Contributions
Abstract: S1550
Type: Oral Presentation
Presentation during EHA23: On Sunday, June 17, 2018 from 08:00 - 08:15
Location: Room A2
Background
Inhibition of protein phosphatase 2A tumor suppressor is essential for chronic myelogenous leukemia stem cell maintenance, evolution and innate anti-cancer immunity. In CML, persistance of quiescent LSCs, disease development and inhibition of NK cell growth and anti-cancer activity depend on cell-autonomous and bone marrow generated signals.
Aims
miR-300 interconnects CSCs, microenvironment and immune cells.
Methods
Exosomes purification, Cell Cycle analysis, LTC-IC and CFC assays, CFSE-mediated tracking of cell division, apoptosis assays, real time RT-PCR.
Results
We show that expression profiling of CD34+ BM progenitors from healthy and CML patients CP and BC phase identified a number of miRs altered in CML and/or during BC transformation. We focused on miR-300 because it directly inhibited expression of multiple components of the PP2A inhibitory pathway and of other factors for LSC maintenance and disease progression, when ectopically expressed in CML but not in normal CD34+ cells. MiR-300 acted as a potent tumor suppressor CML CD34+ progenitors by inducing cell cycle exit and promoting spontaneous and (TKI)-induced apoptosis. In CD34+ CML-BC progenitors miR-300 downregulation required the BCR-ABL1 kinase-dependent inhibition of C/EBPb, which was capable of binding and transcriptionally activating miR-300 promoter. Conversely, low O2 levels and MSC-derived exosomes increased miR-300 expression in a TKI-insensitive manner to induce and/or preserve LSC quiescence and suppress NK cell growth. Indeed, lentiviral-mediated miR-300 inhibition in MSCs significantly prevented the MSC growth inhibitory activity on CD34+ CML-BC and NK cells. Accordingly, miR-300 levels were found higher in CD56+CD3- NK cells from CML patients at diagnosis compared to healthy individuals. Induction/maintenance of high miR-300 levels in LSCs required the hypoxia-induced inhibition of BCR-ABL1 activity and induction of C/EBPb expression/activity. LSCs escaped from the miR-300 apoptotic activity through an autocrine/paracrine mechanism that required the release by CD34+ CML stem/progenitors of TGFb1 that, in turn, induced the expression of TUG1, a lncRNA acting as a miR-300 sponge. In fact, shRNA-mediated TUG1 suppression or Ab-dependent TGFb1 inhibition decreased the quiescent CD34+ LSC number. By contrast, miR-300 inhibition did not alter LSC survival and self-renewal, further supporting a role for TUG1 as a miR-300 sponge. Accordingly, TUG1 was markedly induced in CFSEMAX but not dividing CD34+ CML cells. In fact, low ectopic miR-300 expression induced growth arrest without affecting the number of CFSEMAX LSCs. High doses of miR-300 but not scramble oligonucleotides impaired LSC survival and self-renewal, induced a marked killing of quiescent LSCs and dividing CD34+ CML progenitors, and impaired CML-BC engraftment in NRG-SGM3 mice.
Conclusion
In Summary, loss of miR-300 expression is essential for survival/proliferation of leukemic progenitors and, therefore, increased miR-300 expression is necessary for reduced NK cell number/activity and maintenance of the LSC reservoir. Induction of TUG1 may occur to preserve LSC survival after migration into the BM endosteal niche where quiescence is induced by MSCs and low O2 levels through marked induction of intracellular miR-300 levels. Thus, disrupting the miR-300/TUG1 balance may represent a potential therapeutic approach for treatment/eradication of LSC-derived leukemias and restoration of innate immunity.
Session topic: 7. Chronic myeloid leukemia – Biology & Translational Research
Keyword(s): CML blast crisis, Microenvironment, PP2A, Stem and progenitor cell
Abstract: S1550
Type: Oral Presentation
Presentation during EHA23: On Sunday, June 17, 2018 from 08:00 - 08:15
Location: Room A2
Background
Inhibition of protein phosphatase 2A tumor suppressor is essential for chronic myelogenous leukemia stem cell maintenance, evolution and innate anti-cancer immunity. In CML, persistance of quiescent LSCs, disease development and inhibition of NK cell growth and anti-cancer activity depend on cell-autonomous and bone marrow generated signals.
Aims
miR-300 interconnects CSCs, microenvironment and immune cells.
Methods
Exosomes purification, Cell Cycle analysis, LTC-IC and CFC assays, CFSE-mediated tracking of cell division, apoptosis assays, real time RT-PCR.
Results
We show that expression profiling of CD34+ BM progenitors from healthy and CML patients CP and BC phase identified a number of miRs altered in CML and/or during BC transformation. We focused on miR-300 because it directly inhibited expression of multiple components of the PP2A inhibitory pathway and of other factors for LSC maintenance and disease progression, when ectopically expressed in CML but not in normal CD34+ cells. MiR-300 acted as a potent tumor suppressor CML CD34+ progenitors by inducing cell cycle exit and promoting spontaneous and (TKI)-induced apoptosis. In CD34+ CML-BC progenitors miR-300 downregulation required the BCR-ABL1 kinase-dependent inhibition of C/EBPb, which was capable of binding and transcriptionally activating miR-300 promoter. Conversely, low O2 levels and MSC-derived exosomes increased miR-300 expression in a TKI-insensitive manner to induce and/or preserve LSC quiescence and suppress NK cell growth. Indeed, lentiviral-mediated miR-300 inhibition in MSCs significantly prevented the MSC growth inhibitory activity on CD34+ CML-BC and NK cells. Accordingly, miR-300 levels were found higher in CD56+CD3- NK cells from CML patients at diagnosis compared to healthy individuals. Induction/maintenance of high miR-300 levels in LSCs required the hypoxia-induced inhibition of BCR-ABL1 activity and induction of C/EBPb expression/activity. LSCs escaped from the miR-300 apoptotic activity through an autocrine/paracrine mechanism that required the release by CD34+ CML stem/progenitors of TGFb1 that, in turn, induced the expression of TUG1, a lncRNA acting as a miR-300 sponge. In fact, shRNA-mediated TUG1 suppression or Ab-dependent TGFb1 inhibition decreased the quiescent CD34+ LSC number. By contrast, miR-300 inhibition did not alter LSC survival and self-renewal, further supporting a role for TUG1 as a miR-300 sponge. Accordingly, TUG1 was markedly induced in CFSEMAX but not dividing CD34+ CML cells. In fact, low ectopic miR-300 expression induced growth arrest without affecting the number of CFSEMAX LSCs. High doses of miR-300 but not scramble oligonucleotides impaired LSC survival and self-renewal, induced a marked killing of quiescent LSCs and dividing CD34+ CML progenitors, and impaired CML-BC engraftment in NRG-SGM3 mice.
Conclusion
In Summary, loss of miR-300 expression is essential for survival/proliferation of leukemic progenitors and, therefore, increased miR-300 expression is necessary for reduced NK cell number/activity and maintenance of the LSC reservoir. Induction of TUG1 may occur to preserve LSC survival after migration into the BM endosteal niche where quiescence is induced by MSCs and low O2 levels through marked induction of intracellular miR-300 levels. Thus, disrupting the miR-300/TUG1 balance may represent a potential therapeutic approach for treatment/eradication of LSC-derived leukemias and restoration of innate immunity.
Session topic: 7. Chronic myeloid leukemia – Biology & Translational Research
Keyword(s): CML blast crisis, Microenvironment, PP2A, Stem and progenitor cell