Tumour-specific proline vulnerability uncovered by differential ribosome codon reading.

Tumour growth and metabolic adaptation may restrict the availability of certain amino acids for protein synthesis. It has recently been shown that certain types of cancer cells depend on glycine, glutamine, leucine and serine metabolism to proliferate and survive. In addition, successful therapies using L-asparaginase-induced asparagine deprivation have been developed for acute lymphoblastic leukaemia. However, a tailored detection system for measuring restrictive amino acids in each tumour is currently not available. Here we harness ribosome profiling for sensing restrictive amino acids, and develop diricore, a procedure for differential ribosome measurements of codon reading. We first demonstrate the functionality and constraints of diricore using metabolic inhibitors and nutrient deprivation assays. Notably, treatment with L-asparaginase elicited both specific diricore signals at asparagine codons and high levels of asparagine synthetase (ASNS). We then applied diricore to kidney cancer and discover signals indicating restrictive proline. As for asparagine, this observation was linked to high levels of PYCR1, a key enzyme in proline production, suggesting a compensatory mechanism allowing tumour expansion. Indeed, PYCR1 is induced by shortage of proline precursors, and its suppression attenuated kidney cancer cell proliferation when proline was limiting. High PYCR1 is frequently observed in invasive breast carcinoma. In an in vivo model system of this tumour, we also uncover signals indicating restrictive proline. We further show that CRISPR-mediated knockout of PYCR1 impedes tumorigenic growth in this system. Thus, diricore has the potential to reveal unknown amino acid deficiencies, vulnerabilities that can be used to target key metabolic pathways for cancer treatment.

Antisense oligonucleotide-based treatment of retinitis pigmentosa caused by USH2A exon 13 mutations.

Mutations in USH2A are among the most common causes of syndromic and non-syndromic retinitis pigmentosa (RP). The two most recurrent mutations in USH2A, c.2299delG and c.2276G > T, both reside in exon 13. Skipping exon 13 from the USH2A transcript presents a potential treatment modality in which the resulting transcript is predicted to encode a slightly shortened usherin protein. Morpholino-induced skipping of ush2a exon 13 in zebrafish ush2armc1 mutants resulted in the production of usherinΔexon 13 protein and a completely restored retinal function. Antisense oligonucleotides were investigated for their potential to selectively induce human USH2A exon 13 skipping. Lead candidate QR-421a induced a concentration-dependent exon 13 skipping in induced pluripotent stem cell (iPSC)-derived photoreceptor precursors from an Usher syndrome patient homozygous for the c.2299delG mutation. Mouse surrogate mQR-421a reached the retinal outer nuclear layer after a single intravitreal injection and induced a detectable level of exon skipping until at least 6 months post-injection. In conclusion, QR-421a-induced exon skipping proves to be a highly promising treatment option for RP caused by mutations in USH2A exon 13.

The impact of P-glycoprotein and breast cancer resistance protein on the brain pharmacokinetics and pharmacodynamics of a panel of MEK inhibitors.

Mitogen/extracellular signal-regulated kinase (MEK) inhibitors have been tested in clinical trials for treatment of intracranial neoplasms, including glioblastoma (GBM), but efficacy of these drugs has not yet been demonstrated. The blood-brain barrier (BBB) is a major impediment to adequate delivery of drugs into the brain and may thereby also limit the successful implementation of MEK inhibitors against intracranial malignancies. The BBB is equipped with a range of ATP-dependent efflux transport proteins, of which P-gp (ABCB1) and BCRP (ABCG2) are the two most dominant for drug efflux from the brain. We investigated their impact on the pharmacokinetics and target engagement of a panel of clinically applied MEK inhibitors, in order to select the most promising candidate for brain cancers in the context of clinical pharmacokinetics and inhibitor characteristics. To this end, we used in vitro drug transport assays and conducted pharmacokinetic and pharmacodynamic studies in wildtype and ABC-transporter knockout mice. PD0325901 displayed more promising characteristics than trametinib (GSK1120212), binimetinib (MEK162), selumetinib (AZD6244), and pimasertib (AS703026): PD0325901 was the weakest substrate of P-gp and BCRP in vitro, its brain penetration was only marginally higher in Abcb1a/b;Abcg2-/- mice, and efficient target inhibition in the brain could be achieved at clinically relevant plasma levels. Notably, target inhibition could also be demonstrated for selumetinib, but only at plasma levels far above levels in patients receiving the maximum tolerated dose. In summary, our study recommends further development of PD0325901 for the treatment of intracranial neoplasms.

