treatment in advanced NSCLCs. While chemotherapeutic agents exert beneficial effects, the underlying antitumor mechanism(s) remains unclear. METHODS: Pemetrexed, cisplatin and other chemotherapeutic agents were tested for the potential to induce PD-L1 expression in NSCLC cells by immunoblotting and flow cytometry. The ability to prime the tumor immune microenvironment was then determined by NSCLC/T cell coculture systems and syngeneic mouse models. Subpopulations of NSCLC cells responding differently to pemetrexed were selected and subjected to RNA-sequencing analysis. The key signaling pathways were identified and validated in vitro and in vivo. RESULTS: Pemetrexed induced the transcriptional activation of PD-L1 (encoded by CD274) by inactivating thymidylate synthase (TS) in NSCLC cells and, in turn, activating T-lymphocytes when combined with the anti-PD-1/PD-L1 therapy. Nuclear factor κB (NF-κB) signaling was activated by intracellular reactive oxygen species (ROSs) that were elevated by pemetrexed-mediated TS inactivation. The TS-ROS-NF-κB regulatory axis actively involves in pemetrexed-induced PD-L1 upregulation, whereas when pemetrexed fails to induce PD-L1 expression in NSCLC cells, NF-κB signaling is unregulated. In syngeneic mouse models, the combinatory treatment of pemetrexed with anti-PD-L1 antibody created a more favorable tumor microenvironment for the inhibition of tumor growth. CONCLUSIONS: Our findings reveal novel mechanisms showing that pemetrexed upregulates PD-L1 expression and primes a favorable microenvironment for ICB, which provides a mechanistic basis for the combinatory chemoimmunotherapy in NSCLC treatment.

Monocarboxylate Transporter 4 Is a Therapeutic Target in Non-small Cell Lung Cancer with Aerobic Glycolysis Preference.

Targeting metabolic reprogramming is an emerging strategy in cancer therapy. However, clinical attempts to target metabolic reprogramming have been proved to be challenging, with metabolic heterogeneity of cancer being one of many reasons that causes treatment failure. Here, we stratified non-small cell lung cancer (NSCLC) cells, mainly lung adenocarcinoma, based on their metabolic phenotypes and demonstrated that the aerobic glycolysis-preference NSCLC cell subtype was resistant to the OXPHOS-targeting inhibitors. We identified that monocarboxylate transporter 4 (MCT4), a lactate transporter, was highly expressed in the aerobic glycolysis-preference subtype with function supporting the proliferation of these cells. Glucose could induce the expression of MCT4 in these cells through a ΔNp63α and Sp1-dependent pathway. Next, we showed that knockdown of MCT4 increased intracellular lactate concentration and induced a reactive oxygen species (ROS)-dependent cellular apoptosis in the aerobic glycolysis-preference NSCLC cell subtype. By scanning a panel of monoclonal antibodies with MCT4 neutralizing activity, we further identified a MCT4 immunoglobulin M (IgM) monoclonal antibody showing capable anti-proliferation efficacy on the aerobic glycolysis-preference NSCLC cell subtype. Our findings indicate that the metabolic heterogeneity is a critical factor for NSCLC therapy and manipulating the expression or function of MCT4 can be an effective strategy in targeting the aerobic glycolysis-preference NSCLC cell subtype.

4(1H)-quinolone derivatives overcome acquired resistance to anti-microtubule agents by targeting the colchicine site of ß-tubulin.

Developing new therapeutic strategies to overcome drug resistance of cancer cells is an ongoing endeavor. From among 2 million chemicals, we identified ethyl 4-oxo-2-phenyl-1,4-dihydroquinoline-6-carboxylate (AS1712) as a low-toxicity inhibitor of lung cancer cell proliferation and xenograft tumor growth. We show that AS1712 is active against broad cancer cell lines and is able to bind in the colchicine-binding pocket of ß-tubulin, thereby inhibiting microtubule assembly and, consequently, inducing mitotic arrest and apoptosis. Our cell-based structure-activity relationship study identified a new lead compound, RJ-LC-15-8, which had a greater anti-proliferative potency for H1975 cells than did AS1712, while maintaining a similar mechanism of action. Notably, AS1712 and RJ-LC-15-8 overcame P-glycoprotein efflux pump and ß-tubulin alterations that lead to acquired resistance against microtubule-targeting drugs of cancer cells. AS1712 and RJ-LC-15-8 may be lead compounds that overcome acquired resistance to microtubule-targeting agents of cancer cells.

Involvement of the antioxidative property of morusin in blocking phorbol ester-induced malignant transformation of JB6 P+ mouse epidermal cells.

Chemoprevention has been acknowledged as an important and practical strategy for managing cancer. We have previously synthesized morusin, a prenylated flavonoid that exhibits anti-cancer progression activity. In the present study, we evaluated the anti-cancer promotion potential of morusin by using the mouse epidermal JB6 P+ cell model. Extensive evidence shows that tumor promotion by phorbol esters is due to the stimulation of reactive oxygen species (ROS). Therefore, the effect of morusin on 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced ROS production was assessed. Noncytotoxic concentrations of morusin were found to dose-dependently reduce TPA-induced ROS production. Moreover, morusin inhibited TPA-induced activator protein-1 (AP-1) and nuclear factor-kappa B (NF-κB) activation, which can mediate cell proliferation and malignant transformation. Furthermore, morusin inhibited the TPA upregulation of cyclooxygenase 2 (COX-2), which may be regulated by AP-1 and NF-κB. In addition, noncytotoxic concentrations of morusin reduced the TPA-promoted cell growth of JB6 P+ cells and inhibited TPA-induced malignant properties, such as cytoskeletal rearrangement and cell migration of JB6 P+ cells. Similar to the effects of glutathione (GSH) pretreatment, morusin inhibited TPA-induced expression of N-cadeherin and vimentin, which are malignant cell surface proteins. Finally, morusin treatment dose-dependently suppressed the TPA-induced anchorage-independent cell transformation of JB6 P+ cells. In conclusion, our results evidence that morusin possesses anti-cancer promotion potential because of its antioxidant property, which mediates multiple transformation-associated gene expression.