Cancer Treatment Reviews 41 (2015) 170–178

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Cancer Treatment Reviews journal homepage: www.elsevierhealth.com/journals/ctrv

New Drugs

Emerging therapeutic targets in bladder cancer Benedito A. Carneiro a,c,d,⇑, Joshua J. Meeks b,d, Timothy M. Kuzel c,d, Mariana Scaranti e, Sarki A. Abdulkadir b,d, Francis J. Giles a,c,d a

Northwestern Medicine Developmental Therapeutics Institute, Feinberg School of Medicine, Northwestern University, United States Department of Urology, Feinberg School of Medicine, Northwestern University, United States c Division of Hematology and Oncology, Feinberg School of Medicine, Northwestern University, United States d Robert H. Lurie Comprehensive Cancer Center of Northwestern University, United States e Instituto do Câncer do Estado de São Paulo, Universidade de São Paulo, Brazil b

a r t i c l e

i n f o

Article history: Received 26 September 2014 Received in revised form 14 November 2014 Accepted 15 November 2014

Keywords: Bladder cancer Targeted therapy Urothelial bladder carcinoma Treatment Immunotherapy

a b s t r a c t Treatment of muscle invasive urothelial bladder carcinoma (BCa) remains a major challenge. Comprehensive genomic profiling of tumors and identification of driver mutations may reveal new therapeutic targets. This manuscript discusses relevant molecular drivers of the malignant phenotype and agents with therapeutic potential in BCa. Small molecule pan-FGFR inhibitors have shown encouraging efficacy and safety results especially among patients with activating FGFR mutations or translocations. mTOR inhibitors for patients with TSC1 mutations and concomitant targeting of PI3K and MEK represent strategies to block PI3K/AKT/mTOR pathway. Encouraging preclinical results with ado-trastuzumab emtansine (T-DM1) exemplifies a new potential treatment for HER2-positive BCa along with innovative bispecific antibodies. Inhibitors of cell cycle regulators (aurora kinase, polo-like kinase 1, and cyclin-dependent kinase 4) are being investigated in combination with chemotherapy. Early results of clinical studies with anti-CTLA4 and anti-PDL1 are propelling immune modulating drugs to the forefront of emerging treatments for BCa. Collectively, these novel therapeutic targets and treatment strategies hold promise to improve the outcome of patients afflicted with this malignancy. Ó 2014 Elsevier Ltd. All rights reserved.

Introduction Urothelial bladder carcinoma (BCa) is a major global health challenge with an estimated 429,000 new cases resulting in 165,000 deaths annually [1]. In the United States, approximately 74,000 new cases will be diagnosed, and nearly 16,000 patients will die from bladder cancer in 2014 [2]. In males, BCa represents the fourth most frequent diagnosis of new cancers annually. Over the past two decades, there has been no significant improvement in survival of BCa with 5-year relative survival rates for locally advanced and metastatic disease of 33% and 5%, respectively [2]. The majority of bladder cancers are composed of urothelial carcinoma (90%) with the remaining less common subtypes including squamous cell carcinoma, adenocarcinoma, and small cell carcinoma. Seventy percent of the cases are diagnosed as non-muscle-invasive bladder cancer (NMIBC) with a favorable prognosis following transurethral resection and intravesical ⇑ Corresponding author at: Northwestern Medicine Developmental Therapeutics Institute, 645 N Michigan Ave., Suite 1006, Chicago, IL 60611, United States. Tel.: +1 312 926 3892; fax: +1 312 695 0370. E-mail address: [email protected] (B.A. Carneiro). http://dx.doi.org/10.1016/j.ctrv.2014.11.003 0305-7372/Ó 2014 Elsevier Ltd. All rights reserved.

chemotherapy or immunotherapy with Bacillus Calmette-Guérin (BCG) [3]. Nevertheless, approximately 40% of these patients will progress to muscle-invasive disease at five years depending on tumor pathological features [4]. When considering the prognosis of muscle-invasive bladder cancer, even after optimal treatment with neoadjuvant chemotherapy and surgery, only 60% of these patients will be alive 5 years later due to distant recurrence [5]. This aggressive biological behavior coupled with limited therapeutic options results in a median survival of 15 months for patients with metastatic disease [6]. Therefore, there is an urgent need to improve outcomes with innovation or application of new treatments. Advances in the understanding of the pathophysiology provided by comprehensive genomic profiling of BCa may drastically improve the outcomes for this malignancy [7]. The Cancer Genome Atlas (TCGA) uncovered recurring alterations in 32 genes and three distinct molecular subtypes of BCa providing the foundation for further investigation and drug development [7]. Prominent pathways altered in a significant fraction of BCa include the PI3K/ AKT/mTOR pathway, the MAPK pathway, and chromatin regulatory genes. Novel therapeutic approaches with encouraging pre-clinical results and those undergoing early phase clinical development for

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muscle-invasive bladder carcinoma are discussed on this manuscript. The selection of therapeutic targets was based on the relevance of the pathways to emerging new treatments, and they were organized in the following groups: signal transduction pathway inhibitors, cell cycle regulation, heat shock proteins, and immunotherapy.

