HDAC inhibition unlocks tumor plasticity and enhances immunotherapy response in Myc-Driven Small Cell Lung Cancer

Abstract

Small Cell Lung Cancer (SCLC) is a highly aggressive malignancy, accounting for approximately 15% of all lung cancer cases. Characterized by low immunogenicity, SCLC may utilize epigenetic mechanisms to evade immune detection. Here, we demonstrate that entinostat, a class I histone deacetylase inhibitor (HDACi) upregulates immune-related genes in human SCLC cells. In vivo, we confirmed entinostat treatment increased expression of immunecheckpoint ligands and antigen presentation machinery in Myc-driven tumors in a Rb1/Trp53/Myc^T58A (RPM) SCLC mouse model, while shifting tumors from a neuroendocrine(NE)-high to a NE-low phenotype. Notably, combining entinostat with anti-PD-1 immunotherapy significantly enhances T-cell infiltration, suppresses tumor growth, and prolongs survival in RPM allograft models. These findings underscore the potential of entinostat to reprogram the immunological landscape and NE status of SCLC, enhance immune checkpoint blockade efficacy, and improve therapeutic outcomes.

Keywords: anti-PD-1 therapy; entinostat; histone deacetylase inhibitor (HDACi); immunotherapy; neuroendocrine; small cell lung cancer (SCLC).

Figure 1.

PD-L1 and MHC genes are repressed in SCLC amongst solid malignancies. A. Gene expression levels of PD-L1, HLA-A, HLA-B and HLA-C in different cancer types. Data is from CBioPortal (https://www.cbioportal.org/). **** p < 0.0001. B. Correlation between H3K27ac ChIP-seq signal and gene expression of PD-L1 (CD274), HLA-A, HLA-B, HLA-C in SCLC cell lines. Plot is generated with the sclcCellMinerCDB (https://discover.nci.nih.gov/rsconnect/SclcCellMinerCDB/).

Figure 2.

HDAC inhibitor enthinostat regulates PD-L1 and MHC genes expression in SCLC cell lines. A. The relatve gene expression of D274 (PD-L1), HLA-A, HLA-B and HLA-C on SCLC cell lines H889, H209, H82, H524, and DMS-114 treated with increasing dose (0 uM, 0.2 uM, 1 uM and 5 uM) of entinostat for 24 hours. The relative gene expression was determined by quantitative real-time PCR assay. Error bars are representative of technical triplicates. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. B. Western blot to show the protein level of PD-L1, MHC-1, Acetylated histone 3 (target) and tubulin (control) on the above five SCLC cell lines treated with entinostat for 24 hours in increasing dose of 0uM, 0.2uM, 0.5uM and 1uM.

Figure 3.

Evaluation of immune cell population in SCLC RPM GEM tumor model. A. Rb1/Trp53/Myc^T58A (RPM) mice weer induced at 8 weeks of age with adenoviral Cgrp-Cre (2.5 × 10⁷ pfu). B. Survival of RPM mice was consistent over 2 separate cohorts. C. Immunohistochemistry staining (left) and automated quantification (right) of NCAM(CD56), PD-L1, MHC-II, CD3, CD4 and CD8. Scale bar is 10 uM (20x). Red star mark tumor regions. D. Gene expression heatmap of lungs from two uninduced mice and 4 RPM tumors on the indicated gene sets. Top of the heatmap is the NE score for each sample. E. Immune cell populations in normal and tumor groups analyzed with mMCP-counter. F. Example of the differential distribution of the indicated immune cell populations between normal and tumor samples from mMCP-counter analysis.

Figure 4.

Entinostat change the RPM tumors neuroendocrine status. A. Schematic of the entinostat and anti-PD-1 treatment regimen in RPM mice. PO: Oral gavage. IP: intraperitoneal. MRI: Magnetic resonance imaging. B. Survival curve of the control and treatment groups. P-value is not significant for any treatment compared to vehicle. C. Quantification of IHC staining of Ascl1, Neurod1 and Yap1 in each group of tumors. D. Neuroendocrine score of each sample in four groups. E. Heatmap of NE-associated genes on their expression level and ATAC-seq peak signal in these gene’s promoter region (2kb upstream and downstream of TSS) F. RNA-seq peaks and ATAC-seq peaks track signals on the NE genes.

Figure 5.

Entinostast regulates APM genes and MHC-II genes, enhancing T-cell immune response A. Heatmap of antigen presentation machinery genes on their expression level and ATAC-seq peak signal in these gene’s promoter region (2kb upstream and downstream of TSS) B. Heatmap of MHC-II genes on their expression level and ATAC-seq peak signal in these gene’s promoter region (2kb upstream and downstream of TSS) C. RNA-seq peaks and ATAC-seq peaks track signals on the APM genes and MHC-II genes. D. Comparison between each treatment groups and IgG for the immune cell infiltration differences with limma method. E. Gene Set Enrichment Analysis (GSEA) between ENT + anti-PD-1 and IgG group.

Figure 6.

Entinostat plus anti-PD-1 enhances T-cell infiltration and cytotoxicity in tumors. A. IHC staining of immune cell markers CD3, CD4 and CD8. Scale bar is uM (20x). red star marks tumor regions. B. Quantification of IHC staining on immune cell markers CD3, CD4, CD8 positive cells. C. Gene expression for the chemokine genes. TPM: Transcripts Per Million. D. Model for entinostat function in SCLC immune therapy. Left: SCLC in neuroendocrine-high status have NE-high associated genes highly expressed while MHC genes and APM genes repressed. Antigen processing is blocked. Anti-PD-1 response is limited. T-cell infiltration was minor and not active to kill tumor cell. right: entinostat can regulate key neuroendocrine genes and change NE-high to NE-low while activate MHC and APM genes, enable antigen processing and work together with anti-PD-1 to facilitate the activity of T-cells in killing tumor cells.

Figure 7.

Entinostat plus anti-PD-1 improves survival in the RPM allografts A. Scheme of SCLC RPM allograft mouse model. The GEM Rb1/Trp53/Myc^T58A (RPM) mice was intratracheal injected with viral Cre recombinase and latency after 2 – 3 months when it generates lung tumors. The tumors were transplanted subcutaneously to strain-matched and immunecompetent recipients. After 3 – 5 weeks, treatment commenced according to the study timeline. B. Tumor volume record of allograft mouse control and treatment groups. C. Survival curve of allograft mouse control and treatment groups.