Reema Railkar

Epidermal Growth Factor Receptor (EGFR)-targeted Photoimmunotherapy (PIT) for the Treatment of EGFR-expressing Bladder Cancer

The use of light as a means of therapy for bladder cancer has a long history but has been hampered by a lack of tumor specificity and therefore, damage to the normal bladder mucosa. Here, we describe a targeted form of phototherapy called photoimmunotherapy (PIT), which targets EGFR-expressing bladder cancer. Anti-EGFR antibody panitumumab was labeled with the photoabsorber (PA), IRDye 700Dx (IR700), to create a panitumumab-IR700 antibody–PA conjugate that is activated by near-infrared radiation (NIR). Bladder cancer tissue microarray (TMA) and bladder cancer cell lines were analyzed for expression of EGFR. Mechanism of PIT-induced cell death was studied using proliferation assays, transmission electron microscopy (TEM), and production of reactive oxygen species. Finally, the in vivo effect was studied in xenografts. EGFR staining of TMAs showed that while most bladder cancers have expression of EGFR to a varying degree, squamous cell carcinomas (SCC) have the highest expression of EGFR. Panitumumab-IR700 activated by NIR light rapidly killed UMUC-5 cells, a bladder SCC line. Panitumumab alone, panitumumab-IR700 without NIR, or NIR alone had no effect on cells. TEM demonstrated that cell death is due to necrosis. Singlet oxygen species contributed toward cell death. NIR-PIT with panitumumab-IR700 reduced growth compared with only panitumumab-IR700–treated UMUC-5 xenograft tumors. PIT is a new targeted treatment for bladder cancer. Panitumumab-IR700–induced PIT selectively kills EGFR-expressing bladder cancer cells in vitro and in vivo and therefore warrants further therapeutic studies in orthotopic xenografts of bladder cancer and ultimately in patients. Mol Cancer Ther; 16(10); 2201–14. ©2017 AACR.

Protein kinase D inhibitor CRT0066101 suppresses bladder cancer growth in vitro and xenografts via blockade of the cell cycle at G2/M

The protein kinase D (PKD) family of proteins are important regulators of tumor growth, development, and progression. CRT0066101, an inhibitor of PKD, has antitumor activity in multiple types of carcinomas. However, the effect and mechanism of CRT0066101 in bladder cancer are not understood. In the present study, we show that CRT0066101 suppressed the proliferation and migration of four bladder cancer cell lines in vitro. We also demonstrate that CRT0066101 blocked tumor growth in a mouse flank xenograft model of bladder cancer. To further assess the role of PKD in bladder carcinoma, we examined the three PKD isoforms and found that PKD2 was highly expressed in eight bladder cancer cell lines and in urothelial carcinoma tissues from the TCGA database, and that short hairpin RNA (shRNA)-mediated knockdown of PKD2 dramatically reduced bladder cancer growth and invasion in vitro and in vivo, suggesting that the effect of the compound in bladder cancer is mediated through inhibition of PKD2. This notion was corroborated by demonstrating that the levels of phospho-PKD2 were markedly decreased in CRT0066101-treated bladder tumor explants. Furthermore, our cell cycle analysis by flow cytometry revealed that CRT0066101 treatment or PKD2 silencing arrested bladder cancer cells at the G2/M phase, the arrest being accompanied by decreases in the levels of cyclin B1, CDK1 and phospho-CDK1 (Thr161) and increases in the levels of p27Kip1 and phospho-CDK1 (Thr14/Tyr15). Moreover, CRT0066101 downregulated the expression of Cdc25C, which dephosphorylates/activates CDK1, but enhanced the activity of the checkpoint kinase Chk1, which inhibits CDK1 by phosphorylating/inactivating Cdc25C. Finally, CRT0066101 was found to elevate the levels of Myt1, Wee1, phospho-Cdc25C (Ser216), Gadd45α, and 14-3-3 proteins, all of which reduce the CDK1-cyclin B1 complex activity. These novel findings suggest that CRT0066101 suppresses bladder cancer growth by inhibiting PKD2 through induction of G2/M cell cycle arrest, leading to the blockade of cell cycle progression.

