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Supplementary Figure 6. The effect of tasquinimod on nitric oxide synthase activity in infiltrating myeloid cells.

Supplementary Fig. 6. Histone marks expression in sunitinib resistant RP-R-01 tumors

Supplementary Fig. 5. Histone marks expression associated with resistance to sunitinib

Supplementary Figure S9 shows TIE2 and macrophage markers expression.

Supplementary Figure 4. The effect of tasquinimod on T cell proliferation and regulatory T cells.

Supplementary Fig 4. Intratumoral and plasma sunitinib concentrations

Supplementary Figure 1. Survivin peptide vaccine, SurVaxM, induced antigen-specific CD8 effector cells in the tumor bearing animals.

Supplementary Fig. 7. EZH2 and E-cadherin mRNA expression in 786-0shRNA scramble, 786-0shEZH2 (clone a and d), and 786-0 treated with GSK126

Supplementary Figure legend

Supplementary Figure 2. Tasquinimod modulation of MDSC subpopulations in CR Myc-CaP tumor bearing FVB mice.

Supplementary Fig. 2. Tumor growth of RP-R-01 ccRCC xenograft treated with sunitinib and associated changes in EZH2, H3K27me3 and E-cadherin expression.

Supplementary Figure 3. CD11b+ in tumor and MDSC subpopulations in spleen from B16-h5T4 tumor bearing C57/Bl6 mice.

Supplementary Fig 3. Liver histology and body weights of mice on sunitinib dose escalation.

Supplementary Figure 5. The effect of tasquinimod treatment on CD11c+ dendritic cells.

Supplementary Fig. 1. Characterization of RP-R-01 and RP-R-02 PDX models

Supplementary Figure Legends from Tasquinimod Modulates Suppressive Myeloid Cells and Enhances Cancer Immunotherapies in Murine Models

Receptor tyrosine kinase inhibitors have shown clinical benefit in clear cell renal cell carcinoma (ccRCC), but novel therapeutic strategies are needed. The angiopoietin/Tie2 and MET pathways have been implicated in tumor angiogenesis, metastases, and macrophage infiltration. In our study, we used trebananib, an angiopoietin 1/2 inhibitor, and a novel small-molecule MET kinase inhibitor in patient-derived xenograft (PDX) models of ccRCC. Our goal was to assess the ability of these compounds to alter the status of tumor-infiltrating macrophages, inhibit tumor growth and metastases, and prolong survival. Seven-week-old SCID mice were implanted subcutaneously or orthotopically with human ccRCC models. One month postimplantation, mice were treated with angiopoietin 1/2 inhibitor trebananib (AMG 386), MET kinase inhibitor, or combination. In our metastatic ccRCC PDX model, RP-R-02LM, trebananib alone, and in combination with a MET kinase inhibitor, significantly reduced lung metastases and M2 macrophage infiltration (P = 0.0075 and P = 0.0205, respectively). Survival studies revealed that treatment of the orthotopically implanted RP-R-02LM tumors yielded a significant increase in survival in both trebananib and combination groups. In addition, resection of the subcutaneously implanted primary tumor allowed for a significant survival advantage to the combination group compared with vehicle and both single-agent groups. Our results show that the combination of trebananib with a MET kinase inhibitor significantly inhibits the spread of metastases, reduces infiltrating M2-type macrophages, and prolongs survival in our highly metastatic ccRCC PDX model, suggesting a potential use for this combination therapy in treating patients with ccRCC.

A major barrier for cancer immunotherapy is the presence of suppressive cell populations in patients with cancer, such as myeloid-derived suppressor cells (MDSC) and tumor-associated macrophages (TAM), which contribute to the immunosuppressive microenvironment that promotes tumor growth and metastasis. Tasquinimod is a novel antitumor agent that is currently at an advanced stage of clinical development for treatment of castration-resistant prostate cancer. A target of tasquinimod is the inflammatory protein S100A9, which has been demonstrated to affect the accumulation and function of tumor-suppressive myeloid cells. Here, we report that tasquinimod provided a significant enhancement to the antitumor effects of two different immunotherapeutics in mouse models of cancer: a tumor vaccine (SurVaxM) for prostate cancer and a tumor-targeted superantigen (TTS) for melanoma. In the combination strategies, tasquinimod inhibited distinct MDSC populations and TAMs of the M2-polarized phenotype (CD206+). CD11b+ myeloid cells isolated from tumors of treated mice expressed lower levels of arginase-1 and higher levels of inducible nitric oxide synthase (iNOS), and were less immunosuppressive ex vivo, which translated into a significantly reduced tumor-promoting capacity in vivo when these cells were coinjected with tumor cells. Tumor-specific CD8+ T cells were increased markedly in the circulation and in tumors. Furthermore, T-cell effector functions, including cell-mediated cytotoxicity and IFNγ production, were potentiated. Taken together, these data suggest that pharmacologic targeting of suppressive myeloid cells by tasquinimod induces therapeutic benefit and provide the rationale for clinical testing of tasquinimod in combination with cancer immunotherapies. Cancer Immunol Res; 3(2); 136–48. ©2014 AACR.

Supplementary Figure Legend

Gene list for Fig.5B, C, D

Acquired and intrinsic resistance to receptor tyrosine kinase inhibitors (RTKi) represents a major hurdle in improving the management of clear cell renal cell carcinoma (ccRCC). Recent reports suggest that drug resistance is driven by tumor adaptation via epigenetic mechanisms that activate alternative survival pathways. The histone methyl transferase EZH2 is frequently altered in many cancers, including ccRCC. To evaluate its role in ccRCC resistance to RTKi, we established and characterized a spontaneously metastatic, patient-derived xenograft model that is intrinsically resistant to the RTKi sunitinib, but not to the VEGF therapeutic antibody bevacizumab. Sunitinib maintained its antiangiogenic and antimetastatic activity but lost its direct antitumor effects due to kinome reprogramming, which resulted in suppression of proapoptotic and cell-cycle–regulatory target genes. Modulating EZH2 expression or activity suppressed phosphorylation of certain RTKs, restoring the antitumor effects of sunitinib in models of acquired or intrinsically resistant ccRCC. Overall, our results highlight EZH2 as a rational target for therapeutic intervention in sunitinib-resistant ccRCC as well as a predictive marker for RTKi response in this disease. Cancer Res; 77(23); 6651–66. ©2017 AACR.

Supplemental data on additional models to support the role of EZH2 in RTKi resistance

Receptor tyrosine kinase inhibitors have shown clinical benefit in clear cell renal cell carcinoma (ccRCC), but novel therapeutic strategies are needed. The angiopoietin/Tie2 and MET pathways have been implicated in tumor angiogenesis, metastases, and macrophage infiltration. In our study, we used trebananib, an angiopoietin 1/2 inhibitor, and a novel small-molecule MET kinase inhibitor in patient-derived xenograft (PDX) models of ccRCC. Our goal was to assess the ability of these compounds to alter the status of tumor-infiltrating macrophages, inhibit tumor growth and metastases, and prolong survival. Seven-week-old SCID mice were implanted subcutaneously or orthotopically with human ccRCC models. One month postimplantation, mice were treated with angiopoietin 1/2 inhibitor trebananib (AMG 386), MET kinase inhibitor, or combination. In our metastatic ccRCC PDX model, RP-R-02LM, trebananib alone, and in combination with a MET kinase inhibitor, significantly reduced lung metastases and M2 macrophage infiltration (P = 0.0075 and P = 0.0205, respectively). Survival studies revealed that treatment of the orthotopically implanted RP-R-02LM tumors yielded a significant increase in survival in both trebananib and combination groups. In addition, resection of the subcutaneously implanted primary tumor allowed for a significant survival advantage to the combination group compared with vehicle and both single-agent groups. Our results show that the combination of trebananib with a MET kinase inhibitor significantly inhibits the spread of metastases, reduces infiltrating M2-type macrophages, and prolongs survival in our highly metastatic ccRCC PDX model, suggesting a potential use for this combination therapy in treating patients with ccRCC.

