μM Sunitinib Research Articles

Sunitinib malate induces cell death in adult human cardiac progenitor cells

Sunitinib malate is known to cause cardiotoxicity in a sub-population of patients, with heart failure seen in more severe cases. Cardiac progenitor cells (CPCs) have been identified in adult human myocardium and contribute to overall tissue maintenance, with previous work identifying negative impacts of sunitinib on these cells. This study aimed to characterise the toxic effects of sunitinib in human CPCs, applying sunitinib concentrations equivalent to clinical plasma levels to these cells in vitro. Cell viability was reduced by 26.5 ± 6.6 % by 2 μM sunitinib for 24 h (p < 0.01); this concentration also induced fold-change increases in gene expression of: calpain (3.1 ± 0.73, p < 0.05), FAS (2.3 ± 0.8, p < 0.05) and BAX (1.9 ± 0.2, p < 0.05), and a decrease in BCL-2 (3.5 ± 0.0, p < 0.001), vs. control (1.0 ± 0.0). This was affirmed by sunitinib inducing fold changes in protein expression of: calpain-1 (2.5 ± 0.5, p < 0.05); FAS receptor (2.1 ± 0.2, p < 0.05) and BAX (2.1 ± 0.2, p < 0.05) vs. control (1.0 ± 0.0). These results indicated that sunitinib induced apoptosis in CPCs, but negative annexin V staining and lack of protection by caspase inhibitors indicated this was not the cell death pathway activated. Further investigation found sunitinib was concentrated in the lysosomes and autophagosomes within CPCs, but did not induce accumulation of acidic organelles. In conclusion, these data confirm that cell death is caused by sunitinib in CPCs at concentrations equivalent to clinical plasma levels, inducing cell death pathway signals that lead to non-apoptotic cell death.

Metformin Protects Against Sunitinib-induced Cardiotoxicity: Investigating the Role of AMPK.

Sunitinib is associated with cardiotoxicity through inhibition of AMP-protein kinase (AMPK) signaling. By contrast, the common antidiabetic agent metformin has demonstrated cardioprotection through indirect AMPK activation. In this study, we investigate the effects of metformin during sunitinib-induced cytotoxicity. Left ventricular developed pressure, coronary flow, heart rate, and infarct size were measured in Langendorff-perfused rat hearts treated with 1 µM sunitinib ±50 µM metformin ±1 µM human equilibrative nucleoside transporter inhibitor S-(4-Nitrobenzyl)-6-thioinosine (NBTI). Western blot analysis was performed for p-AMPKα levels. Primary isolated cardiac myocytes from the left ventricular tissue were used to measure live cell population levels. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was used to assess adjunctive treatment of and metformin in human hepatoma G2 and promyelocytic leukemia (HL-60) cells treated with 0.1-100 µM sunitinib ±50 µM metformin. In the perfused hearts, coadministration of metformin attenuated the sunitinib-induced changes to left ventricular developed pressure, infarct size, and cardiac myocyte population. Western blot analysis revealed a significant decrease in p-AMPKα during sunitinib treatment, which was attenuated after coadministration with metformin. All metformin-induced effects were attenuated, and NBTI was coadministered. The MTT assay demonstrated an increase in the EC50 value during coadministration of metformin with sunitinib compared with sunitinib monotherapy in hepatoma G2 and HL-60 cell lines, demonstrating the impact and complexity of metformin coadministration and the possible role of AMPK signaling. This study highlights the novel cardioprotective properties of metformin and AMPK activation during sunitinib-induced cardiotoxicity when administered together in the Langendorff heart model.

Toxicity of anticancer drugs in human placental tissue explants and trophoblast cell lines

The application of anticancer drugs during pregnancy is associated with placenta-related adverse pregnancy outcomes. Therefore, it is important to study placental toxicity of anticancer drugs. The aim of this study was to compare effects on viability and steroidogenesis in placental tissue explants and trophoblast cell lines. Third trimester placental tissue explants were exposed for 72 h (culture day 4–7) to a concentration range of doxorubicin, paclitaxel, cisplatin, carboplatin, crizotinib, gefitinib, imatinib, or sunitinib. JEG-3, undifferentiated BeWo, and syncytialised BeWo cells were exposed for 48 h to the same drugs and concentrations. After exposure, tissue and cell viability were assessed and progesterone and estrone levels were quantified in culture medium. Apart from paclitaxel, all compounds affected both cell and tissue viability at clinically relevant concentrations. Paclitaxel affected explant viability moderately, while it reduced cell viability by 50% or more in all cell lines, at 3–10 nM. Doxorubicin (1 µM) reduced viability in explants to 83 ± 7% of control values, whereas it fully inhibited viability in all cell types. Interference with steroid release in explants was difficult to study due to large variability in measurements, but syncytialised BeWo cells proved suitable for this purpose. We found that 1 µM sunitinib reduced progesterone release to 76 ± 6% of control values, without affecting cell viability. While we observed differences between the models for paclitaxel and doxorubicin, most anticancer drugs affected viability significantly in both placental explants and trophoblast cell lines. Taken together, the placenta should be recognized as a potential target organ for toxicity of anticancer drugs.

