Oleksii Dubrovskyi

Patient-Derived Tumor Xenografts Are Susceptible to Formation of Human Lymphocytic Tumors

Patient-derived xenograft (PDX) tumor models have emerged as a new approach to evaluate the effects of cancer drugs on patients’ personalized tumor grafts enabling to select the best treatment for the cancer patient and providing a new tool for oncology drug developers. Here, we report that human tumors engrafted in immunodeficient mice are susceptible to formation of B-and T-cell PDX tumors. We xenografted human primary and metastatic tumor samples into immunodeficient mice and found that a fraction of PDX tumors generated from patients’ samples of breast, colon, pancreatic, bladder and renal cancer were histologically similar to lymphocytic neoplasms. Moreover, we found that the first passage of breast and pancreatic cancer PDX tumors after initial transplantation of the tumor pieces from the same human tumor graft could grow as a lymphocytic tumor in one mouse and as an adenocarcinoma in another mouse. Whereas subcutaneous PDX tumors resembling human adenocarcinoma histology were slow growing and non-metastatic, we found that subcutaneous PDX lymphocytic tumors were fast growing and formed large metastatic lesions in mouse lymph nodes, liver, lungs, and spleen. PDX lymphocytic tumors were comprised of B-cells which were Epstein-Barr virus positive and expressed CD45 and CD20. Because B-cells are typically present in malignant solid tumors, formation of B-cell tumor may evolve in a wide range of PDX tumor models. Although PDX tumor models show great promise in the development of personalized therapy for cancer patients, our results suggest that confidence in any given PDX tumor model requires careful screening of lymphocytic markers.

Identification of a New Epitope in uPAR as a Target for the Cancer Therapeutic Monoclonal Antibody ATN-658, a Structural Homolog of the uPAR Binding Integrin CD11b (αM)

The urokinase plasminogen activator receptor (uPAR) plays a role in tumor progression and has been proposed as a target for the treatment of cancer. We recently described the development of a novel humanized monoclonal antibody that targets uPAR and has anti-tumor activity in multiple xenograft animal tumor models. This antibody, ATN-658, does not inhibit ligand binding (i.e. uPA and vitronectin) to uPAR and its mechanism of action remains unclear. As a first step in understanding the anti-tumor activity of ATN-658, we set out to identify the epitope on uPAR to which ATN-658 binds. Guided by comparisons between primate and human uPAR, epitope mapping studies were performed using several orthogonal techniques. Systematic site directed and alanine scanning mutagenesis identified the region of aa 268–275 of uPAR as the epitope for ATN-658. No known function has previously been attributed to this epitope Structural insights into epitope recognition were obtained from structural studies of the Fab fragment of ATN-658 bound to uPAR. The structure shows that the ATN-658 binds to the DIII domain of uPAR, close to the C-terminus of the receptor, corroborating the epitope mapping results. Intriguingly, when bound to uPAR, the complementarity determining region (CDR) regions of ATN-658 closely mimic the binding regions of the integrin CD11b (αM), a previously identified uPAR ligand thought to be involved in leukocyte rolling, migration and complement fixation with no known role in tumor progression of solid tumors. These studies reveal a new functional epitope on uPAR involved in tumor progression and demonstrate a previously unrecognized strategy for the therapeutic targeting of uPAR.

Identification of HDAC6-Selective Inhibitors of Low Cancer Cell Cytotoxicity

The histone deacetylases (HDACs) occur in 11 different isoforms, and these enzymes regulate the activity of a large number of proteins involved in cancer initiation and progression. The discovery of isoform‐selective HDAC inhibitors (HDACIs) is desirable, as it is likely that such compounds would avoid some of the undesirable side effects found with the first‐generation inhibitors. A series of HDACIs previously reported by us were found to display some selectivity for HDAC6 and to induce cell‐cycle arrest and apoptosis in pancreatic cancer cells. In the present work, we show that structural modification of these isoxazole‐based inhibitors leads to high potency and selectivity for HDAC6 over HDAC1–3 and HDAC10, while unexpectedly abolishing their ability to block cell growth. Three inhibitors with lower HDAC6 selectivity inhibit the growth of cell lines BxPC3 and L3.6pl, and they only induce apoptosis in L3.6pl cells. We conclude that HDAC6 inhibition alone is insufficient for disruption of cell growth, and that some degree of class 1 HDAC inhibition is required. Moreover, the highly selective HDAC6Is reported herein that are weakly cytotoxic may find use in cancer immune system reactivation.

