Targeting HER2 with T-DM1, an Antibody Cytotoxic Drug Conjugate, is Effective in HER2 Over Expressing Bladder Cancer.

PURPOSE: Systemic therapy for advanced bladder cancer has not changed substantially in more than 2 decades and mortality rates remain high. The recognition of HER2 over expression in bladder cancer has made HER2 a promising therapeutic target. T-DM1, a new drug consisting of the HER2 antibody trastuzumab conjugated with a cytotoxic agent, has been shown in breast cancer to be superior to trastuzumab. We tested T-DM1 in preclinical models of bladder cancer. MATERIALS AND METHODS: We evaluated the effect of T-DM1 compared to trastuzumab in different in vitro and in vivo models of HER2 over expressing bladder cancer. RESULTS: RT4V6 was the highest HER2 expressing bladder cancer cell line and it showed higher growth inhibition with T-DM1 compared to trastuzumab. T-DM1 but not trastuzumab induced apoptosis of RT4V6 cells after G2/M arrest on cell cycle analysis. HER2 expression was higher in cell lines with acquired cisplatin resistance compared to the corresponding parental cell lines. Resistant cells showed higher sensitivity to T-DM1 by the induction of apoptosis. In addition, cells cultured in anchorage independent conditions increased HER2 expression compared to cells cultured in adherent conditions and T-DM1 significantly inhibited colony formation in soft agar compared to trastuzumab. In an orthotopic bladder cancer xenograft model tumor growth of cisplatin resistant RT112 was significantly inhibited by T-DM1 via the induction of apoptosis compared to treatment with control IgG or trastuzumab. CONCLUSIONS: T-DM1 has promising antitumor effects in preclinical models of HER2 over expressing bladder cancer.

Hiding in plain view: genetic profiling reveals decades old cross contamination of bladder cancer cell line KU7 with HeLa.

PURPOSE: KU7 is a popular urothelial carcinoma cell line that was isolated from the bladder of a patient at Keio University in 1980. It has subsequently been widely used in laboratories around the world. We describe how routine cell line authentication revealed that KU7 was cross contaminated almost 30 years ago with HeLa, a cervical carcinoma cell line. MATERIALS AND METHODS: Presumed KU7 clones dating from 1984 to 1999 were provided by M.D. Anderson Cancer Center, Vancouver Prostate Centre, Kyoto University, Tokyo Medical University and Keio University. HeLa was obtained from ATCC. Genomic DNA was isolated and short tandem repeat analysis was performed at the M.D. Anderson Cancer Center Characterized Cell Line Core Facility, Johns Hopkins University Fragment Analysis Facility and RIKEN BioResource Center, Ibaraki, Japan. Comparative genomic hybridization was performed on a platform (Agilent Technologies, Santa Clara, California) at Vancouver Prostate Centre. RESULTS: The short tandem repeat profile of all KU7 clones was an exact match with that of HeLa. Comparative genomic hybridization of all samples revealed an abundance of shared chromosomal aberrations. Slight differences in some genomic areas were explained by genomic drift in different KU7 clones separated by many years. CONCLUSIONS: Our analysis identified that cross contamination of KU7 with HeLa occurred before 1984 at the source institution. All KU7 clones in the urological literature should be considered HeLa and experimental results should be viewed in this light. Our results emphasize the need to authenticate cell lines in oncological research.

A Carbonic Anhydrase IX/SLC1A5 Axis Regulates Glutamine Metabolism Dependent Ferroptosis in Hypoxic Tumor Cells.

The ability of tumor cells to alter their metabolism to support survival and growth presents a challenge to effectively treat cancers. Carbonic anhydrase IX (CAIX) is a hypoxia-induced, metabolic enzyme that plays a crucial role in pH regulation in tumor cells. Recently, through a synthetic lethal screen, we identified CAIX to play an important role in redox homeostasis. In this study, we show that CAIX interacts with the glutamine (Gln) transporter, solute carrier family 1 member 5 (SLC1A5), and coordinately functions to maintain redox homeostasis through the glutathione/glutathione peroxidase 4 (GSH/GPX4) axis. Inhibition of CAIX increases Gln uptake by SLC1A5 and concomitantly increases GSH levels. The combined inhibition of CAIX activity and Gln metabolism or the GSH/GPX4 axis results in an increase in lipid peroxidation and induces ferroptosis, both in vitro and in vivo. Thus, this study demonstrates cotargeting of CAIX and Gln metabolism as a potential strategy to induce ferroptosis in tumor cells.

