Forkhead box O3 (FOXO3) and forkhead box M1 (FOXM1) axis contains important transcription factors for cell proliferation, differentiation, cell survival, senescence, and DNA damage repair.61 FOXO3 has been shown to act as a tumor suppressor gene in many cancers including PCa. In transgenic adenocarcinoma of the mouse prostate (TRAMP) mice, prostate progression was increased when FOXO3a activity was blocked.62 FOXO3a is regulated by the phosphorylation by PI3K-AKT, RAS-ERK, serum glucocorticoid inducible kinases (SGK), and IkB kinase beta (IKKB) which prevents translocation into the nucleus.61,63,64 When FOXO3 is able to translocate to the nucleus it inhibits the oncogene FOXM1 (Figure 3). Similarly to CDK4/6, FOXM1 is involved in cell proliferation and cell cycle progression by promoting entry into S-phase and M-phase; thereby, leading to tumorigenesis.65,66 FOXM1 has been identified as a critical phosphorylation target of CDK4/6 allowing expression of G1/S phase genes which protects cells from senescence. It was also demonstrated that FOXM1 had involvement with regulation of genes in cell cycle DNA replication (cycle E2, MYB, MCM2, MCM10, CDT1) as well as DNA repair (XRCC2, SFRS4).60 Since FOXM1 is overexpressed in many solid tumors including ovarian, breast, prostate, melanoma, hepatoma, angiosarcoma, colorectal cancer, lung cancer and gastric cancer,67 targeting the FOXO3-FOXM1 axis could be a synergistic approach to further prevent tumorigenesis by cell cycle mechanisms. When CDK4/6 is inhibited, using palbociclib, it decreased FOXM1 protein levels by 70%, indicating blockade of CDK4/6 can down-regulate FOXM1.60 Multiple therapeutics have been shown to have effects on this axis to either increase the activity of FOXO3 or inhibit FOXM1 including: chemotherapy (paclitaxel,68 doxorubicin,69 cisplatin),70 CDK4/6i’s (ribociclib,71 palbociclib),60 tyrosine kinase inhibitors (gefitinib),72 PI3K-AKT inhibitors,61 endocrine receptor modulators,73 aurora kinase inhibitors,74 thiazole antibiotics (thiostrepton),75,76 natural compounds like Honokiol,77 and potentially TP 53 activators.78 Combining these agents with CDK4/6i’s may provide synergistic activity against tumorigenesis given FOXO3-FOXM1 role in cell proliferation in mCRPC.
In mCRPC, phosphatidylinositol 3-kinase (PI3K) alterations are seen in almost 50% of patients, with loss of PTEN tumor suppressor occurring in approximately 40% of these patients (Table 1).30 When PTEN loss occurs, it allows PI3K/AKT activation to promote PCa growth in the absence of AR signaling and is associated with worse outcomes.79–81 Single agents targeting PI3K, AKT and mTOR have failed to progress in PCa as a result of toxicity, inadequate target inhibition, and limited efficacy.79 However, combination therapy has shown some activity. AKT inhibitor, ipatasertib, was combined with abiraterone in a randomized phase II study and showed superior antitumor activity with improved OS and time to PSA progression, although not statistically significant, compared to abiraterone alone in patients with mCRPC and PTEN loss.82 Since the PI3K axis can be a means of developing CDK4/6i resistance, combining these agents or sequential treatment is under investigation. In breast cancer, targeting CDK4/6 and PI3K in vitro and in patient-derived tumor xenografts (PDTX) resulted in tumor regression.83 As a result, clinical trials with this combination are ongoing in breast cancer. As more studies are being completed investigators are developing a better understanding of resistance for these agents. It has been shown that chronic exposure to CDK4/6i’s upregulates cyclin D and promotes emergence of PIK3CA driver mutations.34 In breast cancer, PTEN loss has been shown to cause resistance to CDK4/6i’s or PI3K inhibitors such as alpelisib.84,85 This CDK4/6 resistance develops as a result of increased AKT activation. However, selective AKT inhibitors have been shown to restore sensitivity in vitro and in vivo.84 Since PTEN loss is frequent in mCRPC (40%), using AKT inhibitors with CDK4/6i’s, instead of PI3K inhibitors, may result in better efficacy and synergy. There are no clinical trials investigating PI3K or AKT inhibitors with CDK4/6i’s in PCa.
