ROLE OF THE MSC-DERIVED EXOSOMAL AND ENDOGENOUS JAK2-SET/PP2A-?-CATENIN-MODULATOR MIR-300 IN LEUKEMIC STEM/PROGENITOR PROLIFERATION AND SURVIVAL IN CML.
(Abstract release date: 05/19/16)
EHA Library. Perrotti D. 06/11/16; 135247; S491
Dr. Danilo Perrotti
Contributions
Contributions
Abstract
Abstract: S491
Type: Oral Presentation
Presentation during EHA21: On Saturday, June 11, 2016 from 16:00 - 16:15
Location: Hall C11
Background
Nanostring arrays and RNAseq analysis of total, CD34+, CD34+CD38- bone marrow (BM) cells from healthy individuals (n=6) and CML patients (n=16) showed gradual inhibition of miR-300 expression (CML-CPmiR-300>CML-BCmiR-300). MiR-300 is a human intergenic microRNA predicted to target multiple components of the BCR-ABL1/JAK2/hnRNPA1/SET/PP2A/β-catenin pathway that, as reported is essential for survival/self-renewal of leukemic progenitors and quiescent TKI-resistant Ph+ hematopoietic stem cells (HSCs).
Aims
This work is aimed at understanding the mechanisms regulating miR-300 in CML and assessing the importance of its downregulation for proliferation, survival and self-renewal of CML (CP and BC) cells.
Methods
Lentiviral-mediated modulation of miR-300 expression was used in CML cells and cell lines to assess effect of miR-300 on its mRNA targets, and on the clonogenic potential, LTC-IC, and number of quiescent leukemic stem and/or progenitor cells, and on the leukemogenic potential in animal models of CML.MSC-derived exosomes and conditioned medium, hypoxic/normoxic conditions and BCR-ABL1 inhibition were used to determine the mechanisms of regulation of miR-300 expression.
Results
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 >20-fold higher in CFSEMAX compared to dividing CMLCD34+ cells (n=4). Furthermore, it appears that forced miR-300 expression decreases engraftment of BCR-ABL+ cells (32D-BCR-ABL) in immunocompromised mice. Bone marrow transplant experiments in NSG mice with miR-300-transduced human CD34+ CML-BC cells, and with miR-300-transduced Lin- and LSK cells from SCL-tTA-BCR-ABL mice are ongoing and will allow us to determine the role of miR-300 on leukemic HSC engraftment and ability to propagate disease.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. Interestingly, culturing of leukemic cells in hypoxic conditions dramatically induced miR-300 expression that, in turn, impaired proliferation of primary CD34+ CML cells and BCR-ABL cell lines.
Conclusion
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.
Session topic: Chronic myeloid leukemia - Biology
Keyword(s): Chronic myeloid leukemia, Microenvironment, PP2A, Stem and progenitor cell
Type: Oral Presentation
Presentation during EHA21: On Saturday, June 11, 2016 from 16:00 - 16:15
Location: Hall C11
Background
Nanostring arrays and RNAseq analysis of total, CD34+, CD34+CD38- bone marrow (BM) cells from healthy individuals (n=6) and CML patients (n=16) showed gradual inhibition of miR-300 expression (CML-CPmiR-300>CML-BCmiR-300). MiR-300 is a human intergenic microRNA predicted to target multiple components of the BCR-ABL1/JAK2/hnRNPA1/SET/PP2A/β-catenin pathway that, as reported is essential for survival/self-renewal of leukemic progenitors and quiescent TKI-resistant Ph+ hematopoietic stem cells (HSCs).
Aims
This work is aimed at understanding the mechanisms regulating miR-300 in CML and assessing the importance of its downregulation for proliferation, survival and self-renewal of CML (CP and BC) cells.
Methods
Lentiviral-mediated modulation of miR-300 expression was used in CML cells and cell lines to assess effect of miR-300 on its mRNA targets, and on the clonogenic potential, LTC-IC, and number of quiescent leukemic stem and/or progenitor cells, and on the leukemogenic potential in animal models of CML.MSC-derived exosomes and conditioned medium, hypoxic/normoxic conditions and BCR-ABL1 inhibition were used to determine the mechanisms of regulation of miR-300 expression.
Results
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 >20-fold higher in CFSEMAX compared to dividing CMLCD34+ cells (n=4). Furthermore, it appears that forced miR-300 expression decreases engraftment of BCR-ABL+ cells (32D-BCR-ABL) in immunocompromised mice. Bone marrow transplant experiments in NSG mice with miR-300-transduced human CD34+ CML-BC cells, and with miR-300-transduced Lin- and LSK cells from SCL-tTA-BCR-ABL mice are ongoing and will allow us to determine the role of miR-300 on leukemic HSC engraftment and ability to propagate disease.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. Interestingly, culturing of leukemic cells in hypoxic conditions dramatically induced miR-300 expression that, in turn, impaired proliferation of primary CD34+ CML cells and BCR-ABL cell lines.
