Targeting KDM2A Enhances T-cell Infiltration in NSD1-Deficient Head and Neck Squamous Cell Carcinoma

Abstract

In head and neck squamous cell carcinoma (HNSCC), a significant proportion of tumors have inactivating mutations in the histone methyltransferase NSD1. In these tumors, NSD1 inactivation is a driver of T-cell exclusion from the tumor microenvironment (TME). A better understanding of the NSD1-mediated mechanism regulating infiltration of T cells into the TME could help identify approaches to overcome immunosuppression. Here, we demonstrated that NSD1 inactivation results in lower levels of H3K36 dimethylation and higher levels of H3K27 trimethylation, the latter being a known repressive histone mark enriched on the promoters of key T-cell chemokines CXCL9 and CXCL10. HNSCC with NSD1 mutations had lower levels of these chemokines and lacked responses to PD-1 immune checkpoint blockade. Inhibition of KDM2A, the primary lysine demethylase that is selective for H3K36, reversed the altered histone marks induced by NSD1 loss and restored T-cell infiltration into the TME. Importantly, KDM2A suppression decreased growth of NSD1-deficient tumors in immunocompetent, but not in immunodeficient, mice. Together, these data indicate that KDM2A is an immunotherapeutic target for overcoming immune exclusion in HNSCC.

Significance: The altered epigenetic landscape of NSD1-deficient tumors confers sensitivity to inhibition of the histone-modifying enzyme KDM2A as an immunotherapeutic strategy to stimulate T-cell infiltration and suppress tumor growth.

Figure 1:. NSD1 inactivation downregulates the expression of CXCL9 and CXCL10 in HNSCC.

RNA expression and genomic sequencing data from a multicenter, phase II, window-of-opportunity trial (NCT02296684) (23) was examined for CXCL9 and CXCL10 expression and NSD1 mutations. In this trial, neoadjuvant pembrolizumab was administered to patients with locally advanced, resectable, HPV-unrelated HNSCC two to three weeks prior to definitive surgical resection. (A) Comparison of CXCL9 and CXCL10 baseline expression to pathologic tumor response (pTR), as defined in (23), to the anti-PD-1 antibody pembrolizumab. 〇 represent responders. ▲ represent non-responders with wild-type NSD1. ▲ represent non-responders with mutated NSD1. (B) Tumor histology from the two patients in the trial that had tumors with NSD1 mutations. Histology of NSD wild-type tumors is also shown. Red = CD3 staining of T cells. Yellow = pancytokeratin staining of the tumor cells. Blue = DAPI. White scale bars represent ~100 μm. (C) Comparison of the CXCL9 and CXCL10 expression in NSD1 wild-type and mutated HPV-negative HNSCC tumors in the Cancer Genome Atlas. ****p<0.0001. (D) Expression of CXCL9 and CXCL10 in malignant cells of NSD1 wild-type and mutated primary and metastatic HNSCCs. Box plots show expression (Normalized counts) of CXCL9 and CXCL10 in malignant single cells (points) of HNSCC samples, including primary and metastatic (lymph node metastasis) tumors. Black and red boxes and points represent NSD1 wild-type and NSD1 mutated HNSCCs, respectively. Single cell RNA-Sequencing data was accessed from a previous study by Puram et al. (24).

Figure 2:. KDM2A inhibition restores CXCL9 and CXCL10 expression in the absence of NSD1.

