Page 188 - Haematologica - Vol. 105 n. 6 - June 2020

C. Bonolo de Campos et al.
HMCL. Further evaluation with the LC3-fluorescent pro- tein showed a reduction of the acid-sensitive green fluo- rescent protein and maintenance of the acid-insensitive red fluorescent protein in the representative resistant HMCL, EJM, when compared to the sensitive HMCL, JJN3, after 18 h of treatment with APY0201, indicating maturation of autophagosomes to autophagolysosomes in the resistant HMCL (Figure 3D, E).
Finally, we performed a cellular viability assay treating the two representative resistant HMCL, RPMI-8226 and EJM, with 1 nM and 2 nM of the autophagy inhibitor bafilomycin A1, respectively, showing synergistic anti-
MM activity (Figure 3F, G). In summary, these findings demonstrate the key role of autophagic flux disruption in MM cytotoxicity caused by PIKfyve inhibitors (Figure 4). Resistant HMCL maintain functional autophagy more efficiently, representing a mechanism of resistance to these drugs.
APY0201 sensitivity in vitro and ex vivo was predicted with the autophagy detection kit
Since anti-MM activity of APY0201 was associated with disruption of lysosome function and autophagy, we explored an autophagy detection assay, as a biomarker to
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Figure 3. APY0201 treatment leads to an increase in inactive precursors of lysosomal proteases and in autophagy organelles but decreased autophagic flux in sensitive human myeloma cell lines. Four human myeloma cell lines (HMCL) were treated with dimethylsulfoxide (DMSO) or 100 nM of APY0201. Cell lysates were harvested after 48 h of drug incubation and immunoblotting was performed to determine the levels of cathepsin A and D proteins (A) and Beclin-1, LC3 A/B, lyso- some-associated membrane protein 1 (Lamp-1), and sequestosome 1 (SQSTM1)/p62 protein levels (B). (C) The ration of SQSTM1:Beclin1 protein levels in two sen- sitive (KMS26 and JJN3) and two resistant (RPMI-8226 and EJM) HMCL following 48 h of incubation with APY0201. (D) LC3 acid-sensitive green fluorescent protein (GFP) and -insensitive red fluorescent protein (RFP) in representative sensitive and resistant HMCL (JJN3 and EJM, respectively) after 18 h of treatment with 100 nM of APY0201; images were obtained with a Zeiss LSM 800 confocal microscope (63X) and processed with Zen Blue software. (E) Mean LC3 RFP:GFP ratio in JJN3 and EJM. (F) Treatment of the RPMI-8226 resistant HMCL with APY0201 with and without 1 nM of bafilomycin A1 after 48 h. (G) Treatment of the EJM-resistant HMCL with APY0201 with and without 2 nM of bafilomycin A1 after 48 h.
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