D-type cyclins form a complex with CDK4 and CDK6, and promote tumorigenesis by inducing cell cycle transition from G1 phase to S phase. Cyclin D1 is overexpressed in more than 50% of all breast cancers, and CDK4 and cyclin D1 amplification have been reported in up to 25% of breast cancers.

Because CDK4 and CDK6 regulate the cell cycle, they have been attractive targets for cancer treatment for many years. First-generation CDK inhibitors were broad and nonselective, which likely explained why they did not exhibit activity in clinical trials. Expanding knowledge of the cyclin D:CDK4 and CDK6 complex led to the development of second-generation CDK inhibitors, which selectively target CDK4 and CDK6, leading to greater clinical success.

The D-type cyclins were first discovered in 1991. In budding yeast cells and murine macrophages, D-type cyclins were found to induce the transition from G1 phase to S phase of the cell cycle. Subsequently, the corresponding genes and amino acid sequences were identified for cyclins D1, D2, and D3 in humans. The D-type cyclins were determined to have potential oncogenic properties because they responded to mitogen signals and were found to be involved in parathyroid adenoma pathogenesis.

CDK4 was the first cyclin-dependent kinase that was discovered to bind to and be activated by the 3 D-type cyclins. Shortly thereafter, CDK6 was identified and was found to be similar in structure and function to CDK4. Once activated by D-type cyclins, CDK4 and CDK6 phosphorylated the retinoblastoma protein (pRb), causing it to inactivate and induce the cell to transition from G1 to S phase.

CDK4 in particular has nearly exclusive specificity for pRb.

Other CDKs were subsequently discovered, including transcriptional, mitotic, and regulatory CDKs.

Because the cyclin D:CDK axis regulates the cell cycle, several attempts have been made to target this pathway in the treatment of cancer since the early 1990s. Flavopiridol, also known as alvocidib, was the first CDK inhibitor to enter clinical trials.

It was also the most studied first-generation CDK inhibitor, with more than 60 clinical trials over the course of 15 years.

Flavopiridol is derived from the chromone alkaloid, rohitukine, and is administered intravenously.

Although flavopiridol inhibited CDK4 and CDK6, it also suppressed multiple other CDKs, including transcriptional CDK9. As a result, flavopiridol induced not only cell cycle arrest, but also cytotoxicity by inhibiting transcription.

Despite the fact that flavopiridol demonstrated in vitro activity against cancer, preclinical success did not translate into clinical activity.

Flavopiridol had low-to-minimal efficacy in phase I and II trials in patients with cancer who experienced toxicities similar to those of conventional chemotherapy agents, including infusion site irritation and gastrointestinal toxicity. In addition, patients with breast cancer in a phase I trial experienced overly high rates of neutropenia.

Despite these disappointing results, flavopiridol demonstrated clinical activity in chronic lymphocytic leukemia, with a 40% response rate in one early-phase trial. However, subsequent phase III studies were not pursued.

Seliciclib, or R-roscovitine, is a purine-based, nonselective CDK inhibitor. In a phase I trial, seliciclib induced a partial response in only 1 of 56 patients. In APPRAISE, aphase II trial in non-small cell lung cancer (NSCLC), seliciclib did not improve efficacy, and the study was terminated.

UCN-01, a staurosporine analog and broad CDK inhibitor, showed efficacy in preclinical studies, but had significant toxicities in a phase I trial, including arrhythmia and hyperglycemia. In a phase II trial of patients with breast cancer, UCN-01 did not demonstrate efficacy.

The poor specificity of early CDK inhibitors was likely the primary reason for their lack of efficacy. Broad coverage combined with off-target effects resulted in a small therapeutic window, with significant toxicities such as diarrhea, neutropenia, and anemia that limited further dose escalation. Finally, the study populations were nonselective in the absence of any biomarkers to identify patients who might be likely to respond to treatment.

After the failures of multiple CDK inhibitors, efforts were made to develop more selective agents to achieve greater efficacy and reduce off-target effects. From 1993 to 2000, multiple attempts to synthesize a CDK4 inhibitor were unsuccessful.

Then, in the early 2000s, PD0332991 entered development and eventually became the CDK4&6-selective inhibitor, palbociclib. Success in clinical trials led the US Food and Drug Administration (FDA) to approve palbociclib plus fulvestrant for treatment of estrogen receptor (ER)‒positive, human epidermal growth factor receptor (HER2)‒negative, advanced breast cancer that has progressed on prior endocrine therapy.

Other CDK4 and CDK6 inhibitors have emerged as well, including abemaciclib and ribociclib, both of which have demonstrated clinical activity in the clinical setting and have entered phase III trials.

TABLE. Phase III Trials Exploring CDK 4&6 Inhibitors

Abemaciclib plus fulvestant in HR+/HER2- MBC (not recruiting)

Abemaciclib plus an NSAI in HR+/HER2- MBC (not recruiting)

Abemaciclib versus erlotinib in KRAS-mutant NSCLC (currently recruiting)

Palbociclib with exemestane versus capecitabine in HR+/HER- MBC following NSAI resistance (currently recruiting)

Palbociclib plus endocrine therapy following neoadjuvant chemotherapy in HR+/HER2- breast cancer (currently recruiting)

Palbociclib with adjuvant endocrine therapy in HR+/HER2- breast cancer (currently recruiting)

Ribociclib with fulvestrant in HR+/HER2- MBC (currently recruiting)

Ribociclib plus letrozole for HR+/HER2- MBC (not recruiting)

Ribociclib or placebo with an NSAI/goserelin or tamoxifen/goserelin in HR+/HER2- MBC (currently recruiting)

D-type cyclins and CDKs regulate the cell cycle via the Rb pathway and underlie the tumorigenic mechanism for several types of cancer. When cells in G1 phase encounter mitogenic and adhesion signals, they synthesize cyclin D1, which binds to and activates CDK4 and CDK6, known as interphase CDKs. D2 or D3 cyclins may also activate CDK4 and CDK6 by a similar process.

The cyclin D:CDK4 and CDK6 complex then goes on to phosphorylate and inactivate pRb, a tumor suppressor that sequesters the E2F family of transcription factors. Thus released, E2F facilitates the transcription of S-phase target genes, driving the cell from G1 into S phase. The product of one transcription target, cyclin E, binds to CDK2, forming a complex that further phosphorylates, and deactivates pRb and other molecules that regulate the G1/S checkpoint. A positive feedback loop results, and cell cycle progression and proliferation proceed independent of further mitogen signals. Cyclin D:CDK4 and CDK6 may also phosphorylate components of other proliferation and migration pathways, such as SMAD2 and FOXM1.

Several negative regulators, known as cyclin kinase inhibitors, block the cyclin D:CDK4 and CDK6 complex. The INK family, which includes p16INK4a, and the CIP/KIP family, which includes p21 and p27, are upregulated in the presence of inhibitory signals, such as transforming growth factor-&beta; and senescence. By binding to CDK4 and CDK6, p16 INK4a prevents D1 from forming a complex with CDK4 and CDK6, thereby inducing cell cycle arrest and senescence. Dysregulation of p16 INK4a activity has been found to drive the pathogenesis of many cancers. In breast cancer, loss of p21 and p27 may mediate resistance to estrogen treatments.

