Prostate Cancer Cell Function Assessments Reveal Differences Between Gedatolisib and Single-Node PI3K/AKT/mTOR Inhibitors

Academic Background

Prostate cancer (PC) is one of the most common cancers among men worldwide, particularly in advanced stages where patients often develop resistance to androgen deprivation therapy (ADT), progressing to castration-resistant prostate cancer (CRPC). The progression of CRPC is typically accompanied by the inactivation of the androgen receptor (AR) signaling pathway and the abnormal activation of the PI3K/AKT/mTOR (PAM) signaling pathway. The PAM pathway plays a critical role in cell growth, metabolism, and survival, and its abnormal activation is closely related to the progression and treatment resistance of prostate cancer.

In recent years, inhibitors targeting the PAM pathway have emerged as a potential strategy for treating CRPC. However, single-node inhibitors (such as PI3Kα, AKT, or mTOR inhibitors) often have limited efficacy due to feedback loops or compensatory mechanisms. Therefore, researchers have proposed a new strategy: using multi-node inhibitors, such as Gedatolisib, to simultaneously inhibit PI3K and mTORC1/mTORC2, with the aim of overcoming the limitations of single-node inhibitors.

Source of the Paper

This paper was authored by Adrish Sen, Salmaan Khan, Stefano Rossetti, and others from Celcuity, Inc., and published in the journal Molecular Oncology on July 18, 2024, with the DOI 10.¹⁰⁰²⁄₁₈₇₈-0261.13703. The research team evaluated the effects of Gedatolisib and other single-node PAM inhibitors in prostate cancer cells through a series of functional and metabolic experiments.

Research Process and Results

1. Research Process

a) Cell Culture and Treatment

The research team used seven prostate cancer cell lines with different PTEN/PIK3CA statuses (such as 22Rv1, DU145, LNCaP, etc.), representing various PAM pathway mutation states and androgen sensitivities. The cells were cultured in medium containing 10-20% fetal bovine serum (FBS) and maintained at 37°C with 5% CO2.

b) Drug Treatment and Cell Viability Assessment

Cells were exposed to Gedatolisib (a multi-node PAM inhibitor) and four single-node PAM inhibitors (Alpelisib, Capivasertib, Everolimus, and Samotolisib) for 72 hours. Cell viability was assessed using the RT-Glo MT luciferase assay, and the anti-proliferative and cytotoxic effects of the drugs were analyzed using the Growth Rate (GR) metric.

c) Cell Cycle and DNA Replication Analysis

Cell cycle distribution and DNA replication were analyzed using flow cytometry. Cells were labeled with 5-ethynyl-2’-deoxyuridine (Edu) and FXCycle Violet dye to assess the effects of the drugs on the cell cycle.

d) Cell Death and Apoptosis Analysis

Cell death and apoptosis were detected using Zombie dye and cleaved caspase 3 antibody through flow cytometry.

e) PAM Pathway Activity and Protein Synthesis Analysis

The activity of the PAM pathway was assessed by detecting phosphorylated 4EBP1 (p4EBP1) and phosphorylated RPS6 (pRPS6) levels. Additionally, newly synthesized proteins were labeled with O-propargyl-puromycin (OPP) to evaluate the effects of the drugs on protein synthesis.

f) Metabolic Function Analysis

The effects of the drugs on cell metabolism were assessed by measuring lactate production and oxygen consumption rate (OCR). Lactate levels were measured using the Biosen R-line instrument, and OCR was monitored in real-time using the Resipher instrument.

2. Main Results

a) Cell Viability and Growth Rate Analysis

Gedatolisib demonstrated stronger anti-proliferative and cytotoxic effects in all tested cell lines, with a GR50 (concentration required for 50% growth inhibition) range of 6-17 nM, significantly lower than that of other single-node inhibitors. The effect was particularly notable in PTEN-deficient cell lines.

b) Cell Cycle and DNA Replication

Gedatolisib significantly inhibited cell cycle progression, particularly in the G0/G1 and S phases. In 22Rv1 and LNCaP cells, Gedatolisib increased the proportion of G0/G1 phase cells to 73% and 84%, respectively, at a concentration of 1000 nM, while reducing the proportion of S phase cells to 2% and 0%.

c) Cell Death and Apoptosis

Gedatolisib significantly induced cell death and apoptosis within 48 hours, particularly in PTEN-deficient LNCaP cells, where the cell death rate reached 77%. In contrast, the effects of single-node inhibitors were weaker.

d) PAM Pathway Activity and Protein Synthesis

Gedatolisib significantly inhibited the levels of p4EBP1 and pRPS6, indicating strong suppression of the PAM pathway. Additionally, Gedatolisib significantly reduced the synthesis of new proteins, outperforming other inhibitors.

e) Metabolic Function

Gedatolisib significantly reduced lactate production and oxygen consumption rate, indicating its potent regulation of cell metabolism. The effect was particularly pronounced in DU145 and PC3 cells, which have high lactate production, where Gedatolisib outperformed single-node inhibitors.

3. Conclusions and Significance

The results demonstrate that Gedatolisib, as a multi-node PAM inhibitor, can more effectively inhibit the proliferation, survival, and metabolic functions of prostate cancer cells, with its efficacy unaffected by PTEN status. This finding provides new insights for the treatment of CRPC, particularly for patients who have developed resistance to single-node inhibitors.

4. Research Highlights

• Advantages of Multi-Node Inhibition: Gedatolisib overcomes the feedback loop issues of single-node inhibitors by simultaneously inhibiting PI3K and mTORC1/mTORC2.
• Broad Applicability: Gedatolisib exhibits potent anti-cancer effects in both PTEN-positive and PTEN-negative cells, indicating its suitability for a broader patient population.
• Metabolic Regulation: Gedatolisib significantly inhibits the metabolic functions of cancer cells, further enhancing its anti-cancer efficacy.

5. Other Valuable Information

Gedatolisib is currently undergoing multiple clinical trials, including a Phase I/II trial in combination with Darolutamide for the treatment of CRPC, and a Phase III trial in combination with Palbociclib and Fulvestrant for the treatment of HR+/HER2- advanced breast cancer. Preliminary results from these trials have shown promising efficacy and safety profiles for Gedatolisib.

Summary

This study not only reveals the potential of Gedatolisib in prostate cancer treatment but also provides important experimental evidence for the development of more effective PAM pathway inhibitors. In the future, Gedatolisib is expected to become a key drug in the treatment of CRPC, particularly for patients who have developed resistance to existing therapies.