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Author Manuscript Published OnlineFirst on August 16, 2018; DOI: 10.1158/1078-0432.CCR-18-2177 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

CDK4/6 inhibitors: what is the best cocktail?

Marcos Malumbres

Cell Division and group, Spanish National Centre (CNIO)

Madrid

Correspondence to: M.M., Centro Nacional de Investigaciones Oncológicas, Melchor

Fernández Almagro 3, E-28029 Madrid, Spain, Tel. +34 917328000, Fax +34

917328033; [email protected].

Running title: Synergism between CDK4/6 and mTOR inhibitors

Disclosure of Potential Conflicts of Interest

M. Malumbres is principal investigator of research collaborative agreements funded by

Pfizer and Lilly.

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Summary

CDK4/6 inhibitors have shown a great potential in the new armamentarium against

cancer. However, their effect as single agents is limited and the hopes are on new

combinatory strategies. Recent data suggest that inhibiting mTOR may significantly

cooperate with arrest in a variety of .

Text

In this issue of Clinical Cancer Research, Song and colleagues (1) report the synergistic

effect of combining CDK4/6 and mTOR inhibitors in intrahepatic cholangiocarcinoma

(ICC), a highly aggressive tumor with no FDA-approved . ICC is the

second most common in the liver and complete surgical resection remains

the only option for cure. Unfortunately, ICC is not resectable in most patients and

current treatments are based on systemic with nucleoside analogs in

combination with . A number of different oncogenic pathways, including the

EGFR-RAS or the PI3K-mTOR signaling routes, are mutated in cholangiocarcinomas

(2) and recent preclinical data suggest that mTOR inhibitors may have a significant

therapeutic potential in ICC (3). These studies, however, reveal that the therapeutic

effect is mostly mediated by the apoptotic effect of inhibiting mTOR and this strategy

has little impact in preventing tumor cell proliferation.

Three different cell cycle inhibitors, , and , that

target CDK4/6 have recently been approved for the treatment of advanced

hormone-positive breast cancers. CDK4 and CDK6 are two closely related kinases

whose activity depends on binding to their partner cyclin D (D1, D2 or D3). These

cyclins are a major hub that multiple mitogenic pathways, including estrogen receptor,

RAS-ERK or PI3K-AKT-mTOR signaling routes, use to trigger cell cycle progression 2

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(Figure 1). Not surprisingly, the first approved indication for CDK4/6 inhibitors targets

estrogen-dependent mammary gland tumors, in which any of these three inhibitors is

combined with a variety of drugs that either lower estrogen levels (aromatase inhibitors

such as letrozole) or block the estrogen receptor (tamoxifen, fulvestrant; Figure 1). The

effect of CDK4/6 inhibitors as single agents is still unclear and their combination with

hormonotherapy is preferred in current treatments against advanced .

The cooperation between CDK4/6 inhibitors and endocrine therapy has been

quite a success in estrogen-positive breast cancers. Could this combination strategy be

translated to other signaling pathways and tumor types? The RAS-ERK and PI3K-AKT-

mTOR pathways are good candidates (Figure 1), as supported by preclinical evidence

showing the dependence that these pathways have on cyclin-CDK complexes to active

cell proliferation in a variety of tumor types (melanoma, , breast and

pancreatic cancer, etc.). Multiple clinical trials are currently on-going to explore the

effect of these combinatorial strategies in melanoma as well as breast, lung, pancreatic,

or , among other solid tumors. Additional putative combinations

with CDK4/6 inhibitors involve classical chemotherapeutic agents targeting DNA

replication or mitosis, as well as (Figure 1), although the rationale

supporting these combinatorial strategies is less established.

