The fibroblast growth factor receptor (FGFR) is regarded as a promising locus for targeted therapy in UC. The FGFR family is composed of 4 transmembrane receptors (FGFR1–4). The structural domain spanning the cell membrane connects the extracellular and cytoplasmic regions of the cell. This domain comprises approximately 20 non-conserved hydrophobic amino acids, followed by basic residues. These components of the domain facilitate the attachment of the receptor to the membrane, thereby leading to translocation. TKDs and tyrosine phosphorylation sites are present within the intracellular region, whereas the juxtamembrane region of FGFR facilitates receptor dimerization [12]. Fibroblast growth factors (FGF) bind to the inactive monomeric conformation of FGFR and undergo conformational changes that promote dimerization and trans-phosphorylation of the structural domain of the intracellular TK. Activated FGFRs trigger downstream signaling through various pathways [13]. The FGF/FGFR physiological signaling axis is pivotal in organ development and metabolism. Broad-spectrum tumorigenesis that facilitates tumor growth, proliferation, differentiation, and survival is associated with dysregulation of this axis.

Helsten et al. [14] reported the presence of FGFR variants in multiple human cancers. These variants were reported in approximately 7.1% of the nearly 4,000 analyzed tumors. Specifically, UC involved approximately 15% of cases with somatic FGFR3 mutations, approximately 7% with FGFR1 amplification, and approximately 6% with gene fusions. FGFR fusions are of two types. Type 1 fusions, commonly observed in hematological tumors, involve chromosomal translocations affecting the structural domains of kinases. Type 2 fusions mostly cause rearrangements in solid tumors, thereby forming chimeric transmembrane FGFRs [15]. Younger, non-smoking Asian patients are more prone to the FGFR3-transforming acidic coiled-coil containing protein 3 (TACC3) gene fusion, and this fusion is associated with MIBC [16]. TACC3 is involved in stabilizing and organizing the mitotic spindle [17]. When the FGFR3-TACC3 fusion is formed, the TACC coiled-coil structural domain persistently phosphorylates crucial tyrosine residues. This fusion event improves downstream signaling pathways, including mitogen-activated protein kinase (MAPK), PI3K/protein kinase B (Akt), and signal transducer and activator of transcription 3 (STAT3) [18, 19]. Preclinical studies on the FGFR3-TACC3 fusion-harboring cell line observed an enhancement in cell proliferation. Furthermore, in vivo studies conducted on nude mice evidenced that this fusion augments the activation of downstream proteins, including extracellular signal-regulated kinase (ERK) and STAT3 [20]. The FGFR3-TACC3 fusion also leads to an altered TACC3 function, potentially inducing mitotic defects and aneuploidy [21].

FGFR3 dysregulation through mutation, overexpression, or both is observed in 54% of aUC cases [22]. Of note, the luminal-papillary urothelial subtype was recently found to possess a high frequency of FGFR3 genomic alterations (65.2%) [7, 8]. Moreover, FGFR3 mutations are associated with 5–20% of MIBC [22, 23]. Typically, ligand-dependent dimerization, activation, and signaling are actuated by aberrations that incorporate point mutations in extracellular regions [24]. Aggressive tumors exhibit overexpression of wild-type FGFR3, which induces ligand-dependent dimerization and activation [24].

FGFR1 genomic aberrations have received relatively less attention than FGFR3 genomic aberrations, but they are reported to be prevalent in 7–14% of cases [25]. FGFR1 promotes tumor proliferation and survival by activating MAPK and inducing epithelial-to-mesenchymal transition [26].

In a phase I study, rogaratinib (BAY1163877), a pan-FGFR inhibitor, exhibited promising efficacy and safety in aUC patients with FGFR1–3 mRNA overexpression [34, 35]. A phase II/III study (NCT03410693) investigated the effectiveness and safety of rogaratinib in FGFR mRNA-positive aUC patients who had received a platinum-based regimen [36]. The patients were assigned randomly in a 1:1 ratio to receive rogaratinib (n = 87) or chemotherapy (n = 88). In this trial, OS was the primary outcome. The interim analysis suggested that the combination of FGFR-targeted therapy with chemotherapy resulted in comparable effectiveness and a well-tolerated safety profile in FGFR-altered UC patients. According to exploratory trials, FGFR3 DNA alterations associated with FGFR1–3 mRNA overexpression may be a more reliable predictor of response to rogaratinib [36].