PNFLBA-12 TARGETING EPIDERMAL GROWTH FACTOR RECEPTOR (EGFR) AND HUMAN EPIDERMAL GROWTH FACTOR RECEPTOR 2 EXPRESSING BLADDER CANCER USING COMBINATION PHOTOIMMUNOTHERAPY (PIT)

refusal. In the primary ITT analysis (n1⁄4406), there was a significant 34% reduction in risk of recurrence in the G arm compared to S (HR1⁄40.66, 95% CI 0.48, 0.90, p1⁄40.010). For the per-protocol target population, LG NMI UCs, TTR even more strongly favored G (HR 0.50 95% CI 0.33, 0.76; p1⁄40.001). Few muscle invasive events (G 2.5%, S 4.9%) or deaths from any cause (G 8.5%, S 12.2%) occurred. Adverse events (AEs) were infrequent. There were no Grade 4 or 5 complications, and no difference in Grade 3 AEs (G 2.4%, S 3.4%). CONCLUSIONS: Immediate post TURBT intravesical instillation of G was safe, well tolerated and significantly reduced recurrence of LG NMI UC in these participants.

MP98-10 UTILITY OF HIGH THROUGHPUT SCREENING IN IDENTIFYING AND REPURPOSING SMALL MOLECULE INHIBITORS FOR UROTHELIAL CARCINOMA

INTRODUCTION AND OBJECTIVES: We developed a novel treatment for localized and metastatic bladder cancer comprised of gold nanoparticle-based photothermal therapy and immunotherapy (SYMPHONY). We demonstrate that it effectively ablates primary tumors, destroys metastases abscopally, and induces potent anti-tumor immunity. METHODS: MB49 murine bladder cancer cells were injected into the bilateral flanks of C57BL/6 mice and grown until 100 mm3 in size. PEG-functionalized gold nanostars, developed and manufactured by our team, were administered intravenously. A 808-nm laser (0.6 W/ cm2) was used to trigger plasmonic heat production from the gold nanostars in the left flank 24 hours after injection, while the contralateral flank was left untreated. Anti-PD-L1 antibody immunotherapy was coadministered intraperitoneally and repeated q3days. Mice were assessed for ipsilateral and contralateral tumor response and survival. Flow cytometry, multiplex cytokine profiling, and T cell receptor sequencing were used to characterize the immune response. Mice achieving a complete response were rechallenged with an additional injection of MB49 tumor cells 90 days later. RESULTS: Gold nanostar-mediated phototherapy alone completely ablated ipsilateral tumors in 4/5 of mice (pT0 at necropsy) but contralateral tumors grew and all 5 mice required sacrifice within 14 days. Anti-PD-L1 therapy alone slowed tumor growth in 3/5 mice, but tumors rapidly began growing again and 5/5 mice required sacrifice by 45 days. Combined treatment (i.e. SYMPHONY) ablated 5/5 ipsilateral tumors and resulted in partial (3/5) and complete responses (2/5) of untreated contralateral tumors, demonstrating a strongabscopal effect. After 90days of follow-up, the two mice achieving a complete response with SYMPHONYwere rechallengedwithMB49and neither developed a tumor over the ensuing 4weeks indicating strong and effective immunememory. Flow cytometry showed CD4 and CD8 T cell proliferation, decreased myeloid derived suppressor cells, and increased IL2 with SYMPHONY. CONCLUSIONS: SYMPHONY treatment resulted not only in effective ablation of primary tumors but also in immune-mediated abscopal destruction of untreated distant tumors. Strong and permanent anti-tumor immunity developed in some mice, indicating that with further optimization, SYMPHONY may be able to cure more advanced bladder cancers.

