Heterogeneity in NECTIN4 Expression Across Molecular Subtypes of Urothelial Cancer Mediates Sensitivity to Enfortumab Vedotin

Abstract

Purpose: Enfortumab vedotin (EV) is an antibody-drug conjugate (ADC) targeting NECTIN4 (encoded by the PVRL4/NECTIN4 gene) approved for treatment-refractory metastatic urothelial cancer. Factors that mediate sensitivity or resistance to EV are unknown. In this study, we sought to (i) examine heterogeneity of NECTIN4 gene expression across molecular subtypes of bladder cancer and (ii) determine whether NECTIN4 expression mediates EV sensitivity or resistance.

Experimental design: Molecular subtyping and NECTIN4 expression data from seven muscle-invasive bladder cancer clinical cohorts (n = 1,915 total specimens) were used to assess NECTIN4 expression across molecular subtypes. The outcome of the transcriptomic analysis was relative NECTIN4 expression in the consensus molecular subtypes of bladder cancer. Expression of NECTIN4 was validated in bladder cancer cell lines. NECTIN4 was stably overexpressed or knocked down in basal and luminal bladder cancer cell lines and EV drug sensitivity assays were performed, as measured by cell proliferation and clonogenic assays.

Results: NECTIN4 expression is heterogenous across molecular subtypes of bladder cancer and significantly enriched in luminal subtypes. NECTIN4 expression is positively correlated with luminal markers GATA3, FOXA1, and PPARG across all cohorts. NECTIN4 expression is both necessary and sufficient for EV sensitivity in luminal and basal subtypes of urothelial bladder cancer cells. Downregulation of NECTIN4 leads to EV resistance.

Conclusions: Sensitivity to EV is mediated by expression of NECTIN4, which is enriched in luminal subtypes of bladder cancer. These findings may have implications for biomarker development, patient selection, and the inclusion of molecular subtyping in ongoing and future EV clinical trials.See related commentary by Teo and Rosenberg, p. 4950.

FIGURE 1:

NECTIN4 mRNA expression across the consensus molecular subtypes of muscle invasive bladder cancers (MIBC) and association with luminal markers GATA3, FOXA1, PPARG. (A) Violin plots showing NECTIN4 mRNA expression levels by consensus molecular subtypes in four public clinical cohorts of MIBC. (B) NECTIN4 mRNA expression in the commercial Decipher cohort (n=529) by consensus molecular subtype. (C) NECTIN4 mRNA expression across consensus clustering (CC) subtypes in the post-neoadjuvant chemotherapy (NAC) cohort (n=133). The p values from Kruskal-Wallis testing for each cohort are shown in A-C, and pairwise comparisons using the Wilcoxon rank-sum test are shown in Supplementary Table 2. (D) Scatter plot showing the mRNA expression of the luminal markers GATA3 (top), FOXA1 (middle), and PPARG (bottom) versus NECTIN4 in the Decipher cohort, color coded by molecular subtype. Spearman’s rank correlation is shown for NECTIN4 versus GATA3 (r = 0.39), FOXA1 (r = 0.36), and PPARG (r = 0.43).

FIGURE 2:

NECTIN4 expression varies across bladder cancer cell lines and is associated with phenotypic differences and variable EV sensitivity. (A) NECTIN4 mRNA expression between luminal (n=10) and basal (n=13) bladder cancer subtypes (Source: DepMap database) (****p<0.0001). (B) NECTIN4 mRNA expression for individual bladder cancer cell lines are shown. Red denotes luminal cell lines and blue denotes basal cell lines (Source: DepMap database). (C) Surface staining for NECTIN4 protein in representative luminal (HT-1376, HT-1197, and UMUC-9) and basal (UMUC-3, 253JBV, and TCCSUP) bladder cancer cell lines. (D) Western blot demonstrating NECTIN4 protein expression in whole cell lysates across representative luminal (HT-1376, HT-1197, and UMUC-9) and basal (UMUC-3, 253JBV, and TCCSUP) bladder cancer cell lines. GAPDH is shown as the loading control. (E) Brightfield microscopy of luminal (HT-1197 and HT-1376) and basal (TCCSUP and UMUC-3) cells depicting the morphologic differences between basal cells (left) and luminal cells (right). Scale bar = 400 mm. (F) EV dose-response curves for basal cells (UMUC-3 and TCCSUP) and luminal cells (HT-1376 and HT-1197).

FIGURE 3:

NECTIN4 knockdown in HT-1376 cells decreases sensitivity to EV. (A) NECTIN4 surface protein expression in HT-1376 control and CRISPRi NECTIN4 knockdown (KD) HT-1376 cells. (B) EV dose-response WST proliferation assay in HT-1376 control and NECTIN4 KD cells, normalized to the mean absorbance of untreated cells. (C) Clonogenic assay quantification of EV sensitivity in HT-1376 control and HT-1376 NECTIN4 KD cells. **p<0.01 (D) Representative images of the clonogenic assay in HT-1376 control and HT-1376 NECTIN4 KD cells treated with EV.

FIGURE 4:

NECTIN4 overexpression in UMUC-3 cells increases sensitivity to EV. (A) NECTIN4 surface protein expression in UMUC-3 control and NECTIN4 overexpressing (OE) cells. (B) NECTIN4 RNA expression in UMUC-3 Control and UMUC-3 NECTIN4 OE cells by qPCR. **p<0.001 (C) EV dose-response WST assay in UMUC-3 control and NECTIN4 OE cells, normalized to mean survival of untreated cells. (D) Clonogenic assay quantification of EV sensitivity in UMUC-3 Control and UMUC-3 NECTIN4 OE cells. **p<0.01 (E) Representative images of the clonogenic assay in UMUC-3 Control and NECTIN4 OE cells treated with EV.