Preeti Shah

Reversible Epithelial to Mesenchymal Transition and Acquired Resistance to Sunitinib in Patients with Renal Cell Carcinoma: Evidence from a Xenograft Study

Tyrosine kinase inhibitors (TKI) targeting angiogenesis via inhibition of the vascular endothelial growth factor pathway have changed the medical management of metastatic renal cell carcinoma. Although treatment with TKIs has shown clinical benefit, these drugs will eventually fail patients. The potential mechanisms of resistance to TKIs are poorly understood. To address this question, we obtained an excisional biopsy of a skin metastasis from a patient with clear cell renal carcinoma who initially had a response to sunitinib and eventually progressed with therapy. Tumor pieces were grafted s.c. in athymic nude mice. Established xenografts were treated with sunitinib. Tumor size, microvascular density, and pericyte coverage were determined. Plasma as well as tissue levels for sunitinib were assessed. A tumor-derived cell line was established and assessed in vitro for potential direct antitumor effects of sunitinib. To our surprise, xenografts from the patient who progressed on sunitinib regained sensitivity to the drug. At a dose of 40 mg/kg, sunitinib caused regression of the subcutaneous tumors. Histology showed a marked reduction in microvascular density and pericyte dysfunction. More interestingly, histologic examination of the original skin metastasis revealed evidence of epithelial to mesenchymal transition, whereas the xenografts showed reversion to the clear cell phenotype. In vitro studies showed no inhibitory effect on tumor cell growth at pharmacologically relevant concentrations. In conclusion, the histologic examination in this xenograft study suggests that reversible epithelial to mesenchymal transition may be associated with acquired tumor resistance to TKIs in patients with clear cell renal carcinoma. Mol Cancer Ther; 9(6); 1525–35. ©2010 AACR.

High ALDH Activity Identifies Chemotherapy-Resistant Ewing's Sarcoma Stem Cells That Retain Sensitivity to EWS-FLI1 Inhibition

Background Cancer stem cells are a chemotherapy-resistant population capable of self-renewal and of regenerating the bulk tumor, thereby causing relapse and patient death. Ewings sarcoma, the second most common form of bone tumor in adolescents and young adults, follows a clinical pattern consistent with the Cancer Stem Cell model – remission is easily achieved, even for patients with metastatic disease, but relapse remains frequent and is usually fatal. Methodology/Principal Findings We have isolated a subpopulation of Ewings sarcoma cells, from both human cell lines and human xenografts grown in immune deficient mice, which express high aldehyde dehydrogenase (ALDHhigh) activity and are enriched for clonogenicity, sphere-formation, and tumor initiation. The ALDHhigh cells are resistant to chemotherapy in vitro, but this can be overcome by the ATP binding cassette transport protein inhibitor, verapamil. Importantly, these cells are not resistant to YK-4-279, a small molecule inhibitor of EWS-FLI1 that is selectively toxic to Ewings sarcoma cells both in vitro and in vivo. Conclusions/Significance Ewings sarcoma contains an ALDHhigh stem-like population of chemotherapy-resistant cells that retain sensitivity to EWS-FLI1 inhibition. Inhibiting the EWS-FLI1 oncoprotein may prove to be an effective means of improving patient outcomes by targeting Ewings sarcoma stem cells that survive standard chemotherapy.

A dose-finding study of temsirolimus and liposomal doxorubicin for patients with recurrent and refractory bone and soft tissue sarcoma.