Although PI3K/AKT inhibitors remain a viable option, negative feedback loops are present between AR and PI3K/AKT in which inhibition of one activates the other;81,86 therefore, other targets in this pathway have been investigated. Histone deacetylase 3 (HDAC3), class I, known to be upregulated in PCa, has roles in S phase progression, DNA damage control, maintenance of genomic stability and T cell development.87 Blocking HDAC3 has inhibitory effects on both, AKT and AR, through inhibition of AKT phosphorylation and histone deacetylation and condensed chromatin, respectively (Figure 4).86 It was hypothesized that patients with loss of PTEN or mutated SPOP, which results in aberrant activation of AR and AKT, would respond to HDAC3 inhibitors. This was shown with a selective HDAC3 inhibitor, RGFP966, when it inhibited growth in PTEN or SPOP mutated PCa cells in culture and PDTX models.87 Given the inhibitory effects as a single agent, combining HDAC inhibitors with CDK4/6i’s could decrease PI3K associated CDK4/6i resistance. HDAC inhibitors have also been shown to upregulate FOXO1 mRNA and protein levels which have effects on the cell cycle. FOXO1 is a tumor suppressor that induces cell cycle arrest at G1 by modulating p27kip1, p21, Rb protein, cyclin D1/D2 and induces cell cycle arrest at G2 via GADD45.88 Therefore, this HDAC inhibitor effect may add additional inhibition to the cell cycle when added to CDK4/6i’s. Combining CDK4/6i’s with HDAC inhibitors was investigated in preclinical breast cancer models when abemaciclib and vorinostat were combined and revealed synergy with significant diminished tumor growth.89 Otherwise, this combination has not been studied extensively. Further studies with this combination in PCa are warranted.
Fibroblast growth factor pathway aberrancy has been described in many cancers including bladder, breast, endometrial, lung, rhabdomyosarcoma, melanoma, head and neck, brain and PCa.90 FGF and fibroblast growth factor receptor (FGFR) have been shown to be dysregulated in the development of prostate intraepithelial neoplasia, epithelial-mesenchymal transition, angiogenesis, promotion of bone metastases and emergence of CRPC.91 In a cohort of 101 patients with mCRPC, FGFR1 was amplified in 10% of patients.92 The FGF-FGFR signaling pathway leads to activation of STAT, PI3K-AKT-mTOR and RAS-RAF-MEK pathway (Figure 5). Each of these pathways have downstream effects on the cell cycle by increasing cyclin D1.93,94 In breast cancer, the FGF-FGFR axis, specifically FGFR1 over expression, is a way for breast cancer cells to become resistant to CDK4/6i’s and have resulted in shorter progression-free survival.95 As a result of this, combining a CDK4/6i with an FGFR inhibitor has been pursued. The resistance was overcome in breast cancer xenograft models when FGFR1 amplified tumors were given FGFR tyrosine kinase inhibitor, lucitanib and complete responses were seen in xenografts when the FGFR inhibitor, erdafitinib, was given with CDK4/6i and endocrine therapy.95 This triple therapy is being used in a phase Ib trial in patients with metastatic ER+/HER2-/FGFR amplified breast cancer (NCT03238196). In PCa, erdafitinib is being pursued in combination with androgen signaling inhibitors in patients who are androgen receptor negative and no neuroendocrine differentiation (NCT03999515). Dovitinib, a pan class inhibitor targeting FGFR along with PDGFR and VEGF, was used in patients with mCRPC and showed modest activity with PFS of 3.67 months and mOS 13.7 months.96 However, no other clinical trials are ongoing in PCa with this agent. Other FGFR inhibitors under investigation include futibatinib (FGFR 1–4 inhibitor) in a mutation-specific and solid tumor agnostic basket trial (NCT04189445). Further investigation is warranted by combining CDK4/6i’s with FGFR inhibitors in PCa.
