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Introduced mutations in BAP1 that are predicted to disrupt the helix structure of the BAP1-ULD domain dramatically disassociate its binding to the ASXL2-AB box. The GST-UCH association assay was performed with highly purified recombinant ULD-WT, L-P, or F-P mutant proteins and/or ASXL2-AB box proteins.

Deleterious mutations of the ubiquitin carboxy-terminal hydrolase BAP1 found in cancers are predicted to encode inactive truncated proteins, suggesting that loss of enzyme function is a primary tumorigenic mechanism. However, many tumors exhibit missense mutations or in-frame deletions or insertions, often outside the functionally critical UCH domain in this tumor suppressor protein. Thus, precisely how these mutations inactivate BAP1 is unknown. Here, we show how these mutations affect BAP1 interactions with the Polycomb group-like protein, ASXL2, using combinations of computational modeling technology, molecular biology, and in vitro reconstitution biochemistry. We found that the BAP1–ASXL2 interaction is direct and high affinity, occurring through the ASXH domain of ASXL2, an obligate partner for BAP1 enzymatic activity. The ASXH domain was the minimal domain for binding the BAP1 ULD domain, and mutations on the surfaces of predicted helices of ASXH abolished BAP1 association and stimulation of BAP1 enzymatic activity. The BAP1-UCH, BAP1-ULD, and ASXH domains formed a cooperative stable ternary complex required for deubiquitination. We defined four classes of alterations in BAP1 outside the UCH domain, each failing to productively recruit ASXH to the wild-type BAP1 catalytic site via the ULD, resulting in loss of BAP1 ubiquitin hydrolase activity. Our results indicate that many BAP1 mutations act allosterically to inhibit ASXH binding, thereby leading to loss of enzyme activity. Small-molecule approaches to reactivate latent wild-type UCH activity of these mutants might be therapeutically viable.Significance: Combined computational and biochemical approaches demonstrate that the BAP1–ASXL2 interaction is direct and high affinity and that many BAP1 mutations act allosterically to inhibit BAP1–ASXL2 binding. Cancer Res; 78(5); 1200–13. ©2017 AACR.

The deletion R666-H669 in the ULD domain of BAP1 corresponding to that observed in a uveal melanoma completely abolished ASXL2 binding in vivo. HEK 293 cells were co-transiently transfected with plasmids expressing WT or mutant BAP1 and ASXL2 (261-649aa) proteins. At 48 h post-transfection, the cells were collected and whole cell lysates were used for co-IP with ANTI-Flag M2 Affinity Gel. Western blot analyses were performed with BAP1 (rAb) and Flag (rAb).

Supplemental figure legends

Both aLIMD2 (rAb) and aLIMD2 (mAb) detected the exogenous and endogenous LIMD2 protein in cell lines and human cancer specimens.

PDF file - 415K, Supplementary Figure 4. A and B, strong staining for the MM marker mesothelin seen in tumor tissues from both WT and Bap1+/- mice. C, Bap1 staining in MM tissue from WT mouse. MM, malignant mesothelioma; NI, normal intestinal mucosa, both stained positively for Bap1. D, lack of staining for Bap1 in MM from Bap1+/- mouse.

PDF file - 576K, Supplementary Figure 3. A, lower magnification view showing intestinal architecture of MM invasion into smooth muscle of intestinal wall. B, higher magnification view of invading tumor cells corresponding to area within rectangle in panel A.

Data collection and refinement statistics for the LIMD2 protein.

Proteins that communicate signals from the cytoskeleton to the nucleus are prime targets for effectors of metastasis as they often transduce signals regulating adhesion, motility, and invasiveness. LIM domain proteins shuttle between the cytoplasm and the nucleus, and bind to partners in both compartments, often coupling changes in gene expression to extracellular cues. In this work, we characterize LIMD2, a mechanistically undefined LIM-only protein originally found to be overexpressed in metastatic lesions but absent in the matched primary tumor. LIMD2 levels in fresh and archival tumors positively correlate with cell motility, metastatic potential, and grade, including bladder, melanoma, breast, and thyroid tumors. LIMD2 directly contributes to these cellular phenotypes as shown by overexpression, knockdown, and reconstitution experiments in cell culture models. The solution structure of LIMD2 that was determined using nuclear magnetic resonance revealed a classic LIM-domain structure that was highly related to LIM1 of PINCH1, a core component of the integrin-linked kinase–parvin–pinch complex. Structural and biochemical analyses revealed that LIMD2 bound directly to the kinase domain of integrin-linked kinase (ILK) near the active site and strongly activated ILK kinase activity. Cells that were null for ILK failed to respond to the induction of invasion by LIMD2. This strongly suggests that LIMD2 potentiates its biologic effects through direct interactions with ILK, a signal transduction pathway firmly linked to cell motility and invasion. In summary, LIMD2 is a new component of the signal transduction cascade that links integrin-mediated signaling to cell motility/metastatic behavior and may be a promising target for controlling tumor spread. Cancer Res; 74(5); 1390–403. ©2014 AACR.