Acquired and intrinsic resistance to receptor tyrosine kinase inhibitors (RTKi) represents a major hurdle in improving the management of clear cell renal cell carcinoma (ccRCC). Recent reports suggest that drug resistance is driven by tumor adaptation via epigenetic mechanisms that activate alternative survival pathways. The histone methyl transferase EZH2 is frequently altered in many cancers, including ccRCC. To evaluate its role in ccRCC resistance to RTKi, we established and characterized a spontaneously metastatic, patient-derived xenograft model that is intrinsically resistant to the RTKi sunitinib, but not to the VEGF therapeutic antibody bevacizumab. Sunitinib maintained its antiangiogenic and antimetastatic activity but lost its direct antitumor effects due to kinome reprogramming, which resulted in suppression of proapoptotic and cell-cycle–regulatory target genes. Modulating EZH2 expression or activity suppressed phosphorylation of certain RTKs, restoring the antitumor effects of sunitinib in models of acquired or intrinsically resistant ccRCC. Overall, our results highlight EZH2 as a rational target for therapeutic intervention in sunitinib-resistant ccRCC as well as a predictive marker for RTKi response in this disease. Cancer Res; 77(23); 6651–66. ©2017 AACR.

Background: Class II histone deacetylase (HDAC) inhibitors induce hypoxia-inducible factor-1 and -2α degradation and have antitumour effects in combination with vascular endothelial growth factor (VEGF) inhibitors. In this study, we tested the safety and efficacy of the HDAC inhibitor vorinostat and the VEGF blocker bevacizumab in metastatic clear-cell renal cell carcinoma (ccRCC) patients previously treated with different drugs including sunitinib, sorafenib, axitinib, interleukin-2, interferon, and temsirolimus. Methods: Patients with up to two prior regimens were eligible for treatment, consisting of vorinostat 200 mg orally two times daily × 2 weeks, and bevacizumab 15 mg kg−1 intravenously every 3 weeks. The primary end points were safety and tolerability, and the proportion of patients with 6 months of progression-free survival (PFS). Correlative studies included immunohistochemistry, FDG PET/CT scans, and serum analyses for chemokines and microRNAs. Results: Thirty-six patients were enrolled, with 33 evaluable for toxicity and efficacy. Eighteen patients had 1 prior treatment, 13 patients had 2 prior treatments, and 2 patients were treatment naïve. Two patients experienced grade 4 thrombocytopenia and three patients had grade 3 thromboembolic events during the course of exposure. We observed six objective responses (18%), including one complete response and five partial responses. The proportion of patients with PFS at 6 months was 48%. The median PFS and overall survival were 5.7 months (confidence interval (CI): 4.1–11.0) and 13.9 months (CI: 9.8–20.7), respectively. Correlative studies showed that modulation of specific chemokines and microRNAs were associated with clinical benefit. Conclusions: The combination of vorinostat with bevacizumab as described is relatively well tolerated. Response rate and median PFS suggest clinical activity for this combination strategy in previously treated ccRCC.

// Jianmin Wang 1,* , Antonios Papanicolau-Sengos 2,* , Sreenivasulu Chintala 3,10,* , Lei Wei 1 , Biao Liu 1 , Qiang Hu 1 , Kiersten Marie Miles 2 , Jeffrey M. Conroy 2 , Sean T. Glenn 4 , Manuela Costantini 5,8,9 , Cristina Magi-Galluzzi 6 , Sabina Signoretti 7 , Toni Choueiri 7 , Michele Gallucci 5 , Steno Sentinelli 5 , Vito M. Fazio 8 , Maria Luana Poeta 9 , Song Liu 1 , Carl Morrison 2 and Roberto Pili 3,10 1 Department of Biostatistics & Bioinformatics, Roswell Park Cancer Institute, Buffalo, NY, USA 2 Department of Pathology and Center for Personalized Medicine, Roswell Park Cancer Institute, Buffalo, NY, USA 3 Genitourinary Program, Roswell Park Cancer Institute, Buffalo, NY, USA 4 Cancer Genetics, Roswell Park Cancer Institute, Buffalo, NY, USA 5 Department of Urology, Regina Elena National Cancer Institute of Rome, Rome, Italy 6 Department of Pathology, Cleveland Clinic, Cleveland, OH, USA 7 Department of Pathology and Kidney Cancer Program, Dana Farber, Boston, MA, USA 8 Laboratory of Genetic and Clinical Pathology, University Campus BioMedico of Rome, Rome, Italy 9 Department of Biosciences, Biotechnologies and Biopharmaceutics, University of Bari, Bari, Italy 10 Genitourinary Program, Indiana University-Simon Cancer Center, Indianapolis, IN, USA * These authors have contributed equally to this work Correspondence to: Roberto Pili, email: // Keywords : collecting duct carcinoma, CDKN2A, solute carrier family genes Received : December 04, 2015 Accepted : April 16, 2016 Published : April 28, 2016 Abstract The genetic landscape and molecular features of collecting duct carcinoma (CDC) of the kidney remain largely unknown. Herein, we performed whole exome sequencing (WES) and transcriptome sequencing (RNASeq) on 7 CDC samples (CDC1 -7). Among the 7 samples, 4 samples with matched non-tumor tissue were used for copy number analysis by SNP array data. No recurrent somatic SNVs were observed except for MLL , which was found to be mutated (p.V297I and p.F407C) in 2 samples. We identified somatic SNVs in 14 other cancer census genes including: ATM, CREBBP, PRDM1, CBFB, FBXW7, IKZF1, KDR, KRAS, NACA, NF2, NUP98, SS18, TP53 , and ZNF521 . SNP array data identified a CDKN2A homozygous deletion in 3 samples and SNV analysis showed a non-sense mutation of the CDKN2A gene with unknown somatic status. To estimate the recurrent rate of CDKN2A abnormalities, we performed FISH screening of additional samples and confirmed the frequent loss (62.5%) of CDKN2A expression. Since cisplatin based therapy is the common treatment option for CDC, we investigated the expression of solute carrier (SLC) family transporters and found 45% alteration. In addition, SLC7A11 (cystine transporter, xCT), a cisplatin resistance associated gene, was found to be overexpressed in 4 out of 5 (80%) cases of CDC tumors tested, as compared to matched non-tumor tissue. In summary, our study provides a comprehensive genomic analysis of CDC and identifies potential pathways suitable for targeted therapies.

Current standard of care for muscle-invasive urothelial cell carcinoma (UCC) is surgery along with perioperative platinum-based chemotherapy. UCC is sensitive to cisplatin-based regimens, but acquired resistance eventually occurs, and a subset of tumors is intrinsically resistant. Thus, there is an unmet need for new therapeutic approaches to target chemotherapy-resistant UCC. Yes-associated protein (YAP) is a transcriptional co-activator that has been associated with bladder cancer progression and cisplatin resistance in ovarian cancer. In contrast, YAP has been shown to induce DNA damage associated apoptosis in non-small cell lung carcinoma. However, no data have been reported on the YAP role in UCC chemo-resistance. Thus, we have investigated the potential dichotomous role of YAP in UCC response to chemotherapy utilizing two patient-derived xenograft models recently established. Constitutive expression and activation of YAP inversely correlated with in vitro and in vivo cisplatin sensitivity. YAP overexpression protected while YAP knock-down sensitized UCC cells to chemotherapy and radiation effects via increased accumulation of DNA damage and apoptosis. Furthermore, pharmacological YAP inhibition with verteporfin inhibited tumor cell proliferation and restored sensitivity to cisplatin. In addition, nuclear YAP expression was associated with poor outcome in UCC patients who received perioperative chemotherapy. In conclusion, these results suggest that YAP activation exerts a protective role and represents a pharmacological target to enhance the anti-tumor effects of DNA damaging modalities in the treatment of UCC.

