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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 Figure legend

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 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

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

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

Nanotechnology involves the engineering of functional systems at nanoscale, thus being attractive for disciplines ranging from materials science to biomedicine. One of the most active research areas of the nanotechnology is nanomedicine, which applies nanotechnology to highly specific medical interventions for prevention, diagnosis, and treatment of diseases, including cancer disease. Over the past two decades, the rapid developments in nanotechnology have allowed the incorporation of multiple therapeutic, sensing, and targeting agents into nanoparticles, for detection, prevention, and treatment of cancer diseases. Nanoparticles offer many advantages as drug carrier systems since they can improve the solubility of poorly water-soluble drugs, modify pharmacokinetics, increase drug half-life by reducing immunogenicity, improve bioavailability, and diminish drug metabolism. They can also enable a tunable release of therapeutic compounds and the simultaneous delivery of two or more drugs for combination therapy. In this review, we discuss the recent advances in the use of different types of nanoparticles for systemic and topical drug delivery in the treatment of skin cancer. In particular, the progress in the treatment with nanocarriers of basal cell carcinoma, squamous cell carcinoma, and melanoma has been reported.

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.

Several reports indicate that chemo-resistant cancer cells become highly adapted to intrinsic oxidative stress by up-regulating their antioxidant systems, which causes an increase of intracellular GSH content. Doxorubicin is one of the most widely used drugs for tumor treatment, able to kill cancer cells through several mechanisms. However, doxorubicin use is limited by its toxicity and cancer resistance. Therefore, new therapeutic strategies able to reduce doses and to overcome chemo-resistance are needed. A new class of glutathione-responsive cyclodextrin nanosponges (GSH-NS), is able to release anticancer drugs preferentially in cells having high GSH content. Doxorubicin-loaded GSH-NS, in the cancer cells with high GSH content, inhibited clonogenic growth, cell viability, topoisomerase II activity and induced DNA damage with higher effectiveness than free drug. Moreover, GSH-NS reduced the development of human tumor in xenograft models more than free drug. These characteristics indicate that GSH-NS can be a suitable drug delivery carrier for future applications in cancer therapy.

Redox adaptation plays an important role in cancer cells drug resistance. The antioxidant response is principally mediated by the transcription factor Nrf2, that induces the transcriptional activation of several genes involved in GSH synthesis, chemoresistance, and cytoprotection. YAP is emerging as a key mediator of chemoresistance in a variety of cancers, but its role in controlling the antioxidant status of the cells is yet elusive. Here, we show that impairing YAP protein expression reduced GSH content and Nrf2 protein and mRNA expression in bladder cancer cells. Moreover, in YAP knocked down cells the expression of FOXM1, a transcription factor involved in Nrf2 transcription, was down-regulated and the silencing of FOXM1 reduced Nrf2 expression. On the other hand, the silencing of Nrf2, as well as the depletion of GSH by BSO treatment, inhibited YAP expression, suggesting that cross-talk exists between YAP and Nrf2 proteins. Importantly, we found that silencing either YAP or Nrf2 enhanced sensitivity of bladder cancer cells to cytotoxic agents and reduced their migration. Furthermore, the inhibition of both YAP and Nrf2 expressions significantly increased cytotoxic drug sensitivity and synergistically reduced the migration of chemoresistant bladder cancer cells. These findings provide a rationale for targeting these transcriptional regulators in patients with chemoresistant bladder cancer, expressing high YAP and bearing a proficient antioxidant system.

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.

Nanotechnology involves the engineering of functional systems at nanoscale, thus being attractive for disciplines ranging from materials science to biomedicine. One of the most active research areas of the nanotechnology is nanomedicine, which applies nanotechnology to highly specific medical interventions for prevention, diagnosis, and treatment of diseases, including cancer disease. Over the past two decades, the rapid developments in nanotechnology have allowed the incorporation of multiple therapeutic, sensing, and targeting agents into nanoparticles, for detection, prevention, and treatment of cancer diseases. Nanoparticles offer many advantages as drug carrier systems since they can improve the solubility of poorly water-soluble drugs, modify pharmacokinetics, increase drug half-life by reducing immunogenicity, improve bioavailability, and diminish drug metabolism. They can also enable a tunable release of therapeutic compounds and the simultaneous delivery of two or more drugs for combination therapy. In this review, we discuss the recent advances in the use of different types of nanoparticles for systemic and topical drug delivery in the treatment of skin cancer. In particular, the progress in the treatment with nanocarriers of basal cell carcinoma, squamous cell carcinoma, and melanoma has been reported.

