Shoukat Dedhar

A dual role for integrin-linked kinase in platelets: regulating integrin function and alpha-granule secretion.

Integrin-linked kinase (ILK) has been implicated in the regulation of a range of fundamental biological processes such as cell survival, growth, differentiation, and adhesion. In platelets ILK associates with β1- and β3-containing integrins, which are of paramount importance for the function of platelets. Upon stimulation of platelets this association with the integrins is increased and ILK kinase activity is up-regulated, suggesting that ILK may be important for the coordination of platelet responses. In this study a conditional knockout mouse model was developed to examine the role of ILK in platelets. The ILK-deficient mice showed an increased bleeding time and volume, and despite normal ultrastructure the function of ILK-deficient platelets was decreased significantly. This included reduced aggregation, fibrinogen binding, and thrombus formation under arterial flow conditions. Furthermore, although early collagen stimulated signaling such as PLCγ2 phosphorylation and calcium mobilization were unaffected in ILK-deficient platelets, a selective defect in α-granule, but not dense-granule, secretion was observed. These results indicate that as well as involvement in the control of integrin affinity, ILK is required for α-granule secretion and therefore may play a central role in the regulation of platelet function.

Discovery of 4-Hydroxy-3-(3-(phenylureido)benzenesulfonamides as SLC-0111 Analogues for the Treatment of Hypoxic Tumors Overexpressing Carbonic Anhydrase IX

Herein we report the 2-aminophenol-4-sulfonamide 1 and its ureido derivatives 2-23 as inhibitors of the carbonic anhydrase (CA, EC 4.2.1.1) enzymes as analogues of the hypoxic tumor phase II entering drug SLC-0111. This scaffold may determine preferential rotational isomers to selectively interact within the tumor-associated CAs. Most of the compounds indeed showed in vitro selective inhibition of the tumor associated CA isoforms IX and XII. The most potent derivative within the series was 11 ( KIs of 2.59 and 7.64 nM on hCA IX and XII, respectively), which shares the 4-fluorophenylureido tail with the clinical candidate. We investigated by means of X-ray crystallographic studies the binding modes of three selected compounds of this series to CA I. The evaluation of therapeutic efficacy of compound 11 in an orthotopic, syngeneic model of CA IX-positive breast cancer in vivo showed close matching antitumoral effects and tolerance with SLC-0111.

Abstract 841: Therapeutic targeting of cancer cells in the hypoxic microenvironment using an orally bioavailable small molecule inhibitor of carbonic anhydrase IX

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CAnnCarbonic anhydrase IX (CAIX) is a membrane-bound, hypoxia-inducible enzyme that is highly expressed in many types of solid tumors, is an independent marker of poor prognosis and functions as a critical component of the pH regulatory machinery required by hypoxic cancer cells for survival, invasion and metastasis. These attributes make CAIX an attractive therapeutic target for cancer therapy. The development of pharmacologic inhibitors that selectively target tumor-associated, extracellular carbonic anhydrases (CAs) without “off-target” inhibition of cytosolic isoforms is critical for their use as cancer therapeutics. Here, we characterize an orally bioavailable, highly selective small molecule inhibitor of CAIX and CAXII, SLC-0111, and investigate its efficacy when used as a monotherapy and in combination with conventional chemotherapy in vivo. SLC-0111 exhibited a favorable in vitro ADME profile, including IC50 values of >100 mM against most CYP isoforms, an IC50 for hERG inhibition of >30 mM and absence of mutagenic properties. Incubation of the compound with 67NR cells constitutively expressing human CAIX significantly suppressed, in a dose-dependent fashion, a drop in extracellular pH in a cell-based CAIX activity assay. The active compound was formulated as a self-emulsifying liquid for oral administration in vivo and evaluation of plasma levels showed good oral bioavailability of 40%. Toxicity studies involving repeat dosing by oral gavage for 7 days established maximum-tolerated doses in excess of 1,000 and 750 mg/kg for rats and dogs, respectively. Daily oral administration of SLC-0111 to mice harboring established MDA-MB-231 LM2-4 orthotopic human breast tumors, a model of hypoxic, CAIX-positive triple negative metastatic breast cancer, resulted in a significant, dose-dependent reduction in tumor growth, with 30% inhibition at a dose of 50 mg/kg, no toxicity and a 10-fold therapeutic window. Treatment also reduced viable tumor volume as determined by 18F-deoxyglucose uptake detected by positron emission tomography. Furthermore, treatment of mice with SLC-0111 in combination with paclitaxel resulted in significantly reduced tumor growth compared to either treatment administered alone, with no additional toxicity and plasma levels of SLC-0111 similar to those observed with its use as a monotherapy. Plasma levels of SLC-0111 showed dose proportionality and analysis of tumor levels demonstrated a greater than proportional relationship, with a 4-fold increase in dose (25-100 mg/kg) resulting in more than a 13-fold increase in tumor levels of the compound. Collectively, these data demonstrate the potential utility of SLC-0111 as a safe, target-specific inhibitor of CAIX for the treatment of cancer, particularly in combination with conventional chemotherapy.nnCitation Format: Paul C. McDonald, Jasbinder Sanghera, Madhu Singh, Yuanmei Lou, Marylou Vallejo, Claudiu T. Supuran, Shoukat Dedhar. Therapeutic targeting of cancer cells in the hypoxic microenvironment using an orally bioavailable small molecule inhibitor of carbonic anhydrase IX. [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 841. doi:10.1158/1538-7445.AM2014-841

