Highlights • PI3K is an important target for innovative anticancer drug development and precision medicine. • Over 30 small molecule PI3K inhibitors are currently in clinical trial testing. • These ...drugs include dual PI3K/mTOR, pan-Class I PI3K and isoform-selective PI3K inhibitors. • The PI3Kδ inhibitor idelalisib has received FDA approval for the treatment of B-cell malignancies. • Drug resistance, patient selection and development of targeted combinations remain challenges.
Children and young adults with glioblastoma (GBM) have a median survival rate of only 12 to 15 months, and these GBMs are clinically and biologically distinct from histologically similar cancers in ...older adults. They are defined by highly specific mutations in the gene encoding the histone H3.3 variant H3F3A , occurring either at or close to key residues marked by methylation for regulation of transcription—K27 and G34. Here, we show that the cerebral hemisphere-specific G34 mutation drives a distinct expression signature through differential genomic binding of the K36 trimethylation mark (H3K36me3). The transcriptional program induced recapitulates that of the developing forebrain, and involves numerous markers of stem-cell maintenance, cell-fate decisions, and self-renewal.Critically, H3F3A G34 mutations cause profound upregulation of MYCN , a potent oncogene that is causative of GBMs when expressed in the correct developmental context. This driving aberration is selectively targetable in this patient population through inhibiting kinases responsible for stabilization of the protein.
We provide the mechanistic explanation for how the fi rst histone gene mutation inhuman disease biology acts to deliver MYCN, a potent tumorigenic initiator, into a stem-cell compartment of the developing forebrain, selectively giving rise to incurable cerebral hemispheric GBM. Using synthetic lethal approaches to these mutant tumor cells provides a rational way to develop novel and highly selective treatment strategies
Extensive evidence implicates activation of the lipid phosphatidylinositide 3-kinase (PI3K) pathway in the genesis and progression of various human cancers. PI3K inhibitors thus have considerable ...potential as molecular cancer therapeutics. Here, we detail the pharmacologic properties of a prototype of a new series of inhibitors of class I PI3K. PI103 is a potent inhibitor with low IC50 values against recombinant PI3K isoforms p110alpha (2 nmol/L), p110beta (3 nmol/L), p110delta (3 nmol/L), and p110gamma (15 nmol/L). PI103 also inhibited TORC1 by 83.9% at 0.5 micromol/L and exhibited an IC50 of 14 nmol/L against DNA-PK. A high degree of selectivity for the PI3K family was shown by the lack of activity of PI103 in a panel of 70 protein kinases. PI103 potently inhibited proliferation and invasion of a wide variety of human cancer cells in vitro and showed biomarker modulation consistent with inhibition of PI3K signaling. PI103 was extensively metabolized, but distributed rapidly to tissues and tumors. This resulted in tumor growth delay in eight different human cancer xenograft models with various PI3K pathway abnormalities. Decreased phosphorylation of AKT was observed in U87MG gliomas, consistent with drug levels achieved. We also showed inhibition of invasion in orthotopic breast and ovarian cancer xenograft models and obtained evidence that PI103 has antiangiogenic potential. Despite its rapid in vivo metabolism, PI103 is a valuable tool compound for exploring the biological function of class I PI3K and importantly represents a lead for further optimization of this novel class of targeted molecular cancer therapeutic.
Gliomas are primary brain tumors with poor prognosis that exhibit frequent abnormalities in phosphatidylinositol 3-kinase (PI3 kinase) signaling. We investigated the molecular mechanism of action of ...the isoform-selective Class I PI3 kinase and mTOR inhibitor PI-103 in human glioma cells. The potent inhibitory effects of PI-103 on the PI3 kinase pathway were quantified. PI-103 and the mTOR inhibitor rapamycin both inhibited RPS6 phosphorylation but there were clear differences in the response of upstream components of the PI3 kinase pathway, such as phosphorylation of Thr308-AKT, that were inhibited by PI-103 but not rapamycin. Gene expression profiling identified altered expression of genes encoding regulators of the cell cycle and cholesterol metabolism, and genes modulated by insulin or IGF1 signaling, rapamycin treatment or nutrient starvation. PI-103 decreased expression of positive regulators of G1/S phase progression and increased expression of the negative cell cycle regulator p27kip1. A reversible PI-103-mediated G1 cell cycle arrest occurred without significant apoptosis, consistent with the altered gene expression detected. PI-103 induced vacuolation and processing of LC-3i to LC-3ii, which are features of an autophagic response. In contrast to PI-103, LY294002 and PI-387 induced apoptosis, indicative of likely off-target effects. PI-103 interacted synergistically or additively with cytotoxic agents used in the treatment of glioma, namely vincristine, BCNU and temozolomide. Compared to individual treatments, the combination of PI-103 with temozolomide significantly improved the response of U87MG human glioma xenografts. Our results support the therapeutic potential for PI3 kinase inhibitors with PI-103-like profile as therapeutic agents for the treatment of glioma.