Overexpression of D-type cyclins appears to be required to initiate tumorigenesis in breast cancer. In fact, mouse models that lack D1 or D3 are resistant to cancers driven by RAS, ErbB-2/HER2/neu, and Notch1 oncogenes. In addition, the cyclin D:CDK4 and CDK6 complex is necessary for continued oncogenesis and tumor proliferation in breast cancer. Inhibiting CDK4 and CDK6 or cyclin D1 in vivo blocks further tumor growth and leads to senescence.

In a genomic analysis performed by The Cancer Genome Atlas in 510 tumor specimens, CDK4 amplification and cyclin D1 overexpression were most frequently present in luminal A, B, and HER2-enriched tumors. Basal-like tumors were more likely to have pRb loss and less concomitant cyclin D:CDK4 and CDK6 dysregulation, reflecting the dependence of the cyclin D:CDK4 and CDK6 complex on functional pRb.

In both ER- and HER2-positive breast cancer types, endocrine signaling has downstream effects on the cyclin D:CDK4 and CDK6 axis. In particular, estrogen signaling in ER-positive cancer amplifies cyclin D1 levels and CDK4 and CDK6 activity.

Antiestrogen agents have been shown to induce cell cycle arrest and subsequent decreased expression of cyclin D1.

Among patients with breast cancer who do not respond or develop resistance to hormone-based treatment, other dysregulated mitogenic pathways, including HER2, may enhance cyclin D1:CDK4 and CDK6 activity independent of ER signaling. Thus, the cyclin D1:CDK4 and CDK6 complex may be a reasonable target for treatment of ER-positive breast cancer that is resistant to standard endocrine therapy.

Palbociclib is an orally bioavailable, selective, reversible CDK4&6 inhibitor. In preclinical trials, palbociclib achieved tumor stasis by blocking CDK4 and CDK6-mediated pRb phosphorylation in cell lines with functional pRb. Lack of pRb phosphorylation led to downregulation of E2F target genes and loss of proliferation marker Ki67. Among patients with mantle cell lymphoma, posttreatment biopsies showed decreased fluorothymidine-PET uptake compared with pretreatment samples. Xenograft models treated with palbociclib did not exhibit significant toxicity. Models lacking CDKN2A, which encodes tumor suppressor p16, responded to palbociclib, whereas those without functional pRb did not. When palbociclib was withdrawn and then reintroduced, tumors did not exhibit resistance, but they did resume proliferation in the absence of palbociclib, demonstrating the reversible nature of palbociclib-mediated CDK4&6 inhibition.

In clinical trials, palbociclib significantly prolonged progressionfree survival (PFS) among patients with ER-positive, HER2-negative genetic status. In the phase III trial, PALOMA-3, among women with breast cancer that had progressed on prior endocrine therapy, those who were treated with palbociclib plus fulvestrant had a longer PFS than those treated with fulvestrant only. The results of PALOMA-3 led to FDA approval of palbociclib plus fulvestrant for this indication.

PALOMA-2 is a phase III trial that was reported to have met its primary endpoint of improved PFS in women with previously untreated breast cancer who received palbociclib plus letrozole versus letrozole alone. Final results are pending at this time.

The high specificity of palbociclib for cyclin D:CDK4 and CDK6 complexes may explain the efficacy and tolerable toxicity profiles seen in clinical trials. The half-maximal inhibitory concentration (IC50) of palbociclib for D1:CDK4, D3:CDK4, and D2:CDK6 are 11 nmol/L, 9 nmol/L, and 15 nmol/L, respectively. In contrast, palbociclib is far less selective for other CDKs, with the IC50 for CDK1 and CDK2 greater than 10 &mu;mol/L for each target. The major dose-limiting toxicity (DLT) for palbociclib is myelosuppression, particularly neutropenia. &ldquo;Given the reliance of myeloid development in the mouse on cyclin D2- and D3-driven CDK6, these results might have been anticipated,&rdquo; noted Charles Sherr, MD, PhD, with Howard Hughes Medical Institute in Chevy Chase, MD, and colleagues, in an article reviewing CDK4&6 inhibitors.

As a result, palbociclib must be dosed intermittently at 125 mg daily for 3 weeks followed by 1 week off treatment, or 200 mg daily for 2 weeks with 1 week off treatment. Other adverse effects (AEs) are mild and include anemia, nausea, and fatigue.

Ribociclib is also an oral selective CDK4&6 inhibitor that primarily targets cyclin D1:CDK4 and cyclin D3:CDK6, with IC50 10 nmol/L and 39 nmol/L, respectively.

Like palbociclib, it has poor specificity for CDK1 and CDK2, with a corresponding IC50 of greater than 50 &mu;mol/L each. Preclinical data from paired tumor biopsies demonstrated lower Rb phosphorylation and Ki67 expression posttreatment. As with palbociclib, ribociclib has DLTs of myelosuppression and neutropenia, possibly because ribociclib also targets the cyclin D3:CDK complex. Thus, ribociclib requires intermittent dosing with 600 mg daily for 3 weeks followed by 1 week off per cycle. Other AEs include mucositis and a prolonged QTc interval on electrocardiogram.

Ongoing phase I/II clinical trials in patients with breast cancer have demonstrated preliminary clinical activity of ribociclib in combination with exemestane and everolimus, as well as with letrozole and PI3K inhibitor, BYL719.

Ribociclib development has been expedited based on the success with palbociclib, and is currently in phase III trials for treatment of ER-positive, HER2-negative breast cancer (NCT01958021, NCT02422615, NCT02278120).

Similar to palbociclib and ribociclib, abemaciclib is an oral, selective, small-molecule inhibitor of CDK4&6. Compared with the other agents, abemaciclib is a more potent inhibitor of CDK4 and CDK6 via their complexes with cyclin D1, with IC50 0.6 to 2 nmol/L and 2.4 to 5 nmol/L, respectively. In contrast, the IC50 of palbociclib and ribociclib for their targets are 10 nmol/L or higher. The greater potency of abemaciclib for its targets and its selectivity for cyclin D1 may explain the lack of bone marrow toxicity as a major dose-limiting factor. As a result, abemaciclib may be dosed continuously at 200 mg daily without any off-treatment weeks. Other AEs include diarrhea and neutropenia, which may be alleviated by reducing the dosage.

In preclinical trials, tumor biopsies after abemaciclib treatment have demonstrated decreased pRb phosphorylation and topoisomerase Ii&alpha; expression compared with pretreatment samples.

Phase I studies have shown clinical benefit and disease control rates of more than 70% with abemaciclib monotherapy, and these results led to breakthrough therapy designation by the FDA in 2015.

A phase II trial is investigating abemaciclib as monotherapy for refractory, ER-positive, HER2-negative breast cancer (NCT02102490). As with ribociclib, abemaciclib development has also been expedited to phase III trials, which are evaluating abemaciclib in combination with fulvestrant or nonsteroidal aromatase inhibitors in patients with breast cancer (NCT02107703, NCT02246621).

