Inhibiting the Core Autophagy Enzyme ATG4B with Novel Drugs Sensitizes Resistant Leukemic Stem/Progenitor Cells to Standard Targeted Therapy

Leukemic stem cell (LSC) persistence is a major cause of therapy failure and relapse in patients, thus warranting further investigations into the cells' molecular properties to aid in meaningful clinical decisions. Chronic myeloid leukemia (CML) is a clear example and excellent study model with rare, propagating LSCs that are not eradicated by BCR-ABL1-directed tyrosine kinase inhibitor (TKI) monotherapy such as Imatinib Mesylate (IM), since the LSCs do not exclusively rely on BCR-ABL1 for their survival.

We and others have shown that the persistence of primitive leukemic cells is mediated by macroautophagy (autophagy), a catabolic cellular recycling process. In particular, we discovered that transcript and protein expressions of all ATG4 family members, including ATG4A, B, C, and D, are significantly increased in CD34⁺ CML cells compared to CD34⁺ normal bone marrow (BM) cells. ATG4B expression is also significantly higher in pre-treatment CD34⁺ CML cells from IM-nonresponders vs. IM-responders, including LSCs. Moreover, we revealed that stable suppression of ATG4B significantly suppressed autophagy, impaired survival of IM-nonresponder stem/progenitor cells and sensitized leukemic cells to TKI treatment. Thus, we identified ATG4B as a critical therapeutic target in CML (Rothe et al., Blood, 2014).

To further explore whether targeting of ATG4B would be a viable strategy in the successful treatment of various leukemia, two lead ATG4B inhibitors were recently developed for pre-clinical proof-of-concept studies. Compound 4-28, a styrylquinoline, was identified by in silico screening and high content cell-based screening. Its structure-activity relationship (SAR)-based optimization led to a more stable and potent compound, LV-320. LV-320 was further evaluated by Microscale Thermophoresis and showed consistent Kd values for binding to the ATG4B enzyme (Kd=16±1 μM). LV-320 can inhibit autophagic flux, shows excellent tolerability and a good PK profile. It is also characterised as a non-competitive inhibitor of ATG4B and displays similar potency against ATG4A. Interestingly, inhibition of ATG4B with compound 4-28 decreased viability by 40-60% and increased apoptosis by 30-40% in different BCR-ABL1⁺ leukemic cell lines upon serum-deprivation as compared to the same cells without treatment of 4-28 (p<0.05). 4-28 treatment also efficiently inhibited autophagic flux in these cells as shown by Western blot analysis of LC3-II/I and p62 accumulation. In addition, compound 4-28 was able to inhibit the clonal growth of patient-derived CML stem/progenitor cells from IM-nonresponders, in particular when combined with various TKIs compared to single agents (20 vs. 50%, p<0.05). However, this combination approach also showed slight toxic effects on healthy BM cells.

We then tested the more stable and potent compound LV-320 with superior results. Treatment of several drug-resistant and mutated CML and aggressive BCR-ABL1⁺ B-ALL cell lines with LV-320 increased apoptosis up to 90% compared to controls and reached almost 100% when combined with various TKIs (p<0.05). Moreover, LV-320 sensitized IM-nonresponder stem and progenitor cells to TKIs in colony-forming short-term and long-term cell assays as compared to single agents (10 % vs. 40 %, p<0.05), but was not toxic to primitive BM cells from healthy donors (up to 10µM). Mechanistically, we found that LV-320 effectively inhibited autophagic flux in leukemic cells: Confocal microscopy demonstrated an increase in LC3-II-positive punctae (autophagosomes) and the presence of yellow punctae (blocked autophagosome-lysosome fusion) when leukemic cells where transduced with a mRFP-GFP-LC3 construct and treated with LV-320 or a combination of LV-320 and TKIs. Drug interaction analysis further indicated synergy between LV-320 plus IM (CI value ≤ 0.9). A novel in vivo model is currently being investigated to validate our proof-of-concept. Together, our results suggest that targeting of ATG4B with novel autophagy inhibitors in combination with TKIs may be able to circumvent drug resistance in CML and possibly other aggressive leukemia.