The BRD9/7 Inhibitor TP-472 Blocks Melanoma Tumor Growth by Suppressing ECM-Mediated Oncogenic Signaling and Inducing Apoptosis.

Melanoma accounts for the majority of all skin cancer-related deaths and only 1/3rd of melanoma patients with distal metastasis survive beyond five years. However, current therapies including BRAF/MEK targeted therapies or immunotherapies only benefit a subset of melanoma patients due to the emergence of intrinsic or extrinsic resistance mechanisms. Effective treatment of melanoma will thus require new and more effective therapeutic agents. Towards the goal of identifying new therapeutic agents, we conducted an unbiased, druggable epigenetic drug screen using a library of 32 epigenetic inhibitors obtained from the Structural Genome Consortium that targets proteins encoding for epigenetic regulators. This chemical genetic screening identified TP-472, which targets bromodomain-7/9, as the strongest inhibitor of melanoma growth in both short- and long-term survival assays and in mouse models of melanoma tumor growth. Mechanistically, using a transcriptome-wide mRNA sequencing profile we identified TP-472 treatment downregulates genes encoding various extracellular matrix (ECM) proteins, including integrins, collagens, and fibronectins. Reactome-based functional pathway analyses revealed that many of the ECM proteins are involved in extracellular matrix interactions required for cancer cell growth and proliferation. TP-472 treatment also upregulated several pro-apoptotic genes that can inhibit melanoma growth. Collectively, our results identify BRD7/9 inhibitor TP-472 as a potentially useful therapeutic agent for melanoma therapy.

Acidic mammalian chitinase is not a critical target for allergic airway disease.

The expression of acidic mammalian chitinase (AMCase) is associated with Th2-driven respiratory disorders. To investigate the potentially pathological role of AMCase in allergic airway disease (AAD), we sensitized and challenged mice with ovalbumin or a combination of house dust mite (HDM) plus cockroach allergen. These mice were treated or not treated with small molecule inhibitors of AMCase, which significantly reduced allergen-induced chitinolytic activity in the airways, but exerted no apparent effect on pulmonary inflammation per se. Transgenic and AMCase-deficient mice were also submitted to protocols of allergen sensitization and challenge, yet we found little or no difference in the pattern of AAD between mutant mice and wild-type (WT) control mice. In a separate model, where mice were challenged only with intratracheal instillations of HDM without adjuvant, total bronchoalveolar lavage (BAL) cellularity, inflammatory infiltrates in lung tissues, and lung mechanics remained comparable between AMCase-deficient mice and WT control mice. However BAL neutrophil and lymphocyte counts were significantly increased in AMCase-deficient mice, whereas concentrations in BAL of IL-13 were significantly decreased compared with WT control mice. These results indicate that, although exposure to allergen stimulates the expression of AMCase and increased chitinolytic activity in murine airways, the overexpression or inhibition of AMCase exerts only a subtle impact on AAD. Conversely, the increased numbers of neutrophils and lymphocytes in BAL and the decreased concentrations of IL-13 in AMCase-deficient mice challenged intratracheally with HDM indicate that AMCase contributes to the Th1/Th2 balance in the lungs. This finding may be of particular relevance to patients with asthma and increased airway neutrophilia.

Altered regulation of aquaporin gene expression in allergen and IL-13-induced mouse models of asthma.

IL-13 is known to affect many processes that contribute to an asthmatic phenotype, including inflammation, fibrosis, and mucus production. Members of the aquaporin (AQP) family of transmembrane water channels are targets of regulation in models of lung injury and inflammation. Therefore, we examined AQP mRNA and protein expression in allergen and IL-13-induced mouse models of asthma. Lungs from ovalbumin sensitized and ovalbumin challenged (OVA/OVA) and IL-13 treated mice showed airway thickening, increased mucus production, and pulmonary eosinophilia. Pulmonary function tests showed a significant increase in methacholine-induced airway hyperreactivity in OVA/OVA and IL-13-treated mice as compared with controls. Quantitative PCR analysis revealed differential regulation of AQPs in these two models. AQP1 and AQP4 mRNA expression was downregulated in the OVA/OVA model, but not in the IL-13 model. AQP5 mRNA was reduced in both models, whereas AQP3 was upregulated only in the IL-13 model. Western analysis showed that diminished expression of an apically localized aquaporin, (AQP5), and concomitant upregulation of a basolateral aquaporin (AQP3 or AQP4) are characteristic features of both inducible asthma models. These results demonstrate that aquaporins are common targets of gene expression in both allergen and IL-13 induced mouse models of asthma.

