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Malignant mesothelioma is a locally aggressive and highly lethal neoplasm. Dysregulation and activation of Gas6/AXL tyrosine kinase signaling are associated with mesothelioma progression, but the mechanisms of these AXL tumorigenic roles are poorly understood. p53 mutants in lung carcinoma upregulate AXL expression by binding and acetylating the AXL promoter. Although TP53 mutations are uncommon in mesothelioma, we hypothesized that these tumors might have alternative feedback mechanisms between AXL and p53. In the current report, we investigated AXL regulation of TP53 transcription, expression, and biological function in mesothelioma. AXL expression was stronger in mesothelioma than most of the other tumor types from the TCGA gene expression profile dataset. AXL knockdown by shRNA induced wild-type and mutant p53 expression in mesothelioma cell lines, suggesting that AXL pro-tumorigenic roles result in part from the suppression of p53 function. Likewise, induced AXL inhibited expression of wild type p53 in COS-7 cells and 293T cells. Immunofluorescence staining showed nuclear colocalization of AXL and p53, however, association of AXL and p53 was not demonstrated in immunoprecipitation complexes. The AXL effects on p53 expression resulted from the inhibition of TP53 transcription, as demonstrated by qRT-PCR after AXL silencing and TP53 promotor dual luciferase activity assays. Chromatin immunoprecipitation-qPCR and sequencing showed that AXL bound to the initial 600 bp sequence at the 5&prime, end of the TP53 promoter. AXL inhibition (shRNA or R428) reduced mesothelioma cell viability, migration, and invasion, whereas TP53 shRNA knockdown attenuated antiproliferative, migration, and invasive effects of AXL silencing or AXL inactivation in these cells. These studies demonstrate a novel feedback regulation loop between AXL and p53, and provide a rationale for mesothelioma therapies targeting AXL/p53 signaling.

// Bin Li 1, 2, 3 , Guangsen Zhang 2 , Cui Li 1 , Ruijuan Li 2 , Jingchen Lu 3 , Zhengxi He 3 , Quan Wang 3 , Zhenzi Peng 1 , Jun Wang 1 , Yeping Dong 1 , Chunfang Zhang 1 , Jie Qiong Tan 4 , Nacef Bahri 5 , Yuexiang Wang 5, 6 and Chaojun Duan 1 1 Medical Research Center, Key Laboratory of Cancer Proteomics of Chinese Ministry of Health, Xiangya Hospital, Central South University, Changsha, People’s Republic of China 2 Division of Hematology, Institute of Molecular Hematology, The Second Xiang Ya Hospital, Central South University, Changsha, People’s Republic of China 3 Division of Oncology, Xiangya Hospital, Central South University, Changsha, People’s Republic of China 4 State Key Laboratory of Medical Genetics, Xiangya Medical School, Central South University, Changsha, People’s Republic of China 5 Department of Pathology, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts, USA 6 The Institute of Health Sciences, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences/Shanghai Jiao Tong University School of Medicine, Shanghai, People’s Republic of China Correspondence to: Chaojun Duan, email: duancjxy@126.com Keywords: Lyn, CEL Received: October 04, 2015 Accepted: July 26, 2016 Published: August 19, 2016 ABSTRACT The Fip1-like1 (FIP1L1)–platelet-derived growth factor receptor alpha (PDGFRA) (F/P) oncogene can cause chronic eosinophilic leukemia (CEL), but requires IL-5 cytokine participation. In this study, we investigate the mechanism of F/P in collaboration with IL-5 in CEL. The results showed that Lyn, a key effector in the IL-5-motivated eosinophil production, is extensively activated in F/P-positive CEL cells. Lyn can associate and phosphorylate IL-5 receptor α (IL-5RA) in F/P-positive cells. Moreover, the activation of Lyn and IL-5R kinase were strengthened when the cells were stimulated by IL-5. Lyn inhibition in F/P-positive CEL cells attenuated cellular proliferation, induced apoptosis, and blocked cell migration and major basic protein (MBP) release. We identified the FIP1L1-PDGFRA/JAK2/Lyn/Akt complex in the F/P-expressing cells which can be disrupted by dual inhibition of JAK2 and Lyn, repressing cell proliferation in both EOL-1(F/P-positive human eosinophilic cell line) and imatinib-resistance (IR) cells. Altogether, our data demonstrate that Lyn is a vital downstream kinase activated by F/P converged with IL-5 signals in CEL cells. Lyn activate and expand IL-5RA intracellular signaling through FIP1L1-PDGFRA/JAK2/Lyn/Akt network complex, provoking eosinophils proliferation and exaggerated activation manifested as CEL.