A major barrier for cancer immunotherapy is the presence of suppressive cell populations in patients with cancer, such as myeloid-derived suppressor cells (MDSC) and tumor-associated macrophages (TAM), which contribute to the immunosuppressive microenvironment that promotes tumor growth and metastasis. Tasquinimod is a novel antitumor agent that is currently at an advanced stage of clinical development for treatment of castration-resistant prostate cancer. A target of tasquinimod is the inflammatory protein S100A9, which has been demonstrated to affect the accumulation and function of tumor-suppressive myeloid cells. Here, we report that tasquinimod provided a significant enhancement to the antitumor effects of two different immunotherapeutics in mouse models of cancer: a tumor vaccine (SurVaxM) for prostate cancer and a tumor-targeted superantigen (TTS) for melanoma. In the combination strategies, tasquinimod inhibited distinct MDSC populations and TAMs of the M2-polarized phenotype (CD206+). CD11b+ myeloid cells isolated from tumors of treated mice expressed lower levels of arginase-1 and higher levels of inducible nitric oxide synthase (iNOS), and were less immunosuppressive ex vivo, which translated into a significantly reduced tumor-promoting capacity in vivo when these cells were coinjected with tumor cells. Tumor-specific CD8+ T cells were increased markedly in the circulation and in tumors. Furthermore, T-cell effector functions, including cell-mediated cytotoxicity and IFNγ production, were potentiated. Taken together, these data suggest that pharmacologic targeting of suppressive myeloid cells by tasquinimod induces therapeutic benefit and provide the rationale for clinical testing of tasquinimod in combination with cancer immunotherapies. Cancer Immunol Res; 3(2); 136–48. ©2014 AACR.

Acquired resistance to anti-angiogenic tyrosine kinase inhibitors is an important clinical problem in treating various cancers. To what extent acquired resistance is determined by microenvironmental host-factors or by tumor cells directly is unknown. We previously found that tumor cells can become resistant to sunitinib in vitro. Here, we studied to what extent in vitro induced resistance of tumor cells determines in vivo resistance to sunitinib. In severe combined immunodeficient mice, tumors were established from HT-29 parental colon cancer cells (HT-29PAR) or the in vitro induced sunitinib resistant HT-29 cells (HT-29SUN). Treatment with sunitinib (40mg/kg/day) inhibited tumor growth of HT-29PAR tumors by 71±5%, while no inhibition of HT-29SUN tumor growth was observed. Intratumoral sunitinib concentrations and reduced MVD were similar in both groups. Ki67 staining revealed that tumor cell proliferation was significantly reduced with 30% in HT-29PAR tumors, but unaffected in HT-29SUN tumors upon sunitinib treatment. The lysosomal capacity reflected by LAMP-1 and -2 expression was higher in HT-29SUN compared to HT-29PAR tumors indicating an increased sequestration of sunitinib in lysosomes of resistant tumors. In conclusion, we demonstrate that tumor cells rather than host-factors may play a crucial role in acquired resistance to sunitinib in vivo.

Background Vascular endothelial growth factor (VEGF) is produced by bladder cancer cell lines in vitro and expressed in human bladder tumor tissues. Pazopanib is a vascular endothelial receptor tyrosine kinase inhibitor with anti-angiogenesis and anti-tumor activity in several preclinical models. A 2-stage phase II study was conducted to assess the activity and toxicity profile of pazopanib in patients with metastatic, urothelial carcinoma. Methods Patients with one prior systemic therapy for metastatic urothelial carcinoma were eligible. Patients received pazopanib at a dose of 800 mg orally for a 4-week cycle. Results Nineteen patients were enrolled. No grade 4 or 5 events were experienced. Nine patients experienced 11 grade 3 adverse events. Most common toxicities were anemia, thrombocytopenia, leucopenia, and fatigue. For stage I, none of the first 16 evaluable patients were deemed a success (complete response or partial response) by the Response Evaluation Criteria In Solid Tumors criteria during the first four 4-week cycles of treatment. Median progression-free survival was 1.9 months. This met the futility stopping rule of interim analysis, and therefore the trial was recommended to be permanently closed. Conclusions Pazopanib did not show significant activity in patients with urothelial carcinoma. The role of anti-VEGF therapies in urothelial carcinoma may need further evaluation in rational combination strategies.

Purpose: Data from epidemiological and experimental studies suggest that dietary protein intake may play a role in inhibiting prostate and breast cancer by modulating the IGF/AKT/mTOR pathway. In this study we investigated the effects of diets with different protein content or quality on prostate and breast cancer. Experimental Design: To test our hypothesis we assessed the inhibitory effect of protein diet restriction on prostate and breast cancer growth, serum PSA and IGF1 concentrations, mTOR activity and epigenetic markers, by using human xenograft cancer models. Results: Our results showed a 70% inhibition of tumor growth in the castrateresistant LuCaP23.1 prostate cancer model and a 56% inhibition in the WHIM16 breast cancer model fed with a 7% protein diet when compared to an isocaloric 21% protein diet. Inhibition of tumor growth correlated, in the LuCaP23.1 model, with decreased serum PSA and IGF-1 levels, down-regulation of mTORC1 activity, decreased cell proliferation as indicated by Ki67 staining, and reduction in epigenetic markers of prostate cancer progression, including the histone methyltransferase EZH2 and the associated histone mark H3K27me3. In addition, we observed that modifications of dietary protein quality, independently of protein quantity, decreased tumor growth. A diet containing 20% plant protein inhibited tumor weight by 37% as compared to a 20% animal dairy protein diet. Conclusions: Our findings suggest that a reduction in dietary protein intake is highly effective in inhibiting tumor growth in human xenograft prostate and breast cancer models, possibly through the inhibition of the IGF/AKT/mTOR pathway and epigenetic modifications.

