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Objectives We aimed to identify transcriptional gene signatures predictive of clinical response, for pharmacodynamic evaluation, and to provide mechanistic insight into JNJ-55920839, a human IgG1κ neutralizing mAb targeting IFN-α/IFN-ω, in participants with systemic lupus erythematosus (SLE). Methods Blood samples were obtained from SLE participants at baseline and up to Day 130, who received six 10 mg/kg IV doses of JNJ-55920839/placebo every 2 weeks. Participants with mild-to-moderate SLE who achieved clinical responses using SLE Disease Activity Index 2000 Responder Index 4-point change were considered responders. Transcriptional signatures from longitudinally collected blood were generated by RNA-Seq; signatures were generated by microarray from baseline blood samples exposed in vitro to JNJ-55920839 versus untreated. Results Two gene signatures (IFN-I Signaling and Immunoglobulin Immune Response) exhibited pharmacodynamic changes among JNJ-55920839 responders. The Immunoglobulin signature, but not the IFN-I signature, was elevated at baseline in JNJ-55920839 responders. A gene cluster associated with neutrophil-mediated immunity was reduced at baseline in JNJ-55920839 responders, substantiated by lower neutrophil counts in responders. An IFN-I signature was suppressed by JNJ-55920839 in vitro treatment versus untreated blood to a greater extent in responders before in vivo dosing. Conclusions These signatures may enable enrichment for treatment responders when using IFN-I-suppressing treatments in SLE.

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.

Objective: In a previously reported phase II randomized, placebo-controlled, interventional trial, we demonstrated that treatment with ustekinumab, an anti–interleukin-12 (IL-12)/IL-23 p40 neutralizing monoclonal antibody, improved global and organ-specific measures of disease activity in patients with active systemic lupus erythematosus (SLE). Utilizing the biomarker data from this phase II clinical study, we sought to determine whether modulation of the expression of IL-12, IL-23, or both cytokines by ustekinumab is associated with clinical efficacy in patients with SLE. Methods: This phase II randomized, placebo-controlled study enrolled 102 patients with autoantibody-positive SLE whose disease remained active despite standard-of-care therapy. Patients were randomized at a 3:2 ratio to receive ~6 mg/kg ustekinumab intravenously or placebo at week 0, followed by subcutaneous injections of 90 mg ustekinumab or placebo every 8 weeks, with placebo crossover to 90 mg ustekinumab every 8 weeks. The SLE Responder Index 4 (SRI-4) at week 24 was used to determine which patients could be classified as ustekinumab responders and which could be classified as nonresponders. In addition to measurements of p40 and IL-23, serum levels of interferon-γ (IFNγ), IL-17A, IL-17F, and IL-22, as a proxy for the IL-12 and IL-23 pathways, were quantified by immunoassay. Results: Changes in the serum levels of IL-17A, IL-17F, and IL-22 at different time points after treatment were not consistently significantly associated with an SRI-4 clinical response to ustekinumab in patients with SLE. In contrast, an SRI-4 response to ustekinumab was significantly associated (P < 0.01) with durable reductions in the serum IFNγ protein levels at several time points relative to baseline, which was not observed in ustekinumab nonresponders or patients who received placebo. Conclusion: While not diminishing a potential role of IL-23, these serum biomarker assessments indicate that IL-12 blockade has an important role in the mechanism of action of ustekinumab treatment in patients with SLE.