Signal transduction pathway inhibitors Fibroblast growth factor signaling Fibroblast growth factors (FGF), a family comprised of eighteen growth factors and four FGF-homologous factors, play an important role in several cellular processes including development, wound-healing, proliferation and angiogenesis [8]. These growth factors signal through four transmembrane glycoprotein receptors (FGFR1–4) that share a general structure of three extracellular immunoglobulin (Ig) domains, a transmembrane domain and an intracellular tyrosine-kinase domain. Ligand binding leads to receptor dimerization, phosphorylation of the cytoplasmic tyrosine kinase domain and activation of phospholipase C c1 (PLCc1), FGFR substrate 2 (FRS2), Ras/Raf/Mek/Erk, Jnk/Mapk, PI3K and STAT3 pathways [9]. FGF-1 and FGF-2 can also be internalized through receptor-mediated endocytosis and exert direct biological functions in the cytoplasm and nucleus [10]. The signaling complexity of the FGF/FGFR system relates to FGFs affinity to various FGFRs, isoforms of FGFRs with distinct ligand specificity and modulation of FGF by the sprouty family of proteins and FGF-binding proteins [11,12]. Another FGFR receptor has been described (FGFR5 or FGFR-like 1), but it does not have intrinsic tyrosine kinase activity and it might function as a decoy receptor inhibiting canonical FGFR signaling [13]. FGFRs gain-of-function mutations have a pivotal role on syndromes causing craniosynostosis (premature fusion of skull bones; Pfeiffer’s and Apert’s syndromes), Kallman’s syndrome, achondroplasia (most common genetic form of dwarfism associated with FGFR3 mutations), and they have been documented in several malignancies including glioblastoma, multiple myeloma, head and neck, prostate, breast and bladder cancers [14–22]. The high frequency of FGFR genetic alterations encountered in urothelial carcinomas fostered significant interest in this pathway as a potential therapeutic target [22,23]. Results from TCGA documented multiple genetic lesions resulting in FGFR3 activation in 17% of muscle-invasive BCa [7]. These mutations are commonly located in exons 7, 10 and 15 encoding the extracellular domain of the receptor causing ligand-independent receptor activation [24]. Next-generation sequencing analysis of 126 cases of urothelial cancers revealed FGFR aberrations in 33% of the tumors [25]. FGFR3 was the most common receptor altered with activating mutations, amplification and fusions (FGFR3-TACC3) detected in 16%, 10% and 3% of the cases, respectively. In addition, overexpression of FGFR3 can be present in 40% of tumors without FGFR mutations [26]. A number of preclinical studies involving both bladder cancer cells lines and xenograft models of bladder cancer demonstrated the anti-proliferative activity of FGFR inhibitors and their ability to block FGFR-mediated signaling pathways [27–29]. Collectively, these results highlighted the potentially pivotal role of FGFR in urothelial carcinogenesis. Small molecule inhibitors of FGFR 1, 2, 3 and 4 have shown encouraging results in bladder cancer. A phase I trial testing the oral pan-FGFR inhibitor JNJ-42756493 demonstrated a favorable toxicity profile with early signals of efficacy [30]. Thirty-seven patients with advanced solid tumors irrespective of their FGFR mutational status were treated during the dose escalation phase. Most of the adverse effects (AEs) were mild to moderate and

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included hyperphosphatemia (60%), asthenia (46%), dry mouth (30%), vomiting (22%) and constipation (27%). One patient developed grade 3 AST/ALT (aspartate aminotransferase/alanine aminotransferase) elevation classified as a dose-limiting toxicity. The drug also caused a dose-dependent increase in calcium, fibroblast growth factor 23 (FGF23), and phosphate coupled with a decrease in parathyroid hormone (PTH) that support the FGF/FGFR role in bone metabolism [31]. One patient with metastatic bladder cancer carrying the FGFR3-TACC3 translocation had a partial response, and another patient with renal pelvis tumor with FGFR2 truncation had a near complete response. Four additional patients achieved stable disease (breast cancer, lung cancer, and chondrosarcoma). Enrollment to the expansion cohort is ongoing (NCT01962532). Another phase I study tested the pan-FGFR inhibitor BGJ398 specifically in patients with solid tumors carrying FGFR genetic alterations [32]. Dose-limiting toxicities observed among the ninety-four patients enrolled included grade 3 elevation of aminotransferases, grade 3 hyperphosphatemia, and grade 1 corneal toxicity. Other milder adverse effects were fatigue, decreased appetite, alopecia, and stomatitis. Frequent hyperphosphatemia (occurring in up to 78% of patients) was controlled with diet and phosphate-binding agents. Of note, four out of five patients with FGFR3-mutated urothelial carcinomas had tumor responses. Clinical activity was also observed in small cell lung cancer, breast cancer, and cholangiocarcinoma cases. An expansion cohort in bladder cancer with this compound is ongoing (NCT01004224). A third FGFR inhibitor (LY2874455) was evaluated in 36 patients with advanced solid tumors including 17 Asian patients [33]. Adverse effects (AEs) reported were gastrointestinal toxicity, hyperphosphatemia, and thrombosis. Dose-limiting toxicities were not observed, and dose expansion cohort is ongoing. Efficacy results have not been reported (NCT01212107). In contrast to the encouraging results with specific FGFR inhibitors, multi-targeted tyrosine kinase inhibitors (TKIs) have not shown significant efficacy in BCa. Dovitinib, a TKI against VEFG and FGFR, was evaluated in 44 patients with advanced urothelial carcinomas without tumor responses even among patients with FGFR3 mutations [34]. The reasons for lack of activity is unclear, but may be related to non-selective pharmacological properties and potency of this compound. Nevertheless, further development of FGFR inhibitors might lead to effective strategies to treat BCa as single agents or in combination with chemotherapy.

