Combined use of CDK4/6 and mTOR inhibitors induce synergistic growth arrest of diffuse intrinsic pontine glioma cells via mutual downregulation of mTORC1 activity.

BACKGROUND: Diffuse 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 METHODS: We 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. RESULTS: Immunoblot 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. CONCLUSION: These 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.

The distribution, clearance, and brainstem toxicity of panobinostat administered by convection-enhanced delivery.

OBJECTIVE The pan-histone deacetylase inhibitor panobinostat has preclinical efficacy against diffuse intrinsic pontine glioma (DIPG), and the oral formulation has entered a Phase I clinical trial. However, panobinostat does not cross the blood-brain barrier in humans. Convection-enhanced delivery (CED) is a novel neurosurgical drug delivery technique that bypasses the blood-brain barrier and is of considerable clinical interest in the treatment of DIPG. METHODS The authors investigated the toxicity, distribution, and clearance of a water-soluble formulation of panobinostat (MTX110) in a small- and large-animal model of CED. Juvenile male Wistar rats (n = 24) received panobinostat administered to the pons by CED at increasing concentrations and findings were compared to those in animals that received vehicle alone (n = 12). Clinical observation continued for 2 weeks. Animals were sacrificed at 72 hours or 2 weeks following treatment, and the brains were subjected to neuropathological analysis. A further 8 animals received panobinostat by CED to the striatum and were sacrificed 0, 2, 6, or 24 hours after infusion, and their brains explanted and snap-frozen. Tissue-drug concentration was determined by liquid chromatography tandem mass spectrometry (LC-MS/MS). Large-animal toxicity was investigated using a clinically relevant MRI-guided translational porcine model of CED in which a drug delivery system designed for humans was used. Panobinostat was administered at 30 µM to the ventral pons of 2 juvenile Large White-Landrace cross pigs. The animals were subjected to clinical and neuropathological analysis, and findings were compared to those obtained in controls after either 1 or 2 weeks. Drug distribution was determined by LC-MS/MS in porcine white and gray matter immediately after CED. RESULTS There were no clinical or neuropathological signs of toxicity up to an infused concentration of 30 µM in both small- and large-animal models. The half-life of panobinostat in rat brain after CED was 2.9 hours, and the drug was observed to be distributed in porcine white and gray matter with a volume infusion/distribution ratio of 2 and 3, respectively. CONCLUSIONS CED of water-soluble panobinostat, up to a concentration of 30 µM, was not toxic and was distributed effectively in normal brain. CED of panobinostat warrants clinical investigation in patients with DIPG.

Repurposing the anti-epileptic drug sodium valproate as an adjuvant treatment for diffuse intrinsic pontine glioma.

Targeting epigenetic changes in diffuse intrinsic pontine glioma (DIPG) may provide a novel treatment option for patients. This report demonstrates that sodium valproate, a histone deacetylase inhibitor (HDACi), can increase the cytotoxicity of carboplatin in an additive and synergistic manner in DIPG cells in vitro. Sodium valproate causes a dose-dependent decrease in DIPG cell viability in three independent ex vivo cell lines. Furthermore, sodium valproate caused an increase in acetylation of histone H3. Changes in cell viability were consistent with an induction of apoptosis in DIPG cells in vitro, determined by flow cytometric analysis of Annexin V staining and assessment of apoptotic markers by western blotting. Subsequently, immunofluorescent staining of neuronal and glial markers was used to determine toxicity in normal rat hippocampal cells. Pre-treatment of cells with sodium valproate enhanced the cytotoxic effects of carboplatin, in three DIPG cell lines tested. These results demonstrate that sodium valproate causes increased histone H3 acetylation indicative of HDAC inhibition, which is inversely correlated with a reduction in cell viability. Cell viability is reduced through an induction of apoptosis in DIPG cells. Sodium valproate potentiates carboplatin cytotoxicity and prompts further work to define the mechanism responsible for the synergy between these two drugs and determine in vivo efficacy. These findings support the use of sodium valproate as an adjuvant treatment for DIPG.

Polymorphisms near TBX5 and GDF7 are associated with increased risk for Barrett's esophagus.

BACKGROUND & AIMS: Barrett's esophagus (BE) increases the risk of esophageal adenocarcinoma (EAC). We found the risk to be BE has been associated with single nucleotide polymorphisms (SNPs) on chromosome 6p21 (within the HLA region) and on 16q23, where the closest protein-coding gene is FOXF1. Subsequently, the Barrett's and Esophageal Adenocarcinoma Consortium (BEACON) identified risk loci for BE and esophageal adenocarcinoma near CRTC1 and BARX1, and within 100 kb of FOXP1. We aimed to identify further SNPs that increased BE risk and to validate previously reported associations. METHODS: We performed a genome-wide association study (GWAS) to identify variants associated with BE and further analyzed promising variants identified by BEACON by genotyping 10,158 patients with BE and 21,062 controls. RESULTS: We identified 2 SNPs not previously associated with BE: rs3072 (2p24.1; odds ratio [OR] = 1.14; 95% CI: 1.09-1.18; P = 1.8 × 10(-11)) and rs2701108 (12q24.21; OR = 0.90; 95% CI: 0.86-0.93; P = 7.5 × 10(-9)). The closest protein-coding genes were respectively GDF7 (rs3072), which encodes a ligand in the bone morphogenetic protein pathway, and TBX5 (rs2701108), which encodes a transcription factor that regulates esophageal and cardiac development. Our data also supported in BE cases 3 risk SNPs identified by BEACON (rs2687201, rs11789015, and rs10423674). Meta-analysis of all data identified another SNP associated with BE and esophageal adenocarcinoma: rs3784262, within ALDH1A2 (OR = 0.90; 95% CI: 0.87-0.93; P = 3.72 × 10(-9)). CONCLUSIONS: We identified 2 loci associated with risk of BE and provided data to support a further locus. The genes we found to be associated with risk for BE encode transcription factors involved in thoracic, diaphragmatic, and esophageal development or proteins involved in the inflammatory response.