There are few effective therapies for high‐risk sarcomas. Initial chemosensitivity is often followed by relapse. In vitro, mammalian target of rapamycin (mTOR) inhibition potentiates the efficacy of chemotherapy on resistant sarcoma cells. Although sarcoma trials using mTOR inhibitors have been disappointing, these drugs were used as maintenance. We conducted a Phase I/II clinical trial to test the ability of temsirolimus to potentiate the cytotoxic effect of liposomal doxorubicin and present here the dose‐finding portion of this study. Adult and pediatric patients with recurrent or refractory sarcomas were treated with increasing doses of liposomal doxorubicin and temsirolimus using a continual reassessment method for escalation, targeting a dose‐limiting toxicity rate of 20%. Blood samples were drawn before and after the first dose of temsirolimus in Cycles 1 and 2 for pharmacokinetic analysis. The maximally tolerated dose combination was liposomal doxorubicin 30 mg/m2 monthly with temsirolimus 20 mg/m2 weekly. Hematologic toxicity was common but manageable. Dose‐limiting toxicities were primarily renal. Concurrent administration of liposomal doxorubicin resulted in increased exposure to sirolimus, the active metabolite of temsirolimus. Thus, the combination of liposomal doxorubicin and temsirolimus is safe for heavily pretreated sarcoma patients. Co‐administration with liposomal doxorubicin did not alter temsirolimus pharmacokinetics, but increased exposure to its active metabolite.

Vascular Disruption in Combination with mTOR Inhibition in Renal Cell Carcinoma

Renal cell carcinoma (RCC) is an angiogenesis-dependent and hypoxia-driven malignancy. As a result, there has been an increased interest in the use of antiangiogenic agents for the management of RCC in patients. However, the activity of tumor-vascular disrupting agents (tumor-VDA) has not been extensively examined against RCC. In this study, we investigated the therapeutic efficacy of the tumor-VDA ASA404 (DMXAA, 5,6-dimethylxanthenone-4-acetic acid, or vadimezan) in combination with the mTOR inhibitor everolimus (RAD001) against RCC. In vitro studies were carried out using human umbilical vein endothelial cells and in vivo studies using orthotopic RENCA tumors and immunohistochemical patient tumor-derived RCC xenografts. MRI was used to characterize the vascular response of orthotopic RENCA xenografts to combination treatment. Therapeutic efficacy was determined by tumor growth measurements and histopathologic evaluation. ASA404/everolimus combination resulted in enhanced inhibition of endothelial cell sprouting in the 3-dimensional spheroid assay. MRI of orthotopic RENCA xenografts revealed an early increase in permeability 4 hours posttreatment with ASA404, but not with everolimus. Twenty-four hours after treatment, a significant reduction in blood volume was observed with combination treatment. Correlative CD31/NG2 staining of tumor sections confirmed marked vascular damage following combination therapy. Histologic sections showed extensive necrosis and a reduction in the viable rim following combination treatment compared with VDA treatment alone. These results show the potential of combining tumor-VDAs with mTOR inhibitors in RCC. Further investigation into this novel combination strategy is warranted. Mol Cancer Ther; 11(2); 383–92. ©2011 AACR.

Development of two novel benzoylphenylurea sulfur analogues and evidence that the microtubule-associated protein tau is predictive of their activity in pancreatic cancer.

In this work, we evaluated two lead compounds, referred to as SG410 and SG430, obtained from a screen of sulfur benzoylphenylurea analogues, against in vitro and in vivo models of pancreas cancer. Both drugs showed a similar mechanism of action profile, with SG410 being more potent as an inhibitor of tubulin assembly. We determined the best in vivo administration schedule and tested SG410 and SG430 in nine cases of a novel platform of direct pancreas cancer xenografts. Both compounds had antiproliferative activity in vitro in the low nanomolar range, but only SG410 showed significant activity in vivo. Administration of SG410 resulted in significant tumor growth delay in five of nine groups tested. In a direct comparison in three of the cases, SG410 was at least as efficacious as docetaxel. We also sought markers that would be predictive of the efficacy of these agents, and we found such a marker in microtubule-associated protein tau (MAPT). This protein enhances the assembly and stability of microtubules. In both the cell lines and the direct human xenografts, MAPT mRNA and protein levels correlated well. There was also a statistically significant inverse correlation between MAPT expression and sensitivity to the tested agents. In summary, the novel sulfur benzoylphenylurea SG410 showed activity inversely related to MAPT expression in a preclinical model of pancreatic cancer comparable with that observed with docetaxel, another microtubule-targeting agent. [Mol Cancer Ther 2007;6(5):1509–16]

Abstract 2919: Effect of selumetinib and AZD8055 on the growth of anastrozole resistant tumors