ATP-binding cassette transporters limit the brain penetration of Wee1 inhibitors.

Introduction Wee1 is an important kinase involved in the G2 cell cycle checkpoint and frequently upregulated in intracranial neoplasms such as glioblastoma (GBM) and diffuse intrinsic pontine glioma (DIPG). Two small molecules are available that target Wee1, AZD1775 and PD0166285, and clinical trials with AZD1775 have already been started. Since GBM and DIPG are highly invasive brain tumors, they are at least to some extent protected by the blood-brain barrier (BBB) and its ATP-binding cassette (ABC) efflux transporters. Methods We have here conducted a comprehensive set of in vitro and in vivo experiments to determine to what extent two dominant efflux transporters in the BBB, P-gp (ABCB1) and BCRP (ABCG2), exhibit affinity towards AZD1775 and PD0166285 and restrict their brain penetration. Results Using these studies, we demonstrate that AZD1775 is efficiently transported by both P-gp and BCRP, whereas PD0166285 is only a substrate of P-gp. Nonetheless, the brain penetration of both compounds was severely restricted in vivo, as indicated by a 5-fold (PD0166285) and 25-fold (AZD1775) lower brain-plasma ratio in wild type mice compared to Abcb1a/b;Abcg2-/- mice. Conclusion The brain penetration of these Wee1 inhibitors is severely limited by ABC transporters, which may compromise their clinical efficacy against intracranial neoplasms such as DIPG and GBM.

ABCB1 Attenuates the Brain Penetration of the PARP Inhibitor AZD2461.

Poly (ADP-ribose) polymerase (PARP) inhibitors are a relatively new class of anticancer agents that have attracted attention for treatment of glioblastoma because of their ability to potentiate temozolomide chemotherapy. Previous studies have demonstrated that sufficient brain penetration is a prerequisite for efficacy of PARP inhibitors in glioma mouse models. Unfortunately, however, most of the PARP inhibitors developed to date have a limited brain penetration due to the presence of P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP) at the blood-brain barrier. AZD2461 is a novel PARP inhibitor that is unaffected by P-gp mediated resistance in breast cancer models and thus appears to have promising characteristics for brain penetration. We here use a comprehensive set of in vitro and in vivo models to study the brain penetration and oral bioavailability of AZD2461. We report that AZD2461 has a good membrane permeability. However, it is a substrate of P-gp and BCRP, and P-gp in particular limits its brain penetration in vivo. We show that AZD2461 has a low oral bioavailability, although it is not affected by P-gp and BCRP. Together, these findings are not in favor of further development of AZD2461 for treatment of glioblastoma.

P-Glycoprotein (MDR1/ABCB1) Restricts Brain Penetration of the Bruton's Tyrosine Kinase Inhibitor Ibrutinib, While Cytochrome P450-3A (CYP3A) Limits Its Oral Bioavailability.

Ibrutinib (Imbruvica), an oral tyrosine kinase inhibitor (TKI) approved for treatment of B-cell malignancies, irreversibly inhibits the Bruton's tyrosine kinase (BTK). Its abundant metabolite, dihydrodiol-ibrutinib (ibrutinib-DiOH), which is primarily formed by CYP3A, has a 10-fold reduced BTK inhibitory activity. Using in vitro transport assays and genetically modified mouse models, we investigated whether the multidrug efflux transporters ABCB1 and ABCG2 and the multidrug-metabolizing CYP3A enzyme family can affect the oral bioavailability and tissue disposition of ibrutinib and ibrutinib-DiOH. In vitro, ibrutinib was transported moderately by human ABCB1 and mouse Abcg2 but not detectably by human ABCG2. In mice, Abcb1 markedly restricted the brain penetration of ibrutinib and ibrutinib-DiOH, either alone or in combination with Abcg2, resulting in 4.5- and 5.9-fold increases in ibrutinib brain-to-plasma ratios in Abcb1a/1b-/- and Abcb1a/1b;Abcg2-/- mice relative to wild-type mice. Abcb1 and/or Abcg2 did not obviously restrict ibrutinib oral bioavailability, but Cyp3a deficiency increased the ibrutinib plasma AUC by 9.7-fold compared to wild-type mice. This increase was mostly reversed (5.1-fold reduction) by transgenic human CYP3A4 overexpression, with roughly equal contributions of intestinal and hepatic CYP3A4 metabolism. Our results suggest that pharmacological inhibition of ABCB1 during ibrutinib therapy might benefit patients with malignancies or (micro)metastases positioned behind an intact blood-brain barrier, or with substantial expression of this transporter in the malignant cells. Moreover, given the strong in vivo impact of CYP3A, inhibitors or inducers of this enzyme family will likely strongly affect ibrutinib oral bioavailability and, thus, its therapeutic efficacy, as well as its toxicity risks.

