Kwanghee Kim

Targeting the differential addiction to anti-apoptotic BCL-2 family for cancer therapy

BCL-2 family proteins are central regulators of mitochondrial apoptosis and validated anti-cancer targets. Using small cell lung cancer (SCLC) as a model, we demonstrated the presence of differential addiction of cancer cells to anti-apoptotic BCL-2, BCL-XL or MCL-1, which correlated with the respective protein expression ratio. ABT-263 (navitoclax), a BCL-2/BCL-XL inhibitor, prevented BCL-XL from sequestering activator BH3-only molecules (BH3s) and BAX but not BAK. Consequently, ABT-263 failed to kill BCL-XL-addicted cells with low activator BH3s and BCL-XL overabundance conferred resistance to ABT-263. High-throughput screening identified anthracyclines including doxorubicin and CDK9 inhibitors including dinaciclib that synergized with ABT-263 through downregulation of MCL-1. As doxorubicin and dinaciclib also reduced BCL-XL, the combinations of BCL-2 inhibitor ABT-199 (venetoclax) with doxorubicin or dinaciclib provided effective therapeutic strategies for SCLC. Altogether, our study highlights the need for mechanism-guided targeting of anti-apoptotic BCL-2 proteins to effectively activate the mitochondrial cell death programme to kill cancer cells.

Neu-induced retroviral rat mammary carcinogenesis: a novel chemoprevention model for both hormonally responsive and nonresponsive mammary carcinomas.

Clinically relevant animal models of mammary carcinogenesis are crucial for the development and evaluation of new breast cancer chemopreventive agents. The neu-induced retroviral rat mammary carcinogenesis model is based on the direct in situ transfer of the activated neu oncogene into the mammary epithelium using a replication-defective retroviral vector. The resulting mammary carcinomas in intact Wistar-Furth rats exhibit a mixed hormonal response in the same proportion as has been observed in women. In intact rats, approximately 50% of mammary carcinomas can be prevented by tamoxifen treatment. In ovariectomized animals, the mammary carcinomas are hormonally nonresponsive and cannot be prevented by tamoxifen. We evaluated the efficacy of retinoic X receptor-selective retinoids (rexinoids) in this novel model of mammary carcinogenesis. The rexinoids LG100268 and bexarotene (LG1069, Targretin) were highly efficacious in the prevention of neu-induced mammary carcinomas. Dietary LG100268 at 100 mg/kg diet decreased tumor multiplicity by 32% (P = 0.0114) in intact rats and 50% (P < 0.0001) in ovariectomized rats. Bexarotene treatment at a dose of 250 mg/kg diet was associated with reductions in tumor multiplicity of 84% (P < 0.0001) and 86% (P < 0.0001) in intact and ovariectomized animals, respectively. In addition to tumor multiplicity, proliferation and apoptosis were modulated by bexarotene treatment independently of estrogen signaling. The neu-induced retroviral rat mammary carcinogenesis model represents a valuable addition to existing rodent chemoprevention models. The model is useful for assessing the efficacy of chemopreventive agents, specifically those compounds that target hormonally nonresponsive tumors.

Bombesin Antagonist-Based Radiotherapy of Prostate Cancer Combined with WST-11 Vascular Targeted Photodynamic Therapy

