CELL CYCLE PATHWAY AND CDK4/6 INHIBITORS IN CANCER
Cellular proliferation begins with a stimulus allowing cells to enter G1 phase from the quiescent G0 phase. In order for a cell to continue through the cell cycle, it must pass checkpoints controlled by regulatory proteins. The regulatory proteins involved in the transition from G1 phase (growth/metabolism) to S phase (replication) include: cyclin D, CDK4, CDK6, Retinoblastoma gene product (Rb) and E2 transcription factor (E2F).7 Cyclin D is induced by growth factors and binds to CDK4 and CDK6 converting them to an active state. The cyclin-D-CDK4/6 complex phosphorylates the active tumor suppressor, Rb, sending it in to its hyper-phosphorylated inactive state. When Rb is in the active state it binds and inhibits the activating E2F transcription factors. However, when Rb is in the inactive hyper-phosphorylated state, Rb dissociates from the activating E2F transcription factor and drives E2F targeted genes involved in DNA replication allowing the cell to enter S phase.8 The transition from G1 to S is highly regulated by the CDK pathway involving cyclin D-CDK4/6-p16-Rb making CDK4/6 inhibitors prime agents for disrupting the cell cycle. There are multiple CDK4/6i’s that are FDA approved as anti-neoplastic agents including palbociclib, ribociclib, and abemaciclib for metastatic breast cancer.
Palbociclib (PD 0332991, Ibrance) was the first CDK4/6i to gain FDA approval in 2015. It was approved for combination therapy with letrozole in postmenopausal women with locally advanced or metastatic HER2-negative, estrogen receptor positive breast cancer.9 In PALMOA-1 and PALMOA-2, letrozole with and without palbociclib was given as first-line treatment to patients with ER-positive/HER2 negative advanced breast cancer and showed a mPFS prolongation of 10 months (20.2 months vs 10.2 months) and 10.3 months (24.8 months vs 14.5 months), respectively.10,11 The most common grade 3 and 4 adverse events seen were neutropenia (66%), leukopenia (24.8%), anemia and fatigue.11 Palbociclib is given on a 28-day cycle with daily dosing on days 1–21 and off on days 22–28. Clinical trials which are currently recruiting or active for PCa include: palbociclib in patients with mCRPC (NCT02905318) and a Phase II study of ADT with or without palbociclib in Rb-positive (ie, wild type) metastatic PCa (NCT02059213).
Ribociclib (LEE011, Kisqali) gained its first FDA approval in 2017 for treatment of postmenopausal hormone receptor (HR) positive HER2 negative metastatic breast cancer in combination with an aromatase inhibitor. MONALEESA-2 was a Phase III randomized placebo controlled trial investigating letrozole ± ribociclib in post-menopausal patients with HR positive and HER2 negative advanced breast cancer. The ribociclib group had an improved 18 month PFS, 63% vs 42.2% (Hazard Ratio 0.56), leading to its approval.12 The dosing regimen is the same as palbociclib with once-daily dosing for 21 days followed by 7 days off. Adverse effects are similar to palbociclib and may also cause prolong the QT interval (1–6%), limiting its use in patients with cardiac comorbidities.
Abemaciclib (LY2835219, Verzenio) was first approved in 2017 in women with HR positive, HER2 negative advanced breast cancer after recurrence following endocrine therapy. The phase III MONARCH-2, investigated abemaciclib or placebo with fulvestrant (anti-estrogen) in patients who are HR positive HER2 negative and progressed following endocrine therapy. The mPFS was 16.4 months compared to 9.3 months in the placebo group, leading to its approval. Due to its lower toxicity profile, dosing for abemaciclib is continuous, unlike the other two approved CDK4/6i’s. Common adverse effects of abemaciclib include: neutropenia (lower rate compared to palbociclib and ribociclib), diarrhea, nausea and fatigue. Tolerability of CDK 4/6 inhibitors and the benefit of oral administration provides an exceptional alternative to standard chemotherapy and is now first-line treatment for estrogen receptor (ER) positive metastatic breast cancer.
ANDROGEN RECEPTOR INFLUENCE ON CELL CYCLE IN PROSTATE CANCER
ADT has been the gold standard for PCa therapy due to androgen receptor (AR) activation causing PCa cell proliferation and survival. When androgens (ie, testosterone, dihydrotestosterone) enter the cell and bind to cytoplasmic AR, this leads to AR dimerization and subsequent nuclear translocation for genomic signaling. Activated AR dimers bind to DNA androgen response elements in promoter regions of genes to induce gene transcription such as prostate-specific antigen (PSA) and transmembrane protease serine 2 (TMPRSS2).13 This genomic AR signaling pathway influences prostate cancer proliferation, invasion and survival. Rapid non-genomic AR signaling by way of ligand-transformed AR associating with molecular substrates in the cytoplasm and inner leaflet of the cell membrane activating kinase cascades also occurs resulting in enhancement of cell proliferation and survival with many of these pathways regulating the cell cycle (Figure 1).14 This non-genomic AR signaling by the Src, Ras/Raf, protein kinase C, and AKT/PI3K pathways activate mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) to cause increase cell proliferation.15,16 At low androgen concentrations (0.01 −10 nM), AR N-terminal domain binds to Src homology domain 3 (SH3) which results in Src unfolding and autophosphorylation. This activates Src and causes enhanced cell proliferation via MAPK/ERK.17 As prostate cancer progresses, aberrant Src activation occurs, independent of androgens, as a result of increased Src expression or stimulation via growth factors and interleukins.18,19 In breast cancer, c-Src suppression resulted in down regulation of cyclin D1 and increase p27kip1 (intrinsic CDK4/6 inhibitor) suggesting the Src pathway regulates cell cycle progression.20 AR has also been found to activate the PKC pathway, which is regulated by modulation of intracellular calcium leading to activation of MAPK/ERK.21 While the activation of MAPK/ERK by PI3K/AKT pathway relies on AR’s activation of the p85α subunit of PI3K and AR’s interactions with Src leading to activation of AKT and subsequent MAPK/ERK activation.22 Non-genomic AR signaling independent of MAPK/ERK also occurs leading to increase cell proliferation. This is seen with AR causing increase intracellular calcium which not only activates PKC, but also activates protein kinase A which interacts with transcription factors promoting cell proliferation.21 Another AR signaling effect not reliant on MAPK/ERK includes AR interacting with PI3K/AKT causing phosphorylation of the tumor suppressor transcription factor Forkhead box O1 (FOXO1) causing it to be retained in the cytoplasm for degradation preventing its pro-apoptotic effects,23 in addition, PI3K/AKT phosphorylation of mTOR also causes increase cell proliferation.