Pemigatinib (INCB054828) is an ATP-competitive inhibitor selectively and reversibly targeting FGFR1–3. Pemigatinib significantly inhibited FGFR1, 2, and 3 at half-maximal inhibitory concentrations (IC50) of 0.4 nmol/L, 0.5 nmol/L, and 1.0 nmol/L, respectively. However, its activity against FGFR4 was comparatively lower, with its IC50 being 30 nmol/L [37]. In preclinical investigations, pemigatinib demonstrated promising antitumor activity by effectively inhibiting the growth of FGFR-overexpressing tumor cell lines [37]. In the FIGHT-101 study, pemigatinib used against UC demonstrated a good tolerance and safety profile [38]. In that study, hyperphosphatemia was the most repeatedly observed treatment-related AE (TRAE) with an incidence of 75.0%. Conversely, fatigue was the most common grade ≥ 3 TRAE with an incidence of 10.2% [38]. The FIGHT-201 study (NCT02872714) is a completed phase II, open-label, multicenter study that evaluated the effectiveness and security of pemigatinib for aUC patients featuring FGF/FGFR alterations. Patients with FGFR3 mutations or fusions who received pemigatinib in an intermittent dose (ID, 2-weeks-on/1-week-off therapy) or a continuous dose (CD, no planned dose hold) were classified into cohort A-ID and cohort A-CD, respectively. Patients who had other FGF/FGFR alterations and received pemigatinib as an ID were assigned to B-ID. The published findings of this trial revealed that the ORR in cohort A-CD, including 101 patients was 17.8% (95% CI: 10.92–26.70%). In cohort A-ID, including 103 patients, the ORR was 23.3% (95% CI: 15.54–32.66%). Additionally, cohort B-ID, including 44 patients, had an ORR of 6.8% (95% CI: 1.43–18.66%). The combined cohort (n = 147) under the ID regimen (A-ID + B-ID) had an ORR of 18.4% (95% CI: 12.47–25.59%). The combined cohort (A-ID and A-CD, n = 204) included patients with FGFR3 mutations or fusions and exhibited an ORR of 20.6% (95% CI: 15.26–26.79%). Additionally, the combined cohort (n = 248), including patients with FGFR3 mutations or fusions in cohort A and those with other FGF/FGFR alterations in cohort B, had an ORR of 18.1% (95% CI: 13.55–23.52%). The PFS was 4.27 months (95% CI: 3.91–6.05) and 4.04 months (95% CI: 3.45–4.17) in the A-ID and A-CD groups, respectively, while that in the B-ID group was 2.04 months (95% CI: 1.87–2.17). The incidences of serious AEs in cohorts A-ID, B-ID, and A-CD were 43.69%, 59.09%, and 47.52%, respectively. The other AEs (excluding serious) observed in the cohorts were weakness, fatigue, hyperphosphatemia, alopecia, dry eye, abdominal pain, constipation, diarrhea, dry mouth, nausea, stomatitis, urinary tract infection, and taste disorders.

Infigratinib (BGJ398) is an oral FGFR1–3 inhibitor with selectivity. In preclinical trials, infigratinib exhibited a dose-dependent ability to reduce tumor growth in UC xenograft models. Additionally, it effectively lowered phosphorylated FRS2 (pFRS2) and phosphorylated MAPK (pMAPK) levels, thus displaying significant antitumor activity [39]. A phase I study revealed that infigratinib has a tolerable safety and exerts antitumor effects in FGFR3-altered UC patients [40]. Of the 67 infigratinib-treated patients who were ineligible for platinum-based chemotherapy, the remission rate was 25.4% and the disease control rate (DCR) was 64.2%. The chief treatment-emergent toxicities in these patients were hyperphosphatemia, elevated creatinine levels, fatigue, and constipation [41]. In a retrospective study analyzing 67 infigratinib-treated patients, the response rate in patients with hyperphosphatemia was as high as 33.3% (95% CI: 20.4–48.4), whereas that in patients without hyperphosphatemia was only 5.3% (95% CI: 0.1–26.0). Additionally, the DCR was also higher in patients with hyperphosphatemia (75.0%, 95% CI: 60.4–86.4) than in those without hyperphosphatemia (36.8%, 95% CI: 16.3–61.6). These findings suggest that elevated blood phosphorus levels and improved clinical outcomes are potentially correlated [42]. However, larger cohort studies and prospective trials are warranted to further validate these observations and to assess the clinical significance of hyperphosphatemia as a biomarker. The PROOF 302 trial (NCT 04197986) is currently being conducted to address this issue [43]. Although the sponsor terminated the study prematurely, genomic analysis and evaluations of correlations to primary and secondary endpoints are ongoing. The ongoing genomic analysis offers valuable insights into the frequency of FGFR3 alterations [43].