Phosphatidylinossitol 3-kinase/v-akt murine thymoma viral oncogene homolog 1/mammalian target of rapamycin (PI3K/AKT/mTOR) The PI3K/AKT/mTOR pathway is involved in cancer cell survival, motility and metabolism. Forty percent of urothelial carcinomas have genetic alterations of the PI3K/AKT/mTOR pathway. The spectrum of mutations include activating mutations of PIK3, AKT1 or inactivating deletions of critical regulators such as PTEN, TSC1 (tuberous sclerosis complex 1) and TSC2 [35–38]. Thus, multiple drugs have been designed to target this pathway (e.g. PI3K inhibitors, mTOR inhibitors, PI3K/mTOR dual inhibitors, AKT inhibitors, and PDK1 inhibitors). Several compounds targeting individual or all isoforms of class I PI3K (i.e. p110a, p110b, p110c) are in clinical development. The pan-PI3K inhibitor buparlisib (BKM-120) has been investigated in advanced solid tumors with partial responses observed in breast cancer and epithelioid hemangiothelioma [39]. Most frequent grade 3 and 4 AEs included rash, hyperglycemia, and increased transaminases. Based on these results and preclinical evidence of anti-proliferative effect in bladder cancer cells with PI3K blockade [40], buparlisib is being investigated as a second-line treatment for patients with metastatic urothelial carcinoma (NCT01551030).

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Given the similar structures of PI3K p110 subunit and mTOR, some molecules are able to block both components (dual PI3K/ mTOR inhibitors such as NVP-BEZ235) and can avoid the feedback activation of PI3K caused by mTOR inhibition [41]. NVP-BEZ235 showed significant anti-tumor effect on cisplatin-resistant bladder cancer cells lines, but it caused MEK/ERK pathway activation [42]. Since 10–15% of advanced bladder cancers display mutant RAS leading to downstream MEK activation, a combination of a dual PI3K/mTOR inhibitor (PF-04691502) with a MEK inhibitor (PD0325901) was tested in patient-derived xenograft models of bladder cancer with marked reduction of tumor growth [43]. This strategy targeting multiple pathways can decrease the emergence of resistance, a limitation with isolated PI3K blockade. In fact, the combination of PI3K (BYL719) with MEK (MEK162) inhibitors is being investigated in RAS- or BRAF-mutant ovarian and endometrial cancers, and preliminary tumor responses have been observed [44]. This approach is also relevant for bladder cancer given the high frequency of RAS mutations in these tumors. Several AKT inhibitors such as perifosine have been developed but no significant efficacy has been observed in a phase I trial that included cases of BCa [45,46]. However, knowing that HER2 activates downstream AKT, the AKT inhibitor AZD5363 was tested in HER2 positive breast cancer and showed significant activity [47]. These findings raise the possibility that this class of drugs can be active in HER2 positive bladder cancers, particularly in light of the TGCA results showing the involvement of HER2 in disease pathogenesis [7]. Approved and novel mTOR inhibitors represent an area of active investigation in BCa as well. A single arm phase II study evaluated the mTOR inhibitor everolimus in 45 patients with advanced urothelial carcinoma in second-line treatment. This trial did not meet the primary endpoint of PFS at 2 months P70% and significant responses were seen in only 3 patients [48]. However, one patient achieved a durable complete response lasting greater than 26 months. Interestingly, next-generation sequencing of the tumor from this patient identified a loss-of-function mutations in TSC1 and NF2 (neurofibromatosis type 2) genes suggesting that both alterations were associated with everolimus sensitivity [49]. While NF2 mutations were not identified in additional 96 cases of BCa, TSC1 mutations and loss of heterozygosity have been detected in up to 16% and 57% of bladder cancers, respectively [35]. Thus,