3062 Background: MET kinase is a therapeutic target in a range of cancer indications; it is a primary oncogenic driver and a mechanism of therapy resistance. OMO-1 is a highly potent, selective oral inhibitor of MET kinase and Organic Cation Transporter 2 (OCT2). Methods: This study assesses the safety, tolerability, pharmacokinetics (PK) and preliminary activity of OMO-1 in patients (pts) with advanced malignancies (NCT03138083). Module 1 data, evaluating ascending doses of OMO-1 monotherapy, are reported here. Results: As of January 16, 2019, 34 pts were enrolled at 5 twice-daily (BD) dose levels of OMO-1: 100, 200, 250, 350, and 400 mg, including 10 with MET gene amplified or mutated tumours. OMO-1 was generally well tolerated between 100 - 250 mg BD; pts were in the study for an average of 94 days (range: 15-291 days) and 20/34 pts discontinued due to disease progression. Most frequently-reported AEs were nausea (17/34), vomiting (14/34) and fatigue (14/34), mainly G1-2. Notably, no peripheral oedema, cardiovascular events or non-malignancy related LFT abnormalities were observed. A total of 36 SAEs were reported: 17 in 11 subjects were considered related to OMO-1, and included nausea (3/17), vomiting (4/17), chills, diarrhoea, influenza-like illness (2/17), increased blood bilirubin, blood creatinine (3/17) and neutrophil count, and sepsis. A dose of 250 mg BD was determined as the recommended Phase 2 dose (RP2D); doses ≥350mg BD were not in keeping with optimum long-term dosing: at 400 mg BD, 2/3 subjects experienced influenza-like illness (G2 and G3) and at 350 mg BD 2/5 subjects had G2 fatigue and nausea/vomiting. OMO-1 has a half-life of 2.5-3 hrs and plasma exposure is dose-proportional without accumulation. Elevated creatinine was observed across all dose levels, consistent with OCT2 inhibition. IHC analysis on paired tumour biopsies from a MET-mutated NSCLC pt dosed at 200 mg BD showed near-complete inhibition of phosphorylated MET, without affecting total MET. Conclusions: OMO-1 has a favourable safety profile at a RP2D of 250mg BD. Expansion cohorts for MET mutated/amplified tumour types are enrolling. Clinical trial information: NCT03138083.

e14674 Background: The MET kinase is an established therapeutic target in a range of cancer indications, being a primary oncogenic and therapy resistance driver. Methods: OMO-1 is a highly potent, selective oral inhibitor of MET kinase and Organic Cation Transporter 2 (OCT2) that is currently being explored in a Phase I/II study (NCT03138083). Results: Pre-clinical data for OMO-1 indicates that anti-tumor efficacy against MET driven tumor models does not require 24/7 exposure in the plasma. PK/PD assessments indicate that OMO-1 efficacy is AUC driven. Exposures above a trough concentration of ~600ng/ml for a period of 6-8hr and an AUC of 3000ng.h/ml result in complete target inhibition sufficient to induce regression or stasis of MET driven in-vivo models. Dosing of OMO-1 at 12.5mg/kg BD 4hr apart, or split over 4 doses 2hr apart, with a dosing-free period of 18-20hr was sufficient to obtain the effects preclinically. The ongoing Phase I/II study in patients with solid tumours utilised learnings from the pre-clinical and FIH study (NCT01964872) where ascending single doses and multiple doses were evaluated. The selected starting dose for the patient study (100mg BD, 4 hr apart) achieved exposures above the predicted MBAD. Paired tumor biopsies were obtained from a higher dose level (200mg BD). A MET exon 14 skipping mutation NSCLC patient at this dosed demonstrated near-complete inhibition of phospho MET accompanied by signs of clinical benefit and lesion shrinkage, thereby matching the preclinical data. Certain ‘class effect’ adverse events, such as edema, were not observed. Conclusions: OMO-1 is an oral, potent MET TKI with a novel dosing regime, identified during preclinical optimization (BD 4hr apart), that demonstrates encouraging signs of clinical activity without certain ‘class-specific’ adverse events. Further evaluation of this differentiated and efficacious agent is warranted and ongoing.

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.