Melanoma Cells Block PEDF Production in Fibroblasts to Induce the Tumor-Promoting Phenotype of Cancer-Associated Fibroblasts

Loss of pigment epithelium-derived factor (PEDF, SERPINF1) in cancer cells is associated with poor prognosis and metastasis, but the contribution of stromal PEDF to cancer evolution is poorly understood. Therefore, we investigated the role of fibroblast-derived PEDF in melanoma progression. We demonstrate that normal dermal fibroblasts expressing high PEDF levels attenuated melanoma growth and angiogenesis in vivo, whereas PEDF-depleted fibroblasts exerted tumor-promoting effects. Accordingly, mice with global PEDF knockout were more susceptible to melanoma metastasis. We also demonstrate that normal fibroblasts in close contact with PEDF-null melanoma cells lost PEDF expression and tumor-suppressive properties. Further mechanistic investigations underlying the crosstalk between tumor and stromal cells revealed that melanoma cells produced PDGF-BB and TGFβ, which blocked PEDF production in fibroblasts. Notably, cancer-associated fibroblasts (CAF) isolated from patient-derived tumors expressed markedly low levels of PEDF. Treatment of patient CAF and TGFβ-treated normal fibroblasts with exogenous PEDF decreased the expression of CAF markers and restored PEDF expression. Finally, expression profiling of PEDF-depleted fibroblasts revealed induction of IL8, SERPINB2, hyaluronan synthase-2, and other genes associated with tumor promotion and metastasis. Collectively, our results demonstrate that PEDF maintains tumor-suppressive functions in fibroblasts to prevent CAF conversion and illustrate the mechanisms by which melanoma cells silence stromal PEDF to promote malignancy. Cancer Res; 76(8); 2265-76. ©2016 AACR.

Abstract 3287: Targeting GSK-3: a new approach for the treatment of neuroblastoma

Neuroblastoma is a devastating pediatric cancer and most patients older than 18 months present with multi-organ metastatic disease. High grade or recurrent disease is refractory to treatment with chemotherapy and almost uniformly fatal. Because GSK-3beta has been shown to be a positive regulator of NF-kappaB-mediated survival and chemoresistance in cancer cells, we hypothesize that the inhibition of GSK-3 may have potential therapeutic effects in neuroblastoma. To test this premise we used small molecule inhibitors, Western blotting, apoptotic and MTS assays to study the effect(s) of GSK-3 inactivation in neuroblastoma cell lines SK-N-DZ and SK-N-BE(2). Using chemically distinct GSK-3 inhibitors (AR-A014418, TDZD8 and 9-ING-41), we found that pharmacological inhibition of GSK-3 led to a decrease in proliferation and survival of neuroblastoma cells. We observed that inhibition of GSK-3 results in decreased expression of the anti-apoptotic molecules Bcl-XL and XIAP (NF-kappaB target genes) and a subsequent increase in neuroblastoma cell apoptosis. Moreover, we show that our novel GSK-3 inhibitor 9-ING-41 enhances the antitumor effects of irinotecan in neuroblastoma cells. Our results demonstrate that GSK-3 positively regulates neuroblastoma cell survival and proliferation and suggest that the inhibition of GSK-3 is a promising new approach for the treatment of neuroblastoma. Citation Format: Oleksii Dubrovskyi, Andrey Ugolkov, Irina Gaisina, Gennadiy Bondarenko, Luigi Strizzi, Naira Margaryan, Thomas O9Halloran, Alan Kozikowski, Mary Hendrix, Andrew Mazar. Targeting GSK-3: a new approach for the treatment of neuroblastoma. [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 3287. doi:10.1158/1538-7445.AM2015-3287

Abstract 2699: Targeting GSK-3: a novel approach to enhance glioblastoma chemosensitivity

Glioblastoma (GBM) is, in essence, an incurable cancer, with most patients surviving 12-15 months following initial diagnosis. Previously published studies identified Glycogen Synthase Kinase-3 (GSK-3) as a new therapeutic target in GBM. Because GSK-3beta is a positive regulator of NF-kappaB-mediated survival and chemoresistance in cancer cells, we hypothesize that the inhibition of GSK-3 may overcome NF-kappaB-mediated chemoresistance to conventional drugs in human GBM. Using IVIS imaging of live mice, we found that: 1) NF-kappaB is constitutively active in orthotopic GBM patient-derived xenograft (PDX) tumors expressing an NF-kappa luciferase reporter; and 2) a single intravenous injection of our novel GSK-3 inhibitor 9-ING-41 (25 mg/kg) significantly reduced NF-kappaB transcriptional activity in orthotopic GBM PDX. Using 3 different orthotopic GBM PDX- tumor models, enabled for bioluminescence imaging through luciferase modification, we evaluated antitumor effects of 9-ING-41, alone as well as in combination with irinotecan, CCNU and temozolomide. Our in vivo results revealed that treatment with a combination of 9-ING-41 and irinotecan delays GBM PDX tumor growth, relative to either agent as a monotherapy, and, even more promising, 9-ING-41+CCNU leads to a complete tumor regression. Histological evaluation of mouse brain confirmed the absence of cancer cells and a formation of cyst at the location of intracranial tumor in mice treated with 9-ING-41+CCNU. Our results provide a rationale to advance 9-ING-41 for clinical evaluation in treating GBM, especially when combined with CCNU cytotoxic therapy. Citation Format: Andrey Ugolkov, Oleksii Dubrovskyi, Irina Gaisina, Alex Yemelyanov, Gennadiy Bondarenko, Charles James, James Chandler, Thomas O9Halloran, Alan Kozikowski, Jeffry Raizer, Andrew Mazar. Targeting GSK-3: a novel approach to enhance glioblastoma chemosensitivity. [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 2699. doi:10.1158/1538-7445.AM2015-2699