Stanislaw Skora

Drug-tolerant cancer cells show reduced tumor-initiating capacity: depletion of CD44 cells and evidence for epigenetic mechanisms.

Cancer stem cells (CSCs) possess high tumor-initiating capacity and have been reported to be resistant to therapeutics. Vice versa, therapy-resistant cancer cells seem to manifest CSC phenotypes and properties. It has been generally assumed that drug-resistant cancer cells may all be CSCs although the generality of this assumption is unknown. Here, we chronically treated Du145 prostate cancer cells with etoposide, paclitaxel and some experimental drugs (i.e., staurosporine and 2 paclitaxel analogs), which led to populations of drug-tolerant cells (DTCs). Surprisingly, these DTCs, when implanted either subcutaneously or orthotopically into NOD/SCID mice, exhibited much reduced tumorigenicity or were even non-tumorigenic. Drug-tolerant DLD1 colon cancer cells selected by a similar chronic selection protocol also displayed reduced tumorigenicity whereas drug-tolerant UC14 bladder cancer cells demonstrated either increased or decreased tumor-regenerating capacity. Drug-tolerant Du145 cells demonstrated low proliferative and clonogenic potential and were virtually devoid of CD44+ cells. Prospective knockdown of CD44 in Du145 cells inhibited cell proliferation and tumor regeneration, whereas restoration of CD44 expression in drug-tolerant Du145 cells increased cell proliferation and partially increased tumorigenicity. Interestingly, drug-tolerant Du145 cells showed both increases and decreases in many “stemness” genes. Finally, evidence was provided that chronic drug exposure generated DTCs via epigenetic mechanisms involving molecules such as CD44 and KDM5A. Our results thus reveal that 1) not all DTCs are necessarily CSCs; 2) conventional chemotherapeutic drugs such as taxol and etoposide may directly target CD44+ tumor-initiating cells; and 3) DTCs generated via chronic drug selection involve epigenetic mechanisms.

Novel small molecular inhibitors disrupt the JAK/STAT3 and FAK signaling pathways and exhibit a potent antitumor activity in glioma cells

JAK (Janus kinase)/STAT (signal transducers and activators of transcription) signaling is involved in the regulation of cell growth, differentiation and apoptosis. Constitutive activation of STATs, in particular STAT3, is observed in a large number of human tumors, including gliomas and may contribute to oncogenesis by stimulating cell proliferation and preventing apoptosis, thus it emerges as a promising target for anti-cancer therapy. To investigate the therapeutic potential of blocking STAT3 in glioma cells a set of small synthetic molecules - caffeic acid derivatives, structurally related to AG490 was screened for its ability to inhibit STAT3. Inhibitor 2 (E)-2-cyano-N-[(S)-1-phenylethyl]-3-(pyridin-2-yl)acrylamide was the most effective in inhibition of JAK/STAT3 signaling and at doses ≥ 25 μM significantly reduced the level of phosphorylated JAK1, JAK2 and STAT3 (at Tyr705) and downregulated the expression of known STAT3 targets. In treated cells we observed rapid detachment and rounding of cells associated with reduction of focal adhesion kinase phosphorylation and activity, followed by upregulation of phosphorylated p38, JNK and ERK1/2 levels. Accumulation of cells with fragmented DNA, increases of the cleaved caspase 3 and fragmented PARP levels were detected 24 h after the treatment suggesting ongoing apoptotic cell death. Three human malignant glioblastoma cell lines defective in tumor suppressors TP53 and/or PTEN were susceptible to inhibitor 2 that induced the programmed cell death. Global gene expression profiling revealed modulation of numerous genes in cells treated with inhibitor 2 revealing novel, potential JAK/STAT targets. Our study demonstrates that suitably modified caffeic acid molecules exhibit significant cytotoxic potential toward glioma cells.

D-glucose- and D-mannose-based antimetabolites. Part 2. Facile synthesis of 2-deoxy-2-halo-D-glucoses and -D-mannoses.