ABCB1 and ABCG2 restrict the brain penetration of a panel of novel EZH2-Inhibitors.

Enhancer of Zeste Homolog 2 (EZH2) has emerged as a promising therapeutic target for treatment of a broad spectrum of tumors including gliomas. We explored the interactions of five novel, structurally similar EZH2 inhibitors (EPZ005687, EPZ-6438, UNC1999, GSK343 and GSK126) with P-glycoprotein (P-gp/ABCB1) and breast cancer resistance protein (BCRP/ABCG2). The compounds were screened by in vitro transwell assays and EPZ005687, EPZ-6438 and GSK126 were further tested in vivo using wild-type (WT), Abcb1 and/or Abcg2 knockout mice. All EZH2 inhibitors are transported by P-gp and BCRP, although in vitro the transporter affinity of GSK126 was obscured by very low membrane permeability. Both P-gp and Bcrp1 restrict the brain penetration of EPZ005687 and GSK126, whereas the brain accumulation of EPZ-6438 is limited by P-gp only and efflux of EPZ-6438 was completely abrogated by elacridar. Intriguingly, an unknown factor present in all knockout mouse strains causes EPZ005687 and EPZ-6438 retention in plasma relative to WT mice, a phenomenon not seen with GSK126. In WT mice, the GSK126 tissue-to-plasma ratio for all tissues is lower than for EPZ005687 or EPZ-6438. Moreover, the oral bioavailability of GSK126 is only 0.2% in WT mice, which increases to 14.4% in Abcb1;Abcg2 knockout mice. These results are likely due to poor membrane permeability and question the clinical usefulness of GSK126. Although all tested EZH2 inhibitors are substrates of P-gp and BCRP, restricting the brain penetration and potential utility for treatment of glioma, EPZ-6438 would be the most suitable candidate of this series.

P-glycoprotein and breast cancer resistance protein restrict the brain penetration of the CDK4/6 inhibitor palbociclib.

INTRODUCTION: Palbociclib is a cyclin dependent kinase (CDK) 4/6 inhibitor with nanomolar potency and was recently approved for treatment of breast cancer. The drug may also be useful in glioblastoma (GBM) and diffuse intrinsic pontine gliomas (DIPG), which often have an activated CDK4/6-retinoblastoma signaling pathway. However, GBM and DIPG spread widely into the surrounding brain, which calls for a CDK4/6 inhibitor with sufficient blood-brain barrier penetration. METHODS: We first performed in vitro transwell assays and demonstrate that palbociclib is a substrate of both P-gp and BCRP. Next, we conducted pharmacokinetic studies using wildtype, Abcg2(-/-), Abcb1a/b(-/-) and Abcg2; Abcb1a/b(-/-) mice. RESULTS: The plasma levels were about 3000 and 500 nM and similar in all genotypes at 1 and 4 h after i.v. administration of 10 mg/kg. At 4 h the brain-to-plasma ratios were 0.3 in WT and Abcg2(-/-) mice versus 5.5 and 15 in Abcb1a/b(-/-) and Abcg2; Abcb1a/b(-/-) mice, respectively. The oral bioavailability of palbociclib was high (63 %) in WT mice and increased only modestly and non-significantly in Abcg2; Abcb1a/b(-/-) mice. The plasma level after oral dosing of 150 mg/kg was already much higher than observed in patients (200-400 nM) and exceeded 2500 nM for up to 24 h. This latter dose is commonly used in preclinical studies, which calls into question their predictive value as they were conducted at dose levels causing a clinically non-relevant systemic drug exposure. CONCLUSION: Thus, the brain penetration of palbociclib is restricted by P-gp and BCRP, which may restrict the efficacy against GBM and DIPG. Moreover, preclinical studies with this agent should be conducted at a more clinically relevant dose level.

