Neutralization of BCL-2/XL Enhances the Cytotoxicity of T-DM1 In Vivo

Abstract

One of the most recent advances in the treatment of HER2⁺ breast cancer is the development of the antibody-drug conjugate, T-DM1. T-DM1 has proven clinical benefits for patients with advanced and/or metastatic breast cancer who have progressed on prior HER2-targeted therapies. However, T-DM1 resistance ultimately occurs and represents a major obstacle in the effective treatment of this disease. Because anti-apoptotic BCL-2 family proteins can affect the threshold for induction of apoptosis and thus limit the effectiveness of the chemotherapeutic payload, we examined whether inhibition of BCL-2/X_L would enhance the efficacy of T-DM1 in five HER2-expressing patient-derived breast cancer xenograft models. Inhibition of BCL-2/X_L via navitoclax/ABT-263 significantly enhanced the cytotoxicity of T-DM1 in two of three models derived from advanced and treatment-exposed metastatic breast tumors. No additive effects of combined treatment were observed in the third metastatic tumor model, which was highly sensitive to T-DM1, as well as a primary treatment-exposed tumor, which was refractory to T-DM1. A fifth model, derived from a treatment naïve primary breast tumor, was sensitive to T-DM1 but markedly benefited from combination treatment. Notably, both PDXs that were highly responsive to the combination therapy expressed low HER2 protein levels and lacked ERBB2 amplification, suggesting that BCL-2/X_L inhibition can enhance sensitivity of tumors with low HER2 expression. Toxicities associated with combined treatments were significantly ameliorated with intermittent ABT-263 dosing. Taken together, these studies provide evidence that T-DM1 cytotoxicity could be significantly enhanced via BCL-2/X_L blockade and support clinical investigation of this combination beyond ERBB2-amplified and/or HER2-overexpressed tumors.

Figure 1.

Comparison of HER2, BCL-2 and BCL-X_L expression levels. Tumor sections were visualized via H&E (a–e) and were evaluated for HER2 (f–j), BCL-2 (k–o) and BCL-X_L (p–t) via IHC. Representative IHC images are presented for PDX12, PDX8, BCM-3963, BCM-4888 and BCM-3613. IHC or H-scores for each marker are summarized in Table 1. Scale bar, ~90 μm.

Figure 2.

Microscopic analysis of treatment effects. Representative H&E images are presented to compare PDX12 (a–j) and PDX8 (k–t) treatment groups. Note significant elimination of tumor cells post combination treatment (d and n or i and s). H&E images presented in a–e and k–o are magnified in f–j and p–t, respectively. The presence of notable intraductal carcinoma (ID) is indicated. Scale bar, ~480 μm.

Figure 3.

Tumor cellular and stromal characterization post-treatment. Representative Masson’s trichrome stained tumor sections from PDX12 (a and c) and PDX8 (e and g) comparing vehicle controls, single agents and combination treatment. Masson’s trichrome stains the cell nuclei (black), cell cytoplasm (red) and collagen (blue). Note the emergence of scar-like reactive stroma at the tumor bed in response to combination treatment (c and g). Representative EMA stained tumor sections from PDX12 (b and d) and PDX8 (f and h) comparing treatment groups. Note the substantial elimination of invasive tumor cells in response to combination treatment (d and h). The elimination of invasive tumor cells and the emergence of a desmoplastic stroma are characteristic features of effective treatment response. The images presented in a and b or e and f are magnified in c and d or g and h, respectively. The presence of notable intraductal carcinoma (ID) is indicated. Scale bar, ~525 μm.

Figure 4.