While the importance of the cyclin D:CDK pathway in the pathogenesis of many cancers has been recognized for decades, early attempts to develop effective CDK inhibitors have been unsuccessful. In recent years, advances in our understanding of the cyclin D:CDK4 and CDK6 axis have refined our ability to produce agents that selectively target key components while minimizing off-target toxicity.

Continuing to elucidate the intricacies of the cyclin D:CDK4 and CDK6 pathway and its interactions with other molecular drivers of cancer will enable us to identify additional, more-specific targets, and to discover biomarkers that will help guide clinical decision making and personalize treatment.

&ldquo;Despite the decades required for drug discovery and clinical applications, much remains to be learned,&rdquo; Sherr concluded in his CDK4&6 inhibitor review article. &ldquo;Future work will indicate whether the promise of CDK4&6 inhibitors, most advanced for breast cancer, can be validated and extended to other cancers.&rdquo;

Sherr CJ, Beach D, Shapiro GI. Targeting CDK4 and CDK6: from discovery to therapy. Cancer Discov. 2016;6(4):353-367.

Finn RS, Aleshin A, Slamon DJ. Targeting the cyclin-dependent kinases (CDK) 4/6 in estrogen receptor-positive breast cancers. Breast Cancer Res. 2016;18(1):17.

Asghar U, Witkiewicz AK, Turner NC, Knudsen ES. The history and future of targeting cyclin-dependent kinases in cancer therapy. Nat Rev Drug Discov. 2015;14(2):130-146.

Murphy CG, Dickler MN. The role of CDK4/6 inhibition in breast cancer. Oncologist. 2015;20(5):483-490.

US Food and Drug Administration. Palbociclib (IBRANCE Capsules). 2016. http:// www.fda.gov/Drugs/InformationOnDrugs/ApprovedDrugs/ucm487080.htm. Accessed May 10, 2016.

Articles

Clinical Articles

More than 20 years have passed since the cyclin-dependent kinases 4 (CDK4) and CDK6 were discovered to drive oncogenesis in several cancer types.

Overcoming Obstacles: Long Road to Success for CDK Inhibition

CDK4 and CDK6-related oncogenesis is not limited to breast cancer. Recent data from preclinical trials reveal that the cyclin D:CDK4 and CDK6 complex drives dysregulated cell proliferation in other cancer types. In addition, these preclinical findings shed light on the interactions between CDK4 and CDK6 and other oncogenes, which may hold relevance as potential biomarkers and provide new marks for targeted therapies.

CDK4 belongs to the family of serine and threonine protein kinases, and is involved in regulating the checkpoint between the G1 and S phases of the cell cycle.

When D-type cyclins are activated by mitogenic signals, they bind to and activate CDK4 and CDK6, which have similar amino acid makeup and functions.

CDK4 has a high and nearly exclusive specificity for the retinoblastoma tumor suppressor gene product (pRb).

By phosphorylating pRb, CDK4 allows the release of previously sequestered E2F transcription factors, which then drive the transition from G1 to S phase. E2F transcription factors also phosphorylate pRb, leading to further release of E2F, creating a positive feedback loop. As a result, upregulated CDK4 activity induces cells to undergo an irreversible transition to the S phase, and to continue propagating independent of mitogen signals.

The activity of the cyclin D:CDK4 and CDK6 axis is inhibited, however, by p16INK4a and the CIP/KIP family of proteins (p21, p27, p56). When the cell encounters inhibitory signals, such as transforming growth factor-&beta;, p16INK4a and CIP/KIP protein synthesis rapidly increases. These negative regulatory molecules then bind to and inhibit the cyclin D:CDK4 and CDK6 complex. Functional p16INK4a appears to be necessary to activate cellular senescence, and loss of p16INK4a is involved in the pathogenesis of many cancers.

While dysregulation of the cyclin D:CDK4 and CDK6 axis mediates tumorigenesis in several types of cancer, the role of CDK4 in oncogenesis has been most comprehensively studied in breast cancer. Preclinical studies have shown that the ER and HER2 signaling pathways both exert downstream effects on the cyclin D:CDK4 and CDK6 process. Because 60% of breast cancers are ER-positive and 25% are HER2-positive, the cyclin D:CDK4 and CDK6 complex is central to tumorigenesis for the majority of breast cancers.

CDK4 also plays an important role in developing resistance to therapy for both ER-positive and HER2- positive breast cancers. In HER2-positive tumors, cyclin D1:CDK4 mediates resistance to hormonal treatment. In HER2-positive mouse models and human breast cancer cell lines, CDK4&6 inhibitors have been shown to resensitize tumor cells to anti-HER2 treatment. CDK4&6 inhibitors attenuated mTORC1 activity by decreasing TSC2 phosphorylation, thereby reducing feedback inhibition of upstream epidermal growth factor receptor (EGFR) kinases and restoring tumor sensitivity to anti- EGFR/HER2 agents.

In ER-positive breast cancer, ER signaling upregulates CDK4 and CDK6 levels by increasing cyclin D1 levels and enhancing other signaling pathways. The link between D-type cyclins and breast cancer is further illuminated by the finding that most breast cancers with upregulated cyclin D1 are ER-positive. Acquired resistance to hormone therapy may occur as a result of ER-independent dysregulation of mitogenic pathways that potentiate the cyclin D1:CDK4 and CDK6 axis.

Increased CDK4 activity is essential for oncogenesis in other cancer types, as well. In melanoma, CDK4&6 inhibition destabilized FOXM1 and induced senescence in mouse models and human cell lines, suggesting that FOXM1 acts as an alternate substrate for CDK4 and CDK6. Nonsmall cell lung cancer (NSCLC) models with KRAS-driven disease demonstrated apoptosis after CDK4 inhibition. This effect was sustained in the clinical setting: A subsequent phase I study of CDK4&6 inhibitors in NSCLC showed that patients with the KRAS mutation had higher disease control rates (DCRs) than those who did not.

In liposarcoma cell lines with chromosome 12q14 amplification of linked CDK4 and MDM2, CDK4&6 inhibitor palbociclib induced G1 cell cycle arrest within 48 hours. After palbociclib was removed, only the cells that expressed senescence biomarkers remained quiescent. In addition, proteolytic turnover of MDM2 was necessary to maintain senescence. Among patients from whom the cell lines were acquired, those with significantly reduced MDM2 expression had the greatest clinical benefit, with progression-free survival (PFS) ranging from 160 days to more than 800 days. Of note, pRb phosphorylation was reduced in clinical responders, as well.

Finally, preclinical models of glioblastoma multiforme found that the presence of simultaneous deletions in the genes that encode p16INK4a and p18INK4c (CDKN2A and CDKN2C, respectively) was associated with increased sensitivity to CDK4&6 inhibitors.

The wealth of new information from preclinical studies indicates that CDK4 plays a significant role in driving oncogenesis in many cancer types outside of breast cancer. Further elucidating the mechanisms of CDK4-induced oncogenesis in these cancers will provide the key to developing more-effective targeted therapies, and clinical trials will help determine the potential of CDK4&6 inhibitors in treating malignancies other than breast cancer.