Structure-Based Study to Overcome Cross-Reactivity of Novel Androgen Receptor Inhibitors.

The mutation-driven transformation of clinical anti-androgen drugs into agonists of the human androgen receptor (AR) represents a major challenge for the treatment of prostate cancer patients. To address this challenge, we have developed a novel class of inhibitors targeting the DNA-binding domain (DBD) of the receptor, which is distanced from the androgen binding site (ABS) targeted by all conventional anti-AR drugs and prone to resistant mutations. While many members of the developed 4-(4-phenylthiazol-2-yl)morpholine series of AR-DBD inhibitors demonstrated the effective suppression of wild-type AR, a few represented by 4-(4-(3-fluoro-2-methoxyphenyl)thiazol-2-yl)morpholine (VPC14368) exhibited a partial agonistic effect toward the mutated T878A form of the receptor, implying their cross-interaction with the AR ABS. To study the molecular basis of the observed cross-reactivity, we co-crystallized the T878A mutated form of the AR ligand binding domain (LBD) with a bound VPC14368 molecule. Computational modelling revealed that helix 12 of AR undergoes a characteristic shift upon VPC14368 binding causing the agonistic behaviour. Based on the obtained structural data we then designed derivatives of VPC14368 to successfully eliminate the cross-reactivity towards the AR ABS, while maintaining significant anti-AR DBD potency.

Genome-wide synthetic lethal screen unveils novel CAIX-NFS1/xCT axis as a targetable vulnerability in hypoxic solid tumors.

The metabolic mechanisms involved in the survival of tumor cells within the hypoxic niche remain unclear. We carried out a synthetic lethal CRISPR screen to identify survival mechanisms governed by the tumor hypoxia-induced pH regulator carbonic anhydrase IX (CAIX). We identified a redox homeostasis network containing the iron-sulfur cluster enzyme, NFS1. Depletion of NFS1 or blocking cyst(e)ine availability by inhibiting xCT, while targeting CAIX, enhanced ferroptosis and significantly inhibited tumor growth. Suppression of CAIX activity acidified intracellular pH, increased cellular reactive oxygen species accumulation, and induced susceptibility to alterations in iron homeostasis. Mechanistically, inhibiting bicarbonate production by CAIX or sodium-driven bicarbonate transport, while targeting xCT, decreased adenosine 5'-monophosphate-activated protein kinase activation and increased acetyl-coenzyme A carboxylase 1 activation. Thus, an alkaline intracellular pH plays a critical role in suppressing ferroptosis, a finding that may lead to the development of innovative therapeutic strategies for solid tumors to overcome hypoxia- and acidosis-mediated tumor progression and therapeutic resistance.

Hydrogen-deuterium exchange mass spectrometry reveals three unique binding responses of mAbs directed to the catalytic domain of hCAIX.

Human carbonic anhydrase (hCAIX), an extracellular enzyme that catalyzes the reversible hydration of CO2, is often overexpressed in solid tumors. This enzyme is instrumental in maintaining the survival of cancer cells in a hypoxic and acidic tumor microenvironment. Absent in most normal tissues, hCAIX is a promising therapeutic target for detection and treatment of solid tumors. Screening of a library of anti-hCAIX monoclonal antibodies (mAbs) previously identified three therapeutic candidates (mAb c2C7, m4A2 and m9B6) with distinct biophysical and functional characteristics. Selective binding to the catalytic domain was confirmed by yeast surface display and isothermal calorimetry, and deeper insight into the dynamic binding profiles of these mAbs upon binding were highlighted by bottom-up hydrogen-deuterium exchange mass spectrometry (HDX-MS). Here, a conformational and allosterically silent epitope was identified for the antibody-drug conjugate candidate c2C7. Unique binding profiles are described for both inhibitory antibodies, m4A2 and m9B6. M4A2 reduces the ability of the enzyme to hydrate CO2 by steric gating at the entrance of the catalytic cavity. Conversely, m9B6 disrupts the secondary structure that is necessary for substrate binding and hydration. The synergy of these two inhibitory mechanisms is demonstrated in in vitro activity assays and HDX-MS. Finally, the ability of m4A2 to modulate extracellular pH and intracellular metabolism is reported. By highlighting three unique modes by which hCAIX can be targeted, this study demonstrates both the utility of HDX-MS as an important tool in the characterization of anti-cancer biotherapeutics, and the underlying value of CAIX as a therapeutic target.