Traumatized lipomas with degenerative change may demonstrate histopathologic features that mimic atypical lipomatous tumor (ALT). Previously reported series of ALT involving the oral cavity preceded routine use of MDM2 and CDK4 immunohistochemistry. Our aim is to evaluate MDM2 and CDK4 immunohistochemical expression in adipocytic tumors arising in this site, in conjunction with the histiocytic marker PU.1, to determine whether MDM2 and CDK4 impacts classification. 17 cases originally diagnosed as ALT were retrieved and immunohistochemical studies for MDM2, CDK4 and PU.1 were performed. FISH analysis for MDM2 amplification was performed in select cases. For this study group, the male:female ratio was 9:8 and the median age was 62 (range 41–88). All 17 cases presented as well- or predominantly well-circumscribed proliferations of variably sized, mature adipocytes exhibiting uni- or multi-vacuolation with occasional scalloped nuclei and mild nuclear atypia. Variable amounts of fibrous stroma with focal myxoid change and bland spindle cells were identified in 14/17 cases. Lipoblasts or atypical hyperchromatic stromal cells were not identified in any cases. 14 of 17 cases were negative for MDM2 and CDK4 in tumor cells and 11 of these 14 showed weak nuclear positivity for MDM2 in histiocytes. 3 of 17 cases showed weak, multifocal immunohistochemical expression of MDM2 and CDK4. PU.1 highlighted histiocytes in all 17 cases. FISH analysis for MDM2 amplification was negative in all 3 cases with weak MDM2/CDK4 expression. All cases were reclassified as lipoma with degenerative changes. ALT, in all likelihood, is less common than previously thought in this anatomic location and best diagnosed with ancillary studies. MDM2 expression in histiocytes is best interpreted in conjunction with CDK4 immunohistochemistry and confirmatory FISH for MDM2 amplification.

Dermatofibrosarcoma protuberans (DFSP) is an aggressive PDGFB-dependent cutaneous sarcoma characterized by infiltrative growth and frequent local recurrences. Some DFSP progress to a higher-grade fibrosarcomatous form, with rapid growth and increased risk of metastasis. Imatinib provides clinical benefit in approximately 50% of patients with unresectable or metastatic DFSP. However, efficacious medical therapies have not been developed for imatinib-resistant DFSP. We established a model of imatinib-resistant DFSP and evaluated CDK4/6 inhibition as a genomically credentialed targeted therapy. DFSP105, an imatinib-resistant human cell line, was established from a fibrosarcomatous DFSP (FS-DFSP), and was studied by SNP arrays and sequencing to identify targetable genomic alterations. Findings were validated in vitro and in vivo, and confirmed in a series including 12 DFSP and 6 FS-DFSP. SNP analysis of DFSP105 revealed a homozygous deletion encompassing CDKN2A and CDKN2B. The resultant p16 loss implicated CDK4/6 as a potential therapeutic target in DFSP. We further demonstrated CDKN2A homozygous deletion in 1 of 12 conventional DFSP and 2 of 6 FS-DFSP, whereas p16 expression was lost in 4 of 18 DFSP. In vitro treatment of DFSP105 with two structurally distinct selective CDK4/6 inhibitors, PD-0332991 and LEE011, led to inhibition of RB1 phosphorylation and inhibition of proliferation (GI50 160 nmol/L and 276 nmol/L, respectively). In vivo treatment of DFSP105 with PD-0332991 (150 mg/kg) inhibited xenograft growth in mice, in comparison with imatinib-treated or -untreated tumors. In conclusion, CDKN2A deletion can contribute to DFSP progression. CDK4/6 inhibition is a preclinically effective treatment against p16-negative, imatinib-resistant FS-DFSP, and should be evaluated as a therapeutic strategy in patients with unresectable or metastatic imatinib-resistant DFSP. Mol Cancer Ther; 14(6); 1346–53. ©2015 AACR.