// Lei Wei 1,* , Sreenivasulu Chintala 2,5,7,* , Eric Ciamporcero 2,* , Swathi Ramakrishnan 2,5 , May Elbanna 2,7 , Jianmin Wang 1 , Qiang Hu 1 , Sean T. Glenn 3 , Mitsuko Murakami 4 , Lu Liu 4 , Eduardo Cortes Gomez 4 , Yuchen Sun 4 , Jacob Conroy 4 , Kiersten Marie Miles 4 , Kullappan Malathi 6 , Sudha Ramaiah 6 , Anand Anbarasu 6 , Anna Woloszynska-Read 2,5 , Candace S. Johnson 2,5 , Jeffrey Conroy 4 , Song Liu 1 , Carl D. Morrison 4 and Roberto Pili 2,7 1 Department of Biostatistics & Bioinformatics, Roswell Park Cancer Institute, Buffalo, NY, USA 2 Genitourinary Program, Roswell Park Cancer Institute, Buffalo, NY, USA 3 Department of Cancer Genetics, Roswell Park Cancer Institute, Buffalo, NY, USA 4 Center for Personalized Medicine, Roswell Park Cancer Institute, Buffalo, NY, USA 5 Department of Pharmacology and Therapeutics, Roswell Park Cancer Institute, Buffalo, NY, USA 6 Medical & Biological Computing Laboratory, School of Biosciences and Technology, VIT University, Vellore, Tamil Nadu, INDIA 7 Genitourinary Program, Indiana University-Simon Cancer Center, Indianapolis, IN, USA * These authors have contributed equally to this work Correspondence to: Roberto Pili, email: // Keywords : urothelial carcinoma, patient-derived xenograft, PI3KCA Received : October 15, 2016 Accepted : October 22, 2016 Published : November 03, 2016 Abstract Purpose: Effective systemic therapeutic options are limited for bladder cancer. In this preclinical study we tested whether bladder cancer gene alterations may be predictive of treatment response. Experimental design: We performed genomic profiling of two bladder cancer patient derived tumor xenografts (PDX). We optimized the exome sequence analysis method to overcome the mouse genome interference. Results: We identified a number of somatic mutations, mostly shared by the primary tumors and PDX. In particular, BLCAb001, which is less responsive to cisplatin than BLCAb002, carried non-sense mutations in several genes associated with cisplatin resistance, including MLH1 , BRCA2, and CASP8 . Furthermore, RNA-Seq analysis revealed the overexpression of cisplatin resistance associated genes such as SLC7A11 , TLE4, and IL1A in BLCAb001. Two different PIK3CA mutations, E542K and E545K, were identified in BLCAb001 and BLCAb002, respectively. Thus, we tested whether the genomic profiling was predictive of response to a dual PI3K/mTOR targeting agent, LY3023414. Despite harboring similar PIK3CA mutations, BLCAb001 and BLCAb002 exhibited differential response, both in vitro and in vivo . Sustained target modulation was observed in the sensitive model BLCAb002 but not in BLCAb001, as well as decreased autophagy. Interestingly, computational modelling of mutant structures and affinity binding to PI3K revealed that E542K mutation was associated with weaker drug binding than E545K. Conclusions: Our results suggest that the presence of activating PIK3CA mutations may not necessarily predict in vivo treatment response to PI3K targeted therapies, while specific gene alterations may be predictive for cisplatin response in bladder cancer models and, potentially, in patients as well.

Background Class I histone deacetylases (HDACs) have been reported to be overexpressed in clear cell renal cell carcinoma (ccRCC), whereas the expression of class II HDACs is unknown. Methods Four isogenic cell lines C2/C2VHL and 786-O/786-OVHL with differential VHL expression are used in our studies. Cobalt chloride is used to mimic hypoxia in vitro. HIF-2α knockdowns in C2 and 786-O cells is used to evaluate the effect on HDAC 1 expression and activity. Invasion and migration assays are used to investigate the role of HDAC 1 and HDAC 6 expression in ccRCC cells. Comparisons are made between experimental groups using the paired T-test, the two-sample Student’s T-test or one-way ANOVA, as appropriate. ccRCC and the TCGA dataset are used to observe the clinical correlation between HDAC 1 and HDAC 6 overexpression and overall and progression free survival. Results Our analysis of tumor and matched non-tumor tissues from radical nephrectomies showed overexpression of class I and II HDACs (HDAC6 only in a subset of patients). In vitro, both HDAC1 and HDAC6 over-expression increased cell invasion and motility, respectively, in ccRCC cells. HDAC1 regulated invasiveness by increasing matrix metalloproteinase (MMP) expression. Furthermore, hypoxia stimulation in VHL-reconstituted cell lines increased HIF isoforms and HDAC1 expression. Presence of hypoxia response elements in the HDAC1 promoter along with chromatin immunoprecipitation data suggests that HIF-2α is a transcriptional regulator of HDAC1 gene. Conversely, HDAC6 and estrogen receptor alpha (ERα) were co-localized in cytoplasm of ccRCC cells and HDAC6 enhanced cell motility by decreasing acetylated α-tubulin expression, and this biological effect was attenuated by either biochemical or pharmacological inhibition. Finally, analysis of human ccRCC specimens revealed positive correlation between HIF isoforms and HDAC. HDAC1 mRNA upregulation was associated with worse overall survival in the TCGA dataset. Conclusions Taking together, these results suggest that HDAC1 and HDAC6 may play a role in ccRCC biology and could represent rational therapeutic targets. Electronic supplementary material The online version of this article (doi:10.1186/s12885-016-2604-7) contains supplementary material, which is available to authorized users.

Granular cell tumors are an uncommon soft tissue neoplasm. Malignant granular cell tumors comprise T transitions, particularly when immediately preceded by a 5′ G. A loss-of-function mutation was detected in a newly recognized tumor suppressor candidate, BRD7. No mutations were found in known targets of pazopanib. However, we identified a receptor tyrosine kinase pathway mutation in GFRA2 that warrants further evaluation. To the best of our knowledge, this is only the second reported case of a malignant granular cell tumor exhibiting a response to pazopanib, and the first whole-genome sequencing of this uncommon tumor type. The findings provide insight into the genetic basis of malignant granular cell tumors and identify potential targets for further investigation.

Background: Advanced renal cell carcinoma (RCC) responds initially to anti-angiogenic therapies but eventually develop resistance which may be driven by the expression of key intratumoral pro-survival protein and overall kinome reprogramming. Androgen receptor (AR) expression has been reported in clear cell renal cell carcinoma (ccRCC) but its biological role remains elusive and its association with resistance to tyrosine kinase inhibitors (TKIs) has not been studied. Here we report the role of AR and its association with resistance to TKIs in advanced RCC. Methods: Human RCC cell lines; 786-0, 786-0R (with induced sunitinib resistance), ACHN, URMC2 (with intrinsic sunitinib resistance) were used to assess the combination effect of sunitinib and the AR antagonist enzalutamide in vitro. In vivo studies utilizing 786-0 tumors assessed the combination effect of enzalutamide and sunitinib following acquired resistance to sunitinib. AR expression was detected by qRT-PCR and Western blot analysis, and AR localization by immunofluorescence in treated and non-treated cells. Gene array was used to assess 93 AR associated genes in sensitive and resistant cells. Results: Quantitative RT-PCR data revealed a significant increase in AR expression with sunitinib resistance (>100 folds; p>0.001). In vitro and in vivo studies indicated significant increase in nuclear and cytoplasmic expression of AR with sunitinib treatment in resistant cells which was abrogated with single agent enzalutamide and combination treatment. More interestingly, combination treatment of enzalutamide and sunitinib significantly decreased cell growth and induced tumor regression in vitro and in vivo, respectively. Conclusion: Our data suggest the role of AR both in intrinsic and acquired sunitinib resistance in ccRCC and provide a rationale for combination strategies to restore TKI sensitivity that can be tested in the clinical setting. Citation Format: Remi M. Adelaiye-Ogala, Sreenivasulu Chintala, Ashley Orillion, May Elbanna, Ben Elzey, Nur Damayanti, Kiersten M. Miles, Chinghai Kao, Piergiorgio Pettazzoni, Giulio F. Draetta, Roberto Pili. Targeting androgen receptor overcomes resistance to tyrosine kinase inhibitors in advanced 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 4170. doi:10.1158/1538-7445.AM2017-4170

Sunitinib is considered a first-line therapeutic option for patients with advanced clear cell renal cell carcinoma (ccRCC). Despite sunitinib's clinical efficacy, patients eventually develop drug resistance and disease progression. Herein, we tested the hypothesis whether initial sunitinib resistance may be transient and could be overcome by dose increase. In selected patients initially treated with 50 mg sunitinib and presenting with minimal toxicities, sunitinib dose was escalated to 62.5 mg and/or 75 mg at the time of tumor progression. Mice bearing two different patient-derived ccRCC xenografts (PDX) were treated 5 days per week with a dose-escalation schema (40–60–80 mg/kg sunitinib). Tumor tissues were collected before dose increments for immunohistochemistry analyses and drug levels. Selected intrapatient sunitinib dose escalation was safe and several patients had added progression-free survival. In parallel, our preclinical results showed that PDXs, although initially responsive to sunitinib at 40 mg/kg, eventually developed resistance. When the dose was incrementally increased, again we observed tumor response to sunitinib. A resistant phenotype was associated with transient increase of tumor vasculature despite intratumor sunitinib accumulation at higher dose. In addition, we observed associated changes in the expression of the methyltransferase EZH2 and histone marks at the time of resistance. Furthermore, specific EZH2 inhibition resulted in increased in vitro antitumor effect of sunitinib. Overall, our results suggest that initial sunitinib-induced resistance may be overcome, in part, by increasing the dose, and highlight the potential role of epigenetic changes associated with sunitinib resistance that can represent new targets for therapeutic intervention. Mol Cancer Ther; 14(2); 513–22. ©2014 AACR.

