ALK ligand ALKAL2 potentiates MYCN-driven neuroblastoma in the absence of ALK mutation (2020)
Figure 7

Figure 7 - ALKAL2-driven NB is sensitive to treatment with the ALK TKI lorlatinib in vivo.

A. Cells derived from tumours arising in Rosa26_Alkal2;Th-MYCN (#4953) and Alk-F1178S;Th-MYCN (#4938) mice are sensitive to both lorlatinib and brigatinib. The effect of increasing concentrations of each ALK TKI (as indicated) on cell confluence was analyzed by IncuCyte Live Cell Analysis. Data is presented as mean ± SEM from three independent experiments. *p<0.05, **p<0.005; two-tailed paired student t-test.

B. Tumour volume changes over time for Rosa26_Alkal2;Th-MYCN mice treated with lorlatinib (10 mg/kg; twice daily) or vehicle control. Tumour volume was measured by ultrasound on Day 0 and 7, and by direct measurement at Day 14. Day 0 (lorlatinib n=7, Ctrl n=6), Day 7 (lorlatinib n=2, Ctrl n=5) and Day 14 (lorlatinib n=7, Ctrl n=6). Data shown represents mean ± SD. **p<0.005; two-tailed unpaired student t-test.

C. Rosa26_Alkal2;Th-MYCN animals treated with lorlatinib did not display any significant loss of body weight compared with vehicle controls. Data shown represents mean ± SD.

D. Representative ultrasound images of tumours observed in Rosa26_Alkal2;Th-MYCN mice with annotated measurements at Day 0 and Day 7. Tumours arise in the retroperitoneal space ventral to the aorta, Ao.

E. Representative MRI imaging of Rosa26_Alkal2;Th-MYCN tumours in response to lorlatinib at 4d and 14d.

F. Rosa26_Alkal2;Th-MYCN tumours from lorlatinib or vehicle controls were analysed for pHis. A representative field of view for each tumour at 40x (175740μm2) were manually counted. Data shown represents mean ± 95% CI. p=0.0286; Mann-Whitney test.

Acknowledgments: The authors thank Anne Uv for critical comments on the manuscript. The authors would like to acknowledge support of Science for Life Laboratory/Clinical Genomics, Gothenburg for providing assistance in computational infrastructure and of the National Genomics Infrastructure (NGI)/Science for Life Laboratory, Stockholm for performing massive parallel sequencing. Work performed at NGI has been funded by RFI/VR and Science for Life Laboratory, Sweden. Funding: This work has been supported by grants from the Swedish Cancer Society (RHP CAN18/729; B.H.: CAN18/718), the Swedish Childhood Cancer Foundation (RHP:PR2015-0096; SF:NCp2015-0061 and PR2018-0099; JG TJ2016-0088 and PR2016-2011; GU: TJ2018-0056; BH: PR2017-0110), the Swedish Research Council (RHP 2015-04466; BH:2017-01324), the Swedish Foundation for Strategic Research (RB13-0204), the Göran Gustafsson Foundation (RHP2016) and the Knut and Alice Wallenberg Foundation (KAW 2018.0057). Author contributions: M.B., D.L., B.W., T.M., A.E.W. and B.A. performed the mouse experiments and tumour imaging, J.V.d.E. and A.C. carried out the bioinformatics analysis. J.V.d.E. and J.K. performed the R2 database analysis. G.U., W-Y.L., TP.C., P.M.-G., and J.G. carried out the Drosophila, cell and biochemical analyses. S.F, J.G. and M.J. performed and analyzed the WGS of mouse tumours. R.H.P., J.V.d.E. and B.H. supervised the project. R.H.P. wrote the first draft of the manuscript with input from all authors. Conflict of interest: The authors declare that they have no conflict of interest.