Overcoming Adaptive Resistance to KRAS and MEK Inhibitors by Co-targeting mTORC1/2 Complexes in Pancreatic Cancer.

Activating KRAS mutations are found in over 90% of pancreatic ductal adenocarcinomas (PDACs), yet KRAS has remained a difficult target to inhibit pharmacologically. Here, we demonstrate, using several human and mouse models of PDACs, rapid acquisition of tumor resistance in response to targeting KRAS or MEK, associated with integrin-linked kinase (ILK)-mediated increased phosphorylation of the mTORC2 component Rictor, and AKT. Although inhibition of mTORC1/2 results in a compensatory increase in ERK phosphorylation, combinatorial treatment of PDAC cells with either KRAS (G12C) or MEK inhibitors, together with mTORC1/2 inhibitors, results in synergistic cytotoxicity and cell death reflected by inhibition of pERK and pRictor/pAKT and of downstream regulators of protein synthesis and cell survival. Relative to single agents alone, this combination leads to durable inhibition of tumor growth and metastatic progression in vivo and increased survival. We have identified an effective combinatorial treatment strategy using clinically viable inhibitors, which can be applied to PDAC tumors with different KRAS mutations.

Harnessing Induced Essentiality: Targeting Carbonic Anhydrase IX and Angiogenesis Reduces Lung Metastasis of Triple Negative Breast Cancer Xenografts.

Triple Negative Breast Cancer (TNBC) is aggressive, metastatic and drug-resistant, limiting the spectrum of effective therapeutic options for breast cancer patients. To date, anti-angiogenic agents have had limited success in the treatment of systemic breast cancer, possibly due to the exacerbation of tumor hypoxia and increased metastasis. Hypoxia drives increased expression of downstream effectors, including Carbonic Anhydrase IX (CAIX), a critical functional component of the pro-survival machinery required by hypoxic tumor cells. Here, we used the highly metastatic, CAIX-positive MDA-MB-231 LM2-4 orthotopic model of TNBC to investigate whether combinatorial targeting of CAIX and angiogenesis impacts tumor growth and metastasis in vivo to improve efficacy. The administration of a small molecule inhibitor of CAIX, SLC-0111, significantly reduced overall metastatic burden, whereas exposure to sunitinib increased hypoxia and CAIX expression in primary tumors, and failed to inhibit metastasis. The administration of SLC-0111 significantly decreased primary tumor vascular density and permeability, and reduced metastasis to the lung and liver. Furthermore, combining sunitinib and SLC-0111 significantly reduced both primary tumor growth and sunitinib-induced metastasis to the lung. Our findings suggest that targeting angiogenesis and hypoxia effectors in combination holds promise as a novel rational strategy for the effective treatment of patients with TNBC.

Not all NOTCH Is Created Equal: The Oncogenic Role of NOTCH2 in Bladder Cancer and Its Implications for Targeted Therapy.

PURPOSE: Recent molecular analyses of bladder cancer open the door to significant advances in targeted therapies. NOTCH has been identified as a tumor suppressor in bladder cancer, but prior reports have focused on NOTCH1 Here we hypothesized that NOTCH2 is an oncogene suitable for therapeutic targeting in bladder cancer. EXPERIMENTAL DESIGN: We studied genomic aberrations of NOTCH, compared survival and tumor progression according to NOTCH2 expression levels, and studied NOTCH2 function in vitro and vivo RESULTS: We report a high rate of NOTCH2 copy number gain in bladder cancer. High NOTCH2 expression was identified especially in the basal subtype and in mesenchymal tumors. NOTCH2 activation correlated with adverse disease parameters and worse prognosis by immunohistochemistry. Forced overexpression of the intracellular domain of NOTCH2 (N2ICD) induced cell growth and invasion by cell-cycle progression, maintenance of stemness and epithelial-to-mesenchymal transition (EMT). These effects were abrogated by silencing of CSL, indicating that the effects were mediated through the canonical NOTCH signaling pathway. In an orthotopic xenograft model, forced overexpression of N2ICD increased growth, invasion, and metastasis. To explore the potential for therapeutic targeting of NOTCH2, we first silenced the receptor with shRNA and subsequently treated with a specific inhibitory antibody. Both interventions decreased cell growth, invasion, and metastasis in vitro and in the orthotopic xenograft model. CONCLUSIONS: We have demonstrated that NOTCH2 acts as an oncogene that promotes bladder cancer growth and metastasis through EMT, cell-cycle progression, and maintenance of stemness. Inhibition of NOTCH2 is a rational novel treatment strategy for invasive bladder cancer. Clin Cancer Res; 22(12); 2981-92. ©2016 AACR.