A preclinical study by Xiaofeng Dai, with the School of Biotechnology and National Engineering Laboratory for Cereal Fermentation Technology at Jiangnan University in Wuxi, China, and colleagues demonstrated the link between deleted in liver cancer- 1 (DLC1) and CDK6. DLC1 is a tumor suppressor gene that is inactivated by deletions or DNA methylation in many cancers. When functioning normally, DLC1 deactivates RhoGTP by catalyzing its conversion to RhoGDP. As a Rho family GTPase, DLC1 acts as a molecular switch that promotes tumorigenesis via cell cycle progression and malignant transformation. Preclinical data shows that reactivating DLC1 suppresses tumor cell proliferation.

Synergy between DLC1 and CDK6 has been demonstrated in lung, colon, and breast cancers. The combination of high DLC1 and low CDK6 expression correlates with a good prognosis, and the converse denotes poor prognosis. When the DLC1 intronic single nucleotide polymorphism (SNP) rs561681 is heterozygous and occurs with the rare homozygous variant of CDK6 intronic SNP rs3731343, the effect is deleterious; however, when heterozygous rs561681 occurs with the common homozygote of SNP, rs3731343, the effect is protective. The link between DLC1 and CDK6 is also demonstrated by the function of caveolin 1, which enhances the tumor suppressive function of DLC1 while inhibiting CDK6 via downstream effects.

Results from this study also demonstrated a connection between ER, DLC1, and CDK6. In ER-positive breast cancer models, DLC1 enabled anchorage-independent cell growth via estrogen-induced transactivation of liganded ER. Estrogen also induced DLC1 transcription and expression. D-type cyclins may act upstream of the ER pathway to potentiate estrogeninduced transactivation.

In ER-positive breast cancer cells, nonrare homozygotes of DLC1 SNP rs561681 have a protective effect, which is enhanced by the presence of nonrare CDK6 SNP rs3731343 homozygotes. As a result, outcomes tend to be better in patients without mutations, with high DLC1 expression and no CDK6 overexpression. On the other hand, rare homozygotes of DLC1 SNP rs561681 have low copy numbers of DLC1, which correlates with a poor outcome. Finally, heterozygous dimers of DLC1 SNP rs561681 exert a dominant negative effect on CDK6 SNP rs3731343, in which levels of functional DLC1 are lowest in cells with heterozygous DLC1 SNP rs561681 and rare homozygous CDK6 SNP rs3731343.

The authors concluded that, &ldquo;In this study, we have linked the germline genetic polymorphisms and their synergistic effect with breast cancer progression, which provides the basis for experimentally elucidating the mechanisms involved in differential progression. The findings are particularly useful in investigating inherited susceptibility and their cooperative roles in tumor progression, with the ultimate goal of tailoring clinical treatments for breast cancer patients based on their genetic susceptibility.&rdquo;

Continuous Inhibition and Cell Senescence

Continuous inhibition of the cyclin D:CDK4 and CDK6 axis appears to be required to maintain cellular senescence. In mouse models, senescence may be reversed by inactivating pRb through phosphorylation. The presence of functional pRband by corollary, inhibition of the cyclin D:CDK4 and CDK6 axis&mdash;appears to be necessary to induce and maintain senescence.

Sara Tolaney, MD, with Dana-Farber Cancer Institute in Boston, MA, noted that, &ldquo;Preclinical data suggests that continuous administration of CDK4&6 inhibitors may allow for continued cellular senescence.&rdquo; In xenograft cancer models, tumors resumed growth after palbociclib was withdrawn, but reintroducing palbociclib halted further tumor progression. In mouse models, pharmacokinetic and pharmacodynamic studies showed that dosing abemaciclib at a frequency of twice daily was necessary to ensure adequate target coverage.

The importance of continuous dosing plays out in the clinical setting as well. Patients with melanoma who were treated with abemaciclib once daily had evidence of partial reversibility in skin biopsies before the next dose. Even when each individual dose of abemaciclib was lowered, maintaining a continuous dosing regimen appeared to be sufficient for achieving target coverage. In phase I and II trials of abemaciclib for breast cancer treatment, pharmacodynamic CDK4&6 inhibition was maintained even when the dosage was decreased from 200 to 150 mg twice daily for side effects.

Unlike palbociclib and ribociclib, which must be dosed on a schedule with a week off each cycle, abemaciclib does not often result in significant neutropenia, and its continuous dosing regimen is well-tolerated.

&ldquo;Being able to administer abemaciclib on a continuous basis may explain its higher response rates as a single agent, compared with palbociclib and ribociclib,&rdquo; Tolaney stated.

Combination Therapy and Treatment Sequencing

The mechanism of action of CDK4&6 inhibitors may present a problem when they are combined with other anticancer agents. &ldquo;Because CDK4&6 inhibitors cause cell cycle arrest, there have been concerns that they may induce senescence in T-cells and therefore dampen the immune system,&rdquo; Tolaney acknowledged. Immune checkpoint inhibitors used as anticancer agents are antibodies that bind to negative regulatory molecules, such as programmed cell death 1 (PD-1)/programmed death ligand 1 (PDL1), in order to induce T-cell proliferation and differentiation. But T-cell expansion is dependent on the cyclin D:CDK4 and CDK6 axis and may be constrained by CDK4&6 inhibition.

Tolaney indicated that CDK4&6 inhibitors may not blunt the effects of immune checkpoint inhibitors. She noted that &ldquo;preclinical data looking at T-cell populations before and after exposure to CDK4&6 inhibitors suggest that the abundance of Tcells is largely preserved with a reduction in regulatory T-cells. Expression of immune checkpoint proteins on CD8 cells was also reduced.&rdquo; These findings provided the rationale for combining abemaciclib with pembrolizumab as a treatment strategy for breast cancer in a phase I trial (NCT02079636).

Another concern is that CDK4&6 inhibitors may counteract the effects of certain cytotoxic cancer treatments by inducing cell cycle arrest in the G1 phase in both cancerous and healthy cells.

As a result, cancer cells may escape apoptosis from ionizing irradiation and DNA-damaging agents, which target cells in the S or M phases. However, sequencing treatments may help circumvent this potential problem.

In addition, CDK4&6 inhibitors may be used to protect normal hematopoietic cells during cytotoxic chemotherapy. For example, palbociclib during carboplatin administration decreased rates of myelosuppression in a Rb-deficient, HER2-driven breast cancer mouse model. Thus, using CDK4&6 inhibitors in Rb-negative tumors may attenuate dose-limiting hematologic toxicities that occur with chemotherapy.

A phase I/II trial in Rb-negative NSCLC is currently studying the potential benefits of combining G1T28, a CDK4&6 inhibitor, with carboplatin and etoposide (NCT02499770).

Whether using CDK4&6 inhibitors with cytotoxic cancer treatments is problematic remains to be determined. Further studies are needed to establish optimal treatment schedules for drugs that may antagonize each other&rsquo;s effects, and to explore the potential hemo-protective effects of combination treatment.

Finn RS, Aleshin A, Slamon DJ. Targeting the cyclin-dependent kinases (CDK) 4/6 in estrogen receptor-positive breast cancers.

Sherr CJ, Beach D, Shapiro GI. Targeting CDK4 and CDK6: from discovery to therapy.

Dai X, Li L, Liu X, Hu W, et al. Cooperation of DLC1 and CDK6 affects breast cancer clinical outcome. G3 (Bethesda). 2014;5(1):81-91.