We have previously demonstrated an increased DNA copy number and expression of IGF1R to be associated with poor outcome in Wilms tumors. We have now tested whether inhibiting this receptor may be a ...useful therapeutic strategy by using a panel of Wilms tumor cell lines. Both genetic and pharmacological targeting resulted in inhibition of downstream signaling through PI3 and MAP kinases, G1 cell cycle arrest, and cell death, with drug efficacy dependent on the levels of phosphorylated IGF1R. These effects were further associated with specific gene expression signatures reflecting pathway inhibition, and conferred synergistic chemosensitisation to doxorubicin and topotecan. In the in vivo setting, s.c. xenografts of WiT49 cells resembled malignant rhabdoid tumors rather than Wilms tumors. Treatment with an IGF1R inhibitor (NVP-AEW541) showed no discernable antitumor activity and no downstream pathway inactivation. By contrast, Wilms tumor cells established orthotopically within the kidney were histologically accurate and exhibited significantly elevated insulin-like growth factor–mediated signaling, and growth was significantly reduced on treatment with NVP-AEW541 in parallel with signaling pathway ablation. As a result of the paracrine effects of enhanced IGF2 expression in Wilms tumor, this disease may be acutely dependent on signaling through the IGF1 receptor, and thus treatment strategies aimed at its inhibition may be useful in the clinic. Such efficacy may be missed if only standard ectopic models are considered as a result of an imperfect recapitulation of the specific tumor microenvironment.
We collated data from 157 unpublished cases of pediatric high-grade glioma and diffuse intrinsic pontine glioma and 20 publicly available datasets in an integrated analysis of >1,000 cases. We ...identified co-segregating mutations in histone-mutant subgroups including loss of FBXW7 in H3.3G34R/V, TOP3A rearrangements in H3.3K27M, and BCOR mutations in H3.1K27M. Histone wild-type subgroups are refined by the presence of key oncogenic events or methylation profiles more closely resembling lower-grade tumors. Genomic aberrations increase with age, highlighting the infant population as biologically and clinically distinct. Uncommon pathway dysregulation is seen in small subsets of tumors, further defining the molecular diversity of the disease, opening up avenues for biological study and providing a basis for functionally defined future treatment stratification.
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•Pediatric HGG and DIPG comprise a diverse set of clinical and biological subgroups•Somatic coding mutations per tumor range from none to among the highest seen in human cancer•Histone mutations co-segregate with distinct alterations and downstream pathways•H3/IDH1 WT tumors may resemble low-grade lesions and have targetable alterations
Mackay et al. perform an integrated analysis of >1,000 cases of pediatric high-grade glioma and diffuse intrinsic pontine glioma. They identify co-segregating mutations in histone-mutant subgroups and show that histone wild-type subgroups are molecularly more similar to lower-grade tumors.
Novel therapies for paediatric-type diffuse high-grade glioma (PDHGG) are urgently required. Orthotopic models using patient-derived material are invaluable tools in preclinical drug development as ...they retain key genetic/epigenetic features, eg. histone H3G34 or H3K27 alterations. Their evaluation
in situ
is vital and requires sensitive imaging techniques such as MRI. 12 diffuse hemispheric glioma (DHG; 2 DHG-G34) and 21 diffuse midline glioma (DMG; 17 DMG-K27M) tumours have been characterised using MRI following site-specific orthotopic implantation of patient-derived cells directly from tumour material or after minimal expansion as stem cell cultures. Of the 62 models implanted; 3 DHG and 10 DMG samples were not tumourigenic and 13 DHG/3 DMG models are currently under MRI surveillance. Tumours identified on T
2
-weighted (T
2
w)-images varied from a diffuse hyperintense signal to well-defined high contrast masses. Tumour growth in 5 DMG models was too diffuse for longitudinal monitoring with T
2
w-MRI. Once established, diffusion-weighted, T
1
/T
2
mapping and contrast-enhanced MRI were used to further assess tumour phenotype. Quantitative data from 15 DMG models demonstrated higher water diffusivity and T
2
than 10 DHG tumours, which suggests less tightly packed tumour cells but may also reflect the closer proximity of tumours growing in the thalamus/pons/cerebellum to the ventricular system. Lack of contrast-agent enhancement in 11 DMG and 6/10 DHG models indicated an intact blood-brain barrier (BBB), with heterogeneous disruption observed in 4 DHGs; H3-G34 had no bearing on BBB integrity. Upon serial re-implantation survival was shortened in 3/4 DHG and 2/6 DMG models, while quantitative MRI parameters remained similar. Likewise, when 2 DHG and 2 DMG models grown in 2D/3D
in vitro
were implanted in parallel, poorer survival/improved penetrance was associated with 3D-cultured cells with no difference in imaging phenotype. The study highlights the potential of non-invasive MRI to accurately evaluate the efficacy of novel therapeutics in these PDHGG models.