CAT-2 amplifies the agonist-evoked force of airway smooth muscle by enhancing spermine-mediated phosphatidylinositol-(4)-phosphate-5-kinase-gamma activity.

We investigated the effect the loss of the CAT-2 gene (CAT-2-/-) has on lung resistance (R(L)) and tracheal isometric tension. The R(L) of CAT-2-/- mice at a maximal dose of acetylcholine (ACh) was decreased by 33.66% (P = 0.05, n = 8) compared with that of C57BL/6 (B6) mice. The isometric tension of tracheal rings from CAT-2-/- mice showed a significant decrease in carbachol (CCh)-induced force generation (33.01%, P < 0.05, n = 8) compared with controls. The isoproterenol- or the sodium nitroprusside-induced relaxation was not affected in tracheal rings from CAT-2-/- mice. The activity of iNOS and arginase in lung tissue lysates of CAT-2-/- mice was indistinguishable from that of B6 mice. Furthermore, the expression of phospholipase-Cbeta (PLC-beta) and phosphatidylinositol-(4)-phosphate-5-kinase-gamma (PIP-5K-gamma) was examined in the lung tissue of CAT-2-/- and B6 mice. The expression of PIP-5K-gamma but not PLC-beta was significantly reduced in CAT-2-/- compared with B6 mice. The reduced airway smooth muscle (ASM) contractility to CCh seen in the CAT-2-/- tracheal rings was completely reversed by pretreating the rings with 100 muM spermine. This increase in the CAT-2-/- tracheal ring contraction upon spermine pretreatment correlated with a recovery of the expression of PIP-5K-gamma. Our data indicates that CAT-2 exerts control over ASM force development through a spermine-dependent pathway that directly correlates with the expression level of PIP-5K-gamma in the lung.

A role for endothelial selectins in allergic and nonallergic inflammatory disease.

BACKGROUND: Several studies indicate that selectin-mediated leukocyte migration may depend on the types of initiating inflammatory stimuli or on the vascular beds involved in the inflammatory response. Thus, targeting selectin interactions to treat inflammation may have variable effects depending on the site and origin of the inflammatory response. OBJECTIVE: To address whether selectin-mediated leukocyte recruitment is stimulus or tissue dependent. METHODS: We examined pulmonary and cutaneous allergic inflammatory responses and silica-induced nonallergic lung inflammation and fibrosis in wild-type and P- and E-selectin-deficient (P/E-/-) double knockout mice. Allergen-sensitized wild-type and P/E-/- double knockout mice were challenged either intradermally or via the airways to induce allergic responses in the skin or lung, respectively. Other animals were subjected to intranasal silica administration to induce a nonallergic lung inflammatory/fibrotic response. RESULTS: The P/E-/- mice exhibited significantly reduced allergic inflammation in the skin and lung. Allergic late-phase ear swelling and allergic lung airway hyperresponsiveness were also significantly attenuated in the P/E-/- mice compared with identically treated wild-type animals. In contrast, pulmonary inflammation and fibrosis induced by intranasal administration of silica particles resulted in a more severe phenotype in the P/E-/- mice. CONCLUSIONS: Selectin interactions drive allergic inflammation in the lung and skin. Silica-induced pulmonary inflammation and fibrosis, however, was more pronounced in the absence of selectin interactions, suggesting that selectin-mediated leukocyte migration may depend on the types of initiating inflammatory stimuli.

Three-dimensional sonography of the endometrium and adjacent myometrium: preliminary observations.

