CDK4/6 inhibitors have shown 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 cell-cycle arrest in a variety of cancers.

See related article by Song et al., p. 403

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 targeted therapy. ICC is the second most common malignancy 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 chemotherapy with nucleoside analogues in combination with cisplatin. 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, palbociclib, ribociclib, and abemaciclib, which target CDK4/6 kinases, 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 (Fig. 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; Fig. 1). The effect of CDK4/6 inhibitors as single agents is still unclear and their combination with hormone therapy is preferred in current treatments against advanced breast cancer.

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 being 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 antibodies or inhibitors targeting growth factors, receptor tyrosine 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 being tested in clinical trials in combination with inhibitors of the p53 destabilizing protein HDM2 (HDM201), as well as classical chemotherapeutic agents (e.g., DNA-damaging agents targeting S-phase or microtubule poisons targeting mitosis), and new antibodies targeting PD-1 or PD-L1 for immunotherapy. Red boxes list only those agents currently being 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 apoptosis by BCL2 or PIM kinases, as well as JAK or Hedgehog-dependent signaling pathways) are not shown for clarity. 5-FU, 5-fluorouracil.

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 being 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 antibodies or inhibitors targeting growth factors, receptor tyrosine 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 being tested in clinical trials in combination with inhibitors of the p53 destabilizing protein HDM2 (HDM201), as well as classical chemotherapeutic agents (e.g., DNA-damaging agents targeting S-phase or microtubule poisons targeting mitosis), and new antibodies targeting PD-1 or PD-L1 for immunotherapy. Red boxes list only those agents currently being 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 apoptosis by BCL2 or PIM kinases, as well as JAK or Hedgehog-dependent signaling pathways) are not shown for clarity. 5-FU, 5-fluorouracil.

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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 (Fig. 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, glioblastoma, breast and pancreatic cancer, etc.). Multiple clinical trials are currently ongoing to explore the effect of these combinatorial strategies in melanoma as well as breast, lung, pancreatic, or head and neck cancer, among other solid tumors. Additional putative combinations with CDK4/6 inhibitors involve classical chemotherapeutic agents targeting DNA replication or mitosis, as well as immunotherapy (Fig. 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 the retinoblastoma protein (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 biomarkers 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 phosphorylation of pRb are 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 in vitro 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 oncogenes. 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 have developed lethal disease and only small tumors are observed (1).

Mechanistically, the combination of 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 cyclin D1 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 and colleagues (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 with 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 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 (Fig. 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 shut down this signaling pathway.

Although these molecular interactions have previously been reported in other tumor types, the data by Song and colleagues (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 mutations 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 toxicities, 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 (Fig. 1) will tell us what is the best cocktail for specific malignancies.

M. Malumbres reports receiving commercial research grants from Lilly and Pfizer. No other potential conflicts of interest were disclosed.

This work was supported by grants from the Spanish Ministry of Science, Innovation and Universities (SAF2015-69920-R and ERA-NET PCIN 2015-007), and Comunidad de Madrid (iLUNG, B2017/BMD-3884).

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