TSC1 genetic alterations might represent a predictive marker in this disease and epitomize the potential of targeted therapies when used in appropriately selected patients. Another trial with everolimus in a similar patient population showed a disease control rate (stable disease plus partial response) of 27% among 37 patients [50]. Limited efficacy was observed with single-agent temsirolimus [51], but results from the combination of gemcitabine/cisplatin and temsirolimus or everolimus in first-line treatment of advanced disease are pending (NCT01090466; NCT01182168). Inhibitors of both mTORC1 and mTORC2 complexes (dual TORC inhibitors) are also being investigated and preclinical studies have shown synergistic anti-tumor effects with lapatinib (see below) in bladder cancer cells (NCT01058707) [52] (Table 1). Epidermal growth factor receptor 2 (HER2)/ERBB2 Several mutations in the extracellular and tyrosine kinases domains of HER2, receptor overexpression and gene amplifications have been described in urothelial cancers. The TCGA data set detected amplification or mutations in 9% of bladder cancer specimens [7]. Other reports also showed HER2 amplifications in 9% of primary bladder tumors associated with higher frequency in matched lymph node metastases (15%) [53]. Of interest, activating HER2 mutations have also been identified in the absence of gene amplification in both breast and bladder cancers [54,55]. Some of these mutations predicted response via irreversible inhibition with the TKI neratinib and/or resistance to lapatinib (dual EGFR/HER2 TKI) [54]. While most of the HER2 mutations described in other diseases were associated with the tyrosine kinase domain, activating mutations in the HER2 extracellular domain was described in 40% of micropapillary urothelial carcinoma, a subtype associated with worse outcomes and high risk for early metastatic disease [55]. Despite conflicting results regarding the prognostic relevance of HER2 expression, HER2 RNA levels correlated with inferior recurrence-free survival among patients treated with cystectomy [56,57]. Taken together, these results reinforce the role of this pathway in bladder carcinogenesis, and highlight the importance of comprehensive genetic analysis of HER2 pathway beyond protein expression and gene amplification. Identification of HER2 mutations or translocations involving HER2 might ultimately identify bladder cancer subtypes that rely more heavily on this

Table 1 Emerging therapies for muscle-invasive bladder carcinoma. Target

Stage of clinical development

Clinical trials.gov Identifier

Signal transduction pathway inhibitors FGFR JNJ-42756493 BGJ398 LY2874455 Dovitinib PI3K Buparlisib PI3K/mTOR NVP-BEZ235 PF-04691502 mTORC1/mTORC2 MLN0128 HER2 Ado-trastuzumab emtansine MM-111 Afatinib

Agent(s)

Phase I Phase I Phase I Phase II Phase II Preclinical Preclinical Phase I Preclinical Phase I Phase II

NCT01962532 NCT01004224 NCT01212107 Completed NCT01551030

Cell cycle regulation Aurora kinase A Polo-like kinase I

Alisertib (MLN8237) Volasertib (BI6727)

Phase II Phase II

NCT02109328 Completed

Heat shock proteins HSP27 HSP90

OGX-427 SNX-5422

Phase II Phase I

NCT01454089; NCT01780545 NCT01848756

Immunotherapy PD-1 CTLA4 IL-2/p53

MPDL3280A Ipilimumab ALT801

Phase II Phase II Phase II

NCT02108652 NCT01524991 NCT01326871

NCT01058707 NCT01304784 NCT02122172

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pathway and might be responsive to the expanding armamentarium of anti-HER2 agents (e.g. trastuzumab, lapatinib, adotrastuzumab emtansine, and pertuzumab). In a phase II study, patients with metastatic urothelial carcinoma with over-expression or amplification of HER2 were treated with the first-line combination of trastuzumab (antibody against the extracellular domain IV), paclitaxel, carboplatin and gemcitabine. These patients achieved a remarkable response rate of 70% (31 out of 44 patients; 5 complete responses and 26 partial responses), and a median time to progression and overall survival of 9.3 and 14.1 months, respectively [58]. Lapatinib, an oral EGFR and HER2 TKI, has been investigated as second-line treatment for advanced disease with limited responses among this pre-treated patient population that included tumors without or with only 1 + HER2 expression [59]. Nevertheless, novel strategies targeting HER2 hold promise to improve the outcomes of HER2 positive bladder cancer. Ado-trastuzumab emtansine (T-DM1), an antibody-drug conjugate composed of trastuzumab linked to the cytotoxic agent mertansine approved for the HER2 positive metastatic breast cancer progressing after trastuzumab, has shown encouraging preliminary results. This compound allows delivery of a potent cytotoxic agent specific to HER2 expressing tumor cells. Preclinical studies of T-DM1 with bladder cancer cell lines and xenograft models showed significant anti-tumor effects including among cisplatin-resistant cells [60]. Another innovative anti-HER2 strategy utilizing a bispecific HER2/HER3 antibody fusion protein (MM-111) showed promising safety and early efficacy signals. A phase I study enrolled 86 subjects with advanced HER2 positive solid tumors including 11 bladder cancer cases that were treated with MM-111 plus chemotherapy regimens containing trastuzumab +/ lapatinib [61]. Seventeen patients (20%) achieved a partial response that included 2 patients with bladder cancer, 29 patients had stable disease (34%), and one patient with gastric cancer had a complete response. Adverse events included anemia, neutropenia, stomatitis, nausea, and vomiting. Afatinib, a TKI with specificity against EGFR, HER2 and HER4, is also being evaluated in an ongoing phase II trial for patients with advanced urothelial cancers (NCT02122172). Lastly, an autologous cellular immunotherapy consisting of antigen presenting cells cultured with recombinant HER2-derived antigen (DN24-02) is being tested as a potential adjuvant treatment for patients with high risk HER2 positive urothelial cancer, and preliminary results have shown significant immune responses and a favorable toxicity profile (NCT01353222) [62]. The success of anti-HER2 strategies in bladder cancer will rely upon clear identification of tumor subtypes appropriate for HER2 targeting. These might include specimens with HER2 overexpression or amplification and those with activating HER2 mutations.

