Dataset Information

Loss of RET promotes mesenchymal identity in neuroblastoma cells

ABSTRACT: Aberrant activation of Anaplastic Lymphoma Kinase (ALK) drives neuroblastoma (NB). Previous work has identified the RET receptor tyrosine kinase (RTK) as a downstream target of ALK activity in NB models. We show here that ALK activation in response to ALKAL2 ligand results in the rapid phosphorylation of RET in NB cells, providing additional insight into the contribution of RET to the ALK driven gene signature in NB. To further address the role of RET in NB, RET knock-out (KO) SK-N-AS cells were generated by CRISPR/Cas9 genome engineering. Gene expression analysis of RET KO NB cells identified a reprogramming of NB cells to a mesenchymal (MES) phenotype that was characterized by increased migration and upregulation of the AXL and MET RTKs as well as integrins and extracellular matrix components. Strikingly, the upregulation of AXL in the absence of RET reflects the development timeline observed in the neural crest as progenitor cells undergo differentiation during embryonic development. Together, these findings suggest that a MES phenotype is promoted in mesenchymal NB cells in the absence of RET, reflective of a less differentiated developmental status.

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cell Culture

SUBMITTER: Proteomics Core Facility

LAB HEAD: Ruth Palmer

PROVIDER: PXD024551 | Pride | 2021-09-10

REPOSITORIES: Pride

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Dataset's files

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			Action	DRS
	4055-set2_sA1-A16_SPhuman-0mc_TMTpro_Lumos_200817_42-71.msf	Msf
	Lumos_200817_42.raw	Raw
	Lumos_200817_43.raw	Raw
	Lumos_200817_44.raw	Raw
	Lumos_200817_45.raw	Raw

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Loss of RET Promotes Mesenchymal Identity in Neuroblastoma Cells.

Siaw Joachim T JT Gabre Jonatan L JL Uçkun Ezgi E Vigny Marc M Zhang Wancun W Van den Eynden Jimmy J Hallberg Bengt B Palmer Ruth H RH Guan Jikui J

Cancers 20210415 8

Aberrant activation of anaplastic lymphoma kinase (ALK) drives neuroblastoma (NB). Previous work identified the RET receptor tyrosine kinase (RTK) as a downstream target of ALK activity in NB models. We show here that ALK activation in response to ALKAL2 ligand results in the rapid phosphorylation of RET in NB cells, providing additional insight into the contribution of RET to the ALK-driven gene signature in NB. To further address the role of RET in NB, <i>RET</i> knockout (KO) SK-N-AS cells we ...[more]

PMID: 33921066

Similar Datasets

Project description:The crizotinibâresistant ALKF1174L mutation arises de novo in neuroblastoma (NB) and is acquired in ALK translocation-driven cancers, lending impetus to the development of novel ALK inhibitors with different modes of action. The diaminopyrimidine TAE684 and its derivative ceritinib (LDK378), which are structurally distinct from crizotinib, are active against NB cells expressing ALKF1174L. Here we demonstrate acquired resistance to TAE684 and LDK378 in ALKF1174L-driven human NB cells that is linked to overexpression and activation of the AXL tyrosine kinase and epithelial-to-mesenchymal transition (EMT). AXL phosphorylation conferred TAE684 resistance to NB cells through upregulated ERK signaling. Inhibition of AXL partly rescued TAE684 resistance, resensitizing these cells to this compound. AXL activation in resistant cells was mediated through increased expression of the active form of its ligand, GAS6, which also served to stabilize the AXL protein. Although ectopic expression of AXL and TWIST2 individually in TAE684-sensitive parental cells led to the elevated expression of mesenchymal markers and invasive capacity, only AXL overexpression induced resistance to TAE684 as well. TAE684-resistant cells showed greater sensitivity to HSP90 inhibition than did their parental counterparts, with downregulation of AXL and AXL-mediated ERK signaling. Our studies indicate that aberrant AXL signaling and development of an EMT phenotype underlie resistance of ALKF1174L-driven NB cells to TAE684 and its derivatives. We suggest that the combination of ALK and AXL or HSP90 inhibitors be considered to delay the emergence of such resistance. In order to understand the molecular mechanisms driving resistance to ALK inhibition in ALK-mutated neuroblatoma, we established cell line models of resistance to TAE684, an ALK inhibitor, by treating SH-SY5Y cells (bearing the ALKF1174L mutation) with increasing concentration of this compound over time. We then performed an analysis of gene expression changes genome wide using Affymetrix U133 Plus 2 arrays, by comparing the TAE684-sensitive parental SH-SY5Y cells to the TAE684-resistant SH-SY5Y cells (named SY5Y-TR1). For that experiment, we analyzed gene expression variations by comparing the parental SH-SY5Y (control sample) to the resistant SY5Y-TR1 cells. So 2 samples were analyzed, with 3 replicates run for each.

Project description:The crizotinib–resistant ALKF1174L mutation arises de novo in neuroblastoma (NB) and is acquired in ALK translocation-driven cancers, lending impetus to the development of novel ALK inhibitors with different modes of action. The diaminopyrimidine TAE684 and its derivative ceritinib (LDK378), which are structurally distinct from crizotinib, are active against NB cells expressing ALKF1174L. Here we demonstrate acquired resistance to TAE684 and LDK378 in ALKF1174L-driven human NB cells that is linked to overexpression and activation of the AXL tyrosine kinase and epithelial-to-mesenchymal transition (EMT). AXL phosphorylation conferred TAE684 resistance to NB cells through upregulated ERK signaling. Inhibition of AXL partly rescued TAE684 resistance, resensitizing these cells to this compound. AXL activation in resistant cells was mediated through increased expression of the active form of its ligand, GAS6, which also served to stabilize the AXL protein. Although ectopic expression of AXL and TWIST2 individually in TAE684-sensitive parental cells led to the elevated expression of mesenchymal markers and invasive capacity, only AXL overexpression induced resistance to TAE684 as well. TAE684-resistant cells showed greater sensitivity to HSP90 inhibition than did their parental counterparts, with downregulation of AXL and AXL-mediated ERK signaling. Our studies indicate that aberrant AXL signaling and development of an EMT phenotype underlie resistance of ALKF1174L-driven NB cells to TAE684 and its derivatives. We suggest that the combination of ALK and AXL or HSP90 inhibitors be considered to delay the emergence of such resistance. In order to understand the molecular mechanisms driving resistance to ALK inhibition in ALK-mutated neuroblatoma, we established cell line models of resistance to TAE684, an ALK inhibitor, by treating SH-SY5Y cells (bearing the ALKF1174L mutation) with increasing concentration of this compound over time. We then performed an analysis of gene expression changes genome wide using Affymetrix U133 Plus 2 arrays, by comparing the TAE684-sensitive parental SH-SY5Y cells to the TAE684-resistant SH-SY5Y cells (named SY5Y-TR1).