Futibatinib (TAS120) is an orally administered novel inhibitor potent, selective, and irreversible against FGFR1–4. In preclinical studies, futibatinib displayed the potential to inhibited UC growth effectively [44]. Furthermore, a phase I trial involving 19 UC patients evidenced the clinical activity and tolerability of futibatinib during bladder tumor treatment [45]. The trial reported an ORR of 15.8% (95% CI: 3.4–39.6%), including 3 cases achieving a PR and 6 cases of stable disease (SD). The DCR was 47.4%, and the DCR, including SD lasting for > 16 weeks, was 20.5%. Importantly, these results were observed in patients who had received multiple lines of treatment, with > 50% of the enrolled patients having undergone more than three lines of therapy [45]. Hyperphosphatemia was the primary reason for the dose adjustment of futibatinib in two studies (NCT02052778, JapicCTI-142552) on patients administered with 20 mg/day futibatinib [46]. Of note, 85% of patients with hyperphosphatemia received phosphate-binding agents, whereas 30% received phosphate-solubilizing agents. However, no significant difference was noted in response time. Additionally, analgesics (55% of patients with nail lesions; 71% of patients with palmoplantar erythrodysesthesia) and corticosteroids (37% of patients with rash) were the commonly administered concomitant medications. Overall, based on the analysis, futibatinib can be concluded to demonstrate a consistent and manageable safety profile [46].

Epidermal growth factor receptor [EGFR, also known as erythroblastic oncogene B-1 (ErbB-1)or human epidermal growth factor receptor 1 (HER1)], ErbB-2 (HER2/neu), ErbB-3 (HER3), and ErbB-4 (HER4) are the four receptors of the ErbB family. By activating downstream pathways such as MAPK and PI3K/Akt, these cell membrane-bound RTKs become a crucial player in cell proliferation [52–55]. EGFR, ErbB-2, and ErbB-3 mutations or amplifications have been reported in MIBC. EGFR aberrations account for approximately 6–14% of MIBC cases, ErbB-2 mutations for 6–23% of cases, and ErbB-3 mutations for 6% of MIBC cases [8, 56, 57]. Because of the prevalence of these genetic aberrations, the ErbB family has become a promising target for anticancer drug development.

Angiogenesis is a physiological process exploited by malignant tissues to promote tumor development. Using this pathway, tumors stimulate new blood vessel formation, which in turn offers oxygen and nutrients critical for their continued proliferation [90, 91].

Vascular endothelial growth factor-A (VEGF-A), a member of the VEGF protein family, interacts with VEGFR2 on endothelial cells to promote neovascularization [92]. Elevated VEGF levels are usually associated with adverse outcomes in UC patients [93]. For instance, Kanayama et al. [94] identified a correlation between serum VEGF levels and neoplasm characteristics such as stage, grade, vascular invasion, and other factors, and an inverse correlation with disease-free survival [94].

Given the significance of the relationship between tumor progression and angiogenesis, new therapeutic approaches are being developed in this area, including anti-VEGF/VEGFR antibodies, TKIs, and other drugs.