Cell cycle regulation Aurora kinases The family of serine/threonine aurora kinases (human homologs A, B and C) plays an important role in cell cycle progression through regulation of the mitotic spindle. Disruption of aurora kinases results in aneuploidy, genomic instability, and increased expression of aurora kinase A leads to transformation of fibroblasts [63–65]. Overexpression of aurora kinases A and B have also been associated with pathogenesis of several malignancies such as breast cancer, gliomas, and colon cancer [64,66,67]. Furthermore, higher tumor grade and stage of urothelial carcinoma correlates with expression of aurora kinase A and B [68,69]. These findings have fostered the development of aurora kinase inhibitors as

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targeted therapies for solid tumors and hematologic malignancies [70]. Comparative genomic analysis of normal bladder epithelium and muscle-invasive bladder carcinoma showed a relevant disruption of mitotic spindle checkpoint genes in human BCa specimens [71]. Relative to other mitotic spindle genes, aurora kinase A and B were overexpressed in bladder cancer cell lines and prompted testing of aurora kinase A inhibitor MLN8237 (alisertib) in vitro and in vivo. MLN8237 caused reduction in phosphorylation of aurora kinase A, cell cycle arrest, aneuploidy, and apoptosis of bladder cancer cell lines [71]. Based on distinct IC50 values, carcinoma cells were significantly more sensitive to aurora kinase A inhibition compared to normal bladder epithelium (IC50: 30 nmol/L vs. 119 nmol/L). MLN8237 also caused marked reduction of tumor growth in a mouse xenograft model of bladder cancer. Furthermore, sequential treatment of malignant cells with MLN8237 followed by paclitaxel or gemcitabine resulted in synergistic cytotoxic effects. In contrast, simultaneous administration of MLN8237 with each of these agents showed antagonistic effect. This important schedule-dependent synergism suggests that disruption of mitotic spindle machinery can make bladder cancer cells more sensitive to paclitaxel or gemcitabine. Taken together, these results implicate the aurora kinases as potentially relevant and novel therapeutic targets in bladder cancer fueling development of clinical trials. A recently registered clinical trial will evaluate the combination of the orally bioavailable agent alisertib with paclitaxel as a second-line regiment for advanced bladder cancer (NCT02109328). The results of this study will be extremely relevant to the future development of this promising class of drugs in urothelial malignancies. Special attention will be given to the frequency of somnolence and elevation of liver enzymes since they represent doselimiting toxicities of first-generation aurora kinase A inhibitors [72,73]. Other aurora kinases inhibitors such as danusertib and AMG900 are undergoing clinical development for treatment of solid tumors, leukemia, lymphoma, and multiple myeloma (NCT01380756; NCT01034553; NCT01567709). Polo-like kinase 1 The family of polo-like kinases (PLK 1–5) are involved in the regulation of cell cycle progression and mitosis in tandem with aurora kinases. PLKs are overexpressed in several malignancies including bladder cancer [74–77]. Specifically, PLK1 regulates the mitotic spindle through centrosome maturation and degradation of cohesion [78,79]. PLK1 overexpression was demonstrated in 43% of urothelial carcinomas, whereas no significant expression was seen in normal bladder mucosa [77]. Further, bladder cancers overexpressing PLK1 displayed significant chromosomal instability, DNA aneuploidy, centrosome amplification, higher pathological grade, risk for metastasis, and recurrence [77,78]. Promising bladder tumor regressions were seen in preclinical models using intravesical anti-PLK1 strategies, and a potent inhibitor of PLK 1, 2 and 3 (BI 6727; volasertib) showed significant antitumor effect in xenograft models [76,80]. These results supported the clinical developed of volasertib. The first in human phase I study of volasertib enrolled 65 patients and showed preliminary antitumor activity [81]. Among three confirmed partial responses, there was one patient with advanced urothelial carcinoma who achieved a prolonged progression free survival of 403 days. Another phase I study of volasertib in Asian patients also showed one partial response in a patient with metastatic ureteral urothelial carcinoma [82]. A phase II study evaluated the efficacy and safety of single-agent volasertib as second-line treatment for advanced urothelial cancers, a patient population without standard therapeutic options [83]. The treatment