Abstract 811: Histological heterogeneity contributes to sunitinib resistance in clear cell renal cell carcinoma

Abstract Introduction: The receptor tyrosine kinase (RTK) inhibitor sunitinib is the first line treatment for advanced clear cell renal cell carcinoma (ccRCC). Sunitinib inhibits angiogenesis via blocking signaling through VEGFR. About 80% of patients develop resistance after a drug-sensitive period. Molecular changes early in treatment may impact drug resistance, but are poorly understood. Experimental Procedures: ACHN, 786-O and Renca cell lines were treated with 1 µM sunitinib. NSG mice were s.c. xenografted with the model cell lines and were treated with sunitinib at 40 mg/kg/day dose. mRNA expression was screened using Illumina HT-12 bead chip array and miRNA expression was assessed by Nanostring nCounter assay. R statistical packages were used for data processing. Reactome and miRPath softwares were used for downstream analysis. Results: Sunitinib treatment of ccRCC xenografts led to several early changes in tumor histology, such as the emergence of live tumor areas within the necrotic spaces. These areas showed membranous staining for E-cadherin, and β-catenin, while the rest of the tumor and vehicle-treated tumors were negative. In vitro model cell lines developed cancer spheroids when treated with sunitinib. Cancer spheroids were highly tumorigenic and metastatic, and expressed several established cancer stem cell markers. ccRCC cancer spheres, but not the 2D adherent cells, showed membranous staining for E-cadherin and β-catenin; similarly to the live tumor areas observed in in vivo sunitinib treatment. In vitro inhibition of E-cadherin by EGTA or by siRNA, interfered with viability of sunitinib treated ccRCC cell lines. Conclusions: Sunitinib treatment causes early phenotypic changes of the tumor in vivo and in vitro. The formation of highly metastatic and tumorigenic cancer spheres in model cell lines is the most prominent effect in vitro. We provide preliminary evidence that sunitinib induced in vitro cancer spheres and the live tumor areas that survive within necrotic patches of the sunitinib-treated xenografts, are related. Finally, membranous expression of E-cadherin enhances the survival of ccRCC cell lines under sunitinib treatment. Citation Format: Zsuzsanna Lichner, Rola Saleeb, Henriett Butz, Roy Nofech-Mozes, Sara Riad, Mina Farag, Andras Kapus, George Yousef. Histological heterogeneity contributes to sunitinib resistance in clear cell renal cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 811. doi:10.1158/1538-7445.AM2017-811

Alternative scheduling of pulsatile, high dose sunitinib efficiently suppresses tumor growth.

BackgroundIncreased exposure to multitargeted kinase inhibitor sunitinib is associated with improved outcome, emphasizing the importance of maintaining adequate dosing and drug levels. The currently approved schedule (50 mg daily, four weeks on, two weeks off) precludes further dose-intensification. Recent data suggest that sunitinib, although initially developed as an antiangiogenic agent, has direct antitumor activity.MethodsIn this study, we tested whether a chemotherapy-like schedule of pulsatile high dose sunitinib would result in improved antitumor activity.ResultsIn vitro, a single exposure to 20 μM sunitinib for 6-9 h resulted in complete inhibition of tumor cell growth and cell death conveyed through activation of caspases and autophagy upregulation. Notably, repeated exposure of tumor cells to pulses of high concentrations of sunitinib did not induce resistance. In vivo, once-weekly treatment with high dose sunitinib of tumors growing on the chorioallantoic membrane (CAM) of the chicken embryo significantly impaired tumor growth by 57 % compared to vehicle, outperforming the daily, standard scheduling.ConclusionsThese results prompted the initiation of a phase I clinical trial, where intermittent, high dose sunitinib is being investigated in patients with advanced solid tumors (registration number and date: NCT02058901, 30 September 2013, respectively). The trial is actively recruiting patients and promising preliminary indications of antitumor activity have been observed.

Abstract 3340: Doublecortin-like kinase 1 marks cancer stem-like cells and modulates drug-resistance, self-renewal, and tumorigenesis in renal cancer

Abstract Introduction Doublecortin-like kinase 1 (DCLK1), is a tumor stem cell-specific marker, expressed in many cancers and highly correlated to cancer initiation, EMT and progression. We recently reported DCLK1's epigenetic dysregulation and specific overexpression in clear cell renal carcinoma (RCC). In this study, we assessed DCLK1's value as a therapeutic target in RCC progression. Materials and Methods RCC cell line Caki-2 was infected with Lentivirus to overexpress DCLK1. Moreover, DCLK1 gene expression was knocked down in ACHN and Caki-2 cell lines using small interfering RNA. Gene and protein expression levels were measured by real time RT-PCR and Western blotting respectively. Proliferation and drug resistance was determined by MTT assay. Cell cycle analysis and sorting of DCLK1+/DCLK1- cells were completed by FACS. Clonogenicity was evaluated using colony formation assay in matrigel. Results Overexpression of DCLK1 increases Caki-2 cell proliferation, but despite DCLK1's microtubule localization, there was no evidence of altered cell cycle status. Aldehyde dehydrogenase (ALDH), one of only a few known stem cell markers in RCC, was highly upregulated (&amp;gt;5-fold) by DCLK1 overexpression and downregulated (2-fold) by knockdown of DCLK1 in RCC cells. FACS results also indicated the existence of small ALDH and DCLK1 double-positive populations in both Caki-2 (1.51%) and ACHN (0.54%) RCC cells. Furthermore, there was approximately 7% more ALDH+ cells in DCLK1 overexpressing Caki-2 cells compared to control. In further support of molecular hallmarks, we observed increased expression of HIF-1α and Vimentin mRNA and protein as well as enhanced clonogenic capacity of ACHN and Caki-2 RCC cell lines in matrigel at baseline and after 72 h treatment with 0.5 μM Sunitinib. Immunocytochemistry of the spheroids suggested that DCLK1 enhances colony formation ability by triggering expression of pluripotency factors NANOG and POU5F1/OCT4. Since DCLK1 is known to have an extracellular domain we performed FACS sorting to determine if we could isolate a viable DCLK1+ population from the ACHN cell line. ACHN-DCLK1+ cells had &amp;gt;1.5 fold higher relative colony number and larger sphere size in three-dimensional clonogenic assay compared to ACHN-DCLK1- cells. Finally, we performed MTT assays demonstrating that overexpression of DCLK1 caused Caki-2 cells to become resistant to Sunitinib, Everolimus, and Temsirolimus compared to controls after 48 h treatment. Converesely, knockdown of DCLK1 sensitized both ACHN and Caki-2 cells to Sunitinib and Sorafenib after 48 hours treatment. Conclusion DCLK1 modulates proliferation, stemness, tumorigenesis and resistance to FDA-approved drugs, and marks a population of stem-like cells in RCC. These findings suggest that targeting DCLK1 may be a novel primary or adjuvant therapeutic strategy in RCC. Citation Format: Yang Ge, Nathaniel Weygant, Dongfeng Qu, Randal May, William L. Berry, Parthasarathy Chandrakesan, James J. Tomasek, Guangyu An, Courtney W. Houchen. Doublecortin-like kinase 1 marks cancer stem-like cells and modulates drug-resistance, self-renewal, and tumorigenesis in renal 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 3340.

