TRIM28 Facilitates Chronic Myeloid Leukemia Progression By Enhancing Sumoylation and Stability of BCR-ABL

Chronic myeloid leukemia (CML) is a hematopoietic malignancy caused by the BCR-ABL fusion gene. Although tyrosine kinase inhibitors (TKIs) targeting the oncoprotein BCR-ABL have drastically improved the prognosis of patients, TKI resistance has become a major issue, which is caused by mutations in the BCR-ABL kinase domain and other kinase activity-independent mechanisms. Eliminating BCR-ABL at the protein level is a potential therapeutic target for CML. As a posttranslational modification (PTM), SUMOylation plays a critical role in regulating the interaction, stability, and activity of proteins. It is reported that dysregulation of SUMOylation is involved in tumorigenesis. However, the role of SUMOylation in BCR-ABL-induced CML development remains uncertain.

To explore the function of SUMOylation in CML, we treated CML cells with SUMOylation inhibitors, 2-D08 and TAK-981. Treatments inhibited the proliferation and promoted the apoptosis of CML cells. Next, we constructed a mouse model by injecting K562 cells into NOD-SCID mice through the tail vein and treating them with 2-D08. The results showed that SUMOylation was essential for leukemia progression in vivo. Moreover, SUMO1 and SUMO2/3 modifications of BCR-ABL were identified in CML cells.

Liquid chromatography coupled with tandem mass spectrometry showed the potential BCR-ABL interactor, E3 SUMO ligase TRIM28. The association between TRIM28 and BCR-ABL was investigated by immunoprecipitation and confocal microscopy. To further investigate the function of TRIM28 in CML, we generated TRIM28-knockdown and TRIM28-knockout CML cells. TRIM28-knockdown and knockout both inhibited the proliferation and colony formation ability of CML cells.

Next, we determined whether TRIM28 regulated BCR-ABL protein level. We found that TRIM28 knockdown attenuated BCR-ABL protein expression but not mRNA expression. Cycloheximide chase assay showed that TRIM28 knockout promoted BCR-ABL protein degradation. BCR-ABL degradation induced by TRIM28 knockdown or knockout was reversed by autophagy-lysosome inhibitor, chloroquine, and 3-methyladenine (3-MA), but not by the proteasome inhibitor MG132. TRIM28 knockdown inhibited SUMO1 and SUMO2/3 modifications of BCR-ABL, while TRIM28 overexpression promoted SUMO1, SUMO2, and SUMO3 modifications of BCR-ABL. These results revealed that TRIM28 inhibited autophagic degradation by inducing BCR-ABL SUMOylation.

It is reported that p62 was essential for autophagy-lysosomal degradation of BCR-ABL. To elucidate whether TRIM28 impacted p62-mediated autophagic degradation by BCR-ABL SUMOylation, we performed immunoprecipitation and confocal microscopy assay. 2-D08 treatment or TRIM28 knockdown enhanced the interaction between BCR-ABL and p62 compared with the control group. To investigate whether TRIM28 inhibited BCR-ABL protein level to overcome TKI resistance, we used the imatinib-resistant cell line, K562/G01. Treatment with SUMOylation inhibitors, or knockdown TRIM28 expression, inhibited the proliferation of K562/G01 cells.

In conclusion, we demonstrate that upregulated TRIM28 facilitated the SUMOylation of BCR-ABL and subsequently inhibited p62-mediated autophagic degradation, resulting in the progression of CML. Our findings reveal a novel PTM regulating BCR-ABL protein expression and a new therapeutic method for CML.