Supplementary Methods, Figures 1-3, Tables 1-2 from In Vivo Profiling of Hypoxic Gene Expression in Gliomas Using the Hypoxia Marker EF5 and Laser-capture Microdissection

Legend for Supplementary Figures S1-S6. Also includes Supplementary References.

PDF file - 64K

PDF file - 326K, Supplementary Figure 2. A and C, H&E staining of MMs near pancreas in a WT mouse and liver in Bap1+/- mouse, respectively. B and D, Ki-67 staining showing higher proliferative index in MM from Bap1+/- mouse (D) than in tumor from WT mouse (B). E, histogram summarizing percentage of Ki-67-stained nuclei in MM tumors from WT (Bap1+/+) and Bap1+/- mice.

PDF file - 472K, Supplementary Figure 1. A, histogram depicting fiber length distribution in microns. B, dot blot illustrating fiber length and width distributions. Red line depicts mean length of fibers (6.78 μm). Mean fiber width was 0.42 microm. C and D, SEM images of fiber preparations at magnifications x2,500 and x5,000, respectively. Size bars indicate 10 μm and 1 μm in corresponding panels C and D.

Radiation therapy is commonly used to treat cancers in the definitive setting, but the local control often remains suboptimal, thereby motivating us to search for novel radiosensitizers. Recently a series of novel quinacrines based upon a previously reported dimeric chloroquine has been synthesized. We studied one of these agents, DQ660. By itself, the drug upregulates both p53 protein and p53 transcriptional targets and also increases apoptosis in p53-wildtype H460 lung cancer cells. Furthermore, we found that DQ660 is a potent radiosensitizer in vitro. This radiosensitization is not contingent upon wildtype p53 as it occurs in cells null for p53 and containing mutant p53. The combination of radiation and DQ660 proded significant growth retardation of human xenografts grown in vivo compared to radiation or DQ660 alone. We saw higher levels of γ-H2AX foci with combined treatment compared to either alone, suggesting that the drug increases DNA damage induction. Using agarose gel electrophoresis and kinetoplast DNA (kDNA) as a substrate, we found that DQ660 can inhibit topoisomerase II activity. DQ660 did not potentiate the phosphorylation of ATM at S1981 seen after radiation. However, the drug did lead to an increase in Ser428 phosphorylation of ATR. This phosphorylation was not seen in response to radiation alone, suggesting that DQ660 and radiation activate different signaling pathways in response to DNA damage. We saw similar results with another dimeric quinacrine, DQ550, suggesting that this class of agents may be radiosensitizers. Our findings have uncovered a novel class of radiosensitizers that work at nanomolar concentrations and will spur investigations for its use in combination therapy for tumors treated with radiation Citation Format: Ilias V. Karagounis, Jayashree Karar, Christoforos Thomas, Constantinos Koumenis, Jeffrey Winkler, Ravi Amaravadi, Amit Maity. A novel class of dimeric quinacrine compounds induce DNA damage and increase the anticancer activity of radiation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 217.

