Volatile organic compound analysis of malignant pleural mesothelioma chorioallantoic membrane xenografts.

Malignant pleural mesothelioma (MPM) is an aggressive cancer associated with asbestos exposure. MPM is often diagnosed late, at a point where limited treatment options are available, but early intervention could improve the chances of successful treatment for MPM patients. Biomarkers to detect MPM in at-risk individuals are needed to implement early diagnosis technologies. Volatile organic compounds (VOCs) have previously shown diagnostic potential as biomarkers when analysed in MPM patient breath. In this study, chorioallantoic membrane (CAM) xenografts of MPM cell lines were used as models of MPM tumour development for VOC biomarker discovery with the aim of generating targets for investigation in breath, biopsies or other complex matrices. VOC headspace analysis of biphasic or epithelioid MPM CAM xenografts was performed using solid-phase microextraction and gas chromatography-mass spectrometry. We successfully demonstrated the capture, analysis and separation of VOC signatures from CAM xenografts and controls. A panel of VOCs was identified that showed discrimination between MPM xenografts generated from biphasic and epithelioid cells and CAM controls. This is the first application of the CAM xenograft model for the discovery of VOC biomarkers associated with MPM histological subtypes. These findings support the potential utility of non-invasive VOC profiling from breath or headspace analysis of tissues for detection and monitoring of MPM.

BAP1 Loss Is Associated with Higher ASS1 Expression in Epithelioid Mesothelioma: Implications for Therapeutic Stratification.

The nuclear deubiquitylase BRCA1-associated protein 1 (BAP1) is frequently inactivated in malignant pleural mesothelioma (MPM) and germline BAP1 mutation predisposes to cancers including MPM. To explore the influence on cell physiology and drug sensitivity, we sequentially edited a predisposition mutation (w-) and a promoter trap (KO) into human mesothelial cells. BAP1w-/KO MeT5A cells express less BAP1 protein and phenocopy key aspects of BAP1 loss in MPM. Stable isotope labeling with amino acids in cell culture-mass spectrometry revealed evidence of metabolic adaptation, with concomitant alteration of cellular metabolites. In MeT5A, BAP1 deficiency reduces glycolytic enzyme levels but increases enzymes involved in the tricarboxylic acid cycle and anaplerotic pathways. Notably both argininosuccinate synthase 1 (ASS1), essential for cellular synthesis of arginine, and its substrate aspartate, are elevated in BAP1w-/KO MeT5A cells. Likewise, ASS1 expression is higher in BAP1-altered MPM cell lines, and inversely correlates with BAP1 in The Cancer Genome Atlas MESO dataset. Elevated ASS1 is also evident by IHC staining in epithelioid MPM lacking nuclear BAP1 expression, with improved survival among patients with BAP1-negative/ASS1-expressing tumors. Alterations in arginine metabolism may sensitize cells to metabolic drugs and we find that BAP1-negative/ASS1-expressing MPM cell lines are more sensitive to ASS1 inhibition, although not to inhibition of purine synthesis by mizoribine. Importantly, BAP1w-/KO MeT5A become desensitized to arginine deprivation by pegylated arginine deiminase (ADI-PEG20), phenocopying BAP1-negative/ASS1-expressing MPM cell lines. IMPLICATIONS: Our data reveal an interrelationship between BAP1 and arginine metabolism, providing a potential means of identifying patients with epithelioid MPM likely to benefit from ADI-PEG20.

The Chick Embryo Xenograft Model for Malignant Pleural Mesothelioma: A Cost and Time Efficient 3Rs Model for Drug Target Evaluation.