Background:Systemic Lupus Erythematous (SLE) is a clinically and biologically diverse disease, for which only one new therapy has been approved in the past 60 years. In a phase 2 trial on patients with mild-to-moderate SLE, ustekinumab (UST) improved clinical and laboratory measures of disease activity compared with placebo (PBO).1Objectives:We previously reported an association of IFN-γ reduction with response to UST,2suggesting an impact on the IL12/Th1 axis. To extend these findings, we performed unbiased transcriptomic analysis from baseline whole blood samples to identify genes that discriminate UST responders (UST-R) from non-responders (UST-NR) using the primary endpoint of Systemic Lupus Erythematosus Responder Index (SRI)-4 at week 24 to define response.Methods:UST was studied in a Ph2 PBO-controlled study of 102 patients with seropositive SLE and active disease despite standard therapy. Patients were randomized 3:2 to receive IV UST 6 mg/kg or placebo followed by subcutaneous injections of UST 90 mg or PBO every 8 weeks. Whole blood gene expression at baseline was measured via microarray using RNA samples from 100 patients, as samples from 2 patients failed quality control. An unbiased approach was used to identify gene signatures present at baseline that associate with UST response. Recombinant IL-12 or IL-23 was incubatedin vitrowith whole blood from 6 healthy donors for 24h and RNA-Seq was performed to determine the effect of these treatments on representative genes comprising the UST response signature.Results:A non-biased machine learning algorithm identified a 9-gene whole blood signature composed primarily of cytotoxic cell-associated transcripts (PRF1, KLRD1, GZMH, NKG7, GNLY, FGFBP2, TRGC2, TARP, TRGV2) that was enriched at baseline in UST-R vs UST-NR. By Gene Set Variation Analysis, the cytotoxic signature enrichment in UST-NR was less at baseline than both UST-R and a healthy control cohort (P=0.0087, P=0.056, respectively), whereas UST-R cytotoxic gene enrichment was similar to healthy controls (P=0.31). No significant difference in cytotoxic signature enrichment was observed at baseline between PBO responders and PBO non-responders or healthy controls (Figure). Enrichment levels of the cytotoxic gene signature remained stable over time in PBO and UST-NR groups while a trend of decreased cytotoxic signature was observed in UST-R, although never reaching levels seen in UST-NR. To begin to understand the relationship between IL-12 and IL-23, the targets of UST, and the cytotoxic signature, whole blood was stimulated with these cytokinesin vitro. Recombinant IL-12, but not IL-23, resulted in increased expression of representative members of this cytotoxic gene signature.Conclusion:We identified a novel cytotoxic signature in baseline blood samples that associated with UST response in SLE. The observation that IL-12 can increase this signaturein vitroand that IL-12 is a robust inducer of cytotoxic cell activity3as well as IFN-γ3suggests an important role of IL-12 blockade in the mechanism of action of UST in SLE.References:[1]van Vollenhoven RF. Lancet. 2018;392:1330-39[2]Jordan. ACR 2018 Abstract # 2951[3]G. Trinchieri. Nat Rev Immunol. 2003;3:133-46Figure.Disclosure of Interests:Loqmane Seridi Employee of: Janssen Research & Development, LLC, Matteo Cesaroni Employee of: Janssen Research & Development, LLC, Matthew J Loza Employee of: Janssen Research & Development, LLC, Jessica Schreiter Employee of: Janssen Research & Development, LLC, Kristen Sweet Employee of: Janssen Research & Development, LLC, Yevgeniya Orlovsky Employee of: Janssen Research & Development, LLC, Spring House, PA, United States of America, Isabelle Baribaud Employee of: Janssen Research & Development, LLC, Ashley Orillion Employee of: Janssen Research & Development, LLC, Peter Lipsky Consultant of: Horizon Therapeutics, Ronald van Vollenhoven Grant/research support from: AbbVie, Amgen, Arthrogen, Bristol-Myers Squibb, GlaxoSmithKline (GSK), Janssen Research & Development, LLC, Lilly, Pfizer, Roche, and UCB, Consultant of: AbbVie, AstraZeneca, Biotest, Bristol-Myers Squibb, Celgene, Crescendo Bioscience, GSK, Janssen, Lilly, Medac, Merck, Novartis, Pfizer, Roche, UCB and Vertex, Speakers bureau: AbbVie, AstraZeneca, Biotest, Bristol-Myers Squibb, Celgene, Crescendo Bioscience, GlaxoSmithKline, Janssen, Lilly, Merck, Novartis, Pfizer, Roche, UCB, Vertex, Bevra H. Hahn Grant/research support from: Janssen Research & Development, LLC, George Tsokos Grant/research support from: Janssen Research & Development, LLC, Marc Chevrier Employee of: Janssen Research & Development, LLC, Shawn Rose Employee of: Janssen Research & Development, LLC, Frederic Baribaud Shareholder of: Janssen Research & Development, LLC, Employee of: Janssen Research & Development, LLC, Jarrat Jordan Employee of: Janssen Research & Development, LLC

