Tumor pharmacokinetics and pharmacodynamics of the CDK4/6 inhibitor ribociclib in patients with recurrent glioblastoma

Abstract

Introduction

We conducted a phase Ib study () to determine whether ribociclib, an inhibitor of cyclin-dependent kinases 4 and 6 (CDK4/6), penetrates tumor tissue and modulates downstream signaling pathways including retinoblastoma protein (Rb) in patients with recurrent glioblastoma (GBM).

Methods

Study participants received ribociclib (600 mg QD) for 8–21 days before surgical resection of their recurrent GBM. Total and unbound concentrations of ribociclib were measured in samples of tumor tissue, plasma, and cerebrospinal fluid (CSF). We analyzed tumor specimens obtained from the first (initial/pre-study) and second (recurrent/on-study) surgery by immunohistochemistry for Rb status and downstream signaling of CDK4/6 inhibition. Participants with Rb-positive recurrent tumors continued ribociclib treatment on a 21-day-on, 7-day-off schedule after surgery, and were monitored for toxicity and disease progression.

Results

Three participants with recurrent Rb-positive GBM participated in this study. Mean unbound (pharmacologically active) ribociclib concentrations in plasma, CSF, MRI-enhancing, MRI-non-enhancing, and tumor core regions were 0.337 μM, 0.632 μM, 1.242 nmol/g, 0.484 nmol/g, and 1.526 nmol/g, respectively, which exceeded the in vitro IC₅₀ (0.04 μM) for inhibition of CDK4/6 in cell-free assay. Modulation of pharmacodynamic markers of ribociclib CDK 4/6 inhibition in tumor tissues were inconsistent between study participants. No participants experienced serious adverse events, but all experienced early disease progression.

Conclusions

This study suggests that ribociclib penetrated recurrent GBM tissue at concentrations predicted to be therapeutically beneficial. Our study was unable to demonstrate tumor pharmacodynamic correlates of drug activity. Although well tolerated, ribociclib monotherapy seemed ineffective for the treatment of recurrent GBM.

Introduction

Anaplastic glioma (AG; grade III) and glioblastoma (GBM; grade IV) are primary malignant brain tumor sub-types and account for the majority of high-grade gliomas. Standard treatment includes surgical resection followed by radiation and chemotherapy with temozolomide (TMZ) [1]. Despite these interventions, patients with high-grade gliomas invariably exhibit tumor progression, with a 5-year survival rate of less than 10% [2, 3]. Thus, there is an urgent need for improved treatment.

The retinoblastoma protein (Rb), which binds and inhibits E2F transcription factors in the absence of mitogens, regulates the transition from G1 to S phase of the cell cycle. Upon stimulation with growth signals, D-type cyclins increase in abundance and bind cyclin-dependent kinases 4 and 6 (CDK4/6). Cyclin D-bound CDK4 or CDK6 phosphorylates Rb, releasing E2F proteins that drive cell cycle progression [4–6]. Alterations in this CDK4/6-Rb-E2F pathway, which promotes cell proliferation, often occur in human tumors [7]. While only a small number of tumors exhibit loss or mutation of the gene encoding Rb (RB1), an increase in CDK4/6 activity functionally inactivates this protein. Glioblastomas frequently exhibit loss or mutation of CDKN2A, which encodes the p16^INK4A and p14^ARF upstream regulators of CDK4/6 and predicts sensitization to CDK4/6 inhibition in preclinical models [8–10]. Thus, CDK4/6 inhibition as a means of restoring Rb activity is an attractive therapeutic strategy for the treatment of high-grade gliomas.

Preclinical data demonstrate that small-molecule inhibitors of CDK4/6 induce G₀ arrest of malignant cells, including GBM cells, both in vitro and pre-clinically in vivo [10–14]. The orally bioavailable small-molecule CDK4/6 inhibitor ribociclib (LEE011) has anti-tumor activity dependent upon the presence of functional Rb. Although the safety of ribociclib has been established in patients with cancer, the phase I trial excluded those with central nervous system tumors or brain metastases [15].

