Lynn Bjerke

Pharmacologic Characterization of a Potent Inhibitor of Class I Phosphatidylinositide 3-Kinases

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.

Histone H3.3 Mutations Drive Pediatric Glioblastoma through Upregulation of MYCN

UNLABELLED 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. SIGNIFICANCE 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

Drugging PI3K in cancer: refining targets and therapeutic strategies

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.

Molecular pharmacology of phosphatidylinositol 3-kinase inhibition in human glioma.

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.

Recurrent MET fusion genes represent a drug target in pediatric glioblastoma

Pediatric glioblastoma is one of the most common and most deadly brain tumors in childhood. Using an integrative genetic analysis of 53 pediatric glioblastomas and five in vitro model systems, we identified previously unidentified gene fusions involving the MET oncogene in ∼10% of cases. These MET fusions activated mitogen-activated protein kinase (MAPK) signaling and, in cooperation with lesions compromising cell cycle regulation, induced aggressive glial tumors in vivo. MET inhibitors suppressed MET tumor growth in xenograft models. Finally, we treated a pediatric patient bearing a MET-fusion-expressing glioblastoma with the targeted inhibitor crizotinib. This therapy led to substantial tumor shrinkage and associated relief of symptoms, but new treatment-resistant lesions appeared, indicating that combination therapies are likely necessary to achieve a durable clinical response.

Integrated Molecular Meta-Analysis of 1,000 Pediatric High-Grade and Diffuse Intrinsic Pontine Glioma

Summary 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.

Dependence of Wilms tumor cells on signaling through insulin-like growth factor 1 in an orthotopic xenograft model targetable by specific receptor inhibition

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.

The prognostic role of intragenic copy number breakpoints and identification of novel fusion genes in paediatric high grade glioma

BackgroundPaediatric high grade glioma (pHGG) is a distinct biological entity to histologically similar tumours arising in older adults, and has differing copy number profiles and driver genetic alterations. As functionally important intragenic copy number aberrations (iCNA) and fusion genes begin to be identified in adult HGG, the same has not yet been done in the childhood setting. We applied an iCNA algorithm to our previously published dataset of DNA copy number profiling in pHGG with a view to identify novel intragenic breakpoints.ResultsWe report a series of 288 iCNA events in pHGG, with the presence of intragenic breakpoints itself a negative prognostic factor. We identified an increased number of iCNA in older children compared to infants, and increased iCNA in H3F3A K27M mutant tumours compared to G34R/V and wild-type. We observed numerous gene disruptions by iCNA due to both deletions and amplifications, targeting known HGG-associated genes such as RB1 and NF1, putative tumour suppressors such as FAF1 and KIDINS220, and novel candidates such as PTPRE and KCND2. We further identified two novel fusion genes in pHGG – CSGALNACT2:RET and the complex fusion DHX57:TMEM178:MAP4K3. The latter was sequence-validated and appears to be an activating event in pHGG.ConclusionsThese data expand upon our understanding of the genomic events driving these tumours and represent novel targets for therapeutic intervention in these poor prognosis cancers of childhood.

Abstract 1932: Mutations in ATRX increase genetic instability and sensitivity to PARP inhibitors in paediatric glioblastoma cells