Sunitinib Reduces Acute Myeloid Leukemia Clonogenic Cells &lt;i&gt;in Vitro&lt;/i&gt; and Has Potent Inhibitory Effect on Sorted AML ALDH+ Cells

Sunitinib is an orally administered, multi-target tyrosine kinase inhibitor that has been approved by the FDA for the treatment of renal cell carcinoma and imatinib resistant gastro-intestinal tumors. Anti-leukemic activity of sunitinib has been examined in early clinical trials with limited success. However, recent trials on acute myeloid leukemia (AML) patients carrying FLT3 mutations have shown promising results. Effects of sunitinib on leukemic clonogenic cells and potential leukemic stem cells have not been examined so far. We analyzed the anti-proliferative and apoptotic properties of sunitinib on AML-derived cell lines. We also tested the effect of sunitinib on AML patient derived clonogenic cells (AML-CFC), as well as flow-sorted potential leukemic progenitors. Peripheral blood or bone marrow samples were obtained from newly diagnosed AML patients and flow sorted for CD34+ CD133+ or ALDH+ cells. Umbilical cord blood derived CD34+ cells were used as normal controls. Sunitinib induced growth arrest and apoptosis in AML derived cell lines. In addition, 7 μM sunitinib induced 75% reduction of AML-CFC as compared to DMSO treated control (±6.79%; n = 4). In contrast, 7 μM sunitinib treatment of umbilical cord blood derived normal CD34+ cells showed 29% reduction in AML-CFC (±6.77%; n = 5). Treatment of ALDH+ cells sorted from 2 AML cases and CD34+ CD133+ cells from one patient showed reduction of AML-CFC on treatment with sunitinib. Our study highlighted a potent anti-proliferative and proapoptotic effect of sunitinib on AML cell lines, AML patient derived clonogenic cells and potential leukemic stem cells.

Abstract 5375: Combined treatment of trichostatin A enhances cytotoxic effects of sunitinib on renal cell carcinoma cells

Abstract Introduction: Sunitinib (SU) is one of the multi-targeted tyrosine kinase inhibitors (TKIs) and currently approved for use in advanced renal cell carcinoma (RCC) by inhibition of angiogenic signaling. Although it is a really attractive drug for patients who cannot have surgical treatment, SU treatment hasn't been always successful because of frequent resistance and severe side effects. Combination therapy is one of the strategies to solve these problems. The combinations of SU and IFN-α or mTOR inhibitors in the treatment of RCC have been already explored. However, both of which resulted in failure due to dose-limiting toxicities. Therefore, it is important to establish a novel combination strategy for RCC treatment. In the context of seeking for a prominent combination agent for SU, we paid attention to epigenetic agents. There has been several reports where epigenetic agents were proven to induce apoptotic cells in combination with chemotherapeutic agents, such as gemcitabine and sorafenib. To investigate whether epigenetic agents could be a sensitizer of SU as well in RCC cells, we used trichostain A (TSA), a well-known and strong inhibitor of histone deacetylase (HDAC), and also examined what the mechanism is. Methods: We used 786-O, ACHN and Caki-1 RCC cell lines. The cells were treated with SU alone, TSA alone or the combination, then cytotoxicity was measured by the MTT cytotoxicity method under normoxic or hypoxic conditions. To explore the effect on cell cycle after the combination treatment, flowcytometry was performed. The expression of RTK signaling proteins and p21, which is one of the major targets of TSA, were detected by western blotting. Results: TSA significantly enhanced SU cytotoxicity in all of RCC cells, especially in 786-O. VEGF protein expression, a major angiogenic factor showed increasing trend in 786-O after 8 h of hypoxic exposure, which was decreased by the combined treatment. Flowctometry has revealed that suggested apoptotic cell population (sub-G1) was significantly higher in 1 μM SU and 0.5 μM TSA combination group compared with control or single SU treatment group. On the other hand, ACHN cells didn't show significant increase of sub-G1 population although cell cycle-arrest at S and G2/M phase was observed in combined treatment group (2.5 μM SU and 0.25 μM TSA). Additionally, cell cycle regulator protein, p21 was significantly increased in both 786-O and ACHN cells in TSA concentration dependent manner, which was accompanied with increase of acetylated histone H3. Conclusion: These findings suggest that combined treatment of SU and TSA is effective against RCC cells. The combination possibly enhances apoptosis or growth inhibition, where increase of p21 by TSA might be involved. Citation Format: Hiromi Sato, Tatsuro Kashiba, Miaki Uzu, Takuya Fujiwara, Yukihiro Shibata, Rina Suzuki, Katsunori Yamaura, Akihiro Hisaka. Combined treatment of trichostatin A enhances cytotoxic effects of sunitinib on renal cell carcinoma cells. [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 5375. doi:10.1158/1538-7445.AM2015-5375