Purpose: Diet and healthy weight are established means of reducing cancer incidence and mortality. However, the impact of diet modifications on the tumor microenvironment and antitumor immunity is not well defined. Immunosuppressive tumor-associated macrophages (TAMs) are associated with poor clinical outcomes and are potentially modifiable through dietary interventions. We tested the hypothesis that dietary protein restriction modifies macrophage function toward antitumor phenotypes. Experimental Design: Macrophage functional status under different tissue culture conditions and in vivo was assessed by Western blot, immunofluorescence, qRT-PCR, and cytokine array analyses. Tumor growth in the context of protein or amino acid (AA) restriction and immunotherapy, namely, a survivin peptide–based vaccine or a PD-1 inhibitor, was examined in animal models of prostate (RP-B6Myc) and renal (RENCA) cell carcinoma. All tests were two-sided. Results: Protein or AA-restricted macrophages exhibited enhanced tumoricidal, proinflammatory phenotypes, and in two syngeneic tumor models, protein or AA-restricted diets elicited reduced TAM infiltration, tumor growth, and increased response to immunotherapies. Further, we identified a distinct molecular mechanism by which AA-restriction reprograms macrophage function via a ROS/mTOR-centric cascade. Conclusions: Dietary protein restriction alters TAM activity and enhances the tumoricidal capacity of this critical innate immune cell type, providing the rationale for clinical testing of this supportive tool in patients receiving cancer immunotherapies.

Background: Immune checkpoint inhibitors targeting the PD-1/PD-L1 axis have shown clinical benefit in solid tumor patients, including renal cell carcinoma (RCC). However, the rate of clinical response remains modest. Growing evidence suggests that epigenetic modifying agents may have an immunomodulatory role. Our group has previously demonstrated that the selective class I histone deacetylase (HDAC) inhibitor entinostat decreases the function of regulatory T-cells (Treg) and myeloid-derived suppressor cells (MDSC), and synergizes with PD-1 blockade. In this study, we assessed the immunomodulatory activity and efficacy of combining PD-1 blockade with the pan-HDAC inhibitor vorinostat in a RCC model. Methods: To test the efficacy of a combination therapy with a PD-1 inhibitor, mDX-400 (10 and 20 mg/kg I.P) (Merck & Co., Inc.) and a pan-HDAC inhibitor, vorinostat (100 and 150 mg/kg I.P) (Merck & Co., Inc.), we utilized a syngeneic mouse model of metastatic RCC following orthotopic implantation of luciferase expressing RENCA cells in immunocompetent BALB/c mice. Antitumor activity was assessed by bioluminescence technique as well as end point measurements of tumor weights. Immune landscape profiling of tumor infiltrating lymphocytes (TILs) was performed by flow cytometry, immunohistochemistry, and immunofluorescence. Survival analysis was performed by Kaplan–Meier estimates and log-rank statistic. Differences in chromatin accessibility in peripheral blood mononuclear cells (PBMC) were assessed by Assay for Transposase-Accessible Chromatin with high-throughput sequencing (ATAC-seq). Results: Statistically significant reductions in end point tumor weights, as well as lung metastases nodules, were observed in mice treated with the combination of vorinostat (100 mg/kg P=0.0391; 150 mg/kg P=0.0165) and mDX-400 (20 mg/kg) compared to vehicle, while no statistical significant reduction was observed in those treated with single-agent mDX-400. Combination therapy also significantly lengthened the survival of the mice (median survival = 60 days; P=0.009) compared to treatment with the single agent mDX-400 (median survival=42 days). Immune landscape profiling did not demonstrate a significant increase in CD8+ tumor infiltration (P=0.479), but a statistically significant increase in natural killer cell infiltration (P=0.048) was observed. Though the CD8+ tumor infiltration was unchanged, a significant reduction (P=0.049) of exhausted CD8+ T-cells (CD8+PD1+) was observed in the combination treatment compared to mDX400 alone. Furthermore, a decrease was observed in the immunosuppressive Tregs (CD4+FOXP4+) and MDSC (CD11b+Gr1+) in the combination group compared to mDX400 alone. Bioinformatic analyses of ATAC-seq data from the PBMC cells of mice in the combination treatment and mDX400 alone showed increased chromatin accessibility between the two conditions. Pathway analysis of genes associated with more accessible chromatin in the combination treatment than mDX400 treatment identified enrichment of cell cycle control and immune cell activation pathways. Conclusions: Our results demonstrate that the pan-HDAC inhibitor vorinostat augments the antitumor effect of immune checkpoint inhibitor mDX-400 and prolongs survival in the RENCA model. This combination advantage was achieved by changing the immune landscape in TILs, especially by decreasing the exhausted subset of T-cells. The combination of these drugs is associated with higher chromatin accessibility near genes involved in cell cycle progression and immune cell activation. Taken together, our results support the clinical testing of pan-HDAC inhibitors in combination of PD-1 inhibitors and provide a novel potential immunomodulatory effect of epigenetic drugs. Citation Format: Nur P. Damayanti, Justin A. Budka, Josue D. Ordaz, Ashley Orillion, Khunsha Ahmed, Xioana Chu, Yue Wang, Yunlong Liu, Roberto Pili. T-cell rejuvenation is associated with vorinostat-induced immune response in combination with immune checkpoint blockade [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A184.