Ras/Raf/MEK/ERK is an important signaling pathway involving MAPKs that are involved in regulating proliferation, differentiation and apoptosis.97 Amplification of members within the MAPK pathway is as high as 32% in patients with mCRPC.92 Phosphorylated ERK1/2 is associated with biochemical recurrence, rapid progression to CRPC and reduced disease-specific survival.92,98 The MAPK pathway influence on the cell cycle has been investigated in multiple studies. It has been suggested that Ras signaling is required for inactivation of tumor suppressor pRb within the cell cycle when Ras neutralizing antibodies caused G1 arrest.99 Ras signaling is also essential in inactivation of p53-mediated induction of p21Cip1 (Figure 5). This was demonstrated when loss of Ras resulted in transcriptional activation of p53.100 If p53 is activated, then this tumor suppressor leads to increase p21Cip1 and G1/S cell cycle arrest.100 p21 causes this arrest by suppressing activity of cyclin A/CDK2, cyclin A/CDK1, and decreasing transcription of E2F1, STAT3, and MYC.101 Paradoxically, p21 can bind CDK4/6 to increase kinase activity to promote progression through G1.101 The MAPK pathway has also been a means of CDK4/6i resistance. Resistant cells have been shown to have increased MAPK activation leading to CDK4/6-Rb bypass to induce aggressive phenotypes and metastasis.35 In PCa, these CDK4/6 resistant cells were sensitized to MEK inhibitors.35 Therefore, this suggests that MEK inhibitors could be used in combination with CDK4/6i’s to delay resistance or used sequentially after resistance occurs. Combining MEK inhibitors with CDK4/6i’s is being investigated in multiple tumor types including: KRAS mutant non-small cell lung cancer (NCT03170206),102 KRAS mutant colorectal cancer,103 pancreatic cancer,104 and melanoma.105 The MEK inhibitor, trametinib, is being investigated as single agent in mCRPC (NCT02881242); however, no combination trials are underway providing an opportunity to combine MEKi with CDK4/6i.
The guardian of the genome, tumor suppressor p53, has many roles essential for protecting against oncogenic transformation including: cell cycle arrest, DNA repair, senescence and cell death, modulation of autophagy, and cancer metabolism.106 G1 cell cycle arrest by p53 is mainly the result of transcriptional activation of p21 which binds and inhibits cyclin E/CDK2 and cyclin D/CDK4, thereby preventing Rb phosphorylation (Figure 5).107 The main regulator of p53 is a ubiquitin ligase, mouse double minute 2 (MDM2), which results in proteasome ubiquitination of the tumor suppressor.108 As mentioned earlier, Ras signaling is also essential in inactivation of p53-mediated induction of p21Cip1.100 Therapeutic targets within this pathway are needed since TP53 is mutated in over 50% of cancers and in mCRPC, an aberration causing loss of function is found in 36–53% of patients (Table 1).30–32 Mutated TP53 results in loss of function of wild-type p53 and gain of function in other aspects that lead to oncogenic activity.109 Patients with these mutations are found to have worse outcomes, progression and high rate of recurrence.110,111 Treatment strategies targeting TP53 mutated cancers are focused on restoration of wild-type p53 function (APR-246, MIRA-1, JNJ-26854165, Calcein AM, NSC59984), direct attack on p53 deficient cells, enhancement of normal p53 function (MDM2 inhibitors Nutlin-3a, RITA) and mimicking DNA damage with a virus.109 APR-246 is being investigated in multiple clinical trials in hematologic malignancies along with solid tumors including: ovarian (NCT02098343) and prostate (NCT00900614). In patients with mCRPC, a phase I trial with APR-246 was shown to be safe and tolerable when given to 7 patients intravenously for 4 consecutive days.112 Adverse effects included fatigue, dizziness, headache and confusion. Combining therapeutics that target p53 and CDK4/6 has been investigated in sarcoma cancer cells when MDM2 antagonists were combined with CDK4/6i’s in the preclinical setting.113 It resulted in reduced MDM2 antagonistic activity and diminished RNA polymerase II recruitment and decreased transcription of p53 target genes (MDM2 and p21).113 These effects led to antagonistic cytotoxic effects suggesting combining these agents may not be beneficial. This suggests CDK4-cyclin D1 complex has a positive impact on p53 as more checks and balances are needed when cells are entering the cell cycle. However, a separate study with liposarcoma xenografts showed synergy when MDM2 and CDK4/6 targeting agents were combined.114 There are no clinical trials ongoing combining APR-246 with CDK4/6i’s. Given the varying results, additional studies are warranted in combining these agents.
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