TNG260 Is a Small-Molecule CoREST Inhibitor That Sensitizes STK11-Mutant Tumors to Anti-PD-1 Immunotherapy

Abstract

Patients with non-small cell lung cancer (NSCLC) with loss of the tumor suppressor gene STK11 are resistant to immune checkpoint therapies like anti-PD-1. In this study, we conducted an in vivo CRISPR screen that identified histone deacetylase 1 as a target to reverse anti-PD-1 resistance driven by loss of STK11 and developed TNG260, a potent small-molecule inhibitor of the CoREST complex with selectivity exceeding previously generated inhibitors in this class in preclinical studies. Treatment with TNG260 led to increased expression of immunomodulatory genes in STK11-deficient cancer cells. When combined with anti-PD-1, TNG260 induced immune-mediated stasis and/or regression in STK11-deficient syngeneic tumor models and autochthonous NSCLC models. In the tumors of patients with STK11-deficient cancers in a clinical trial (NCT05887492), treatment with a combination of TNG260 and pembrolizumab increased intratumoral histone acetylation, PD-L1 tumor proportion scores, and T-cell infiltration into the tumor microenvironment. This study illustrates a promising treatment strategy for addressing immune evasion in patients with STK11-mutant NSCLC.

Significance: Targeting CoREST with TNG260 sensitizes STK11-deficient non-small cell lung cancer to anti-PD-1 immunotherapy, offering a potential treatment for patients not served by existing therapies. See related commentary by Lin and Shen, p. 3821.

Figure 1.

HDAC1 knockout resensitizes STK11-mutant tumors to immune checkpoint blockade. A, Average volumes of MC38_sgNTC tumors in the indicated mouse strain; anti–PD-1 antibody was administered biweekly (N = 8 mice/group). B, Average volume of MC38_sgStk11 tumors in the indicated mouse strain; anti–PD-1 antibody was administered biweekly (N = 8 mice/group). Values are mean ± SEM. One-way ANOVA was used to determine statistical significance. **, P < 0.01; ***, P < 0.001; ns, not significant. C, Scheme describing CRISPR screen using STK11 isogenic MC38 tumors. D, Volcano plot of sgRNAs comparing MC38_sgStk11 tumors in C57BL/6 mice treated with anti–PD-1 with MC38_sgStk11 tumors in athymic nude mice. The green point indicates average sgRNAs for HDAC1. E, Volcano plot of sgRNAs comparing MC38_sgStk11 tumors with MC38_sgNTC tumors in C57BL/6 mice treated with anti–PD-1. The green point indicates average sgRNAs for HDAC1. C, Created in BioRender. Ahronian, L. (2025) https://BioRender.com/mndvf20.

Figure 2.

TNG260 is a CoREST-selective deacetylase inhibitor. A, Structure of TNG260 aligned with structures of class-I HDAC small-molecule inhibitors; the fluorophenyl substituent is highlighted by the dotted lines. B, Crystal structure of TNG260 bound to HDAC2, with the key hydrogen bond and metal interactions illustrated as dotted lines. C, TNG260’s selectivity for HDAC1/2 over HDAC3 can be understood from the overlay of the reported HDAC3 crystal structure (PDB 4A69, blue) onto the TNG260-bound HDAC2 structure (PDB 9NTB, white). HDAC3’s Leu133 position clashes with the TNG260 fluorophenyl group, whereas HDAC2’s Leu140 position allows tight ligand binding but would clash with HDAC3’s Tyr107. D, Inhibition of the indicated HDAC enzyme in Fluor de Lys deacetylase assay by 3 hours of incubation with a dose response of TNG260. Plots are averages of N = 2 experiments. E, Western blotting of acetyl-H3K9 in the MC38 cell line treated with a dose response of TNG260 for 72 hours. F, Cellular HDAC1, HDAC2, and HDAC3 NanoBRET assay with a dose response of TNG260 incubated for 3 hours. Plots are averages of N = 2 experiments. G, Heat map showing IC₅₀s of purified HDAC complexes profiled in an in vitro deacetylase assay with the indicated compounds.

Figure 3.