A particular concern for the treatment of brain tumors is the ability of systemically administered agents to cross the blood–brain barrier (BBB). A number of human studies have attempted to measure drug penetration into brain tumors with mixed results [16–25]. Additionally, the degree of tumor tissue penetration does not necessarily predict treatment efficacy [26]. While investigators often contend that most drugs do not efficiently cross the BBB, brain metastases and primary brain tumors frequently respond to systemically administered therapies [2, 27–29]. This suggests that the BBB is compromised within tumor tissue, leading to a leaky “blood-tumor barrier” that allows entry of therapeutic agents into the tumor tissue. In support of this concept, animal studies with the CDK4/6 inhibitor palbociclib demonstrated that concentrations of this drug were higher in intracerebral tumor tissue than normal brain tissue [14]. We therefore undertook a phase Ib study in patients with recurrent high-grade gliomas to assess the ability of ribociclib to penetrate brain tumor tissue and inhibit CDK4/6-Rb signaling and growth in tumor cells.

Materials and methods

Study population

Eligible study participants were 18 years or older, candidates for surgical resection following diagnosis of recurrent GBM or AG, and had available archived formalin-fixed, paraffin embedded (FFPE) or frozen tumor tissue from the initial surgery for molecular analysis. Additional eligibility criteria included participants having a Karnofsky performance scale score ≥ 70, adequate hematopoietic, renal and hepatic function, and the ability to provide written informed consent. Exclusion criteria included impaired gastro-intestinal drug absorption, a history of any cardiovascular disease, including long QT syndrome, and inability to comply with protocol procedures. Pregnant and lactating women and patients taking any strong inducers or inhibitors of CYP3A4/5 were also excluded. The clinical trial was approved by the University of Virginia Institutional Review Board () and by the FDA (IND #125168).

Study design

This single-institution (University of Virginia Health System) study was an open-label phase Ib clinical trial (Fig. 1). Baseline blood samples for ribociclib concentrations were collected prior to initiation of ribociclib treatment. Study participants were treated with ribociclib at the recommended phase II dose of 600 mg/day [15], taken once orally for 8–21 days prior to surgery. Novartis Pharmaceuticals generously provided 200 mg ribociclib capsules. Subjects received the last pre-surgical ribociclib dose 4–8 h prior to surgery to allow sufficient time for any modulation of downstream effectors of CDK4/6. We obtained blood samples within 1 h prior to surgery, and separated into plasma and buffy coat fractions. When feasible during surgery, CSF was also collected. Brain tumor core and infiltrating tumor were identified by co-localization with stealth MRI, and tissue samples of each area were obtained for FFPE or frozen (Fig. 2).

Fig. 1.

Study overview

Fig. 2.

Axial contrast-enhanced T1-weighted images. Images were obtained using a 3D magnetization prepared rapid acquisition gradient recalled-echo sequence) (a), and an axial FLAIR image (b) of recurrent glioblastoma (Subject 003). In (a), enclosed within the solid line is the necrotic core; enclosed between the dashed and solid lines is the enhancing neoplasm. Arrows indicate non-enhancing neoplasm

Immunohistochemistry (IHC) for Rb was performed on recurrent GBM specimens (detailed below); participants with Rb-negative tumors were excluded from further treatment as previous work suggested that Rb-deficient tumors are unlikely to respond to CDK4/6 inhibition [10, 12]. Study participants with Rb-positive tumors continued ribociclib treatment on a 21-days-on, 7-days-off schedule of a 28-day cycle until disease progression or the development of unacceptable toxicity.

MRI assessment

All patients had a post-operative brain MRI within 72 h post-surgery to determine the extent of resection and to have as a baseline for further measurement of tumor size. Subjects with Rb-positive tumors who continued ribociclib treatment post-surgery had follow-up MRI performed (including per-fusion and diffusion-weighted images) after every two cycles of treatment. The Response Assessment in Neuro-Oncology (RANO) Working Group criteria were used to determine disease progression [30].

Safety monitoring

Complete blood counts and blood chemistry, 12-lead electrocardiogram, vital signs, Karnofsky performance status, and physical exam were monitored throughout the course of treatment. Toxicities were graded using the National Cancer Institute (NCI) Common Terminology Criteria for Adverse Events (CTCAE) version 4.0, and adverse events were evaluated according to expectedness and attribution to drug treatment.

Measurement of ribociclib concentrations in tissues

The total concentrations of ribociclib in plasma, CSF, and frozen tumor tissue samples were determined using a validated liquid chromatography coupled with tandem mass spectrometry (LC–MS/MS) method as previously described [31]. The unbound fraction of ribociclib in plasma and tumor homogenate was determined using equilibrium dialysis with a 5-kDa cutoff regenerated cellulose membrane (96-well Equilibrium DIALYZER, Harvard Apparatus), as previously described [31]. Unbound ribociclib concentration in plasma or tumor tissue samples was calculated as the product of the fraction unbound and the total drug concentration. The concentration of unbound ribociclib in plasma and tumor tissue samples was calculated as the product of the unbound fraction and the total concentration.