Malignant pleural mesothelioma (MPM) has limited treatment options and poor prognosis. Frequent inactivation of the tumour suppressors BAP1, NF2 and P16 may differentially sensitise tumours to treatments. We have established chick chorioallantoic membrane (CAM) xenograft models of low-passage MPM cell lines and protocols for evaluating drug responses. Ten cell lines, representing the spectrum of histological subtypes and tumour suppressor status, were dual labelled for fluorescence/bioluminescence imaging and implanted on the CAM at E7. Bioluminescence was used to assess viability of primary tumours, which were excised at E14 for immunohistological staining or real-time PCR. All MPM cell lines engrafted efficiently forming vascularised nodules, however their size, morphology and interaction with chick cells varied. MPM phenotypes including local invasion, fibroblast recruitment, tumour angiogenesis and vascular remodelling were evident. Bioluminescence imaging could be used to reliably estimate tumour burden pre- and post-treatment, correlating with tumour weight and Ki-67 staining. In conclusion, MPM-CAM models recapitulate important features of the disease and are suitable to assess drug targets using a broad range of MPM cell lines that allow histological or genetic stratification. They are amenable to multi-modal imaging, potentially offering a time and cost-efficient, 3Rs-compliant alternative to rodent xenograft models to prioritise candidate compounds from in vitro studies.

Combinatorial approaches for mitigating resistance to KRAS-targeted therapies.

Approximately 15% of all cancer patients harbor mutated KRAS. Direct inhibitors of KRAS have now been generated and are beginning to make progress through clinical trials. These include a suite of inhibitors targeting the KRASG12C mutation commonly found in lung cancer. We investigated emergent resistance to representative examples of different classes of Ras targeted therapies. They all exhibited rapid reactivation of Ras signaling within days of exposure and adaptive responses continued to change over long-term treatment schedules. Whilst the gene signatures were distinct for each inhibitor, they commonly involved up-regulation of upstream nodes promoting mutant and wild-type Ras activation. Experiments to reverse resistance unfortunately revealed frequent desensitization to members of a panel of anti-cancer therapeutics, suggesting that salvage approaches are unlikely to be feasible. Instead, we identified triple inhibitor combinations that resulted in more durable responses to KRAS inhibitors and that may benefit from further pre-clinical evaluation.

Aggressive uveal melanoma displays a high degree of centrosome amplification, opening the door to therapeutic intervention.

Uveal melanoma (UM) is the most common intraocular cancer in adults. Whilst treatment of primary UM (PUM) is often successful, around 50% of patients develop metastatic disease with poor outcomes, linked to chromosome 3 loss (monosomy 3, M3). Advances in understanding UM cell biology may indicate new therapeutic options. We report that UM exhibits centrosome abnormalities, which in other cancers are associated with increased invasiveness and worse prognosis, but also represent a potential Achilles' heel for cancer-specific therapeutics. Analysis of 75 PUM patient samples revealed both higher centrosome numbers and an increase in centrosomes with enlarged pericentriolar matrix (PCM) compared to surrounding normal tissue, both indicative of centrosome amplification. The PCM phenotype was significantly associated with M3 (t-test, p < 0.01). Centrosomes naturally enlarge as cells approach mitosis; however, whilst UM with higher mitotic scores had enlarged PCM regardless of genetic status, the PCM phenotype remained significantly associated with M3 in UM with low mitotic scores (ANOVA, p = 0.021) suggesting that this is independent of proliferation. Phenotypic analysis of patient-derived cultures and established UM lines revealed comparable levels of centrosome amplification in PUM cells to archetypal triple-negative breast cancer cell lines, whilst metastatic UM (MUM) cell lines had even higher levels. Importantly, many UM cells also exhibit centrosome clustering, a common strategy employed by other cancer cells with centrosome amplification to survive cell division. As UM samples with M3 display centrosome abnormalities indicative of amplification, this phenotype may contribute to the development of MUM, suggesting that centrosome de-clustering drugs may provide a novel therapeutic approach.

Loss of BAP1 expression is associated with an immunosuppressive microenvironment in uveal melanoma, with implications for immunotherapy development.