Alternative pathways to the VEGF, such as hepatocyte growth factor or HGF/c-met, are emerging as key players in tumor angiogenesis and resistance to anti-VEGF therapies. The aim of this study was to assess the effects of a combination strategy targeting the VEGF and c-met pathways in clear cell renal cell carcinoma (ccRCC) models. Male SCID mice (8/group) were implanted with 786-O tumor pieces and treated with either a selective VEGF receptor tyrosine kinase inhibitor, axitinib (36 mg/kg, 2×/day); a c-met inhibitor, crizotinib (25 mg/kg, 1×/day); or combination. We further tested this drug combination in a human ccRCC patient–derived xenograft, RP-R-01, in both VEGF-targeted therapy-sensitive and -resistant models. To evaluate the resistant phenotype, we established an RP-R-01 sunitinib-resistant model by continuous sunitinib treatment (60 mg/kg, 1×/day) of RP-R-01–bearing mice. Treatment with single-agent crizotinib reduced tumor vascularization but failed to inhibit tumor growth in either model, despite also a significant increase of c-met expression and phosphorylation in the sunitinib-resistant tumors. In contrast, axitinib treatment was effective in inhibiting angiogenesis and tumor growth in both models, with its antitumor effect significantly increased by the combined treatment with crizotinib, independently from c-met expression. Combination treatment also induced prolonged survival and significant tumor growth inhibition in the 786-O human RCC model. Overall, our results support the rationale for the clinical testing of combined VEGF and HGF/c-met pathway blockade in the treatment of ccRCC, both in first- and second-line setting. Mol Cancer Ther; 14(1); 101–10. ©2014 AACR.

Background The Notch ligand Delta-like 4 (Dll4) is highly expressed in vascular endothelium and has been shown to play a pivotal role in regulating tumor angiogenesis. Blockade of the Dll4-Notch pathway in preclinical cancer models has been associated with non-productive angiogenesis and reduced tumor growth. Given the cross-talk between the vascular endothelial growth factor (VEGF) and Delta-Notch pathways in tumor angiogenesis, we examined the activity of a function-blocking Dll4 antibody, REGN1035, alone and in combination with anti-VEGF therapy in renal cell carcinoma (RCC). Methods and Results Severe combined immunodeficiency (SCID) mice bearing patient-derived clear cell RCC xenografts were treated with REGN1035 and in combination with the multi-targeted tyrosine kinase inhibitor sunitinib or the VEGF blocker ziv-aflibercept. Immunohistochemical and immunofluorescent analyses were carried out, as well as magnetic resonance imaging (MRI) examinations pre and 24 hours and 2 weeks post treatment. Single agent treatment with REGN1035 resulted in significant tumor growth inhibition (36–62%) that was equivalent to or exceeded the single agent anti-tumor activity of the VEGF pathway inhibitors sunitinib (38–54%) and ziv-aflibercept (46%). Importantly, combination treatments with REGN1035 plus VEGF inhibitors resulted in enhanced anti-tumor effects (72–80% growth inhibition), including some tumor regression. Magnetic resonance imaging showed a marked decrease in tumor perfusion in all treatment groups. Interestingly, anti-tumor efficacy of the combination of REGN1035 and ziv-aflibercept was also observed in a sunitinib resistant ccRCC model. Conclusions Overall, these findings demonstrate the potent anti-tumor activity of Dll4 blockade in RCC patient-derived tumors and a combination benefit for the simultaneous targeting of the Dll4 and VEGF signaling pathways, highlighting the therapeutic potential of this treatment modality in RCC.

Background: Alterations in epigenetic mechanisms including histone modification and hyper-methylation at gene promoter regions have been implicated as mechanisms of drug resistance in cancer. Alternation of epigenetic regulators such histone methyltransferase, EZH2, has been reported in numerous cancer types including advanced renal cell carcinoma (RCC). Previous studies suggest that sunitinib may have a direct anti-tumor effect and that acquired sunitinib resistance may be induced in tumor cells rather than just in endothelial cells. In our study, we investigated the role of EZH2 in sunitinib resistance in clear cell renal cell carcinoma. Methods: Human RCC cell lines 786-0 were treated and exposed to increasing concentrations of sunitinib to develop a resistant cell line, 786-0R. Parental and resistant cell lines were treat with either sunitinib, GSK126 (EZH2 inhibitor) or both. In parallel, EZH2 was knocked down in 786-0 cells and exposed to increasing concentrations of sunitinib. Cell viability was quantitated by absorbance of crystal violet stained cells using a spectrometer at 570nm. In a second set of experiments, control and treated cells were collected for western analysis. Mice bearing human ccRCC patient derived xenograft (PDXs); RP-R-01, RP-R-02 and RP-R-02LM (a metastatic ccRCC model established from RP-R-02) were implanted into SCID mice either subcutaneously or orthotopic in the kidney (sub-renal). When tumors reached an average volume of 50mm3, mice were randomly grouped into 2 arms; control and sunitinib treatment (40mg/kg, 5days/week). Tumors volumes and body weight were assessed once per week. Tumor tissues and lungs were collected for immunohistochemistry analysis. All assessments and quantification were done blindly. Results: Our in vitro and in vivo data showed an increased expression of EZH2 with resistance to sunitinib. Furthermore, inhibition of EZH2 in our in vitro studies correlated with a significant decrease in the anti-tumor effect of sunitinib in both parental and resistant cell lines. Conclusion: Overall our data suggest the potential role of epigenetic alterations, specifically EZH2 overexpression and its association with resistance to sunitinib. We are currently assessing the effect of EZH2 inhibition with sunitinib resistance in our in vivo system using metastatic ccRCC PDX models. Citation Format: Remi M. Adelaiye-Ogala, Sreenivasulu Chintala, Li Shen, Ashley Orillion, Eric Ciamporcero, May Elbanna, Kiersten Marie Miles, Bryan Gillard, Michael Buck, Roberto Pili. Inhibition of EZH2 overcomes resistance to sunitinib in clear cell renal cell carcinoma models. [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 3508. doi:10.1158/1538-7445.AM2015-3508

