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Functional RNAi screen targeting cytokine and growth factor receptors reveals oncorequisite role for interleukin-2 gamma receptor in JAK3-mutation-positive leukemia.

ABSTRACT: To understand the role of cytokine and growth factor receptor-mediated signaling in leukemia pathogenesis, we designed a functional RNA interference (RNAi) screen targeting 188 cytokine and growth factor receptors that we found highly expressed in primary leukemia specimens. Using this screen, we identified interleukin-2 gamma receptor (IL2R?) as a critical growth determinant for a JAK3(A572V) mutation-positive acute myeloid leukemia cell line. We observed that knockdown of IL2R? abrogates phosphorylation of JAK3 and downstream signaling molecules, JAK1, STAT5, MAPK and pS6 ribosomal protein. Overexpression of IL2R? in murine cells increased the transforming potential of activating JAK3 mutations, whereas absence of IL2R? completely abrogated the clonogenic potential of JAK3(A572V), as well as the transforming potential of additional JAK3-activating mutations such as JAK3(M511I). In addition, mutation at the IL2R? interaction site in the FERM domain of JAK3 (Y100C) completely abrogated JAK3-mediated leukemic transformation. Mechanistically, we found IL2R? contributes to constitutive JAK3 mutant signaling by increasing JAK3 expression and phosphorylation. Conversely, we found that mutant, but not wild-type JAK3, increased the expression of IL2R?, indicating IL2R? and JAK3 contribute to constitutive JAK/STAT signaling through their reciprocal regulation. Overall, we demonstrate a novel role for IL2R? in potentiating oncogenesis in the setting of JAK3-mutation-positive leukemia. In addition, our study highlights an RNAi-based functional assay that can be used to facilitate the identification of non-kinase cytokine and growth factor receptor targets for inhibiting leukemic cell growth.

SUBMITTER: Agarwal A

PROVIDER: S-EPMC4324389 | biostudies-literature | 2015 Jun

REPOSITORIES: biostudies-literature

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Functional RNAi screen targeting cytokine and growth factor receptors reveals oncorequisite role for interleukin-2 gamma receptor in JAK3-mutation-positive leukemia.

Agarwal A A MacKenzie R J RJ Eide C A CA Davare M A MA Watanabe-Smith K K Tognon C E CE Mongoue-Tchokote S S Park B B Braziel R M RM Tyner J W JW Druker B J BJ

Oncogene 20140811 23

To understand the role of cytokine and growth factor receptor-mediated signaling in leukemia pathogenesis, we designed a functional RNA interference (RNAi) screen targeting 188 cytokine and growth factor receptors that we found highly expressed in primary leukemia specimens. Using this screen, we identified interleukin-2 gamma receptor (IL2Rγ) as a critical growth determinant for a JAK3(A572V) mutation-positive acute myeloid leukemia cell line. We observed that knockdown of IL2Rγ abrogates phosp ...[more]

PMID: 25109334

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Project description:Activating alternative cell death pathways, including autophagic cell death, is a promising direction to overcome the apoptosis resistance observed in various cancers. Yet, whether autophagy acts as a death mechanism by over consumption of intracellular components is still controversial and remains undefined at the ultrastructural and the mechanistic levels. Here we identified conditions under which resveratrol-treated A549 lung cancer cells die by a mechanism that fulfills the previous definition of autophagic cell death. The cells displayed a strong and sustained induction of autophagic flux, cell death was prevented by knocking down autophagic genes and death occurred in the absence of apoptotic or necroptotic pathway activation. Detailed ultrastructural characterization revealed additional critical events, including a continuous increase over time in the number of autophagic vacuoles, in particular autolysosomes, occupying most of the cytoplasm at terminal stages. This was followed by loss of organelles, disruption of intracellular membranes including the swelling of perinuclear space and, occasionally, a unique type of nuclear shedding. A signalome-wide shRNA-based viability screen was applied to identify positive mediators of this type of autophagic cell death. One top hit was GBA1, the Gaucher disease-associated gene, which encodes glucocerebrosidase, an enzyme that metabolizes glucosylceramide to ceramide and glucose. Interestingly, glucocerebrosidase expression levels and activity were elevated, concomitantly with increased intracellular ceramide levels, both of which correlated in time with the appearance of the unique death characteristics. Transfection with siGBA1 attenuated the increase in glucocerebrosidase activity and the intracellular ceramide levels. Most importantly, GBA1 knockdown prevented the strong increase in LC3 lipidation, and many of the ultrastructural changes characteristic of this type of autophagic cell death, including a significant decrease in cytoplasmic area occupied by autophagic vacuoles. Together, these findings highlight the critical role of GBA1 in mediating enhanced self-consumption of intracellular components and endomembranes, leading to autophagic cell death.

Dataset Information

Functional RNAi screen targeting cytokine and growth factor receptors reveals oncorequisite role for interleukin-2 gamma receptor in JAK3-mutation-positive leukemia.

Publications

Functional RNAi screen targeting cytokine and growth factor receptors reveals oncorequisite role for interleukin-2 gamma receptor in JAK3-mutation-positive leukemia.

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