Immunotherapy using immune checkpoint inhibitors (ICIs) induces durable responses in many metastatic cancers. Metastatic uveal melanoma (mUM), typically occurring in the liver, is one of the most refractory tumours to ICIs and has dismal outcomes. Monosomy 3 (M3), polysomy 8q, and BAP1 loss in primary uveal melanoma (pUM) are associated with poor prognoses. The presence of tumour-infiltrating lymphocytes (TILs) within pUM and surrounding mUM - and some evidence of clinical responses to adoptive TIL transfer - strongly suggests that UMs are indeed immunogenic despite their low mutational burden. The mechanisms that suppress TILs in pUM and mUM are unknown. We show that BAP1 loss is correlated with upregulation of several genes associated with suppressive immune responses, some of which build an immune suppressive axis, including HLA-DR, CD38, and CD74. Further, single-cell analysis of pUM by mass cytometry confirmed the expression of these and other markers revealing important functions of infiltrating immune cells in UM, most being regulatory CD8+ T lymphocytes and tumour-associated macrophages (TAMs). Transcriptomic analysis of hepatic mUM revealed similar immune profiles to pUM with BAP1 loss, including the expression of IDO1. At the protein level, we observed TAMs and TILs entrapped within peritumoural fibrotic areas surrounding mUM, with increased expression of IDO1, PD-L1, and ß-catenin (CTNNB1), suggesting tumour-driven immune exclusion and hence the immunotherapy resistance. These findings aid the understanding of how the immune response is organised in BAP1 - mUM, which will further enable functional validation of detected biomarkers and the development of focused immunotherapeutic approaches. © 2020 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Targeting centrosome amplification, an Achilles' heel of cancer.

Due to cell-cycle dysregulation, many cancer cells contain more than the normal compliment of centrosomes, a state referred to as centrosome amplification (CA). CA can drive oncogenic phenotypes and indeed can cause cancer in flies and mammals. However, cells have to actively manage CA, often by centrosome clustering, in order to divide. Thus, CA is also an Achilles' Heel of cancer cells. In recent years, there have been many important studies identifying proteins required for the management of CA and it has been demonstrated that disruption of some of these proteins can cause cancer-specific inhibition of cell growth. For certain targets therapeutically relevant interventions are being investigated, for example, small molecule inhibitors, although none are yet in clinical trials. As the field is now poised to move towards clinically relevant interventions, it is opportune to summarise the key work in targeting CA thus far, with particular emphasis on recent developments where small molecule or other strategies have been proposed. We also highlight the relatively unexplored paradigm of reversing CA, and thus its oncogenic effects, for therapeutic gain.

Isoform-specific Ras signaling is growth factor dependent.

HRAS, NRAS, and KRAS isoforms are almost identical proteins that are ubiquitously expressed and activate a common set of effectors. In vivo studies have revealed that they are not biologically redundant; however, the isoform specificity of Ras signaling remains poorly understood. Using a novel panel of isogenic SW48 cell lines endogenously expressing wild-type or G12V-mutated activated Ras isoforms, we have performed a detailed characterization of endogenous isoform-specific mutant Ras signaling. We find that despite displaying significant Ras activation, the downstream outputs of oncogenic Ras mutants are minimal in the absence of growth factor inputs. The lack of mutant KRAS-induced effector activation observed in SW48 cells appears to be representative of a broad panel of colon cancer cell lines harboring mutant KRAS. For MAP kinase pathway activation in KRAS-mutant cells, the requirement for coincident growth factor stimulation occurs at an early point in the Raf activation cycle. Finally, we find that Ras isoform-specific signaling was highly context dependent and did not conform to the dogma derived from ectopic expression studies.

SRSF1 modulates PTPMT1 alternative splicing to regulate lung cancer cell radioresistance.