Modified D-glucose and D-mannose analogs are potentially clinically useful metabolic inhibitors. Biological evaluation of 2-deoxy-2-halo analogs has been impaired by limited availability and lack of efficient methods for their preparation. We have developed practical synthetic approaches to 2-deoxy-2-fluoro-, 2-chloro-2-deoxy-, 2-bromo-2-deoxy-, and 2-deoxy-2-iodo derivatives of D-glucose and D-mannose that exploit electrophilic addition reactions to a commercially available 3,4,6-tri-O-acetyl-D-glucal.

d-Glucose and d-mannose-based metabolic probes. Part 3: Synthesis of specifically deuterated d-glucose, d-mannose, and 2-deoxy-d-glucose

Altered carbohydrate metabolism in cancer cells was first noted by Otto Warburg more than 80 years ago. Upregulation of genes controlling the glycolytic pathway under normoxia, known as the Warburg effect, clearly differentiates malignant from non-malignant cells. The resurgence of interest in cancer metabolism aims at a better understanding of the metabolic differences between malignant and non-malignant cells and the creation of novel therapeutic and diagnostic agents exploiting these differences. Modified d-glucose and d-mannose analogs were shown to interfere with the metabolism of their respective monosaccharide parent molecules and are potentially clinically useful anticancer and diagnostic agents. One such agent, 2-deoxy-d-glucose (2-DG), has been extensively studied in vitro and in vivo and also clinically evaluated. Studies clearly indicate that 2-DG has a pleiotropic mechanism of action. In addition to effectively inhibiting glycolysis, 2-DG has also been shown to affect protein glycosylation. In order to better understand its molecular mechanism of action, we have designed and synthesized deuterated molecular probes to study 2-DG interference with d-glucose and d-mannose metabolism using mass spectrometry. We present here the synthesis of all desired probes: 2-deutero-d-glucose, 2-deutero-d-mannose, 6-deutero-d-glucose, 6-deutero-d-mannose, and 2-deutero-2-deoxy-d-glucose as well as their complete chemical characterization.

IGF-1R and mTOR Blockade: Novel Resistance Mechanisms and Synergistic Drug Combinations for Ewing Sarcoma

BACKGROUND Therapies cotargeting insulin-like growth factor receptor 1 (IGF-1R) and mammalian target of rapamycin (mTOR) have demonstrated remarkable, albeit short-lived, clinical responses in a subset of Ewing sarcoma (ES) patients. However, the mechanisms of resistance and applicable strategies for overcoming drug resistance to the IGF-1R/mTOR blockade are still undefined. METHODS To elucidate predominant mechanism(s) of acquired drug resistance while identifying synergistic drug combinations that improve clinical efficacy, we generated more than 18 ES cell lines resistant to IGF-1R- or mTOR-targeted therapy. Two small-molecule inhibitors of IGF-1R were chosen, NVP-ADW-742 (IGF-1R-selective) and OSI-906 (a dual IGF-1R/insulin receptor alpha [IR-α] inhibitor). Reverse-phase protein lysate arrays (RPPAs) revealed proteomic changes linked to IGF-1R/mTOR resistance, and selected proteins were validated in cell-based assays, xenografts, and within human clinical samples. All statistical tests were two-sided. RESULTS Novel mechanisms of resistance (MOR) emerged after dalotuzumab-, NVP-ADW-742-, and OSI-906-based targeting of IGF-1R. MOR to dalotuzumab included upregulation of IRS1, PI3K, and STAT3, as well as p38 MAPK, which was also induced by OSI-906. pEIF4E(Ser209), a key regulator of Cap-dependent translation, was induced in ridaforolimus-resistant ES cell lines. Unique drug combinations targeting IGF-1R and PI3K-alpha or Mnk and mTOR were synergistic in vivo and vitro (P < .001) as assessed respectively by Mantel-Cox and isobologram testing. CONCLUSIONS We discovered new druggable targets expressed by chemoresistant ES cells, xenografts, and relapsed human tumors. Joint suppression of these newfound targets, in concert with IGF-1R or mTOR blockade, should improve clinical outcomes.

