Targeting LSD1 inhibits gastric cancer by reprogramming macrophages and restoring T-cell response

Technical Summary: Targeting LSD1 Inhibits Gastric Cancer by Reprogramming Macrophages and Restoring T-cell Response

Problem Statement
Gastric cancer (GC) remains a leading cause of cancer mortality globally, with advanced-stage disease carrying a five-year survival rate of under 10%. While multimodal treatments including immunotherapy have improved outcomes, clinical benefits are often limited by late diagnosis, drug resistance, and low response rates to immune checkpoint inhibitors. A critical barrier is the tumor's ability to evade the immune system. Lysine-specific demethylase 1 (LSD1/KDM1A) is overexpressed in various malignancies and drives tumor progression, yet its specific role in shaping the GC tumor immune microenvironment (TIME) remains incompletely defined. Furthermore, existing LSD1 inhibitors, particularly phenylcyclopropylamine-based agents, face challenges regarding selectivity and toxicity, while reversible inhibitors often lack sufficient potency. There is a pressing need for novel, potent, and selective LSD1-targeting agents that can effectively reprogram the immune microenvironment to overcome GC immune evasion.

Methodology
The study employed a multi-faceted approach combining computational chemistry, molecular biology, and immunology:

1. Drug Discovery and Design: Starting from a quinazoline-based lead (Z-1), the researchers utilized bioisosteric replacement (substituting a ring nitrogen with carbon) to generate a quinoline scaffold. Systematic structure-activity relationship (SAR) studies were conducted on the R1, R2, R3, and R4 positions of the scaffold. Molecular docking simulations (using PDB 2V1D) guided the optimization of binding interactions with the LSD1 active site, specifically mimicking FAD binding.
2. Genetic Manipulation: LSD1 knockout (KO) mouse forestomach carcinoma (MFC) cells and human GC cell lines (AGS, MKN45) were generated using CRISPR-Cas9 to study the intrinsic effects of LSD1 loss.
3. Single-Cell RNA Sequencing (scRNA-seq): CD45+ tumor-infiltrating lymphocytes (TILs) from subcutaneous tumors in immunocompetent mice were profiled to characterize immune cell populations and their states (e.g., M1/M2 macrophages, exhausted CD8+ T cells) in the presence or absence of tumor-intrinsic LSD1.
4. In Vitro Co-culture Systems: Conditioned media (CM) from GC cells (WT vs. KO vs. drug-treated) was used to treat PMA-induced THP-1 macrophages and primary T cells. This assessed macrophage polarization (M0 to M1/M2) and T-cell activation/exhaustion markers (CD69, CD25, PD-1, LAG-3, TIM-3).
5. In Vivo Models: Syngeneic subcutaneous tumor models in C57BL/6 (615) mice were established. The study utilized selective depletion strategies using clodronate liposomes, PLX3397, or anti-CSF1R antibodies for macrophages, and anti-CD8α antibodies for T cells. The lead compound, KL-1, was administered via intragastric gavage to evaluate tumor growth inhibition and immune modulation.

Key Contributions

• Mechanistic Insight: The study identifies a novel mechanism where tumor-intrinsic LSD1 promotes immune evasion by orchestrating a crosstalk loop: it drives M0 macrophages toward a pro-tumor M2 phenotype, which in turn induces CD8+ T-cell exhaustion. Conversely, LSD1 loss skews macrophages toward an anti-tumor M1 phenotype and restores CD8+ T-cell function.
• Novel Inhibitor Development: The authors designed and synthesized a series of quinoline-based reversible LSD1 inhibitors. The lead compound, KL-1 (Compound 29), demonstrated high potency (IC50 = 18 nM) and a specific binding mode involving FAD-competitive interactions and hydrogen bonding with key residues (Glu801, Ser289, Gly287).
• Therapeutic Validation: The study validates that pharmacological inhibition of LSD1 with KL-1 recapitulates the genetic knockout phenotype, effectively suppressing GC growth by reprogramming the immune microenvironment.

Results

• Immune Reprogramming by LSD1 Loss: scRNA-seq of MFC tumors revealed that LSD1 KO tumors exhibited a significant increase in M1 macrophages and activated CD8+ T cells, alongside a reduction in M2 macrophages and exhausted CD8+ T cells (CD8+ Tex).
• Macrophage-T Cell Axis: In vitro experiments confirmed that CM from LSD1 KO cells drove M0 macrophages toward an M1 phenotype (high CD80/CD86, low CD206/CD163) and reduced M2 polarization. Crucially, this M1 skewing was required to prevent CD8+ T-cell exhaustion; co-culture with M2 macrophages induced by control cells led to high expression of exhaustion markers (LAG-3, PD-1, TIM-3), whereas M2 macrophages treated with CM from LSD1 KO cells failed to induce this exhaustion.
• KL-1 Efficacy: KL-1 treatment in GC cells reproduced the effects of genetic deletion, promoting M1 polarization and CD8+ T-cell activation while suppressing exhaustion markers.
• In Vivo Efficacy and Dependency: In syngeneic mouse models, KL-1 treatment significantly inhibited tumor growth. This efficacy was dependent on the immune system:
  • Depletion of macrophages partially reversed the anti-tumor effect of KL-1.
  • Depletion of CD8+ T cells completely abrogated the therapeutic efficacy of KL-1.
  • KL-1 treatment increased intratumoral M1 macrophages and functional CD8+ T cells while decreasing M2 macrophages.
  • Depletion of CD8+ T cells abolished the KL-1-induced shift in macrophage polarization (M1/M2 ratio), suggesting a reciprocal regulatory loop where T cells also influence macrophage phenotypes in this context.
• Safety: Histological analysis (H&E staining) of major organs from KL-1-treated mice showed no signs of in vivo toxicity.

Significance
The paper positions LSD1 as a tractable epigenetic node for immune reprogramming in gastric cancer. By elucidating the mechanism where LSD1 suppresses anti-tumor immunity via the M2 macrophage-mediated exhaustion of CD8+ T cells, the study provides a rationale for targeting LSD1 not just for direct tumor suppression but for immune modulation. The identification of KL-1 as a potent, reversible, and selective inhibitor that successfully engages this mechanism in vivo establishes it as a promising clinical candidate. The findings suggest that combining LSD1 inhibition with existing immunotherapies could overcome resistance in GC by restoring the cytotoxic potential of T cells through macrophage reprogramming. The authors acknowledge limitations, including the lack of patient-derived scRNA-seq data and the need for further causality studies using OT-I mice, but emphasize that their work offers a foundational understanding of the LSD1-M2-CD8+ T-cell axis in GC.