Purpose: DOTA-AR, a bombesin-antagonist peptide, has potential clinical application for targeted imaging and therapy in gastrin-releasing peptide receptor (GRPr)–positive malignancies when conjugated with a radioisotope such as 90Y. This therapeutic potential is limited by the fast washout of the conjugates from the target tumors. WST-11 (Weizmann STeba-11 drug; a negatively charged water-soluble palladium-bacteriochlorophyll derivative, Tookad Soluble) vascular targeted photodynamic therapy (VTP) is a local ablation approach recently approved for use in early-stage prostate cancer. It generates reactive oxygen/nitrogen species within tumor blood vessels, resulting in their instantaneous destruction followed by rapid tumor necrosis. We hypothesize that the instantaneous arrest of tumor vasculature may provide a means to trap radiopharmaceuticals within the tumor, thereby improving the efficacy of targeted radiotherapy. Experimental Design: GRPr-positive prostate cancer xenografts (PC-3 and VCaP) were treated with 90Y-DOTA-AR with or without VTP. The uptake of radioisotopes was monitored by Cherenkov luminescence imaging (CLI). The therapeutic efficacy of the combined VTP and 90Y-DOTA-AR in PC-3 xenografts was assessed. Results: CLI of 90Y-DOTA-AR demonstrated longer retention of radiotracer within the VTP-treated PC-3 xenografts compared with the non–VTP-treated ones (P < 0.05) at all time points (24–144 hours) after 90Y-DOTA-AR injection. A similar pattern of retention was observed in VCaP xenografts. When 90Y-DOTA-AR administration was combined with VTP, tumor growth delay was significantly longer than for the control or the monotherapy groups. Conclusions: Tumor vascular arrest by VTP improves 90Y-DOTA-AR retention in the tumor microenvironment thereby enhancing therapeutic efficacy. Clin Cancer Res; 23(13); 3343–51. ©2017 AACR.

Internalization of secreted antigen–targeted antibodies by the neonatal Fc receptor for precision imaging of the androgen receptor axis

A radiolabeled antibody against a secreted antigen uses Fc receptor–mediated internalization for cancer imaging and therapy. Prostate cancer hide-and-seek Prostate cancer is typically treated by targeting the androgen receptor, at least initially, but there is no convenient way to monitor the receptor’s activity or to determine when a tumor is becoming resistant to treatment. Although the androgen receptor cannot be imaged directly at this time, Thorek et al. identified an enzyme called human kallikrein-related peptidase 2 (hK2), whose activation requires signaling through the androgen receptor pathway. The authors used a radiolabeled antibody against hK2 in mouse models and human tissues to accurately detect prostate cancer lesions, including bone metastases, and to monitor their status during the course of treatment. Targeting the androgen receptor (AR) pathway prolongs survival in patients with prostate cancer, but resistance rapidly develops. Understanding this resistance is confounded by a lack of noninvasive means to assess AR activity in vivo. We report intracellular accumulation of a secreted antigen–targeted antibody (SATA) that can be used to characterize disease, guide therapy, and monitor response. AR-regulated human kallikrein-related peptidase 2 (free hK2) is a prostate tissue–specific antigen produced in prostate cancer and androgen-stimulated breast cancer cells. Fluorescent and radio conjugates of 11B6, an antibody targeting free hK2, are internalized and noninvasively report AR pathway activity in metastatic and genetically engineered models of cancer development and treatment. Uptake is mediated by a mechanism involving the neonatal Fc receptor. Humanized 11B6, which has undergone toxicological tests in nonhuman primates, has the potential to improve patient management in these cancers. Furthermore, cell-specific SATA uptake may have a broader use for molecularly guided diagnosis and therapy in other cancers.

Systemic antitumor immunity by PD-1/PD-L1 inhibition is potentiated by vascular-targeted photodynamic therapy of primary tumors.

Purpose: PD-1/PD-L1 pathway inhibition is effective against advanced renal cell carcinoma, although results are variable and may depend on host factors, including the tumor microenvironment. Vascular-targeted photodynamic (VTP) therapy with the photosensitizer WST11 induces a defined local immune response, and we sought to determine whether this could potentiate the local and systemic antitumor response to PD-1 pathway inhibition. Experimental Design: Using an orthotopic Renca murine model of renal cell carcinoma that develops lung metastases, we treated primary renal tumors with either VTP alone, PD-1/PD-L1 antagonistic antibodies alone, or a combination of VTP and antibodies and then examined treatment responses, including immune infiltration in primary and metastatic sites. Modulation of PD-L1 expression by VTP in human xenograft tumors was also assessed. Results: Treatment of renal tumors with VTP in combination with systemic PD-1/PD-L1 pathway inhibition, but neither treatment alone, resulted in regression of primary tumors, prevented growth of lung metastases, and prolonged survival in a preclinical mouse model. Analysis of tumor-infiltrating lymphocytes revealed that treatment effect was associated with increased CD8+:regulatory T cell (Treg) and CD4+FoxP3-:Treg ratios in primary renal tumors and increased T-cell infiltration in sites of lung metastasis. Furthermore, PD-L1 expression is induced following VTP treatment of human renal cell carcinoma xenografts. Conclusions: Our results demonstrate a role for local immune modulation with VTP in combination with PD-1/PD-L1 pathway inhibition for generation of potent local and systemic antitumor responses. This combined modality strategy may be an effective therapy in cancers resistant to PD-1/PD-L1 pathway inhibition alone. Clin Cancer Res; 24(3); 592–9. ©2017 AACR.