BACKGROUND: Radioresistance is the major cause of cancer treatment failure. Additionally, splicing dysregulation plays critical roles in tumorigenesis. However, the involvement of alternative splicing in resistance of cancer cells to radiotherapy remains elusive. We sought to investigate the key role of the splicing factor SRSF1 in the radioresistance in lung cancer. METHODS: Lung cancer cell lines, xenograft mice models, and RNA-seq were employed to study the detailed mechanisms of SRSF1 in lung cancer radioresistance. Clinical tumor tissues and TCGA dataset were utilized to determine the expression levels of distinct SRSF1-regulated splicing isoforms. KM-plotter was applied to analyze the survival of cancer patients with various levels of SRSF1-regulated splicing isoforms. FINDINGS: Splicing factors were screened to identify their roles in radioresistance, and SRSF1 was found to be involved in radioresistance in cancer cells. The level of SRSF1 is elevated in irradiation treated lung cancer cells, whereas knockdown of SRSF1 sensitizes cancer cells to irradiation. Mechanistically, SRSF1 modulates various cancer-related splicing events, particularly the splicing of PTPMT1, a PTEN-like mitochondrial phosphatase. Reduced SRSF1 favors the production of short isoforms of PTPMT1 upon irradiation, which in turn promotes phosphorylation of AMPK, thereby inducing DNA double-strand break to sensitize cancer cells to irradiation. Additionally, the level of the short isoform of PTPMT1 is decreased in cancer samples, which is correlated to cancer patients' survival. CONCLUSIONS: Our study provides mechanistic analyses of aberrant splicing in radioresistance in lung cancer cells, and establishes SRSF1 as a potential therapeutic target for sensitization of patients to radiotherapy.

RAS variant signalling.

RAS proteins are small GTPases that regulate signalling networks that control cellular proliferation and survival. They are frequently mutated in cancer and a commonly occurring group of developmental disorders called RASopathies. We discuss recent findings describing how RAS isoforms and different activating mutations differentially contribute to normal and disease-associated biology and the mechanisms that have been proposed to underpin this.

The deubiquitylase USP15 regulates topoisomerase II alpha to maintain genome integrity.

Ubiquitin-specific protease 15 (USP15) is a widely expressed deubiquitylase that has been implicated in diverse cellular processes in cancer. Here we identify topoisomerase II (TOP2A) as a novel protein that is regulated by USP15. TOP2A accumulates during G2 and functions to decatenate intertwined sister chromatids at prophase, ensuring the replicated genome can be accurately divided into daughter cells at anaphase. We show that USP15 is required for TOP2A accumulation, and that USP15 depletion leads to the formation of anaphase chromosome bridges. These bridges fail to decatenate, and at mitotic exit form micronuclei that are indicative of genome instability. We also describe the cell cycle-dependent behaviour for two major isoforms of USP15, which differ by a short serine-rich insertion that is retained in isoform-1 but not in isoform-2. Although USP15 is predominantly cytoplasmic in interphase, we show that both isoforms move into the nucleus at prophase, but that isoform-1 is phosphorylated on its unique S229 residue at mitotic entry. The micronuclei phenotype we observe on USP15 depletion can be rescued by either USP15 isoform and requires USP15 catalytic activity. Importantly, however, an S229D phospho-mimetic mutant of USP15 isoform-1 cannot rescue either the micronuclei phenotype, or accumulation of TOP2A. Thus, S229 phosphorylation selectively abrogates this role of USP15 in maintaining genome integrity in an isoform-specific manner. Finally, we show that USP15 isoform-1 is preferentially upregulated in a panel of non-small cell lung cancer cell lines, and propose that isoform imbalance may contribute to genome instability in cancer. Our data provide the first example of isoform-specific deubiquitylase phospho-regulation and reveal a novel role for USP15 in guarding genome integrity.

Patterns of BAP1 protein expression provide insights into prognostic significance and the biology of uveal melanoma.

