Abstract
A thorough understanding of the signaling pathways required for AML formation is necessary in order to develop much needed therapeutic treatments for AML patients. While there are many known mutations that contribute to AML, additional unknown genes likely exist, since the leukemic cells of a significant number of AML patients are cytogenetically normal and AML likely forms as a result of two mutations in different classes of oncogenes. Therefore, in order to identify novel mutations and signaling pathways that contribute to AML, we undertook functional genetic screening for genes, expressed in the leukemic cells of AML patients, that could transform myeloid cells in culture. To do this we generated cDNA libraries from cytogenetically normal AML patients and screened 32D myeloid cells for cytokine independent growth. We did not identify any genes capable of transforming 32D cells to cytokine independence by this approach, likely because the apoptotic response upon cytokine removal is very rapid. We then performed screens in 32D cells that exogenously express the anti-apoptotic Bcl2 protein in order to attempt to sensitize these cells to transformation by unknown oncogenes. These cells undergo cell death much slower than parental 32D cells in the absence of cytokine. From these screens, we identified IL27Ra (also known as TCCR and WSX1) as a gene that can induce the cytokine-independent growth of 32D cells. Interestingly, IL27R is capable of transforming 32D cells that lack exogenous Bcl2 expression, suggesting that our use of Bcl2 sensitized 32D cells to transformation in the context of expressing a library of genes in these cells. Therefore, this approach may allow for the identification of AML oncogenes in a relevant cell system. IL27Ra (IL27R) is a type I cytokine receptor that functions as the ligand-binding component for the receptor for IL-27 and functions with the gp130 co-receptor to induce signal transduction in response to IL-27. Cytokine independent 32D/IL27R cells contain elevated levels of activated forms of various signaling proteins, including JAK1, JAK2, STAT1, STAT5, ERK1/2, and SHP-2. Activation of these signaling proteins is dependent on the kinase activity of JAK family proteins as the pan JAK inhibitor, JAK inhibitor I, blocked activation of these signaling proteins. JAK inhibitor I also induced apoptotic cell death in 32D cells transformed to cytokine independence by IL27R, suggesting the transforming properties of IL27R are dependent on the activity of JAK family members. In addition, IL27R can transform BaF3 pro-B cells to cytokine independence. Since BaF3 cells lack expression of the gp130 co-receptor for IL-27, this suggests that IL27R-mediated transformation of hematopoietic cells is gp130-independent. This is a novel finding for IL27R-dependent signal transduction. IL27R has primarily been studied in the context of T-cell regulation. Our results are very exciting because our work is the first to suggest that IL27R may function as a hematopoietic cell oncogene. This is also interesting because type I cytokine receptors are required for activation of a JAK2 mutant (V617F) found in various myeloproliferative disorders (MPDs). We also show that IL27R is capable of activating JAK2-V617F in cells. Our data demonstrate that IL27R possesses hematopoietic cell transforming properties and suggest that type I cytokine receptors, such as IL27R, may play unappreciated roles in MPDs, including AML.
Signals in motion: Determining how signal transduction is mechanically coupled through type-I cytokine receptors