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

Program: Oral and Poster Abstracts
Type: Oral
Session: 631. Chronic Myeloid Leukemia: Biology and Pathophysiology, excluding Therapy: CML Stem Cells And Their Environment
Hematology Disease Topics & Pathways:
Adult, Biological, Diseases, Therapies, CML, Technology and Procedures, enzyme inhibitors, Xenograft models, Study Population, Clinically relevant, Quality Improvement , Myeloid Malignancies, imaging, TKI, stem cells
Monday, December 3, 2018: 5:00 PM
Room 7B (San Diego Convention Center)

Katharina Rothe, PhD1,2*, Akie Watanabe3*, Donna L. Forrest, MD4, Sharon Gorski, PhD5,6*, Robert Young, PhD7* and Xiaoyan Jiang, MD-PhD1,8,9

1Department of Medical Genetics, University of British Columbia, Vancouver, BC, Canada
2BC Cancer, Terry Fox Laboratory, Vancouver, BC, Canada
3BC Cancer Terry Fox Laboratory, Vancouver, BC, Canada
4Leukemia/BMT Program of BC, BC Cancer, Vancouver, BC, Canada
5Genome Sciences Centre, BC Cancer, Vancouver, Canada
6Department of Molecular Biology and Biochemistry, Simon Fraser University, Burnaby, Canada
7Department of Chemistry, Simon Fraser University, Burnaby, Canada
8BC Cancer Research Centre, Vancouver, BC, Canada
9Department of Medicine, University of British Columbia, Vancouver, BC, Canada

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.

Disclosures: No relevant conflicts of interest to declare.

*signifies non-member of ASH