Buparlisib (BKM120), a highly specific and potent pan-class PI3K inhibitor, was evaluated to determine the viability and proof of concept in patients with advanced tumors in a phase I study [136]. Subsequently, a phase II study determined the effectiveness of buparlisib in patients with a platinum refractory aUC [137]. In the initial cohort of 16 patients, genetic alterations in the PI3K/Akt/mTOR pathway were not considered while selecting patients. The reported PFS at 2 months was 54%. Afterward, a cohort was screened for four patients with mutations in phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA), Akt1, or TSC1. Regrettably, the study was terminated early by the sponsors because of under-recruitment. Of note, none of these patients demonstrated progression at the 8-week assessment, and 38% of the patients encountered substantial AEs necessitating dose reductions, which ultimately resulted in the premature withdrawal of 2 patients from the study.

In preclinical studies, the pan-class I PI3K inhibitor copanlisib could increase the effectiveness of ICIs in syngeneic mouse models of UC, irrespective of whether activation alterations are present in the PI3K pathway [138]. A current phase II study is assessing the combination of copanlisib and avelumab as maintenance therapy for mUC patients after chemotherapy. In this trail, the primary objective is to measure PFS [139].

MPT0L145, a novel PI3K catalytic subunit type 3 (PIK3C3)/FGFR inhibitor, exerts significant antitumor effects against UC cells, which makes it a promising first-in-class candidate for UC treatment [140, 141]. Alpelisib, which has a manageable safety profile and promising initial efficacy, may be useful as a therapeutic option for solid tumors with PIK3CA alterations. This rationalizes combining selective PI3Kα inhibition with other drugs for effectively controlling PIK3CA-mutant tumors [142]. Marqués et al. [143] revealed that the nintedanib + alpelisib combination exerts synergistic antitumor activity. Simultaneous administration of nintedanib and PI3K inhibitors not only overcame UC resistance to nintedanib but also increased its antiangiogenic effects [143].

According to preclinical modeling data, pictilisib significantly inhibits the growth of PIK3CA mutation-harboring UC cells [144, 145]. However, no clinical evidence is currently available for evaluating its effectiveness in this context further.

Eganelisib (IPI-549) is a novel PI3Kγ inhibitor that is administered orally. In preclinical studies, it exhibited antitumor activity as both monotherapy and in combination with ICIs [146]. The MARIO-1 trial aimed to determine the efficacy of eganelisib as a once-daily single agent and in combination with nivolumab in patients with solid tumors. The trial further determined the security and tolerability of eganelisib compared with that of nivolumab (Figure 3). In the monotherapy arm, the predominant grade ≥ 3 treatment-related toxicities included elevated ALT levels (18%), aspartate aminotransferase (18%), and alkaline phosphatase (5%). During the first 28 days, dose-limiting toxicities (DLTs) were not reported. However, subsequent treatment cycles using 60 mg eganelisib resulted in toxic reactions according to the criteria for DLTs, with the primary occurrence being reversible grade 3 elevations in liver enzymes. In the combination therapy arm, the major grade ≥ 3 treatment-related toxicities included elevated levels of glutamine aminotransferase (13%), ALT, and rash (10%). Overall, 5% of patients receiving monotherapy had serious TRAEs, including 1 case each of grade 4 bilirubin elevation and liver enzyme elevation. Serious TRAEs were observed in 13% of the patients receiving combination therapy. The combination therapy exerted a significant antitumor effect, even in patients whose disease had progressed with the ICIs. Nonetheless, acknowledging that these findings stem from the MARIO-1 trial is crucial, and additional clinical assessments are imperative to validate these results [147].

Using human UC cell lines, preclinical studies have reported that ectopic pan Akt inhibitors, such as MK-2206 and AZ7328, are potent and selective Akt inhibitors with very low toxicity [148, 149]. Moreover, in preclinical studies, Akt and ERK1/2 phosphorylation in UC cells was significantly increased by the chemotherapeutic agent piroxicam. MK-2206 used as a single agent or in combination with the ERK1/2 inhibitor AZD6244 exhibited significant sensitization of UC cells to the chemotherapeutic agent pirarubicin [150]. Additionally, capivasertib (AZD5363) exhibited UC inhibition in preclinical studies [151]. The ATP-competitive pan-Akt inhibitor ipatasertib (GDC-0068) was evaluated in a phase Ib clinical trial involving aUC patients [152]. The trial revealed that ipatasertib, when combined with chemotherapy or hormonal therapy, exhibits a good tolerability and safety profile consistent with those of other ATP-competitive Akt inhibitors [152].