WST11 Vascular Targeted Photodynamic Therapy Effect Monitoring by Multispectral Optoacoustic Tomography (MSOT) in Mice

Objective: Monitoring emerging vascular-targeted photodynamic therapy (VTP) and understanding the time-dynamics of treatment effects remains challenging. We interrogated whether handheld multispectral optoacoustic tomography (MSOT) could noninvasively monitor the effect of VTP using WST11, a vascular-acting photosensitizer, on tumor tissues over time using a renal cell cancer mouse model. We also investigated whether MSOT illumination can induce VTP, to implement a single-modality theranostic approach. Materials and Methods: Eight BalB/c mice were subcutaneously implanted with murine renal adenocarcinoma cells (RENCA) on the flank. Three weeks later VTP was performed (10 min continuous illumination at 753 nm following intravenous infusion using WST11 or saline as control. Handheld MSOT images were collected prior to VTP administration and subsequently thereafter over the course of the first hour, at 24 and 48 h. Data collected were unmixed for blood oxygen saturation in tissue (SO2) based on the spectral signatures of deoxy- and oxygenated hemoglobin. Changes in oxygen saturation over time, relative to baseline, were examined by paired t-test for statistical significance (p < 0.05). In-vivo findings were corroborated by histological analyses of the tumor tissue. Results: MSOT is shown to prominently resolve changes in oxygen saturation in tumors within the first 20 min post WST11-VTP treatment. Within the first hour post-treatment, SO2 decreased by more than 60% over baseline (p < 0.05), whereas it remained unchanged (p > 0.1) in the sham-treated group. Moreover, unlike in the control group, SO2 in treated tumors further decreased over the course of 24 to 48 h post-treatment, concomitant with the propagation of profound central tumor necrosis present in histological analysis. We further show that pulsed MSOT illumination can activate WST11 as efficiently as the continuous wave irradiation employed for treatment. Conclusion: Handheld MSOT non-invasively monitored WST11-VTP effects based on the SO2 signal and detected blood saturation changes within the first 20 min post-treatment. MSOT may potentially serve as a means for both VTP induction and real-time VTP monitoring in a theranostic approach.

MP65-19 A MURINE MODEL OF OX-40 AND PD-1 IMMUNOTHERAPY IN COMBINATION WITH VASCULAR TARGET PHOTODYNAMIC THERAPY (VTP) ON UROTHELIAL CANCER TREATMENT