In vivo disposition of doxorubicin is affected by mouse Oatp1a/1b and human OATP1A/1B transporters.

Organic anion-transporting polypeptides (OATPs) are important drug uptake transporters, mediating distribution of substrates to several pharmacokinetically relevant organs. Doxorubicin is a widely used anti-cancer drug extensively studied for its interactions with various drug transporters, but not OATPs. Here, we investigated the role of OATP1A/1B proteins in the distribution of doxorubicin. In vitro, we observed ∼ 2-fold increased doxorubicin uptake in HEK293 cells overexpressing human OATP1A2, but not OATP1B1 or OATP1B3. In mice, absence of Oatp1a/1b transporters led to up to 2-fold higher doxorubicin plasma concentrations and 1.3-fold higher plasma AUC. Conversely, liver AUC and liver-to-plasma ratios of Oatp1a/1b(-/-) mice were 1.4-fold and up to 4.1-fold lower than in wild-type mice, respectively. Decreased doxorubicin levels in the small intestinal content reflected those in the liver, indicating a reduced biliary excretion of doxorubicin in Oatp1a/1b(-/-) mice. These results demonstrate important control of doxorubicin plasma clearance and hepatic uptake by mouse Oatp1a/1b transporters. This is unexpected, as the fairly hydrophobic weak base doxorubicin is an atypical OATP1A/1B substrate. Interestingly, transgenic liver-specific expression of human OATP1A2, OATP1B1 or OATP1B3 could partially rescue the increased doxorubicin plasma levels of Oatp1a/1b(-/-) mice. Hepatic uptake and bile-derived intestinal excretion of doxorubicin were completely reverted to wild-type levels by OATP1A2, and partially by OATP1B1 and OATP1B3. Thus, doxorubicin is transported by hepatocyte-expressed OATP1A2, -1B1 and -1B3 in vivo, illustrating an unexpectedly wide substrate specificity. These findings have possible implications for the uptake, disposition, therapy response and toxicity of doxorubicin, also in human tumors and tissues expressing these transporters.

Acquired and intrinsic resistance to vemurafenib in BRAFV600E -driven melanoma brain metastases.

BRAFV600 -mutated melanoma brain metastases (MBMs) are responsive to BRAF inhibitors, but responses are generally less durable than those of extracranial metastases. We tested the hypothesis that the drug efflux transporters P-glycoprotein (P-gp; ABCB1) and breast cancer resistance protein (BCRP; ABCG2) expressed at the blood-brain barrier (BBB) offer MBMs protection from therapy. We intracranially implanted A375 melanoma cells in wild-type (WT) and Abcb1a/b;Abcg2-/- mice, characterized the tumor BBB, analyzed drug levels in plasma and brain lesions after oral vemurafenib administration, and determined the efficacy against brain metastases and subcutaneous lesions. Although contrast-enhanced MRI demonstrated that the integrity of the BBB is disrupted in A375 MBMs, vemurafenib achieved greater antitumor efficacy against MBMs in Abcb1a/b;Abcg2-/- mice compared with WT mice. Concordantly, P-gp and BCRP are expressed in MBM-associated brain endothelium both in patients and in A375 xenografts and expression of these transporters limited vemurafenib penetration into A375 MBMs. Although initially responsive, A375 MBMs rapidly developed therapy resistance, even in Abcb1a/b;Abcg2-/- mice, and this was unrelated to pharmacokinetic or target inhibition issues. Taken together, we demonstrate that both intrinsic and acquired resistance can play a role in MBMs.

Dual mTORC1 and mTORC2 inhibitor Palomid 529 penetrates the blood-brain barrier without restriction by ABCB1 and ABCG2.