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was well tolerated, but volasertib showed limited efficacy with overall response rate of 14% and median progression free survival of 1.4 months. While these results reduced the enthusiasm with volasertib for treatment of bladder cancer, a phase I trial demonstrated the safety and possible enhancement of efficacy when volasertib was combined with cisplatin or carboplatin in the treatment of solid tumors suggesting a possible role for this drug in combination with chemotherapy protocols used for advanced bladder cancer [84]. Moreover, recent results suggest that acquired resistance to PLK1 inhibitors (BI 2536, volasertib and GSK641364) is mediated by ATP-binding cassette (ABC) transporters including ABCG2 that can be overexpressed in bladder cancer and correlate with recurrence [85,86]. PLK1 is also being targeted through a lipid nanoparticle formulation of a small interfering RNA directed against PLK1 (TKM 080301) for advanced solid tumors and lymphoma with preliminary efficacy results in patients with colon cancer and carcinoid [87]. An ongoing phase I study is evaluating the compound in neuroendocrine tumors and adrenocortical carcinoma (NCT01262235), and other PLK1 inhibitors have shown promising preclinical activity [88]. Other preclinical studies based on the principle of synthetic lethality have identified cancer genes whose expression sensitizes tumors to the effects of PLK1 inhibitors [89].

Cyclin-dependent kinase 4 (CDK4)/cyclin D1/retinoblastoma pathway Cell cycle progression is tightly regulated by cyclin-dependent kinases and cyclins at the checkpoints between the 4 phases of cell cycle (G1, S phase, G2, and mitosis). The transition between G1 and S phase is controlled by CDK4 that forms a complex with cyclin D1 leading to phosphorylation and inactivation of retinoblastoma (Rb) protein. In turn, phosphorylation of Rb results in activation of the transcription factor E2F that plays a critical role on cell cycle progression [90]. Another important regulator of this CDK4/cyclin D1/Rb pathway is p16. This tumor suppressor encoded by the CDKN2A gene directly inhibits CDK4. This pathway is frequently altered in several malignancies by overexpression of Cyclin D1, amplification or activating mutations of CDK4, and loss of p16 by CDKN2A gene deletions or promoter hypermethylation [91–93]. Thus, significant efforts have been directed to develop novel therapies targeting this pathway with CDK4 inhibitors to restore the cell cycle control machinery. Several disruptions of this pathway have been demonstrated in bladder cancer. Cyclin D1 mRNA and protein expression are increased in muscle-invasive bladder cancer compared to normal bladder tissue, and these findings correlate with the risk for lymph node metastasis [93,94]. Expression of cyclin D1 is also associated with higher risk of progression from non-muscle-invasive disease to muscle-invasive cancer according to a multicenter cohort of 859 cases of Ta/T1 urothelial carcinomas [95]. Homozygous deletions of the CDKN2A gene causing loss of p16 and p14 (tumor suppressor involved with stabilization of p53) have also been detected in 10–35% of bladder cancers [96,97]. Notably, the frequency of CDKN2A homozygous deletions was higher among tumors with FGFR3 mutations compared to wild type tumors (28% vs. 13%) [98]. While there was no association between CDKN2A deletions and tumor stage in general, there was a significant correlation when considering only tumors with FGFR3 mutations. Further, non-muscle-invasive tumors carrying FGFR3 mutations and CDKN2A deletions showed a greater risk of progression to muscle-invasive disease compared to tumors without CDKN2A deletions. These findings suggest an important biological role for CDKN2A in the progression of tumors with FGFR3 mutations and/ or a critical cooperation between these two pathways in bladder cancer carcinogenesis.

Recent characterization of molecular subtypes of bladder cancer enhances the clinical relevance of these results and highlights possible new treatments. Whole-genome expression profiling of muscle-invasive bladder cancer identified three subgroups: basal, luminal and ‘‘p53-like’’ [99]. Basal tumors were enriched with sarcomatoid and squamous histologies, displayed features of undifferentiated cells and mesenchymal markers, and were associated with shorter survival. Similar to breast cancer luminal subtype, bladder cancer luminal tumors expressed epithelial biomarkers (i.e. CD24, FOXA1, GATA3, HER2) and high levels of FGFR3 coupled with frequent activating FGFR3 mutations. The p53-like group shared some of the epithelial markers with luminal subtype, but displayed a wild-type p53 gene signature. Therefore, it is conceivable that CDK4/cyclin D1/Rb pathway plays an important role in the pathogenesis of luminal bladder cancers that frequently harbor FGFR3 mutations. Considering the common pathways driving luminal subtypes of bladder and breast cancers and the remarkable results with the novel CDK4/6 inhibitor palbociclib in metastatic breast cancer [100], CDK4/6 inhibitors may yet emerge as an attractive class of drugs to be tested in bladder cancer. Although there is a soon to be opened trial with the CDK4/6 inhibitor LEE011 for solid tumors and hematological malignancies with disruption on this pathway (i.e. CDK4 or CDK6 amplification or mutation, Cyclin D1 amplification, Cyclin D3 amplification, or p16 mutation) (NCT02187783), there are no ongoing clinical trials in bladder cancer with CDK4/6 inhibitors to our knowledge.