Abstract 3251: Blockade of HIF-1 with a small molecule inhibitor WP1066 in melanoma.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC WP1066 was shown to exert its potent inhibitory activity against several types of human cancer including melanoma in vitro and in vivo via suppression of JAK2/STAT3/STAT5 activation. However, if there is any other additional molecular mechanism(s) by which WP1066 generates its anti-tumor effects remain largely unknown. Here, we report a novel function for WP1066 in inducing the degradation of both HIF-1α and HIF-1β subunits in human melanoma cells. We found constitutive activation of HIF-1α and an increased expression of hexokinase II, a down target of HIF-1α, in normoxia in 6/6 of melanoma cell lines, but not in other tumor types. Both hypoxia and cobalt chloride, a transition metal that mimics hypoxia, further increased HIF-1α protein accumulation in a time-dependent manner in WM35 and SKMEL-1 melanoma cells. WP1066 selectively decreased the constitutive and hypoxia and cobalt chloride induced increase in the level of HIF-1α in a dose-dependent manner with a half-life (T1/2) of ∼13, ∼10, and ∼10 min in WM35 cells. Moreover, WP1066 decreased the T1/2 of HIF-1α following cycloheximide treatment from ∼13 to ∼7 min suggesting that WP1066 induced HIF-1α instability is not mediated via inhibition of its translation. Further, inhibition of HIF-1α protein accumulation by WP1066 was not abolished in the presence of MG132 and proteasome inhibitor I, a potent inhibitor of the 26S proteasome, suggesting that the 26S proteasome-system is not responsible for WP1066-induced HIF-1α instability. Immunoprecipitation, confocal microscopy, and cellular fractionation analyses revealed that WP1066 induced ubiquitinylation, aggregation and degradation of HIF-1α in WM35 cells. Finally, WP1066 directly inhibits activity of rhUSP5 and rUCH-L1. Altogether, our study identified a novel function for WP1066 in inducing HIF-1 instability that may also contribute to the anti-cancer effects of WP1066 in human melanoma. Citation Format: Arumugam Jayakumar, Jana Rauvolfova, Hanying Bao, Izabela Fokt, Stanislaw Skora, Amy Heimberger, Waldemar Priebe. Blockade of HIF-1 with a small molecule inhibitor WP1066 in melanoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3251. doi:10.1158/1538-7445.AM2013-3251

Abstract C65: Drug-tolerant cancer cells show reduced tumor-initiating capacity: Depletion of CD44+ cells and evidence for epigenetic mechanisms

Abstract Cancer stem cells (CSCs) possess high tumor-initiating capacity and have been reported to be resistant to therapeutics. Vice versa, therapy-resistant cancer cells seem to manifest CSC phenotypes and properties. It has been generally assumed that drug-resistant cancer cells may all be CSCs although the generality of this assumption is unknown. Here, we chronically treated Du145 prostate cancer cells with etoposide, paclitaxel and some experimental drugs (i.e., staurosporine and 2 paclitaxel analogs), which led to populations of drug-tolerant cells (DTCs). Surprisingly, these DTCs, when implanted either subcutaneously or orthotopically into NOD/SCID mice, exhibited much reduced tumorigenicity or were even nontumorigenic. Drug-tolerant DLD1 colon cancer cells selected by a similar chronic selection protocol also displayed reduced tumorigenicity whereas drug-tolerant UC14 bladder cancer cells demonstrated either increased or decreased tumor-regenerating capacity. Drug-tolerant Du145 cells demonstrated low proliferative and clonogenic potential and were virtually devoid of CD44+ cells. Prospective knockdown of CD44 in Du145 cells inhibited cell proliferation and tumor regeneration whereas restoration of CD44 expression in drug-tolerant Du145 cells increased cell proliferation and partially increased tumorigenicity. Interestingly, drug-tolerant Du145 cells showed both increases and decreases in many ‘stemness’ genes. Finally, evidence was provided that chronic drug exposure generated DTCs via epigenetic mechanisms involving molecules such as CD44 and KDM5A. Our results thus reveal that 1) not all DTCs are necessarily CSCs; 2) conventional chemotherapeutic drugs such as taxol and etoposide may directly target CD44+ tumor-initiating cells; and 3) DTCs generated via chronic drug selection involve epigenetic mechanisms. Citation Format: Xin Chen, Izabela Fokt, Stanislaw Skora, Waldemar Priebe, Yongyi Bi, Dean G. Tang, Hong Yan, Qiuping Zhang, Jichao Qin, Hangwen Li, Can Liu, Tammy Davis, Luis D. Coletta, Jim Klostergaard. Drug-tolerant cancer cells show reduced tumor-initiating capacity: Depletion of CD44+ cells and evidence for epigenetic mechanisms [abstract]. In: Proceedings of the AACR Special Conference on Advances in Prostate Cancer Research; 2012 Feb 6-9; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2012;72(4 Suppl):Abstract nr C65.

