Critical Role Of Interleukin Receptor Signaling In Acute Myeloid Leukemia Identified Using An RNAi Functional Screen

Acute myeloid leukemia (AML) is one of the most common hematologic malignancies affecting both children and adults. Targeted therapy represents a promising approach in AML with the potential for increased efficacy and reduced toxicity. However, the molecular abnormalities in AML are extraordinarily heterogeneous. Numerous genetic changes are reported which require careful functional analyses to distinguish “driver mutations” from “passengers.” Because cytokine/growth factor receptors contribute to cancer pathogenesis by regulating growth, differentiation, senescence and survival, we designed a functional screen focusing on these molecules. This RNAi-based screen was designed to reveal the functional and prognostic relevance of cytokine/growth factor receptors in leukemia. Using this approach, we identified several interleukin receptors that play critical roles in promoting leukemia cell growth. Importantly, we show the novel roles of two interleukin receptors, IL2Rγ and IL1R1, in promoting AML cell growth.

We designed an siRNA-based functional screen targeting 188 growth factor receptors that were found highly expressed in mononuclear cells of 140 primary leukemia samples by gene expression microarray analysis. We tested 72 leukemia patient samples with AML, ALL and MPN and 10 cell lines for dependence on these receptors. Cells were electroporated with siRNAs and cell viability was quantified after 72 hrs. Novel candidate targets found in AML patient samples include IL17R, IL9R, IL4R IL1R1, IL2Rγ, IL2Rα and IL15Rα. We found a variety of genetic abnormalities in these target genes, including splice variation (IL2Rα) and intron retention (IL15Rα).

We found that IL2Rγ is a potential target in a 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, JAK1, STAT5, MAPK and 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 were rescued by co-expressing IL2Rγ with JAK3^A572V but not by co-expressing IL2Rγ with JAK3^Y100C, an inactivating mutation. Additionally, we found that 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. Similarly, IL2Rγ receptor is critical for transforming potential of additional JAK3 activating mutations such as JAK3^M511I and JAK3^A573V. Overall these results demonstrate an oncogenic potentiating role of IL2Rγ.

Additionally, we found that silencing IL1R1 reduces the viability of 25% of AML primary samples. Most IL1R1 knockdown-sensitive AML samples exhibit monocytic and myelomonocytic features; however, no correlation with particular somatic mutations has thus far been observed. Confirming the importance of IL1R1 using a genetic model, we found that the absence of IL1R1 in murine bone marrow leads to the significant ablation of clonogenic potential (80% reduction) induced with AML1-ETO9a, NRAS^G12D and MLL-ENL oncogenes as compared to wild-type marrow in a ligand-dependent manner. Similarly knocking down IL1R1 in CD34+ AML cells reduces cell growth and phosphorylation of p38. Conversely, the presence of exogenous IL1 promotes AML cell survival by increasing p38 phosphorylation, which is significantly inhibited with p38 inhibitors such as Doramapimod. These results support a novel role for IL1R1-mediated signaling in promoting AML cell growth and targeting of this pathway in AML.

RNAi-based functional screening for leukemia cell dependence on cytokine/growth factor receptors led to the identification of novel oncogenic pathophysiological mechanisms. Specifically, (A) IL2Rγ is essential for the growth of leukemia cells harboring activating JAK3 mutations and (B) IL1R1 is involved in regulating growth and survival of AML cells, particularly with monocytic differentiation. These findings underscore the importance of interleukin receptors in leukemia pathogenesis and suggest that targeting these pathways in AML will be beneficial.

Tyner:Incyte Corporation: Research Funding. Bagby:NIH: Membership on an entity’s Board of Directors or advisory committees.