Heat shock proteins Heat shock proteins (HSPs) comprise a family of chaperone proteins mediating several carcinogenesis processes through regulation of protein folding and degradation. Overexpression of HSPs is intrinsically involved with chemotherapy and radiotherapy resistance and thus, has been pursued as a therapeutic target. Among the several HSPs, the importance of HSP27 and HSP90 in bladder cancer was shown by their increased expression in tumors compared to normal epithelium, and inhibitory growth effects of HSP blockade [101–103]. Furthermore, pathway analysis of the TCGA bladder data set identified HSP90 as a relevant signaling hub [7]. Preclinical studies with the antisense oligonucleotide targeting HSP27 (OGX-427) showed significant growth inhibition of bladder cancer cell lines and orthotopic tumors [103]. OGX-427 also enhanced the sensitivity of cell lines to chemotherapy drugs such as gemcitabine, cisplatin, and paclitaxel [104,105]. Based on these findings, a phase I trial of intravesical OGX-427 was performed that revealed a favorable toxicity profile with a 38% rate of complete responses [106]. A phase II study is testing OGX-427 plus gemcitabine and cisplatin for first-line treatment of advanced disease, and another trial is combining OGX-427 with docetaxel for relapsed metastatic bladder cancer (NCT01454089; NCT01780545). Treatment of bladder cancer cells with the HSP90 first-generation ansamycin inhibitor 17-allylamino-17-demethoxygeldanamycin (17-AAG) induced apoptosis, cell cycle arrest, and sensitized cells to chemoradiotherapy [107,108]. Another HSP90 inhibitor, 17-(dimethylaminoethylamino)-17-demethoxygeldanamycin (17DMAG), also demonstrated preclinical activity in bladder cancer and potentiated the effects of cisplatin [109]. Moreover, the resorcinol-based second generation HSP90 inhibitor ganetespib showed potent cytotoxicity of bladder cancer cell lines carrying FGFR3TACC3 and FGFR3-BAI1AP2L1 translocations with efficacy comparable to pan-FGFR inhibitors (e.g. BGJ398) in vitro and in vivo [110]. Further, ganetespib was also effective against cells with FGFR

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mutations that confer resistance to BGJ398 suggesting an alternative inhibitory strategy potentially very useful given the frequent development of resistance to TKIs. These results also suggest a potential role of combination of anti-FGFR small molecule inhibitors with HSP90 blockers. Among the several HSP90 inhibitors in clinical development, SNX-5422 is being tested in a phase I trial for HER2 positive tumors that include breast, esophagogastric, lung, and bladder cancers (NCT01848756).

Immunotherapy Checkpoint PD-1 pathway Several lines of evidence documenting the immune dysfunction associated with bladder cancer support the hypothesis that immunotherapy can alter the process of carcinogenesis. Earlier studies investigating the immunological profile of patients with bladder cancer showed significant impairment of lymphocyte function and a predominance of T-regulatory cells and Th1 inhibitory cytokines in the tumor micro-environment, and some of the immune dysfunction was reversed after cystectomy [111,112]. The number of tumor-infiltrating CD8+ T lymphocytes correlates with bladder cancer-specific survival, and further supports the important role of immune surveillance in this disease. One of the critical mediators of immune response is the B7 family of immune co-regulatory proteins (B7-H1 or PD-L1, B7-H3, PD1), which may be involved in the immune evasion by urothelial cancer [113]. The B7 homolog 3 (B7-H3) displays higher expression in bladder cancer cells compared to normal urothelium (70% vs. 20%) irrespective of tumor stage suggesting its involvement in early stages of carcinogenesis [114]. PD-L1 is expressed by 12% of bladder tumor cells, 27% of tumor infiltrating immune cells, and in up to 50% of malignant urothelial cells in carcinoma in situ [115,116]. In addition, 95% of lymphocytes that invade bladder tumors express the PD-1 receptor. Urothelial expression of PD-L1 was also predictive of mortality following cystectomy in patients with organ-limited disease. These results have identified the PDL1/PD-1 pathway as an attractive therapeutic target for the treatment of bladder cancer similar to the findings in other epithelial tumors such as renal cell cancer, lung cancer, and melanoma [117]. The relevance of PD-1 blockade in urothelial cancer was recently confirmed by a phase I study evaluating a human monoclonal antibody directed against PD-L1 (MPDL3280A) which included sixty-seven patients with metastatic bladder cancer [115]. The treatment was well tolerated among this pre-treated patient population (79% of patients had received cisplatin and 34% had received carboplatin; 48% had undergone cystectomy) including patients with renal insufficiency. There were no treatment related grade 4 or 5 adverse events or immune-related toxicities. Grade 3–4 adverse events occurred in three patients and included asthenia, thrombocytopenia and hypophosphatemia. Other frequent grade 1 and 2 adverse events were decreased appetite, fatigue, and nausea. An overall response rate of 52% was documented for patients with PD-L1 positive tumor-infiltrating immune cells with P12 weeks of follow up and it included two complete responses. There was also a higher response rate among patients with PD-L1 positive tumor-infiltrating immune cells compared to PD-L1 negative specimens (43% vs. 11%). Median time to first response was 42 days and median duration of response had not been reached at the presentation of the results [115]. Taken together, these results validated the checkpoint PD-1 pathway as a promising therapeutic target in bladder cancer. An ongoing phase II trial is testing MPDL3280A as first-line treatment for advanced bladder cancer in patients not candidates for cisplatin-containing regimens or as a second-line option (NCT02108652).