Abstract 5449: Transfection of connexin 43 enhances sunitinib-induced apoptosis in malignant mesothelioma cells possibly via its interaction with Bax

Abstract Introduction: Malignant mesothelioma (MM) is an aggressive tumor arising from mesothelial cells. MM is an increasing tumor worldwide with poor prognosis due to its chemo and radioresistant characteristics. We are aiming to enhance chemosensitivity of MM, and focus our attention on connexin (Cx), which is a component of intercellular channel called gap junction (GJ). Various evidences suggest that cellular physiology is maintained via GJ function. However, in cancer cells, GJ function is often aberrant, associating with the decreased Cx expression. Previously, we have shown that the sensitivity of MM cells to cisplatin was enhanced by increasing expression of Cx43, which is the major Cx subtype in mesothelial cells. Recently, it is suggested that MM is a highly angiogenic tumor and vascular endothelial growth factor (VEGF) signaling pathway plays a key role in pathogenesis of MM. Therefore, tyrosine kinase inhibitors (TKIs) are thought to be new prominent therapeutic agents for this tumor; however, they displayed a limited therapeutic activity in MM patients. Hence, the restoration of Cx expression would be beneficial to treatments with sunitinib (SU), a representative TKI, to exert an antiproliferative effect in MM patients. The present study aims to evaluate the effect of Cx43 transfection on the cytotoxicity of SU in MM cells. Methods: We used H28, a human MM cell line, to transfect Cx43 gene. The transfected cell was named H28-T. Cell viability was assessed by MTT assay. SubG1 population (suggested apoptotic cell population) was measured by flow cytometry. Protein interaction and expression level were analyzed by western blotting after immunoprecipitation. Results: At first, sensitivity to SU was compared between H28 and H28-T. Cell viability of H28-T was significantly lower than that of H28 in the presence of 0.38-10 μM SU. Moreover, the rate of SubG1 population was significantly higher in H28-T than in H28 in the presence of 10 μM SU. Next, the mechanism of enhanced cytotoxicity of SU was investigated using H28-T. To examine the involvement of GJ function, the combined treatment with SU and 18β-glycyrrhetinic acid, a GJ inhibitor, was performed; however, cell viability was not altered as compared to SU treatment alone. The interaction between Cx43 and Bax, a proapoptotic factor, was then investigated. It was shown that Bax was immunoprecipitated with an anti-Cx43 antibody. Protein expression of Bax was higher in H28-T than in H28. Furthermore, a cleaved (active) form of Bax was detected by treatment with 10 μM SU only in H28-T. Conclusion: These findings indicate that Cx43 directly interacts with Bax, and this interaction may be involved in enhancement of SU-induced apoptosis in MM cells through a GJ function-independent mechanism. Citation Format: Miaki Uzu, Hiromi Sato, Tatsuro Kashiba, Takuya Fujiwara, Yukihiro Shibata, Katsunori Yamaura, Akihiro Hisaka. Transfection of connexin 43 enhances sunitinib-induced apoptosis in malignant mesothelioma cells possibly via its interaction with Bax. [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 5449. doi:10.1158/1538-7445.AM2015-5449

Molecular analysis of sunitinib resistant renal cell carcinomacells after sequential treatment with RAD001(everolimus) or sorafenib.

Sequential application of target drugs is standard procedure after renal cell carcinoma (RCC) patients develop resistance. To optimize the sequence, antitumour effects of the mTOR inhibitor RAD001 or the tyrosine kinase inhibitor (TKI) sorafenib on RCC cells with acquired resistance to the TKI sunitinib was evaluated. RCC cells were exposed to 1 μM sunitinib for 24 hrs (as control) and for 8 weeks (to induce resistance) and then switched to RAD001 (5 nM) or sorafenib (5 μM) for a further 8 weeks. Tumour cell growth, cell cycle progression, cell cycle regulating proteins and intracellular signalling were then investigated. Short-term application of sunitinib (24 hrs) induced cell growth blockade with accumulation in the G2/M phase. RCC cells became resistant to sunitinib after 8 weeks, demonstrated by accelerated cell growth along with enhanced cdk1, cdk2, loss of p27, activation of Akt, Rictor and Raptor. Switching to sorafenib only slightly reduced growth of the sunitinib resistant RCC cells and molecular analysis indicated distinct cross-resistance. In contrast, full response was achieved when the cancer cells were treated with RAD001. p19 and p27 strongly increased, phosphorylated Akt, Rictor and Raptor decreased and the tumour cells accumulated in G0/G1. It is concluded that an mTOR-inhibitor for second-line therapy could be the strategy of choice after first-line sunitinib failure.

