Functional RNAi Screen Identifies Novel Cytokine and Growth Factor Receptors Critical for Leukemia Cell Growth

Despite the great strides that have been made in the treatment of acute myeloid leukemia (AML), one-third of patients are resistant to treatment. In many cases, disease-causing genetic targets still need to be elucidated. Cytokine and growth factor receptors contribute to cancer pathogenesis by regulating various downstream signaling cascades, including tyrosine kinase driven pathways, which represent amenable therapeutic targets. However, additional investigation is needed to understand the functional and therapeutic implications of cytokine and growth factor receptors in cancer pathogenesis. We have developed 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. Importantly, we show proof-of-principle data where IL2Rγ is found to be essential for JAK3 mutant mediated leukemogenesis.

To identify non-kinase cytokine and growth factor receptors that are critical for leukemia cell viability, we have designed a novel RNAi based functional screen targeting 188 growth factor receptors that were found to be highly expressed by gene microarray analysis of primary AML patient specimens. We have tested 40 AML patient samples and 5 AML cell lines for dependence on these receptors by electroporating cells with 188 individual siRNAs. After culturing cells for four days, effects on cell viability were measured using an MTS assay. Candidate targets found in primary patient samples include CD24, NCOA4, IL2Rα, IL15Rα and IL2Rγ. 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 and thus contributes to oncogenesis. For instance, we identified IL2Rγ as 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. In a converse experiment, overexpression of IL2Rγ enhanced JAK3 A572V mediated signaling and increased its transformation potential in a ligand-independent manner. Similarly, IL2Rγ overexpression also increased the oncogenic potential of other JAK3 mutants such as M511I, which have previously been identified in AML patients. 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 (IC₅₀is increased from 150 nM to 625 nM). Mechanistically, IL2Rγ contributes to constitutive JAK3 mutant signaling by increasing JAK3 protein levels and phosphorylation. In fact, 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.

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. These findings underscore the importance of cytokine and growth factor receptors in leukemia pathogenesis. 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. Future studies integrating this RNAi screen with deep sequencing will lend additional power to this assay by accelerating our understanding of the genetic mechanisms underlying these functional gene targets such that these findings can be rapidly translated into novel therapeutic strategies.

No relevant conflicts of interest to declare.