Uveal melanoma (UM) is a rare aggressive intraocular tumour with a propensity for liver metastases, occurring in ∼50% of patients. The tumour suppressor BAP1 is considered to be key in UM progression. Herein, we present the largest study to date investigating cellular expression patterns of BAP1 protein in 165 UMs, correlating these patterns to prognosis. Full clinical, histological, genetic, and follow-up data were available for all patients. BAP1 gene sequencing was performed on a subset of 26 cases. An independent cohort of 14 UMs was examined for comparison. Loss of nuclear BAP1 (nBAP1) protein expression was observed in 54% (88/165) UMs. nBAP1 expression proved to be a significant independent prognostic parameter: it identified two subgroups within monosomy 3 (M3) UM, which are known to have a high risk of metastasis. Strikingly, nBAP1-positiveM3 UMs were associated with prolonged survival compared to nBAP1-negative M3 UMs (Log rank, p = 0.014). nBAP1 protein loss did not correlate with a BAP1 mutation in 23% (6/26) of the UMs analysed. Cytoplasmic BAP1 protein (cBAP1) expression was also observed in UM: although appearing 'predominantly diffuse' in most nBAP1-negative UM, a distinct 'focal perinuclear' expression pattern - localized immediately adjacent to the cis Golgi - was seen in 31% (18/59). These tumours tended to carry loss-of-function BAP1 mutations. Our study demonstrates loss of nBAP1 expression to be the strongest prognostic marker in UM, confirming its importance in UM progression. Our data suggest that non-genetic mechanisms account for nBAP1 loss in a small number of UMs. In addition, we describe a subset of nBAP1-negative UM, in which BAP1 is sequestered in perinuclear bodies, most likely within Golgi, warranting further mechanistic investigation.

New Perspectives, Opportunities, and Challenges in Exploring the Human Protein Kinome.

The human protein kinome comprises 535 proteins that, with the exception of approximately 50 pseudokinases, control intracellular signaling networks by catalyzing the phosphorylation of multiple protein substrates. While a major research focus of the last 30 years has been cancer-associated Tyr and Ser/Thr kinases, over 85% of the kinome has been identified to be dysregulated in at least one disease or developmental disorder. Despite this remarkable statistic, for the majority of protein kinases and pseudokinases, there are currently no inhibitors progressing toward the clinic, and in most cases, details of their physiologic and pathologic mechanisms remain at least partially obscure. By curating and annotating data from the literature and major public databases of phosphorylation sites, kinases, and disease associations, we generate an unbiased resource that highlights areas of unmet need within the kinome. We discuss strategies and challenges associated with characterizing catalytic and noncatalytic outputs in cells, and describe successes and new frontiers that will support more comprehensive cancer-targeting and therapeutic evaluation in the future. Cancer Res; 78(1); 15-29. ©2017 AACR.

Kinome-wide transcriptional profiling of uveal melanoma reveals new vulnerabilities to targeted therapeutics.

Metastatic uveal melanoma (UM) is invariably fatal, usually within a year of diagnosis. There are currently no effective therapies, and clinical studies employing kinase inhibitors have so far demonstrated limited success. This is despite common activating mutations in GNAQ/11 genes, which trigger signalling pathways that might predispose tumours to a variety of targeted drugs. In this study, we have profiled kinome expression network dynamics in various human ocular melanomas. We uncovered a shared transcriptional profile in human primary UM samples and across a variety of experimental cell-based models. The poor overall response of UM cells to FDA-approved kinase inhibitors contrasted with much higher sensitivity to the bromodomain inhibitor JQ1, a broad transcriptional repressor. Mechanistically, we identified a repressed FOXM1-dependent kinase subnetwork in JQ1-exposed cells that contained multiple cell cycle-regulated protein kinases. Consistently, we demonstrated vulnerability of UM cells to inhibitors of mitotic protein kinases within this network, including the investigational PLK1 inhibitor BI6727. We conclude that analysis of kinome-wide signalling network dynamics has the potential to reveal actionable drug targets and inhibitors of potential therapeutic benefit for UM patients.

Regulation of the cell cycle and centrosome biology by deubiquitylases.