Abstract 5641: Sunitinib inhibits AXL phosphorylation in tumor cells.

Abstract Background Sunitinib is a multi-targeted tyrosine kinase inhibitor (TKI), that is used for treatment of patients with advanced renal cell cancer (RCC) Drug resistance to sunitinib is a major clinical problem. We recently reported that continuous exposure to sunitinib causes acquired resistance of tumor cells. The receptor tyrosine kinase Axl has been implicated in acquired resistance to multiple TKIs, including erlotinib and lapatinib. In addition, sunitinib was previously reported to be a potential inhibitor of Axl catalytic activity. We therefore investigated the potential role of Axl in sunitinib resistance. Methods 786-O cells resistant to sunitinib were obtained by continuous exposure to the drug as previously published (Gotink et al. Clin Cancer Res 2011). AXL expression was studied in 786-O cell lines and EC-RF24 endothelial cells by RT-PCR and Western blot. Sunitinib and AXL inhibitor R428 sensitivity of cells transfected with siRNAs against AXL or scrambled siRNAs was determined by MTT cell viability assays. For kinase activity experiments recombinant AXL and tyrosine peptide arrays (PamGene, ‘s Hertogenbosch, the Netherlands) were used. Phospho-AXL and total AXL in cell lysates were measured by ELISA. Results Axl phosphorylation in vitro in both 786-O and EC-RF24 cells was dose-dependently inhibited by sunitinib, with a 50% inhibitory concentration of 5 μM. Activity of recombinant Axl kinase was measured on tyrosine containing peptide arrays in presence of 0, 1 or 4 μM sunitinib or 2 μM R428. Sunitinib induced dose-dependent inhibition of Axl activity, where 2 μM R428 blocked all peptide phosphorylations. In addition, 786-O sunitinib resistant cells (786-O SUN) showed a 4-fold overexpression of Axl protein compared to 786-O parental cells. However, gene silencing of AXL in 786-O SUN cells by RNA interference did not sensitize the cells to sunitinib. Sunitinib resistant cells were shown to be cross-resistant to the Axl inhibitor R428 with respective IC50 values of 0.9 μM (±0.1 μM) and 1.8 μM (±0.1 μM). Conclusion Phosphorylation of Axl is inhibited by sunitinib in both 786-O RCC and EC-RF24 endothelial cells. Despite its overexpression in sunitinib resistant cells, gene silencing of AXL did not sensitize 786-O resistant cells to sunitinib, indicating that sunitinib resistance is not mediated by AXL. Citation Format: Johannes C. Van der Mijn, Kristy J. Gotink, Richard R. de Haas, Henk Dekker, Connie R. Jimenez, Henk J. Broxterman, Henk M.w. Verheul. Sunitinib inhibits AXL phosphorylation in tumor cells. [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 5641. doi:10.1158/1538-7445.AM2013-5641

Abstract 1621: Short-time exposure to high concentrations of sunitinib causes tumor cell death.

Abstract Background: Sunitinib, although initially developed as an antiangiogenic agent, has been shown to exert direct anti-tumor effects. Previously we demonstrated that sunitinib inhibits proliferation of various tumor cell lines in a dose-dependent manner. We here studied whether short exposure to high peak concentrations of sunitinib would induce tumor cell death. Methods: 786-O renal cancer cells were incubated with increasing concentrations of sunitinib (5, 10 and 20 μM) for various time intervals (1 hour, 3, 6, 9, 24, 48, 72 hours). At prespecified time points, sunitinib was removed and cells were subsequently allowed to grow in drug-free culture medium. Viability of these cells one week after short time sunitinib exposure and sensitivity of recovered cells after repetitive treatment with high sunitinib concentrations were determined by MTT assays. Flow cytometry with Propidium Iodide staining was employed to determine apoptosis. Results: Short-time exposure to high concentrations of sunitinib significantly induced cell death. Exposure to 20 μM of sunitinib for 9 hours provoked cell death in a manner identical to the prolonged exposure of 24, 48 or 72 hours. Flow cytometry confirmed the significant increase in cell death (60%) after 9-hour treatment with 20 μM sunitinib, compared to the untreated control (2%). Tumor cells that were repetitively treated with high concentrations of sunitinib retain their sensitivity to the drug identical to its parental cell line. Analysis of the key signaling pathways involved in sunitinib-induced cell death is ongoing. Conclusion: We here report that repetitive short-time exposure to high concentration sunitinib causes cell death without inducing resistance. Interestingly, single-dose sunitinib up to 300 mg was previously reported to be well tolerated by patients. Based on these findings, administration of high dose intermittent sunitinib may result in cytotoxic antitumor activity irrespective of angiogenesis inhibition. A phase I trial to test this hypothesis in patients with cancer will start shortly. Citation Format: Maria Rovithi, Dennis Poel, Jens Voortman, Mariette Labots, Henk Dekker, Kristy J. Gotink, Henk J. Broxterman, Henk M.W. Verheul. Short-time exposure to high concentrations of sunitinib causes tumor cell death. [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 1621. doi:10.1158/1538-7445.AM2013-1621

Abstract A54: Potential mechanisms of resistance to targeted agents in human clear cell renal cell carcinoma

