Jinfei Xu

Germline mutation of Bap1 accelerates development of asbestos-induced malignant mesothelioma

Malignant mesotheliomas are highly aggressive tumors usually caused by exposure to asbestos. Germline-inactivating 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(+/-) knockout mouse model to assess its susceptibility to mesothelioma upon chronic exposure to asbestos. Bap1(+/-) 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(+/-) mice than in WT animals (median survival, 43 weeks vs. 55 weeks after initial exposure, respectively) and showed increased invasiveness and proliferation. No spontaneous mesotheliomas were seen in unexposed Bap1(+/-) mice followed for up to 87 weeks of age. Mesothelioma cells from Bap1(+/-) mice showed biallelic inactivation of Bap1, consistent with its proposed role as a recessive cancer susceptibility gene. Unlike in WT mice, mesotheliomas from Bap1(+/-) mice did not require homozygous loss of Cdkn2a. However, normal mesothelial cells and mesothelioma cells from Bap1(+/-) mice showed downregulation of Rb through a p16(Ink4a)-independent mechanism, suggesting that predisposition of Bap1(+/-) 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.

Losses of Both Products of the Cdkn2a/Arf Locus Contribute to Asbestos-Induced Mesothelioma Development and Cooperate to Accelerate Tumorigenesis

The CDKN2A/ARF locus encompasses overlapping tumor suppressor genes p16(INK4A) and p14(ARF), which are frequently co-deleted in human malignant mesothelioma (MM). The importance of p16(INK4A) loss in human cancer is well established, but the relative significance of p14(ARF) loss has been debated. The tumor predisposition of mice singly deficient for either Ink4a or Arf, due to targeting of exons 1α or 1β, respectively, supports the idea that both play significant and nonredundant roles in suppressing spontaneous tumors. To further test this notion, we exposed Ink4a(+/−) and Arf(+/−) mice to asbestos, the major cause of MM. Asbestos-treated Ink4a(+/−) and Arf(+/−) mice showed increased incidence and shorter latency of MM relative to wild-type littermates. MMs from Ink4a(+/−) mice exhibited biallelic inactivation of Ink4a, loss of Arf or p53 expression and frequent loss of p15(Ink4b). In contrast, MMs from Arf(+/−) mice exhibited loss of Arf expression, but did not require loss of Ink4a or Ink4b. Mice doubly deficient for Ink4a and Arf, due to deletion of Cdkn2a/Arf exon 2, showed accelerated asbestos-induced MM formation relative to mice deficient for Ink4a or Arf alone, and MMs exhibited biallelic loss of both tumor suppressor genes. The tumor suppressor function of Arf in MM was p53-independent, since MMs with loss of Arf retained functional p53. Collectively, these in vivo data indicate that both CDKN2A/ARF gene products suppress asbestos carcinogenicity. Furthermore, while inactivation of Arf appears to be crucial for MM pathogenesis, the inactivation of both p16(Ink4a) and p19(Arf) cooperate to accelerate asbestos-induced tumorigenesis.

Group I p21-Activated Kinases (PAKs) Promote Tumor Cell Proliferation and Survival through the AKT1 and Raf–MAPK Pathways

Group I p21–activated kinases (PAK) are important effectors of the small GTPases Rac and Cdc42, which regulate cell motility/migration, survival, proliferation, and gene transcription. Hyperactivation of these kinases have been reported in many tumor types, making PAKs attractive targets for therapeutic intervention. PAKs are activated by growth factor–mediated signaling and are negatively regulated by the tumor suppressor neurofibromatosis type 2 (NF2)/Merlin. Thus, tumors characterized by NF2 inactivation would be expected to show hyperactivated PAK signaling. On the basis of this rationale, we evaluated the status of PAK signaling in malignant mesothelioma, an aggressive neoplasm that is resistant to current therapies and shows frequent inactivation of NF2. We show that group I PAKs are activated in most mesotheliomas and mesothelioma cell lines and that genetic or pharmacologic inhibition of PAKs is sufficient to inhibit mesothelioma cell proliferation and survival. We also identify downstream effectors and signaling pathways that may contribute mechanistically to PAK-related tumorigenesis. Specifically, we show that inhibition of PAK results in attenuation of AKT and Raf–MAPK signaling and decreased tumor cell viability. Collectively, these data suggest that pharmacologic inhibition of group I PAKs may have therapeutic efficacy in tumors characterized by PAK activation. Mol Cancer Res; 10(9); 1178–88. ©2012 AACR.