Conclusion
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.
Session topic: Chronic myeloid leukemia - Biology
Keyword(s): Chronic myeloid leukemia, Microenvironment, PP2A, Stem and progenitor cell
Abstract: S491
Type: Oral Presentation
Presentation during EHA21: On Saturday, June 11, 2016 from 16:00 - 16:15
Location: Hall C11
Background
Nanostring arrays and RNAseq analysis of total, CD34+, CD34+CD38- bone marrow (BM) cells from healthy individuals (n=6) and CML patients (n=16) showed gradual inhibition of miR-300 expression (CML-CPmiR-300>CML-BCmiR-300). MiR-300 is a human intergenic microRNA predicted to target multiple components of the BCR-ABL1/JAK2/hnRNPA1/SET/PP2A/β-catenin pathway that, as reported is essential for survival/self-renewal of leukemic progenitors and quiescent TKI-resistant Ph+ hematopoietic stem cells (HSCs).
Aims
This work is aimed at understanding the mechanisms regulating miR-300 in CML and assessing the importance of its downregulation for proliferation, survival and self-renewal of CML (CP and BC) cells.
Methods
Lentiviral-mediated modulation of miR-300 expression was used in CML cells and cell lines to assess effect of miR-300 on its mRNA targets, and on the clonogenic potential, LTC-IC, and number of quiescent leukemic stem and/or progenitor cells, and on the leukemogenic potential in animal models of CML.MSC-derived exosomes and conditioned medium, hypoxic/normoxic conditions and BCR-ABL1 inhibition were used to determine the mechanisms of regulation of miR-300 expression.
Results
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 >20-fold higher in CFSEMAX compared to dividing CMLCD34+ cells (n=4). Furthermore, it appears that forced miR-300 expression decreases engraftment of BCR-ABL+ cells (32D-BCR-ABL) in immunocompromised mice. Bone marrow transplant experiments in NSG mice with miR-300-transduced human CD34+ CML-BC cells, and with miR-300-transduced Lin- and LSK cells from SCL-tTA-BCR-ABL mice are ongoing and will allow us to determine the role of miR-300 on leukemic HSC engraftment and ability to propagate disease.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. Interestingly, culturing of leukemic cells in hypoxic conditions dramatically induced miR-300 expression that, in turn, impaired proliferation of primary CD34+ CML cells and BCR-ABL cell lines.
Conclusion
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.
Session topic: Chronic myeloid leukemia - Biology
Keyword(s): Chronic myeloid leukemia, Microenvironment, PP2A, Stem and progenitor cell
Type: Oral Presentation
Presentation during EHA21: On Saturday, June 11, 2016 from 16:00 - 16:15
Location: Hall C11
Background
Nanostring arrays and RNAseq analysis of total, CD34+, CD34+CD38- bone marrow (BM) cells from healthy individuals (n=6) and CML patients (n=16) showed gradual inhibition of miR-300 expression (CML-CPmiR-300>CML-BCmiR-300). MiR-300 is a human intergenic microRNA predicted to target multiple components of the BCR-ABL1/JAK2/hnRNPA1/SET/PP2A/β-catenin pathway that, as reported is essential for survival/self-renewal of leukemic progenitors and quiescent TKI-resistant Ph+ hematopoietic stem cells (HSCs).
Aims
This work is aimed at understanding the mechanisms regulating miR-300 in CML and assessing the importance of its downregulation for proliferation, survival and self-renewal of CML (CP and BC) cells.
Methods
Lentiviral-mediated modulation of miR-300 expression was used in CML cells and cell lines to assess effect of miR-300 on its mRNA targets, and on the clonogenic potential, LTC-IC, and number of quiescent leukemic stem and/or progenitor cells, and on the leukemogenic potential in animal models of CML.MSC-derived exosomes and conditioned medium, hypoxic/normoxic conditions and BCR-ABL1 inhibition were used to determine the mechanisms of regulation of miR-300 expression.
Results
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 >20-fold higher in CFSEMAX compared to dividing CMLCD34+ cells (n=4). Furthermore, it appears that forced miR-300 expression decreases engraftment of BCR-ABL+ cells (32D-BCR-ABL) in immunocompromised mice. Bone marrow transplant experiments in NSG mice with miR-300-transduced human CD34+ CML-BC cells, and with miR-300-transduced Lin- and LSK cells from SCL-tTA-BCR-ABL mice are ongoing and will allow us to determine the role of miR-300 on leukemic HSC engraftment and ability to propagate disease.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. Interestingly, culturing of leukemic cells in hypoxic conditions dramatically induced miR-300 expression that, in turn, impaired proliferation of primary CD34+ CML cells and BCR-ABL cell lines.
Conclusion
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.
Session topic: Chronic myeloid leukemia - Biology
Keyword(s): Chronic myeloid leukemia, Microenvironment, PP2A, Stem and progenitor cell
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