Abstract Targeted therapy with multiple receptor tyrosine kinase inhibitors (RTKI) has led to a substantial improvement in the standard of care for patients with advanced or metastatic clear cell renal cell carcinoma (CCRCC). We have previously reported that using a phosphoproteomics approach, Ras and Rab interactor 1 (RIN1) was identified as a potential target modulating the antiproliferative effects of sorafenib and sunitinib in a panel of human CCRCC (Proc. AACR 51: Abst. #1626, 2010). While both 10 μM sorafenib and 7.5 μM sunitinib were effective in down-regulating RIN1 protein levels in CCRCC following treatment for 48h, during subsequent recovery for 48h in drug free medium, recovery of RIN1 protein levels was observed in sorafenib but not sunitinib treated cells. mRNA expression profiling revealed that: (a) VEGF-A and HIF-1α levels were unaffected during 48h treatment with sorafenib or sunitinib or subsequent recovery; and (b) while HIF-2α levels were increased &amp;gt;2-fold following 48h exposure to sorafenib or sunitinib, during subsequent recovery in drug-free medium, the increased levels of HIF-2α persisted in cells treated with sorafenib but not sunitinib. Compared to CCRCC cells that were growth inhibited in vitro at concentrations of 5–10 μM sorafenib or sunitinib, in 27 patient CCRCC samples evaluated, the median mRNA levels of RIN1 was 7.2-fold lower in 22 samples (Range 1.6–22.3) and 2.2-fold higher in 5 samples (Range 1.2–27.1). Stable over-expression and down-regulation of RIN1 in CCRCC led to resistance or sensitivity respectively, to the anti-proliferative effects of sorafenib and sunitinib but not the mTOR inhibitor temsirolimus. In contrast, CCRCC cells adapted to proliferate in 4 μM everolimus exhibited a &amp;gt;3-fold increase in IC50, and cross-resistance to temsirolimus but not sorafenib or sunitinib. Evaluation in vitro of other targeted agents, demonstrated dose-dependent anti-proliferative effects with the PI3K/mTOR dual inhibitor BEZ235 (&amp;gt;80% growth inhibition at 10nM) but not the pan-PI3K inhibitor BKM120 (&amp;lt;30% growth inhibition at 10–500 nM) with CCRCC cells. The observed anti-proliferative effects of BEZ235 or BKM120 were comparable in CCRCC cells harboring the wild type or mutant von Hippel-Lindau gene. While BEZ235 (1–5 nM) or everolimus (100 nM) alone led to &amp;lt;40% growth inhibition, the combination significantly enhanced anti-proliferative effects (&amp;gt;85% growth inhibition) in CCRCC cells expressing resistance to everolimus. These results suggest the PI3K/mTOR dual inhibitor BEZ235 may possibly improve the treatment efficacy of mTOR inhibitors for CCRCC. The findings suggest RTKI and mTOR inhibitors have distinct mechanisms of resistance, and combinations may be useful to improve disease control of metastatic CCRCC.

Inhibition of RNase L and RNA-dependent Protein Kinase (PKR) by Sunitinib Impairs Antiviral Innate Immunity

RNase L and RNA-dependent protein kinase (PKR) are effectors of the interferon antiviral response that share homology in their pseudokinase and protein kinase domains, respectively. Sunitinib is an orally available, ATP-competitive inhibitor of VEGF and PDGF receptors used clinically to suppress angiogenesis and tumor growth. Sunitinib also impacts IRE1, an endoplasmic reticulum protein involved in the unfolded protein response that is closely related to RNase L. Here, we report that sunitinib is a potent inhibitor of both RNase L and PKR with IC(50) values of 1.4 and 0.3 μM, respectively. In addition, flavonol activators of IRE1 inhibited RNase L. Sunitinib treatment of wild type (WT) mouse embryonic fibroblasts resulted in about a 12-fold increase in encephalomyocarditis virus titers. However, sunitinib had no effect on encephalomyocarditis virus growth in cells lacking both PKR and RNase L. Furthermore, oral delivery of sunitinib in WT mice resulted in 10-fold higher viral titers in heart tissues while suppressing by about 2-fold the IFN-β levels. In contrast, sunitinib had no effect on viral titers in mice deficient in both RNase L and PKR. Also, sunitinib reduced mean survival times from 12 to 6 days in virus-infected WT mice while having no effect on survival of mice lacking both RNase L and PKR. Results indicate that sunitinib treatments prevent antiviral innate immune responses mediated by RNase L and PKR.

Abstract 2852: Lysosomal sequestration of sunitinib may play a role in its resistance