Immunohistochemistry

Methods provided in Supplementary Information.

Results

Patient characteristics, safety, and efficacy

The planned enrollment for this study was ten evaluable study participants with Rb-positive recurrent GBM or AG. The study, however, was closed due to slow accrual after three participants completed treatment. Table 1 shows the patient demographics. All study participants received ribociclib for between 8 and 21 days, followed by surgical resection of the recurrent tumor. All enrolled participants had Rb-positive recurrent GBM and continued ribociclib treatment after surgery. All observed adverse events were grade 2 or less and were considered unlikely to be related to ribociclib treatment (Table 2). No participants withdrew from the study due to adverse events.

Table 1.

Patient demographics and study outcomes

	001	002	003
Age	81 years	53 years	35 years
Sex	Male	Male	Male
Date of first surgery (resection)	9/26/2014 (subtotal)	11/20/2015 (gross total)	3/29/2012 (gross total)
Pathology	GBM	GBM	GBM
MGMT status	Hypermethylated	Unmethylated	Unmethylated
IDH-1 (R132H)	Negative	Negative	Positive
PFS from first surgery	21 months	9 months	54 months
KPS at recurrence	70	80	90
Ribociclib treatment prior to surgery	8 days	12 days	14 days
Date of second surgery (resection)	8/16/2016 (subtotal)	9/7/2016 (gross total)	10/19/2016 (subtotal)
Pathology	Recurrent GBM	Recurrent GBM	Recurrent GBM
PFS from start of Ribociclib	2 months	5 months	2 months
OS from second surgery	10 months	19 months	12 months
OS from first surgery	32 months	28 months	66 months

GBM glioblastoma, MGMT methyl-guanine-methyl-transferase, IDH isocitrate dehydrogenase, PFS progression-free- survival, KPS Karnofsky performance scale score, OS overall survival

Table 2.

Adverse events while receiving ribociclib prior to surgery and during treatment phase

Toxicity	Grade 1	Grade 2	Total
Gastrointestinal disorders
Nausea	1	0	1
General disorders and administration site conditions
Gait disturbance	0	1	1
Edema, limbs	1	0	1
Renal and urinary disorders
Creatinine increased	0	1	1
Metabolism and nutrition disorders
Hyperglycemia	0	1	1
Musculoskeletal and connective tissue disorders
Generalized muscle weakness	0	1	1
Musculoskeletal and connective tissue disorder—other: muscle pain	1	0	1
Nervous system disorders
Stroke	1	0	1
Peripheral sensory neuropathy	1	0	1
Headaches	0	1	1
Seizure	1	1
Psychiatric disorders
Psychosis	1	0	1
Insomnia	0	1	1
Respiratory, thoracic and mediastinal disorders
Cough	1	0	1

Study participants, 001, 002 and 003 experienced tumor progression based on MRI at 2, 5, and 2 months, respectively, following surgical resection of their recurrent tumor (Table 1). They were treated with other regimens off-study following disease progression on ribociclib. Survival times from the date of second surgery were 10, 19, and 12 months, respectively. Subject 002, who had longer progression-free and overall survival than the other study participants following the second surgery, was the only participant who had gross total resection of the recurrent tumor, which may have contributed to his improved outcome.

Ribociclib concentrations in tissues

Total and unbound (pharmacologically active) ribociclib concentrations were measured in plasma, CSF, and tumor tissue obtained 4–8 h following the last dose of ribociclib administered on the day of surgery (Table 3). Ribociclib was not detected in plasma samples obtained at baseline (prior to initiation of ribociclib treatment). Day of surgery plasma concentrations of total and unbound ribociclib ranged from 1.26–5.44 μM to 0.15–0.49 μM, respectively. The only CSF sample obtained in one patient revealed a CSF ribociclib concentration of 0.63 μM, which provided a CSF/unbound plasma concentration ratio of 1.29 (i.e., 0.63/0.49), suggesting that the CSF and unbound plasma ribociclib concentrations were similar.

Table 3.

