ccr-21-3521_supplementary_figure_3_suppfs3.jpeg (2.07 MB)

Supplementary Figure 3 from ALK Amplification and Rearrangements Are Recurrent Targetable Events in Congenital and Adult Glioblastoma

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posted on 2023-04-01, 00:26 authored by Anne-Florence Blandin, Ross Giglio, Maya Srikanth Graham, Guadalupe Garcia, Seth Malinowski, Jared K. Woods, Shakti Ramkissoon, Lori Ramkissoon, Frank Dubois, Kathleen Schoolcraft, Jessica Tsai, Dayle Wang, Robert Jones, Jayne Vogelzang, Kristine Pelton, Sarah Becker, Fiona Watkinson, Claire Sinai, Elizabeth F. Cohen, Matthew A. Booker, Michael Y. Tolstorukov, Veerle Haemels, Liliana Goumnerova, Karen Wright, Mark Kieran, Katie Fehnel, David Reardon, Arnault Tauziede-Espariat, Rishi Lulla, Benjamin Carcamo, Stanley Chaleff, Alain Charest, Frederik De Smet, Azra H. Ligon, Adrian Dubuc, Melanie Pages, Pascale Varlet, Patrick Y. Wen, Brian M. Alexander, Susan Chi, Sanda Alexandrescu, Ralf Kittler, Robert Bachoo, Pratiti Bandopadhayay, Rameen Beroukhim, Keith L. Ligon

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.

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ARTICLE ABSTRACT

Anaplastic lymphoma kinase (ALK) aberrations have been identified in pediatric-type infant gliomas, but their occurrence across age groups, functional effects, and treatment response has not been broadly established. We performed a comprehensive analysis of ALK expression and genomic aberrations in both newly generated and retrospective data from 371 glioblastomas (156 adult, 205 infant/pediatric, and 10 congenital) with in vitro and in vivo validation of aberrations. ALK aberrations at the protein or genomic level were detected in 12% of gliomas (45/371) in a wide age range (0–80 years). Recurrent as well as novel ALK fusions (LRRFIP1–ALK, DCTN1–ALK, PRKD3–ALK) were present in 50% (5/10) of congenital/infant, 1.4% (3/205) of pediatric, and 1.9% (3/156) of adult GBMs. ALK fusions were present as the only candidate driver in congenital/infant GBMs and were sometimes focally amplified. In contrast, adult ALK fusions co-occurred with other oncogenic drivers. No activating ALK mutations were identified in any age group. Novel and recurrent ALK rearrangements promoted STAT3 and ERK1/2 pathways and transformation in vitro and in vivo. ALK-fused GBM cellular and mouse models were responsive to ALK inhibitors, including in patient cells derived from a congenital GBM. Relevant to the treatment of infant gliomas, we showed that ALK protein appears minimally expressed in the forebrain at perinatal stages, and no gross effects on perinatal brain development were seen in pregnant mice treated with the ALK inhibitor ceritinib. These findings support use of brain-penetrant ALK inhibitors in clinical trials across infant, pediatric, and adult GBMs.