Role of the MSC-Derived Exosomal and Endogenous JAK2-SET/PP2A-Beta Catenin-Modulator Mir-300 in Leukemic Stem/Progenitor Proliferation and Survival in CML
Authors: Silvestri Giovannino , Stramucci Lorenzo , Ellis Justin , Yu Justine , Harb Jason , Neviani Paolo , Marcucci Guido , Srutova Klara , Machova Polakova Katerina , Roy Denis-Claude , Hokland Peter , Deininger Michael W. , Bhatia Ravi , Gambacorti-Passerini Carlo , Milojkovic Dragana , Reid Alistair G , Apperley Jane F. , Livak Ferenc , Baer Maria R. , Trotta Rossana , Perrotti Danilo 
Published: October 2019

Abstract
MiR-300 is a microRNA predicted to target multiple components of the BCR-ABL1 / JAK2 / hnRNPA1 / SET / PP2A / β-catenin pathway, which is essential for survival/self-renewal of leukemic progenitors and quiescent TKI-resistant Ph+ hematopoietic stem cells (HSCs). Nanostring arrays analysis of bone marrow (BM) cells from healthy individuals (n=5) and CML patients (n=10) showed gradual inhibition of miR-300 expression (CML-CPmiR-300>CML-BCmiR-300).
MiR-300 transduction in CMLCD34+ cells and BCR-ABL1+ cell lines decreased JAK2, β-catenin, hnRNPA1 and SET expression and increased PP2A activity. Targets were confirmed by miR-300 expression in BCR-ABL1+ cells expressing Flag-tagged miR-300-targets lacking or carrying a wild-type or mutated 3'UTR. Restored miR-300 expression in CMLCD34+ cells and/or BCR-ABL1+ cell lines impaired cell cycle progression, proliferation and clonogenic potential, markedly reduced LTC-ICs, and increased TKI sensitivity. Notably, miR-300 expression was inhibited by BCR-ABL1 in proliferating cells. Accordingly, imatinib restored miR-300 expression in CD34+ dividing progenitors and BCR-ABL1+ cell lines without altering miR-300 levels in quiescent (CFSEMAX) CMLCD34+ cells (n=3), consistent with the BCR-ABL1 kinase-dependent activation of the Jak2/SET/PP2A/β-catenin pathway in CML progenitors but not quiescent Ph+ HSCs. Indeed, ectopic SET expression counteracted the negative effects of mir-300 on cell proliferation and survival. Surprisingly, miR-300 levels were increased in CD34+CD38- compared to CD34+CD38+ CML cells, and >20-fold higher in CFSEMAX compared to dividing CMLCD34+ cells (n=4).
To determine whether enhanced miR-300 expression in quiescent cells depends on cell autonomous events or is induced by the BM microenvironment, we exposed BCR-ABL+ cells to conditioned medium (CM) of HS-5 or hTERT mesenchymal stem cells (MSC). CM strongly decreased proliferation, induced imatinib but not FTY720 (PP2A activator) resistance, increased miR-300 levels, decreased BCR-ABL1 activity and Jak2 expression but not its activity, and did not alter b-catenin levels or PP2A activity. Interestingly, miR-300 was found in MSC-derived exosomes, and its expression increased in BCR-ABL1+ cells exposed to exosomes. Accordingly, proliferation of CML-BCCD34+ and LAMA-84 cells was strongly reduced upon exposure to MSC-derived exosomes. These effects were abolished when we used CM from MSCs transduced with a miR-300 antagomir.
Altogether our results indicate that downregulation of miR-300 appears necessary for the activation of JAK2/SET/PP2A/b-catenin survival signals in CML progenitors. Conversely, increased miR-300 levels (endogenous and MSC-derived) seem to be required for HSC quiescence.

Disclosures
Deininger: Novartis: Other: Consulting or Advisory Role, Research Funding; BMS: Other: Consulting & Advisory Role, Research Funding; Celgene: Research Funding; Genzyme: Research Funding; Gilead: Research Funding; ARIAD Pharmaceutical Inc.: Other: Consulting or Advisory Role; Incyte: Other: Consulting or Advisory Role; Pfizer: Other: Consulting or Advisory Role. Milojkovic:BMS: Honoraria; ARIAD Pharmaceuticals Inc.: Honoraria; Novartis: Honoraria; Pfizer: Honoraria.