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Despite significant improvement in the treatment outcome of hormone responsive post-menopausal breast cancer, some eventually acquire resistance to AIs. Using our MCF-7Ca xenograft model, we observed that although, AI anastrozole inhibited tumor growth, tumors eventually began to grow. Our previous data shows that anastrozole resistant tumors upregulate growth factor receptor pathways as they adapt to grow in the low estrogen environment. In the current study, we investigated the effect of inhibiting the growth factor receptor pathways with two signal transduction inhibitors. We treated the mice with anastrozole resistant tumors with selumetinib (AZD6244, ARRY-142866); an MEK 1/2 inhibitor and AZD8055; a dual mTORC1 and mTORC2 inhibitor, alone or in combination with anastrozole. MCF-7Ca cells were inoculated sc into ovariectomized athymic nude mice supplemented with androstenedione (100 μg/d), the substrate for aromatase conversion to estrogen. Once the tumors reached a size of ∼300mm3, the mice were assigned to one of two treatment groups so that mean tumor volumes were not significantly different (p=0.98) at the start of treatment: control, anastrozole (200μg/d). All animals continued to be supplemented with androstenedione (≥4A). The tumors in the anastrozole group doubled after 6 weeks. At week 6, the animals were regrouped into six groups such that the mean tumor volumes were not significantly different (p=0.18). The mice received the following treatments (i) anastrozole, (ii) anastrozole withdrawal (≥4A alone), (iii) selumetinib (25mg/kg/d, bid, po), (iv) selumetinib + anastrozole, (v) AZD8055 (20mg/kg/d, po), (vi) AZD8055 + anastrozole (n=10 mice/group). The treatments were given for 6 weeks (till week 12) and then the mice were euthanized, tumors were collected and analyzed. Using mixed effects model, the growth rates of tumors (≥i) between different groups of treatments were compared. The tumors of mice treated with selumetinib + anastrozole had significantly lower growth rate than both single agents (p=0.008). The growth rate of tumors of mice treated with AZD8055 + anastrozole was marginally lower than the single agents (p=0.058). Western blotting analysis of the tumors showed that treatment with anastrozole resulted in upregulation of proteins in the growth factor receptor cascade such as p-mTOR, pAkt, pMEK and pMAPK. This was accompanied by downregulation of ERα protein, consistent with previous findings. This suggests that anastrozole resistance results in adaptation of the tumors to growth factor receptor pathway. When the tumors were treated with signal transduction inhibitors such as selumetinib or AZD8055, the respective pathways were effectively inhibited, which resulted in upregulation of ERα. Our results suggest that inhibition of growth factor receptor pathway with selumetinib or AZD8055 can reverse anastrozole resistance. 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 2919. doi:1538-7445.AM2012-2919

Abstract LB-110: Establishment of a chordoma xenograft and in vivo testing of compounds identified by high-throughput screening

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Chordoma is a rare primary bone malignancy that arises in the skull base, spine and sacrum and originates from remnants of the notochord. Therapy primarily consists of surgical resection and radiation. These tumors are typically resistant to conventional chemotherapy, and to date there are no FDA-approved agents for chordoma. The lack of in vivo models of chordoma has impeded the development of new therapies for this tumor. We have established a chordoma xenograft growing in NOD/SCID/IL-2R gamma-null mice. The histology of the xenograft is consistent with a dedifferentiated chordoma. The xenograft expresses the chordoma marker T brachyury, which is aberrantly localized in the cytoplasm. SNP array shows loss of heterozygosity at the p16 locus, and gene expression profiling shows high expression of numerous cancer-related genes, such as ROS1 and CPA4 The NIH Chemical Genomics Center performed high-throughput screening of 2,816 compounds. Two established chordoma cell lines, U-CH1 and UCH2B, were treated and cell viability measured by CellTiter-Glo assay. Cells were incubated for 48 hours with drug concentrations ranging from 0.5nM to 46uM. The screen yielded several compounds that showed activity and two were tested in the xenograft. Compared with mice treated with vehicle alone, both drugs showed a dramatic inhibition of tumor growth. In conclusion, we have established a xenograft model of dedifferentiated chordoma. High-throughput screening of compounds identified compounds that show activity against chordoma. In vivo testing of two identified compounds showed a dramatic reduction of tumor growth. 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 LB-110. doi:1538-7445.AM2012-LB-110

