Recent advances of highly selective CDK4/6 inhibitors in breast cancer

Breast cancer is the most common female tumor type and accounts for the leading cancer mortality in women worldwide [1]. In spite of the great achievement in diagnosis and treatment, breast cancer remains a significant global burden [1]. Sequencing of breast cancer genome and transcriptome has identified breast cancer as a malignant disease with vast heterogeneity which is categorized into five distinct molecular subtypes including luminal A, luminal B, human epidermal growth factor receptor 2 (HER2)-enriched, basal-like, and claudin-low [2]. Among these, luminal-type accounts for the most part of breast cancer and is characterized with the typical expression of estrogen receptor (ER) and/or progesterone receptor (PR), which can be effectively targeted with hormone therapy. However, some patients have intrinsic resistance or acquired tolerance to hormone or endocrine therapy, which hampers the survival prolongation of these patients. Basal-like breast cancer, which is characterized with comparatively aggressive phenotype and the absent status of ER, PR and HER2, still lacks efficient treatment strategy. Thus, novel effective therapies are urgently required for breast cancer population.

Disordered cell cycle regulation is induced by complex mechanisms including the functional imbalance of oncogene and anti-oncogene, and contributes to uncontrolled cell proliferation resulting in cancer formation [3‐8]. The past decades have witnessed the great progress in developing novel effective therapies [9‐12], especially through diverting tumor cells from a proliferation phenotype towards a non-division state. Among the emerging therapies, cyclin-dependent kinase 4/6 (CDK4/6) inhibitors are the most attractive findings. CDK4/6 coordinates the cell cycle progression by reversible combination with cyclin D [13], and the bipartite complex of these elements phosphorylates pivotal tumor suppressors and transcription factors, contributing to cell cycle progression [14‐16]. The essential roles of CDK4/6 in cell cycle regulation make them effective targets for cancer therapeutic intervention, especially in breast cancer [17‐19]. The orally highly selective inhibitors of CDK4/6 are currently under active investigation, including palbociclib (PD0332991), ribociclib (LEE011), and abemaciclib (LY2835219). Among these, palbociclib and ribociclib remarkably prolonged the progression-free survival (PFS) in combination with letrozole for patients with ER-positive/HER2-negative advanced breast cancer, and have gained accelerated approval from Food and Drug Administration (FDA) as initial endocrine-based therapy for these patients.

In this review, we will first elaborate the anti-tumor mechanisms of CDK4/6 inhibitors, and then trace the preclinical and clinical evidence of these three highly specific inhibitors. At last, we will discuss the possible future directions in this field.

Cancer derives from uncontrolled cell division which results from the dysregulation of cell cycle progression including four stages of G1 (Gap phase 1), S phase (DNA synthesis), G2 (Gap phase 2), and M phase (mitosis) (Fig. 1). Cell cycle is monitored by a wide range of pathways including the retinoblastoma (RB)-E2F signaling [20]. RB, a well-known tumor suppressor, plays switching roles in cell cycle [20]. E2F is an evolutionarily conserved family of transcription factors, which functions in cell cycle control and contributes to tumor development [21]. The combination of RB and E2F makes E2F transcription modules in a suppressed state through inducing the recruitment of chromatin remodeling proteins, histone modifiers, and repressive chromatin marks, resulting in cell cycle block [22] (Fig. 1). The CDKs-RB axis is essential to cell cycle entry. CDK4/6 in combination with cyclin D, phosphorylates and inactivates RB [23], and then releases E2F, resulting in the recruitment of transcriptional activators, the alter transcription of genes involved during cell cycle process and subsequent G1-S block [22] (Fig. 1). Furthermore, the active combination of CDK4/6 and cyclin D is also involved in the phosphorylation of the cell proliferation-specific transcription factor forkhead box M1 (FOXM1), inducing the expression of genes which drive cell division and suppress cellular senescence in a FOXM1-dependent manner [24] (Fig. 1). However, the kinase activity of CDK4/6 is suppressed by p16^INK4A [25‐27] (Fig. 1), and cyclin D is regulated by a complex network such as ER/PR/androgen receptor (AR), nuclear factor kB (NF-kB), mitogen activated protein kinases (MAPKs), signal transducers and activators of transcription (STATs), Wnt/β-catenin and phosphatidylinositol 3-kinase (PI3K)/AKT/mTOR [24] (Fig. 1).