Collecting duct renal cell carcinoma (CDC) is a rare (∼1-3%) type of kidney cancer, aggressive in nature, associated with poor prognosis and limited response to treatments such as cisplatin/gemcitabine and tyrosine kinase inhibitors. Accordingly, there is an unmet need for understanding the molecular alterations associated with CDC and the development of novel effective therapeutic agents for this disease. Since cisplatin based therapy is widely used to manage CDC and the response is often very limited, we investigated the possible molecular alteration(s) associated with cisplatin resistance in CDC. RNA sequence analysis was performed on 5 CDC cases to evaluate gene expression profiles. Immunohistochemical analysis was performed on 6 cases of CDC tumor samples (2 cases included in sequencing) and their matched normal kidneys arranged in a tissue microarray to assess the representative overexpressed genes. Clinical annotation was evaluated to determine the association of gene expression with cisplatin based therapy response and survival. RNA sequencing analysis data revealed the upregulation of 2879 genes and the downregulation of 1951 genes in CDC tumors, as compared to matched normals. Among the altered genes, many of them were transporters. Since transporters play a significant role in drug resistance mechanism(s), particularly in cisplatin resistance, we investigated the Solute Carrier (SLC) family group of membrane transporter genes and found 41 genes upregulated (log2 fold change of 6.4 to 1.0) and 95 genes downregulated (log2 fold change of -8.4 to -0.8). The number of altered SLC family genes was 136 (41 upregulated and 95 downregulated), which is 3.8 percent (136 out of 4830) of the total gene alterations and 45.3% (136 out of 300) of the SLC group of family members, indicating their significance in CDC. The critical analysis of individual amino acid transporters of SLC family genes from the upregulated group led us to investigate three transporters: SLC7A11 (xCT, cystine transporter), SLC1A3 (GLAST, glutamate and aspartate transporter), and SLC6A7 (PROT, proline transporter), which are known to be drug resistance markers. RNA sequence data showed the upregulation of SLC7A11 and SLC1A3 mRNA in 75% of the cases (3 out of 4), with SLC6A7 upregulated in 100% of the cases (4 out of 4). Immunohistochemical analysis of 6 CDC tumors revealed the overexpression of xCT, a known marker for cisplatin resistance in 66% (4 out of 6) of the cases. Kaplan Meyer survival analysis revealed the worst survival for CDC patients with xCT overexpression. Our results demonstrate for the first time that overexpression of xCT, a known cisplatin resistance marker, is associated with CDC. These results suggest that xCT targeted combination therapies may be beneficial to CDC patients. Citation Format: Sreenivasulu Chintala, Jianmin Wang, Lei Wei, Biao Liu, Eric Ciamporcero, Kiersten Marie Miles, May Elbanna, Remi Adelaiye, Li Shen, Ashley Orillion, Sheng Yu Ku, Antonios Papanicolau-Sengos, Carl D. Morrison, Roberto Pili. Solute carrier family group of membrane transporter gene alteration in collecting duct renal cell carcinoma. [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 4300. doi:10.1158/1538-7445.AM2015-4300

Background: Sunitinib, a multi-tyrosine kinase inhibitor, is considered first-line therapy for patient with advanced renal cell carcinoma. The mechanism by which sunitinib harnesses angiogenesis is by targeting receptors of pro-angiogenic growth factors such as VEGF, PDGF and kit. Recently, it has been speculated that sunitinib may have a direct anti-tumor effect. Our previous studies have shown that tumors resistant to sunitinib still present decreased tumor vasculature compared to untreated tumors, suggesting that sunitinib anti-tumor effect may be in part independent from its anti-angiogenic effect. Hence, we wanted to further investigate the anti-tumor effect of sunitinib in human renal cell carcinoma (RCC) cell lines and a patient derived clear cell RCC xenograft model. In addition, we examined the effect of sunitinib on early and late stage metastasis in a spontaneous metastatic human ccRCC model. Methods: Human ccRCC PDX RP-R-02 was implanted into SCID mice subcutaneously. Spontaneous metastatic ccRCC model, RP-R-02LM was developed from RP-R-02. RP-R-02LM tumor bearing mice were treated with sunitinib (40mg/kg; 5days/week). Tumor tissues and lungs were collected for immunohistochemistry analysis. In parallel, human RCC cell lines were treated in vitro with varying concentrations of sunitinib and cell viability was assessed. Results: Human RCC cells lines treated in vitro with sunitinib at pharmacological achievable concentrations showed a decrease in cell proliferation, suggesting a direct anti-tumor effect of sunitinib in RCC. RP-R-02LM but not parental RP-R-02 implanted either subcutaneous or orthotopically in the kidney, spontaneously metastasize to the lungs and closely mimics what is seen in the clinic. PCR results indicated that both xenografts still maintain human origin. In our in vivo system, RP-R-02LM treated with sunitinib had no anti-tumor effect on tumor at the primary site; however, we observed an inhibition of dissemination to the metastatic lung site as indicated by significantly low numbers of metastasis compared to the controls. Immunohistochemical analysis showed decrease in tumor vasculature with sunitinib treatment compared to the control, with increased tumor cell proliferations. Conclusion: Our studies suggest that sunitinib has a direct anti-tumor in vitro and eventually tumor cells acquire drug resistance. Our in vivo studies show that tumors resistant to sunitinib have decreased vessel density as compared to the untreated tumors, suggesting that sunitinib is still a potent anti-angiogenic agent. In addition, we show the anti-metastatic effect of sunitinib in a spontaneous metastatic human clear cell renal cell carcinoma. Overall our data suggest that sunitinib maintains an anti-metastatic effect in sunitinib resistant tumor bearing animals that is independent from its anti-angiogenic effect. Citation Format: Remi M. Adelaiye-Ogala, Li Shen, Sreenivasulu Chintala, Eric Ciamporcero, Ashley Orillion, May Elbanna, Shengyu Ku, Kiersten Marie Miles, Bryan Gillard, Michael Buck, Roberto Pili. Anti-tumor and anti-metastatic effect of sunitinib in a patient derived metastatic clear cell renal cell carcinoma xenograft model. [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 4132. doi:10.1158/1538-7445.AM2015-4132

Introduction: Histone deacetylases are overexpressed in several tumors including prostate, breast and clear cell renal cell carcinoma (ccRCC). Our group has previously reported that class II HDACs, HDAC4 and HDAC6 regulate HIF-α stability in ccRCC cell lines. Interestingly, HDAC6 overexpression in ER-α positive breast cancer has been shown to correlate with overall and cancer specific survival in response to tamoxifen treatment. In addition, HDAC6 increases cell motility by deacetylating α-tubulin, and HDAC6 interaction with ER-α on the cell membrane increases its deacetylating activity. The objective of our study was to assess whether HDAC6 associates with ER-α in a subset of ccRCC and whether this association can be targeted therapeutically. Methods: Radical nephrectomy tumor samples (n=14) with matched adjacent non tumor tissues were collected and analyzed for HDAC6 expression by Western blot analysis. HDAC6 expression was also assessed in C2, C2VHL and 786-O ccRCC cell lines by Western blot and immunofluorescence analysis. HDAC6 and ER-α colocalization was examined by immunoprecipitation and immunofluorescence. HDAC 6 was overexpressed in cell lines and investigated for cell motility by scratch assay. Cell lines were also treated with hydroxy tamoxifen in short term (4 hours) and long term (24, 48 and 72 hours) culture experiments for evaluation of effects on acetylated α-tubulin and cell proliferation, respectively. Results: Analysis of matched patient tumor samples revealed that a subset of ccRCC had higher HDAC6 expression as compared to the adjacent non tumor tissue. HDAC6 and ER-α examined in ccRCC tumors (n=44) by immunofluorescence showed overexpression in 10% of tumor samples. Immunoprecipitation of HDAC 6 in ccRCC cell lines showed that ER-α is present in the same complex as HDAC 6 as confirmed also by fluorescence microscopy. HDAC6 overexpression in ccRCC cell lines increased cell motility, although overexpression did not affect cell proliferation. Cells treated for short term experiments with hydroxy tamoxifen showed an increase in acetylated α-tubulin when examined by immunofluorescence. Upon long term hydroxy tamoxifen treatment in regular DMEM medium with serum, ccRCC cell proliferation was affected at high concentrations (10-20µM), similar to MCF 7 cells treated under similar conditions. Conclusions: HDAC 6 and ER-α are overexpressed in a subset of ccRCC. HDAC6 overexpression affects cell motility but not proliferation. HDAC6 and ER-α are present in the same immunocomplex and this association may be targeted with therapeutic interventions. Ongoing studies are testing concomitant, either genetic or pharmacological, inhibition of both HDAC6 and ER-α in ccRCC and will provide the rationale for novel targeted therapies for a selected group of patients with ccRCC. Citation Format: Swathi Ramakrishnan, Sheng-Yu Ku, Wendy Swetzig, Dylan Conroy, Li Shen, Sreenivasulu Chintala, Paula Sotomayor, Kiersten M. Miles, Remi Adelaiye, Eric Ciamporcero, Ashley Orillion, Leigh Ellis, Gokul Das, Roberto Pili. Evidence for hdac6 and er-α association in a subset of clear cell renal cell carcinoma. [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 4061. doi:10.1158/1538-7445.AM2014-4061

