CBTTC project 0011: Targeting Alternative Lengthening of Telomeres (ALT) in Pediatric CNS malignancies Kristina A. Cole, MD/PhD The Children’s Hospital of Philadelphia University of Pennsylvania School of Medicine

ALT in pediatric brain tumors (Alternative Maintenance of Telomeres) ALT is a telomere maintenance mechanism required for continued cellular proliferation (vs. telomerase) 20-40% of pediatric high grade gliomas use ALT, including: All H3F3A G34 mutant tumors (Korshunev et al, Gessi et al) 23% H3F3A K27M diffuse midline gliomas often associated with ATRX, DAXX and TP53 mutations 12% of sPNET, 23% of choroid plexus carcinomas use ALT Studies are limited by a lack of pediatric brain tumor models with ALT Tumors that use ALT may have therapeutic vulnerabilities

CBTTC 0011: Downstream studies CBTTC 0011 proposes to generate patient derived (PD) cell lines of known ALT status They will be used for: testing of published proposed inhibitors of ALT tumors (ie ATRi, CHK1i) validation of candidates from drug and CRISPR screens (i.e. isogenic ATRX lines) functional analysis of candidate TS and OG from large genomic studies in primary tumors TS = tumor suppressor OG =oncogene

CBTTC 0011 Proposal and Timeline (proposal approved in the summer 2016) 38 HGG/PNET samples with freezing media Aim 1: Analysis of corresponding primary tumor DNA for prioritization and downstream validation ALT (CCA) assay of primary tumor (3 months) - completed Sequencing of H3F3A for K27M, G34R/V (3 months) – completed Identification of mutations by WGS (n= 17 overlapping sets) - ongoing Aim 2: Generation of Cell lines Adherent and suspension lines (6 m - 1 yr) – 30/38 in various stages of production - ongoing Aim 3: Cell line validation and characterization H3F3A targeted sequencing (9/9 tested to date match primary tumor) Targeted sequencing of known BRAF, IDH1, ATRX, TP53 etc mutations growth kinetics, tumorgenecity, ICC/IHC, ALT assays, Telomerase assays, WB for ATRX etc Sequencing CCA = c-circle assay. This is a rolling circle amplification assay (based on tel sequence) that is read out on a dot blot. It only requires < 50 ng of DNA. WGS = whole genome sequencing ICC = immunocytochemistry and/or IHC immunohistochemistry for typical glioma/PNET markers WB = western blot

Potential Characteristics of Cell lines (based on primary tumor DNA analysis) Of 38 samples 2 are diagnostic / relapse pairs 17 tumor are midline, and 13 are hemispheric gliomas 8 samples pathology is “PNET” ALT status 9/35 primary tumor DNA samples are ALT (CCA) positive Targeted sequencing of H3F3A showed that 3/3 (100%) of G34R mutant, 2/8 (25%) of K27M mutant and 4/24 (17%) of H3F3A wild-type tumors are ALT positive. 17 of 38 had WGS. 4 of these were from ALT positive tumors. The ALT positive tumors have known pathogenic mutations in TP53 (4/4), ATRX (2/4), H3F3A (2/4) and IDH1 (1/4). The reason only 35 out of 38 have CCA (ALT) testing is because the other 3 were added to the project later.

Thank-you ! CBTTC: Martha Williams, Mateusz Koptyra, Jen Mason, Elizabeth Appert, Adam Resnick and Angela Waanders Cole lab: Heba Ijaz U Penn: Robert Dilley and Roger Greenberg Bioinformatics: Pichai Raman, Yuankun Zhu, Bo Zhang All CBTTC investigator sites ! Questions/comments/recommendations, please email Kristina at: colek@email.chop.edu