Abstract

The cyclin D-cyclin-dependent kinase (CDK) 4/6 pathway controls the cell cycle machinery by regulating the G1-to-S-phase transition. Dysregulation of this pathway, resulting in increased cellular proliferation, is frequently observed in a variety of human cancers. Activation of cyclin D-CDK 4/6 pathway can occur through different mechanisms, including gene amplification/rearrangement, loss of negative regulatory factors, epigenetic modifications, and point mutations of different components of this pathway. Quite conspicuously, CDK 4/6 inhibitors have emerged as promising anticancer agents in various tumors in which CDK 4/6 has a pivotal role in the G1-to-S-phase cell cycle transition. The clinical use of first-generation, nonselective pan-CDK inhibitors was not progressed beyond early phase trials, due to unacceptable toxicity and lack of efficacy noted with these agents. The emergence of selective CDK 4/6 inhibitors, including ribociclib, abemaciclib, and palbociclib, has enabled us to effectively target cyclin D-CDK 4/6 pathway, at the cost of acceptable toxicity. The results of landmark phase III trials investigating palbociclib and ribociclib in advanced hormone receptor (HR)-positive breast cancer have demonstrated a substantial clinical benefit with a well-tolerated toxicity profile. Mechanisms of acquired resistance to selective CDK 4/6 inhibitors are beginning to emerge. Clearly, a detailed understanding of these resistance mechanisms is very much essential for the rational development of post-CDK 4/6 inhibitor therapeutic strategies. Extending the use of selective CDK 4/6 inhibitors beyond HR-positive breast cancer is a challenging task and will likely require identification of clinically meaningful biomarkers to predict response and the use of combination approaches to optimize CDK 4/6 targeting.

Introduction

Uncontrolled cellular proliferation, as a result of dysregulated cell division, is one of the key hallmarks of cancer, and identifying appropriate therapeutic targets to block cell division is a widely used strategy of anticancer therapy. Cyclin-dependent kinases (CDKs) control the transition from one stage of the cell cycle to the next, and they are activated upon interaction with their partner cyclins.[1] Therefore, quite conspicuously, CDKs have long been regarded as attractive therapeutic targets for cancer treatment. Unfortunately, many of the early first-generation CDK inhibitors failed in the clinical development because of nonselective pan-CDK inhibition, which was found to be toxic-to-nonmalignant cells.[2] These issues of effectiveness and toxicity of nonselective CDK inhibitors seem to have been overcome in the last decade by the development of selective CDK-targeting agents – which selectively target CDK 4/6.

Dysregulation of cyclin D-CDK 4/6 pathway is frequently observed in human cancers and results in uncontrolled cell cycle progression.[3] CDK 4/6 mediates the transition from G1 to S phase by associating with cyclin-D and regulating the phosphorylation of retinoblastoma (Rb) protein. Increased cyclin D-CDK 4/6 pathway activity can occur through several mechanisms, including overexpression of D-type cyclins, mutation or amplification of CDK 4/6, epigenetic alterations, or loss of negative regulators.[2],[3] Thus, the development of selective CDK 4/6 inhibitors offers a novel therapeutic approach in the field of oncology. Following the encouraging results of early phase clinical trials, three of the selective CDK 4/6 inhibitors (e.g., abemaciclib, palbociclib, and ribociclib) have emerged as agents with promising anticancer activity and acceptable toxicity profile,[4],[5],[6],[7],[8],[9],[10] and among them, palbociclib and ribociclib have already received FDA approval, with landmark phase III data available, in the setting of hormone receptor (HR)-positive, human epidermal growth factor receptor-2 (HER-2)-negative advanced breast cancer.[11],[12],[13],[14]

In this review, we discuss the rationale of selectively targeting CDK 4/6 pathway and the challenges with regard to optimizing their use. We also provide an overview of the currently available clinical data for selective CDK4/6 inhibitors in different human cancers, other than HR-positive, HER-2 negative breast cancer.

Overview of Cyclin D-Cyclin-Dependent Kinase 4/6 pathway Dysregulation

principle mechanisms by which the cyclin D-CDK 4/6 pathway can become dysregulated in various human cancers are amplification of the genes encoding cyclin D1 (CCND1) or deletion of the locus encoding CDKN2A. According to the published data, amplification of CCND1 is frequently found in some human cancers, for example, breast cancer (35% of cases), head-and-neck cancer (26%–39%), endometrial cancer (26%), pancreatic adenocarcinoma (25%), and nonsmall cell lung cancer (NSCLC) (5%–30%).[15],[16] In a recently reported landmark study, which investigated the role of routine molecular screening to identify actionable mutations in advanced refractory cancer patients, Cassier et al. found CCND1 amplification and homozygous deletion of CDKN2A in 17% and 21% of patients, respectively.[17]