Post-translational modification of proteins by ubiquitylation is increasingly recognised as a highly complex code that contributes to the regulation of diverse cellular processes. In humans, a family of almost 100 deubiquitylase enzymes (DUBs) are assigned to six subfamilies and many of these DUBs can remove ubiquitin from proteins to reverse signals. Roles for individual DUBs have been delineated within specific cellular processes, including many that are dysregulated in diseases, particularly cancer. As potentially druggable enzymes, disease-associated DUBs are of increasing interest as pharmaceutical targets. The biology, structure and regulation of DUBs have been extensively reviewed elsewhere, so here we focus specifically on roles of DUBs in regulating cell cycle processes in mammalian cells. Over a quarter of all DUBs, representing four different families, have been shown to play roles either in the unidirectional progression of the cell cycle through specific checkpoints, or in the DNA damage response and repair pathways. We catalogue these roles and discuss specific examples. Centrosomes are the major microtubule nucleating centres within a cell and play a key role in forming the bipolar mitotic spindle required to accurately divide genetic material between daughter cells during cell division. To enable this mitotic role, centrosomes undergo a complex replication cycle that is intimately linked to the cell division cycle. Here, we also catalogue and discuss DUBs that have been linked to centrosome replication or function, including centrosome clustering, a mitotic survival strategy unique to cancer cells with supernumerary centrosomes.

Transcriptomic and epigenetic regulation of disuse atrophy and the return to activity in skeletal muscle.

Physical inactivity and disuse are major contributors to age-related muscle loss. Denervation of skeletal muscle has been previously used as a model with which to investigate muscle atrophy following disuse. Although gene regulatory networks that control skeletal muscle atrophy after denervation have been established, the transcriptome in response to the recovery of muscle after disuse and the associated epigenetic mechanisms that may function to modulate gene expression during skeletal muscle atrophy or recovery have yet to be investigated. We report that silencing the tibialis anterior muscle in rats with tetrodotoxin (TTX)-administered to the common peroneal nerve-resulted in reductions in muscle mass of 7, 29, and 51% with corresponding reductions in muscle fiber cross-sectional area of 18, 42, and 69% after 3, 7, and 14 d of TTX, respectively. Of importance, 7 d of recovery, during which rodents resumed habitual physical activity, restored muscle mass from a reduction of 51% after 14 d TTX to a reduction of only 24% compared with sham control. Returning muscle mass to levels observed at 7 d TTX administration (29% reduction). Transcriptome-wide analysis demonstrated that 3714 genes were differentially expressed across all conditions at a significance of P ≤ 0.001 after disuse-induced atrophy. Of interest, after 7 d of recovery, the expression of genes that were most changed during TTX had returned to that of the sham control. The 20 most differentially expressed genes after microarray analysis were identified across all conditions and were cross-referenced with the most frequently occurring differentially expressed genes between conditions. This gene subset included myogenin (MyoG), Hdac4, Ampd3, Trim63 (MuRF1), and acetylcholine receptor subunit α1 (Chrna1). Transcript expression of these genes and Fboxo32 (MAFbx), because of its previously identified role in disuse atrophy together with Trim63 (MuRF1), were confirmed by real-time quantitative RT-PCR, and DNA methylation of their promoter regions was analyzed by PCR and pyrosequencing. MyoG, Trim63 (MuRF1), Fbxo32 (MAFbx), and Chrna1 demonstrated significantly decreased DNA methylation at key time points after disuse-induced atrophy that corresponded with significantly increased gene expression. Of importance, after TTX cessation and 7 d of recovery, there was a marked increase in the DNA methylation profiles of Trim63 (MuRF1) and Chrna1 back to control levels. This also corresponded with the return of gene expression in the recovery group back to baseline expression observed in sham-surgery controls. To our knowledge, this is the first study to demonstrate that skeletal muscle atrophy in response to disuse is accompanied by dynamic epigenetic modifications that are associated with alterations in gene expression, and that these epigenetic modifications and gene expression profiles are reversible after skeletal muscle returns to normal activity.-Fisher, A. G., Seaborne, R. A., Hughes, T. M., Gutteridge, A., Stewart, C., Coulson, J. M., Sharples, A. P., Jarvis, J. C. Transcriptomic and epigenetic regulation of disuse atrophy and the return to activity in skeletal muscle.