Abstract 50: 2-Deoxy-2-halo-D-glucose derivatives inhibition of glycolysis in pancreatic cancer

Pancreatic ductal adenocarcinoma (PDAC) is one of the most hypoxic and subsequently glycolytic tumors. Thus the inhibition of glycolysis appears to be an attractive therapeutic approach that should be explored. Methods: A series of 2-deoxy analogs of D-glucose, namely, 2-deoxy-D-glucose (2-DG), 2-fluoro-D-glucose (2-FG), 2-chloro-D-glucose (2-CG), 2-bromo-D-glucose (2-BG), and 2,2-difluoro-D-glucose (2,2-diFG) were synthesized in our laboratory. The ability of 2-deoxy-2-halo-D-glucose derivatives to inhibit glycolysis in pancreatic cancer cell lines was tested using Seahorse 96 analyzer and XF Glycolysis Stress Test kit. The cellular ATP level was measured using CellTiter-Glo assay. Viability of cells cultured in normoxic and hypoxic conditions were assessed using MTS assay. Results: 2,2-diFG and 2-FG were significantly more potent inhibitors than 2-DG of the Extracellular Acidification Rate (ECAR) of medium in oligomycin-stimulated PDAC cells (complete inhibition at Conclusion: Both, 2,2-diFG and 2-FG have a superior to 2-DG ability to inhibit glycolysis in vitro and should be further evaluated in vivo in the PDAC models as a potential anticancer agents. Citation Format: Rafal Zielinski, Izabela Fokt, Aleksandra Rusin, Stanislaw Skora, Waldemar Priebe. 2-Deoxy-2-halo-D-glucose derivatives inhibition of glycolysis in pancreatic cancer. [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 50.

Abstract 4540: Development of orally bioavailable formulation of WP1066 and its evaluation in vivo

Background: Our drug discovery program has led to the development of a series of diverse, unique small molecule drugs targeting the key oncogenic transcription factor STAT3 which drives diverse human cancers. WP1066 was selected as a lead drug because of its drug-like properties, therapeutic window and validated in vivo activity in a broad range of tumor models. During the process of WP1066 development as a clinical phase new agent, we developed and further characterized an orally bioavailable formulation of WP1066 using a spray dried oral formulation. Methods: The spray dried formulation of WP1066 (SDD1) was prepared using Hypermellose acetate succinate (HPMCAS) as an enteric coating film. HPMCAS is a mixture of acetic and monosuccinic acid esters of hydroxypropylmethyl cellulose. While insoluble in gastric fluids, the SDD1 formulation swells and leads to rapid dissolution in the higher pH found in upper small intestine, and subsequent release of the drug. For theses studies SDD1 was characterized for size, stability and purity. Single and multiple-dose toxicity studies were done in female CD-1 mice, using 5% dextrose as a vehicle. Single dose PK and organ distribution analysis was performed after oral gavage administration of 200 mg/kg after 12 hour fasting. Animals were sacrifice at different time-points (2 min to 24 hours). Plasma, brain and pancreas were isolated and the apparent concentration of WP1066 was determined using HPLC/MS/MS analysis. For multi-dose PK analysis, animals were fasted for six hours, dosed with SDD1 at 200 mg/kg. Animals were euthanized at different time intervals after the second dose (given six hours after first dose). Results: Mice dosed with SDD1 did not show any apparent toxicity when administered up to 200 mg/kg. For multiple doses, mice received 50, 100 or 200 mg/kg of WP1066 in SDD1 every day for 28 days. No apparent toxicity or significant weight lost was observed. In a similar way, animals dosed twice a day with 200 mg/kg of WP1066 (SDD1) for 15 doses did not show any symptoms of toxicity. Pilot PK analysis confirmed dose-dependent absorption of WP1066. Single-dose PK analysis revealed rapid GI absorption with a TMAX = 45 min and CMAX in plasma 1.5 μg/ml (which corresponds to ∼4 μM). The estimated half-life was approximately 3 hrs. Similar parameters were obtained from PK analysis performed in canines. SDD1 dosing led to significant accumulation of WP1066 in intact brain and pancreatic tissues in mice. These results support further preclinical development of SDD1 formulated WP1066 aimed the initiation of clinical studies in pancreatic, brain cancer and melanoma metastasis to brain patients. Acknowledgment: These studies were supported in part by the grants: Brain SPORE (2 P50 CA127001), Melanoma SPORE (2 P50 CA093459); Viragh Foundation, Houston Pharmaceuticals. Citation Format: Rafal Zielinski, Aleksandra Rusin, Timothy Madden, Charles Conrad, Mary Johansen, Izabela Fokt, Stanislaw Skora, Arumugam Jayakumar, Amy Heimberger, Waldemar Priebe. Development of orally bioavailable formulation of WP1066 and its evaluation in vivo. [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 4540. doi:10.1158/1538-7445.AM2015-4540