DLX5 (Distal-less Homeobox 5) Promotes Tumor Cell Proliferation by Transcriptionally Regulating MYC

The human DLX homeobox genes, which are related to Dll (Drosophila distal-less gene), encode transcription factors that are expressed primarily in embryonic development. Recently, DLX5 was reported to act as an oncogene in lymphomas and lung cancers, although the mechanism is not known. The identification of target genes of DLX5 can facilitate our understanding of oncogenic mechanisms driven by overexpression of DLX5. The MYC oncogene is aberrantly expressed in many human cancers and regulates transcription of numerous target genes involved in tumorigenesis. Here we demonstrate by luciferase assay that the MYC promoter is specifically activated by overexpression of DLX5 and that two DLX5 binding sites in the MYC promoter are important for transcriptional activation of MYC. We also show that DLX5 binds to the MYC promoter both in vitro and in vivo and that transfection of a DLX5 expression plasmid promotes the expression of MYC in a dose-dependent manner in mammalian cells. Furthermore, overexpression of DLX5 results in increased cell proliferation by up-regulating MYC. Knockdown of DLX5 in lung cancer cells overexpressing DLX5 resulted in decreased expression of MYC and reduced cell proliferation, which was rescued by overexpression of MYC. Because DLX5 has a restricted pattern of expression in adult tissues, it may serve as a potential therapeutic target for the treatment of cancers that overexpress DLX5.

A Novel Recurrent Chromosomal Inversion Implicates the Homeobox Gene Dlx5 in T-Cell Lymphomas from Lck-Akt2 Transgenic Mice

The oncogene v-akt was isolated from a retrovirus that induced murine thymic lymphomas. Transgenic mice expressing a constitutively activated form of the cellular homologue Akt2 specifically in immature T cells develop spontaneous thymic lymphomas. We hypothesized that tumors from these mice might exhibit oncogenic chromosomal rearrangements that cooperate with activated Akt2 in lymphomagenesis. Cytogenetic analysis revealed a recurrent clonal inversion of chromosome 6, inv(6), in thymic lymphomas from multiple transgenic founder lines, including one line in which 15 of 15 primary tumors exhibited this same rearrangement. Combined fluorescence in situ hybridization, PCR, and DNA sequence analyses showed that the distal inv(6) breakpoint resides at the T-cell receptor beta chain locus, Tcrb. The proximal breakpoint maps to a region near a locus comprising the linked homeobox/transcription factor genes Dlx5 and Dlx6. Expression analysis of genes translocated to the vicinity of the Tcrb enhancer revealed that Dlx5 and Dlx6 are overexpressed in tumors exhibiting the inv(6). Experimental overexpression of Dlx5 in mammalian cells resulted in enhanced cell proliferation and increased colony formation, and clonogenic assays revealed cooperativity when both Dlx5 and activated Akt2 were coexpressed. In addition, DLX5, but not DLX6, was found to be abundantly expressed in three of seven human T-cell lymphomas tested. These findings suggest that the Dlx5 can act as an oncogene by cooperating with Akt2 to promote lymphomagenesis.

Inactivation of Tp53 and Pten drives rapid development of pleural and peritoneal malignant mesotheliomas: SEMENTINO et al.

Malignant mesothelioma (MM) is a therapy‐resistant cancer arising primarily from the lining of the pleural and peritoneal cavities. The most frequently altered genes in human MM are cyclin‐dependent kinase inhibitor 2A (CDKN2A), which encodes components of the p53 (p14ARF) and RB (p16INK4A) pathways, BRCA1‐associated protein 1 (BAP1), and neurofibromatosis 2 (NF2). Furthermore, the p53 gene (TP53) itself is mutated in ~15% of MMs. In many MMs, the PI3K–PTEN–AKT–mTOR signaling node is hyperactivated, which contributes to tumor cell survival and therapeutic resistance. Here, we demonstrate that the inactivation of both Tp53 and Pten in the mouse mesothelium is sufficient to rapidly drive aggressive MMs. PtenL/L;Tp53L/L mice injected intraperitoneally or intrapleurally with adenovirus‐expressing Cre recombinase developed high rates of peritoneal and pleural MMs (92% of mice with a median latency of 9.4 weeks and 56% of mice with a median latency of 19.3 weeks, respectively). MM cells from these mice showed consistent activation of Akt–mTor signaling, chromosome breakage or aneuploidy, and upregulation of Myc; occasional downregulation of Bap1 was also observed. Collectively, these findings suggest that when Pten and Tp53 are lost in combination in mesothelial cells, DNA damage is not adequately repaired and genomic instability is widespread, whereas the activation of Akt due to Pten loss protects genomically damaged cells from apoptosis, thereby increasing the likelihood of tumor formation. Additionally, the mining of an online dataset (The Cancer Genome Atlas) revealed codeletions of PTEN and TP53 and/or CDKN2A/p14ARF in ~25% of human MMs, indicating that cooperative losses of these genes contribute to the development of a significant proportion of these aggressive neoplasms and suggesting key target pathways for therapeutic intervention.