Total and unbound ribociclib concentrations and ratios in plasma, CSF, and tumor tissue samples

Sample	Subject ID	Fraction unbound (%)	Total ribociclib	Unbound ribociclib
Plasma	RIBO-001	10.2	3.65 μM	0.37 μM
Plasma	RIBO-002	11.9	1.26 μM	0.15 μM
Plasma	RIBO-003	9.0	5.44 μM	0.49 μM
CSF	RIBO-003	–	0.63 μM	–
Enhancing tumor	RIBO-001	5.9	8.73 nmol/g	0.52 nmol/g
Enhancing tumor	RIBO-001	3.9	27.22 nmol/g	1.06 nmol/g
Non-enhancing tumor	RIBO-001	4.2	5.80 nmol/g	0.25 nmol/g
Non-enhancing tumor	RIBO-001	6.1	11.92 nmol/g	0.72 nmol/g
Tumor core	RIBO-001	5.0	16.30 nmol/g	0.82 nmol/g
Enhancing tumor	RIBO-002	5.1	14.63 nmol/g	0.74 nmol/g
Enhancing tumor	RIBO-002	6.4	24.14 nmol/g	1.55 nmol/g
Enhancing tumor	RIBO-002	5.8	26.12 nmol/g	1.53 nmol/g
Enhancing tumor	RIBO-002	5.4	34.03 nmol/g	1.85 nmol/g
Tumor core	RIBO-002	6.4	17.13 nmol/g	1.09 nmol/g
Tumor core	RIBO-002	6.4	48.92 nmol/g	3.12 nmol/g
Enhancing tumor	RIBO-003	4.7	33.94 nmol/g	1.61 nmol/g
Enhancing tumor	RIBO-003	4.1	33.48 nmol/g	1.36 nmol/g
Enhancing tumor	RIBO-003	4.1	29.06 nmol/g	1.19 nmol/g
Enhancing tumor	RIBO-003	4.3	23.46 nmol/g	1.02 nmol/g
Tumor core	RIBO-003	5.7	21.80 nmol/g	1.24 nmol/g
Tumor core	RIBO-003	6.3	21.49 nmol/g	1.36 nmol/g

Subject ID	Sample	Total drug tumora	Unbound drug tumora	Total drug tumor-to-plasma ratiob	Unbound drug tumor-to-plasma ratioc
RIBO-001	Enhancing tumor	17.97	0.79	5.13	2.19
	Non-enhancing tumor	8.86	0.48	2.53	1.35
	Tumor core	16.30	0.82	4.65	2.29
RIBO-002	Enhancing tumor	24.73	1.14	20.43	9.83
	Tumor core	33.03	2.11	27.28	14.60
RIBO-003	Enhancing tumor	29.99	1.29	5.74	2.75
	Tumor core	21.65	1.30	4.14	2.77

^aMean concentrations of total and unbound ribociclib (in nmol/g) in tumor specimens from each subject were calculated

^bTotal drug tumor-to-plasma ratio was calculated as the ratio of total drug concentration in tumor (converted to molarity using an estimated tissue density of 1.04 g/mL) to that in plasma

^cUnbound drug tumor-to-plasma ratio was calculated as the ratio of unbound drug concentration in tumor (converted to molarity using an estimated tissue density of 1.04 g/mL) to that in plasma

Ribociclib concentrations in MRI-enhancing, MRI-non-enhancing, and tumor core regions showed considerable intra- and inter-tumor variability (Table 3). The mean concentrations of total and unbound ribociclib in MRI-enhancing, non-enhancing, and tumor core are summarized in Table 3. The extent of drug-tumor penetration was assessed by the tumor-to-plasma ratio of total ribociclib (Kp) and unbound ribociclib (Kp, uu) in the three subjects. Overall, tumor tissue concentrations of total and unbound ribociclib ranged from 5.80–48.92 nmol/g (mean ± SD of 23.42 ± 10.79 nmol/g) and 0.25–3.12 nmol/g (mean ± SD of 1.24 ± 0.64 nmol/g), respectively. The unbound fraction of ribociclib in tumor specimens (3.9–6.4%) were lower than those found in plasma (9.0–11.9%), which suggested that ribociclib has higher non-specific binding to brain tumor tissue than to plasma proteins.

Drug concentrations in MRI-enhancing, MRI-non-enhancing, and tumor core specimens were similar. Ribociclib penetrated better to enhancing and tumor core as compared to non-enhancing tumor, as demonstrated by higher unbound drug concentrations and higher unbound tumor-to-plasma ratio (Table 3). This was consistent with the notion that MRI-enhancing tumor regions present a disrupted blood–brain barrier. Notably, ribociclib CSF concentration and unbound concentration in non-enhancing tumor were > fivefold of the IC₅₀ for inhibition of CDK4/6 (Table 3), suggesting that ribociclib could penetrate and achieve potentially therapeutic concentration not only in tumor regions with disrupted blood–brain barrier but also in regions with largely intact blood–brain barrier in GBM patients.

