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Combined CDK4/6 and mTOR Inhibition Is Synergistic against Glioblastoma via Multiple Mechanisms.

ABSTRACT: Purpose: Glioblastoma (GBM) is a deadly brain tumor marked by dysregulated signaling and aberrant cell-cycle control. Molecular analyses have identified that the CDK4/6-Rb-E2F axis is dysregulated in about 80% of GBMs. Single-agent CDK4/6 inhibitors have failed to provide durable responses in GBM, suggesting a need to combine them with other agents. We investigate the efficacy of the combination of CDK4/6 inhibition and mTOR inhibition against GBM.Experimental Design: Preclinical in vitro and in vivo assays using primary GBM cell lines were performed.Results: We show that the CDK4/6 inhibitor palbociclib suppresses the activity of downstream mediators of the mTOR pathway, leading to rebound mTOR activation that can be blocked by the mTOR inhibitor everolimus. We further show that mTOR inhibition with everolimus leads to activation of the Ras mediator Erk that is reversible with palbociclib. The combined treatment strongly disrupts GBM metabolism, resulting in significant apoptosis. Further increasing the utility of the combination for brain cancers, everolimus significantly increases the brain concentration of palbociclib.Conclusions: Our findings demonstrate that the combination of CDK4/6 and mTOR inhibition has therapeutic potential against GBM and suggest it should be evaluated in a clinical trial. Clin Cancer Res; 23(22); 6958-68. ©2017 AACR.

SUBMITTER: Olmez I

PROVIDER: S-EPMC5690820 | biostudies-literature | 2017 Nov

REPOSITORIES: biostudies-literature

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Combined CDK4/6 and mTOR Inhibition Is Synergistic against Glioblastoma via Multiple Mechanisms.

Olmez Inan I Brenneman Breanna B Xiao Aizhen A Serbulea Vlad V Benamar Mouadh M Zhang Ying Y Manigat Laryssa L Abbas Tarek T Lee Jeongwu J Nakano Ichiro I Godlewski Jakub J Bronisz Agnieszka A Abounader Roger R Leitinger Norbert N Purow Benjamin B

Clinical cancer research : an official journal of the American Association for Cancer Research 20170816 22

<b>Purpose:</b> Glioblastoma (GBM) is a deadly brain tumor marked by dysregulated signaling and aberrant cell-cycle control. Molecular analyses have identified that the CDK4/6-Rb-E2F axis is dysregulated in about 80% of GBMs. Single-agent CDK4/6 inhibitors have failed to provide durable responses in GBM, suggesting a need to combine them with other agents. We investigate the efficacy of the combination of CDK4/6 inhibition and mTOR inhibition against GBM.<b>Experimental Design:</b> Preclinical < ...[more]

PMID: 28814434

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Project description:BackgroundDiffuse intrinsic pontine glioma (DIPG) is a lethal type of pediatric brain tumor that is resistant to conventional chemotherapies. Palbociclib is a putative novel DIPG treatment that restricts the proliferation of rapidly dividing cancer cells via selective inhibition of cyclin-dependent kinase (CDK) 4 and CDK6. However, implementing palbociclib as a monotherapy for DIPG is unfeasible, as CDK4/6 inhibitor resistance is commonplace and palbociclib does not readily cross the blood-brain barrier (BBB) or persist in the central nervous system. To inhibit the growth of DIPG cells, we aimed to use palbociclib in combination with the rapamycin analog temsirolimus, which is known to ameliorate resistance to CDK4/6 inhibitors and inhibit BBB efflux.Materials and methodsWe tested palbociclib and temsirolimus in three patient-derived DIPG cell lines. The expression profiles of key proteins in the CDK4/6 and mammalian target of rapamycin (mTOR) signaling pathways were assessed, respectively, to determine feasibility against DIPG. Moreover, we investigated effects on cell viability and examined in vivo drug toxicity.ResultsImmunoblot analyses revealed palbociclib and temsirolimus inhibited CDK4/6 and mTOR signaling through canonical perturbation of phosphorylation of the retinoblastoma (RB) and mTOR proteins, respectively; however, we observed noncanonical downregulation of mTOR by palbociclib. We demonstrated that palbociclib and temsirolimus inhibited cell proliferation in all three DIPG cell lines, acting synergistically in combination to further restrict cell growth. Flow cytometric analyses revealed both drugs caused G1 cell cycle arrest, and clonogenic assays showed irreversible effects on cell proliferation. Palbociclib did not elicit neurotoxicity in primary cultures of normal rat hippocampi or when infused into rat brains.ConclusionThese data illustrate the in vitro antiproliferative effects of CDK4/6 and mTOR inhibitors in DIPG cells. Direct infusion of palbociclib into the brain, in combination with systemic delivery of temsirolimus, represents a promising new approach to developing a much-needed treatment for DIPG.

Project description:Background: Few druggable targets have been discovered in glioblastoma (GBM); however, increasing knowledge has uncovered two hallmarks of this disease: epigenetic modifications and cell cycle dysregulation. Methods: Here, we investigated the effects of the EZH2 inhibitor GSK126 alone and in combination with the CDK4/6 inhibitor abemaciclib on morphology, growth, invasiveness, protein and gene expression, mitochondrial function, and induction of cell death in patient-derived GBM cells (GBM03, GBM06, GBM14, and GBM15). Results: EZH2 expression, CDK4 amplification, and CDKN2A deletion were first confirmed. The combined use of GSK126 and abemaciclib resulted in synergistic effects in all patient-derived GBM cell lines. After treatment, the cells appeared swollen with a large flat cytoplasm and cellular stress fibers due to HIF1 upregulation and CalR translocation. Notably, abemaciclib-induced cellular stemness (NANOG+, OCT3/4+, and SOX2+) was antagonized by this combination treatment. The MitoStress Test revealed a massive impairment of mitochondrial function. The basal oxygen consumption rate, ATP synthesis, and maximal respiration of the mitochondria decreased, confirming reduced cellular fitness and disrupted endoplasmic reticulum-mitochondrial homeostasis. This was paralleled by massive mitochondrial ROS production leading to mitochondrial depolarization and upregulation of the UPR sensors PERK, ATF6, and IRE1. Spheroid invasion was reduced in 3/4 cases, again with synergistic effects after dual EZH2 and CDK4/6 blockade. Differentially expressed genes involved mitotic aberrations/spindle assembly (Rb, PLK1, RRM2, PRC1, CENPF, TPX2), histone modification (HIST1H1B, HIST1H3G), DNA damage/replication stress events (TOP2A, ATF4), immuno-oncology (DEPDC1), and a shift in the stemness profile towards a more differentiated state. Notably, the antitumor effect was partially confirmed in ovo as well as in patient-derived organoids. Conclusions: We propose dual blocking of GBM hallmarks as a potential treatment strategy. Biomarker-driven targeted therapies will hopefully guide the development of more refined treatments.

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Combined CDK4/6 and mTOR Inhibition Is Synergistic against Glioblastoma via Multiple Mechanisms.

Publications

Combined CDK4/6 and mTOR Inhibition Is Synergistic against Glioblastoma via Multiple Mechanisms.

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