MP98-17 TARGETING PROTEIN KINASE D2 MAY REPRESENT A THERAPEUTIC STRATEGY FOR BLADDER CANCER

INTRODUCTION AND OBJECTIVES: Chemoresistance to cisplatin is a principal cause of treatment failure and disease progression of advanced bladder cancer. In the present study we explore the novel relationship between cisplatin resistance and pyruvate kinase 2 (PKM2) e a rate-limiting enzyme responsible for Warburg effect in cancer cells, and whether down-regulating PKM2 by RNAi or small molecules reduces chemoresistance and enhances chemosensitivity of bladder cancer cells to cisplatin. METHODS: Cell lines from mouse and human bladder cancer and their derivatives expressing RNAi of PKM2 were assessed for their chemosensitivity to cisplatin or shikonin a chemical inhibitor of PKM2, or both. The effects and mechanisms of PKM2 inhibition on cisplatinresistance were examined. Cisplatin and shikonin as single or dual agents for inhibiting bladder cancer proliferation and metastasis were further tested in syngeneic mice. RESULTS: Shikonin binds PKM2 and inhibits bladder cancer cell proliferation in a dose-dependent but pyruvate kinase activity-independent manner. Down-regulation of PKM2 by shRNA blunts cellular responses to shikonin but enhances the responses to cisplatin. Shikonin and cisplatin together exhibit significantly greater growth inhibition and apoptosis than when used alone. Experimentally induced cisplatinresistance is strongly associated with PKM2 overexpression, and cisplatin-resistant cells respond sensitively to shikonin. In syngeneic mice, shikonin and cisplatin together, but not as single-agents, markedly reduces bladder cancer growth and lung metastases. CONCLUSIONS: PKM2 overexpression is a key mechanism of natural and acquired chemoresistance of bladder cancer to cisplatin. Inhibition of PKM2 via RNAi or chemical inhibitors may be a highly effective approach to overcome chemoresistance and improve the outcome of advanced bladder cancer.

MP65-13 FLAVOPIRIDOL AS A NOVEL AGENT FOR BLADDER CANCER

INTRODUCTION AND OBJECTIVES: Although Bacillus Calmette-Guerin (BCG) is the most effective agent for non-muscleinvasive bladder cancers, approximately 30% of patients treated with intravesical BCG fail to respond to this agent. Previous studies from our lab showed the potential linkage of estrogen/estrogen receptor signaling with the efficacy of BCG, yet the detailed mechanisms remain unclear. Our new data showed the combination of BCG and the anti-estrogen ICI 182,780 (ICI) or tamoxifen could lead to a better suppression of bladder cancer (BCa) than BCG alone. METHODS: We first applied PCR to detect BCG internalization in two ERa positive BCa cell lines to investigate the potential effect of anti-estrogen ICI. Then, we used Q-PCR and western blot and examined the E2/ER effects on the integrin-a5b1 expression and the BCG attachment/internalization to BCa cells. To examine whether ICI can help the recruitment of macrophages toward BCa cells, we applied the transwell migration assay and in vivo mouse BCG model. Q-PCR, Elisa assay and MTT assay were used to detect the cytokine profile changes and BCa cell viability. For our in vivo studies, we applied the BBN-induced mouse BCa model, HE staining, BrdU and F4/80 staining to show the changes of macrophage infiltration and to prove the better efficacy of combining BCG plus anti-estrogen. RESULTS: We found treatment with either 1 mM ICI or tamoxifen significantly increased the BCG attachment/internalization, and the neutralization of integrin-a5b1 could reduce the ability of the ICI enhanced BCG attachment/internalization to BCa cells (Figure 1). Mechanism dissection revealed ICI could promote BCG attachment/ internalization to the BCa cells through targeting ERa and increased the integrin-a5b1 expression and IL-6 secretion. The increased cytokine production may enhance BCG-mediated suppression of BCa cell growth and TNF-a production via recruiting more monocytes/macrophages to BCa cells (Figure 2-3). Consistently, in vivo studies found ICI could potentiate the anti-BCa effects of BCG in the carcinogen-induced mouse BCa models (Figure 4). CONCLUSIONS: Taken together, these in vitro and in vivo results suggest that combining BCG with the anti-estrogen may become a new therapeutic approach with better efficacy to suppress BCa progression and recurrence. Source of Funding: none

Abstract 3758: Quantitative high-throughput screening as a tool to identify novel therapies in bladder cancer: lessons from flavopiridol