Palomid 529, a novel dual mTORC1/2 inhibitor has displayed interesting activities in experimental models and is a candidate for clinical evaluation. We have assessed the interaction of Palomid 529 with ATP-binding cassette (ABC) drug efflux transporters ABCB1 (P-gp/P-glycoprotein) and ABCG2 (BCRP/Breast Cancer Resistant Protein) by in vitro transwell assays, and their effects on the brain penetration using drug disposition analysis of i.v. and oral Palomid 529 in wild-type (WT) and Abcb1 and/or Abcg2 knockout (KO) mice. Palomid 529 lacked affinity for these transporters in vitro, in contrast to GDC-0941, a small molecule PI3K inhibitor, which we used as control substance for in vitro transport. The plasma AUCi.v. of micronized and DMSO formulated Palomid 529 was similar in WT and KO mice. Importantly, the brain and brain tumor concentration of Palomid 529 at a high dose (54 mg/kg) was also similar in both strains, whereas a less than 1.4-fold difference (p < 0.05) was found at the low (5.4 mg/kg) dose. Because of poor solubility, the oral bioavailability of micronized Palomid 529 was only 5%. Olive oil or spray-dried formulation greatly improved the bioavailability up to 50%. Finally, Palomid 529 effectively inhibits the orthotopic U87 glioblastoma growth. In summary, Palomid 529 is the first mTOR targeting drug lacking affinity for ABCB1/ABCG2 and having good brain penetration. This warrants further evaluation of Palomid 529 for treatment of high-grade gliomas and other intracranial malignancies.

A pathogenic proteolysis-resistant huntingtin isoform induced by an antisense oligonucleotide maintains huntingtin function.

Huntington's disease (HD) is a late-onset neurological disorder for which therapeutics are not available. Its key pathological mechanism involves the proteolysis of polyglutamine-expanded (polyQ-expanded) mutant huntingtin (mHTT), which generates N-terminal fragments containing polyQ, a key contributor to HD pathogenesis. Interestingly, a naturally occurring spliced form of HTT mRNA with truncated exon 12 encodes an HTT (HTTΔ12) with a deletion near the caspase-6 cleavage site. In this study, we used a multidisciplinary approach to characterize the therapeutic potential of targeting HTT exon 12. We show that HTTΔ12 was resistant to caspase-6 cleavage in both cell-free and tissue lysate assays. However, HTTΔ12 retained overall biochemical and structural properties similar to those of wt-HTT. We generated mice in which HTT exon 12 was truncated and found that the canonical exon 12 was dispensable for the main physiological functions of HTT, including embryonic development and intracellular trafficking. Finally, we pharmacologically induced HTTΔ12 using the antisense oligonucleotide (ASO) QRX-704. QRX-704 showed predictable pharmacology and efficient biodistribution. In addition, it was stable for several months and inhibited pathogenic proteolysis. Furthermore, QRX-704 treatments resulted in a reduction of HTT aggregation and an increase in dendritic spine count. Thus, ASO-induced HTT exon 12 splice switching from HTT may provide an alternative therapeutic strategy for HD.

ATP-binding cassette transporters restrict drug delivery and efficacy against brain tumors even when blood-brain barrier integrity is lost.

The impact of a compromised blood-brain barrier (BBB) on the drug treatment of intracranial tumors remains controversial. We characterize the BBB integrity in several intracranial tumor models using magnetic resonance imaging, fluorescent dyes, and autoradiography and determine the distribution and efficacy of docetaxel in brain tumors grafted in Abcb1-proficient and Abcb1-deficient mice. Leakiness of the tumor vasculature varies from extensive to absent. Regardless of the extent of leakiness, tumor blood vessels express ATP-binding cassette transporters (Abcb1 and Abcg2). A leaky vasculature results in higher docetaxel tumor levels compared to normal brain. Nevertheless, Abcb1 can reduce drug distribution and efficacy even in leaky models. Thus, BBB leakiness does not ensure the unimpeded access of ATP-binding cassette transporter substrate drugs. Therapeutic responses may be observed, but the full potential of such therapeutics may still be attenuated. Consequently, BBB-penetrable drugs with little to no affinity for efflux transporters are preferred for the treatment of intracranial tumors.