Abstract
Novel therapies for paediatric-type diffuse high-grade glioma (PDHGG) are urgently required. Orthotopic models using patient-derived material are invaluable tools in preclinical drug ...development as they retain key genetic/epigenetic features, eg. histone H3G34 or H3K27 alterations. Their evaluation in situ is vital and requires sensitive imaging techniques such as MRI. 12 diffuse hemispheric glioma (DHG; 2 DHG-G34) and 21 diffuse midline glioma (DMG; 17 DMG-K27M) tumours have been characterised using MRI following site-specific orthotopic implantation of patient-derived cells directly from tumour material or after minimal expansion as stem cell cultures. Of the 62 models implanted; 3 DHG and 10 DMG samples were not tumourigenic and 13 DHG/3 DMG models are currently under MRI surveillance. Tumours identified on T2-weighted (T2w)-images varied from a diffuse hyperintense signal to well-defined high contrast masses. Tumour growth in 5 DMG models was too diffuse for longitudinal monitoring with T2w-MRI. Once established, diffusion-weighted, T1/T2 mapping and contrast-enhanced MRI were used to further assess tumour phenotype. Quantitative data from 15 DMG models demonstrated higher water diffusivity and T2 than 10 DHG tumours, which suggests less tightly packed tumour cells but may also reflect the closer proximity of tumours growing in the thalamus/pons/cerebellum to the ventricular system. Lack of contrast-agent enhancement in 11 DMG and 6/10 DHG models indicated an intact blood-brain barrier (BBB), with heterogeneous disruption observed in 4 DHGs; H3-G34 had no bearing on BBB integrity. Upon serial re-implantation survival was shortened in 3/4 DHG and 2/6 DMG models, while quantitative MRI parameters remained similar. Likewise, when 2 DHG and 2 DMG models grown in 2D/3D in vitro were implanted in parallel, poorer survival/improved penetrance was associated with 3D-cultured cells with no difference in imaging phenotype. The study highlights the potential of non-invasive MRI to accurately evaluate the efficacy of novel therapeutics in these PDHGG models.
We report here on a chemical genetic screen designed to address the mechanism of action of a small molecule. Small molecules that were active in models of urinary incontinence were tested on the ...nematode Caenorhabditis elegans, and the resulting phenotypes were used as readouts in a genetic screen to identify possible molecular targets. The mutations giving resistance to compound were found to affect members of the RGS protein/G-protein complex. Studies in mammalian systems confirmed that the small molecules inhibit muscarinic G-protein coupled receptor (GPCR) signaling involving G-alphaq (G-protein alpha subunit). Our studies suggest that the small molecules act at the level of the RGS/G-alphaq signaling complex, and define new mutations in both RGS and G-alphaq, including a unique hypo-adapation allele of G-alphaq. These findings suggest that therapeutics targeted to downstream components of GPCR signaling may be effective for treatment of diseases involving inappropriate receptor activation.
Abstract
Diffuse intrinsic pontine glioma (DIPG) are incurable childhood brain tumours marked by alterations in histone 3 in the form of somatic K27M mutations and loss of H3K27 trimethylation ...(H3K27me3). These highly aggressive tumours display extensive intratumoral heterogeneity, despite their relatively modest mutational burden. We recently proposed a model whereby co-operative subclonal interactions may exist to promote tumorigenesis in DIPG, and identified rare cancer stem cell populations to harbour mutations in the histone H4 lysine methyltransferase KMT5B. Although not previously described in glioma, loss of H4K20me2/3 caused by abrogated H4 methyltransferase function has been linked to defects in the DNA damage response and enhanced invasion in breast and pancreatic cancer. In order to explore whether dysregulation of H4 methylation may be directly responsible for tumorigenic phenotypes in DIPG, we targeted the methyltransferases by genetically engineering numerous isogenic cells from patient-derived in vitro models using CRISPR/Cas9 to knock out KMT5B and/or KMT5C, alongside pharmacological treatment with the small molecule KMT5B/C inhibitor A-196. We observed the expected reduction in H4K20me2 and H4K20me3 in response to inhibition of KMT5B/C, with an attendant increase in H3K27me3. Knock-out of KMT5B caused a significant increase in tumour cell migration and invasion in vitro, whilst KMT5C-deficient cells had reduced motility. Double knock-outs presented an intermediate phenotype. With H4K20me2/3 playing an important role in DNA repair via 53BP1 recruitment, we further observed a significantly enhanced efficacy of the PARP inhibitors olaparib and talazoparib in knock-out DIPG cells in vitro. Ongoing analysis of differential binding of post-translational modifications by ChIP-seq will provide important insights into the underlying mechanisms of epigenetic dysregulation, particularly in respect of the cross-talk between H3 and H4 marks. Although often present only in small tumour cell populations, H4K20me2/3 and KMT5B/C may represent an important novel aspect of DIPG tumorigenesis.
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