Abstract 3806: STAT3/STAT5 blockade by WP1066 inhibits ovarian cancer cell proliferation and induces cell death

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA BACKGROUND: Ovarian cancer is the most deadly gynecologic malignancy with 22,240 new cases and 14,030 deaths estimated in 2013. In order to improve the clinical management of women with ovarian cancer, it is critical to dissect the molecular drivers of ovarian neoplastic transformation and find efficient means of pharmacologic control. Constitutive activation of the signal transducer and activator of transcription 3 and 5 (STAT3 and STAT5) pathways is found in 70% of ovarian cancer patients. Overexpression of STATs is associated with a malignant phenotype and confers resistance to chemotherapy. Our drug discovery and development program has led to the development of a series of unique, small molecule inhibitors of STATs including a promising lead compound WP1066. The aim of this work was to investigate the antiproliferative and proapoptotic effects of WP1066 in selected ovarian cancer cells with constitutively activated or inducible STAT3 and STAT5. EXPERIMENTAL DESIGN: The effects of WP1066 on the levels of p-STAT3 and p-STAT5 were assessed with Western blotting and the MSD assay. Nuclear translocation of p-STAT3 and p-STAT5 was confirmed with confocal microscopy. Cell viability was determined with the MTS assay and dye exclusion test. Apoptosis induction was assessed on the basis of morphological markers using live-cell imaging (cell rounding, membrane blebbing and chromatin condensation) and biochemical markers using the MSD assay and confocal microscopy (cleaved PARP, cleaved caspase 3). SK-OV-3, SK-OV-3 CR (carboplatin resistant) and OVCAR-5 cells were used for in vitro studies. RESULTS: SK-OV-3 and OVCAR-5 cells grown in vitro show constitutive activation of STAT3, but not STAT5. Treatment of ovarian cancer cells with IL-6 or IFN-α and -β results in high levels of p-STAT3 with subsequent p-STAT3 translocation to the nucleus. Treatment with EGF results in low level STAT3 activation. Cells treated with IFN-α and β or EGF exhibit activation of STAT5, while IL-6 treatment does not affect p-STAT5 level. Importantly, cells extracted from disseminated intraperitoneal ovarian tumors express high levels of phosphorylated STAT3 and clearly show nuclear p-STAT3 localization. WP1066 inhibits both p-STAT3 and p-STAT5 but not STAT3 and STAT5 in the panel of tested ovarian cancer cells. Treatment with WP1066 inhibits cell growth, is cytotoxic to SK-OV-3 and OVCAR-5 cells as well as the carboplatin resistant subline, SK-OV-3 CR, all at low micromolar concentrations. We found that WP1066 induces robust apoptosis, starting 4h after its addition to the culture medium. CONCLUSION: Coordinated inhibition of p-STAT3/p-STAT5 by WP1066 results in profound apoptosis in ovarian cancer cells. Inhibition of STATs in ovarian cancer should be further explored as a potential therapeutic strategy for women with ovarian cancer. Citation Format: Rafal Zielinski, Aleksandra Rusin, Anna Priebe, Jayakumar Arumugam, Radjendirane Venugopal, Stanislaw Skora, Izabela Fokt, Waldemar A. Priebe. STAT3/STAT5 blockade by WP1066 inhibits ovarian cancer cell proliferation and induces cell death. [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 3806. doi:10.1158/1538-7445.AM2014-3806