Tumor tissue pharmacodynamic analyses

As a measure of ribociclib target engagement, we performed IHC for the CDK4/6 phosphorylation targets Rb_S807/811 using paired tumor tissue specimens acquired at baseline (untreated, archived, from first surgical resection) and after 8–21 days of ribociclib treatment (from second surgical resection). Two of the three subjects showed a decrease in the proportion of phospho-Rb-positive tumor cells and phospho-Rb histoscores in ribociclib-treated vs. baseline tumors, but one subject showed the opposite (Fig. S1A).

To determine if ribociclib treatment alters tumor cell fate, we measured tumor cell proliferation by Ki-67 IHC staining. Proportions of tumor cells staining positively for Ki-67 decreased in post-ribociclib-treated tumors compared to baseline in subjects 001 and 002, but in subject 003 Ki-67 staining increased compared to baseline (Fig. S1B). Surprisingly, these results directly contradict the observed changes in phospho-Rb (Fig. S1A).

Discussion

Our results suggest that ribociclib penetrates and accumulates within recurrent enhancing and non-enhancing regions of glioblastomas. In vitro studies have shown that treatment with 200 nM ribociclib (total drug) is sufficient to suppress Rb phosphorylation in cultured cells [13, 32]. Assuming a tumor tissue density of 1.04 g/mL, we observed tumor tissue concentrations of total ribociclib ranging from 6.03 to 50.88 μM (mean ± SD of 24.36 ± 11.22 μM), which exceeds those necessary for suppression of Rb phosphorylation and cell growth in vitro [GI₅₀ = 0.276 μM]. The unbound (pharmacologically active) ribociclib intra-tumor drug concentrations ranged from 0.26 to 3.24 μM (mean ± SD of 1.29 ± 0.67 μM); although tumor drug concentrations varied widely between and within patients, all concentrations measured were predicted to cause strong target inhibition. Despite observing what appeared to be supra-pharmacological ribociclib concentrations in tumors, we found that treatment with the standard dose of ribociclib (600 mg/day) as monotherapy did not consistently inhibit tumor tissue CDK4/6 activity (assessed by phospho-Rb IHC) or cell proliferation (assessed by Ki-67 IHC) when comparing archived tumor tissue with post-ribociclib treated tumor tissue. These findings correlate with the limited clinical efficacy of CDK4/6 inhibitor monotherapy with palbociclib or ribociclib, observed herein, as well as in other types of solid tumors [33]. The wide range of ribociclib concentrations observed within tumor tissues highlights the importance of sampling multiple tissue regions in studies with primarily pharmacokinetic and pharmacodynamic endpoints.

The daily administration of ribociclib for 8 to 21 days before surgery assured enough time to achieve steady state ribociclib concentrations in plasma and meaningful tumor tissue concentrations. Our results are limited by the small number of cases, but are consistent with a prior study in which five patients with recurrent brain tumors received pre-surgical treatment with 900 mg/day of ribociclib for 5 days. In concordance with our results, unbound drug concentrations in non-enhancing and enhancing MRI tumor regions were 0.46 ± 0.24 nmol/g and 2.32 ± 1.79 nmol/g, respectively, and mean unbound ribociclib fractions were 12.7% in plasma (CV = 17.6%) and 4.6% in tumor tissues (CV = 23%) [31]. Therefore, even in non-enhancing GBM, ribociclib achieves concentrations that exceed those felt to be pharmacologically active to inhibit CDK4/6.