Quantitative pathological assessment of treatment effects. Tumor cellular content was summarized across multiple tumors from PDX12 (a) and PDX8 (d) experiments (Mann-Whitney one-tail test PDX12: T-DM1 versus T-DM1 + ABT-263; p value = 0.0286, T-DM1 versus T-DM1 + ^PULSEABT-263; p value = 0.1714 and PDX8: T-DM1 versus T-DM1 + ABT-263; p value = 0.0429, T-DM1 versus T-DM1 + ^PULSEABT-263; p value = 0.0286). Tumor stromal content was summarized across multiple tumors from PDX12 (b) and PDX8 (e) experiments (Mann-Whitney one-tail test PDX12: T-DM1 versus T-DM1 + ABT-263; p value = 0.0286, T-DM1 versus T-DM1 + ^PULSEABT-263; p value = NS and PDX8: T-DM1 versus T-DM1 + ABT-263; p value = 0.0286, T-DM1 versus T-DM1 + ^PULSEABT-263; p value = 0.0571). Tumor cross-sectional area was summarized across multiple tumors from PDX12 (c) and PDX8 (f) experiments (Welch’s one-tail t test PDX12: T-DM1 versus T-DM1 + ABT-263; p value = 0.0418, T-DM1 versus T-DM1 + ^PULSEABT-263; p value = 0.0366 and PDX8: T-DM1 versus T-DM1 + ABT-263; p value = 0.0667, T-DM1 versus T-DM1 + ^PULSEABT-263; p value = 0.0115). p values ≤ 0.05 (*) are indicated as numerical values, p values > 0.05 and ≤ 0.2 are also indicated as numerical values whereas p values > 0.2 are indicated as NS. Additional statistical tests comparing vehicle with each of the treatment groups are summarized in Supplementary Table 2. Each line represents the median (a and d or b and e) or the mean (c and f).

Figure 5.

BCM-3963, BCM-4888 and BCM-3613 response to combination treatment. BCM tumors were administered vehicle controls, single agents or combination treatments for 14 days. ABT-263 was administered continuously days 1–14. Representative H&E images are presented to compare BCM-3963 (a–d), BCM-4888 (g–j) and BCM-3613 (m–p) treatment groups. Note significant elimination of BCM-3963 tumor cells post-combination treatment (d). Tumor cross-sectional area was summarized across multiple tumors from BCM-3963 (e), BCM-4888 (k) and BCM-3613 (q) experiments (Welch’s one-tail t test BCM-3613: vehicle versus T-DM1; p value = 0.0159). Tumor cellular content was summarized across multiple tumors from BCM-3963 (f), BCM-4888 (l) and BCM-3613 (r) experiments (Mann-Whitney one-tail test BCM-3963: vehicle versus T-DM1; p value = 0.0143, T-DM1 versus T-DM1 + ABT-263; p value = 0.0143 and BCM-4888: vehicle versus ABT-263; p value = 0.0357). p values ≤ 0.05 (*) are indicated as numerical values and p values > 0.2 are indicated as NS. Additional statistical tests comparing vehicle with each of the treatment groups are summarized in Supplementary Table 3. Each line represents the mean (e, k and q) or the median (f, l and r). Scale bar, ~185 μm.

Figure 6.

HER2 levels post-treatment. PDX12 (a–e) and PDX8 (g–k) tumor sections were evaluated for HER2 via IHC. Representative HER2 IHC images are presented to compare treatment groups. Note PDX12 and PDX8 T-DM1-treated tumors express lower levels of HER2 (c–e and i–k). Note single agent ABT-263 upregulates HER2 in PDX12 tumors (b). HER2 was quantified via H-score assessment (37). Data were summarized across multiple tumors from PDX12 (f) and PDX8 (l) experiments (Mann-Whitney one-tail test PDX12: vehicle versus ABT-263; p value = 0.0500, vehicle versus T-DM1; p value = 0.0500, vehicle versus T-DM1 + ABT-263; p value = 0.0286, vehicle versus T-DM1 + ^PULSEABT-263; p value = 0.0286 and PDX8: vehicle versus T-DM1; p value = 0.0079, vehicle versus T-DM1 + ABT-263; p value = 0.0079, vehicle versus T-DM1 + ^PULSEABT-263; p value = 0.0179). p values ≤ 0.05 (*) or ≤ 0.01 (**) are indicated as numerical values. Each line represents the median. Scale bar, ~90 μm.