TNG260 reverses resistance to anti–PD-1 in STK11-deficient tumor models. A, Mouse plasma levels of unbound (u) TNG260 after 2 days of once daily oral treatment with TNG260 at the indicated dose (N = 4 mice/group). The IC₅₀ of HDAC1 in the cellular NanoBRET assay is indicated by the dotted red line. B, Relative amounts of acetyl-H3K9 normalized to histone 3 from tumor tissue at the doses indicated, quantified from Western blotting. Each time point is normalized to the vehicle control tumors (N = 4 mice/group). C, Relative levels of acetyl-H3K9 normalized to histone H3 following 7 days of TNG260 treatment at the indicated dose, quantified from Western blotting (N = 3 representative tumors/group). Values are average ± SD. A t test was used for statistical analysis comparing each treatment with vehicle control. D, Individual MC38_sgStk11 tumor volumes treated with anti-IgG2a and TNG260 (once daily orally) at 30 mg/kg, anti–PD-1 (intraperitoneally twice weekly) at 10 mg/kg, or the combination of TNG260 and anti–PD-1 (N = 8 mice/group). ORR was calculated from the sum of mice with a partial regression and complete regression. Statistical analysis was performed by one-way ANOVA for the combination arm versus anti–PD-1. E and F, Survival curve of the indicated treatment groups in the MC38_sgStk11 study from D (E) or CT26_sgStk11 from Supplementary Fig. S4 (F). Statistical analysis was performed comparing the combination TNG260 and anti–PD-1 groups versus the anti–PD-1 groups by log-rank tests. G, Inhibition of HDAC1 and HDAC3 as determined by cellular NanoBRET assay plotted by free exposures of TNG260 in vivo. Shaded box indicates efficacious exposures of TNG260 in mouse. H, Average tumor volumes of animals previously cured of MC38_sgStk11 tumors with combination treatment (N = 5) compared with an untreated, naïve cohort (N = 8). Mice were rechallenged with MC38_sgStk11 tumor cells and tumor volume was evaluated over time. I and J, Average tumor volumes of MC38_sgStk11 tumors in C57BL/6 (I) and BALB/c athymic nude mice (J) treated as indicated (N = 8 mice/group). Statistical comparisons were carried out by one-way ANOVA comparing anti-IgG2a to each treatment group. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001; ns, not significant.

Figure 4.

TNG260 sensitizes STK11-deficient lung tumors to anti–PD-1 treatment through transcriptional reprogramming. A and B, Average tumor volumes of KL tumors (N = 15–20 mice/group; A) and KP tumors (N = 5 mice/group; B) in B6-Albino mice, treated as indicated. C, Tumor volume changes measured by MRI scans of cre-induced KL GEMM mice, treated as indicated (N = 8–10 mice/group). Statistical comparisons were carried out by one-way ANOVA comparing anti-IgG2a with each treatment group. **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001; ns, not significant. D, Bar chart showing fold change of the expression of genes related to antigen presentation and peptide loading in KL and KP cells treated with 1 µmol/L TNG260 for 7 days. E, Heatmap showing acetyl-H3K9 signal in both KL and KP cell lines treated with DMSO (control) or 1 µmol/L TNG260 (N = 2) for 7 days. ChIP signal intensity is shown by red shading. Genomic regions are clustered into seven groups by k-means (k = 7) clustering. F, Genome browser view at the Cxcl3 locus of KL and KP cell lines treated with DMSO or TNG260 (N = 2). G, Enrichment plot showing positive enrichment of differentially accessible acetyl-H3K9 peaks for genes increased by TNG260 treatment. H, GO analysis of genes from KL tumors in clusters 3 and 4 from Supplementary Fig. S7I. Clusters 3 and 4 were defined as genes with increased acetyl-H3K9 and increased expression with TNG260 treatment. I, Heatmap showing acetyl-H3K9 signals in KL and KP tumors treated with the combination of TNG260 and anti–PD-1. ChIP signal intensity is shown by red shading. Genomic regions are clustered into seven groups by k-means (k = 6) clustering (N = 3–5 mice/group).

Figure 5.

TNG260 modifies the tumor immune microenvironment in patients with STK11-deficient tumors. A, Percentage of acetylated histone H4 Lys5 compared with total acetylated and unacetylated histone H4 detected by mass spectrometry in FFPE tissue collected before and in study. B, TPS of PD-L1 from FFPE tissue biopsies collected in A. C and D, FFPE tissue biopsies analyzed by multiplex immunofluorescence for helper T cells (CD3⁺/CD4⁺; C) and cytotoxic T cells (CD3⁺/CD8⁺; D), respectively. Quantifications were performed in tumor regions identified by hematoxylin and eosin. E, Exemplar paired biopsy of a patient with NSCLC (patient 9; Supplementary Table S6) imaged by multiplex immunofluorescence for the indicated markers before and during treatment with 120 mg TNG260 and pembrolizumab. Scale bar, 50 µm. *, P ≤ 0.05.