Susceptibility of AML to Chloroquine Therapy Is Independent of Autophagy
Abstract Introduction: autophagy is a self-recycling and "waste disposal" process that maintains cellular homeostasis. Recently, the autophagy mechanism has evolved as a therapeutic target in cancer treatment due to commonly seen high autophagy activity in cancer cells and its potential role in chemoresistance. To date, inhibition of autophagy has shown therapeutic benefits in several types of solid tumors. However, whether autophagy can be a potentially effective target in AML therapy remains unclear. Here we have used autophagy gene targeted mouse models and drug inhibitors to examine the potential benefits and limitations of autophagy targeting in the treatment of AML. Methods: MLL-AF9 (MA9) oncogene transduced Atg5f/fCreER and Atg5f/fMxCre Lin- mouse bone marrow cells were used for in vitro and in vivo experiments, respectively. Autophagy activity was determined by biochemical Western Blotting and immunofluorescent microscopy against LC3 and electron microscopy (EM). Deletion of FIP200, a gene that is indispensable for both canonical and alternative autophagy pathways, was carried out similarly to further confirm the effect of autophagy-specific targeting on MA9 leukemia. Chloroquine (CQ), a late stage autophagy and lysosome inhibitor, was used at 10μM to 25μM dosages in vitro. Combinatory effects of CQ with chemotherapy, including Doxorubicin (DA) and Cytarabine (AraC), were also analyzed. CQ was also administered to mice through i.p., at 50mg/Kg, bid X 4 days. Leukemia burden, cell survival and CBC counts were analyzed after drug treatment. Results: Primary and clonal MA9 leukemia cells showed a significantly higher level of autophagy flux than normal bone marrow cells. As expected, Atg5-/- MA9 cells showed defective LC3II formation and higher p62 accumulation upon CQ treatment. However, Atg5 deletion did not cause detectable defects in proliferation or survival, or altered ROS or mitochondria levels in leukemia cells. Surprisingly, Atg5-/- MA9 leukemia cells showed reduced sensitivity to either DA or AraC treatment. Transplantation experiments showed that Atg5 deletion in vivo did not reduce leukemia burden in the bone marrow or prolong the survival of the leukemic mice, although it decreased WBC counts in peripheral blood. When examined by EM, no obvious ultrastructural difference was observed between Atg5+/+ and Atg5-/- leukemia cells and both could form endolysosomes upon CQ blockage. Although FIP200 deletion in MA9 leukemia cells caused p62 accumulation at the basal state, similar functional effects were seen as in the case of Atg5 deletion. When compared to wild type controls, FIP200-/- MA9 leukemia cells did not show any proliferation or survival disadvantage, changes in ROS accumulation or mitochondrial level. FIP200 deletion also failed to sensitize leukemia cells to chemotherapy. Finally, CQ independently suppressed leukemia cell proliferation and induced apoptosis, but it did not distinguish Atg5+/+ vs Atg5-/- or FIP200+/+ vs FIP200-/- MA9 leukemia cells in sensitivity. CQ also showed a combinatory effect with DA or AraC in inhibiting MA9 cell proliferation. Treatment of MA9 leukemia xenograft mice with CQ greatly improved anemia in the mice (P<0.01), and we are currently examining the potential effects of CQ on survival of the leukemia bearing mice. Conclusions: MA9 leukemia cells contain a high basal autophagy activity. However, autophagy-specific targeting, either through FIP200-deletion which abolishes autophagy initiation, or via Atg5-deletion which prevents autophagosome membrane elongation, does not affect the survival and proliferation of MA9 leukemia cells nor does it prolong survival of MA9 leukemia xenograft mice. These results suggest that targeting autophagy alone will unlikely produce therapeutic benefit in AML. Loss of Atg5or FIP200 does not sensitize leukemia cells to chemotherapy, further reducing the potential value of targeting autophagy in a combinatory chemotherapy scheme. Mechanistically, neither Atg5 nor FIP200 is required for proper endolysosome-formation or lysosomal degradation in leukemia cells. While CQ displayed an apparent anti-leukemic effect, it acts through an autophagy independent pathway. CQ has a combinatory effect with conventional chemotherapy drugs and may be a useful treatment regimen for MA9 mediated and other types of AML. Disclosures No relevant conflicts of interest to declare.
