A New AHI-1-DNM2-BCR-ABL Complex Regulates Leukemic Properties of Primitive CML Cells through Mediation of Cellular Endocytosis and ROS-Induced Autophagy

Abstract Tyrosine kinase inhibitor (TKI) therapies have been introduced into clinical practice with remarkable effects on chronic phase CML. However, early relapses, acquired drug resistance, and persistence of leukemic stem cells (LSCs) remain problematic. Improved treatment approaches to specifically target key molecular elements active in CML LSCs are needed. One candidate is the adaptor protein AHI-1 (Abelson helper integration site-1), an oncogene that is highly deregulated in LSCs. An AHI-1-mediated protein complex containing BCR-ABL and JAK2 has been shown to modulate transforming activity and TKI-response/resistance of CML LSCs. We have recently identified the large GTPase Dynamin-2 (DNM2) as another AHI-1 interacting protein using the AHI-1 SH3 domain as protein "bait" in immunoprecipitation (IP)/mass spectrometry. DNM2 plays key roles in the regulation of trafficking processes such as endocytosis, and is activated through tyrosine phosphorylation. However, its role in CML pathogenesis is unknown. We have now demonstrated that transcript levels of DNM2 are significantly increased in treatment-naive CD34+ cells from CML patients who were classified retrospectively, after Imatinib (IM) therapy, as IM-responders (n=11) and IM-nonresponders (n=15) in comparison to CD34+ normal bone marrow cells (n=7, p=0.013 and 0.037). In particular, DNM2 is more highly expressed in CML stem-enriched cells (lin-CD34+CD38-) and progenitor cells (lin-CD34+CD38+) than more mature cells (lin+CD34-, 2-fold). Interestingly, BCR-ABL+ human cells with stable knockdown of DNM2 exhibited significantly reduced cell growth and increased apoptosis as compared to control cells. They also showed enhanced sensitivity to single TKIs or MitMAB (DNM2 inhibitor) and these effects were greatly enhanced by combination treatments (2-3 fold). Mechanistically, co-IP and confocal co-localization analysis with mutant forms of AHI-1 and DNM2 (HA-AHI-1, HA-AHI-1 SH3Δ, Myc-DNM2 and Myc-DNM2 PRDΔ) in 293T cells indicated that the PRD domain of DNM2 is mainly responsible for the interaction between the SH3 domain of AHI-1 and DMN2. More importantly, we identified a new protein interaction between DNM2 and BCR-ABL in both BCR-ABL and BCR-ABL/AHI-1 co-transduced hematopoietic cells using co-IP/Western analysis; this interaction is enhanced in BCR-ABL/AHI-1 co-transduced cells. Moreover, DNM2 phosphorylation was decreased upon IM treatment in BCR-ABL-transduced cells, but remained unchanged in control and T315I mutant cells, suggesting that DNM2 is a direct target of BCR-ABL. Interestingly, we further observed that AHI-1 co-localizes with EEA-1 (early endosome marker) and LAMP-1 (late endosome marker) in cells co-transfected with full-length AHI-1 and DNM2, but not in AHI-1 and DNM2 mutant cells. Using transferrin uptake assays, increased transferrin signals were observed in BCR-ABL/AHI-1 co-transduced cells compared to BCR-ABL- and BCR-ABL/AHI-1 SH3Δ-transduced cells, while transferrin signals were much lower in DNM2-knockdown cells than control cells. These results suggest that the AHI-1-DNM2-BCR-ABL complex indeed improves the kinetics and increases efficiency of endocytosis, which may contribute to response/resistance of primitive CML cells to TKIs. This is further supported by observation of increased surface expression levels of CXCR4 in DNM2 knockdown cells, a key endocytotic target and known mediator of TKI response in CML. Moreover, reactive oxygen species (ROS) signals were found to be higher in BCR-ABL/AHI-1 co-transduced cells than BCR-ABL- and BCR-ABL/AHI-1 SH3Δ-transduced cells, while ROS accumulation was significantly decreased in DNM2-knockdown cells compared to control cells. More interestingly, ROS-induced autophagy was further observed in BCR-ABL/AHI-1 co-transduced cells, but reduced in DNM-2-knockdown cells, with protein expression changes in key autophagy proteins, including ULK-1, Beclin-1, LC3 and p62. To the best of our knowledge, this is the first study to implicate a new AHI-1-DNM2-BCR-ABL complex in the deregulation of endocytosis signaling and ROS production/autophagy in CML. This may play a unique and important role in the regulation of cellular properties of primitive CML cells, including their response/resistance to TKI. Disclosures No relevant conflicts of interest to declare.

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