Abstract 2418: Establishment and characterization of a primary chordoma xenograft

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Chordomas are slow growing, malignant cancers believed to originate from notochord remnants. Chordoma accounts for 1% of cancers of the central nervous system with an annual incidence of 0.08 cases per 100,000 individuals. There are 3 different histologic subtypes- classic chordoma, chondroid chordoma, and dedifferentiated chordoma. Chordomas are characterized by the presence of physaliphorous cells with vacuolated cytoplasm and high expression of the transcription factor T Brachury. Management of chordoma is primarily surgical – chemotherapy and radiation therapy have limited effect. The development of effective treatments for this tumor is hampered by the lack of adequate animal models. We report here the establishment of the first primary xenograft model of chordoma. Tumor excised from a patient diagnosed with classic chordoma was implanted adjacent to the right proximal tibia in a NOD/SCID/IL2Rγ (null) mouse. The resulting tumor has been serially transplanted at least 10 times with no diminution in growth rate over time. Histologically, the xenograft does not appear to be a classic chordoma; interestingly, the patient from whom the xenograft was derived relapsed with a dedifferentiated morphology. To confirm the identity of the tumor xenograft, T brachyury expression was demonstrated by immunohistochemistry and PCR. We have begun to characterize this xenograft by gene expression profiling and comparative genomic hybridization. Expression profiling results were verified by RT-PCR. Cells isolated from xenografts were analyzed by the Aldefluor assay to assess the levels of expression of aldehyde dehydrogenase activity. A cell line was generated from this xenograft as well and is being characterized. In summary, we report here the establishment of the first primary chordoma xenograft. Initial molecular characterization of the xenograft and a cell line derived from this tumor is ongoing. These tools will shed valuable light on the pathophysiology of this rare tumor and allow the preclinical development of novel treatment strategies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2418. doi:10.1158/1538-7445.AM2011-2418

Abstract 4274: High aldehyde dehydrogenase activity identifies a chemotherapy-resistant population of ewing's sarcoma cells with a stem cell phenotype that retains sensitivity to EWS-FLI1 inhibition

Ewing9s sarcoma family tumors (ESFT) are the second most common bone tumors in children and young adults. Despite being quite responsive to chemotherapy, patients with localized disease have a 30% recurrence rate, and 80% of patients with metastatic disease die within 5 years of diagnosis. The expression of the EWS-FLI1 fusion protein as a result of chromosomal translocation (11; 22) (q24; q12) is crucial for initiation and maintenance of the tumor; however, the mechanism by which EWS-FLI1 mediates neoplastic transformation is poorly understood. RNA helicase A (RHA) physically interacts with EWS-FLI1, modulating its oncogenic activity. The cancer stem cell hypothesis proposes that tumors originate from and are maintained by a small subset of chemotherapy-resistant stem cells. Thus, identification of Ewing9s sarcoma stem cells will lead to the development of targeted therapies that should improve the treatment of patients with this disease. We attempted to identify a population of Ewing9s sarcoma cells with stem cell properties (self-renewal in vitro, clonogenic activity, and tumor initiating activity in immunodeficient mice) based on high aldehyde dehydrogenase (ALDH high ) activity. The ALDH high population was enriched for stem cell activity (compared to the ALDH low cells) as defined by these assays, and was resistant to standard chemotherapy drugs to treat sarcomas (doxorubicin and etoposide). Both populations expressed EWS-FLI1. We then investigated whether a novel small molecule, YK-4-279, which specifically targets the RHA/EWS-FLI1 interaction, is toxic to the chemotherapy-resistant ALDH high cells. We found that the ALDH high cells retain sensitivity YK-4-279. Our data demonstrate that ESFT cells that express high levels of ALDH have stem-like properties, and, combined with evidence that YK-4-279 inhibits the growth of ESFT xenografts in mice, provide support for a novel approach to ESFT therapy. This approach should improve the survival of patients with these tumors in the future. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4274.