The homeoprotein Dlx5 drives murine T-cell lymphomagenesis by directly transactivating Notch and upregulating Akt signaling

Homeobox genes play a critical role in embryonic development, but they have also been implicated in cancer through mechanisms that are largely unknown. While not expressed during normal T-cell development, homeobox transcription factor genes can be reactivated via recurrent chromosomal rearrangements in human T-cell acute leukemia/lymphoma (T-ALL), a malignancy often associated with activated Notch and Akt signaling. To address how epigenetic reprogramming via an activated homeobox gene might contribute to T-lymphomagenesis, we investigated a transgenic mouse model with thymocyte-specific overexpression of the Dlx5 homeobox gene. We demonstrate for the first time that Dlx5 induces T-cell lymphomas with high penetrance. Integrated ChIP-seq and mRNA microarray analyses identified Notch1/3 and Irs2 as direct transcriptional targets of Dlx5, a gene signature unique to lymphomas from Lck-Dlx5 mice as compared to T-cell lymphomas from Lck-MyrAkt2 mice, which were previously reported by our group. Moreover, promoter/enhancer studies confirmed that Dlx5 directly transactivates Notch expression. Notch1/3 expression and Irs2-induced Akt signaling were upregulated throughout early stages of T-cell development, which promoted cell survival during β-selection of T lymphocytes. Dlx5 was required for tumor maintenance via its activation of Notch and Akt, as tumor cells were highly sensitive to Notch and Akt inhibitors. Together, these findings provide unbiased genetic and mechanistic evidence that Dlx5 acts as an oncogene when aberrantly expressed in T cells, and that it is a novel discovery that Notch is a direct target of Dlx5. These experimental findings provide mechanistic insights about how reactivation of the Dlx5 gene can drive T-ALL by aberrant epigenetic reprogramming of the T-cell genome.

Abstract 2833: Genetic and pharmacologic inhibition of mTOR delays mortality due to thymc lymphoma formation in mice and is associated with decreases in cell cycle proteins

The AKT/mTOR pathway is frequently hyperactivated in T-cell acute lymphoblastic leukemia (T-ALL). To model inhibition of this pathway in lymphoma, mice with T-lymphocyte-specific, constitutively active AKT (Lck-MyrAkt2) were crossed to mice with genetically reduced mTOR expression (knock-down, KD). Mice with genetic reduction of mTOR had increased survival by 10 weeks relative to wild type mTOR mice, though both developed thymic pre-T-cell lymphoblastic leukemia/lymphoma (pre-T LBL). Similarly, when mTOR wild type Lck-MyrAkt2 mice were treated for 8 weeks with the rapamycin analog, everolimus, an inhibitor of the mTOR TORC1 complex, survival was also increased. Gene expression profiling of thymic lymphomas from the mice revealed that mTOR KD was associated with decreased expression of Cdk6, a critical proliferative control node in T-cell development and oncogenic transformation. Pharmacologic inhibition of mTOR in tumor cells also decreased CDK6. The combination of a mTOR inhibitor (rapamycin) and a CDK4/6 inhibitor (PD-0332991, Palbociclib) synergistically decreased the overall viability and signaling downstream of drug targets in mouse lymphoma cells and in human T-ALL/LBL cell lines. This combination was also evaluated in mice using a disseminated leukemia model. In vivo treatment with this combination not only reduced tumor size by inhibiting tumor cell proliferation and arresting tumor cell cycle, but also increased overall survival. We are currently validating upstream regulators of Cdk6 as well as downstream targets in the pre-T LBL tumors from the mTOR deficient mice. Citation Format: Shuling Zhang, Joy M. Gary, John K. Simmons, Jinfei Xu, Benjamin J. Gamache, Ke Zhang, Nicholas Watson, Alexander L. Kovalchuk, Aleksandra M. Michalowski, Jin-Qiu Chen, Michelle A. Herrmann, Tuddow Thaiwong, Matti Kiupel, Wendy Dubois, Joseph R. Testa, Beverly A. Mock. Genetic and pharmacologic inhibition of mTOR delays mortality due to thymc lymphoma formation in mice and is associated with decreases in cell cycle proteins. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2833.