Combined Therapy of AXL and HDAC Inhibition Reverses Mesenchymal Transition in Diffuse Intrinsic Pontine Glioma.

PURPOSE: Diffuse intrinsic pontine glioma (DIPG) is an incurable type of pediatric brain cancer, which in the majority of cases is driven by mutations in genes encoding histone 3 (H3K27M). We here determined the preclinical therapeutic potential of combined AXL and HDAC inhibition in these tumors to reverse their mesenchymal, therapy-resistant, phenotype. EXPERIMENTAL DESIGN: We used public databases and patient-derived DIPG cells to identify putative drivers of the mesenchymal transition in these tumors. Patient-derived neurospheres, xenografts, and allografts were used to determine the therapeutic potential of combined AXL/HDAC inhibition for the treatment of DIPG. RESULTS: We identified AXL as a therapeutic target and regulator of the mesenchymal transition in DIPG. Combined AXL and HDAC inhibition had a synergistic and selective antitumor effect on H3K27M DIPG cells. Treatment of DIPG cells with the AXL inhibitor BGB324 and the HDAC inhibitor panobinostat resulted in a decreased expression of mesenchymal and stem cell genes. Moreover, this combination treatment decreased expression of DNA damage repair genes in DIPG cells, strongly sensitizing them to radiation. Pharmacokinetic studies showed that BGB324, like panobinostat, crosses the blood-brain barrier. Consequently, treatment of patient-derived DIPG xenograft and murine DIPG allograft-bearing mice with BGB324 and panobinostat resulted in a synergistic antitumor effect and prolonged survival. CONCLUSIONS: Combined inhibition of AXL and HDACs in DIPG cells results in a synergistic antitumor effect by reversing their mesenchymal, stem cell-like, therapy-resistant phenotype. As such, this treatment combination may serve as part of a future multimodal therapeutic strategy for DIPG.

Buparlisib is a brain penetrable pan-PI3K inhibitor.

Characterization of the genomic landscapes of intracranial tumours has revealed a clear role for the PI3K-AKT-mTOR pathway in tumorigenesis and tumour maintenance of these malignancies, making phosphatidylinositol 3-kinase (PI3K) inhibition a promising therapeutic strategy for these tumours. Buparlisib is a novel pan-PI3K inhibitor that is currently in clinical development for various cancers, including primary and secondary brain tumours. Importantly however, earlier studies have revealed that sufficient brain penetration is a prerequisite for antitumor efficacy against intracranial tumours. We therefore investigated the brain penetration of buparlisib using a comprehensive set of in vitro and in vivo mouse models. We demonstrate that buparlisib has an excellent brain penetration that is unaffected by efflux transporters at the blood-brain barrier, complete oral bioavailability and efficient intracranial target inhibition at clinically achievable plasma concentrations. Together, these characteristics make buparlisib the ideal candidate for intracranially-targeted therapeutic strategies that involve PI3K inhibition.

Improved Brain Penetration and Antitumor Efficacy of Temozolomide by Inhibition of ABCB1 and ABCG2.

The anticancer drug temozolomide is the only drug with proven activity against high-grade gliomas and has therefore become a part of the standard treatment of these tumors. P-glycoprotein (P-gp; ABCB1) and breast cancer resistance protein (BCRP; ABCG2) are transport proteins, which are present at the blood-brain barrier and limit the brain uptake of substrate drugs. We have studied the effect of P-gp and BCRP on the pharmacokinetics and pharmacodynamics of temozolomide, making use of a comprehensive set of in vitro transport experiments and in vivo pharmacokinetic and antitumor efficacy experiments using wild-type, Abcg2-/-, Abcb1a/b-/-, and Abcb1a/b;Abcg2-/- mice. We here show that the combined deletion of Abcb1a/b and Abcg2 increases the brain penetration of temozolomide by 1.5-fold compared to wild-type controls (P < .001) without changing the systemic drug exposure. Moreover, the same increase was achieved when temozolomide was given to wild-type mice in combination with the dual P-gp/BCRP inhibitor elacridar (GF120918). The antitumor efficacy of temozolomide against three different intracranial tumor models was significantly enhanced when Abcb1a/b and Abcg2 were genetically deficient or pharmacologically inhibited in recipient mice. These findings call for further clinical testing of temozolomide in combination with elacridar for the treatment of gliomas, as this offers the perspective of further improving the antitumor efficacy of this already active agent.