Perhaps a more complicated question is which specific patients may benefit from

these combination therapies. Tumors in which pRb is not present are typically resistant

to CDK4/6 inhibitors, as this oncogenic event is downstream of CDK4/6 activity. Apart

from this negative selection, no clear to identify patients that may benefit

from CDK4/6 inhibitors have been proposed. However, tumors that respond to CDK4/6

inhibitors frequently display cyclin D-activation features (4). Interestingly, both cyclin

D overexpression and of the retinoblastoma (pRb) are

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commonly found in ICC, suggesting functional activation of cyclin D-CDK4/6

complexes in this tumor type. In the study by Song and colleagues (1), treatment of ICC

tumor cells with palbociclib results in slower cell proliferation and delayed

tumor progression in vivo. Strikingly, the combination of palbociclib with the mTOR

inhibitor MLN0128 displays synergistic effects in the proliferation of ICC cells. Even

more impressive is the effect of this combination in tumor growth in vivo in an ICC

model driven by hydrodynamic injection of AKT and YapS127A . By the

time all control mice injected with these oncogenes have succumbed because of ICC,

none of the animals treated with the combination of CDK4/6 and mTOR inhibitors has

developed lethal disease and only small tumors are observed (1).

Mechanistically, the combination between CDK4/6 and mTOR inhibitors seems

to have a stronger effect in the proliferative capacity of ICC cells than in their survival.

Concomitant treatment with these two inhibitors results in a dramatic loss of pRb

phosphorylation and loss of typical cell cycle markers such as Ki67 or cyclins that act

downstream of CDK4/6 in the subsequent phases of the cell cycle (such as cyclins E, A

or B). However, perhaps the most informative result is the loss of expression.

This cyclin is typically upregulated in the presence of CDK4/6 inhibitors most likely as

a consequence of the stabilization of inactive cyclin D-CDK4/6 complexes. Song et al.

(1) demonstrate that silencing of cyclin D1 actually improves the effect of palbociclib,

confirming previous data suggesting that high levels of cyclin D are associated to

resistance to CDK4/6 inhibitors. Interestingly, concomitant treatment of cells with

palbociclib and MLN0128 prevents the accumulation of cyclin D1, very likely

contributing to the more efficient cell cycle arrest observed in these cells. In addition to

the reduction in cell cycle entry, ICC cells treated with palbociclib and MLN0128

display decreased activation of the AKT-mTOR cascade. The molecular mechanism

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behind these observations is not addressed in detail, although cyclin D1-CDK4

complexes have been previously shown to phosphorylate and activate IRS2, and to

phosphorylate and inactivate the mTOR inhibitor TSC2 (Figure 1). Thus, CDK4/6

inhibition could result in decreased PI3K-AKT signaling due to reduced IRS2 signaling,

as well as a TSC2-dependent inhibition of mTOR, thus contributing to shutting-down

this signaling pathway.

Although these molecular interactions have previously been reported in other

tumor types, the data by Song et al. (1) strongly support the possibility that certain ICC

patients, perhaps those with activation of the PI3K-AKT pathway, may benefit from

therapeutic strategies combining CDK4/6 and mTOR inhibitors. The clinical relevance

of this proposal is clear as putative clinical trials in this pathology should consider

CDK4/6 inhibitors together with inhibitors for the other pathways typically activated by

associated to cholangiocarcinomas (2). It is tempting to speculate that

concomitant inhibition of the RAS-ERK pathway should eventually be also taken into

consideration. Recent data in melanoma tumors that become resistant to the treatment

with MEK and CDK4/6 inhibitors suggest a strong dependence on the mTOR pathway

and sensitivity to mTOR inhibitors (5). These triple combination therapies may be quite

effective in a variety of tumors, assuming that proper scheduling protocols are designed

to limit associated thus allowing an acceptable therapeutic window.

Hopefully, the plethora of current clinical trials testing the combination of CDK4/6

inhibitors with a variety of complementary strategies (Figure 1) will tell us what is the

best cocktail for specific .

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Acknowledgements

Funding: Spanish Ministry of Science, Innovation and Universities (SAF2015-69920-R

and ERA-NET PCIN 2015-007); Comunidad de Madrid (iLUNG, B2017/BMD-3884).

References

1. Song X, Liu X, Wang H, Wang J, Qiao Y, Cigliano A, et al. Combined CDK4/6

and pan-mTOR inhibition is synergistic against intrahepatic cholangiocarcinoma.