INTRODUCTION AND OBJECTIVES: WST11-VTP creates a strong antitumor immune reaction by exposing tumor antigens and trigging some systemic immune response. We postulated that the combination of VTP ablation with check point immune modulators OX40 and PD-1 could improve local tumor control and survival in a murine urothelial cancer model. METHODS: 65 Black6 immunocompetent mice were Subcutaneous injected with Luciferase MB-49 cells in the right flank. After 14 days the tumors were measured and image performed. 8 groups were created with similar tumors dimension: controls (5), PD1 (5), OX40 (5), PD1þOX40 (10), VTP (10), VTPþPD1 (10), VTPþOX40 (10), VTPþPD1þOX40 (10). VTP was performed on day 15 and the antibodies on the day after following this schedule: single dose of OX40 and 6 doses of PD1 (one every 3 days). IVIS Spectrum images and tumor measurements were done once week until 90 days. The experiment was repeated twice. Immunohistochemistry and Flow cytometry of the tumor were done in 2 different endpoints for lymphocytes expression: 3 days and 7 days after VTP procedure. Statistical analysis was done using the GraphPad Prism 7 software RESULTS: After 90 days, the survival rate in the VTPs groups was 12.5%, 22.2%, 33% and 66% for VTP þ OX40, VTP þ PD1, VTP and VTPþPD1þOX40 (VPO) respectively (p<0.001). One mouse (10%) from OX40þPD1 combination survived whereas all mice from the others group without VTP died before 60 days (no statistical difference compared to control group). Only VPO group and OX40þPD1 group did not present lung metastasis in IVIS image during the study (p<0.001). Immunohistochemistry (IHC) showed statistical difference comparing CD4 and CD8 expression in VPO and OX40þPD1 with the others group (p<0.001), especially on day 7 after VTP. Similar results were demonstrated by flow cytometry on day 7: VPO increased fourfold in CD4, six fold in CD8 (Graph 1) and 15 times more in CD4þKi67 expression compare to VTP alone (p<0.001). CONCLUSIONS: Ox-40 þ PD-1 immunotherapy in combination with VTP improves local tumor control and survival in MB-49 cell line model. The 2 different pathways combination seems rationale and encouraging approach in VTP treatment of urothelial cancer

Androgen Deprivation Therapy Potentiates the Efficacy of Vascular Targeted Photodynamic Therapy of Prostate Cancer Xenografts

Purpose: WST11 vascular targeted photodynamic therapy (VTP) is a local ablation approach relying upon rapid, free radical-mediated destruction of tumor vasculature. A phase III trial showed that VTP significantly reduced disease progression when compared with active surveillance in patients with low-risk prostate cancer. The aim of this study was to identify a druggable pathway that could be combined with VTP to improve its efficacy and applicability to higher risk prostate cancer tumors. Experimental Design: Transcriptome analysis of VTP-treated tumors (LNCaP-AR xenografts) was used to identify a candidate pathway for combination therapy. The efficacy of the combination therapy was assessed in mice bearing LNCaP-AR or VCaP tumors. Results: Gene set enrichment analysis identifies the enrichment of androgen-responsive gene sets within hours after VTP treatment, suggesting that the androgen receptor (AR) may be a viable target in combination with VTP. We tested this hypothesis in mice bearing LNCaP-AR xenograft tumors by using androgen deprivation therapy (ADT), degarelix, in combination with VTP. Compared with either ADT or VTP alone, a single dose of degarelix in concert with VTP significantly inhibited tumor growth. A sharp decline in serum prostate-specific antigen (PSA) confirmed AR inhibition in this group. Tumors treated by VTP and degarelix displayed intense terminal deoxynucleotidyl transferase–mediated dUTP nick end labeling staining 7 days after treatment, supporting an increased apoptotic frequency underlying the effect on tumor inhibition. Conclusions: Improvement of local tumor control following androgen deprivation combined with VTP provides the rationale and preliminary protocol parameters for clinical trials in patients presented with locally advanced prostate cancer. Clin Cancer Res; 24(10); 2408–16. ©2018 AACR.