Through all these pathways mentioned above, AR has been found to be a key regulator of transcription of genes that allow for G1 to S transition.5 Further investigation of AR’s role in the cell cycle has revealed its effects on cyclin D1, a substrate for activating CDK4 and CDK6. The mechanism for increasing cyclin D1 is through the activation of MAPK and Akt24,25 as well as via mammalian target of rapamycin (mTOR) and subsequent upregulation of protein translation.26 Androgens, via activated AR, also have effects on the intrinsic CDK inhibitors p21, p27 and p16. Androgens have been shown to transcriptionally downregulate the CDK inhibitors p21 and p27.27 Therefore, these pathways via MAPK, Akt, and mTOR increasing cyclin D1 and reduction of intrinsic CDK4/6i’s (p21 and p27), all promote inactivation of Rb tumor suppressor allowing the cell to progress from G1 to S phase. Using ADT will influence these pathways, unfortunately, these androgen sensitive PCa cells will eventually become androgen-independent by way of AR mutations, AR amplification and aberrant activation of AR.28 To circumvent this resistance, CDK 4/6 inhibitors have been investigated to disrupt these AR signaling pathways, in turn, decreasing their cancer cell promoting effects. Preclinical models using palbociclib revealed CDK 4/6 inhibitors can be used in the treatment of PCa. Early models showed this agent limited proliferation in HSPC and CRPC cells in vitro, as well as xenografts and primary human tumors ex vivo.29
In the evolving paradigm of precision medicine, genomic analysis of metastatic PCa has shown many alterations that utilize the cell cycle to promote survival and growth.30–32 Therefore, patients with these alterations, such as amplification of cyclin D1 gene (ie, CCND1), found in 4.7% of mCRPC patients (Table 1), should be more responsive to CDK4/6i’s. However, it is important to note that some genomic alterations found in PCa may make these agents ineffective. For example, patients with Rb gene (ie, RB) loss, p16INKa high, cycle E1/E2 amplification or E2F amplification would likely be resistant.6 In patients with mCRPC, RB loss is found in 9–17% of patients.30,31 Therefore, RB expression and potentially genomic analysis should be monitored to predict response, especially in patients who have CRPC.
CURRENT CLINICAL TRIALS WITH CDK 4/6 INHIBITORS IN PROSTATE CANCER
There are currently five clinical trials (Table 2) involving CDK 4/6 inhibitors in PCa, excluding two mutation-specific, tumor agnostic “basket” trials using palbociclib. A phase II trial (NCT03706365) is evaluating the safety and effectiveness of abiraterone with and without abemaciclib in patients with mCRPC. Ribociclib is being evaluated in PCa in two separate trials. A phase IB/II trial (NCT02555189) is investigating enzalutamide with and without ribociclib in patients with mCRPC who are chemotherapy-naïve and retain Rb expression. The second phase II trial (NCT02494921) is evaluating ribociclib with docetaxel in patients with mCRPC. There are two clinical trials testing palbociclib’s use in PCa. A phase II trial (NCT02905318), using palbociclib in patients with PCa, is recruiting to evaluate side effects, but also determine markers that could predict response to palbociclib. The only CDK4/6i trial with results to date is a phase II trial (NCT02059213) evaluating ADT with and without palbociclib in patients with mHSPC who are RB-positive.33 Twenty patients were randomized to ADT alone and 40 to ADT plus palbociclib. The primary outcome was proportion of patients who achieved a PSA < 4 ng/mL after seven months of treatment. The primary PSA endpoint was met in 80% of patients in both arms with a p-value of 0.87 for superiority. All-cause mortality in both groups was 0%. Serious adverse events occurred in 25% of patients with ADT alone and 17.5% with ADT plus palbociclib. In the palbociclib group, Grade 3 or 4 neutropenia was seen in 33% of patients. The clinical PFS is not mature, but 12-month biochemical PFS in ADT was 69% vs 74% in palbociclib.33 In this study 97% of patients were RB-positive. Further investigation is warranted in PCa in order to find the appropriate patient population based on genomic alterations and potential synergistic combinations to make these agents more efficacious.
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