Goel S, Wang Q, Watt AC, et al. Overcoming therapeutic resistance in HER2-positive breast cancers with CDK4/6 inhibitors.

Takahashi A, Ohtani N, Hara E. Irreversibility of cellular senescence: dual roles of p16INK4a/Rb-pathway in cell cycle control.

Recent preclinical findings have revealed that the activity of CDK4 and CDK6 is interwoven with multiple pathways that drive oncogenesis in many cancers besides breast cancer. In addition, a link between DLC1 and CDK6 has been discovered that predicts breast cancer progression, and may one day provide prognostic information for lung and colon cancers, as well. Preclinical data illustrating the necessity of continuous dosing in CDK4&6 inhibition will likely guide the development of future agents. Understanding the interdependence and synergy between the cyclin D:CDK4 and CDK6 axis and other oncogenic pathways is central to developing effective treatment regimens, identifying potential therapy-related problems, and discovering novel combinations that may enhance existing strategies.

Inhibition of cyclin-dependent kinase 4 (CDK4) and CDK6 has been extensively studied in estrogen receptor (ER)‒driven breast cancer.

Preclinical Findings Illuminate Intricacies in CDK4&6 Inhibition

Abemaciclib is another agent that is rapidly advancing through clinical trials. This potent agent has received a breakthrough therapy designation from the FDA for its potential as a monotherapy for patients with refractory HR-positive advanced breast cancer. In addition to single-agent activity, this agent is also being explored in other subtypes of breast cancer, including the HER2- positive group.

To gain further insight into how these agents will be incorporated into the standard of care for patients with breast cancer,

spoke with Debu Tripathy, MD, Hope S. Rugo, MD, and Sara A. Hurvitz, MD.

The University of Texas MD Anderson Cancer Center

Can you discuss the various CDK4&6 inhibitors that are in development?

: In many other fields of medicine, we have drugs that are similar and &ldquo;me-too&rdquo; drugs but, in cancer, we are finding there is enough difference between one person to another and the biology of their tumor that we have to look at each drug on its own. That is why it&rsquo;s so important for us to look at the whole landscape of CDK4&6 inhibitorsnot only palbociclib, but ribociclib, too. Abemaciclib is another drug that&rsquo;s in clinical trials; these are all unique drugs that we need to explore.

From a community oncologist&rsquo;s perspective, how can CDK4&6 inhibitors be integrated into the management of breast cancer?

The importance of CDK inhibition in the management of hormone receptor-positive cancers is critical. Palbociclib is a drug that more than doubles progression-free survival (PFS). We are still waiting on survival data, and it is a drug that is reasonably well tolerated.

The important take-home message is that it is an important option to delay progression. One issue that comes up a lot is, in the absence of a survival advantage, should we be using the drugand when should we be using it? Most of us, and our patients, feel that something could clearly extend PFS, but when patients progress they might need to move to chemotherapy sooner and their quality of life might decease. This is important and we need to await those data.

However, the fact that it is a well-tolerated drug is important. It does require more monitoring; you have to check more blood counts and you have to adjust doses if needed. There are a small number of patients who have to come off of therapy because of this. Fortunately, the rate of increase and febrile neutropenia is very low, but it is important that clinicians be aware of how to use the drug, what the patient selection would be, and, particularly, how to monitor some of the toxicities and adjust the drugs.

Very recently, palbociclib was also approved in the second-line setting. The first trialthe first approval&mdash;was in combination with letrozole for patients who were candidates for treatment with aromatase inhibitors. Specifically, the trial enrolled patients who either have never seen hormonal therapy in the adjuvant setting or were more than 1 year out from an aromatase inhibitor or hormonal therapy.

The more recent study was in second-line patients who had previously been exposed to hormonal therapy, were in the metastatic setting, or who recurred within 1 year. This compared fulvestrant (Faslodex) to fulvestrant with palbociclib. It also showed a significant PFS improvement.

How has the incorporation of these agents changed secondor third-line treatments?

It has created a lot of dilemmas. This is because, now, a lot of people are getting this as their frontline therapy, and we have very little information on the natural history of these patients. How should we treat them when they progress? What are the different options available for them? These are things that need to be sorted out with future trials.

Clinical Professor, Department of Medicine (Hematology/Oncology)

Director, Breast Oncology Clinical Trials Program

How do you foresee the CDK4&6 inhibitors being used for the treatment of women with breast cancer?

: So you know, the whole world of hormone receptor-positive disease now, in the metastatic setting, is about targeted therapy and how we can improve response. We talk about reverse resistance, but really what we&rsquo;re trying to do is improve response to endocrine therapy and improve the duration of response. I think those are our two biggest goals.

The CDK data is really fascinating because it came from UCLA. Dennis Slamon was, as usual, looking ahead to the next step and where things can come from. They have a series of cell lines at UCLA and they were able to expand this research further and show that where CDK inhibitors were most likely to have a benefit was in estrogen receptor (ER)‒positivelike disease, and HER2-positive disease, which is another area. I remember somebody telling me from one of the companies that has a CDK inhibitor, &ldquo;Well it&rsquo;s only luminal B&rsquo;s,&rdquo; but we don&rsquo;t know that. There are absolutely no data that show it. But there will be data coming out of these trials, I think, that tells us more about it.

Can you discuss the clinical trials for the CDK4&6 inhibitors?

There were very intriguing data that led to the phase I and then the phase II trial, PALOMA-1, which of course, I think, somewhat surprisingly for many of us, led to accelerated approval in the first-line setting with letrozole.

We&rsquo;re going to see results from two other very interesting studies. So there&rsquo;s the MONALEESA Trial with ribociclib, Novartis&rsquo; CDK4&6 inhibitor. And then there&rsquo;s a third CDK4&6 inhibitor, which is a little bit different from the others, abemaciclib, from Lilly, and that drug had single-agent activity, which the other two don&rsquo;t seem to have much of. And it was really fairly impressive single-agent activity in phase I.

So they did a single-agent phase II trial, the MONARCH Trial, that&rsquo;s single-agent drug in refractory hormone receptorpositive breast cancer. And then, of course, they also have their fulvestrant study. Everybody who takes abemaciclib gets diarrhea, but it&rsquo;s not bad diarrhea, and so you don&rsquo;t have to take upfront prophylaxis but you have to be taking Imodium. So my patients who are on abemaciclib for a long time traveled with the Imodium.

Director of the Hematology/Oncology Breast Cancer Program,

Associate professor, Department of Medicine

Are CDK4&6 inhibitors also being studied in HER2-positive patients?

: There is a study that is about to get started, or has just started, that is looking at HR-positive/ HER2-positive metastatic breast cancer. Our own cell line data on CDK4&6 inhibitors in breast cancer shows that not only luminal ER-positive/ HR-positive breast cancer cell lines are sensitive to CDK4&6 inhibition but, indeed, a number of HER2-positive lines are sensitive, as well.

Abemaciclib, which is being explored in this setting, has a different safety profile than other CDK4&6 inhibitors, so it is also going to be interesting to see how that plays out in a larger patient population in these larger, ongoing studies that are going to be presented in the next 6 to 12 months.