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.

Background: Cystine/glutamate exchanger xCT is a catalytic component of system xc- involved in transport of ‘conditionally indispensable’ amino acid cystine. Cystine transport is a rate limiting step for the synthesis of glutathione, a major intracellular redox regulator. Recently, we and other groups reported the overexpression of xCT and its association with drug resistance in several human cancers including bladder, glioma, breast, and colon. There are no studies to show the xCT expression in clear cell renal cell carcinoma (ccRCC) and its association with tyrosine kinase inhibitors resistance and metastasis. In the current study we have evaluated xCT expression in human ccRCC tumors arranged in tissue microarray (TMA) and determined its role in receptor tyrosine kinase inhibitor (RTKI) resistance and metastases using the patient derived tumor xenografts (PDX) models and TKI resistance ccRCC cells. Methods: Human Renal cell carcinoma tumor nephrectomy specimens arranged in tissue microarray (TMA) were used to determine xCT expression by immunohistochemistry. Patient derived tumor xenografts (PDX) of primary, metastasis, and sunitinib resistance were used to determine the role of xCT in RCC. To understand the molecular alterations associated with RTKI resistance, we have generated sunitinib resistance 786 OR ccRCC cells and performed RNAseq analysis. To determine the xCT inhibition effect on ccRCC tumor metastases, sulfasalazine, an inhibitor of xCT was used to treat metastatic ccRCC tumor xenografts transplanted in SCID mice. Results: Immunohistochemical evaluation of xCT in RCC TMA revealed that 70 % (19 out of 27) of the tumors express different levels of xCT. Association with tumor response to RTKI will be presented. RTKI less responsive PDX RP-R-02 was developed using the dose escalation treatment strategy and was found to have an upregulation of xCT when the tumors become less responsive to sunitinib. RNAseq analysis revealed differential gene expression of various pathway genes including lysosome biogenesis and function such as SLC7A11, HPS4, HPS5, CTSB, IFI30, PPT1, SCPEP1, TPP1, ATP6AP1L, MCOLN1, PRKAG2, and VPS18 in sunitinib resistant 786 OR cells compared to parental cells supports the role of lysosomes function in RTKI drug resistance. Furthermore, xCT inhibitor sulfasalazine treatment significantly decreased the metastasis lung nodules of RP-R-02LM in SCID mice demonstrated the xCT role in RCC tumor metastasis. Conclusions: We found preliminary evidence that overexpression of xCT may be associated with RTKI resistance in ccRCC. These results suggest that targeting the xCT in ccRCC may reverse the resistance and enhance the efficacy of RTKI. Additional studies using larger numbers of ccRCC tumors are required to identify xCT as a potential predictive biomarker for response/resistance to RTKI in ccRCC patients. Citation Format: Sreenivasulu Chintala, Remi Adelaiye-Ogala, Ashley Orillion, Sreevani Arisa, May Elbanna, Nur P. Damayanti, Roberto Pili. Association of xCT overexpression with RTKI resistance and metastases 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 94. doi:10.1158/1538-7445.AM2017-94