Functional analysis of androgen receptor mutations that confer anti-androgen resistance identified in circulating cell-free DNA from prostate cancer patients.

BACKGROUND: The androgen receptor (AR) is a pivotal drug target for the treatment of prostate cancer, including its lethal castration-resistant (CRPC) form. All current non-steroidal AR antagonists, such as hydroxyflutamide, bicalutamide, and enzalutamide, target the androgen binding site of the receptor, competing with endogenous androgenic steroids. Several AR mutations in this binding site have been associated with poor prognosis and resistance to conventional prostate cancer drugs. In order to develop an effective CRPC therapy, it is crucial to understand the effects of these mutations on the functionality of the AR and its ability to interact with endogenous steroids and conventional AR inhibitors. RESULTS: We previously utilized circulating cell-free DNA (cfDNA) sequencing technology to examine the AR gene for the presence of mutations in CRPC patients. By modifying our sequencing and data analysis approaches, we identify four additional single AR mutations and five mutation combinations associated with CRPC. Importantly, we conduct experimental functionalization of all the AR mutations identified by the current and previous cfDNA sequencing to reveal novel gain-of-function scenarios. Finally, we evaluate the effect of a novel class of AR inhibitors targeting the binding function 3 (BF3) site on the activity of CRPC-associated AR mutants. CONCLUSIONS: This work demonstrates the feasibility of a prognostic and/or diagnostic platform combining the direct identification of AR mutants from patients' serum, and the functional characterization of these mutants in order to provide personalized recommendations regarding the best future therapy.

Targeting Binding Function-3 of the Androgen Receptor Blocks Its Co-Chaperone Interactions, Nuclear Translocation, and Activation.

The development of new antiandrogens, such as enzalutamide, or androgen synthesis inhibitors like abiraterone has improved patient outcomes in the treatment of advanced prostate cancer. However, due to the development of drug resistance and tumor cell survival, a majority of these patients progress to the refractory state of castration-resistant prostate cancer (CRPC). Thus, newer therapeutic agents and a better understanding of their mode of action are needed for treating these CRPC patients. We demonstrated previously that targeting the Binding Function 3 (BF3) pocket of the androgen receptor (AR) has great potential for treating patients with CRPC. Here, we explore the functional activity of this site by using an advanced BF3-specific small molecule (VPC-13566) that was previously reported to effectively inhibit AR transcriptional activity and to displace the BAG1L peptide from the BF3 pocket. We show that VPC-13566 inhibits the growth of various prostate cancer cell lines, including an enzalutamide-resistant cell line, and reduces the growth of AR-dependent prostate cancer xenograft tumors in mice. Importantly, we have used this AR-BF3 binder as a chemical probe and identified a co-chaperone, small glutamine-rich tetratricopeptide repeat (TPR)-containing protein alpha (SGTA), as an important AR-BF3 interacting partner. Furthermore, we used this AR-BF3-directed small molecule to demonstrate that inhibition of AR activity through the BF3 functionality can block translocation of the receptor into the nucleus. These findings suggest that targeting the BF3 site has potential clinical importance, especially in the treatment of CRPC and provide novel insights on the functional role of the BF3 pocket. Mol Cancer Ther; 15(12); 2936-45. ©2016 AACR.

Patient-derived bladder cancer xenografts in the preclinical development of novel targeted therapies.

Optimal animal models of muscle invasive bladder cancer (MIBC) are necessary to overcome the current lack of novel targeted therapies for this malignancy. Here we report on the establishment and characterization of patient-derived primary xenografts (PDX). Patient tumors were grafted under the renal capsule of mice and subsequently transplanted over multiple generations. Patient tumor and PDX were processed for analysis of copy number variations by aCGH, gene expression by microarray, and expression of target pathways by immunohistochemistry (IHC). One PDX harbouring an FGFR3 mutation was treated with an inhibitory monoclonal antibody targeting FGFR3. Five PDX were successfully established. Tumor doubling time ranged from 5 to 11 days. Array CGH revealed shared chromosomal aberrations in the patient tumors and PDX. Gene expression microarray and IHC confirmed that PDXs maintain similar patterns to the parental tumors. Tumor growth in the PDX with an FGFR3 mutation was inhibited by the FGFR3 inhibitor. PDXs recapitulate the tumor biology of the patients' primary tumors from which they are derived. Investigations related to tumor biology and drug testing in these models are therefore more likely to be relevant to the disease state in patients. They represent a valuable tool for developing precision therapy in MIBC.

