Atezolizumab potentiates Tcell-mediated cytotoxicity and coordinates with FAK to suppress cell invasion and motility in PD-L1+ triple negative breast cancer cells.

Immune check point inhibitors targeting programmed cell death protein-1 (PD-1) and its ligand (PD-L1) have shown clinical success in treatment of human malignancies. Triple negative breast cancer (TNBC), which is primarily characterized by high heterogeneity and presence of tumor infiltrating lymphocytes, remains therapeutic challenge due to unavailability of approved targeted therapy. Therapeutic potential of immune check point inhibitors for TNBC patients is under active clinical investigation. In this study, we show that FDA-approved anti-PD-L1 antibody, atezolizumab (ATE), potentiates T cell-mediated cytotoxicity and apoptosis of TNBC cells that express higher levels of PD-L1, but does not have significant effect on TNBC cells expressing low levels of PD-L1. PD-L1 knockdown further confirmed that ability of ATE to promote T cell-induced cytotoxicity is PD-L1 expression dependent. Combination of ATE with PD-L1 upregulating agents, such as HDAC, proteasomal, and lysosomal inhibitors, further augmented cytotoxic activity of T cells toward TNBC cells. Based on analysis of breast cancer tissue samples deposited in The Cancer Genome Atlas (TCGA), we found a positive correlation between PD-L1 and focal adhesion kinase (FAK) mRNA expression in PD-L1-positive (PD-L1+) TNBC, suggesting a functional association of FAK and immune checkpoints. We further demonstrate that ATE dramatically downregulates phosphorylation status of FAK, an important regulator of cell invasion and migration, and significantly enhances FAK inhibitor mediated inhibition of cell motility and invasion of PD-L1+ TNBC cells independent of T cells. Taken together, our data suggest that ATE shows promising anti-tumor activity in PD-L1+ TNBC via both T cell-dependent and -independent mechanisms.

Trastuzumab-mediated cardiotoxicity: current understanding, challenges, and frontiers.

Trastuzumab, an epidermal growth factor receptor 2 (HER2) targeting humanized monoclonal antibody, has been approved for the treatment HER2-positive breast cancer and HER2-positve metastatic gastric cancer. However, cardiotoxicity associated with its clinical application poses challenges for clinicians and patients, mechanisms of which are still evolving. This review will summarize the current mechanistic understanding of trastuzumab-mediated cardiotoxicity, discuss the novel role of DNA topoisomerase IIB as a shared target for enhanced cardiotoxicity induced by trastuzumab and anthracyclines-based combination regimens, and speculate the potential impact of trastuzumab intervention in immune checkpoint inhibitors-based therapies.

Type IIB DNA topoisomerase is downregulated by trastuzumab and doxorubicin to synergize cardiotoxicity.

Despite heightened risk of cardiotoxicity associated with combination therapy of anthracyclines and trastuzumab in HER2-positive breast cancer patients, little research effort has been invested in exploring the molecular mechanisms of cardiotoxicity induced by this combination therapy. In this study, we demonstrate that trastuzumab downregulates both gene and protein expressions of type IIB DNA topoisomerase/DNA topoisomerase IIB (TOP2B), a major intracellular target mediating doxorubicin-induced cardiotoxicity, in human primary cardiomyocytes. This in turn induces DNA damage activity and DNA double strand breaks, which is indicated by the enhanced phosphorylation of H2AX (γH2AX) and ataxia telangiectasia and Rad3-related protein (ATR pS428) in trastuzumab-treated cardiomyocytes. Furthermore, concurrent or sequential treatment of doxorubicin and trastuzumab significantly increases the downregulation of the protein levels of TOP2B, enhances apoptosis and cell growth inhibition, and promotes production of reactive oxidative and nitrative species in human cardiomyocytes as compared to either trastuzumab or doxorubicin treatment, indicating augmentation of cardiotoxicity in combination therapy. Additionally, our data reveal that doxorubicin treatment increases the levels of ErbB2/HER2 expression in human cardiomyocytes as compared with that in cells not treated with doxorubicin, leading to the enhanced activity downstream of HER2 signaling. Consequently, this may render the cardiomyocytes to become addicted to HER2 signaling for survival under stressed conditions. Enhanced HER2 protein expression leaves cardiomyocytes more sensitive to trastuzumab treatment after doxorubicin exposure. This study provides molecular basis for significantly increased cardiotoxicity in cancer patients who are treated with anthracyclines and trastuzumab-based combination regimens.

Payload of T-DM1 binds to cell surface cytoskeleton-associated protein 5 to mediate cytotoxicity of hepatocytes.

Off-target toxicity is a major cause of dose-limiting toxicity for antibody-drug conjugates (ADCs), mechanisms of which remain poorly understood. Here, we demonstrate that cytoskeleton-associated protein 5 (CKAP5) serves as a cell surface target for T-DM1 and that binding of T-DM1 to CKAP5 is mediated by payload (DM1). This study introduces a novel molecular mechanism of ADC payload-mediated interaction with cell surface molecules to induce cytotoxicity. Upon binding to CKAP5, T-DM1 causes cell membrane damage and leads to calcium influx into the cells, resulting in disorganized microtubule network and apoptosis. While binding of T-DM1 with HER2 is critical for killing HER2-positive tumor cells, our data suggest that cytotoxicity induced by T-DM1 interaction with CKAP5 may preferentially damage normal cells/tissues where HER2 expression is low or missing to cause off-target toxicity. This study provides molecular basis of ADC-induced off-target cytotoxicity and opens a new avenue for developing next generation of ADCs.

Depletion of BBS Protein LZTFL1 Affects Growth and Causes Retinal Degeneration in Mice.

Bardet-Biedl syndrome (BBS) is a heterogeneous disease characterized by deficiencies in various organs that are caused by defects in genes involved in the genesis, structural maintenance, and protein trafficking of cilia. Leucine zipper transcription factor-like 1 (LZTFL1) has been identified as a BBS protein (BBS17), because patients with mutations in this gene exhibit the common BBS phenotypes. In this study, we generated a knockout mouse model to investigate the effects of LZTFL1 depletion. Lztfl1 knockout mice were born with low birth weight, reached similar weight to those of wild-type mice at 10 weeks of age, and later gained more weight than their wild-type counterparts. LZTFL1 was localized to the primary cilium of kidney cells, and the absence of LZTFL1 increased the ciliary localization of BBS9. Moreover, in the retinas of Lztfl1 knockout mice, the photoreceptor outer segment was shortened, the distal axoneme of photoreceptor connecting cilium was significantly enlarged, and rhodopsin was targeted to the outer nuclear layer. TUNEL assay showed that many of these abnormal photoreceptor cells in Lztfl1 knockout mice underwent apoptosis. Interestingly, the absence of LZTFL1 caused an abnormal increase of the adaptor protein complex 1 (AP1) in some photoreceptor cells. Based on these data, we conclude that LZTFL1 is a cilium protein and regulates animal weight and photoreceptor connecting cilium function probably by controlling microtubule assembly and protein trafficking in cilia.

Detecting protein interactions in live cells via complementation of a hydrolysis-deficient ß-lactamase.

Protein-fragment complementation assay using a hydrolysis-deficient ß-lactamase provides a robust tool for studying protein-protein interactions.

Photogenerated quinone methides as protein affinity labeling reagents.

Photogenerated quinine methides (QMs) can act as highly specific light-controllable protein affinity labeling reagents, broadening their applications to the study of protein-ligand and protein-protein interactions. The reactions between QMs and nucleophiles are less heterogeneous than those of other photogenerated active intermediates such as carbenes and nitrenes, making it easier to identify labeled species.