In addition, the active combination of CDK2 and cyclin E also participates in the phosphorylation of RB (Fig. 1). Besides, CDK2 is accumulating and active in stages S, G2, and M in combination with cyclin E and cyclin A, respectively, while CDK1/cyclin A/B complex mediates the transition from G2 to M stage [22] (Fig. 1).

In consideration of the pivotal role of CDK4/6 in cell cycle progression, amounting studies have been conducted to suppress cancer cell proliferation through targeting the CDK4/6 signaling for effective cancer therapies during the past decades. Several CDK inhibitors have been explored for potential tumor treatment and assessed for pharmacokinetics, efficacy, and safety in many clinical trials.

Flavopiridol, also named as alvocidib developed by Sanofi-Aventis, is the most extensively investigated one among the first generation pan-CDK inhibitors, showing inhibitory effects on CDK1, CDK2, CDK4, CDK6, CDK7, and CDK9 [28‐30], with half-maximal inhibitory concentration (IC₅₀) values ranging from 20 to 170 nM (Fig. 2). In addition to cell cycle inhibition, flavopiridol-associated distal cellular effects also include apoptosis, transcriptional suppression, autophagy and endoplasmic reticulum stress [31‐33], leading to several unacceptable high rates of dose-limiting toxicities, including neutropenia, hyperglycemia, cardiac, and pulmonary dysfunction [34]. The development of this non-selective compound was discontinued due to the low specificity for CDKs and narrow therapeutic window [17].

Subsequently, inhibitors highly selective for CDK4/6 were explored and developed, primarily through chemical screening and optimization via adding pyrido [2,3-d] pyrimidin-7-one compounds with a 2-amino pyridine side chain at the C2 position [35]. Chief among this new waves are palbociclib, ribociclib, and abemaciclib, all of which are orally administrated inhibitors and exhibit little or no suppression of other CDK activities at clinically achievable doses. Of these selective inhibitors, palbociclib is the most extensively investigated and has been evaluated in vitro and in vivo till recent times [36, 37]. It potently induces G1-S cell cycle block through blocking the phosphorylation of RB and related proteins and then down-regulating S-phase cyclins and mitotic regulatory genes as well as suppressing nucleotide biosynthesis and DNA replication [38, 39]. But its biological function has been limited because the anti-tumor effects of palbciclib is dependent on the presence of an active RB protein [36, 37]. Scientists at Eli Lilly and Novartis have also developed the parallel drugs ribociclib and abemaciclib, respectively [40‐44].

The highly selective inhibitors are analogous to the pan-CDK inhibitor flavopiridol in structure but different in chemical function. The chemical structures of these four CDK4/6 inhibitors described above are showed in Fig. 2.

Taken together, oral highly selective CDK inhibitors, including palbociclib, ribociclib, and abemaciclib, represent an important therapeutic advancement in breast oncology. Apart from the clinical success of palbociclib and ribociclib, abemaciclib is in active investigation and the favorable effects of this agent on PFS of ER-positive/HER2-negative advanced breast cancer were observed in clinical trials. However, there are still some challenges in the optimization of CDK inhibitors in clinical practice. Firstly, there is still lack of predictive biomarkers to screen appropriate population who can benefit most from these agents. Selection of sensitive patients can improve the cost-effective ratio of these drugs. Although several studies have implied some potential candidates for sensitivity prediction such as the protein levels of RB and p16, further extensive clinical trials are urgently needed before applied as clinically useful biomarkers [94]. In consideration that liquid biopsy is a new technique for monitor tumor progression and treatment response [95‐97], is it possible to identify potential biomarkers for predicting response to CDK inhibitors through analyzing circulating breast cancer cells or cell-free DNA? Secondly, aside from the already investigated combination strategies, whether CDK4/6 inhibitors in combination with other therapeutic regimens including chemotherapy, radiotherapy and immunecheckpoint inhibitors are also more effective than monotherapy is a problem, which is imperatively needed to be solved. For instance, there is some doubt whether palbociclib antagonizes the anti-tumor effects of cytotoxic chemotherapy and radiotherapy which function through killing cancer cells in cell cylce. Thirdly, triple-negative breast cancer, which is characterized with comparatively aggressive phenotype and the absent status of ER, PR, and HER2, is still lack of efficient treatment strategy. Previous study by Asghar U indicated that a subset of triple-negative breast cancer cells with expression of AR and the loss of cyclin E1 could be responsive to CDK4/6 inhibition [86]. Clinical trials are ongoing or in plans to address these questions.