MP88-17 PATIENT DERIVED XENOGRAFTS OF UPPER TRACT UROTHELIAL CARCINOMA: A POTENTIAL TOOL FOR PERSONALIZED MEDICINE

that tumor exosomes play in cancer development, metastasis and drug resistance. Tumor cells have been shown to selectively package certain proteins and RNA material into exosomes for the purpose of cell to cell communication. After internalization, recipient cells show altered gene expression, which in turn, modifies their invasiveness, apoptotic rate and sensitivity to therapeutic drugs. Few studies have examined the role of exosomal microRNAs (miRNA) in transference of drug resistance in bladder cancer. We hypothesize, that specific miRNAs have distinct roles in the establishment of chemoresistance. Here we strive to identify exosomal miRNAs profiles and their roles in Cisplatin, Gemcitabine and Cisplatin/Gemcitabine chemoresistance in bladder cancer. METHODS: Three resistant sublines of the human CUB III bladder carcinoma cell line were developed by gradually exposing the cells to increasing doses of Gemcitabine, Cisplatin or a combination of Gemcitabine and Cisplatin, over a period of 6 months. Exosomes were harvested and characterized by nanoparticle tracking analysis. Exosomal miRNAs were profiled via qRT-PCR array analysis. These distinct exosomal miRNA signatures were investigated in several additional bladder carcinoma resistant cell lines. RESULTS: Chemoresistant CUB III cells exhibit distinct miRNA profiles within their exosomes, which is unique depending on the drug of treatment. Of the 759 miRNA profiled, sixteen were differentially expressed (at least two-fold) across all the CUBIII resistant sublines relative to their parental line. Our data showed that 10 miRs were consistently down-regulated and 6 miRNAs were up regulated. Among the differentially expressed exosomal miRNAs in the resistant sublines, miR-Let-7i-3p was the most significantly down-regulated while miR-215p was the highest up-regulated compared to their chemosensitive counterpart. CONCLUSIONS: Our findings demonstrate that for each chemotherapeutic drug, resistant cells had differentially expressed miRNA profiles within their exosomes. Many of these miRNAs have been shown to play a role in oncogenesis or the development of drug resistance in other tumor types. After further validation these exosomal miRNAs may have utility as predictive biomarkers of treatment response and possibly as therapeutic targets to enhance drug response.

MP100-08 MITOCHONDRIAL – TARGET PEPTIDES ANTIOXIDANTS SS-20 AND SS-31 AS A KIDNEY PROTECTOR AGAINST HIGH DOSE WST-11 VASCULAR TARGET PHOTODYNAMIC THERAPY (VTP)

INTRODUCTION AND OBJECTIVES: WST11-VTP is a promising technology in cancer treatment. Several preclinical models demonstrated higher efficacy in ablation of prostate, urothelial and kidney tumors. Likewise others ablation procedures, some normal tissue beyond the tumor’s area can be affected causing some undesirable effects. Sveto et al showed the capacity of SS-20 and SS-31 peptides in reduce cells damage after ischemic kidney injury in rats. We examined combination of VTP and SS peptides on kidney tissue damage after high-dose VTP application METHODS: 28 black-6 male mice arranged in 4 different groups: VTP alone, VTP plus SS-20, VTP plus SS-31 and VTP plus SS-20 and SS-31. All mice got same high dose of VTP e 200 mW/cm / 100 e and retro-orbital WST11. The SS peptides dose was 2mg/Kg, gave 300 before VTP and daily for 4 days by subcutaneous injection. In the VTP plus SS-20/ SS-31 combination group, a single shot of SS20 was used 300 before VTP and the following daily doses were just SS-31. All VTP application was performed on left kidney after surgical approach by small flank incision and renal externalization. Urea and creatinine blood exam were realized one day before VTP, 24 hrs and 72 hrs after. All mice were euthanized on day 5 after VTP and tissues of interest were collected for histology assessment by a board certificated pathologist RESULTS: After 28 mice submitted a VTP high dose treatment, just one from VTP alone group died. A reduced kidney damageanalyzed by tubular injury scorewas observed in all peptides groups compare to VTP alone, but only the SS-20/SS-31 combination showed a statistical significance ( p < 0.05) . As a single agent, SS-20 seems has a better effect in kidney protection compares to SS-31. The E-Cadherin grades were lowered in all peptides treatment groups compared to VTP only (positive effect), but do not reach significance. This may be mitigated by powering the study to include more animals in each cohort. The same fact was observed in creatinine and urea results analysis. Compare to VTP alone, all peptides groups had lower creatinine and urea values 24 hrs after VTP application, but without statistical importance. Exception for SS-31 and VTP alone groups that reached statistical difference (p1⁄40.04) in the urea measures 24 hrs after VTP. Once again, SS-20/SS31 combination and SS-31 groups presented lowest levels of creatinine and urea on day after VTP. After 72 hrs of VTP application, creatinine and urea returned to baseline values in all groups CONCLUSIONS: The use of mitochondria-target peptides, SS-20 and SS-31, can protect the kidney against high dose VTP in a mouse model. The rationale of combine this drugs before and after VTP seems a promising approach in renal preservation and side effects prevention after VTP ablation