Introduction and Objectives: Bladder cancer (CaB) is the 4th most common cancer among men and 12th most common among women in United States. It is one of the most expensive malignancies to treat from diagnosis to death. No new pharmacological agents have been approved for the treatment of bladder cancer in the last two decades. Therefore, there is an urgent need for development of new treatment therapies. Quantitative high throughput screening (qHTS) of representative cancer cell lines with oncology drugs may identify new treatments and pathways. We utilized this technique to identify new targets and therapies in two primary bladder lines (T24 and UMUC3) and their metastatic derivatives (T24T, SLT3 and FL3 of T24 and LUL-2 for UMUC3). Methods: We screened 7 bladder cancer cell lines (T24T, SLT3, FL3, LUL-2, RT4, T24, and UMUC3) against 1,912 oncology drugs using a 48 hour cell proliferation assay with an ATP−based readout (CellTiterGlo) to determine activity and potency of compounds in a dose response manner. One of the candidate drugs inhibitory in all cell lines tested is flavopiridol, a pan-CDK inhibitor. We further characterized the mechanism of action of flavopiridol using various cell based assays such as cell proliferation, cell cycle analysis, apoptosis assays, and modified colony forming assays. Finally, mouse xenograft studies were carried out to elucidate the in vivo effects of flavopiridol. Results: The initial screen identified 95 compounds active in 7 cell lines. The top 50 compounds were further analyzed for molecular size of >200 g/mol and TPSA Conclusions: qHTS can identify novel compounds. Flavopiridol is an effective inhibitor both in vitro and in vivo. Although it merely caused cytostatic inhibition in a xenograft model when delivered systemically, its physical properties are most suited for intravesical use which may lead to it being more effective as higher doses can be administered into the bladder with minimal/no systemic toxicities. Studies are now underway to evaluate the use of flavopiridol as an intravesical agent. Citation Format: Reema Railkar, Achuth Nair, Keidren Lewi, Spencer Krane, Rajarshi Guha, Marc Ferrer, Craig Thomas, Piyush K. Agarwal. Quantitative high-throughput screening as a tool to identify novel therapies in bladder cancer: lessons from flavopiridol. [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 3758.

Abstract 109: Y chromosome genes contribute to higher male bladder cancer incidence

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Introduction: Clinical reports show that males have a higher bladder cancer (BCa) incidence than females. The gender bias in BCa occurrence suggests that there may be genetic differences between males and females. One apparent factor important in sex differentiation is the existence of the Y chromosome in males. We sought to evaluate whether genes on the Y chromosome play a role in BCa development. Methods: mRNA expression of Y chromosome genes from male and female BCa tissues were analyzed from the Oncomine database ( ). Real-time PCR (RT-PCR) was then used to analyze SMCY mRNA expression in all available BCa cell lines in our lab (RT4, T24, UMUC3, UOBL101, UOBL102, UOBL103, 647v, TCCSUP, 253J, J82, 5637, HT1197, SCaBer, TCCSUP, and SVHUC). Lentiviral vectors with SMCY shRNA were used for SMCY knockdown in 647v and RT4 (male-derived) BCa cell lines. Lentiviral vectors encoding SMCY cDNA were used for SMCY overexpression in T24 (female-derived) and UMUC-3 (Y chromosome-deleted male-derived) BCa cell lines. Cell proliferation was performed using the MTT assay. Anchorage independent cell growth was performed by plating 1% agarose on the bottom of plates, and then seeding the cell suspension in 0.35% of agarose in MEM. Cells were grown in agarose for two weeks and the number of colonies was counted. Results: Oncomine analysis demonstrated that certain genes on the Y chromosome (SMCY, DDX3Y, and USP9Y) have higher expression in male tumors than in female tumors. RT-PCR confirmed this finding in BCa cell lines with respect to SMCY mRNA expression. Furthermore, knockdown of SMCY in the male-derived BCa cell lines reduced cell proliferation rates. Moreover, overexpression of SMCY in BCa cells derived from female patients or from Y chromosome deleted male patients (UMUC-3) increased the cell proliferation rates. Anchorage independent cell growth is a hallmark of higher tumorigenicity. Consistently, 647v with SMCY knockdown grow fewer colonies compared to control cells in anchorage independent cell growth assay. T24 and UMUC-3 with SMCY expression grow more colonies than control cells. SMCY is a histone H3 lysine 4 (H3K4) demethylase and plays a central role in histone code. It demethylates trimethylated and dimethylated but not monomethylated H3 lysine 4. H3K4 methylation is tightly associated with gene activation. Our preliminary data indicated that with SMCY knockdown, trimethylated and dimethylated H3K4 is increased in 647v and RT4 cells, suggesting that SMCY maintains its demethlyase activity in cancer cells. Conclusions: Overall, we hypothesize that Y chromosome genes may contribute to cancer growth and tumorigenesis in men and partially account for higher male bladder cancer incidence rates. The finding that SMCY is implicated in histone modification also suggests the importance of chromatin remodeling genes in bladder cancer. Citation Format: Iawen Hsu, Reema Railkar, Quentin Li, Piyush Agarwal. Y chromosome genes contribute to higher male bladder cancer incidence. [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 109. doi:10.1158/1538-7445.AM2015-109

