iPSC-Based Phenomic Screen Revealed an Impact of Uncontrolled NFkB Activity at the Initiating Stages of AML1-ETO Related Leukemia
Authors: Niwa Akira , Nakahata Tatsutoshi , Saito Megumu K 
Published: October 2019

Onset of acute myeloid leukemia (AML) has been accounted for by cooperation between multiple genetic alterations which induce abnormal control of various cellular pathways. Among the previously listed leukemogenic lesions, AML1-ETO fusion (AE) generated by translocation (8;21) (q22;q22) is one of the common mutations observed in 20-40% of patients. AE affects transcriptional regulation associated with hematopoietic differentiation, while 60% of AE-positive AML cases are shown to have together other types of mutation of genes involved in cell proliferation, such as receptor tyrosine kinase (RTK) c-kit and FLT3. However, the detailed mechanisms of how they work in the very early stages of leukemogenesis and what unknown "cooperative " cues function in those periods. From this viewpoint, in order to identify novel cellular molecules involved in the acquisition of leukemic phenotypes, we have conducted the gene-trap strategy-based phenomic screen in the use of pluripotent stem cell (PSC)-derived hematopoietic culture.

Through our screening, we found that the functional knockdown of a gene NSFL1c, also known as p47, enhanced the activities of hematopoietic progenitor cells harboring AE to show leukemic phenotype both in vivo and in vitro: Cells differentiated from AE-PSCs which have additionally the poly A trapping sequence inserted in NSFL1c locus showed doubled efficiency in engraftment into immunodeficient NOG mice than cells without trapping (3.1% v.s. 1.3%, 16 weeks after intra bone marrow transplantation), and also showed the significantly higher colony replating efficacy in methylcellulose colony forming assay.

In order to clear what lineages of cells were most responsible for those phenomena, we next performed those colony forming experiments after sorting of CD34+CD43+CD13- immature cells, CD34+/-CD43+CD13+ myeloid cells and CD71+CD41+ erythro-megakaryocytic subpopulations of cells. As a result, CD34+/-CD43+CD13+ myeloid precursors showed the strongest tendencies to emerge highly proliferative clones followed by CD34+CD43+CD13- immature progenitors in the presence of NSF1c trapping. Interestingly, those activities were cancelled in the absence of AE expression.

NSFL1c is one of the component of NEMO complex which binds to ubiquitinated NEMO and induces its degradation, resulting in reduced IKK and elevated NFkB activities. In AML, elevated NF-κB pathways have been detected in more than 30% of patients. Although NF-κB signaling networks have proved induced by inflammatory and immune signals, and previous studies in vivo and in vitro showed their abnormal activities make leukemia cells escape from cell death and go into abnormal proliferation, the detailed mechanisms of how they are involved in the pathogenesis, in particular at the very early stages of the leukemogenesis, are well not defined. Our data therefore reflect the novel mechanisms behind the deviation of progenitor cell fate from normal to abnormal pathway leading to the emergence of leukemic initiating cells, and suggested the myeloid-biased leukemogenic potentials.

Disclosures

No relevant conflicts of interest to declare.