Sylvia Jebiwott

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.

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.

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.

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.

Contrast enhanced ultrasound imaging can predict vascular-targeted photodynamic therapy induced tumor necrosis in small animals

AIMS To evaluate the accuracy of contrast-enhanced ultrasound (CEUS) for monitoring tumor necrosis following WST-11 vascular targeted photodynamic therapy (VTP) using imaging-pathology correlation. METHODS Renal adenocarcinoma cells were injected into the hindlimb of 13 BalB/c mice resulting in tumors ranging from 9.8 to 194.3mm3. US guidance was used to place a laser fiber into the tumor, and VTP was performed. CEUS was performed prior to animal sacrifice, 24h post-VTP. Whole tumors were extracted for histopathologic analysis using H&E and TUNEL staining. Pathology samples corresponding to the CEUS imaging plane were prepared in order to compare the size and extents of tumor necrosis. RESULTS Tumor necrosis following VTP appeared as a central region of non-enhancement on CEUS, while viable tumor appeared as patchy regions of enhancement in the tumor periphery. The region of tumor necrosis measured in mean 66% and 64.8% of total tumor area on CEUS and pathology respectively (p=0.2). The size and location of the necrosis on CEUS images and pathology samples were found correlative with no inter-observer difference (weighted kappa of 0.771 and 0.823, respectively). CONCLUSION CEUS allows accurate monitoring of VTP induced tumor necrosis in a small animal model.

Abstract 3555: Targeting the BCL-2 family in small cell lung caner

Small cell lung cancer (SCLC) represents 13% of all lung cancer cases, affecting approximately 30,000 people annually in the United States. The prognosis for patients with SCLC is poor, and cancer-specific mortality of this malignancy is 95% at five years. This dismal prognosis has been further marred by the absence of major improvements in its treatment: there have been no substantial changes in the standard of care for advanced SCLC over the last three decades. Interestingly, small molecule inhibitors of anti-apoptotic BCL-2 family members BCL-2 and BCL-XL, ABT-737 and its orally bioavailable analog ABT-263, were shown to effectively kill some SCLC cell lines in preclinical studies, suggesting that targeting the BCL-2 family proteins may hold promise for the treatment of this dreadful cancer. However, these inhibitors do not inhibit MCL-1, another anti-apoptotic BCL-2 family member, which may explain why multiple clinical trials have not led to meaningful results. Because there are no effective MCL-1 inhibitors developed yet, we propose to identify the best strategy that enhances the apoptotic effect of ABT-737/263. To this end, we have performed a high-throughput screen in ABT-737/263 resistant H196 cell lines using two libraries, a library of FDA-approved anti-cancer agents and a pathway inhibitor library including ∼1000 compounds. As a result, doxorubicin and dinaciclib were identified as synergizers of ABT-737/263 in triggering apoptosis. These two drugs enhanced the death inducing activity of ABT-737/263 through downregulation of MCL-1 mRNA. Interestingly, some of the ABT-737/263 resistant cell lines rely on MCL-1 for survival and thereby were killed by dinaciclib or doxorubicin as a single agent, indicating that predictive biomarkers of cellular addiction to anti-apoptotic BCL-2 family proteins (BCL-2s) can guide treatment decisions and reduce unwanted toxicity. Importantly, we revealed that the expression ratio between BCL-2, BCL-XL, and MCL-1 could predict cellular addiction to BCL-2s. Surprisingly, we demonstrated that BCL-XL overexpression is an another potential mechanism behind ABT-263 resistance. ABT-263 failed to inhibit the interaction between BCL-XL and BAX/BAK in the absence of activator BH3-only molecules and thereby failed to kill one BCL-XL-addicted SCLC cell line with low expression of activator BH3-only molecules. Finally, we showed that BCL-2 selective inhibitor ABT-199 can also cooperate with doxorubicin or dinaciclib to kill ABT-263-resistant cell lines. The in vivo efficacy of combination therapy including ABT-199 and doxorubicin was demonstrated in a SCLC xenograft model. These data have direct translational implications for the treatment of SCLC. Citation Format: Akane Inoue-Yamauchi, Paul Jeng, Kwanghee Kim, Hui-Chen Chen, Song Han, Yogesh Tengarai Ganesan, Yiyu Dong, Sylvia Jebiwott, James J. Hsieh, Emily H. Cheng. Targeting the BCL-2 family in small cell lung caner. [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 3555.

Abstract 3389: Androgen deprivation therapy potentiates the efficacy of vascular targeted photodynamic therapy of prostate cancer xenografts

Focal therapies for prostate cancer offer the possibility of less side effects than more aggressive interventions for organ-confined tumors. Vascular targeted photodynamic therapy (VTP) is being investigated in phase II and phase III clinical trials, and relies upon rapid, free radical-mediated destruction of tumor vasculature following photoactivation of a systemic prodrug. In a phase II trial, >80% of patients did not have detectable cancer in prostate biopsies taken 6 months after VTP treatment. Although these early results are encouraging, VTP efficacy could potentially be improved upon by combining with other therapies. Using the LNCaP-AR prostate cancer model system, we employed microarrays and Gene Set Enrichment Analysis (GSEA) to identify potential druggable pathways active in tumors exposed to VTP. Three to six hours post-VTP, androgen responsive gene sets were enriched, 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 the AR pathway inhibitors degarelix (to achieve pharmacological castration) or enzalutamide (AR antagonist), alone or in combination with VTP. Degarelix was administered as a single 0.5-1 mg dose 3 days prior to initiation of VTP, while enzalutamide was given daily for two weeks total both before and after VTP. Serum measurements of prostate specific antigen (PSA) were used as a readout of AR activity. Compared to either AR pathway inhibitor or VTP used alone, degarelix or enzalutamide in combination with VTP significantly inhibited tumor growth. A sharp decline in serum PSA confirmed AR inhibition. Combined degarelix plus VTP treated tumors displayed intense TUNEL staining 7 days post-treatment, supporting an increased apoptotic frequency underlying the effect on tumor inhibition. In prostate cancer treated with VTP, compensatory acute upregulation of pro-survival AR signaling may occur. These findings may warrant investigation of combination with androgen deprivation therapy in future clinical trials utilizing VTP for prostate cancer. Citation Format: Kwanghee Kim, Philip A. Watson, Sylvia Jebiwott, Alexander J. Somma, Stephen P. La Rosa, Dipti Mehta, Katie S. Murray, Hans Lilja, David Ulmert, Avigdor Scherz, Jonathan Coleman. Androgen deprivation therapy potentiates the efficacy of vascular targeted photodynamic therapy of prostate cancer xenografts. [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 3389.