The goal of targeted therapy is to match a selective drug with a genetic lesion that predicts for drug sensitivity. In a diverse panel of cancer cell lines, we found that the cells most sensitive to focal adhesion kinase (FAK) inhibition are deficient in the expression of the NF2 tumor suppressor gene product, Merlin. Merlin expression is often lost in malignant pleural mesothelioma (MPM), an asbestos-induced aggressive cancer with limited treatment options. Our data demonstrate that low Merlin expression predicts for increased sensitivity of MPM cells to a FAK inhibitor, VS-4718, in vitro and in tumor xenograft models. Disruption of MPM cell-cell or cell-extracellular matrix (ECM) contacts with blocking antibodies suggests that weak cell-cell adhesions in Merlin-negative MPM cells lead to their greater dependence on cell-ECM-induced FAK signaling. This provides one explanation of why Merlin-negative cells are vulnerable to FAK inhibitor treatment. Furthermore, we validated ALDH as a marker of cancer stem cells (CSCs) in MPM, a cell population thought to mediate tumor relapse after chemotherapy. Whereas pemetrexed and cisplatin, standard-of-care agents for MPM, enrich for CSCs, FAK inhibitor treatment preferentially eliminates these cells. These preclinical results provide the rationale for a clinical trial in MPM patients using a FAK inhibitor as a single agent after first-line chemotherapy. With this design, the FAK inhibitor could potentially induce a more durable clinical response due to reduction of CSCs along with a strong antitumor effect. Furthermore, our data suggest that patients with Merlin-negative tumors may especially benefit from FAK inhibitor treatment.

Malignant mesotheliomas are highly aggressive tumors usually caused by exposure to asbestos. Germlineinactivating mutations of BAP1 predispose to mesothelioma and certain other cancers. However, why mesothelioma is the predominate malignancy in some BAP1 families and not others, and whether exposure to asbestos is required for development of mesothelioma in BAP1 mutation carriers are not known. To address these questions experimentally, we generated a Bap1 þ/À knockout mouse model to assess its susceptibility to mesothelioma upon chronic exposure to asbestos. Bap1 þ/À mice exhibited a significantly higher incidence of asbestos-induced mesothelioma than wild-type (WT) littermates (73% vs. 32%, respectively). Furthermore, mesotheliomas arose at an accelerated rate in Bap1 þ/À mice than in WT animals (median survival, 43 weeks vs. 55weeks after initial exposure, respectively) and showed increased invasiveness and proliferation. No spontaneous mesotheliomas were seen in unexposed Bap1 þ/À mice followed for up to 87 weeks of age. Mesothelioma cells from Bap1 þ/À mice showed biallelic inactivation of Bap1, consistent with its proposed role as a recessive cancer susceptibility gene. Unlike in WT mice, mesotheliomas from Bap1 þ/À mice did not require homozygous loss of Cdkn2a. However, normal mesothelial cells and mesothelioma cells from Bap1 þ/À mice showed downregulation of Rb through a p16(Ink4a)-independent mechanism, suggesting that predisposition of Bap1 þ/À mice to mesothelioma may be facilitated, in part, by cooperation between Bap1 and Rb. Drawing parallels to human disease, these unbiased genetic findings indicate that BAP1 mutation carriers are predisposed to the tumorigenic effects of asbestos and suggest that high penetrance of mesothelioma requires such environmental exposure. Cancer Res; 74(16); 4388-97. Ó2014 AACR.

Individuals harboring inherited heterozygous germline mutations in BAP1 are predisposed to a range of benign and malignant tumor types, including malignant mesothelioma, melanoma, and kidney carcinoma. However, evidence to support a tumor suppressive role for BAP1 in cancer remains contradictory. To test experimentally whether BAP1 behaves as a tumor suppressor, we monitored spontaneous tumor development in three different mouse models with germline heterozygous mutations in Bap1, including two models in which the knock-in mutations are identical to those reported in human BAP1 cancer syndrome families. We observed spontaneous malignant tumors in 54 of 93 Bap1-mutant mice (58%) versus 4 of 43 (9%) wild-type littermates. All three Bap1-mutant models exhibited a high incidence and similar spectrum of neoplasms, including ovarian sex cord stromal tumors, lung and mammary carcinomas, and spindle cell tumors. Notably, we also observed malignant mesotheliomas in two Bap1-mutant mice, but not in any wild-type animals. We further confirmed that the remaining wild-type Bap1 allele was lost in both spontaneous ovarian tumors and mesotheliomas, resulting in the loss of Bap1 expression. Additional studies revealed that asbestos exposure induced a highly significant increase in the incidence of aggressive mesotheliomas in the two mouse models carrying clinically relevant Bap1 mutations compared with asbestos-exposed wild-type littermates. Collectively, these findings provide genetic evidence that Bap1 is a bona fide tumor suppressor gene, and offer key insights into the contribution of carcinogen exposure to enhanced cancer susceptibility.

Malignant mesothelioma is a highly aggressive, asbestos-related cancer frequently marked by mutations of both NF2 and CDKN2A. We demonstrate that germline knockout of one allele of each of these genes causes accelerated onset and progression of asbestos-induced malignant mesothelioma compared with asbestosexposed Nf2 þ/À or wild-type mice. Ascites from some Nf2 þ/À ;Cdkn2a þ/À mice exhibited large tumor spheroids, and tail vein injections of malignant mesothelioma cells established from these mice, but not from Nf2 þ/À or wildtype mice, produced numerous tumors in the lung, suggesting increased metastatic potential of tumor cells from Nf2

Exposure to asbestos is causally associated with the development of malignant mesothelioma (MM), a cancer of cells lining the internal body cavities. MM is an aggressive cancer resistant to all current therapies. Once inhaled or ingested, asbestos causes inflammation in and around tissues that come in contact with these carcinogenic fibers. Recent studies suggest that inflammation is a major contributing factor in the development of many types of cancer, including MM. The NALP3/NLRP3 inflammasome, including the component ASC, is thought to be an important mediator of inflammation in cells that sense extracellular insults, such as asbestos, and activates a signaling cascade resulting in release of mature IL-1β and recruitment of inflammatory cells. To determine if inflammasome-mediated inflammation contributes to asbestos-induced MM, we chronically exposed Asc-deficient mice and wild-type littermates to asbestos and evaluated differences in tumor incidence and latency. The Asc-deficient mice showed significantly delayed tumor onset and reduced MM incidence compared to wild-type animals. We also tested whether inflammation-related release of IL-1β contributes to tumor development in an accelerated mouse model of asbestos-induced MM. Nf2+/−;Cdkn2a+/− mice exposed to asbestos in the presence of anakinra, an IL-1 receptor (IL-1R) antagonist, showed a marked delay in the median time of MM onset compared to similarly exposed mice given vehicle control (33.1 weeks versus 22.6 weeks, respectively). Collectively, these studies provide evidence for a link between inflammation-related IL-1β/IL-1R signaling and the development of asbestos-induced MM. Furthermore, these findings provide rationale for chemoprevention strategies targeting IL-1β/IL-1R signaling in high risk, asbestos-exposed populations.