Novel renal medullary carcinoma cell lines, UOK353 and UOK360, provide preclinical tools to identify new therapeutic treatments

Abstract Renal medullary carcinoma (RMC) is a rare, aggressive disease that predominantly afflicts individuals of African or Mediterranean descent with sickle cell trait. RMC comprises 1% of all renal cell carcinoma diagnoses with a median overall survival of 13 months. Patients are typically young (median age—22) and male (male:female ratio of 2:1) and tumors are characterized by complete loss of expression of the SMARCB1 tumor suppressor protein. Due to the low incidence of RMC and the disease's aggressiveness, treatment decisions are often based on case reports. Thus, it is critical to develop preclinical models of RMC to better understand the pathogenesis of this disease and to identify effective forms of therapy. Two novel cell line models, UOK353 and UOK360, were derived from primary RMCs that both demonstrated the characteristic SMARCB1 loss. Both cell lines overexpressed EZH2 and other members of the polycomb repressive complex and EZH2 inhibition in RMC tumor spheroids resulted in decreased viability. High throughput drug screening of both cell lines revealed several additional candidate compounds, including bortezomib that had both in vitro and in vivo antitumor activity. The activity of bortezomib was shown to be partially dependent on increased oxidative stress as addition of the N‐acetyl cysteine antioxidant reduced the effect on cell proliferation. Combining bortezomib and cisplatin further decreased cell viability both in vitro and in vivo that single agent bortezomib treatment. The UOK353 and UOK360 cell lines represent novel preclinical models for the development of effective forms of therapy for RMC patients.


| INTRODUCTION
Renal cell carcinoma (RCC) affects nearly 300 000 individuals worldwide each year with over 100 000 deaths annually. RCC consists of several distinct subtypes that vary in incidence and demonstrate associations with specific histologic, genetic, and clinical features. Renal medullary carcinoma (RMC) is a rare and aggressive subtype of RCC that predominantly afflicts individuals of African or Mediterranean decent with sickle cell trait. 1,2 Although RMC comprises less than 1% of all RCC, it is the deadliest subtype with the median overall survival of 13 months. 1,3,4 This necessitates a concerted effort to develop a mechanistically-driven approach to therapy for this cancer. Patients are most often young, with a median age from 19 to 22 years old and more frequently male with a 2:1 male to female ratio, similar to the ratios observed in other RCC subtypes. 2,4-6 RMC occurs more frequently within the right kidney for reasons that have yet to be elucidated. 2,7 While patients can present with gross hematuria or flank pain, RMC has a propensity to metastasize early, often resulting in the presentation of late stage disease with poor prognosis. 2,4,6 A central feature of RMC is loss of expression of the SWI/SNFrelated matrix-associated actin-dependent regulator of chromatin subfamily B member 1 (SMARCB1) protein, also known as integrase interactor 1 (INI1), BRG1-associated factor 47 (BAF47), or sucrose nonfermenting 5 (SNF5). [8][9][10] The SMARCB1 protein, encoded by SMARCB1 in chromosome band 22q11.23, is a core subunit of the SWI/SNF chromatin remodeling complex. Loss destabilizes the complex and disrupts its equilibrium with the polycomb repressive complex 2 (PRC2) resulting in dysregulation of transcription activity. 11,12 In addition to RMC, malignant rhabdoid tumors (MRTs) also are characterized by complete inactivation of the SMARCB1 gene. 13 MRTs are rare and aggressive pediatric renal tumors with an estimated 80%-90% of children succumbing to the disease, most within a year of diagnosis. 14 It has been shown that tumors with mutation of the SWI/SNF complex subunits have a dependency on EZH2, a component of the PRC2 complex, and studies of EZH2 inhibitors in SMARCB1 mutant MRT mouse xenograft models have shown complete responses. 15,16 Due to the low incidence of RMC and the disease's aggressive nature, large clinical trials have been difficult to perform, and treatment decisions are often based on a limited number of published case reports. A handful of reports have suggested that either bortezomib or platinum-based combination chemotherapies, such as MVAC (methotrexate, vinblastine, doxorubicin, and cisplatin), could provide a therapeutic option. 6,[17][18][19] Currently, there is a need for cell line models for RMC to provide a crucial tool for the investigation of novel therapies for patients affected with RMC. A recent study by Hong et al 20 managed to produce two primary tumor cell lines from two separate patients using a ROCK inhibitor (Y-27632) based methodology 21 to produce a successful culture.
Both cell lines and the tumors from which they were derived had the expected loss of SMARCB1 function and the cell lines demonstrated an in vitro response to bortezomib. 20 Neither of the primary tumor lines developed in this study were capable of producing tumor xenografts and so this study relied on utilizing the SMARCB1-deficient rhabdoid cell line G401 as a substitute for an actual RMC cell line in its in vivo studies. 20 Confirmation of in vitro data by in vivo analysis is fundamentally important for the evaluation of therapeutic agents; thus, a model that allows for in vivo analysis is essential.
We endeavored to establish new RMC cell lines in hopes of elucidating their biology and defining effective treatments. Herein we report the development of two patient tumor-derived cell lines, UOK353 and UOK360, that resulted from spontaneous transformation and represent models for RMC. These cell lines allow the adoption of a two-pronged approach to identification of potential therapeutics using (1) a targeted approach focused on exploiting SMARCB1 loss and (2) high throughput drug screening. Both cell lines produced tumor xenografts in nude mice, with UOK360 more rapidly, providing models for both in vitro and in vivo analysis of potential therapeutic targets.