Camptothecin (CPT) is a potent DNA Topoisomerase I inhibitor with anti-tumor activity in hematological and solid tumors. However, it did not reach clinical use because of its poor solubility and high degrability. β-Cyclodextrin nanosponge (CN) have been demonstrated to be able to increase the solubility of lipophilic compounds and to protect them from degradation. In the present study, we evaluated whether β-Cyclodextrin nanosponge carriers can overcome CPT chemical disadvantages and improve the in vitro anti-tumor efficacy in the androgen refractory models of prostate cancer DU145 and PC-3 and the androgen sensitive model LNCaP. Camptothecin-loaded β-Cyclodextrin nanosponge (CN-CPT) showed sizes of about 400 nm, spherical shape and a drug loading of 38%. HPLC analysis, performed on the cell pellet after treatment with CN-CPT revealed that CPT concentration increased over time indicating a prolonged release of the drug. Moreover, CN-CPT inhibited Topoisomerase I activity, and induced DNA damage, and cell cycle arrest more effectively than CPT, indicating that the CN-CPT formulation does not affect activity of the drug. Moreover, Annexin V/Propidium Iodide staining showed an induction of cell death at low concentrations that were not effective for CTP. LNCaP cells were less sensitive to CPT than PC-3 and DU145 cells, but CN-CPT still exerted higher anti-proliferative activity and DNA damage ability than CPT. The experiments performed in LNCaP cells demonstrated that CN-CPT treatment inhibited expression of the androgen receptor at doses where CPT was ineffective. Our results demonstrated the higher anti-tumor effectiveness of CN-CPT compare to CPT in prostate cancer cells, supporting the relevance of future studies for the use of the β-Cyclodextrin nanosponge to deliver anticancer drugs in vivo.

4-hydroxynonenal (HNE), a lipid peroxidation product, is a promising anti-neoplastic drug due to its remarkable anti-cancer activities. However, this possibility has not been explored, because the delivery of HNE is very challenging as a result of its low solubility and its poor stability. This study intentionally designed a new type of lipid nanocapsules specifically for HNE delivery. They consist of a medium chain triglyceride liquid oil core surrounded by a polymer shell. A β-cyclodextrin-poly(4-acryloylmorpholine) conjugate was selected as the shell component. HNE-loaded nanocapsules were about 350 nm in size with a negative surface charge. They were stable for two years when stored in suspensions at 4 degrees C. In vitro experiments showed that HNE was released from the nanocapsules at a considerable rate. Nanocapsule uptake into cells was evaluated using a fluorescent formulation that revealed rapid internalisation. Cytotoxicity studies demonstrated the safety of the formulation. Enhanced anti-tumoral activity against various cell lines, depending on increased HNE stability, was obtained by using HNE-loaded nanocapsules. In particular, we have demonstrated an increase in anti-proliferative, pro-apoptotic and differentiative activity in several tumour cell lines from different tissues. Moreover, we evaluated the effects of these new nanocapsules on a three-dimensional human reconstructed model of skin melanoma. Interestingly, the encouraging results obtained with topical administration on the epidermal surface could open new perspectives in melanoma treatments.

Heat shock 60 kDa protein 1 (HSP60) is a chaperone and stress response protein responsible for protein folding and delivery of endogenous peptides to antigen-presenting cells and also a target of autoimmunity implicated in the pathogenesis of atherosclerosis. By two-dimensional electrophoresis and mass spectrometry, we found that exposure of human promyelocytic HL-60 cells to a nontoxic concentration (10 μM) of 4-hydroxy-2-nonenal (HNE) yielded a HSP60 modified with HNE. We also detected adducts of HNE with putative uncharacterized protein CXorf49, the product of an open reading frame identified in various cell and tissue proteomes. Moreover, exposure of human monocytic THP-1 cells differentiated with phorbol 12-myristate 13-acetate to 10 μM HNE, and to light density lipoprotein modified with HNE (HNE-LDL) or by copper-catalyzed oxidation (oxLDL), but not to native LDL, stimulated the formation of HNE adducts with HSP60, as detected by immunoprecipitation and western blot, well over basal levels. The identification of HNE-HSP60 adducts outlines a framework of mutually reinforcing interactions between endothelial cell stressors, like oxLDL and HSP60, whose possible outcomes, such as the amplification of endothelial dysfunction, the spreading of lipoxidative damage to other proteins, such as CXorf49, the activation of antigen-presenting cells, and the breaking of tolerance to HSP60 are discussed.

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.