Abstract Resistance to tyrosine kinase inhibitors (TKIs) is a major clinical problem. Mechanisms that mediate drug resistance include gene mutations, multidrug efflux and activation of alternative growth factor pathways. Sunitinib, a multi-targeted antiangiogenic TKI, has demonstrated clinical efficacy in advanced renal cell cancer (RCC) and gastrointestinal stromal tumors, but is hampered by resistance. In this study we aimed to unravel mechanisms of sunitinib resistance. Based on its large volume of distribution, we hypothesized that sunitinib concentrations intratumoral would be higher compared to plasma. Indeed we found that in patients harboring different tumors, intratumoral sunitinib concentrations were &amp;gt;30-fold higher than parallel plasma concentrations: 9.5±2.4 µM versus 0.28±0.06 µM (n=3, mean±SEM), respectively, as measured by LC-MS/MS. Subsequently, we studied the in vitro sensitivity to sunitinib (provided by Pfizer Inc.) of 1 RCC and 8 colorectal cancer cell lines and found clinically relevant inhibitory concentrations (IC) on proliferation of IC50 = 1.3±0.1 µM and IC90 = 4.6±0.6 µM sunitinib. In parallel experiments, we induced resistance in vitro in 786-O and HT-29 cells by continuous exposure to increasing concentrations of sunitinib for &amp;gt; 1 year. These resistant tumor cells continued to stably grow on exposures to clinically achieved intratumoral concentrations of 6 µM (786-O) and 12 µM (HT-29) sunitinib, whereas their parental cells died at these concentrations. Microscopy of sensitive and resistant tumor cells revealed sequestration of sunitinib in specific subcellular compartments. Because sunitinib is a hydrophobic (logP=5.2) weak base (pKa = 8.95), we reasoned that it might preferentially accumulate in acidic lysosomes. Indeed, staining with a lysosome-specific fluorescent dye revealed predominant co-localization of sunitinib in lysosomes. In addition, compared to sensitive cells, lysosomal accumulation capacity was increased in resistant cells, which was confirmed by flow cytometry and Western blot analysis (LAMP-1 and LAMP-2 expression). Intracellular concentrations were increased in the resistant tumor cells compared to their parental cells, 4.6±1.1 mM and 2.3±0.4 mM, respectively, and were up to 1000-fold higher than the micromolar concentrations used for in vitro exposure. Growth of resistant cells in drug-free medium resulted in restoration of drug sensitivity and normalization of lysosomal drug accumulation capacity within 12 weeks. In conclusion, our data show that: 1) sunitinib inhibits proliferation of tumor cells in vitro at clinically relevant intratumoral concentrations, 2) tumor cells acquire a transient drug-resistant phenotype under continuous exposure to sunitinib, 3) sunitinib is sequestered in acidic lysosomes and 4) increased lysosomal sequestration may contribute to sunitinib resistance. 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 2852. doi:10.1158/1538-7445.AM2011-2852

Sunitinib induces PTEN expression and inhibits PDGFR signaling and migration of medulloblastoma cells

We previously showed that inhibition of the platelet-derived growth factor receptor (PDGFR) blocks the survival and migration of medulloblastoma cells. Identification of in vitro PDGFR-targeting pharmacologic agents that are suitable for preclinical testing in medulloblastoma models in vivo will be critical for efficiently translating these agents to clinical investigation in children with medulloblastoma. In this study, we investigated whether the multi-tyrosine kinase inhibitor sunitinib, effectively inhibits PDGFR signaling required for medulloblastoma cell migration. Daoy and D556 human medulloblastoma cells pre-treated for 1 h with 0.2 μM sunitinib demonstrated induction of PTEN expression and significant inhibition of PDGFR signaling activity and transactivation of EGFR, in a RAS-independent manner, in response to PDGF-BB stimulation. Sunitinib pre-treatment markedly reduced medulloblastoma cell migration in response to both PDGF-BB and 10% serum at 4 and 24 h after treatment. Pre-treatment with sunitinib for 1 h also resulted in detachment and decreased viability of D556, but not Daoy, cells and only after 48 h following treatment. However, sunitinib did not induce apoptosis in either cell line at any time point, indicating that the anti-migratory effects of sunitinib were not due to impeding cell survival. Sunitinib similarly inhibited PDGFR signaling and migration of primary murine Smo/Smo medulloblastoma cells, suggesting that the Smo/Smo mouse is an appropriate model for preclinical testing of sunitinib. These results indicate that sunitinib may be an important pharmacologic agent for the treatment of invasive medulloblastoma, particularly given evidence of its ability to cross the blood-brain barrier to target tumor cells, and thus warrants further in vivo testing for confirmation of efficacy.

Abstract 1626: Tyrosine phosphorylation of RIN1 modulates the anti-proliferative effects of sorafenib and sunitinib in renal cell carcinoma (RCC)

Abstract Receptor-tyrosine kinase inhibitors (TKI), sorafenib (SRF) and sunitinib (SNT) are clinically used in treatment of RCC. Both drugs have anti-proliferative effects in several tumor types with inhibition of angiogenesis being a major mechanism of action in RCC. The intracellular molecular effects of SRF and SNT have not been completely described. Thus the identification of targets mediating the anti-proliferative effects of SRF and SNT could be useful to improve efficacy and to reduce toxicity. In this work we used immunoaffinity isolation for enrichment of phosphotyrosine containing proteins in control or SRF-treated CAKI-1 cells. To identify tyrosine-phosphorylated proteins that were differentially affected by SRF treatment, we performed liquid chromatography - tandem mass spectrometry (MS) followed by quantitative analysis using peptide counts and native peptide reference MS methods. In CAKI-1 cells, MS results revealed that signal transducer and activator of transcription 1 (STAT1) and Ras and Rab interactor 1 (RIN1) were the predominant proteins which exhibited tyrosine hypophosphorylation following treatment for 48 h with 10 μM SRF, when compared to the untreated control. Western blot analysis with phosphospecific antibody indicated a potential role for tyrosine 36 phosphorylation in RIN1 (pY36-RIN1) in CAKI-1, RC-45 and 786-O clear cell RCC. Up to a 8-fold SRF dose-dependent (5-15 µM) decrease in pY36-RIN1, but not in total RIN1 expression was also observed in a panel of RCC cell lines. Hypophosphorylation of Y36-RIN1 was also detected in CAKI-1 cells treated with 5 µM SNT but not with imatinib (≤ 10 µM), suggesting that pY36-RIN1 is a selective target for agents SRF and SNT that are also clinically effective in treatment of RCC. A ∼2-fold higher expression of RIN1 (total and phosphorylated) was observed in CAKI-1 cells following continuous exposure (∼ 6 weeks) to 7.5 µM SRF. However, unlike parent CAKI-1 cells, in cells adapted to 7.5 μM SRF, exposure to 10 μM SRF did not completely abrogate phosphorylation of Y36-RIN1. Stable expression of RIN1 targeted shRNA in CAKI-1 cells led to a 2-4 fold down-regulation of RIN1 protein and significantly (p &amp;lt; 0.002) enhanced sensitivity to the anti-proliferative effects of 7.5 μM SRF or SNT compared to scrambled shRNA. In contrast, overexpression of RIN1 in RC-45 or 786-O RCC cells resulted in significantly (p &amp;lt; 0.05) increased resistance to 10 μM SRF or 7.5 μM SNT compared to vector control. These results demonstrate that pY36-RIN1 is targeted by SRF and SNT and strongly suggest that anti-proliferative effects of these TKI drugs in clear cell RCC is mediated by inhibitory effects on RIN1 tyrosine phosphorylation. 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 1626.