Deleterious mutations of the ubiquitin carboxy-terminal hydrolase BAP1 found in cancers are predicted to encode inactive truncated proteins, suggesting that loss of enzyme function is a primary tumorigenic mechanism. However, many tumors exhibit missense mutations or in-frame deletions or insertions, often outside the functionally critical UCH domain in this tumor suppressor protein. Thus, precisely how these mutations inactivate BAP1 is unknown. Here, we show how these mutations affect BAP1 interactions with the Polycomb group-like protein, ASXL2, using combinations of computational modeling technology, molecular biology, and in vitro reconstitution biochemistry. We found that the BAP1–ASXL2 interaction is direct and high affinity, occurring through the ASXH domain of ASXL2, an obligate partner for BAP1 enzymatic activity. The ASXH domain was the minimal domain for binding the BAP1 ULD domain, and mutations on the surfaces of predicted helices of ASXH abolished BAP1 association and stimulation of BAP1 enzymatic activity. The BAP1-UCH, BAP1-ULD, and ASXH domains formed a cooperative stable ternary complex required for deubiquitination. We defined four classes of alterations in BAP1 outside the UCH domain, each failing to productively recruit ASXH to the wild-type BAP1 catalytic site via the ULD, resulting in loss of BAP1 ubiquitin hydrolase activity. Our results indicate that many BAP1 mutations act allosterically to inhibit ASXH binding, thereby leading to loss of enzyme activity. Small-molecule approaches to reactivate latent wild-type UCH activity of these mutants might be therapeutically viable. Significance: Combined computational and biochemical approaches demonstrate that the BAP1–ASXL2 interaction is direct and high affinity and that many BAP1 mutations act allosterically to inhibit BAP1–ASXL2 binding. Cancer Res; 78(5); 1200–13. ©2017 AACR.

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. Cancer Res; 74(16); 4388–97. ©2014 AACR.

Inflammatory breast cancer (IBC) is the most lethal form of breast cancer that accounts for about 10% of breast cancer mortality annually in US. Poor prognosis is largely due to the high propensity of IBC tumors to develop distant metastasis that occurs directly from the gland epithelium and through lymphatic invasion in which dermal lymphatics are filled with tumor emboli. Owing to the complex metastatic process, the molecular basis of IBC aggressiveness is poorly understood, and no specific therapeutic target has been identified. Despite the lack of estrogen receptor α (ERα) in the majority of IBC tumors, estrogen may still play a role in these cancers through pathways that involve ERβ. Our tissue staining reveals expression of ERβ in more than 50% of IBCs that is reproduced in IBC cell lines. Furthermore, analysis of IBC datasets indicates correlation of receptor expression with good prognosis. We studied this association in preclinical models of IBC by knocking out ERβ in IBC cells. This promotes migration and invasion through cytoskeleton remodeling whereas re-expression of the receptor in knockout cells restores the cytoskeletal structure and migration to the levels of control cells. Consistent with increased migration, deletion of ERβ activates large gene networks of cell de-differentiation and cytokine synthesis that trigger tumor microenvironment responses to promote the motile phenotype of IBC cells. In contrast, ligands that activate the receptor inhibit signaling that contributes to metastasis in IBC. Analysis of an orthotopic xenograft model shows that IBC tumors lacking ERβ have higher propensity for metastasis compared with the ERβ-proficient tumors supporting the anti-metastatic activity of the receptor. Our findings point towards a role of ERβ in preventing distant metastases by inhibiting dissemination of IBC cells and maintaining the integrity of emboli. This function combined with distinct expression indicates the potential of ERβ to represent a unique prognostic marker and therapeutic target that can be utilized to repress IBC metastasis and eliminate its associated mortality. Citation Format: Thomas C, Karagounis I, Srivastava RK, Kumar S, Karar J, Chao H-H, Kazimierczak A, Bado I, Nikolos F, Leli N, Koumenis C, Krishnamurthy S, Ueno NT, Chakrabarti R, Maity A. Estrogen receptor β suppresses metastasis of inflammatory breast cancer by regulating cell cytoskeleton and cytokine signaling [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P5-05-10.

Hypoxia is a key determinant of tumor aggressiveness, yet little is known regarding hypoxic global gene regulation in vivo. We used the hypoxia marker EF5 coupled with laser-capture microdissection to isolate RNA from viable hypoxic and normoxic regions of 9L experimental gliomas. Through microarray analysis, we identified several mRNAs (including the HIF targets Vegf, Glut-1, and Hsp27) with increased levels under hypoxia compared with normoxia both in vitro and in vivo. However, we also found striking differences between the global in vitro and in vivo hypoxic mRNA profiles. Intriguingly, the mRNA levels of a substantial number of immunomodulatory and DNA repair proteins including CXCL9, CD3D, and RAD51 were found to be downregulated in hypoxic areas in vivo, consistent with a protumorigenic role of hypoxia in solid tumors. Immunohistochemical staining verified increased HSP27 and decreased RAD51 protein levels in hypoxic versus normoxic tumor regions. Moreover, CD8+ T cells, which are recruited to tumors upon stimulation by CXCL9 and CXCL10, were largely excluded from viable hypoxic areas in vivo. This is the first study to analyze the influence of hypoxia on mRNA levels in vivo and can be readily adapted to obtain a comprehensive picture of hypoxic regulation of gene expression and its influence on biological functions in solid tumors. Cancer Res; 71(3); 779–89. ©2011 AACR.