| Cell line production
Surgically resected tumor tissue from one patient and a tumor biopsy from a second patient were utilized to establish two cell lines, UOK353 and UOK360, in accordance with protocols and techniques previously described by the Urologic Oncology Branch. 22 Sterile tumor tissues were acquired and transferred to tissue culture as rapidly as possible to maximize the viability of the tissue. Both cell lines were propagated for over 20 passages before analysis. UOK353 was found to have a doubling time of approximately 72 hours, while UOK360 had a doubling time of approximately 24 hours. All cells were cultivated in high glucose (25 mM D-glucose) DMEM medium supplemented with 10% fetal calf serum and 2 mM L-glutamine. Short tandem repeat (STR)-based DNAprofiling was performed on the cell lines and the matched peripheral blood DNA to confirm the origins of both cell lines.

| Spectral karyotyping (SKY) and fluorescence in situ hybridization (FISH)
FISH and SKY were performed on both UOK353 and UOK360, on interphase and metaphase spreads, respectively. Interphase cells were hybridized with a SMARCB1 break apart probe (#SMARCB1BA-20-GROR, Empire Genomics, Williamsville, NY). Images were imaged on ×63 objective using a Leica Thunder Imager (Leica Microsystems, Wetzlar, Germany). Metaphase spreads (n = 25) were scored for chromosomal copy number and for structural aberrations as previously described. 23 Spectrum-based classification and analysis of the fluorescent images was performed by using SkyView software (Applied Spectral Imaging, Carlsbad, CA). The chromosome complements of every metaphase spread were analyzed and the karyotypes were described according to human chromosome nomenclature standards described in ISCN 2009. 24

| RNA extraction and real-time PCR analysis
Total RNA was extracted from cell lines grown in 10 cm dishes using

| Drug treatment and growth assay protocol
The cells were seeded onto 96-well plates and treated with the indicated drug with multiple replicates for 48 hours. Cell proliferation was measured by Cell Titer Glo (Promega) at 48 hours posttreatment. The Cell Titer Glo assay was conducted according to the manufacturer's instructions.

| Tumor spheroid generation and drug treatment
Cell lines were evaluated using several EZH2 inhibitors, GSK126, EPZ6438, and JQEZ5, but these agents require an extended time period to be effectively evaluated that is not compatible with 2D in vitro assays in these fast-growing cell lines, so 3D spheroid viability assays were utilized. 28 Tumor spheroids were seeded into Ultra-Low Attachment (ULA) 96 well plates (Corning 4530, Kennebunk, ME, Nexcelom ULA-96U-20, Lawrence, MA) at spheroid forming density. The cells were incubated at 37 C, 5% CO 2 until formation was confirmed by eye under a microscope. UOK353 did not produce usably spheroids, only loose aggregates, and where not analyzed.
However, UOK360 cells produced tight spheroids and these were evaluated with the positive control SMARCB1-deficient MRT cell line G401.
After spheroids had been determined to have formed, drugs were added at ×2 concentration to reach ×1. All drugs were incubated for 48 hours and evaluated using Cell Titer Glo3D. For GSK126, every 3-4 days 100 μL of the 200 μL of media in each well was removed and replaced with 100 μL fresh media containing ×2 the desired drug concentration. Cell Titer Glo 3D assay was conducted according to the manufacturer's instructions.

