CD70 is a therapeutic target upregulated in EMT-associated EGFR tyrosine kinase inhibitor resistance

Abstract

Effective therapeutic strategies are needed for non-small cell lung cancer (NSCLC) patients with epidermal growth factor receptor (EGFR) mutations that acquire resistance to EGFR tyrosine kinase inhibitors (TKIs) mediated by epithelial-to-mesenchymal transition (EMT). We investigate cell surface proteins that could be targeted by antibody-based or adoptive cell therapy approaches and identify CD70 as being highly upregulated in EMT-associated resistance. Moreover, CD70 upregulation is an early event in the evolution of resistance and occurs in drug-tolerant persister cells (DTPCs). CD70 promotes cell survival and invasiveness, and stimulation of CD70 triggers signal transduction pathways known to be re-activated with acquired TKI resistance. Anti-CD70 antibody drug conjugates (ADCs) and CD70-targeting chimeric antigen receptor (CAR) T cell and CAR NK cells show potent activity against EGFR TKI-resistant cells and DTPCs. These results identify CD70 as a therapeutic target for EGFR mutant tumors with acquired EGFR TKI resistance that merits clinical investigation.

Keywords: CD70; EGFR; NSCLC.

Figure 1.. CD70 is elevated in NSCLC cells with acquired, EMT-associated EGFR TKI resistance.

(A) Differential expression of genes transcribing cell surface proteins in erlotinib resistant (ER) and parental cells. (B) Mean CD70 expression ± SD in parental (H1975, HCC4006) and osimertinib-resistant (OR) variants. *p = 0.008. (C) CD70 expression by Western blotting. (D-F) Mean CD70 positivity ± SD by flow cytometry (n = 3–6). (G) Mean CD70 positivity ± SD in HCC827 and YUL-0019 cells as compared to MDA-L-011 and MDA-L-004K (n = 2–5). *p = 0.003. (H) Expression of CD70 and EMT-related genes in patient-derived models of erlotinib resistance. (I) scRNAseq analysis of CD70 in T790M negative resistant cells that had undergone EMT. (J) Change in expression of an EMT gene signature and CD70 in osimertinib-refractory and matched pre-treatment clinical samples. (K) Spearman correlation of differences in EMT-score and CD70 expression for 10 patients with matched pre- and post-osimertinib treatment. Student’s t test was used for B and G. See also Figure S1 and Tables S1 and S2.

Figure 2.. CD70 is increased in osimertinib-resistant NSCLC clinical samples.

(A) Violin plot of CD70 IHC score in specimens collected at baseline (n = 16) or after progression on osimertinib (n = 36). Dashed line = median; dotted lines = first and third quartile. Student’s t test. (B) Representative images of CD70 IHC. 400x magnification; scale bars = 20 μm. (C) CD70 staining intensity among osimertinib-refractory tumors. (D) Overall survival for EGFR TKI refractory NSCLC patients with high or low CD70. (E) Overall survival for NSCLC patients (TCGA-LUAD and GEO databases) with high or low CD70.

Figure 3.. CD70 expression is associated with EMT and epigenetic changes in EGFR mutant NSCLC cells.

(A and B) In NSCLC samples (TCGA-LUAD dataset; A) and across NSCLC cell lines (B), CD70 expression correlated with an EMT expression score (left) and ZEB1 (right). (C) Mean CD70 RNA expression ± SEM in HCC827 cells expressing ZEB1 (n = 3). *p = 0.0096. (D) Mean CD70 positivity ± SD in HCC827 cells with or without ZEB1 (n = 3). *p < 0.0001. (E) GSEA analysis of TGFB expression signature in ER cells that acquired resistance through EMT. (F) Correlation of CD70 and TGFB1 among NSCLC patients (TCGA). (G & H) TGFβ (10 ng/ml) increased ZEB1 (G) and CD70 (H) mRNA. Mean ± SD (n = 3–4). *p < 0.001. (I) Mean CD70 positivity ± SD following TGF-β treatment (n = 2). *p < 0.001. (J) CD70 expression is associated with reduced CD70 promoter methylation. (K) CD70 promoter methylation in epithelial (E) and mesenchymal (M) NSCLC cell lines. Box plots depict the median (line) as well as the 25th and 75th percentile, with whiskers showing 1.5x the interquartile range (IQR). (L) CD70 promoter methylation in parental (P) and osimertinib resistant (OR) and erlotinib resistant (ER) cells. (M) Mean CD70 ± SD expression following exposure to decitabine (DEC; 1 μM; n = 3). *p < 0.0001. Student’s t test was used for C, D, G, H, I, K, M. See also Figure S2.

Figure 4.. CD70 activates signal transduction in EGFR TKI resistant cells and regulates survival and invasive pathways.