The Role of Integrin-Linked Kinase in Cancer Development and Progression

Integrin-Linked Kinase (ILK) is a multifunctional intracellular effector of cell-extracellular matrix interactions. ILK is a central component of focal adhesions and regulates many cellular processes critical for cancer progression, including proliferation, survival, epithelial–mesenchymal transition, migration, invasion, and angiogenesis. The activity and expression of ILK are controlled by a network of intracellular and intercellular processes that result in aberrant ILK expression and signaling in many human malignancies. While the causes of pathologic ILK expression may be varied and remain to be fully elucidated, accumulating evidence supports the targeting of ILK for therapeutic intervention in cancer. Currently, agents designed to interfere with aberrant ILK expression and activity are being evaluated in preclinical models. Interrogation of the ILK interactome using cutting-edge proteomic strategies is also uncovering novel interactions and cellular functions of ILK that may have important implications for the development of effective agents for cancer therapy.

Abstract 2983: Stat3 regulates supernumerary centrosome clustering in cancer cells via Stathmin/PLK1

Centrosome amplification and supernumerary centrosomal content are common features of cancer cells. Such cells must cluster their centrosomes to form a bipolar spindle and achieve productive mitosis. Targeting the mechanisms that allow cells to cluster extra centrosomes is considered a promising cancer therapeutic strategy. We have now identified Stat3, a protein that is frequently activated in many types of cancers and also regulates stem cell function, as a regulator of centrosome clustering in cancer cells. A high content chemical screen for the identification of inhibitors of centrosome clustering identified Stattic, a Stat3 inhibitor, as a centrosome clustering inhibitor. Stat3 depletion in cell lines as well as in tumors in vivo resulted in significant inhibition of centrosome clustering and in decreased tumor viability and growth. Interestingly, we identify a novel, transcription-independent mechanism for Stat3-mediated centrosome clustering that requires activities of Stathmin, a Stat3 interactor involved in microtubule depolymerisation, and polo-like kinase1 (PLK1). Furthermore, stem cell function in PLK4-driven centrosome amplified breast tumor cells is highly sensitive to Stat3 inhibitors, reflected in higher inhibitor sensitivity of tumors derived from these cells in vivo. We have therefore identified a novel role of Stat3 in the regulation of centrosome clustering, and this role of Stat3 may be critical in identifying tumor types that are sensitive to Stat3 inhibitors. Citation Format: Edward J. Morris, Eiko Kawamura, Jordan Gillespie, Paul McDonald, William Muller, Shoukat Dedhar. Stat3 regulates supernumerary centrosome clustering in cancer cells via Stathmin/PLK1. [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 2983.

Abstract 3887: A screen for new cancer specific drugs that target centrosome clustering

Most normal cells have two centrosomes that form bipolar spindles during mitosis, while cancer cells often contain extra centrosomes. Such cancer cells then achieve bipolar division by clustering centrosomes into two functional poles. Our lab has previously shown that inhibition of Integrin-linked kinase, which is a signaling and scaffold protein in focal adhesion and is also a centrosomal protein, results in scattered centrosomes and multipolar spindles, which leads to cancer specific cell death (Fielding et al., 2011, 30:521-534, Oncogene). A major problem with clinically used anti-mitotic drugs, such as taxol, is their toxicity in normal cells. Our goal is to develop new drugs that act specifically on cancer cells through targeting extra centrosomes without affecting normal cells. To discover new drugs, we established a high-content screen that automatically detects cells with de-clustered centrosomes. BT-549, a breast cancer cell line, was used for the screen, since it is known to effectively cluster centrosomes. The Canadian Chemical Biology Network enabled us to test chemical libraries predicted to have drug-like properties. For screening, cells were grown in 96 well plates, treated with test compounds for 5 hours, and subjected to immunofluorescence to examine centrosome arrangement in mitotic cells. Images were taken and analyzed with an automated fluorescence imager, Cellomics Array Scan VTI. We have thus screened over 6000 compounds from which we identified 18 hits. Fifteen of these were confirmed to inhibit centrosome clustering in the cells cultured under standard conditions, and induce an arrest in mitosis. Three compounds are structurally similar, suggesting a common structural motif as preventing centrosome clustering. To compare the effects of these drugs on normal and cancer cells, the viability of several breast and other cancer cell lines, an immortalized model of normal human mammary epithelial cells (MCF10A), and freshly isolated primary normal human mammary epithelial cells were examined after two days of drug treatment using the MTT assay. These comparisons identified some compounds that selectively reduced the viability of cancer cells, but not normal mammary epithelial cells suggesting their potential for cancer-specific therapy. These small molecules are currently being tested in orthotopic and xenograft models, and will be studied for target identification and mechanism of action for further drug development. 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 3887. doi:1538-7445.AM2012-3887