Abstract 2309: Murine model of dual mTORC kinase inhibition identifies CDK6 as a synergistic target in T-ALL

The PI3K/AKT/mTOR pathway is frequently hyperactivated in T- cell acute lymphoblastic leukemia (T-ALL). To model inhibition of this pathway in lymphoma, mice with constitutively-active AKT specific to T-lymphocytes (Lck-MyrAkt2) were crossed with mice that have genetically-reduced mTOR expression (mTOR knock-down, KD). Lck-MyrAkt2 mice with mTOR KD had altered thymic T-lymphocyte development; these mice also had delayed thymic pre-T cell lymphoblastic leukemia/lymphoma (pre-T LBL) progression and increased survival relative to wild type (WT) mTOR/ Lck-MyrAkt2 mice (average survival of 24 versus 14 weeks, respectively). Delayed pre-T LBL formation was also observed when WT mTOR/Lck-MyrAkt2 mice were treated for 8 weeks with the rapamycin analog, everolimus, an inhibitor of the mTOR TORC1 complex. Transcriptional profiling of thymic lymphomas from the WT vs KD mTOR/ Lck-MyrAkt2 mice revealed that mTOR KD was associated with decreased expression of Cdk6, a critical proliferative control node in T- cell development and oncogenic transformation. Pharmacologic inhibition of mTOR in tumor cells also decreased CDK6. Tumor cells from WT mTOR/Lck-MyrAkt2 mice were more sensitive to CDK4/6 inhibitors than tumor cells from KD mTOR/Lck-MyrAkt2 mice. Combining an mTOR inhibitor (rapamycin) with a CDK4/6 inhibitor (PD-0332991) synergistically inhibited the viability and decreased downstream signaling in both mouse lymphoma cells and human T-cell acute lymphoblastic leukemia (T-ALL) cell lines. Combining a dual mTORC inhibitor (PP242) with a CDK4/6 inhibitor decreased downstream signaling to an even greater extent in human T-ALL cell lines. Our results suggest that an mTORi/CDKi combination may be beneficial in the treatment of human T-ALL. Citation Format: Joy M. Gary, Jinfei Xu, John Simmons, Shuling Zhang, Benjamin Gamache, Ke Zhang, Alexander Kovalchuk, Aleksandra Michalowski, Jin-Qiu Chen, Michelle Herrmann, Wendy Dubois, Joseph Testa, Beverly A. Mock. Murine model of dual mTORC kinase inhibition identifies CDK6 as a synergistic target in T-ALL. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2309. doi:10.1158/1538-7445.AM2015-2309

Abstract 1555: Use of a mouse model of constitutive mTOR inhibition to identify molecular modulators of acquired resistance.

The AKT/PI3K/mTOR signaling pathway is hyperactivated in cancers, including both solid tumors and hematologic malignancies. This hyperactivation is often due to an overexpression or mutation in PI3K, which is upstream of AKT, or a down regulation of PTEN, an inhibitor of the PI3K pathway. mTOR is a central downstream target of this pathway and is a critical modulator of cell cycle, size, proliferation and metabolism. Resistance to mTOR inhibition has been reported in many tumors as well as in certain cell lines, resulting in decreased efficacy of cancer therapy involving this class of inhibitor. In these studies, mice (Mtortm1.1Lgm) with constitutive reduction in mTOR protein level were crossed to mice (Lck-MyrAkt2) that spontaneously develop thymic lymphomas due to constitutive AKT activation to simulate mTOR inhibitor treatment of thymic lymphoma development. The Lck-MyrAkt2 mice spontaneously develop thymic lymphomas at 10-20 weeks of age, and frequently harbor a 14;15 translocation that leads to the apposition of the TCRalpha and Myc genes. Tumor formation was significantly delayed in the mTOR knockdown x Lck-MyrAkt2 cohort, and the mice had a median survival of 181 days compared to 88 days in mice with normal mTOR expression. A similar t(14;15) translocation frequency was observed in thymic lymphomas of mice from both groups. To evaluate potential mTOR resistance mechanisms, gene expression profiling (GEP) of tumors arising from both groups of mice was performed. Molecular and functional enrichment analysis of the genes differentially expressed in the tumors arising despite mTOR inhibition was performed. Increased expression of an oncogenic transcription factor was observed, and is the subject of on-going investigation. Citation Format: Joy Gary, Jinfei Xu, John Simmons, Alexander Kovalchuk, Wendy Dubois, Joseph Testa, Beverly Mock. Use of a mouse model of constitutive mTOR inhibition to identify molecular modulators of acquired resistance. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1555. doi:10.1158/1538-7445.AM2013-1555