What work would you like to see done in this field?

We&rsquo;re really beginning to appreciate the fact that HR-positive/ HER2-positive breast cancer behaves quite differently. It has different surgical and long-term outcomes compared with HR-negative/ HER2-positive breast cancer. We are also beginning to see a difference in outcomes with PI3K pathway activation status, so that may help inform decision-making and make treatment options available in the future.

A number of highly effective cyclin-dependent kinase 4 (CDK4) and CDK6 inhibitors are currently in development as treatments for patients with metastatic breast cancer.

Novel CDK4&6 Inhibitors Offer Further Potential in Breast Cancer

To date, palbociclib is the only CDK4&6 inhibitor that has been approved for treatment of breast cancer. Since abemaciclib and ribociclib have demonstrated encouraging results in clinical trials, their eventual approval is also expected. Understanding the evidence behind each agent and the implications of the differences between them will be critical for guiding clinical decision making and determining potential future applications.

Palbociclib is the first orally, bioavailable, CDK4&6-selective inhibitor developed as a treatment for cancer. In preclinical studies, palbociclib induced G1 phase arrest in solid tumor cell lines with cyclin D1:CDK4 dysregulation and leukemia cell lines driven by D2/D3:CDK6. Trials in xenograft models showed that palbociclib achieved tumor stasis and regression, and that the presence of functional retinoblastoma product (pRb) was critical for treatment efficacy. These preclinical results confirmed that upregulation of the cyclin D:CDK4 and CDK6 axis is an important underlying mechanism for pathogenesis in many types of cancer.

Preclinical trial success led to phase I studies in advanced cancers solid tumors and lymphoma&mdash;that were pRb-positive. Doseranging trials found that the maximum tolerated dose (MTD) and dosing schedule was 200 mg daily for 2 weeks, followed by an off-period of 1 week. Myelosuppression, particularly neutropenia, was the primary dose-limiting toxicity (DLT). Another phase I study established that the ideal dosing regimen was 125 mg daily for 3 weeks, followed by off-treatment for 1 week. Investigators found that while the 75-mg dose induced neutropenia in some patients, the neutropenia remained uncomplicated and tolerable even up to the 125-mg dose. The myelosuppressive effects of palbociclib were reversible and resolved after treatment was withdrawn. Other common toxicities tended to be low grade and included nausea, fatigue, and diarrhea.

In phase II studies, palbociclib did not produce encouraging results as a single-agent therapy. Of 28 patients with pRb-positive advanced breast cancer, 6% had partial response (PR) and 14% had stable disease (SD) for more than 6 months. No response occurred in any patients with estrogen receptor (ER)‒negative status. In addition, 1 patient who had undergone multiple prior cycles of chemotherapy experienced febrile neutropenia.

Fortunately, palbociclib fared better as a combination therapy with other anticancer agents. PALOMA-1, a phase I/II study, evaluated palbociclib plus letrozole, a nonsteroidal aromatase inhibitor (AI), versus letrozole alone as a first-line therapy in postmenopausal women with ER-positive, human epidermal growth factor receptor 2 (HER2)‒negative advanced breast cancer. The phase II segment explored the utility of CCND1 and p16 as potential biomarkers, but found that they did not predict treatment response any better than ER status alone.

In the second interim analysis of PALOMA-1, progressionfree survival (PFS) in the palbociclib-plus-letrozole group was more than triple that in the letrozole-only group (26.1 vs 7.5 months; HR, 0.37;

The clinical benefit in the palbociclib arm was sustained in the final analysis: Median PFS was 20.2 months with palbociclib versus 10.2 months with letrozole alone (HR, 0.488;

Although neutropenia and myelosuppression were the most common adverse events (AEs), no instances of febrile neutropenia were noted. However, grade 3/4 toxicities, such as pulmonary embolism and infection, were more frequent in the palbociclib arm than in the control arm (5% vs 0%, respectively). Nevertheless, combination therapy with palbociclib and letrozole was considered to be well tolerated, with similar AE rates in both treatment groups.

The success of PALOMA-1 led the US Food and Drug Administration (FDA) to grant breakthrough therapy designation to palbociclib.

In addition, the marked efficacy of palbociclib in ER-positive breast cancer confirmed the utility of using ER status to determine which patients are most likely to respond to CDK4&6 inhibition.

PALOMA-2 is a phase III trial that was designed to verify the results of PALOMA-1 by assessing the safety and efficacy of palbociclib plus letrozole as a first-line therapy in advanced ER-positive, HER2-negative breast cancer. In April 2016, Pfizer announced that the study met its primary endpoint, with the palbociclib group having a longer PFS than the letrozole-only group. Detailed results will be presented at the American Society of Clinical Oncology 2016 Annual Meeting.

Another phase III trial, PALOMA-3, addressed the treatment of women with advanced ER-positive, HER2-negative breast cancer who had relapsed or progressed on prior endocrine therapy. PALOMA-3 examined the efficacy of palbociclib in combination with fulvestrant, an ER antagonist, in this population. Median PFS was longer in the palbociclib group than in the fulvestrantonly group (9.5 vs 4.6 months; HR, 0.46;

<.0001). Follow-up for overall survival (OS) is ongoing.

The rates of neutropenia and leukopenia in the palbociclib arm (62% and 25%, respectively) were similar to those in the PALOMA-1 trial.

The significant findings from PALOMA-3 led the FDA to approve palbociclib for treatment of advanced HR-positive, HER2-negative breast cancer after progression on endocrine therapy.

&ldquo;Patients in [the PALOMA-3] population are generally thought to be endocrine-refractory, and are traditionally given singledrug cytotoxic chemotherapy&hellip;, which is often of limited benefit, rarely produces meaningful objective responses, and frequently resulted in increased toxicity and reduced quality of life,&rdquo; the PALOMA-3 authors concluded. &ldquo;Our findings confirm the important observation that endocrine monotherapy has limited efficacy in patients with disease progression after previous exposure to endocrine therapy, irrespective of clinical or molecularly defined endocrine sensitivity, suggesting a need for the routine use of more effective combination regimens.&rdquo;

Preclinical trials have shown that ribociclib induces tumor regression and has activity as a single agent in melanoma and breast cancer.

In one study, paired tumor biopsies were obtained from patients before and after ribociclib treatment. Ki67 and phosphorylated pRb were reduced by approximately 50%, although corresponding clinical effect was not reported. Partial responses were seen in 1 patient with ER-positive breast cancer plus PI3K mutation and CCND1 amplification, as well as 1 patient with melanoma of CCND1-amplified, BRAF/NRAS wild type. Overall, 14% had SD for 6 or more cycles.

Early but limited results from ongoing phase I/II trials are available. One study evaluating ribociclib plus exemestane and everolimus in advanced ER-positive, HER2-negative breast cancer showed that the combination appears to be feasible.

In a phase Ib/II study, ribociclib plus PI3K inhibitor BYL719 and letrozole was well tolerated in 15 postmenopausal women with advanced ER-positive, HER2-negative breast cancer. Common AEs included neutropenia, nausea, and hyperglycemia, with 1 instance of dose-limiting neutropenia.

Preliminary clinical activity was seen with both combinations of ribociclib.