Uniquely amongst the major tumor types, the premalignant state in colorectal cancer (CRC) is readily detectable and diagnosed. Indeed, a multi-step process of CRC genesis was defined by the seminal work of Vogelstein et al, who described the mutational events leading from adenoma to adenocarcinoma. In addition to genetic aberrations, recent evidence has highlighted the importance of the gut microbiome and associated inflammation in predicting CRC progression. To date however, these insights have not led to significant changes in the treatment paradigm because efforts continue to be focused on the later stages of disease. We believe CRC presents an exceptional opportunity for disease interception but to design effective interventional strategies, the exact sequence of molecular events underlying progression needs to be better defined and understood. To address these needs, we have assembled a clinically annotated sample database encompassing all stages of CRC (healthy colon, adjacent mucosa, adenomas, high-grade dysplasia, primary CRC and liver metastases), including samples from the conventional, microsatellite stable subtype, as well as from the serrated, microsatellite instable pathway. The progression status of each sample was characterized using standard pathology criteria. In addition, molecular progression was determined by targeted mutation profiling and targeted copy number profiling, as well as genome-wide expression profiling. This analysis confirms previous observations of early mutational events at the adenoma stage, including known tumor suppressor and oncogene driver mutations, e.g. KRAS G12 and G13 are mutated in 15% of the conventional adenomas but not in sessile serrated adenomas, as well as in 23% of the colorectal tumors. Copy number aberrations were observed at the adenoma and carcinoma stage, but with a lower prevalence then somatic mutations. Furthermore, genome wide expression analysis indicates that several pathways known to be affected in colorectal cancer are already disregulated at the adenoma stage. These pathways include Wnt signaling, mucosal barrier defects, bile acid metabolism, and several immune respons genes, again fingerpointing at the interplay between local inflammation, the microbiome, and epithelial events. In addition, we observed that while genetic events are very dissimilar between the serrated/MSI and conventional/MSS pathway, the transcriptional regulation has many similarities, indicating at a possibility at targeting these disease subtypes using the same therapeutics. These initial findings provide rational avenues to intercept CRC at the adenoma stage and efforts are now focused on exploring the added role of the colonic microbiota and immune system modulation. A more comprehensive and integrated view of the changes associated with disease initiation will lead to the identification of new paths for prevention, interception and cure. Citation Format: Joke Reumers, Liesbeth Van Wesenbeeck, Eric Ciamporcero, Gerald Chu, Stan Gaj, Emanuele Palescandolo, Carl Van Hove, Karin Verstraeten, Gary Borzillo, Dianna Wu, Pieter Peeters, Janine Arts. Characterisation of molecular events across the colorectal cancer progression axis [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 4388. doi:10.1158/1538-7445.AM2017-4388

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

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.

Oxidative stress provokes the peroxidation of polyunsaturated fatty acids in cellular membranes, leading to the formation of aldheydes that, due to their high chemical reactivity, are considered to act as second messengers of oxidative stress. Among the aldehydes formed during lipid peroxidation (LPO), 4-hydroxy-2-nonenal (HNE) is produced at a high level and easily reacts with both low-molecular-weight compounds and macromolecules, such as proteins and DNA. In particular, HNE-protein adducts have been extensively investigated in diseases characterized by the pathogenic contribution of oxidative stress, such as cancer, neurodegenerative, chronic inflammatory, and autoimmune diseases.In this review, we describe and discuss recent insights regarding the role played by covalent adducts of HNE with proteins in the development and evolution of those among the earlier mentioned disease conditions in which the functional consequences of their formation have been characterized.Results obtained in recent years have shown that the generation of HNE-protein adducts can play important pathogenic roles in several diseases. However, in some cases, the generation of HNE-protein adducts can represent a contrast to the progression of disease or can promote adaptive cell responses, demonstrating that HNE is not only a toxic product of LPO but also a regulatory molecule that is involved in several biochemical pathways.In the next few years, the refinement of proteomical techniques, allowing the individuation of novel cellular targets of HNE, will lead to a better understanding the role of HNE in human diseases.

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.

A great variety of compounds are formed during lipid peroxidation of polyunsaturated fatty acids of membrane phospholipids. Among them, bioactive aldehydes, such as 4-hydroxyalkenals, malondialdehyde (MDA) and acrolein, have received particular attention since they have been considered as toxic messengers that can propagate and amplify oxidative injury. In the 4-hydroxyalkenal class, 4-hydroxy-2-nonenal (HNE) is the most intensively studied aldehyde, in relation not only to its toxic function, but also to its physiological role. Indeed, HNE can be found at low concentrations in human tissues and plasma and participates in the control of biological processes, such as signal transduction, cell proliferation, and differentiation. Moreover, at low doses, HNE exerts an anti-cancer effect, by inhibiting cell proliferation, angiogenesis, cell adhesion and by inducing differentiation and/or apoptosis in various tumor cell lines. It is very likely that a substantial fraction of the effects observed in cellular responses, induced by HNE and related aldehydes, be mediated by their interaction with proteins, resulting in the formation of covalent adducts or in the modulation of their expression and/or activity. In this review we focus on membrane proteins affected by lipid peroxidation-derived aldehydes, under physiological and pathological conditions.