Mara Vinci

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Summary We collated data from 157 unpublished cases of pediatric high-grade glioma and diffuse intrinsic pontine glioma and 20 publicly available datasets in an integrated analysis of 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.

Functional diversity and cooperativity between subclonal populations of pediatric glioblastoma and diffuse intrinsic pontine glioma cells

The failure to develop effective therapies for pediatric glioblastoma (pGBM) and diffuse intrinsic pontine glioma (DIPG) is in part due to their intrinsic heterogeneity. We aimed to quantitatively assess the extent to which this was present in these tumors through subclonal genomic analyses and to determine whether distinct tumor subpopulations may interact to promote tumorigenesis by generating subclonal patient-derived models in vitro and in vivo. Analysis of 142 sequenced tumors revealed multiple tumor subclones, spatially and temporally coexisting in a stable manner as observed by multiple sampling strategies. We isolated genotypically and phenotypically distinct subpopulations that we propose cooperate to enhance tumorigenicity and resistance to therapy. Inactivating mutations in the H4K20 histone methyltransferase KMT5B (SUV420H1), present in <1% of cells, abrogate DNA repair and confer increased invasion and migration on neighboring cells, in vitro and in vivo, through chemokine signaling and modulation of integrins. These data indicate that even rare tumor subpopulations may exert profound effects on tumorigenesis as a whole and may represent a new avenue for therapeutic development. Unraveling the mechanisms of subclonal diversity and communication in pGBM and DIPG will be an important step toward overcoming barriers to effective treatments.Genomic and functional analysis of intratumor heterogeneity in pediatric glioma uncovers early clonal divergence and stable spontaneous cooperation between subclonal populations throughout tumor evolution.

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.

Abstract 463: MAP4K4 (HGK) plays a key role in tumor progression

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL MAP4K4/HGK is an STE kinase that was identified in an siRNA screen for modulators of tumor cell motility and in a five-gene signature predicting metastasis and survival in colon cancer. Interestingly, its catalytic function is required for anchorage-independent growth in 3D soft agar (but not in 2D monolayers) of 3T3 fibroblasts and rat intestinal epithelial cells. The aim of the present study was to evaluate MAP4K4 expression in a range of human tumor cell lines and further explore its role in cell motility and invasion. Using western blotting analysis, MAP4K4 protein expression was measured in a panel of approximately 50 human tumor cell lines and 3 non-transformed cell lines. Selected high-expressing MAP4K4 cell lines were used for genetic manipulation. Lentiviral shRNA inducible (TRIPZ-RFP) and stable (GIPZ-GFP) knockdowns (KD) permitted the selection of isogenic cell lines for functional studies. Novel high-throughput 96-well plate-based 3D assays were used to analyze: (i) 3D spheroid growth, (ii) tumor cell dissemination over gelatin or collagen type I and (iii) invasion into Matrigel™. A fully automated image analysis system was validated against standard microscopy-based methods and applied to these studies. Cell viability was assessed in 2D and 3D by the Cell Titer Glo assay. Pharmacodynamic biomarkers were analyzed in KD and control cells to correlate MAP4K4 expression to the activation/inhibition status of putative downstream signalling pathways. MAP4K4 protein expression levels were high to moderate in 60% of the cell lines tested (including glioma, neuroblastoma, hepatocellular, prostate, colorectal, ovarian, squamous and breast carcinomas) and undetectable in PNT2 and MCF10A non-transformed prostate and breast epithelial cells. It was also expressed in activated human endothelial cells. The SF188 pediatric glioma cell line was prioritized for further study based on its strong expression of MAP4K4 (and activated downstream signalling pathways), the ability to generate reproducible tumor spheroids and the highly invasive nature of this cancer type. Additional confirmatory studies utilized the metastatic triple-negative human breast cancer cell line MDA MB 231. Both stable and inducible KD were achieved as described above. Functional studies showed that MAP4K4 KD induced between 30-50% inhibition of cell viability, motility and/or invasion. Inhibition of MAP4K4 expression in SF188 cells also down-regulated key downstream signalling molecules such as pJNK, pSTAT3, pTAK1 and MMP2. In MDA MB 231, MAP4K4 was upregulated when cells were cultured in 3D spheroids and again, stable knockdown inhibited 3D migration by ∼ 50%. Conclusions: In the present study we provide evidence that MAP4K4 plays an important role in functions associated with tumor progression (ability to grow in 3D, migration and invasion) in two cancer types where invasion and/or metastasis contribute to their notoriously poor prognosis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 463. doi:1538-7445.AM2012-463