The pharmacodynamic endpoints to assess pathway inhibition, one of the objectives of our window-of-opportunity study, did not yield a consistent pattern. A limitation of our study is the use of paired archived baseline tumor tissue from the first surgery as a comparator to recurrent post-ribociclib tumor tissue. In a similar study, patients with recurrent GBM were treated with 7 days of the CDK4/6 inhibitor palbociclib, before surgery. In this study by Taylor et al., analysis of 4 matched samples comparing archival FFPE tumor tissue from the initial surgery and tumor obtained at the second surgery showed drug concentrations that exceeded those felt to be pharmacologically active, but did not produce significant changes in GBM cell proliferation as determined by Ki-67 IHC [34]. A caveat to the comparison of paired tumor specimens from these time points is that study participants received standard therapies (radiation, chemotherapy) after the first surgical resection, and recurrent tumors may have evolved [e.g. (epi)genetically] in the interim [35, 36]. In contrast, Tien et al. recently reported findings from a study of 12 patients with recurrent GBM (all tumors were Rb-positive with either CDKN2A deletion or amplification of CDK4/6 or CCND1/2/3) treated with 900 mg/day of ribociclib for 5 days before surgery. The authors observed a significant decrease in Rb phosphorylation and cell proliferation when comparing archived tumor samples from initial surgery to those obtained at the time of surgery after ribociclib treatment [37]. The mean concentrations of unbound ribociclib across all subjects observed by Tien et al. were similar to those reported herein in CSF (0.37 μM vs. 0.63 μM), MRI-enhancing tumor or core (2.15 nmol/g vs. 1.33 nmol/g), and MRI-non-enhancing tumor (0.56 nmol/g vs. 0.48 nmol/g). In the Tien et al. study, six patients who had a “responsive” pharmacokinetic/pharmacodynamic signal in the post-ribociclib treatment tumor continued treatment with ribociclib after surgery and achieved a median PFS of 9.7 weeks, indicating that ribociclib monotherapy has limited clinical efficacy in GBM. Interestingly, in the tumor tissue obtained from three subjects during re-resection after progression on ribociclib, they identified a rebound in phospho-Rb levels and mTOR pathway upregulation as a potential mechanism of resistance to CDK4/6 inhibition [37].

Although the optimal study would have a tumor biopsy specimen obtained at the time of recurrence, followed by the experimental treatment and then surgery to obtain tissue for relevant molecular pharmacodynamic studies (the biopsy-treat-biopsy paradigm), this is not feasible in most cases of high-grade gliomas because of the risk-benefit of an additional invasive surgical procedure. Tumor heterogeneity is another factor to consider in the interpretation of results of tumor pharmacodynamic assays because one is comparing tumor obtained at different time points and slightly different locations. However, comparison of phospho-Rb and Ki-67 measurements in primary and recurrent GBM specimens showed no significant difference between time points [37]. Furthermore, in our study, the presence of necrotic tumor precluded adequate assessment of phosphoprotein modulation in some tumor areas. In the assessment of new drugs for the treatment of gliomas, identifying appropriate pharmacodynamic biomarkers that both correlate with clinical outcomes and are amenable to serial non-invasive measurement remains a challenge.

Early disease progression in our small cohort suggested that monotherapy with ribociclib may not have meaningful clinical activity in the treatment of recurrent high-grade glioma. All of our patients had low tumor burdens after their second surgeries, and recurrent tumors were Rb-positive by IHC. In a phase II trial, 22 patients were treated with palbociclib (125 mg/day on a 21-days-on, 7-days-off cycle) for recurrent Rb-positive GBM. That study was stopped prematurely and suggested a lack of efficacy, with a median PFS of 5.1 weeks and an OS of 15.4 weeks [34]. A third CDK4/6 inhibitor (abemaciclib, 150 mg or 200 mg twice daily) showed limited efficacy in a phase I trial including 17 patients with recurrent GBM; the best response occurred in three patients experiencing stable disease for ≥ 24 weeks [38]. These results, when combined with our findings, suggest that monotherapy with current CDK4/6 inhibitors is unlikely to be of substantial benefit in the treatment of high-grade gliomas.

Enthusiasm for GBM treatment with single-agent CDK4/6 inhibition has waned following the observation of negative results from early clinical trials. Although our accrual goal was ten subjects, we closed the study following evaluation of three eligible subjects due to slow accrual, and disappointing emergent results from other trials that tested CDK4/6 inhibitor monotherapies for high-grade gliomas. Preclinical studies from our institution have suggested potential GBM resistance mechanisms to CDK4/6 inhibitors, including rapid proneural-mesenchymal transition [39], mTOR upregulation [40], and MET and TRK upregulation [41]. These reports demonstrate the synergistic potential of combining CDK4/6 inhibitors with mTOR and MET/TRK inhibitors. Along these lines, an ongoing clinical trial is defining and testing the maximum tolerated doses of the combination of ribociclib and everolimus after radiation in children with diffuse infiltrating pontine gliomas and high-grade gliomas (). Combination therapy with CDK4/6 inhibitors may thus represent a viable path forward in the treatment of GBM, and more such trials are warranted. The finding that ribociclib reaches therapeutic concentrations in GBM confirms it as a promising CDK4/6 inhibitor to be studied in such combinations.