Introduction: We previously demonstrated that dystrophin inactivation results from genomic DMD 5-end deletions in advanced GIST and leiomyosarcoma, fostering migration, invasion, anchorage independence, and invadopodia formation. Hence, dystrophin inactivation, the most common cause of muscular dystrophy, is also a tumorigenic mechanism in sarcoma. CDKN2A/CDKN2B inactivation is an early event in peripheral nerve sheath tumor (PNST) progression, associated with progression from neurofibroma (NF) to atypical NF. PRC2 inactivation is a later event during progression of some MPNST. Additional biologic markers are needed to distinguish NF from low-grade MPNST. In the present study, we identify and characterize dystrophin dysregulation at an early phase in PNST progression. Methods: Dystrophin immunohistochemistry (IHC) was performed in FFPE sections using the DYS1 monoclonal antibody. DYS1 and other dystrophin monoclonals were used in immunoblotting (IB) studies of snap-frozen PNST. Genomic analyses were performed by targeted gene capture and sequencing (Haloplex custom panel of 812 cancer-associated genes) and Cytoscan HD SNPs. Dystrophin restorations were performed in MPNST cultures using Dp116 (puromycin resistance) and Dp427 (neomycin resistance), either singly or in combination. Results: Dystrophin IHC against TMAs containing common subtypes of benign and malignant soft-tissue tumors showed that most soft-tissue tumor subtypes lacked detectable dystrophin expression whereas 18 of 19 schwannomas expressed dystrophin strongly and diffusely, at levels comparable to that in non-neoplastic skeletal muscle. Dystrophin was also expressed strongly in the neoplastic Schwann-cell component of neurofibromas (NF), whereas dystrophin expression was absent or weak in each of 17 MPNST. IB studies in a separate series of snap-frozen specimens showed that the dystrophin expressed strongly in schwannomas (classic histology = 4; cellular = 6) was coexpression of 427kDa (so-called myogenic) and 116kDa Schwann-cell specific isoforms. IB studies in snap-frozen MPNST showed loss of dystrophin 116kDa and 427kDa expression in 16 cases, retained expression in 1 case, and retained expression of 427kDa only, in 1 case. Genomic surveys demonstrated intragenic DMD deletions or inactivating mutations in 4 of 11 MPNST. Each of 3 NF transitioning to MPNST had strong dystrophin expression in the NF component but not in the associated MPNST. Conjoined restoration of 116kDa and 427kDa dystrophin isoforms (but not restoration of the 116kDa isoform, on its own) inhibited MPNST migration. IHC and IB studies (in progress) suggest that PNST dystrophin inactivation occurs concurrent with or subsequent to CDKN2A/CDKN2B inactivation during transition from NF to low-grade MPNST, and prior to PRC2 inactivation. Conclusions: We show that Dp116 and Dp427 dystrophin isoforms have tumor-suppressor properties in PNST. Extinction of these isoforms occurs in most MPNST, and in laboratory models is associated with increased cell migration. Dystrophin inactivation coincides with transition from NF to low-grade MPNST; therefore, dystrophin IHC might be useful in diagnostic distinction between atypical NF and MPNST. Citation Format: Inga-Marie Schaefer*, Armelle Dufresne*, Nacef Bahri, Marije A. J. de Rooij, Stacy M. Yanofsky, Yuexiang Wang, Chandrajit P. Raut, Laurence H. Baker, Adrian Marino-Enriquez, Matt van de Rijn, Jonathan A. Fletcher. Dystrophin is a tumor suppressor in peripheral nerve sheath tumors [abstract]. In: Proceedings of the AACR Conference on Advances in Sarcomas: From Basic Science to Clinical Translation; May 16-19, 2017; Philadelphia, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(2_Suppl):Abstract nr B16.