Autophagy Collaborates with Ubiquitination to Down-Regulate Oncoprotein E2A/Pbx1 in B Cell Acute Lymphoblastic Leukemia

Background: B cell acute lymphoblastic leukemia (B-ALL) accounts for the most cancer incidences in children. The t(1;19) translocation leukemia accounts for a quarter of pre-B ALL and up to 5% of all ALL patients, in which the transcriptional activator E2A and homeobox pre-B-cell leukemia transcription factor 1 (PBX1) fuses, resulting in expression of the chimeric transcription factor E2A/PBX1. E2A/PBX1 has been proved to be an oncogene and could induce malignant transformation. Methods: (1) Childhood B-ALL patients were collected and the stem/progenitor cells (CD34+CD38-) and leukemia cells (CD19+) were sorted with BD FACS Aria III. The autophagy level in these cells was measured by real-time Q-PCR, including gene expression of Beclin1, Atg7, Atg5, LC3 and p62. Normal bone marrow cells from healthy donors were used as control. (2) E2A/PBX1 fusion gene positive pre-B ALL 697 cells were used to establish leukemia mouse model and the autophagy activity in the mice was enhanced by administration of rapamycin. Mice were sacrificed three weeks post treatment, leukemia phenotype was then identified and E2A/PBX1 oncoprotein of liver was detected by western blotting. (3) Autophagy and ubiquitination were manipulated with inhibitors or starvation in 697 cells and the degradation mechanism of E2A/PBX1 was explored. Co-localization of E2A/PBX1-LC3 and E2A/PBX1-Ub was observed by confocal microscopy and quantified by Amnis image flow cytometry. Results: (1) B-ALL primary cells from childhood patients show down-regulated level of autophagy. (2) The NOD-SCID mouse model study shows that activating autophagy of mice by rapamycin improved the survival of leukemia animals, prevented leukemiagenesis by inhibition on the transplanted leukemia cells (examined by blood cell counting, liver HE staining and expression of CD 45, 10, 19 from transplanted human 697 cells by flow cytometry), promoted the degradation of oncoprotein E2A/PBX1 (by Western blotting)), and more importantly, restored hematopoietic stem cells (LSKCD34- cell number detected by flow cytometry). (3) The ALL 697 cell line study shows that activation of autophagy by rapamycin and starvation could down-regulate E2A/PBX1 expression detected by flow cytometry and western blotting. The confocal microscopic results show co-localization of E2A/PBX1 with autophagy marker GFP-LC3 in both rapamycin and starvation treatment groups. To confirm the degradation mechanism, autophagy inhibitor (3-MA or Baf-A1) and ubiquitin-proteasome inhibitor (MG132) were used to treat 697 cells. The results show that inhibition of autophagy in the early stage by 3-MA fails to degrade E2A/PBX1 in 697 cells, but ubiquitination also contributes to the degradation of E2A/PBX1. Quantitative analysis shows increased co-localization percentage of E2A/PBX1-LC3 in rapamycin and starvation treatment groups and increased co-localization of E2A/PBX1 with Ubiquitin in starvation group; but MG132 treatment inhibited the co-localization of E2A/PBX1-Ub induced by starvation, indicating a collaborative role between autophagy and ubiquitination in the degradation of E2A/PBX1. Conclusions: B-ALL primary cells from patients show low autophagy activity; Autophagy activation fights against B-ALL by inhibition on transplanted leukemia cells, degradation of oncoprotein E2A/Pbx1 and restoration of hematopoietic stem cells in the NOD-SCID B-ALL mouse model; autophagy collaborates with ubiquitination in the degradation of E2A/PBX1 in the 697 cells, thereby proposing a novel strategy for targeted therapy on childhood B-ALL. Disclosures No relevant conflicts of interest to declare.