Colorectal cancer develops in the context of probably the most complex microenvironment in the human body: the colon microbiota comprised of >10¹³ bacteria, most of which are uncharacterized. Recently there has been great interest in defining the normal colon microbiome and how it is remodeled during disease states. Both commensal and infective enteropathogenic bacteria utilize a vast array of mechanisms to reduce inflammation in the gut, many of which are directed at the innate immune system, though undoubtedly, other cellular homeostasis systems are targeted. To limit inflammation bacterially encoded virulence proteins are injected into colon epithelial cells: many function by disrupting NFκB signaling. We have characterized the NLEE virulence protein which is encoded by EPEC bacteria and following injection by the T3SS, targets the TAB2 scaffold protein resulting in inactivation of the TAK1 kinase in the NFκB pathway. The Shao laboratory discovered that NLEE is an S-adenosyl-methionine (SAM)-dependent cysteine methyltransferase that methylates a single cysteine in the C4 zinc finger of TAB2 thereby unfolding and abolishing its function. The discovery of this first-in-class, Zinc Finger (ZF)-directed cysteine methytransferase, establishes the paradigm that eukaryotic ZFs can be subject to PTMs at the chelation residues that abolish finger function. We hypothesize that cysteine methylation of ZF domains is widespread and as yet, under appreciated PTM which regulates cellular signal transduction. The crystal structure of NLEE was solved and shows a novel SAM binding motif positioned in a deep cleft which confers a very high specificity for ZF binding and methylation. We searched for novel NLEE ZF-containing substrates in EPEC infected colon cells and discovered that the human DNA repair protein ZRANB3, a helicase which participates in the translesion synthesis repair pathway directly at replication forks, is a robust substrate for NLEE. Methylation of the C4 ZF of ZRANB3 is predicted to abolish its binding to K63-linked ubiquitinated-PCNA at stalled replication forks leading to fork collapse and replication stress. Additional NLEE substrates discovered in this DNA repair pathway include a PCNA-directed OTU class deubiquitinase and the NPL4 segregase complex, both of which limit repair functions. Together with recent work showing that bacterial virulence proteins can also down regulate the DNA mismatch repair system, these results suggest the remarkable conclusion that infectious colon bacteria have the capacity to abolish specific DNA repair systems in the colon epithelium. This work provides a unique and unexpected mechanistic link between the colon microbiome and genome stability in the colon. Whether a remodeled colon cancer specific microbiome contributes to tumor progression via induction of genome instability by inactivating DNA repair pathways is interesting to contemplate. Citation Format: Jayashree Karar, Hongzhuang Peng, Li Zhang, Qing Yao, Ilan Rosenshine, Feng Shao, Frank J. Rauscher. Control of DNA repair and genome stability by the colon microbiome: The EPEC bacterially encoded NLEE virulence effector protein methylates and inactivates the human ZRANB3 DNA repair helicase. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-323. doi:10.1158/1538-7445.AM2014-LB-323