OBJECTIVE: By evaluating a series of patients undergoing pelvic sonography with routine 2-dimensional (2D) as well as 3-dimensional (3D) reconstructed images in the coronal plane, we attempted to characterize the types of additional information that can be obtained. METHODS: Ninety randomly selected patients undergoing transvaginal pelvic sonography were imaged according to a standard 2D protocol. A 3D uterine volume was then acquired in the sagittal plane and reconstructed in the coronal plane. The endometrium and surrounding myometrium were evaluated for architecture, masses, the relationship of masses to the endometrial cavity, and the anatomic configuration of the cavity. RESULTS: Ninety-one studies were obtained. Additional findings were obtained on the coronal view in 28 studies (30.8%). No additional findings were obtained in 63 studies (69.2%). Normal endometrial and myometrial findings were obtained by conventional 2D imaging in 42 of 91 patients. Of this group, additional findings were shown in 2 (5%) patients. Forty-nine of the 91 patients had abnormal findings by 2D imaging. Additional information was obtained in 26 (53%) of these patients. Added information included uterine anomalies, better definition of the endometrium, more accurate delineation and location of endometrial polyps, location of leiomyomas, visualization of cystic areas within the myometrium, and confirmation of the location of intrauterine devices. CONCLUSIONS: The 3D reconstructed view of the endometrium and adjacent myometrium appears to be most helpful after a conventional transvaginal study, showing abnormalities within the endometrium and myometrium but being of little added benefit if the conventional findings are normal.

Gob-5 contributes to goblet cell hyperplasia and modulates pulmonary tissue inflammation.

Gob-5 is a member of the calcium-activated chloride channel family and has been associated with allergic response in mouse models of pulmonary inflammation. Gene expression of Gob-5 has been shown to be induced in allergic airways and has been strongly associated with mucin gene regulation and goblet cell hyperplasia. We investigated the physiologic role of Gob-5 in murine models of pulmonary inflammation using mice deficient in Gob-5. After sensitization and aerosol challenge with ovalbumin (OVA), Gob-5 knockout mice exhibit significantly increased bronchoalveolar lavage (BAL) inflammation as compared with wild-type controls. The augmented inflammation in BAL consisted predominantly of neutrophils. Examination of perivascular inflammation revealed that tissue inflammation was decreased in OVA-challenged Gob-5-/- mice. OVA-challenged Gob-5 knockout mice also had decreased goblet cell hyperplasia as well as decreased mucus production. These mice also had decreased airway hypersensitivity after cholinergic provocation with methacholine. Gob-5 knockout mice were also challenged via intranasal LPS, a TLR-4 agonist. Gob-5-/- mice responded with increased neutrophilic BAL inflammation and decreased perivascular tissue inflammation as compared with wild-type controls. There was little effect on goblet cell hyperplasia and mucus production after LPS challenge. These observations reinforce findings that associate Gob-5 with goblet cell hyperplasia and mucus production in the allergic immune response, but also implicate Gob-5 in the regulation of tissue inflammation in the innate immune response.

TH1-mediated airway hyperresponsiveness independent of neutrophilic inflammation.

BACKGROUND: T(H)2-mediated allergic asthma is characterized by eosinophilia, mucus overproduction, and airway hyperresponsiveness (AHR). Although it is clear that T(H)2 cells and their cytokines play an important role in AHR, the roles of T(H)1 cells and neutrophils in AHR are controversial. OBJECTIVE: We sought to determine the roles of T(H)1 cells and neutrophils in AHR. METHODS: Ovalbumin-specific CD4(+) T cells were purified from DO11.10 mice, differentiated into T(H)1 cells, and injected into naive BALB/c, IL-4RalphaKO, or IL-8RKO mice. After ovalbumin antigen challenge, cytokine mRNA levels in lung samples, as well as inflammatory cell types and numbers in bronchoalveolar lavage fluid (BALF), were determined. AHR was assessed by measuring resistance in tracheostomized mice and enhanced pause in freely moving mice. RESULTS: T(H)1 cells induced AHR as robust as T(H)2 cells. They also induced lung inflammation dominated by neutrophils. Neither AHR nor inflammation were reduced when T(H)1 cells were transferred into IL-4RalphaKO mice. When IL-8RKO mice were used as recipients of T(H)1 cells, neutrophilia was greatly reduced, but the AHR was as strong as that seen in wild-type mice. On the other hand, dexamethasone treatment had no effect on neutrophilia but has significantly reduced AHR. Reduction in AHR was accompanied by a reduction in the numbers of lymphocytes and macrophages in BALF. CONCLUSIONS: T(H)1 cells can induce strong AHR independent of IL-4 and IL-13. The AHR is associated with the presence of lymphocytes and macrophages, but not neutrophils, in BALF. Our results point to a pathway whereby T(H)1 cells mediate AHR independent of neutrophilic inflammation.