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Anti-cytotoxic T lymphocyte associated antigen (Anti-CTLA-4) Blockade of CTLA-4 is also under active investigation as another immunotherapy strategy in urothelial cancers. CTLA-4 has an important role in the tumor cell mediated immunosuppression, and its blockade with the anti-CTLA-4 monoclonal antibody ipilimumab enhances T lymphocyte function resulting in meaningful tumor responses in melanoma, renal cell carcinoma, and non-small cell lung cancer [117,118]. Treatment of patients with localized bladder cancer prior to cystectomy with ipilimumab demonstrated the feasibility and safety of this approach [119]. In addition, the treatment increased the population of CD4+ T lymphocytes expressing ICOS (inducible costimulator; CD4 +ICOShi T cells) in both the bladder tissue and peripheral blood serving as an indicator of ipilimumab immunological effects, and as a potential biomarker for therapy monitoring. Supported by these results, an ongoing phase II trial is evaluating the combination of gemcitabine, cisplatin and ipilimumab as first-line treatment of metastatic urothelial carcinoma (NCT01524991). Furthermore, based on preclinical evidence showing the potential for enhancing anti-tumor activity with combinations of immunotherapies (e.g. anti-PD-1, anti-CTLA-4, and vaccines), ongoing clinical trials are starting to investigate the safety and efficacy of combinations including anti-PD-L1, anti-CTLA-4 and OX-40 agonist in advanced solid tumors including bladder cancer (NCT02205333) [120–122]. IL-2/T lymphocyte receptor fusion protein targeting p53 epitope/HLAA⁄0201 complex Another innovative strategy utilizes a fusion protein comprised of the IL-2 molecule combined with a single chain HLA-A2.1restricted T lymphocyte receptor (TCR) specific to p53 epitope (aa264–272) presented by MHC in the cell surface. This fusion protein (ALT-801) activates the IL-2 receptor in infiltrating T-lymphocytes and was hypothesized to target p53 peptide expressing tumor cells [123]. TCR-based therapies represent an alternative to monoclonal antibody based treatments particularly useful when targeting MHC-restricted antigens not accessible to antibodies. This targeted immunotherapy, however, would be suggested to be limited to patients with a specific HLA type but this approach can overcome major limitations of IL-2 therapy (i.e. short half-life and toxicity) and concentrate IL-2 to the tumor microenvironment. In fact, a phase I study with ATL-801 revealed a longer half-life and lower incidence of liver and renal toxicities compared to high dose IL-2, and showed encouraging efficacy in a variety of p53-expressing tumors (e.g. melanoma, renal cell carcinoma, neuroendocrine, head and neck, and prostate cancers) [124]. Another phase I study testing ALT-801 combined with cisplatin and gemcitabine for patients with metastatic urothelial cancer documented 3 complete responses and 5 partial responses among 10 patients [125]. An expansion cohort is ongoing (NCT01326871). Additional immunotherapy strategies in bladder cancer include gene therapy with adenoviral vectors expressing CD40 ligand that functions as an immune activator and induces anti-tumor effects when administered intravesically for muscle-invasive disease [126]. Vaccines utilizing MAGE-A3 as the tumor antigen combined with a proprietary AS15 immunostimulant is also undergoing a phase I trial as a potential adjuvant treatment of muscle-invasive bladder cancer (NCT01435356), a similar clinical setting as the HER2-based vaccine discussed previously. Conclusions The rapidly advancing knowledge of urothelial carcinoma pathophysiology and underlying molecular alterations identified by the Cancer Genome Atlas and other initiatives is providing the

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foundation for novel treatments. This manuscript reviewed some of the most relevant molecular pathways and drivers of the malignant phenotype and agents that might demonstrate therapeutic potential. In addition to the promising inhibition of overactive signaling networks (i.e. FGFR, PI3K/AKT/mTOR and HER2) and modulation of cell cycle machinery, immunotherapy strategies may have a particularly important role given the clear involvement of immune mechanisms in controlling tumor development as demonstrated by the efficacy of BCG in non-muscle-invasive urothelial cancer treatment during the last 30 years. Moreover, the favorable toxicity profile of immunotherapy treatments allows combinations with agents targeting signaling pathways and/or existing chemotherapy protocols. However, an efficient and innovative clinical trial infrastructure providing a supportive environment for collaborative trial design and execution between basic scientists, urologists, and medical oncologists is critical to maximize the therapeutic gains identified to date and improve the clinical outcome for patients.

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Emerging therapeutic targets in bladder cancer.

Treatment of muscle invasive urothelial bladder carcinoma (BCa) remains a major challenge. Comprehensive genomic profiling of tumors and identificatio...
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