Clinical Cancer Research 2018;this issue.

2. Razumilava N, Gores GJ. Cholangiocarcinoma. Lancet 2014;383:2168-79.

3. Zhang S, Song X, Cao D, Xu Z, Fan B, Che L, et al. Pan-mTOR inhibitor

MLN0128 is effective against intrahepatic cholangiocarcinoma in mice. J Hepatol

2017;67:1194-203.

4. Gong X, Litchfield LM, Webster Y, Chio LC, Wong SS, Stewart TR, et al.

Genomic Aberrations that Activate D-type Cyclins Are Associated with Enhanced

Sensitivity to the CDK4 and CDK6 Inhibitor Abemaciclib.

2017;32:761-76.

5. Teh JLF, Cheng PF, Purwin TJ, Nikbakht N, Patel P, Chervoneva I, et al. In Vivo

E2F Reporting Reveals Efficacious Schedules of MEK1/2-CDK4/6 Targeting and

mTOR-S6 Resistance Mechanisms. Cancer Discov 2018;8:568-81.

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Figure Legend

Figure 1. A map of combination strategies testing CDK4/6 inhibitors in clinical trials.

CDK4/6 kinases are a major hub for integrating proliferative signals in the cell. Cyclin

D is induced by multiple mitogenic pathways, leading to CDK4/6 activation,

inactivation of the pRb protein and transcription of the cell cycle machinery required for

DNA replication (S-phase) and mitosis. Several CDK4/6 inhibitors (with differential

activities on other CDK family members; yellow box) are currently tested in clinical

trials for a wide spectrum of tumors either as single agents or in combination with other

agents (red boxes). These agents include inhibitors of hormone synthesis and their

receptors, as well as or inhibitors targeting growth factors, receptor

kinases (RTK) and the downstream RAS-RAF-MEK-ERK or PI3K-AKT-mTOR

pathways. CDK4/6 may in turn regulate the latter pathway by phosphorylating IRS2 and

TSC2. CDK4/6 inhibitors are also tested in clinical trials in combination with inhibitors

of the destabilizing protein HDM2 (HDM201), as well as classical

chemotherapeutic agents (e.g. DNA damage agents targeting S-phase or

poisons targeting mitosis), and new antibodies targeting PD-1 or PD-L1 for

immunotherapy. Red boxes only list those agents currently tested in combination

therapies with CDK4/6 inhibitors. Additional clinical trials in which CDK4/6 inhibitors

are combined with other cellular pathways (e.g. proteasome-dependent protein

degradation, control of by BCL2 or PIM kinases, as well as JAK or

Hedgehog-dependent signaling pathways) are not shown for clarity.

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Figure 1:

Letrozole Anastrozole Growth Xentuzumab Exemestane Pertuzumab factors Goserelin Erdafitinib Leuprolide PD-1/PD-L1 Atezolizumab Spartalizumab RTK RTK Avelumab Hormones

IRS2

Taselisib AR/ER RAS PI3K Pictilisib Fulvestrant Tamoxifen Buparlisib Bicalutamide RAF AKT

Trametinib MEK1/2 TSC2 PD-0325901

Everolimus Ulixertinib ERK1/2 mTOR Gedatosilib Radiation 5-FU Cyclin D Nab-

Palbociclib CDK4/6 HDM201 Ribociclib Abemaciclib S-phase Mitosis Transcription SHR6390 pRb factors P276-00 ---

Cell cycle progression

© 2018 American Association for Cancer Research Downloaded from clincancerres.aacrjournals.org on October 8, 2021. © 2018 American Association for Cancer Research. Author Manuscript Published OnlineFirst on August 16, 2018; DOI: 10.1158/1078-0432.CCR-18-2177 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

CDK4/6 inhibitors: what is the best cocktail?

Marcos Malumbres

Clin Cancer Res Published OnlineFirst August 16, 2018.

Updated version Access the most recent version of this article at: doi:10.1158/1078-0432.CCR-18-2177

Author Author manuscripts have been peer reviewed and accepted for publication but have not yet Manuscript been edited.

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