Abstract 951: Role of the MSC-derived exosomal and endogenous JAK2-SET/PP2A-β-catenin-modulator miR-300 in leukemic stem/progenitor proliferation and survival in CML
Authors: Trotta Rossana , Silvestri Giovannino , Stramucci Lorenzo , Ellis Justin J. , Yu Justine , Harb Jason J. , Neviani Paolo , Marcucci Guido , Srutova Klara , Machova Polakova K. , Roy Denis-Claude , Hokland Peter , Deininger Michael , Bhatia Ravi , Gambacorti-Passerini Carlo , Milojkovic Dragana , Reid Alistair , Apperley Jane , Livak Ferenc , Qi Jianfei , Baer Maria R. , Perrotti Danilo 
Published: July 2016

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&gt;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 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. Surprisingly, miR-300 levels were increased in CD34+CD38- compared to CD34+CD38+ CML cells, and &gt;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 β-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/β-catenin survival signals in CML progenitors. Conversely, increased miR-300 levels (endogenous and MSC-derived) seem to be required for HSC quiescence.

Citation Format: Rossana Trotta, Giovannino Silvestri, Lorenzo Stramucci, Justin J. Ellis, Justine Yu, Jason J. Harb, Paolo Neviani, Guido Marcucci, Klara Srutova, Polakova K. Machova, Denis-Claude Roy, Peter Hokland, Michael Deininger, Ravi Bhatia, Carlo Gambacorti-Passerini, Dragana Milojkovic, Alistair Reid, Jane Apperley, Ferenc Livak, Jianfei Qi, Maria R. Baer, Danilo Perrotti. Role of the MSC-derived exosomal and endogenous JAK2-SET/PP2A-β-catenin-modulator miR-300 in leukemic stem/progenitor proliferation and survival in CML. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 951.