Background: Drug screening is a key component for drug development and optimizing anti-tumor therapies. Traditionally, in vitro drug testing has been conducted in monolayer systems that are not capable of recapitulating the tumor complexity. Recently, the field has witnessed the rise of interest in 3D culture systems which are capable of reproducing tumor complexity while circumventing the cost associated with in vivo drug testing. Our access to fresh patient samples has enabled us to establish a novel 3D culture system consisting of bladder cancer patient derived cell lines. Using a wide range of matrices and co-culture conditions with tumor associated stromal cells we were able to establish a unique high throughput drug testing tool. Methods: Matrigel and collagen based matrices were used to establish 3D culture systems of bladder cancer patient derived cells. Tumor cells were cultured in 3D conditions either alone or in coculture with tumor associated stromal cells. Response to Cisplatin and PI3K pathway targeted agents (i.e. LY LY3023414) was tested in both conditions. High throughput imaging via Thermo ArrayScan XTI was used to assess the biological behavior of spheroids as well as their response to therapies overtime. Confocal microscopy was used to validate the biological mimicry of tumor derived spheroids to the original patient tumors. Integration of RNA-seq data from the patient-derived tumor cells with the biological behavior and therapeutic response in 3D culture is ongoing for the purpose of characterizing the 3D model Results: In 3D culture conditions; bladder cancer derived cells were able to re-express E-cadherin that was suppressed upon propagation in monolayer. The re-expression of the epithelial marker (E-cadherin) observed in 3D accurately mirrors the original tumors; which are of epithelial origin. Phenotypic differences were observed across different matrix conditions and also among different tumor derived cells. Bladder 3D organoids of luminal origin were more sensitive to both cisplatin and PI3K pathway inhibitors as compared to those of basal origin. This drug response profile was reminiscent of what we observed in vivo using patient derived xenograft (PDX) models derived from the same tumors. The phenotypic as well as the drug response variations observed in our 3D culture correlated with variable gene expression profiles (luminal vs basal) that were detected in our RNA-seq data. Conclusion: As compared to monolayer, 3D culture is more capable of recapitulating tumor complexity and accurately reflects the drug resistance / sensitivity profiles that are observed in PDX models in vivo. Therefore, a 3D culture system provides an invaluable tool for high throughput screening of drugs in bladder cancer and providing a better understanding of tumor biology in the search of more effective treatments for bladder cancer patients. Citation Format: May Elbanna, Sreenivasulu Chintala, Eric Ciamporcero, Remi Adelayie, Ashley Orillion, Sreevani Arisa, Nur Damayanti, Michelle Grimard, TJ Puls, Sherry Harbin, Melissa Fishel, Roberto Pili. In vitro modeling of patient derived bladder cancer cell lines in 3D culture systems [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 5783. doi:10.1158/1538-7445.AM2017-5783

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

Background: Recent advances in immunotherapy have highlighted the antitumor effects of immune checkpoint inhibition. Novel anti-PD-1/PD-L1 immunotherapies have been shown to effectively overcome tumor avoidance of immune surveillance in several tumor types including renal cell carcinoma. Our group has recently shown that the selective class I HDAC inhibitor entinostat is effective in suppressing regulatory T cells and enhancing immunotherapies in murine renal and prostate models, RENCA and Myc-Cap respectively. In this study we have evaluated the combination of entinostat with an anti-PD-1 antibody in the RENCA renal cell carcinoma model. Methods: 32 BALB/c female mice were implanted with the syngenic, orthotopic, renal cell carcinoma mouse model, RENCA - luciferase tagged - at day -8. Treatment (8 mice /group) with anti-mouse-PD-1 (aPD-1; 10mg/kg twice a week, I.P.), entinostat (5mg/kg 5 days a week), or combination of the two was begun at day 1. Bioluminescence imaging was performed at days -1, 9 and 19 to assess the orthotopic tumor growth. End point tumor weights were taken to assess the effect of combination treatment. Results: Analysis of tumor growth showed a reduction of bioluminescence across the three time points in the combination group as compared to the vehicle and single agent treatments. Additionally, end point analysis of tumor weights revealed an overall reduction in the size of the tumors in the entinostat/anti-mPD-1 combination group (88% inhibition) as compared to the vehicle (p = 0.0002), aPD-1 alone (25% inhibition)(p = 0.0181), and entinostat alone (63% inhibition)(p = 0.0481) groups. Examination of the status of the infiltrating immune cells of the tumor microenvironment via flow cytometry, qRT-PCR, immunohistochemistry, and/or immunofluorescence analysis is ongoing. Conclusions: Our preliminary results suggest that the immunomodulatory activity of the selective class I HDAC inhibitor entinostat may enhance the antitumor effect of PD-1/PD-L1 inhibition and provide the rationale for the clinical testing of this novel combination in patients with RCC. Citation Format: Ashley R. Orillion, Li Shen, Remi Adelaiye-Ogala, May Elbanna, Sreenivasulu Chintala, Sreevani Arisa, Roberto Pili. The selective class I HDAC inhibitor entinostat enhances the antitumor effect of PD-1 inhibition in a syngeneic orthotopic murine model of renal cell carcinoma. [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 4906.

