Targeting quiescent leukemic stem cells using second generation autophagy inhibitors

Pablo Baquero,Mary T Scott,Leon O Murphy,Rebecca Mitchell,Tessa L Holyoake,Christina Halsey,O Porta Olivares ,Michael C Nicastri,Kirsty Dunn ,Alison M Michie,Jeffrey D Winkler,Kevin M Ryan,Amy Dawson,Arunima Mukhopadhyay,Erin P Keaney,Elodie M Kuntz,Ravi K Amaravadi,Angela Ianniciello,Eyal Gottlieb,G Vignir Helgason

doi:10.1038/s41375-018-0252-4

Abstract

In chronic myeloid leukemia (CML), tyrosine kinase inhibitor (TKI) treatment induces autophagy that promotes survival and TKI-resistance in leukemic stem cells (LSCs). In clinical studies hydroxychloroquine (HCQ), the only clinically approved autophagy inhibitor, does not consistently inhibit autophagy in cancer patients, so more potent autophagy inhibitors are needed. We generated a murine model of CML in which autophagic flux can be measured in bone marrow-located LSCs. In parallel, we use cell division tracing, phenotyping of primary CML cells, and a robust xenotransplantation model of human CML, to investigate the effect of Lys05, a highly potent lysosomotropic agent, and PIK-III, a selective inhibitor of VPS34, on the survival and function of LSCs. We demonstrate that long-term haematopoietic stem cells (LT-HSCs: Lin−Sca-1+c-kit+CD48−CD150+) isolated from leukemic mice have higher basal autophagy levels compared with non-leukemic LT-HSCs and more mature leukemic cells. Additionally, we present that while HCQ is ineffective, Lys05-mediated autophagy inhibition reduces LSCs quiescence and drives myeloid cell expansion. Furthermore, Lys05 and PIK-III reduced the number of primary CML LSCs and target xenografted LSCs when used in combination with TKI treatment, providing a strong rationale for clinical use of second generation autophagy inhibitors as a novel treatment for CML patients with LSC persistence.

Highlights

Electronic supplementary material The online version of this article contains supplementary material, which is available to authorized users.Chronic myeloid leukemia (CML) arises following a reciprocal chromosomal translocation within a haematopoietic stem cell (HSC) leading to expression of the fusion oncoprotein BCR-ABL
In line with previous studies performed in normal haematopoietic cells, autophagy induction was linked to increased degradation of GFP-light chain 3 (LC3) and a reduction in GFP-LC3 levels [25], which was reverted by HCQ-mediated autophagy inhibition (Figure S1D-F)
We use a double transgenic model of CML, where BCR-ABL expression is induced in stem cells [21], and demonstrate that Lys05 treatment consistently inhibits autophagy in CML leukemic stem cells (LSCs) in vivo, while HCQ approximating clinically achievable concentrations fails to do so

Summary

Introduction

Despite the significant increase in life expectancy of CML patients due to the development of BCR-ABL-targeting tyrosine kinase inhibitors (TKIs) [1], a quarter of patients will fail TKI therapy due to BCR-ABL kinase mutations, alternative oncogene activation, or because of progression to accelerated phase or blast crisis [2]. Leukemic stem cells (LSCs) are insensitive to TKIs [3, 4], giving rise to disease persistence and reducing the possibility of successful treatment-free remission (TFR) to only 10– 20% [5]. This figure may rise with second generation TKIs, the majority of CML patients will require lifelong TKI therapy. A key aim is to identify critical survival mechanisms in LSCs, such that LSC-targeting interventions can be developed, increasing the proportion of patients that achieve sustained deep molecular responses and TFR

Methods

Results

Conclusion