Acceptable safety profiles and evidence of clinical activity were also seen with ribociclib as monotherapy for pRb-positive solid tumors and lymphomas, and as combination therapy with BRAF inhibitor LGX818 in advanced melanoma.

Based on the experience with palbociclib, ribociclib development was expedited to phase III trials.4 MONALEESA-2 is evaluating ribociclib plus letrozole in postmenopausal women with advanced ER-positive, HER2-negative breast cancer (NCT01958021). MONALEESA-3 is studying ribociclib plus fulvestrant in a similar population (NCT02422615). Ribociclib in combination with goserelin with or without tamoxifen is being investigated in premenopausal women with HR-positive, HER2- negative breast cancer (NCT02278120).

Unlike palbociclib and ribociclib, abemaciclib had significant single-agent activity in phase I studies. In one trial of women with advanced breast cancer, abemaciclib had activity only in the ER-positive group. Response and disease control rates (DCRs) in ER-positive patients were 31% and 81%, respectively. PFS was also markedly higher in women with ER-positive disease (8.8 vs 1.1 months for ER-negative). In an expansion cohort of the same trial, PR and clinical benefit rates (CBRs) were 28% and 72%, respectively, among 25 patients with advanced, HRpositive, HER2-negative breast cancer who had failed multiple lines of prior treatment.

Clinical benefit was seen with combination therapy, as well. Another cohort of the phase I study evaluated abemaciclib plus fulvestrant in patients with ER-positive disease refractory to previous therapies. The DCR was 72.2%. Diarrhea was the most common AE, occurring in 66% of patients, but nearly all cases were grade 2 or lower. The overall and grade 3/4 rates of neutropenia were 40% and 21%, respectively.

Based on the phase I results, abemaciclib received breakthrough therapy designation from the FDA in 2015. As with ribociclib, abemaciclib has been expedited to phase III trials.

MONARCH 2 is evaluating abemaciclib plus fulvestrant in patients with advanced, HR-positive, HER2-negative breast cancer that has relapsed or is refractory to standard therapy (NCT02107703). MONARCH 3 combines abemaciclib with a nonsteroidal AI as a first-line therapy in advanced, previously untreated, HR-positive, HER2-negative breast cancer (NCT02246621).

Finally, the clinical single-agent activity seen with abemaciclib makes it unique among the CDK4&6 inhibitors. A phase II trial, MONARCH 1, is investigating abemaciclib as a monotherapy in women with refractory, HR-positive, HER2-negative breast cancer (NCT02102490).

The most notable difference between the CDK4&6 inhibitors is their toxicity profiles. Myelosuppression, particularly neutropenia, is the primary DLT for palbociclib and ribociclib.1 Rates of neutropenia are approximately 40% for both agents, and febrile neutropenia in palbociclib is rare (0.6% in PALOMA-3).

&ldquo;Neutropenia, although common, was not accompanied by serious clinical outcomes and is likely to be the result of an ontarget effect of CDK4&6 inhibition on marrow progenitor cells,&rdquo; the authors of PALOMA-1 noted. &ldquo;The absence of serious complications resulting from palbociclib-associated neutropenia probably reflects a cytostatic rather than cytotoxic effect of the drug on bone marrow progenitor cells, different from what is seen with typical cytotoxic drugs.&rdquo;

In addition, patients in the palbociclib studies did not have any mucositis or skin toxicity, which are common portals for infection that induces febrile neutropenia during chemotherapy.

Patients who received abemaciclib had milder hematologic toxicity, specifically neutropenia. The all-grade rate of neutropenia was 49% and grade 3/4 events were seen in 21% of patients. With palbociclib plus letrozole, the overall rate of neutropenia was 75% and 54% of patients had a grade 3/4 event.4 One possible rationale for the difference in neutropenia rates is that abemaciclib is a more potent inhibitor of CDK4 than palbociclib or ribociclib (CDK4 IC50 0.6 to 2 nmol/L vs 11 nmol/L and 10 nmol/L, respectively). In addition, overall hematologic and nonhematologic toxicities may be fewer with abemaciclib because it is more specific for CDK4 than for CDK6.

Neutropenia is not a DLT for abemaciclib, which may be dosed continuously, unlike palbociclib and ribociclib, which each require an off-treatment week per cycle. The ability to continuously dose abemaciclib may explain its success as a single agent in clinical trials when monotherapy with other CDK4&6 inhibitors have shown disappointing results.

Gastrointestinal toxicity, however, is more common with abemaciclib than with the other CDK4&6 inhibitors. In a phase I trial, the incidence of diarrhea was 66%, although only 6% of patients had grade 3 diarrhea and none had grade 4.

Lowering the dosage of abemaciclib may help eliminate or reduce the severity of diarrhea. Supportive medications may also be used to treat this side effect; 2 trials investigating abemaciclib now administer the drug with prophylactic loperamide (NCT02677844, NCT02441946).

Finally, abemaciclib crosses the blood-brain barrier, which makes it an attractive agent for treating primary and secondary central nervous system malignancies.

The relatively impermeable nature of the blood-brain barrier poses a significant challenge for clinicians treating brain tumors. Brain metastases are the most common type of brain tumor, occurring in up to 40% of patients, particularly in the melanoma, lung, and breast cancer populations. In addition, patients with glioblastoma multiforme (GBM) have a 5-year survival rate of less than 3%, indicating a strong unmet need. Rb pathway dysregulation is present in 78% of GBM cases and plays an important role in many peripheral cancers, so CDK4&6 inhibitors may offer an effective treatment in these patients.

Abemaciclib has a wide distribution, with cerebrospinal fluid concentrations similar to those found in plasma.

In GBM xenograft models, abemaciclib had a 10-fold higher increase in brain-to-plasma concentration ratio than palbociclib. As a result, lower dosages of abemaciclib than palbociclib were needed to reach therapeutic unbound concentrations in the brain. In addition, abemaciclib had similar efficacy as temozolomide for increasing survival time, and using abemaciclib in conjunction with temozolomide had an additive and possibly synergistic treatment effect.

Phase I results of abemaciclib in GBM demonstrated decreased tumor size and PFS greater than 16 months in 2 of 17 patients.1 A phase II trial is currently evaluating abemaciclib as a monotherapy for patients with brain metastases from melanoma, non-small cell lung cancer, and HR-positive breast cancer (NCT02308020).

Murphy CG, Dickler MN. The role of CDK4/6 inhibition in breast cancer.

DeMichele A, Clark AS, Tan KS, et al. Cancer Therapy: Clinical CDK 4/6 Inhibitor Palbociclib (PD0332991) in Rb+ Advanced Breast Cancer: Phase II Activity, Safety, and Predictive Biomarker Assessmen [published on December 11, 2014].

Pfizer Announces Positive Top-Line Results for Phase 3 PALOMA-2 Clinical Trial of IBRANCE&reg; (palbociclib) [news release]. New York, NY: Pfizer Inc. April 19, 2016. http://www.pfizer.com/news/press-release/press-release-detail/pfizer_announces_ positive_top_line_results_for_phase_3_paloma_2_clinical_trial_of_ibrance_palbociclib. Accessed May 10, 2016.