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

Background: A major challenge in cancer drug development has been largely attributed to the inability of cell lines to recapitulate the heterogeneity of human tumors. Patient derived xenografts (PDX) represent a major advance as they are more representative of the clinical setting. However, appropriate characterization of PDXs is necessary to guide biomarker driven drug discovery research. Methods: H&E staining was used to compare the original patient tumors with PDX developed from cystectomy specimens to validate morphological similarity. Immunohistochemical (IHC) staining for cytokeratins (CK) 5/6 & 20 and EMT markers E cadherin and vimentin were used to identify whether these tumors represent a basal or a luminal subtype. In addition, Western blot and RT-PCR analysis were performed to assess markers of epithelial-to-mesenchymal transition (EMT). Therapeutic response to cisplatin was evaluated both in vitro and in vivo. Furthermore, expression of xCT (cystine transporter), one of the known cisplatin resistance markers, was determined by RT-PCR, Western blot and IHC in PDXs and cells. Results: Morphologically, we observed that our PDX models (RP-B-01 and RP-B-02) did not drift from the original patient tumors and they still retained their characteristic stromal features. Based on cytokeratin (CK) staining, we found both models to be CK 5/6 positive and CK 20 negative, suggesting a basal subtype of bladder cancer. Both PDXs, similarly to the original tumors, were E cadherin positive. However, cells derived from these tumors and cultured in vitro lost E cadherin and acquired vimentin expression, suggesting an EMT phenotype. When cells from PDX were transplanted back into mice they re-expressed E cadherin and lost vimentin expression, suggesting a reversion to MET. Despite no difference in cytokeratins and EMT markers between the two models, both PDX and cells isolated from PDXs had differential expression of xCT and possessed differential response to cisplatin treatment. RP-B01 expressed higher levels of xCT and was less responsive to cisplatin as compared to RP-B-02 which expressed lower levels of xCT. Conclusion: Differential expression of xCT in bladder cancer PDXs and cells correlated with cisplatin response, suggesting that pharmacological targeting of xCT in combination therapies will enhance cisplatin efficacy in bladder cancer. Citation Format: May F. Elbanna, Eric Ciamporcero, Swathi Ramakrishnan, Remi Adelaiye, Li Shen, Ashley Orillion, Sheng-Yu Ku, Sreenivasulu Chintala, Roberto Pili. Characterization of patient-derived bladder cancer xenografts: role of xCT in response to cisplatin. [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 1462. doi:10.1158/1538-7445.AM2015-1462

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

Renal cell carcinoma (RCC) is the most common type of kidney cancer in adults and represents 80% of the cases. Treatment options for patients with advanced disease are limited. Targeted molecular therapies have shown survival benefit but most patients develop therapy resistance overtime. Preclinical and clinical studies indicate that immunotherapies are effective and may play a role in the treatment of advanced RCC. However, the response rate is ∼ 20%, with a limited number of patients achieving durable remission. A major barrier to vaccine therapy is the presence of immunosuppressive cell populations including regulatory T cells (Tregs), myeloid-derived suppressor cells (MDSCs) and tumor-associated macrophages (TAMs). In the current study, we designed a tumor cell and Tregs dual-targeting vaccine by inducing Foxp3 expression in tumor cells, and tested this vaccine along with an unmodified vaccine in an orthotopic murine model of RCC, RENCA. We observed that both RENCA cell vaccine and RENCA Foxp3 cell vaccine prevented tumor growth in 6 out 10 animals. Interestingly, in an intervention setting, the RENCA Foxp3 tumor cell vaccine was superior to the RENCA tumor cell vaccine with a significant anti-tumor activity (45% inhibition of tumor weights) (p Citation Format: Li Shen, Ashley Orillion, Remi Adelaiye, Eric Ciamporcero, Swathi Ramakrishnan, Roberto Pili. Activity of a novel Foxp3-tumor cell vaccine in a murine model of 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 2514. doi:10.1158/1538-7445.AM2015-2514

