C-CBL Inactivation Promotes Cytokine-Driven Growth of Human CD34+ Cells and AML1-ETO Expressing Cells

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Abstract 2464C-CBL is an E3 ubiquitin ligase and negatively regulates activated receptor tyrosine kinases. C-CBL mutations have been frequently found in a variety of myeloid neoplasms, most frequently in chronic myelomonocytic leukemia (CMML) and juvenile myelomonocytic leukemia (JMML). The mutations occur almost exclusively in exons 8–9 that encode linker/RING finger domains, which result in loss of E3 ligase activity. These mutants are considered to have dominant-negative function over the wild-type C-CBL. In acute myeloid leukemia (AML), C-CBL mutations are associated with core binding factor (CBF) leukemia. Although the physiologic roles of C-CBL and its mutations have been elucidated with several mouse models, the precise roles of C-CBL in primary human hematopoietic cells remain unclear.We have developed a unique culture system to model myeloid leukemogenesis using human CD34+ cord blood (CB) cells cultured in cytokines (SCF, TPO, Flt3 ligand, IL-3, IL-6), and have shown that CBF fusion oncoproteins, AML1-ETO (AE) and CBFB-MYH11 (CM), enable long-term proliferation in vitro by increasing self-renewal of CD34+ cells. Using this system, we assessed functions of C-CBL and its disease-related mutants in primary human CD34+ CB cells. All 3 C-CBL mutants (Gln367Pro, Tyr371Ser, DExon8/9), but not wild-type C-CBL, promoted the growth of CD34+ CB cells. However, unlike cells expressing AE or CM, C-CBL mutant-expressing cells stopped growing at about the same time as normal CB cells (3–4 weeks), suggesting that the mutants do not increase the self-renewal of CD34+ cells. Similar to the results obtained for normal CB cells, all 3 C-CBL mutants accelerated the cytokine-driven expansion of AE cells.Next, to test functions of endogenous C-CBL, we knocked down C-CBL expression in AE-expressing cells using 5 different shRNAs. To our surprise, one shRNA promoted, while the other 4 inhibited the growth of AE cells. The disparate effects of shRNAs were not due to different knockdown efficiencies of each shRNA, because the two best shRNAs exhibited nearly complete C-CBL depletion but showed either growth-promoting (shCBL-1) or growth-inhibitory (shCBL-2) effects. To determine which effect of shRNA is actually due to C-CBL downregulation, we cotransduced shRNA-resistant C-CBL together with shCBL-1 or shCBL-2 in AE cells, and compared the growth of shRNA-transduced cells and shRNA/C-CBL cotransduced cells. C-CBL reintroduction suppressed the enhanced AE cell growth by shCBL-1, while it did not rescue the defective growth of shCBL-2-transduced AE cells. These results indicate that the growth-promoting effect of shCBL-1 is actually due to C-CBL knockdown, while the growth inhibitory effect of shCBL-2 (and possibly the other 3 shRNAs) is caused by off-target and/or nonspecific toxicity of shRNAs. Transduction of the shCBL-1 also promoted the proliferation of normal CB cells. Interestingly, neither expression of C-CBL mutants nor C-CBL depletion by shCBL-1 showed growth-promoting effects on a cytokine-independent AE cell line (Kasumi-1), indicating that C-CBL inactivation promotes cell growth by enhancing the response to cytokines.To characterize the increased cell growth induced by C-CBL mutations/depletion, we assessed expression of a stem cell marker CD34 in the mutant expressing or C-CBL depleted CB and AE cells. In contrast to effects seen with CBF fusion genes, C-CBL inactivation results in decreased expression of CD34. We also performed cell cycle and apoptosis analyses, and found a significant increase in the proportion of S/G2/M phase cells upon C-CBL inactivation. These results indicate that C-CBL inactivation promotes cell growth mainly through increased cell cycle progression.In summary, we found that C-CBL mutant expression and C-CBL depletion promote cytokine-dependent growth, but do not increase self-renewal of human CD34+ CB cells (Fig.1). C-CBL mutations/depletion also showed growth-promoting effects on AE-expressing cells (Fig.1), which support the notion that C-CBL inactivation collaborates with CBF-rearranged genes (AE and CM) to promote AML. Functional cooperation between C-CBL mutations and AE in vivo is currently being tested using a xenotransplantation assay. These unique human cell models for myeloid neoplasms with C-CBL mutations/deletion will be useful to reveal their molecular pathogenesis and to find novel therapeutic targets. [Display omitted] Disclosures:No relevant conflicts of interest to declare.