MELK Inhibition in Diffuse Intrinsic Pontine Glioma.

Purpose: Diffuse intrinsic pontine glioma (DIPG) is a highly aggressive pediatric brain tumor, for which no effective therapeutic options currently exist. We here determined the potential of inhibition of the maternal embryonic leucine zipper kinase (MELK) for the treatment of DIPG.Experimental Design: We evaluated the antitumor efficacy of the small-molecule MELK inhibitor OTSSP167 in vitro in patient-derived DIPG cultures, and identified the mechanism of action of MELK inhibition in DIPG by RNA sequencing of treated cells. In addition, we determined the blood-brain barrier (BBB) penetration of OTSSP167 and evaluated its translational potential by treating mice bearing patient-derived DIPG xenografts.Results: This study shows that MELK is highly expressed in DIPG cells, both in patient samples and in relevant in vitro and in vivo models, and that treatment with OTSSP167 strongly decreases proliferation of patient-derived DIPG cultures. Inhibition of MELK in DIPG cells functions through reducing inhibitory phosphorylation of PPARγ, resulting in an increase in nuclear translocation and consequent transcriptional activity. Brain pharmacokinetic analyses show that OTSSP167 is a strong substrate for both MDR1 and BCRP, limiting its BBB penetration. Nonetheless, treatment of Mdr1a/b;Bcrp1 knockout mice carrying patient-derived DIPG xenografts with OTSSP167 decreased tumor growth, induced remissions, and resulted in improved survival.Conclusions: We show a strong preclinical effect of the kinase inhibitor OTSSP167 in the treatment of DIPG and identify the MELK-PPARγ signaling axis as a putative therapeutic target in this disease. Clin Cancer Res; 24(22); 5645-57. ©2018 AACR.

PI3K-mTOR Pathway Inhibition Exhibits Efficacy Against High-grade Glioma in Clinically Relevant Mouse Models.

Purpose: The PI3K-AKT-mTOR signaling pathway is frequently activated in glioblastoma and offers several druggable targets. However, clinical efficacy of PI3K/mTOR inhibitors in glioblastoma has not yet been demonstrated. Insufficient drug delivery may limit the efficacy of PI3K/mTOR inhibitors against glioblastoma. The presence of the efflux transporters ABCB1/Abcb1 (P-glycoprotein, MDR1) and ABCG2/Abcg2 (BCRP) at the blood-brain barrier (BBB) restricts the brain penetration of many drugs.Experimental Design: We used in vitro drug transport assays and performed pharmacokinetic/pharmacodynamic studies in wild-type and ABC-transporter knockout mice. The efficacy of PI3K-mTOR inhibition was established using orthotopic allograft and genetically engineered spontaneous glioblastoma mouse models.Results: The mTOR inhibitors rapamycin and AZD8055 are substrates of ABCB1, whereas the dual PI3K/mTOR inhibitor NVP-BEZ235 and the PI3K inhibitor ZSTK474 are not. Moreover, ABCG2 transports NVP-BEZ235 and AZD8055, but not ZSTK474 or rapamycin. Concordantly, Abcb1a/b-/-;Abcg2-/- mice revealed increased brain penetration of rapamycin (13-fold), AZD8055 (7.7-fold), and NVP-BEZ235 (4.5-fold), but not ZSTK474 relative to WT mice. Importantly, ABC transporters limited rapamycin brain penetration to subtherapeutic levels, while the reduction in NVP-BEZ235 brain penetration did not prevent target inhibition. NVP-BEZ235 and ZSTK474 demonstrated antitumor efficacy with improved survival against U87 orthotopic gliomas, although the effect of ZSTK474 was more pronounced. Finally, ZSTK474 prolonged overall survival in Cre-LoxP conditional transgenic Pten;p16Ink4a/p19Arf;K-Rasv12;LucR mice, mainly by delaying tumor onset.Conclusions: PI3K/mTOR inhibitors with weak affinities for ABC transporters can achieve target inhibition in brain (tumors), but have modest single-agent efficacy and combinations with (BBB penetrable) inhibitors of other activated pathways may be required. Clin Cancer Res; 23(5); 1286-98. ©2016 AACR.