Background: Clear cell renal cell carcinoma (ccRCC) is the most common form of kidney cancer and diagnoses have been on the rise since the 1990s. While patients with early stage ccRCC have more than a 50% survival rate over four years, those with advanced stage disease have a less than 10% chance of 5 year survival. In our current study, we utilized an angiopoietin 1/2 inhibitor (Amgen 386) and a novel c-MET inhibitor in murine models grafted with human derived xenografts (PDX) with a high metastatic penetrance. Our goal was to assess the ability of these compounds to prolong survival via metastatic inhibition. Methods: Seven week old SCID mice were implanted either subcutaneously or orthotopically with human ccRCC PDX RP-R-02LM. Approximately one month post implantation, mice were started on treatment with Angiopoietin 1/2 inhibitor (Amgen 386), c-met inhibitor (compound A) or a combination of the two. Amgen 386 was administered twice a week subcutaneously at 5.6mg/kg. Compound A was given daily via oral gavage at 30mg/kg. As a survival study, treatment was continued until the mice reached a point where they needed to be euthanized or they were found dead. Results: In our metastatic PDX model, RP-R-02LM, Amgen386 single agent treatment and combination c-MET inhibitor and Amgen386 treatment significantly reduced metastases significantly, p = 0.0075 and p = 0.0001 respectively. Further, an ongoing survival study has shown that at four and a half months post implantation both single agent and combination treatments prolong the survival of mice implanted with RP-R-02LM, a highly metastatic PDX. Conclusions: Our preliminary results suggest that Amgen386 in combination with a novel c-MET inhibitor are significantly inhibiting the spread of metastases and prolonging survival. This indicates a potential use for combination therapy in treating patients with ccRCC in advanced stages of the disease. Citation Format: Ashley Orillion, Remi Adelaiye-Ogala, Li Shen, Eric Ciamporcero, Sheng Yu Ku, Swathi Ramakrishnan, May Elbanna, Sreenivasulu Chintala, Roberto Pili. Inhibition of angiopoietin 1/2 and c-MET impairs metastatic potential in a patient derived xenograft of renal 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 4110. doi:10.1158/1538-7445.AM2015-4110

Renal clear cell carcinoma (RCC) is the most common type of kidney cancer in adults and represents 80% of cases. At the time of diagnosis, approximately 30% of patients have distant metastases. In addition, one third of patients who have surgical resection of the primary tumor eventually relapse and develop metastases. Effective agents that help to prevent the development of metastatic RCC are not available. Tasquinimod is a novel agent in clinical development for metastatic castration-resistant prostate cancer that has been shown to have immunomodulatory, anti-angiogenic, and anti-metastatic properties. In the current study, we tested whether tasquinimod effectively inhibits local invasion and spontaneous lung metastases in RCC. Two preclinical mouse models of RCC were used for this purpose: a xenograft nude model A498 and a recently established patient-derived xenograft model, RP-R-02 LM. In both models, tasquinimod had a modest effect on the growth of the primary tumors implanted subcutaneously. Interestingly, in the A498 RCC model, gene expression analysis in tumors revealed that tasquinimod increased E-cadherin and decreased vimentin expression, suggesting an effect on reversal of epithelial-mesenchymal transition. In addition, tasquinimod significantly decreased CXCR4 expression, suggesting a reduction in invasiveness and metastatic phenotype. To further test this hypothesis, we used the RP-R-02 LM model that is able to develop lung metastases. Tasquinimod induced a 66% reduction of lung metastases in comparison with the control group (p Citation Format: Li Shen, Valerie Pierron, Anne-Laure Bauchet, Jessica Nakhle, Ashley Orillion, Remi Adelaiye, Eric Ciamporcero, Florence Meyer-Losic, Helena Eriksson, Nathalie Lassau, Ingrid Leguerney, Fabien Schmidlin, Anders Olsson, Roberto Pili. Tasquinimod inhibits local invasion and metastases in two preclinical models of 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 4120. doi:10.1158/1538-7445.AM2015-4120

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: 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

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