Extended Data Fig. 7: p95HER2 abrogates T-DXd-induced immune infiltration but is exquisitely sensitive to neratinib.
(a,b) T-DXd is effective against FL-HER2+ EMT6 tumorgrafts, regardless of their size at time of treatment initiation. Cells were orthotopically implanted into BALB/c mice. After 13 days, mice were divided into two groups based on tumor size and received two doses of T-DXd. Tumor growth curves in (a), terminal tumor weights in (b) along with percent growth inhibition. Large tumors at the start of treatment: IgG n = 10, T-DXd n = 10; Small tumors at the start of treatment: IgG n = 10, T-DXd n = 10. Data represent mean ± SEM. Statistical analyses by unpaired, two-tailed Student’s t test. P values are indicated within the plots. (c) qRT-PCR quantifying the levels of immune-related genes in bulk RNA harvested from the T-DXd-treated tumors described in Fig. 7b. N = 8 EV tumors and n = 10 p95HER2+ tumors. Data represent mean ± SEM. Statistical analyses by unpaired, two-tailed Student’s t test. P values are indicated within the plots. (d) qRT-PCR analysis of dendritic cell marker Cd11c in tumors treated with IgG only. N = 10 EV tumors and n = 8 p95HER2+ tumors. Data represent mean ± SEM. Statistical analyses by unpaired, two-tailed Student’s t test (p = 0.0031). (e) mIF analysis of dendritic cell subtypes to complement mIF analysis shown in Fig. 7d. N = 5 EV tumors and n = 5 p95HER2+ tumors. Data presented as truncated violin plots. Statistical analyses by ordinary one-way ANOVA with Tukey’s correction for multiple comparisons. P values are indicated within the plots. (f) The glycosylated form of p95HER2 is selectively targeted for proteasomal degradation by irreversible TKIs (neratinib, pyrotinib, and afatinib) but not reversible TKIs (tucatinib, lapatinib). Both classes of TKI inhibit PD-L1 expression. (g,h) Western blotting demonstrates that proteasome inhibitors Bortezomib (g) and MG132 (h) prevent the downregulation of glycosylated p95HER2 induced by irreversible TKIs. For all panels (f-h), western blot data results are representative of n = 3 biological repeats.
