Autophagic modulation of DNA damage signalling as a therapeutic avenue in leukemia

Acute Myeloid Leukemia (AML) is the most frequent adult leukemia characterised by a poor overall survival rate of just over 30%. Elderly patients in particular have poor treatment outcomes stimulating investigation into finding alternative treatment strategies. Leukemic stem cells (LSC) in the bone marrow microenvironment, alike hematopoietic stem cells (HSC), drive AML initiation, maintenance, and relapse and are protected through autophagy. Autophagy is a lysosomal degradation mechanism that recycles damaged organelles and protein aggregates to maintain genomic stability and hematopoiesis. Modulation of autophagy in principle could make leukemia cells especially the LSC more amenable to cell death in conjunction with more established chemotherapeutics. Building on previous work identifying biomarkers for PARP inhibitors, this study aims to understand the role of modulated autophagy and DNA repair in AML. Rapamycin (autophagy inducer) and Chloroquine (autophagy inhibition) were used to modulate autophagy in AML and normal fibroblasts (IMR90) and assessed the Base Excision Repair (BER), Homologous Recombination (HR) and Non-Homologous End Joining (NHEJ) repair using in vitro assays. The competitive ELISA study revealed increased BER activity upon autophagy induction and decreased activity with autophagy inhibition. IMR90 cells showed lower corrected 8OHdG concentrations (treated and untreated with H2O2) compared to Kasumi1, NB4, and U937, indicating greater H2O2 susceptibility or compromised BER in AML cell lines. H2O2-treated samples had higher 8OHdG levels (~3.0 ng/mL) with significant differences: IMR90 vs. Kasumi1 (p ≤ 0.05), U937 (p ≤ 0.01), and NB4 (p ≤ 0.001). Rapamycin treatment reduced corrected 8OHdG in AML cells (~2.0 ng/mL, p ≤ 0.001) and in IMR90 (~3.0 ng/mL, p ≤ 0.01), highlighting autophagy's impact on BER activity suggesting AML cell lines are either more susceptible to H2O2-induced damage or exhibit more compromised BER efficiency compared to IMR90 cells. A qPCR analysis of rapamycin-induced autophagy revealed elevated HR activity in both AML and IMR90 cell lines. Among these, Kasumi1 showed the most pronounced increase, with a 5% elevation in HR activity (p ≤ 0.05), followed by U937 at 4% (p ≤ 0.05) and NB4 at 1% (p ≤ 0.0001), compared to untreated controls. Cytotoxicity assays further demonstrated heightened sensitivity of leukemia cells to autophagy inhibitors, either alone or in combination with PARP inhibitors and antimetabolites. Increased cell death specifically, chloroquine-mediated autophagy inhibition profoundly enhanced the cytotoxic effects of Olaparib and Gemcitabine in AML cell lines. Kasumi1 cells exhibited the greatest sensitivity, with % Relative Survival dropping to 17% when treated with 0.5µM Olaparib combined with 75 µM chloroquine. Similarly, U937 cells showed reduced survival rates of 49% and 44% at 0.1µM and 0.5µM Olaparib, respectively, under autophagy-inhibited conditions. In contrast, IMR90 fibroblasts displayed greater resilience, maintaining 83% relative survival at 1µM Olaparib and 25µM chloroquine, underscoring the selective vulnerability of AML cells. In conclusion, this study highlights a promising therapeutic strategy by combining the synthetic lethality approach of PARP inhibitors and antimetabolites with autophagy modulation. These findings provide a compelling foundation for further exploration of this combined therapeutic approach for treating AML.