Supplementary Figure 3 from <i>ALK</i> Amplification and Rearrangements Are Recurrent Targetable Events in Congenital and Adult Glioblastoma
Authors: Blandin Anne-Florence , Giglio Ross , Graham Maya Srikanth , Garcia Guadalupe , Malinowski Seth , Woods Jared K. , Ramkissoon Shakti , Ramkissoon Lori , Dubois Frank , Schoolcraft Kathleen , Tsai Jessica , Wang Dayle , Jones Robert , Vogelzang Jayne , Pelton Kristine , Becker Sarah , Watkinson Fiona , Sinai Claire , Cohen Elizabeth F. , Booker Matthew A. , Tolstorukov Michael Y. , Haemels Veerle , Goumnerova Liliana , Wright Karen , Kieran Mark , Fehnel Katie , Reardon David , Tauziede-Espariat Arnault , Lulla Rishi , Carcamo Benjamin , Chaleff Stanley , Charest Alain , De Smet Frederik , Ligon Azra H. , Dubuc Adrian , Pages Melanie , Varlet Pascale , Wen Patrick Y. , Alexander Brian M. , Chi Susan , Alexandrescu Sanda , Kittler Ralf , Bachoo Robert , Bandopadhayay Pratiti , Beroukhim Rameen , Ligon Keith L. 
Published: April 2023

<p>Supplementary Figure 3: PPP1CB-ALK fusion is oncogenic and sensitive to targeted therapy. A. Immunoblot analysis of ALK downstream signaling proteins in cortical mNSC (CTX#6) and brainstem mNSC (BS#3) expressing PPP1CB-ALK. Cells were treated for 4 hours with ceritinib and lorlatinib at the indicated concentrations.B. Left panel: Soft agar colony-forming assay using stable mNSC PPP1CB-ALK demonstrated colony formation with dose-dependent inhibition by ceritinib. mNSC KRAS did not respond to ceritinib validating the drug specificity. Right panel: Quantification of colony formation under targeted drug inhibition using CellProfiler. Values represent colony counts relative to the untreated group ± s.d. The mean of three independent replicates is shown. Significance between treatments determined by the Mann-Whitney test. *P < 0.05, **P < 0.01.C. Cell viability of ceritinib-treated PPP1CB-ALK NIH-3T3 relative to eGFP-positive cells by CellTiterGlo.D. Relative viability of the ALK wild-type GBM line (BT164) and BT1857 lorlatinib-treated cells versus DMSO control.E. Relative viability of BT164 and BT1857 ceritinib-treated cells versus DMSO control.F. Relative viability of BT164 and BT1857 cells treated with an EGFR tyrosine kinase inhibitor neratinib.G. Pharmacodynamic analysis of NIH 3T3-PPP1CB-ALK s.c. tumors treated with vehicle or ceritinib at 30mg/kg for 5 days. Representative H&E stain, ALK, pSTAT3, pAKT S473 and Ki-67 IHC.H. Quantification of Ki-67-positive cells in vehicle and ceritinib-treated tumors using CellProfiler software. Error bars show standard error of the mean. *P < 0.05.I. Quantification of pSTAT3-positive cells in vehicle and ceritinib-treated tumors using CellProfiler software. Error bars show standard error of the mean. *P < 0.05.J. Tumor growth following subcutaneous implantation of PPP1CB-ALK and eGFP-expressing fibroblasts.K. Kaplan-Meier survival curves of SCID mice injected subcutaneously with mNSC CTX-PPP1CB-ALK (red, n = 5) or mNSC CTX-eGFP (black, n = 4) and treated with ceritinib at 30mg/kg/d for 15 days. Mice were euthanized at endpoint (tumor volume of 2000mm3). The grey area represents the treatment period.L. Vehicle or ceritinib-treated mouse weights at start and end of treatment.</p>