Antibody dependent cell-mediated cytotoxicity selection pressure induces diverse mechanisms of resistance.

Targeted monoclonal antibody therapy has emerged as a powerful therapeutic strategy for cancer. However, only a minority of patients have durable responses and the development of resistance remains a major clinical obstacle. Antibody-dependent cell-mediated cytotoxicity (ADCC) represents a crucial therapeutic mechanism of action; however, few studies have explored ADCC resistance. Using multiple in vitro models of ADCC selection pressure, we have uncovered both shared and distinct resistance mechanisms. Persistent ADCC selection pressure yielded ADCC-resistant cells that are characterized by a loss of NK cell conjugation and this shared resistance phenotype is associated with cell-line dependent modulation of cell surface proteins that contribute to immune synapse formation and NK cell function. We employed single-cell RNA sequencing and proteomic screens to interrogate molecular mechanisms of resistance. We demonstrate that ADCC resistance involves upregulation of interferon/STAT1 and DNA damage response signaling as well as activation of the immunoproteasome. Here, we identify pathways that modulate ADCC sensitivity and report strategies to enhance ADCC-mediated elimination of cancer cells. ADCC resistance could not be reversed with combinatorial treatment approaches. Hence, our findings indicate that tumor cells utilize multiple strategies to inhibit NK cell mediated-ADCC. Future research and development of NK cell-based immunotherapies must incorporate plans to address or potentially prevent the induction of resistance.

Fibroblast activation protein regulates natural killer cell migration, extravasation and tumor infiltration.

Natural killer (NK) cells play a critical role in physiologic and pathologic conditions such as pregnancy, infection, autoimmune disease and cancer. In cancer, numerous strategies have been designed to exploit the cytolytic properties of NK cells, with variable success. A major hurdle to NK-cell focused therapies is NK cell recruitment and infiltration into tumors. While the chemotaxis pathways regulating NK recruitment to different tissues are well delineated, the mechanisms human NK cells employ to physically migrate are ill-defined. We show for the first time that human NK cells express fibroblast activation protein (FAP), a cell surface protease previously thought to be primarily expressed by activated fibroblasts. FAP degrades the extracellular matrix to facilitate cell migration and tissue remodeling. We used novel in vivo zebrafish and in vitro 3D culture models to demonstrate that FAP knock out and pharmacologic inhibition restrict NK cell migration, extravasation, and invasion through tissue matrix. Notably, forced overexpression of FAP promotes NK cell invasion through matrix in both transwell and tumor spheroid assays, ultimately increasing tumor cell lysis. Additionally, FAP overexpression enhances NK cells invasion into a human tumor in immunodeficient mice. These findings demonstrate the necessity of FAP in NK cell migration and present a new approach to modulate NK cell trafficking and enhance cell-based therapy in solid tumors.

The Next Generation of Cellular Immunotherapy: Chimeric Antigen Receptor-Natural Killer Cells.

The advent of chimeric antigen receptor (CAR) engineering has led to the development of powerful cellular therapies for cancer. CAR T cell-based treatments have had notable clinical success, but logistical issues and associated toxicities are recognized limitations. There is emerging interest in using other immune effector cell types for CAR therapy. Natural killer (NK) cells are part of the innate immune system, and these lymphocytes play major roles in immunosurveillance and antitumor immune responses. Incorporating CARs into NK cells provides the opportunity to harness and enhance their innate cytotoxic potential toward malignancies. In this review, we discuss the production of CAR-engineered NK cells, highlight data on their preclinical and clinical efficacy, and examine the obstacles and strategies to overcome them.

TORC1/2 kinase inhibition depletes glutathione and synergizes with carboplatin to suppress the growth of MYC-driven medulloblastoma.

Medulloblastoma is the most common malignant pediatric brain tumor. Tumors having high levels of c-MYC have the worst clinical prognosis, with only a minority of patients surviving. To address this unmet clinical need, we generated a human neural stem cell model of medulloblastoma that recapitulated the most aggressive subtype phenotypically and by mRNA expression profiling. An in silico analysis of these cells identified mTOR inhibitors as potential therapeutic agents. We hypothesized that the orally bioavailable TORC1/2 kinase inhibitor TAK228 would have activity against MYC-driven medulloblastoma. TAK228 inhibited mTORC1/2, decreased cell growth and caused apoptosis in high-MYC medulloblastoma cell lines. Comprehensive metabolic profiling of medulloblastoma orthotopic xenografts showed upregulation of glutathione compared to matched normal brain. TAK228 suppressed glutathione production. Because glutathione is required to detoxify platinum-containing chemotherapy, we hypothesized that TAK228 would cooperate with carboplatin in medulloblastoma. TAK228 synergized with carboplatin to inhibit cell growth and induce apoptosis and extended survival in orthotopic xenografts of high-MYC medulloblastoma. Brain-penetrant TORC1/2 inhibitors and carboplatin may be an effective combination therapy for high-risk medulloblastoma.

Disrupting LIN28 in atypical teratoid rhabdoid tumors reveals the importance of the mitogen activated protein kinase pathway as a therapeutic target.

Atypical teratoid rhabdoid tumor (AT/RT) is among the most fatal of all pediatric brain tumors. Aside from loss of function mutations in the SMARCB1 (BAF47/INI1/SNF5) chromatin remodeling gene, little is known of other molecular drivers of AT/RT. LIN28A and LIN28B are stem cell factors that regulate thousands of RNAs and are expressed in aggressive cancers. We identified high-levels of LIN28A and LIN28B in AT/RT primary tumors and cell lines, with corresponding low levels of the LIN28-regulated microRNAs of the let-7 family. Knockdown of LIN28A by lentiviral shRNA in the AT/RT cell lines CHLA-06-ATRT and BT37 inhibited growth, cell proliferation and colony formation and induced apoptosis. Suppression of LIN28A in orthotopic xenograft models led to a more than doubling of median survival compared to empty vector controls (48 vs 115 days). LIN28A knockdown led to increased expression of let-7b and let-7g microRNAs and a down-regulation of KRAS mRNA. AT/RT primary tumors expressed increased mitogen activated protein (MAP) kinase pathway activity, and the MEK inhibitor selumetinib (AZD6244) decreased AT/RT growth and increased apoptosis. These data implicate LIN28/RAS/MAP kinase as key drivers of AT/RT tumorigenesis and indicate that targeting this pathway may be a therapeutic option in this aggressive pediatric malignancy.