Quantitative RT-PCR (qRT-PCR) analysis of NSD1 expression after inhibition of NSD1 expression by shRNA transduction in human (FaDu) (A) and mouse (MOC1) (B) HNSCC cell lines. Representative Western blots of H3K36me2 and H3K27me3 levels in FaDu (C) and MOC1 (D) cells after inhibition of NSD1 expression by shRNA transduction. Quantification of mRNA expression by qRT-PCR of CXCL9 and CXCL10 in FaDu (E) and MOC1 (F) cells transduced to express NSD1 shRNA. Data shown are representative of experiments repeated at least three times. Error bars represent standard error of the mean (SEM), ** p <0.01, *** p ≤0.001. (G) Reciprocal relationship of H3K36me2 and H3K27me3. NSD1 catalyzes the di-methylation of H3K36 (H3K36me2). KDM2A is a lysine demethylase with specificity for H3K36me2. Tri-methylation of H3K27 (H3K27me3) is directly antagonized by H3K36me2 (and H3K36me3). (H) KDM2A mRNA expression (assessed by qRT-PCR and normalized to HPRT1) in FaDu and MOC1 cell lines, transduced to express shRNA targeting NSD1 with and without shRNA targeting KDM2A. H3K36me2 and H3K27me3 levels were assessed by Western blot analysis of FaDu (I) and MOC1 (J) cells, transduced to express shRNA targeting NSD1 with and without shRNA targeting KDM2A. All experiments were repeated at least three times. Error bars represent standard error of the mean (SEM), *p<0.05, ** p <0.01, *** p ≤0.001. CXCL9 and CXCL10 mRNA expression levels were assessed by qRT-PCR and normalized to expression of HPRT1 in FaDu (K) and MOC1 (L) cell lines, transduced to express shRNA targeting NSD1 with and without shRNA targeting KDM2A. Data shown are representative of experiments repeated at least three times. Error bars represent standard error of the mean (SEM), * p <0.05, *** p ≤0.001.

Figure 3.. KDM2A inhibition reverses the immune cold phenotype induced by NSD1 inactivation and induces T cell infiltration into the tumor microenvironment.

(A) Overview and illustration of experiment workflow. Tumor spheroids were established from HNSCC tumor cells transfected to express truncated CD19 on the cell surface and grown in submerged Matrigel. The spheroids were co-cultured with CD19-CAR-T cells for three days and then isolated for analysis by confocal microscopy. (B) Assessment by qRT-PCR of CXCL9 and CXCL10 expression after CXCL9 or CXCL10 overexpression (OE) in FaDu cells transduced to express shRNA targeting NSD1 (NSD1-sh). (C) Representative images of tumor spheroids after co-cultured with CD19-CAR-T cells for 3 days. Ctrl = vector control; NSD1-sh = shRNA targeting NSD1; CXCL9-OE = overexpression construct for CXCL9; CXCL10-OE = overexpression construct for CXCL10; KDM2A-sh = shRNA targeting KDM2A. Scale bar=50um. (D) Representative images of tumor spheroids stained with anti-CD3 antibody (red) and DAPI (blue). Scale bar=50 um. (E) Quantification of CD3+ cells per 100 cells in the spheroids. (F) Representative images of FaDu tumor spheroids expressing control or NSD1 targeted shRNA with or without the KDM2A inhibitor (daminozide) treatment. The spheroids were stained with anti-CD3 antibody (red) and DAPI (blue). Scale bar=50 um. (G) Quantification of CD3⁺ cells per 100 cells in the spheroids. Data shown are representative of experiments repeated at least three times. Error bars represent standard error of the mean (SEM). * p <0.05, ** p <0.01, *** p ≤0.001.

Figure 4.. KDM2A inhibition induces T cell infiltration of the tumor microenvironment in vivo.

Growth curves of MOC1 control (vector control) and NSD1 knockdown (NSD1-sh) tumors in syngeneic Rag1^–/– (Rag1 KO) mice (n=6 for each group) (A) and wild-type mice (n=8 per group) (B). (C) Tumor infiltrating T cells (CD45⁺CD3⁺) were quantified by flow cytometry of dissociated tumors. Growth curves of MOC1 control, NSD1 knockdown (NSD1-sh) and NSD1 and KDM2A double-knockdown (NSD1-sh & KDM2A-sh) cells in Rag1 KO mice (n=6 for each group) (D) and wild-type mice (n=5 for each group) (E). (F) Representative immunofluorescence images of CD3, CXCL9, and CXCL10 expression (red) in tumors formed from MOC1 cells transduced with vector control (Ctrl), NSD1 shRNA (NSD1-sh), or both NSD1 shRNA and KDM2A shRNA (NSD1-sh&KDM2A-sh). Scale bar=50 um. Blue stain=DAPI. Error bars represent standard error of the mean (SEM). *** p ≤0.001. ns = not significant.