Rapamycin represents the initial discovery of a mTOR inhibitor [153]. This compound specifically targets the mTOR complex 1 (mTORC1), a key regulatory factor in translation and cell growth [153]. In addition to rapamycin, the FDA has approved three other rapamycin analogs, namely everolimus, temsirolimus, and ridaforolimus, which are commonly used. Although, in preclinical studies, everolimus demonstrated antitumor activity, clinical trials have usually reported unsatisfactory results in aUC patients [154]. In a phase II trial, all 45 mUC patients, who had previously received at least one cytotoxic drug and experienced disease progression, received everolimus [155]. The PFS rate at the 2-month mark was 51%. Two patients, however, attained PR, with 1 patient achieving CR for a duration of > 2 years. A post-analysis of the tumor genome in this patient revealed the presence of TSC1 inactivating mutations alongside neurofibromatosis type 2 mutations [156].

In a phase II trial, everolimus was assessed in aUC patients who had experienced progression after chemotherapy [157]. The 2-month DCR in this trial was 27%. PTEN was expressed in all patients with controlled disease (n = 6) and 6 of the 14 patients with uncontrolled disease (43%). In a subsequent analysis of archival tissue, Akt activation in PTEN-deficient cells increased during treatment with mTOR inhibitors [157]. Tumors lacking PTEN exhibited heightened Akt activation after treatment with mTOR inhibitors, potentially serving as a mechanism of resistance to everolimus. In addition to PTEN deficiency, increased Akt activity may occur because everolimus selectively inhibits a mTORC1 subunit, leading to increased activation of mTORC2, a known Akt activator [158]. Another clinical study of everolimus in UC reported unfavorable outcomes [159]. However, some case reports have indicated the significant effectiveness of everolimus treatment in patients with PIK3CA mutant UC. This suggests that the rare M1043I mutation variant could potentially serve as a biomarker for everolimus sensitivity [160]. Xia et al. [161] revealed that high-dose everolimus monotherapy causes tumor regression but also induces immunosuppression. The combination of low-dose everolimus with ICIs effectively suppressed UC growth by boosting the antitumor immune response in the tumor microenvironment and peripheral area [161].

In preclinical models, temsirolimus improved the cytotoxic effectiveness of cisplatin and gemcitabine against UC cell lines [162]. A phase II trial used temsirolimus in aUC patients undergoing first-line chemotherapy [163]. Of the 45 patients included, 48.9% patients demonstrated a progression-free status at the 2-month mark. Importantly, > 50% of the patients experienced severe toxicity, and 11 patients had to be discontinued because of the development of adverse effects, which resulted in the discontinuation of recruitment. In addition, another trial investigating temsirolimus as a treatment for aUC patients was prematurely terminated because of insufficient observed efficacy [164].

Second-generation mTOR inhibitors include small-molecule ATP analogs and dual PI3K/mTOR inhibitors. By directly binding to the ATP-binding site of mTOR or PI3K, or by acting upon it, these inhibitors cause competitive inhibition [165]. Three UC cell lines were treated with OSI-027 to effectively hinder the phosphorylation of components within both mTORC1 and mTORC2 pathways, thereby causing a noticeable reduction in cell proliferation. Furthermore, in an in vitro assay of UC, OSI-027 combined with lapatinib exerted synergistic antitumor effects. The study also exhibited antitumor synergy in UC in vitro [166].

Sapanisertib (TAK-228) inhibits mTORC1 and mTORC2 complexes, and when combined with paclitaxel or the PI3Kα inhibitor TAK-117, sapanisertib exerts synergistic antitumor effects in preclinical UC models [167]. Sapanisertib demonstrated a manageable safety profile in a trial [168]. A phase II clinical trial assessing the efficacy of sapanisertib in aUC patients carrying TSC1 or TSC2 mutations was prematurely terminated because of a lack of effectiveness and a high incidence of AEs [169].

Dactolisib, an oral dual mTOR/PI3K inhibitor, increases the antitumor effect of cisplatin by exerting synergistic effects [170]. A phase II study determined the effectiveness of dactolisib in UC [171]. The study revealed a modest clinical effect, with 10% of patients achieving PFS after 16 weeks. However, the trial also reported a significant level of toxicity.