Background: Sunitinib is first-line therapy for patients with advanced clear cell renal cell carcinoma (ccRCC). Despite the clinical efficacy of sunitinib, in the majority of patients the disease eventually develops resistance and progresses. Epigenetic modifications of histone protein in chromatins have been shown to play a role in the regulation of gene transcription patterns in cells primarily by the catalytic activity of histone methyltransferase. EZH2 has been shown to contribute to tumor angiogenesis by inactivating anti-angiogenic factors via methylation at their promoter region. In this study, we examined epigenetic changes that may be associated with response and the resistant phenotype. Methods: Human ccRCC xenograft models RP-01 (isolated from a skin metastasis in a patient with sporadic ccRCC) and RP-02 (isolated from a skin metastasis in a patient with hereditary ccRCC) were implanted (∼1mm pieces) subcutaneously into SCID mice and were randomly assigned into two groups (sunitinib and vehicle). Mice were treated 5 days/week with sunitinib at 40mg/kg. Tumor volumes and body weights were assessed weekly by caliper measurements and weigh scale, respectively. Tumor tissues and blood were collected prior treatments and when tumors became resistant to treatment. Tissues collected were used for immunohistochemistry studies and RNA analysis. Results: Our results showed that RP-01 and RP-02 tumors, although initially responsive to treatment at 40 mg/kg, eventually became resistant to treatment. Immunohistochemistry analysis of RP-01 tumor samples indicated tumors to be hypo-vasculature when responding to sunitinib, but then became hyper-vascularized at point of resistance by CD31 staining. Analysis also revealed an associated increase in the expression of the methyltransferase EZH2 when tumors became resistant to sunitinib. However, there was no change in the expression levels of its EZH2 associated histone make H3K27me3. In addition, we noticed an increase in the levels of H3k9me2 and H3k4me2 in tumors resistant to sunitinib as compared to controls. Conclusion: Overall, our results suggest the potential role of epigenetic changes that that may be associated with sunitinib-induced resistance in ccRCC Citation Format: REMI ADELAIYE, Kiersten Marie Miles, Eric Ciamporcero, Dylan Conroy, Swathi Ramakrishnan, Ashley Orillion, Sheng Yu Ku, May Elbanna, Li shen, Sreenivasulu Chintala, Roberto Pili. Epigenetic changes associated with resistance to sunitinib in human clear cell renal cell carcinoma models. [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 1375. doi:10.1158/1538-7445.AM2014-1375

Background: Transitional cell carcinoma of the bladder (TCC) arises from the cells lining the bladder and accounts for 90% of bladder cancers. Current standard of care for muscle-invasive TCC is surgery with perioperative cisplatin-based chemotherapy regimens. Some tumors are quite sensitive to cisplatin but others either acquire resistance or are refractory. A reduced drug availability, an increased efficacy of the DNA damage repair machinery or defects in the apoptotic process are the main mechanisms involved in cisplatin resistance. The Hippo tumor suppressor pathway controls multiple cellular functions that are important for tumorigenesis and drug-resistance, including proliferation, apoptosis and oxidative stress regulation. Specifically, the overexpression of Yes-associated protein (YAP), one of the two repressed-targets of this pathway, prevents tumor necrosis factor-induced apoptosis in mouse liver and protects cancer cells from chemotherapy-induced apoptosis. Intriguingly, this transcriptional co-activator has been shown to be involved in bladder cancer progression and to be a biomarker for poor prognosis of patients with TCC. Furthermore, in ovarian cancer, YAP has been shown to confer resistance to cisplatin. Thus, we hypothesized that YAP may be involved in cisplatin resistance in bladder cancer and could predict drug response in TCC patients. Methods: Cisplatin activity and YAP protein expression and localization were analyzed in different in vitro and in vivo models of TCC: 1) Collection of bladder cancer cell lines, genetically manipulated by knocking-down or overexpressing YAP; 2) Cell line models of cisplatin resistance, obtained by long term continuous in vitro exposure to increasing drug concentrations; 3) Two TCC patient derived xenograft (PDX) models and derived primary cells (RP-B-01 and RP-B-02) with different intrinsic sensitivity to cisplatin. Furthermore, by continuous in vivo cisplatin treatment (2.5 to 5 mg/Kg, 1x/week, i.p.), we are currently establishing a cisplatin resistant RP-B-02 model. In vitro cisplatin effect was evaluated in apoptosis, proliferation and colony formation assays. Results: YAP expression and nuclear localization was inversely correlated with cisplatin sensitivity in TCC cell lines. Cisplatin-resistant TCC cell lines expressed higher YAP than parental counterparts. Cisplatin-induced apoptosis was significantly increased by YAP knockdown in T24 cell line. In vivo cisplatin treatment showed a greater anti-tumor effect in the low YAP-expressing RP-B-02 model as compared to the high YAP-expressing RP-B-01 tumors. Expression of YAP in human TCC specimens will be also presented. Summary: Overall, these data suggest that YAP has a role in bladder cancer response to cisplatin and highlights the potential therapeutic application of YAP as a biomarker of cisplatin sensitivity in TCC patients. Citation Format: Eric Stefano Ciamporcero, He Shen, Sreenivasulu Chintala, Swathi Ramakrishnan, Sheng Yu Ku, Kiersten M. Miles, Stefania Pizzimenti, Candace Johnson, Jianmin Zhang, Giuseppina Barrera, Roberto Pili. Hippo pathway effector Yes-associated protein and cisplatin resistance in transitional cell carcinoma of the bladder. [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 3754. doi:10.1158/1538-7445.AM2014-3754