Proteins that communicate signals from the cytoskeleton to the nucleus are prime targets for effectors of metastasis as they often transduce signals regulating adhesion, motility and invasiveness. LIM domain proteins shuttle between the cytoplasm and the nucleus, and bind to partners in both compartments, often coupling changes in gene expression to extracellular cues and hence are a prime target for deregulation during tumor progression and metastasis. The LIM domain is a modular Zn finger structure, often found tandemly repeated in proteins. These LIM arrays often serve as scaffolds for assembling signal transduction apparatus. In this work, we characterize LIMD2 which is unique in that it encodes a single LIM domain. LIMD2 was originally identified as a transcript overexpressed in metastatic lesions but absent in the matched primary tumor from the same patient suggesting that it may be either a marker or effector of metastatic spread. We have shown that LIMD2 levels in fresh and archival tumors positively correlate with cell motility, metastatic potential and tumor grade in many different tumor types including bladder, melanoma, breast and thyroid tumors. LIMD2 directly contributes to these cellular phenotypes as shown by overexpression, knockdown and reconstitution experiments in cell culture models. Tumor cells with poor metastatic capability are converted to highly motile, invasive cells by expression of LIMD2 suggesting a dominant gain of function action. To understand the molecular mechanisms of its biological effects we determined its solution structure using NMR. The structure studies of LIMD2 revealed a classic LIM-domain structure containing a rigid hydrophobic core which bound 2 molecules of Zn. The 3D structure of LIMD2 was most highly related to the LIM1 domain of PINCH1, a core component of the Integrin Linked Kinase-Parvin-Pinch (IPP) complex. The IPP complex plays a key role in cell-cell and cell matrix interaction by transducing signals from membrane bound integrins to the nucleus. Structural and biochemical analyses revealed that LIMD2 bound directly to the kinase domain of ILK near the active site and strongly activated ILK kinase activity in vitro. Immunolocalization studies showed that LIMD2 and components of the IPP complex co-existed in focal adhesion plaques. Cells which were null for ILK failed to respond to the induction of motility and invasion by ectopic expression of LIMD2. This strongly suggests that LIMD2 potentiates its biological effects through direct interactions with ILK, a signal transduction pathway firmly linked to cell motility and invasion. In summary, we have defined LIMD2 as a new component of the signal transduction cascade that links integrin-mediated signaling to cell motility/metastatic behavior and may be a promising target for controlling tumor spread. Citation Format: Hongzhuang Peng, Mehdi Taleb Zadeh Farrooji, Michael J. Osborne, Jeremy W. Prokop, Paul C. McDonald, Jayashree Karar, Zhaoyuan Hou, Mei He, Electron Kebebew, Torben Orntoft, Meenhard Herlyn, Andrew J. Caton, William Fredericks, Bruce Malkowicz, Christopher S. Paterno, Alexandra S. Carolin, David W. Speicher, Emmanuel Skordalakes, Qihong Huang, Shoukat S. Dedhar, Katherine L. B. Borden, Frank J. Rauscher. LIMD2 is a small LIM-only protein overexpressed in metastatic lesions which regulates cell motility and tumor progression by directly binding to and activating the integrin-linked-kinase. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 992. doi:10.1158/1538-7445.AM2014-992

Our goal was to investigate the effects of the Novartis compound NVP-BEZ235, a dual PI3K/mTOR inhibitor, on hypoxia-inducible factor-1 alpha (HIF-1α) expression. NVP-BEZ235 was much more potent than either the mTOR inhibitor rapamycin or the PI3K inhibitor LY294002 in blocking the hypoxic induction of HIF-1α in vitro, due to decreased protein translation. 7-methyl GTP chromatography showed that NVP-BEZ235 led to a robust recruitment of 4E-BP1 to eIF4E and a near absence of binding of eIF4G, consistent with inhibition of protein translation. NVP-BEZ235 also decreased expression of several proteins regulated by eIF4E including cyclin B1 and D1, survivin, and vascular endothelial growth factor. NVP-BEZ235 specifically decreased eIF4G but not eIF4E expression. As HIF-1α has been associated with adaptation under hypoxia, we examined the effect of NVP-BEZ235 on cell survival in low pO2 conditions. The drug increased killing of cells under hypoxia as measured by both short-term (MTT) and long-term (clonogenic) assays. To understand the mechanism behind this observation, we performed immunoblotting for factors associated with cell survival. In normoxia, Akt S473 phosphorylation decreased within an hour of NVP-BEZ235 treatment, but then increased by 24 hours. In contrast, under hypoxia, NVP-BEZ235 caused a prolonged suppression of Akt phosphorylation. Furthermore, we found a greater increase in PARP cleavage in hypoxic cells treated 1-16 hours after drug treatment than in normoxic cells, consistent with increased apoptosis. This was confirmed using an ELISA assay that measures cytoplasmic oligonucleosomes. We also found that NVP-BEZ235 increases autophagy as measured by (i) LC3-I to LC3-II conversion on immunoblotting, (ii) degradation of p62 and (iii) GFP-LC3 subcellular localization detected by immunofluorescence. Treatment of cells with the drug under hypoxia further increased autophagy, although it is unclear at this point whether this actually contributes to cell death or is a pro-survival mechanism. In conclusion, we have found that NVP-BEZ235 blocks HIF-1α induction and increases cell killing under hypoxia, which may have important implications for the use of this drug in cancer therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2800. doi:1538-7445.AM2012-2800