Crizotinib Resistance Mediated by Autophagy Is Higher in the Stem-Like Cell Subset in ALK-Positive Anaplastic Large Cell Lymphoma, and This Effect Is MYC-Dependent

Abstract

Previously it was shown that autophagy contributes to crizotinib resistance in ALK-positive anaplastic large cell lymphoma (ALK + ALCL). We asked if autophagy is equally important in two distinct subsets of ALK + ALCL, namely Reporter Unresponsive (RU) and Reporter Responsive (RR), of which RR cells display stem-like properties. Autophagic flux was assessed with a fluorescence tagged LC3 reporter and immunoblots to detect endogenous LC3 alongside chloroquine, an autophagy inhibitor. The stem-like RR cells displayed significantly higher autophagic response upon crizotinib treatment. Their exaggerated autophagic response is cytoprotective against crizotinib, as inhibition of autophagy using chloroquine or shRNA against BECN1 or ATG7 led to a decrease in their viability. In contrast, autophagy inhibition in RU resulted in minimal changes. Since the differential protein expression of MYC is a regulator of the RU/RR dichotomy and is higher in RR cells, we asked if MYC regulates the autophagy-mediated cytoprotective effect. Inhibition of MYC in RR cells using shRNA significantly blunted crizotinib-induced autophagic response and effectively suppressed this cytoprotective effect. In conclusion, stem-like RR cells respond with rapid and intense autophagic flux which manifests with crizotinib resistance. For the first time, we have highlighted the direct role of MYC in regulating autophagy and its associated chemoresistance phenotype in ALK + ALCL stem-like cells.

Holistic View of ALK TKI Resistance in ALK-Positive Anaplastic Large Cell Lymphoma

“…Although there have been only a few clinical studies examining ALK TKI combinations in ALK+ ALCL, clinical trials are currently underway ( Table 2 ). ALK TKIs also cause adaptive changes in tumor cells that favor their survival such as the upregulation of Bcl-2 and inhibition of autophagy; therapeutic strategies that target these changes may be effective in the treatment of ALK+ ALCL ( 56 , 77 , 79 ).…”

“…Potentiating the effect of miR-7-5P using an miRNA mimic enhanced crizotinib-induced autophagic flux and cytotoxicity ( 76 ). Interestingly, in ALK+ ALCL stem cells, crizotinib more potently induced autophagy and enhanced tumor cell resistance to the drug, an effect mediated by MYC ( 77 ).…”

The Dual Role of Autophagy in Crizotinib-Treated ALK+ ALCL: From the Lymphoma Cells Drug Resistance to Their Demise

“…With this background, we tested if the different protein levels of MYC between RU and RR cells contributed to their difference in the autophagic flux triggered by crizotinib. This turned out to be the case [18]. Specifically, inhibition of MYC in RR cells significantly dampened the crizotinib-induced autophagic response and its cytoprotective effect.…”

“…This led to both the development of a new generation of TKI inhibitors [10,11] as well as a diverse range of combined therapies, in an attempt to preempt relapses and to eradicate the malignant cells [12,13]. In this context, and to improve crizotinib therapy, we investigated the role and regulation of macro-autophagy (hereafter referred to as autophagy) in crizotinib-treated ALK + ALCL [14][15][16][17][18].…”

“…Importantly, inhibition of autophagy using chloroquine significantly sensitized RR cells to crizotinib, suggesting that autophagy is directly responsible for the cytoprotection against crizotinib. Details of these experiments can be found in reference [18].…”