Quantification of spatiotemporal patterns of Ras isoform expression during development.

Ras proteins are important signalling hubs frequently dysregulated in cancer and in a group of developmental disorders called Rasopathies. Three Ras genes encode four proteins that differentially contribute to these phenotypes. Using quantitative real-time PCR (qRT-PCR) we have measured the gene expression profiles of each of the Ras isoforms in a panel of mouse tissues derived from a full developmental time course spanning embryogenesis through to adulthood. In most tissues and developmental stages we observe a relative contribution of KRas4B > > NRas ≥ KRas4A > HRas to total Ras expression with KRas4B typically representing 60-99% of all Ras transcripts. KRas4A is the most dynamically regulated Ras isoform with significant up-regulation of expression observed pre-term in stomach, intestine, kidney and heart. The expression patterns assist interpretation of the essential role of KRas in development and the preponderance of KRas mutations in cancer.

Combined Analyses of the VHL and Hypoxia Signaling Axes in an Isogenic Pairing of Renal Clear Cell Carcinoma Cells.

The loss of function of the Von Hippel-Lindau (VHL) tumor suppressor leads to the development of hypervascular tumors, exemplified by clear-cell-type renal cell carcinoma (RCC). VHL governs the adaptive responses to fluctuation of oxygen levels largely through the regulated suppression of hypoxia inducible factors (HIFs). Here, we combine proteome and phospho-proteomic analysis of isogenic 786-O RCC (±VHL) cells to compare signatures that reflect hypoxia and/or loss of VHL. VHL-independent hypoxic responses, notably include up-regulation of phosphorylation at Ser232 on the pyruvate dehydrogenase α subunit that is known to promote glycolysis. Hypoxic responses governed by VHL include up-regulation of known biomarkers of RCC (e.g., GLUT1, NDRG1) and the signaling adaptor molecule IRS-2. Notably, we also observe down-regulation of linked-components associated with the Jacobs-Stewart cycle, including the intracellular carbonic anhydrase II (CA2), which governs cellular response to CO2 fluctuations that often accompany hypoxia in tumors. Further studies indicate an unusual mechanism of control for CA2 expression that, at least in part, reflects enhanced activity of the NFκB pathway, which is associated with loss of VHL.

Loss of the deubiquitylase BAP1 alters class I histone deacetylase expression and sensitivity of mesothelioma cells to HDAC inhibitors.

Histone deacetylases are important targets for cancer therapeutics, but their regulation is poorly understood. Our data show coordinated transcription of HDAC1 and HDAC2 in lung cancer cell lines, but suggest HDAC2 protein expression is cell-context specific. Through an unbiased siRNA screen we found that BRCA1-associated protein 1 (BAP1) regulates their expression, with HDAC2 reduced and HDAC1 increased in BAP1 depleted cells. BAP1 loss-of-function is increasingly reported in cancers including thoracic malignancies, with frequent mutation in malignant pleural mesothelioma. Endogenous HDAC2 directly correlates with BAP1 across a panel of lung cancer cell lines, and is downregulated in mesothelioma cell lines with genetic BAP1 inactivation. We find that BAP1 regulates HDAC2 by increasing transcript abundance, rather than opposing its ubiquitylation. Importantly, although total cellular HDAC activity is unaffected by transient depletion of HDAC2 or of BAP1 due to HDAC1 compensation, this isoenzyme imbalance sensitizes MSTO-211H cells to HDAC inhibitors. However, other established mesothelioma cell lines with low endogenous HDAC2 have adapted to become more resistant to HDAC inhibition. Our work establishes a mechanism by which BAP1 loss alters sensitivity of cancer cells to HDAC inhibitors. Assessment of BAP1 and HDAC expression may ultimately help identify patients likely to respond to HDAC inhibitors.