ABCB1, ABCG2, and PTEN determine the response of glioblastoma to temozolomide and ABT-888 therapy.

PURPOSE: Little is known about the optimal clinical use of ABT-888 (veliparib) for treatment of glioblastoma. ABT-888 is a PARP inhibitor undergoing extensive clinical evaluation in glioblastoma, because it may synergize with the standard-of-care temozolomide (TMZ). We have elucidated important factors controlling ABT-888 efficacy in glioblastoma. EXPERIMENTAL DESIGN: We used genetically engineered spontaneous glioblastoma mouse models and allograft models that were orthotopically transplanted into wild-type (WT) and Abcb1/Abcg2-deficient (KO) recipients. RESULTS: ABT-888/TMZ is not efficacious against p53;p16(Ink4a)/p19(Arf);K-Ras(v12);LucR allografts in wild-type recipients, indicating inherent resistance. Abcb1/Abcg2 mediated efflux of ABT-888 at the blood-brain barrier (BBB) causes a 5-fold reduction of ABT-888 brain penetration (P < 0.0001) that was fully reversible by elacridar. Efficacy studies in WT and KO recipients and/or concomitant elacridar demonstrate that Abcb1/Abcg2 at the BBB and in tumor cells impair TMZ/ABT-888 combination treatment efficacy. Elacridar also markedly improved TMZ/ABT-888 combination treatment in the spontaneous p53;p16(Ink4a)/p19(Arf);K-Ras(v12);LucR glioblastoma model. Importantly, ABT-888 does enhance TMZ efficacy in Pten deficient glioblastoma allografts and spontaneous tumors, even in Abcb1/Abcg2 proficient wild-type mice. Loss of PTEN occurs frequently in glioblastoma (36%) and in silico analysis on patient with glioblastoma samples revealed that it is associated with a worse overall survival (310 days vs. 620 days, n = 117). CONCLUSIONS: The potential of ABT-888 in glioblastoma can best be demonstrated in patients with PTEN null tumors. Therefore, clinical trials with ABT-888 should evaluate these patients as a separate group. Importantly, inhibition of ABCB1 and ABCG2 (by elacridar) may improve the efficacy of TMZ/ABT-888 therapy in all glioblastoma patients.

Sildenafil is not a useful modulator of ABCB1 and ABCG2 mediated drug resistance in vivo.

AIM: Recently, sildenafil was reported to be an inhibitor of P-glycoprotein (P-gp/ABCB1) and breast cancer resistance protein (BCRP/ABCG2) in vitro. We have now investigated the in vivo potency of sildenafil. METHODS: By using wild-type and Abcb1; Abcg2 knockout mice we have investigated the effect of sildenafil on the brain penetration of two substrate drugs (docetaxel and topotecan). Next we have investigated if sildenafil was able to improve the efficacy of doxorubicin against P-glycoprotein expressing CT26 colon cancer cells in syngeneic Balb/c mice. RESULTS: Sildenafil administered orally at a dose of 50mg/kg did not improve the brain penetration of docetaxel and topotecan, although the plasma level of sildenafil was already much higher than can be achieved in humans. On the other hand, sildenafil increased the plasma levels of the cytotoxic drugs, but not by inhibition of Abcb1 or Abcg2, since this effect was also seen in Abcb1;Abcg2 knockout mice. The brain penetration of sildenafil was more than 20-fold higher in Abcb1;Abcg2 mice versus wild-type mice, indicating that sildenafil is a good substrate of the two transporters. Sildenafil was also not able to improve the efficacy of doxorubicin against subcutaneous CT26 tumours. The doxorubicin level in tumour tissue did increase, but so did the concentration of doxorubicin in plasma and heart. CONCLUSION: These results demonstrate that the potency and specificity of sildenafil as an inhibitor of ABCB1 and ABCG2 is not sufficient to warrant further clinical testing of this agent in combination with anticancer drugs.