Abstract 2333: Functional RNAi screen identifies novel cytokine and growth factor receptors critical for the growth of leukemia patient samples.
Authors: Agarwal Anupriya , MacKenzie Ryan , Braziel Rita , Tyner Jeffrey W. , Druker Brian J. 
Published: August 2014

Background: Cytokine and growth factor receptors contribute to cancer pathogenesis by regulating variety of signaling pathways. However, additional investigation is needed to understand the functional and therapeutic implications of cytokine and growth factor receptors in cancer. Here we present a novel RNAi-based screen to uncover the functional and prognostic relevance of growth factor/cytokine receptors for leukemia pathogenesis. This assay enables us to discover novel mechanisms of leukemogenesis involving various non-kinase receptors.

Methods and Results: We have designed a novel RNAi based functional screen targeting 188 growth factor receptors that were found highly expressed in primary leukemia specimens by gene microarray analysis. We have tested primary cells from 72 leukemia patients with AML, ALL and MPN and 8 leukemia cell lines for dependence on these receptors. Novel candidate targets found in primary patient samples include CD24, NCOA4, IL1R1, IL2Rα and IL15Rα. In many cases, we found that these targets have genetic abnormalities ranging from splice variation (IL2Rα) to intron retention (IL15Rα). In a few cases, the receptor was found to serve as a scaffold for regulating downstream oncogenic signaling. Importantly, we show proof-of-principle data where IL2Rγ is a potential target in the JAK3 A572V mutation positive AML cell line (CMK). Validation experiments demonstrated that knockdown of IL2Rγ significantly reduces the viability of CMK cells (90% decrease) and abrogates phosphorylation of JAK3 and downstream signaling molecules, STAT5, MAPK and the pS6 ribosomal protein. Intriguingly, the absence of IL2Rγ in murine bone marrow completely abrogated the clonogenic potential of JAK3 A572V as compared to IL2Rγ-wild type marrow. These effects can be rescued by co-expressing IL2Rγ with JAK3 A572V but not by co-expressing IL2Rγ with JAK3 Y100C, a FERM domain inactivating mutation. Additionally, overexpression of IL2Rγ made JAK3 A572V cells less sensitive to the JAK family inhibitor, JAK Inhibitor I. Mechanistically, IL2Rγ contributes to constitutive JAK3 mutant signaling by increasing JAK3 protein levels and phosphorylation. Conversely, mutant but not wild type JAK3 increased the expression of IL2Rγ, indicating IL2Rγ contributes to constitutive JAK3 signaling through a feedback mechanism. Overall these results demonstrate an oncogenic potentiating role of IL2Rγ.

Conclusions: RNAi-based functional screening for AML cell dependence on non-kinase cytokine and growth factor receptors led to the identification of novel oncogenic etiologies for AML. This assay can identify genes that are crucial for malignant cell growth, regardless of the mutational status both in cell lines and in primary samples. These findings underscore the importance of targeting cytokine and growth factor receptors in leukemia pathogenesis.

Citation Format: Anupriya Agarwal, Ryan MacKenzie, Rita Braziel, Jeffrey W. Tyner, Brian J. Druker. Functional RNAi screen identifies novel cytokine and growth factor receptors critical for the growth of leukemia patient samples. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2333. doi:10.1158/1538-7445.AM2013-2333