Cristofanilli M, Turner NC, Bondarenko I, et al. Fulvestrant plus palbociclib versus fulvestrant plus placebo for treatment of hormone-receptor-positive, HER2- negative metastatic breast cancer that progressed on previous endocrine therapy (PALOMA-3): final analysis of the multicentre, double-blind, phase 3 randomised controlled trial [published correction appears in Lancet Oncol. 2016;17:429].

Turner NC, Ro J, Andr&eacute; F, et al. Palbociclib in hormone-receptor-positive advanced breast cancer.

Juric D, Hamilton E, Garcia Est&eacute;vez L, et al. Abstract P5-19-24: Phase Ib/II study of LEE011 and BYL719 and letrozole in ER+, HER2 breast cancer: safety, preliminary efficacy and molecular analysis.

Patnaik A, Shapiro GI, et al. Efficacy and safety of abemaciclib, an inhibitor of CDK4 and CDK6, for patients with breast cancer, nonsmall cell lung cancer, and other solid tumors [published online May 23, 2016].

Finn RS, Crown JP, Lang I, et al. The cyclin-dependent kinase 4/6 inhibitor palbociclib in combination with letrozole versus letrozole alone as first-line treatment of oestrogen receptor-positive, HER2-negative, advanced breast cancer (PALOMA-1/ TRIO-18): a randomised phase 2 study.

Raub TJ, Wishart GN, Kulanthaivel P, et al. Brain Exposure of two selective dual CDK4 and CDK6 inhibitors and the antitumor activity of CDK4 and CDK6 inhibition in combination with temozolomide in an intracranial glioblastoma xenograft.

CDK4&6 inhibitors have the potential to be effective treatments for advanced breast cancer. Differences in pharmacokinetics, potency, and efficacy have important clinical implications. These differences will affect patient selection as well as provide new directions for future investigation, including the development of therapies for other cancer types.

Cyclin-dependent kinase 4 (CDK4) and CDK6 inhibitors are a promising class of anticancer agents that may present effective alternatives to standard therapy for women with advanced, refractory, or relapsed hormone receptor (HR)‒positive breast cancer.

Differences Between CDK4&6 Inhibitors Predict Future Approaches

Evidence from recent studies suggests that CDK4&6 inhibitors may be effective in other cancer types, as well. Preclinical trials show that CDK4&6 inhibitors demonstrate activity in human epidermal growth factor receptor 2 (HER2)‒positive breast cancer cells, and may prove effective in overcoming resistance to standard therapy. Data from preclinical and early-phase clinical trials in melanoma and mantle cell lymphoma (MCL) have produced encouraging results that point to CDK4&6 inhibitors as potential alternative therapies. Clinical studies of CDK4&6 inhibitors in breast cancer subtypes and other cancers are currently ongoing.

CDK4&6 Inhibitors in ER-Positive Breast Cancer

The development of CDK4&6 inhibitors is most advanced in the area of breast cancer treatment, particularly for the ERpositive, HER2-negative type. Based on the results of PALOMA-3, palbociclib was approved by the FDA in February 2016 for treating advanced or metastatic breast cancer in combination with fulvestrant.

In another phase III trial, PALOMA-2, palbociclib plus letrozole was reported to prolong progression-free survival (PFS) compared with letrozole alone, and results will be presented at the 2016 ASCO Annual Meeting.

Abemaciclib has demonstrated high disease control rates (DCRs) and prolonged PFS as a single agent and in combination with fulvestrant in phase I/II studies.

The lack of hematologic and other serious dose-limiting adverse events (AEs) fuels the hope that abemaciclib may be an effective monotherapy for ER-positive, HER2-negative breast cancer.

Abemaciclib is being studied as a single-agent treatment in a phase II study, MONARCH 1 (NCT02102490). Phase III studies of abemaciclib in combination with nonsteroidal aromatase inhibitors (AIs; MONARCH 3, NCT02246621) and fulvestrant (MONARCH 2, NCT02107703) are ongoing.

Preclinical and phase I studies have shown promising results for ribociclib as a treatment for ER-positive, HER2-negative breast cancer. Single-agent activity has been demonstrated in retinoblastoma (Rb)‒positive tumors, although myelosuppression remains a dose-limiting toxicity (DLT).

Ribociclib has advanced to phase III trials as combination treatment with letrozole (MONALEESA-2, NCT01958021), fulvestrant (MONALEESA- 3, NCT02422615), and goserelin with either tamoxifen or an AI (MONALEESA-7, NCT02278120).

CDK4&6 inhibitors have shown efficacy for treating ER-positive breast cancer that is refractory to standard therapy, but many patients who initially respond to CDK4&6 inhibitors will develop resistance to this treatment modality, as well.

Multiple pathways exist that enable ER-positive tumors to become resistant, and most patients treated with palbociclib were eventually found to progress on therapy. CCNE1 amplification and RB1 gene loss are 2 mechanisms that have been demonstrated to mediate resistance. Notably, cells with CCNE1 amplification were sensitive to CDK2 inhibition, whereas those with RB1 loss were not. Thus, CDK2 may serve as a target for future therapies to overcome resistance to CDK4&6 inhibitors.

Resistance to CDK4&6 inhibition may also develop via the PI3K pathway. Constitutive activation of the PI3K pathway is associated with relapses in ER-positive breast cancer, and up to 40% of all breast cancer types have a mutation in the PIK3CA gene, which encodes the catalytic site of PI3K&alpha;. In addition, more than 50% of patients with breast cancer have abnormalities in components of the PI3K pathway, such as PTEN and AKT1-3, that lead to increased PI3K signaling. In both preclinical studies and the clinical setting, persistent retinoblastoma product (pRb) phosphorylation, the result of incomplete PI3K pathway inhibition, predicted treatment failure with CDK4&6 inhibitors. Mutations in PI3KCA may serve as a biomarker to identify the patients who are most likely to progress on CDK4&6 inhibitors.

In a breast cancer cell line study by Sadhna Vora, MD, with Massachusetts General Hospital Cancer Center in Boston, MA, and colleagues, 1 of 4 models responded to CDK4 and 6 plus PI3K inhibitor (PI3Ki) and did not regrow after treatment was discontinued. However, the other 3 models were resistant to treatment, suggesting that more biomarkers are needed to appropriately select patients for targeted therapy.

Interestingly, fulvestrant was found to have synergy with CDK4 and CDK6 and PI3Ki in 1 of the resistant models, which highlights residual ER signaling as an alternate pathway of resistance in tumors with PI3K dysregulation.

In other preclinical studies, both palbociclib and ribociclib have demonstrated synergy with PI3Ki.

Significant strides have been made in developing cyclin-dependent kinase 4 (CDK4) and CDK6 inhibitors to treat estrogen receptor (ER)‒positive breast cancer.

CDK4&6 Inhibition

Overcoming Obstacles: Long Road to Success for CDK Inhibition

Preclinical Findings Illuminate Intricacies in CDK4&6 Inhibition

Novel CDK4&6 Inhibitors Offer Further Potential in Breast Cancer

Differences Between CDK4&6 Inhibitors Predict Future Approaches

Future Studies Hope to Move CDK4&6 Inhibition Into New Terrain