The cyclin D-CDK 4/6 pathway can be dysregulated by multiple other mechanisms also, for example, mutations in the genes encoding various components of this pathway, epigenetic alterations, and mutations in the upstream factors. Haluska and Hodi found that about 20% of familial malignant melanoma cases harbor CDKN2A mutations.[18],[19] Epigenetic modifications of the CDKN2A gene have been reported in human ovarian cancer.[20] Jackson et al. highlighted the importance of mutations in the upstream factors as a mechanism of cyclin D-CDK 4/6 pathway dysregulation in malignant rhabdoid tumors, where the INI1/SMARCB1 gene is frequently mutated.[21]

Biologic Rationale of Selectively Inhibiting Cyclin-Dependent Kinase 4/6 in Human Cancers

The ideal CDK-targeted agents should block CDK-mediated signaling in malignant cells and at the same time should spare the aspects of CDK activity which are critical for the survival of nonmalignant cells, thus avoiding toxicity. Inhibition of CDK1 by nonspecific inhibitors could affect all cell types and result in toxicity, as evidenced by the reported fact that mouse embryos lacking CDK1 fail to develop beyond the blastocyst stage.[22] In addition, nonspecific targeting of CDKs might also result in inhibition of CDKs 7, 8, and 9, the exact functions of which are less well established.[23] Clearly, toxicity is a major concern regarding nonselective CDK-targeted agents because CDKs play a critical role in the proliferation of both normal cells and cancer cells.

The difficulty in finding a therapeutic window wherein CDK inhibition is both safe and effective was reflected in the early clinical experience with various nonselective CDK inhibitors, for example, flavopiridol and seliciclib. To date, the most well-studied nonselective CDK inhibitor is flavopiridol, which showed limited clinical benefit, mainly because of its complex pharmacokinetics and high levels of off-target effects.[24] Seliciclib, a purine-based compound that inhibits CDKs 1, 2, 5, 7, and 9, failed to demonstrate effective clinical activity in phase I studies.[25]

It is possible that cancers with known aberrations in the cyclin D-CDK 4/6 pathway will be more sensitive to CDK 4/6 inhibition than normal cells.[26] Furthermore, selective inhibitors spare CDK2 activity which allows normal cells to continue to function and proliferate. In addition, in contrast to the cytotoxic effects of pan-CDK inhibitors, selective CDK 4/6 inhibitors are usually found to have cytostatic effects, which might further limit the potential of these agents to cause significant clinical toxicity.[27]

Selective Cyclin-Dependent Kinase 4/6 Inhibitors in Cancer Therapy

As discussed earlier, after the encouraging results from preclinical studies, three CDK4/6 inhibitors have currently reached early phase clinical trials – abemaciclib, palbociclib, and ribociclib with published phase III data available for palbociclib and ribociclib, in the setting of HR-positive, HER-2-negative advanced breast cancer.[11],[12],[13],[14]

The next part of this review will focus on the currently available preclinical and clinical data of selective CDK 4/6 inhibitors in different human cancers, other than the archetypal model of ER-positive, HER-2-negative luminal breast cancer.

preclinical Data

Abemaciclib

It has been shown to reduce the phosphorylation of Rb1 in colorectal cancer and melanoma xenografts, thus inducing G1 arrest.[28] Abemaciclib has also been demonstrated to induce growth regression in vemurafenib-resistant melanoma models, in which expression of cyclin D1 was noted to be elevated in conjunction with mitogen-activated protein kinase (MApK) pathway reactivation in vitro.[29]

Ribociclib

Single-agent ribociclib has been shown to inhibit the growth of neuroblastoma and liposarcoma cell lines, by inducing G1 arrest and reducing Rb1 phosphorylation.[30] It inhibits CDK 4/6 effectively even at nanomolar concentrations.

palbociclib

It has been shown to be active in mantle cell lymphoma xenografts[31] and glioblastoma cell lines.[32] Moreover, activity of palbociclib in combination with bortezomib has been demonstrated in both acute myeloid leukemia and myeloma.[33],[34] In ovarian cancer cell lines, a response to palbociclib was found to be most marked in Rb1-proficient cell lines with low p16INK4A expression, and amplification of cyclin E1 was associated with resistance.[35]

Data from Early phase Clinical Trials

After the publication of promising results from preclinical research, quite conspicuously, selective CDK 4/6 inhibitors have been investigated in early phase clinical trials also.

Abemaciclib

The first-in-human phase I trial of abemaciclib enrolled 75 patients with advanced solid tumors.[4] The dose-limiting toxicity was Grade 3 fatigue. The most common treatment-related adverse events (AEs) included diarrhea (52%), nausea (32%), fatigue (21%), vomiting (21%), and neutropenia (19%). pharmacodynamic evidence of targeted CDK4/6 inhibition was observed, as shown by a decrease in Rb phosphorylation in the skin. In an expansion cohort of this trial in patients with NSCLC, 51

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