The observed upregulation of RTKs along with the inhibition of the PI3K/Akt/mTOR pathway suggests that inhibitors targeting both pathways may together increase efficacy. Everolimus and pazopanib, a VEGFR inhibitor, were assessed in a phase I trial of aUC patients. Only 21% of the participants had a response. TSC1/2 or mTOR gene alterations were observed among patients who experienced clinical effects. Specifically, of the 5 patients who benefited from the treatment, 4 patients had the aforementioned mutations, whereas the other patient had an FGFR3-TACC3 fusion [112]. A phase Ib study assessed the combined inhibition of PIK3CA and FGFR in patients with different PIK3CA-mutant solid tumors [172]. In that study, 60% of patients encountered severe AEs. Notably, only 10% of patients demonstrated DLTs. Akt and mTOR inhibitors together exert an inhibitory effect on UC cell lines [151]. Specifically, the J82 cell line, which has PI3KCA and mTOR mutations, was sensitive to AZD5363, AZD2014, and BEZ235 individually, as well as the combinations of AZD5363/AZD2014 and AZD5363/BEZ235. Although all single agents exert inhibitory effects on cell proliferation, the combinations exert a synergistic effect on cell viability and colony formation.

Before ICIs were introduced, the standard second-line therapy remained undefined for aUC patients. HDAC inhibitors have demonstrated anticancer effects in various tumor models, including the modulation of apoptosis of UC cell lines. Vorinostat is an enzyme inhibitor that significantly impedes cell survival, growth, and apoptosis regulation, all of which are essential in cancer. Consequently, this drug can disrupt a tumor’s capability to sustain its growth. A phase II study determined the response rate to vorinostat in aUC patients. The patients received 200 mg vorinostat [suberoylanilide hydroxamic acid (SAHA)] orally each day on a 3-week cycle. All 14 patients completed the study. The OS was 4.3 months (95% CI: 2.1–8.3), while the PFS was 1.1 months (95% CI: 1.0–2.1). The study was terminated early after the first stage of a two-stage design, which allowed for discontinuation because of unsatisfactory results (NCT00363883). A phase I study investigated the combination of vorinostat and docetaxel in patients with advanced and relapsed solid malignancies, including UC. The trial determined whether this combination treatment yielded outcomes superior to those of docetaxel alone (NCT00565227). However, the study was unfortunately terminated prematurely, and no results have been published. The trial (NCT00363883) assessed the effectiveness and toxicity of vorinostat in aUC patients who experienced treatment failure with first-line therapy, in the adjuvant/neoadjuvant setting, or for recurrent/advanced tumors [190]. The main endpoint was the response rate. A response rate of > 20% was considered notable for the two-stage study design, which required at least one response in the initial 12 patients so as to proceed to the second stage, with a total of 37 subjects. In the absence of any observed response, the initial phase of recruitment was terminated. Among the patients, the best response of SD was achieved in three individuals. Because of the limited efficacy and the notable toxicity associated with vorinostat at this specific dosage regimen, the risk-benefit ratio was unsatisfactory for aUC patients.

A phase I/Ib study determined the effectiveness of the combination of pembrolizumab and vorinostat for aUC patients, but the study results have not been presented at this time (NCT02619253).

Through preclinical experiments, Michael et al. [191] first demonstrated the ability of the HDAC inhibitor belinostat to effectively suppress UC cell growth. Alterations in COMPASS-associated proteins were noted in approximately two-thirds of UC patients, which suggested that histone regulation is associated with tumor survival [192]. Administering the enhancer of zeste homolog 2 inhibitor tazemetostat to mice reduced UC disease. Moreover, tazemetostat administered in vivo boosted the immune response by directly affecting cytokines and antigen recognition mechanisms, particularly major histocompatibility complex I (MHC-I) and MHC-II [192]. Consequently, a pilot study (ETCTN 10183; NCT03854474) was conducted to evaluate the effectiveness of the tazemetostat and pembrolizumab combination against aUC (Table 7) [192]. The study comprised two cohorts: cohort A involving patients refractory to cisplatin, while cohort B included patients non-eligible for chemotherapy. Each cohort consisted of 12 patients. The treatment regimen involved 800 mg tazemetostat administered twice daily in conjunction with 200 mg pembrolizumab every 3 weeks. The median treatment duration was 12 weeks (IQR: 7–30; range: 3–107 weeks). In this study, 1 case of grade 4 sepsis and 2 cases of grade 3 lymphopenia were reported. Other observed TRAEs included 1 case each of anemia, elevated alkaline phosphatase, and herpes simplex virus oral infections. In this study, three patients (25%) achieved PR, while three (25%) experienced SD [192]. The median survival was 3.1 months (95% CI: 2.3–NA), with a median OS of 8.0 months (95% CI: 4.7–NE). The study successfully determined that RP2D is the combination of 800 mg tazemetostat and 200 mg pembrolizumab administered every 3 weeks. This therapy demonstrated durable effects and was feasible and well tolerated in patients with low-risk chemotherapy-refractory UC.