| Combination index calculation
Samples were treated with equimolar concentration of the indicated compounds and viability as determined by Cell Titer Glo was utilized as the endpoint. The combination index calculation was performed using CompuSyn software (ComboSyn, Inc., Paramus, NJ).

| Invasion assay
Invasion assays were performed using the xCELLigence system as previously described 27   ( Figure 2C). In addition, UOK360 was revealed to have a heterozygous missense (c.542G>C) mutation in TP53, resulting in an altered protein (p.R181H) at a known cancer mutation hotspot ( Figure 2D).   Figure 3A,B). However, the mRNA and protein expression for other SWI/SNF subunits, such as PBRM1 and SMARCA4/BRG1, were retained ( Figure 3A,B). Conversely, the expression of several PRC2 subunits, EZH2, SUZ12, and EED, were upregulated at both the mRNA and protein levels in both cell lines in comparison to RPTEC cells ( Figure 3A,B). Due to the similar loss of SMARCB1 and upregulation of EZH2 shared between MRTs and RMCs, the EZH2 inhibitors GSK126, EPZ6438, and JQEZ5 were investigated as potential targeted therapies. EZH2 inhibition requires an extended time period to be effectively evaluated, 28 Figure 3D). While the GSK126 EZH2 inhibitor had a limited effect on the UOK360 cells, it significantly reduced viability of the G401 cells (<10% of untreated cells) at all drug concentrations suggesting variability in response to EZH2 inhibitor between MRT and RMC cells that highlights the importance of evaluating representative cell line models ( Figure S2).

| High-throughput drug screening of UOK353 and UOK360
In collaboration with the National Center for Advancing Translational   Figure 5E). 30 In the UOK353 cells the combination increased the degree of cell death achievable and in UOK360 lowered the IC50 to 5.49 nM compared to 7.07 nM with bortezomib alone. Cisplatin was also able to increase ROS as a single agent and the equimolar combination of bortezomib and cisplatin increased the ROS levels more than the same concentration of bortezomib alone ( Figure S4). an improved suppression of the tumor growth rate compared to single agent treatment, but tumors were not eliminated ( Figure 6B). A repeat study for the combination was performed using a larger number of mice (n = 13) injected with fewer cells (~1 million) and treated by IP with either 1 mg/kg bortezomib and 5 mg/kg cisplatin or 0.9% saline twice weekly for 6 weeks beginning 1 week after initial injection. This demonstrated the same trend that the combination reduced the growth rate of UOK360 xenografts but did not eliminate the tumors ( Figure S5).  was seen in both cell lines, suggesting a common mechanism for down regulation in RMC and a potential cause for sensitivity to ROS-induction.

| DISCUSSION
Although the frequency of NQO1 loss in RMC tumors is currently unknown, it could provide a biomarker for specific sensitivity to treatment with ROS inducing agents.
This study also identified a response in these cell line models to panobinostat, an HDAC inhibitor, that could affect the gene expression dysregulation resulting from loss of SMARCB1/SNF5. Panobinostat inhibited proliferation and may represent a potential component of a combination therapy rather than a single agent treatment but highlights the potential for using preclinical models for drug discovery.
In summary, UOK353 and UOK360 represent two novel human cell line models for RMC and the first in vivo mouse xenograft models of RMC. These models will provide an invaluable tool for research and preclinical drug testing. Initial analysis of these models confirmed the potential for combination therapy of bortezomib and cisplatin in RMC and highlighted other potential therapeutic options for patients with advanced renal medullary carcinoma.

ACKNOWLEDGMENTS
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