Abstract 3311: Molecular targeted photoimmunotherapy as a treatment for bladder cancer

Introduction: Bladder cancer (BCa) is a common cancer and is the most expensive malignancy to treat from diagnosis to death. Although no new therapies have been introduced in the past two decades, data from ‘The Cancer Genome Atlas (TCGA)’ confirms that Epidermal Growth Factor Receptor (EGFR) family and its downstream signaling molecules are altered in almost 72% of muscle invasive bladder cancer (MIBC) cases. However, anti-EGFRs to date have failed to demonstrate any benefit over standard of care chemotherapy. We have applied a technique of employing monoclonal antibodies (mAbs) conjugated with photo-activatable compounds (IRDye 700Dx, a phthalocyanine dye) that are activated by near-infrared light (NIR). When incubated with the conjugate, exposure to NIR destroys only targeted cells. We explored photoimmunotherapy (PIT) in a panel of BCa cell lines. The purpose of this study was to study the efficacy and mechanism of action of PIT using the anti-EGFR panitumumab (Pan)-IR700 immunoconjugate as a selective therapeutic strategy for bladder cancer. Methods: Using flow cytometry, the surface expression of EGFR was profiled in several BCa cell lines. The cytotoxicity of Pan-IR700 was analyzed using LIVE/DEAD and IC50 was measured using the MTS assay. The type of cell death was examined by fluorometric caspase assay, Annexin V-PI staining, and TEM. The mechanism of cell death was elucidated using fluorometric measurement of reactive and singlet oxygen species. Results: Using flow cytometry, the surface expression of EGFR was profiled in several BCa cell lines and cell lines with increasing amounts of surface EGFR (RT4, TCCSUP, 5637 and UMUC-5) were selected for further studies. Pan-IR700 rapidly killed UMUC-5 cells (high EGFR expressions) with an IC50 of 4 nM at 4 J/cm2 NIR. In TCCSUP cells (low surface EGFR), the same IC50 of Pan-IR700 could be achieved at 64 J/cm2 of NIR. No significant cytotoxicity was observed in the presence of IR700 or NIR alone, or in cell lines without any EGFR expression. Absence of any caspases and presence of most cells in the late apoptosis/necrosis quadrant of Annexin V-PI staining suggested that PIT kills cells by necrosis. In TEM, these cells showed classic features of necrotic cell death such as swollen organelles, disintegrated plasma membrane and depleted cytoplasm and nucleoplasm. Cells undergoing PIT showed production of ROS and singlet oxygen species in large amounts immediately after exposure to NIR. However, only the singlet oxygen quencher (NaN3) and not the ROS quencher (Trolox) was able to protect the cells from PIT induced cell death indicating partial involvement of singlet oxygen in PIT-induced necrosis of cells. Conclusions: PIT is a new targeted treatment for bladder cancer. Our data demonstrate that Pan-IR700-induced PIT selectively and efficiently kills EGFR-expressing bladder cancer cells in vitro and therefore warrants further preclinical therapeutic studies in in vivo bladder cancer models. Citation Format: Reema Railkar, Quentin Li, Srinivas Vourganti, Sam J. Brancato, Peter L. Choyke, Hisataka Kobayashi, Piyush K. Agarwal. Molecular targeted photoimmunotherapy as a treatment for bladder cancer. [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 3311. doi:10.1158/1538-7445.AM2015-3311