Cell cycle regulation is another crucial target of UC. The cyclin-dependent kinase inhibitor 2A/B (CDKN2A/B) gene, responsible for inhibiting p14 and p16 of the cyclin-dependent kinases 4 and 6 (CDK4/6), is commonly altered in 5–23% of MIBC cases [8, 56]. CDK4/6 inhibition has emerged as a therapeutic target in UC. Palbociclib is a highly selective, orally administered CDK4 and CDK6 inhibitor. Inhibition of these proteins facilitates the restoration of the tumor suppressor effects of retinoblastoma protein (RB), thereby leading to cell cycle arrest. The crucial role of an intact RB in the mechanism underlying CDK4/6 inhibitor therapy in cancer is well-established. The presence of CDKN2A deletion with an intact RB mechanistically predicts sensitivity to CDK4/6 inhibitors. According to preclinical data from UC cell lines, RB1 inactivation leads to resistance, while CDKN2A inactivation results in sensitivity to palbociclib [193]. An open-label, single-arm, multicenter phase II trial was conducted to determine the effectiveness of palbociclib in previously treated UC patients exhibiting p16 deletion and an intact RB. Regrettably, palbociclib exhibited ineffectiveness, as only 17% of patients achieved PFS at 4 months [194]. Preclinical studies have reported that the combination of palbociclib and talazoparib enhances treatment efficacy in UC, although additional clinical studies are warranted to validate these findings [195]. Trilaciclib and abemaciclib have also entered clinical trials and are being assessed for their effects on UC.

Evidence suggests that the transforming growth factor β (TGF-β) is a critical factor for cancer that influences various aspects, including resistance to cell death, evasion of growth inhibitors, induction of angiogenesis, and activation of invasion and metastasis [196]. Furthermore, the TGF-β pathway serves as a modulator of immune cell rejection and a contributor to ICI resistance [196]. The effectiveness of TGF-β pathway inhibitors in combination with ICIs against various tumor types, including UC, has been assessed. Unfortunately, two clinical trials investigating the combination of SAR439459 and cemiplimab for treating patients with advanced solid tumors were prematurely terminated because of drug toxicity. An ongoing phase II study (NCT04064190) aims to assess the potential of the vactosertib + durvalumab combination in significantly improving the ORR among UC patients who have not achieved remission with an anti-PD-1/PD-L1 based regimen. The aryl hydrocarbon receptor (AHR), a transcription factor, binds to diverse endogenous and exogenous ligands and thus modulates the function of various innate and adaptive immune cells. Elevations in AHR levels have been linked to resistance against ICIs. In preclinical studies, the selective orally active AHR antagonist called IK-175 could impede tumor growth and reverse immunosuppression in mouse tumor models [197]. According to preliminary analyses, both the monotherapy and combination therapy arms of IK-175 demonstrated promising antitumor activity and good tolerability in eligible UC patients [198]. During a phase II trial, aUC patients whose disease had recurred or progressed after chemotherapy received a combination therapy involving the soluble ephrin type-B receptor 4-human serum albumin (sEphB1-HSA) and pembrolizumab. The combined use of sEphB1-HSA and pembrolizumab had synergistic effects, which improved OS and ORR [197]. Additionally, clinical trials evaluating the combination of arginase inhibitor INCB001158 and the burton TKI acalabrutinib with ICIs for UC patients are currently in progress.