Paediatric glioblastomas (pGBM) are amongst the most common causes of cancer-related deaths in children, and are defined by highly recurrent mutations in H3 histones. Mutations affecting the chromatin remodeling protein ATRX have been reported in 30% of pGBM cases, and are strongly associated with the alternative lengthening of telomeres (ALT) phenotype, but their precise interaction with histone mutations and their role in tumorigenesis remain unclear. We collected sequence data from 262 published and 64 unpublished cases of pGBM and identified somatic ATRX mutations in 54/326 (17%) of cases. ATRX mutations are mainly loss of function mutations, with the majority of frameshift mutations (37/54, 68,5%) found upstream of the helicase domain resulting in truncation of the main functional domain of ATRX. Missense mutations (16/54, 29,6%) reside almost exclusively in the helicase domain (11/54, 20,4%), whereas nonsense mutations are a less common event (7/54, 13%) but present in both the helicase (4/7, 57,1%) and ADD domains (3/7, 42,9%). ATRX mutations commonly co-segregate with H3.3 G34 (16/54) and TP53 (42/54) mutations, and define a subgroup of patients with a longer overall survival (16 months median overall survival in mutant ATRX cases versus 11 months in wild-type ATRX cases, COXPH p = 0.079), though with a greater number of somatic mutations (MWU p = 0.023) and copy number alterations (MWU p = 0.0011) than wild-type cases. We screened a series of 21 primary patient-derived pGBM cell cultures for histone and ATRX mutation status in addition to ATRX protein expression and ALT, and subjected the panel to a high-throughput in vitro cell viability screen of >400 chemotherapeutics and small molecules. We identified a specific genetic dependency for ATRX mutation and sensitivity to distinct PARP inhibitor chemotypes, including olaparib and rucaparib (PARP catalytic inhibitors), and talazoparib (PARP trapper inhibitor). These data were validated using CRISPR-Cas9-engineered ATRX knockout, targeting either the ADD or helicase domain, in SF188 pGBM cells. Gene editing was confirmed by IonTorrent sequencing and Western blot. ATRX mutant clones were also more sensitive to drugs targeting DNA damage response pathways such as bleomycin and sapacitabine. Gene expression analysis of ATRX mutant pGBM samples confirmed an intact homologous recombination pathway and overexpression of PARP1, suggesting an underlying mechanism distinct from that observed in BRCA-mutant breast and ovarian cancers. Ongoing work is aimed at unravelling the specific pathways involved, and evaluating the utility of PARP inhibition in orthotopic pGBM xenografts in vivo. These data suggest a synthetic lethality for PARP inhibitors in ATRX-deficient pGBM cells, and may represent a novel therapeutic strategy for these highly aggressive tumours in children. Citation Format: Janat Fazal-Salom, Mara Vinci, Diana Carvalho, Helen Pemberton, Stephen J. Pettitt, Christopher J. Lord, Alan Mackay, Lynn Bjerke, Chris Jones. Mutations in ATRX increase genetic instability and sensitivity to PARP inhibitors in paediatric glioblastoma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1932. doi:10.1158/1538-7445.AM2017-1932

Abstract 2962: Histone H3.3 mutations drive paediatric glioblastoma through upregulation of MYCN.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Glioblastomas of children and young adults (pGBM) have a median survival of only 12-15 months and 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. The G34 mutation is specific to tumours of the cerebral hemispheres and is associated with a distinct age of incidence (16 yrs) and gene expression signature compared to K27 and wild-type tumours. ChIP-Seq for the activating K36 trimethylation mark (H3K36me3) mark of G34V mutant KNS42 pGBM cells identified 156 genes differentially bound and expressed compared to wild-type pGBM control. 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 which is causative of glioblastomas when expressed in the correct developmental context. A synthetic lethality siRNA screen revealed this driving aberration to be selectively targetable in this patient population by inhibiting kinases responsible for stabilisation of the protein such as AURKA and CHK1. We thus provide the mechanistic explanation for how the first histone gene mutation in human 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 glioblastoma. Employing synthetic lethal approaches to these mutant tumour cells provides a rational way to develop novel and highly selective treatment strategies. Citation Format: Lynn Bjerke, Alan Mackay, Meera Nandhabalan, Anna Burford, Alexa Jury, Sergey Popov, Dorine Bax, Diana Carvalho, Katy Taylor, Mara Vinci, Illirjana Bajrami, Imelda McGonnell, Chris Lord, Rui Reis, Darren Hargrave, Alan Ashworth, Paul Workman, Chris Jones. Histone H3.3 mutations drive paediatric glioblastoma through upregulation of MYCN. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2962. doi:10.1158/1538-7445.AM2013-2962 Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.