Minimally invasive establishment of murine orthotopic bladder xenografts.

Orthotopic bladder cancer xenografts are the gold standard to study molecular cellular manipulations and new therapeutic agents in vivo. Suitable cell lines are inoculated either by intravesical instillation (model of nonmuscle invasive growth) or intramural injection into the bladder wall (model of invasive growth). Both procedures are complex and highly time-consuming. Additionally, the superficial model has its shortcomings due to the lack of cell lines that are tumorigenic following instillation. Intramural injection, on the other hand, is marred by the invasiveness of the procedure and the associated morbidity for the host mouse. With these shortcomings in mind, we modified previous methods to develop a minimally invasive approach for creating orthotopic bladder cancer xenografts. Using ultrasound guidance we have successfully performed percutaneous inoculation of the bladder cancer cell lines UM-UC1, UM-UC3 and UM-UC13 into 50 athymic nude. We have been able to demonstrate that this approach is time efficient, precise and safe. With this technique, initially a space is created under the bladder mucosa with PBS, and tumor cells are then injected into this space in a second step. Tumor growth is monitored at regular intervals with bioluminescence imaging and ultrasound. The average tumor volumes increased steadily in in all but one of our 50 mice over the study period. In our institution, this novel approach, which allows bladder cancer xenograft inoculation in a minimally-invasive, rapid and highly precise way, has replaced the traditional model.

Ultrasound-guided intramural inoculation of orthotopic bladder cancer xenografts: a novel high-precision approach.

Orthotopic bladder cancer xenografts are essential for testing novel therapies and molecular manipulations of cell lines in vivo. Current xenografts rely on tumor cell inoculation by intravesical instillation or direct injection into the bladder wall. Instillation is limited by the lack of cell lines that are tumorigenic when delivered in this manner. The invasive model inflicts morbidity on the mice by the need for laparotomy and mobilization of the bladder. Furthermore this procedure is complex and time-consuming. Three bladder cancer cell lines (UM-UC1, UM-UC3, UM-UC13) were inoculated into 50 athymic nude mice by percutaneous injection under ultrasound guidance. PBS was first injected between the muscle wall and the mucosa to separate these layers, and tumor cells were subsequently injected into this space. Bioluminescence and ultrasound were used to monitor tumor growth. Contrast-enhanced ultrasound was used to study changes in tumor perfusion after systemic gemcitabine/cisplatin treatment. To demonstrate proof of principle that therapeutic agents can be injected into established xenografts under ultrasound guidance, oncolytic virus (VSV) was injected into UM-UC3 tumors. Xenograft tissue was harvested for immunohistochemistry after 23-37 days. Percutaneous injection of tumor cells into the bladder wall was performed efficiently (mean time: 5.7 min) and without complications in all 50 animals. Ultrasound and bioluminescence confirmed presence of tumor in the anterior bladder wall in all animals 3 days later. The average tumor volumes increased steadily over the study period. UM-UC13 tumors showed a marked decrease in volume and perfusion after chemotherapy. Immunohistochemical staining for VSV-G demonstrated virus uptake in all UM-UC3 tumors after intratumoral injection. We have developed a novel method for creating orthotopic bladder cancer xenograft in a minimally invasive fashion. In our hands this has replaced the traditional model requiring laparotomy, because this model is more time efficient, more precise and associated with less morbidity for the mice.

Fibroblast growth factor receptor 3 is a rational therapeutic target in bladder cancer.

Activating mutations of fibroblast growth factor receptor-3 (FGFR3) have been described in approximately 75% of low-grade papillary bladder tumors. In muscle-invasive disease, FGFR3 mutations are found in 20% of tumors, but overexpression of FGFR3 is observed in about half of cases. Therefore, FGFR3 is a particularly promising target for therapy in bladder cancer. Up to now, most drugs tested for inhibition of FGFR3 have been small molecule, multityrosine kinase inhibitors. More recently, a specific inhibitory monoclonal antibody targeting FGFR3 (R3Mab) has been described and tested preclinically. In this study, we have evaluated mutation and expression status of FGFR3 in 19 urothelial cancer cell lines and a cohort of 170 American patients with bladder cancer. We have shown inhibitory activity of R3Mab on tumor growth and corresponding cell signaling in three different orthotopic xenografts of bladder cancer. Our results provide the preclinical proof of principle necessary to translate FGFR3 inhibition with R3Mab into clinical trials in patients with bladder cancer.