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.

Large homozygous deletions of 9p21 that inactivate CDKN2A, ARF, and MTAP are common in a wide variety of human cancers. The role for CDKN2A and ARF in tumorigenesis is well established, but whether MTAP loss directly affects tumorigenesis is unclear. MTAP encodes the enzyme methylthioadenosine phosphorylase, a key enzyme in the methionine salvage pathway. To determine if loss of MTAP plays a functional role in tumorigenesis, we have created an MTAP-knockout mouse. Mice homozygous for a MTAP null allele (Mtap lacZ ) have an embryonic lethal phenotype dying around day 8 postconception. Mtap/Mtap lacZ heterozygotes are born at Mendelian frequencies and appear indistinguishable from wild-type mice during the first year of life, but they tend to die prematurely with a median survival of 585 days. Autopsies on these animals reveal that they have greatly enlarged spleens, altered thymic histology, and lymphocytic infiltration of their livers, consistent with lymphoma. Immunohistochemical staining and fluorescence-activated cell sorting analysis indicate that these lymphomas are primarily T-cell in origin. Lymphoma-infiltrated tissues tend to have reduced levels of Mtap mRNA and MTAP protein in addition to unaltered levels of methyldeoxycytidine. These studies show that Mtap is a tumor suppressor gene independent of CDKN2A and

Pleural malignant mesothelioma is a therapy-resistant cancer affecting the serosal lining of the thoracic cavity. Mutations/deletions of BAP1, CDKN2A, and NF2 are the most frequent genetic lesions in human malignant mesothelioma. We introduced various combinations of these deletions in the pleura of conditional knockout (CKO) mice, focusing on the contribution of Bap1 loss. While homozygous CKO of Bap1, Cdkn2a, or Nf2 alone gave rise to few or no malignant mesotheliomas, inactivation of Bap1 cooperated with loss of either Nf2 or Cdkn2a to drive development of malignant mesothelioma in approximately 20% of double-CKO mice, and a high incidence (22/26, 85%) of malignant mesotheliomas was observed in Bap1; Nf2;Cdkn2a (triple)-CKO mice. Malignant mesothelioma onset was rapid in triple-CKO mice, with a median survival of only 12 weeks, and malignant mesotheliomas from these mice were consistently high-grade and invasive. Adenoviral-Cre treatment of normal mesothelial cells from Bap1;Nf2; Cdkn2a CKO mice, but not from mice with knockout of one or any two of these genes, resulted in robust spheroid formation in vitro, suggesting that mesothelial cells from Bap1;Nf2;Cdkn2a mice have stem cell-like potential. RNAseq analysis of malignant mesotheliomas from triple-CKO mice revealed enrichment of genes transcriptionally regulated by the polycomb repressive complex 2 (PRC2) and others previously implicated in known Bap1-related cellular processes. These data demonstrate that somatic inactivation of Bap1, Nf2, and Cdkn2a results in rapid, aggressive malignant mesotheliomas, and that deletion of Bap1 contributes to tumor development, in part, by loss of PRC2-mediated repression of tumorigenic target genes and by acquisition of stem cell potential, suggesting a potential avenue for therapeutic intervention. Significance: Combinatorial deletions of Bap1, Nf2, and Cdkn2a result in aggressive mesotheliomas, with Bap1 loss contributing to tumorigenesis by circumventing PRC2mediated repression of oncogenic target genes.

We report a high-risk cancer family with multiple mesotheliomas, cutaneous melanomas, basal cell carcinomas, and meningiomas segregating with a germline nonsense mutation in BAP1 (c.1938T>A; p.Y646X). Notably, most (four of five) mesotheliomas were peritoneal rather than the usually more common pleural form of the disease, and all five mesothelioma patients also developed second or third primary cancers, including two with meningiomas. Another family member developed both cutaneous melanoma and breast cancer. Two family members had basal cell carcinomas, and six others had melanocytic tumors, including four cutaneous melanomas, one uveal melanoma, and multiple benign melanocytic tumors. The family resides in a subtropical area, and several members had suspected exposure to asbestos either occupationally or in the home. We hypothesize that the concurrence of a genetic predisposing factor and environmental exposure to asbestos and UV irradiation contributed to the high incidence of multiple cancers seen in this family, specifically mesothelioma and various skin tumors, respectively.