There have been several elegant studies showing non coding resident microRNA and circulating extracellular microRNA alterations in ccRCC. MicroRNAs have been reported to be epigenetically regulated. Histone deacetylase (HDAC) inhibitors are currently in clinical development for ccRCC. The objectives of the study were i) to determine the effect of the HDAC inhibitor vorinostat and the VEGF blocker bevacizumab on circulating microRNAs in serum of ccRCC patients with either objective response (Responders) or progression (Progressors); ii) To evaluate serum levels of secreted growth factors (VEGF, FGF2, HGF), apoptotic markers (sFAS-L and TRAIL), invasion and metastatic markers (SDF and OPN), and cytokines (IL8). MicroRNAs were isolated from the serum of ccRCC patient treated with vorinostat and bevacizumab and determined their expression by Exiqon's miRCURY LNATM microarray system. ELISA and Luminex analysis methods were used to measure the secreted growth factors, apoptosis markers, metastasis and invasive makers, and cytokines. In silico bioinformatics analysis was performed to determine the target molecules of the altered microRNAs. Additionally, we have evaluated the possible role of altered microRNA on the regulation of secreted factors in the serum to determine if microRNA could serve as a biomarker of the response to treatment. Exiqon microarray analysis revealed the differential regulation of 134 microRNAs in the post-vs pre-treatment of vorinostat and bevacizumab. The analysis of microRNA expression in Responders and Progressors at the baseline, pre, and post treatment revealed the differential expression of 10 microRNAs in the serum. We found downregulation of miR-20a, miR-142-3p, miR-154, miR-199a-5p, and miR-let-7b and up regulation of miR-99a, miR-605, miR-485-3p, miR-365, and miR-451 in the Responders at baseline. MicroRNA miR-20a and miR-let7b were significantly up regulated after treatment. In addition, miR-605, miR-485-3p, and miR-365 were found to be significantly downregulated after treatment. These results suggest the role of vorinostat and bevacizumab in the alteration of microRNAs miR-20a, miR-let7b, miR-605, miR-485-3p, and miR-365. In patients who progressed there was no downregulation of miR-let7b or upregulation of miR-605, miR-485-3p, and miR-365 following treatment, suggesting the potential role of these microRNAs in the observed response. We also observed downregulation of secreted VEGF, FGF2, SDF, OPN, sFAS-L, and IL8 in Responders following treatment. Bioinformatics analysis revealed the role of microRNAs in the regulation of these markers. Our results suggest the regulation of selected circulating microRNAs in serum of ccRCC patients treated with vorinostat and bevacizumab and the potential role of microRNAs as biomarkers for response in ccRCC patients receiving epigenetic therapies in combination with antiangiogenics. Citation Format: Sreenivasulu Chintala, Kiersten M. Miles, Remi Adelaiye, Roberto Pili. Modulation of circulating microRNAs in serum of clear cell renal cell carcinoma (ccRCC) patients treated with vorinostat and bevacizumab. [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 4395. doi:10.1158/1538-7445.AM2014-4395

Background: Acquired and/or intrinsic resistance to sunitinib, a multikinase inhibitor, is one of the major obstacles for the treatment of renal cell carcinoma (RCC). Recently, lysosomal sequestration of sunitinib has been shown as one of the potential mechanisms of resistance in renal cancer. The current study was initiated to understand the role of the Cystine Transporter xCT (encoded product of Solute Carrier family 7A member 11, SLC7A11 gene), a lysosome regulatory gene, on intracellular sequestration of sunitinib and to evaluate the cytotoxic effects of the xCT inhibitor sulfasalazine alone and in combination with sunitinib on RCC cells. Methods: Intracellular sequestration of sunitinib was evaluated using the fluorescent microscopy technique by determining the autofluorescence of sunitinib. Cytotoxic effects of sulfasalazine alone and in combination with sunitinib were assessed by sulforhodamine B assay. Quantitative RT-PCR analysis and Western blot analysis were utilized to determine the expression of xCT in normal renal tubular HK2 cells, human umbilical vein endothelial cells (HUVEC), and RCC 786-O cells, and to determine the association of sunitinib sequestration and xCT expression. Expression of xCT interacting partner CD44s and its variant forms CD44v6 and CD44v8, which influence the expression of xCT, was determined in 786-O and HK2 cells. Additionally, we utilized The Cancer Genome Atlas (TCGA) data to determine the expression of xCT in ccRCC patients and evaluated the significance of its expression and correlation of patient survival. Results: Our results revealed higher intracellular sequestration of sunitinib in RCC 786-O cells compared to HK2 and HUVEC cells. Furthermore, we found higher expression of xCT in 786-O cells compared to HK2 and HUVEC cells, which was associated with high intracellular sequestration of sunitinib. The pharmacological inhibition of xCT by sulfasalazine revealed the reversal of intracellular sequestration of sunitinib in 786-O cells. The SRB assay showed the enhanced cytotoxic effects of sunitinib in combination with sulfasalazine, which indicates that, the reversal of intracellular sequestration of sunitinib may led to the enhanced cytotoxic effects. Moreover, there was a significant decrease of xCT stabilizing isoform CD44v6 mRNA in 786-O cells compared to HK2 and no change was observed in CD44s and CD44v8. Bioinformatics analysis using cBioPortal Cancer Genomics from MSKCC data of TCGA revealed the significant up regulation of xCT in ccRCC patients correlated with poor prognosis. Conclusions: Our results indicate that lysosome regulatory protein xCT inhibition by sulfasalazine leads to the reversal of intracellular sequestration of sunitinib and enhances the efficacy of sunitinib in renal cancer. These results provide the rationale for the preclinical and clinical testing of sulfasalazine in combination with sunitinib in the treatment of ccRCC. Citation Format: Sreenivasulu Chintala, Hillary Nguyen, Swathi Ramakrishnan, Sheng-Yu Ku, Eric Ciamporcero, Kiersten M. Miles, Roberto Pili. Regulation of intracellular sequestration of sunitinib by cystine transporter xCT in renal cancer. [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 686. doi:10.1158/1538-7445.AM2014-686

Background: Clear cell renal cell carcinoma (ccRCC), the most common form of renal cell carcinoma, is characterized VEGF driven tumor vascularization. However, in patients treated with anti-VEGF therapies inevitably tumor progression occurs, likely driven by up-regulation of alternative proangiogenic pathways including angiopoietins and c-MET. We hypothesized that angiopoietin inhibition has anti-tumor effect on RCC and may affect the development of resistance to anti-VEGF therapies. In the current study we utilized the dual angiopoietin inhibitor AMG 386 in murine models of RCC, and assessed the preclinical impact of this strategy. Methods: Seven week old female Balb/c mice were injected with 250,000 RENCA cells (luciferase expressing), one week later these mice were randomized, using bioluminescence imaging into two treatment groups: vehicle or Amgen 386 (5.6mg/kg/day, twice a week, subQ injection). Body weight measurements, tumor growth rate, and end point tumor weight measurements were used to evaluate possible side effects and the progression of RENCA tumors. Four weeks of treatment culminated in collection of tumor tissues for immunohistochemistry analysis of Ki67 and CD31. In addition we have ongoing studies utilizing patient derived xenografts (PDX) of ccRCC developed in our lab. Results: In the RENCA model AMG 386 induced a significant decrease of tumor weight by 44.0 percent (p = 0.0123). In addition, histological analysis of tumor sections revealed a high proportion of necrosis and decreased blood vessel density. Studies are ongoing with sunitinib sensitive PDXs and matched sunitinib resistant PDXs treated with AMG 386 in combination with a selective c-MET inhibitor. Results from these models will be available at the time of the meeting. Conclusions: Our preliminary results suggest that AMG 386 as a single agent is inhibiting both vascularization and tumor growth in an orthotopic renal cell carcinoma model. Ongoing studies with AMG 386 in combination with a selective c-MET inhibitor in sunitinib sensitive and resistant PDX models will provide guidance for the optimal clinical development of angiopoietin inhibitors in patients with ccRCC. Citation Format: Ashley Orillion, Kiersten Marie Miles, Li Shen, Remi Adelaiye, Eric Ciamporcero, Swathi Ramakrishnan, Sheng Yu Ku, May Elbanna, Sreenivasulu Chintala, Roberto Pili. Angiopoietin 1/2 inhibition impairs tumor growth in an orthotopic model of renal cell carcinoma. [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 1013. doi:10.1158/1538-7445.AM2014-1013