Target identification by phosphoproteomics: RIN1 modulation of sorafenib-induced cytotoxicity in renal cell carcinoma

e14543 Background: Sorafenib (SFB) is a multi-tyrosine kinase inhibitor clinically useful in treatment of metastatic renal cancer. While the inhibition of angiogenesis is considered a major mechanism of action, identification of targets regulating growth inhibitory effects of SFB is necessary to further improve its efficacy and reduce toxicity. Methods: In this study we used targeted phosphoproteomics to identify tyrosine phosphorylated proteins that are differentially affected in control and SFB-treated human CAKI-1 renal cell carcinoma cells. The strategy involved immunoaffinity isolation of phosphotyrosine containing proteins and liquid chromatography - tandem mass spectrometry (MS) for identification of candidate proteins. Results: Among identified proteins, signal transducer and activator of transcription 1 (STAT1) and Ras and Rab interactor 1 (RIN1) were found to be hypophosphorylated in SFB-treated compared to untreated CAKI-1 cells based on quantitative MS analysis, by peptide counts and native peptide reference method. A ∼4-fold decrease in expression and phosphorylation of STAT1 was observed in cells treated with 10 μM SFB for 48h. Up to 8-fold SFB dose-dependent (5–15 μM) decrease in phosphorylation of RIN1 at tyrosine 36, but not in total RIN1 expression, was observed. Similar effects on decreased phosphorylation of STAT1 and RIN1 were also observed in 786-O renal cell carcinoma treated with SFB. Hypophosphorylation of RIN1 at tyrosine 36 was observed in CAKI-1 cells treated with 5 μM sunitinib but not with imatinib (≤ 10 μM). Treatment of CAKI-1 cells with RIN1 targeted, but not control si-RNA led to down-regulation of RIN1 expression and attenuation of antiproliferative effects of SFB. Notably, ∼2-fold higher expression of RIN1 protein (total and phosphorylated) was observed in CAKI-1 cells selected for resistance following continuous exposure to 7.5 μM SFB. However, unlike parent CAKI-1 cells, prolonged exposure of these SFB-resistant CAKI-1 cells to 7.5 μM SFB did not completely abrogate phosphorylation of RIN1 at tyrosine 36. Conclusions: These results demonstrate that RIN1, a Ras effector protein with multiple biochemical functions, is a target for the anti-tumor effects of SFB in kidney cancer cells. [Table: see text]

6511 ORAL Phase II study of erlotinib (E) with or without sunitinib (SU) in the treatment of metastatic non-small cell lung cancer (NSCLC)

The multitargeted tyrosine-kinase inhibitor sunitinib is a highly effective anti-angiogenic and cytostatic agent in the therapy of various tumours. While malignant gliomas have been shown to be responsive to sunitinib, detailed studies analysing human meningiomas are missing. We therefore analysed the effects of sunitinib in two benign (BenMen-1, HBL52) and two malignant (IOMM-Lee, KT21MG) human meningioma cell lines and found that DNA synthesis was significantly (p ⩽ 0.001) inhibited following 1, 2 or 5 μM sunitinib, with IC50 values between 2 and 5 μM in all cell lines. This effect was associated with a G2M-arrest at 10 μM for BenMen-1, HBL52 and IOMM-Lee, and 20 μM in KT21MG cells. Nuclear bisbenzimide staining revealed chromatin condensation following treatment with sunitinib concentrations of 10 μM or higher. Corresponding, cell viability assays showed a significant (p ⩽ 0.001) short term decrease of viable cells (24 h) only for high sunitinib concentrations with IC50-values between 10 and 20 μM. However, pre-irradiated meningioma cells (5 Gy) showed a sensitivity shift towards IC50-values around 5 μM sunitinib. We also found that 5 μM strongly reduced meningioma cell migration in vitro. Western blot analyses showed abolished platelet derived growth factor receptor (PDGFR)-autophosphorylation after sunitinib. Interestingly, the drug also inhibited the autophosphorylation of the receptor tyrosine kinase fms-like tyrosine kinase 3 (Flt3) in a dose-dependent manner. Taken together, the present data show that micromolar sunitinib has strong cytostatic and anti-migratory effects on human meningioma cells.