(A-C) Growth rate of H1975, H1975 OR5, and H1975 OR16 following CD70 knockdown (n = 4). Data points are mean viability ± SD. p < 0.001. (D & E) Representative images and high-powered field (HPF) quantification of mean number of migrating H1975 OR16 cells ± SD after CD70 knockdown. *p < 0.0001. (n=3) Scale bars = 1000 μm. (F – I) p-AKT and p-ERK1/2 following stimulation with rhsCD27. (J) Mean number of migrating cells ± SEM following stimulation with rhsCD27 (n = 3). *p < 0.05; student’s t-test. (K) Mean CD70 positivity ± SD in HCC827 cells engineered to overexpress CD70. (L & M) Osimertinib (OSI) dose response curve for HCC827 cells with or without CD70 or rhsCD27. Mean ± SD (n = 3). One-way ANOVA was applied for A, B, C, E. See also Figure S3.

Figure 5.. CD70 upregulation is an early event in the evolution of EGFR TKI resistance.

(A-C) Western blotting of HCC4006 (A), HCC827 (B), and H1975 (C) cells following treatment with osimertinib (OSI). (D & E) ZEB1 (D) and CD70 (E) RNA levels following OSI treatment (n = 3). *p < 0.05; **p < 0.001. (F & G) Protein expression by RPPA in control and DTPCs following erlotinib (ERL; F) and osimertinib treatment (OSI; G) (n = 3). (H & I) ZEB1 and CD70 RNA in osimertinib-treated DTPCs (n = 3). *p < 0.05; **p < 0.001. (J) CD70 positivity on osimertinib-derived DTPC (n = 3). *p = 0.016, HCC827; p = 0.0086, HCC4006. (K) Representative flow cytometry data for CD70 expression on H1975 DTPCs. (L) CD70 positivity on osimertinib-derived DTPC (n = 3). *p <0.0001. (M) CD70 expression in H1975 parental and DTPCs. All bars are mean ± SD. One-way ANOVA was applied for D and E; Student’s t test was applied for H, I, J, L.

Figure 6.. CD70 expression can targeted in EGFR TKI resistant cells.

(A) HCC827 cells with or without CD70 expression treated with cusatuzumab (Cus)-MMAE. *p ≤ 0.01. (B) H1975 and OR cells treated with cusatuzumab-MMAE. *p ≤ 0.01. (C) Viability of H1975 and OR variants treated with cusatuzumab (Cus)-MMAE (3 μg/ml). *p < 0.5; **p < 0.0007. (D - G) Activity of CD70 CAR T cells against HCC827 cells with or without CD70 expression (D), HCC4006 OR cells (E), HCC4006 parental cells (F), or MDA-L-011 cells (G). *p < 0.01; *p < 0.001. (H) CD107a expression on CD70 CAR T cells following incubation with HCC827 cells, HCC827 cells expressing CD70, and DTPCs. *p < 0.01; *p < 0.001. (I) Activity of CD70 CAR T cells against HCC827 cells and HCC827 DTPCs. (J &K) Activity of CD70-targeting CAR NK cells against HCC827 cells with or without CD70 (J) or HCC4006 parental cells and HCC4006 OR variants (K). (L & M) Viability (L) and clonogenic growth (M) of HCC4006 (GFP+) and HCC4006 OR7 (GFP-) cells grown as a mixed culture and treated with CD70-CAR NK (trCD27) cells, and osimertinib (OSI; 200 nM). *p < 0.0001. For all graphs, data shown as mean ± SD (n = 3). Statistics were applied using multiple t tests (A, B, C), Student’s t test (H), or one-way ANOVA (M). See also Figures S4 and S5.

Figure 7.. CD70 targeting has potent anti-tumor cell activity in vivo.

(A) Growth rate of HCC827-CD70 xenografts treated with CD70-targeting cusatuzumab (Cus)-MMAE, control IgG antibodies, or an irrelevant ADC - brentuzimab (Bren)-MMAE) (n = 9 mice/group). *p < 0.0001 vs control. (B) Effect of wild-type (WT) NK cells or CD70-targeting CAR NK cells on HCC827-CD70 xenografts (n = 9 mice/group). *p < 0.0001. (C) Growth rate of H1975 OR17 xenografts treated with IgG antibodies, osimertinib (OSI), Cus-MMAE, or the combination (n = 7–8 mice/group). *p < 0.0001 vs control. (D) Growth H1975 OR17 xenografts treated with wild-type NK cells or CD70-targeting CAR NK cells (n = 7–8mice/group). *p < 0.0001 vs control; p<0.0001 vs control NK cells. For all graphs, data are mean ± SEM.