Leukemia Supplements (2014) 3, S1–S2 © 2014 Macmillan Publishers Limited All rights reserved 2044-5229/14 www.nature.com/leusup
SPECIAL REPORT
Altered microenvironmental regulation of CML stem cells R Bhatia Leukemia Supplements (2014) 3, S1–S2; doi:10.1038/leusup.2014.1 Keywords: TKI resistance; CML stem cells; LSC heterogeneity; BM microenvironment
Despite the undeniable success of tyrosine kinase inhibitors (TKI) in treatment of Bcr-Abl-dependent leukemia, the issue of BcrAbl-positive stem cells that persist in the blood of patients with major molecular remission or complete molecular remission remains unresolved. Our data indicate that the frequency of BCRABL+CD34+CD38 − cells was 1–2% in the blood of patients in remission.1 This is with agreement with clinical data showing that ~ 60% of patients relapse after TKI discontinuation.2 A subset of patients will achieve deep molecular remission, and in these patients TKI treatment will be discontinued. Forty to fifty percent of these patients will maintain treatment-free remission; however, BCR-ABL+ cells are still detectable.3 We believe that heterogeneity of leukemia stem cell (LSC) potential and immune/microenvironmental regulation allow these cells to be present even without the recurrence of leukemia. Co-culture with BM mesenchymal cells (MSC) enhances survival and maintenance of CML stem cells as assessed by in vitro analysis of CML CD34+CD38 − cells or by engraftment of CML CD34+ cells in NSG mice.4 We found that the N-cadherin receptor plays an important role in MSC-mediated protection of CML stem cells from TKI. N-cadherin-mediated adhesion to MSC was associated with increased cytoplasmic N-cadherin–β-catenin complex formation, as well as enhanced β-catenin nuclear translocation and transcriptional activity. Increased exogenous Wnt-mediated β-catenin signaling played an important role in MSC-mediated protection of CML progenitors from TKI treatment. These results reveal novel mechanisms of CML LSC resistance to TKI treatment, and suggest new targets for treatment. We have also used an SCL-tTA /TRE-BCR-ABL transgenic mouse model based on targeted Bcr-Abl expression in murine hematopoietic stem cells (HSC) via a Tet-regulated SCL promoter to study CML stem cell regulation and targeting. In this model, withdrawal of tetracycline leads to BCR-ABL expression in murine HSC and development of a CML-like disorder with leukocytosis and splenomegaly.5 We have used this model to characterize the main features of the LSC. Long-term hematopoietic stem cell (LT-HSC) numbers were markedly reduced in the BM of BCR-ABL mice, but were increased in the spleen compared with controls. Long-term engrafting and leukemia-initiating capacity is restricted to BCR-ABL cells with LT-HSC phenotype. Cell cycling of BCR-ABL LT-HSC was increased compared to control mice. We observed that both reduced homing to the BM and enhanced egress from the BM to the spleen contribute to altered LT-HSC localization in BCR-ABL mice. However, we also found that there are altered chemokine and cytokine levels in the BM of BCR-ABL mice compared to control mice, and we confirmed that altered chemokine and cytokine expression in CML BM may persist after TKI treatment.6 CML LT-HSC demonstrated reduced homing and
retention in the bone marrow (BM), related to decreased CXCL12 expression in CML BM, resulting from increased G-CSF production by leukemia cells. In transplantation experiments, we observed that CML stem cells had a competitive advantage in the CML BM microenvironment in comparison to normal LT-HSC. Leukemia-induced abnormalities in cytokine expression selectively suppressed normal stem cell growth and enhanced LSC growth in CML BM. This observation was confirmed in an in vitro setting, where CML stem cells had a growth advantage over the normal HSC upon growth in CML versus normal conditioned medium. Our data indicated that IL-1α, IL-1β, TNF-α, MIP-1α and MIP-1β contributed to enhanced growth of leukemic compared to normal stem cells. We are currently assessing whether inhibition of the major pro-inflammatory cytokine IL-1 can lead to reduced leukemia development and prolonged survival of CML mice, as well as target primary CML CD34+ CD38 − CD90+ cells. Our data show that maintenance of HSC depends on interaction with their niche, and these interactions become abnormal in the pathophysiology of leukemia and contribute to persistence of leukemic stem cells in the niche. Interestingly, in limiting dilution transplantation experiments, we have observed that Bcr-Abl+ LT-HSC have a heterogeneous leukemogenic capacity. In gene expression profiling we identified MPL as one of the genes that are differentially expressed in LT-HSC isolated from leukemic and non-leukemic mice. We are currently assessing the role of MPL in the leukemogenic potential of Bcr-Abl+ LT-HSC, and as a potential target for LSC-directed therapeutics. CONFLICT OF INTEREST The author declares no conflict of interest.
ACKNOWLEDGEMENTS The symposium and publication of this supplement were sponsored by the Division of Hematology/Oncology at the Warren Alpert Medical School of Brown University and NIH Center of Biomedical Research Excellence (COBRE) for Stem Cells Biology at Rhode Island Hospital.
REFERENCES 1 Chu S, McDonald T, Lin A, Chakraborty S, Huang Q, Snyder DS et al. Persistence of leukemia stem cells in chronic myelogenous leukemia patients in prolonged remission with imatinib treatment. Blood 2011; 118: 5565–5572. 2 Mahon FX, Rea D, Guilhot J, Guilhot F, Huguet F, Nicolini F et al. Discontinuation of imatinib in patients with chronic myeloid leukaemia who have maintained complete molecular remission for at least 2 years: the prospective, multicentre Stop Imatinib (STIM) trial. Lancet Oncol 2010; 11: 1029–1035.
Division of Hematopoietic Stem Cell and Leukemia Research, City of Hope National Medical Center, Duarte, CA, USA. Correspondence: Dr R Bhatia, Division of Hematopoietic Stem Cell and Leukemia Research, City of Hope National Medical Center, Duarte, CA 91010, USA. E-mail: [email protected]
Microenvironmental regulation of CML cells R Bhatia
S2 3 Ross DM, Branford S, Seymour JF, Schwarer AP, Arthur C, Yeung DT et al. Safety and efficacy of imatinib cessation for CML patients with stable undetectable minimal residual disease: results from the TWISTER study. Blood 2013; 122: 515–522. 4 Zhang B, Li M, McDonald T, Holyoake TL, Moon RT, Campana D et al. Microenvironmental protection of CML stem and progenitor cells from tyrosine kinase inhibitors through N-cadherin and Wnt-beta-catenin signaling. Blood 2013; 121: 1824–1838.
Leukemia Supplements
5 Koschmieder S, Gottgens B, Zhang P, Iwasaki-Arai J, Akashi K, Kutok JL et al. Inducible chronic phase of myeloid leukemia with expansion of hematopoietic stem cells in a transgenic model of BCR-ABL leukemogenesis. Blood 2005; 105: 324–334. 6 Zhang B, Ho YW, Huang Q, Maeda T, Lin A, Lee SU et al. Altered microenvironmental regulation of leukemic and normal stem cells in chronic myelogenous leukemia. Cancer Cell 2012; 21: 577–592.
SPECIAL REPORT
Altered microenvironmental regulation of CML stem cells R Bhatia Leukemia Supplements (2014) 3, S1–S2; doi:10.1038/leusup.2014.1 Keywords: TKI resistance; CML stem cells; LSC heterogeneity; BM microenvironment
Despite the undeniable success of tyrosine kinase inhibitors (TKI) in treatment of Bcr-Abl-dependent leukemia, the issue of BcrAbl-positive stem cells that persist in the blood of patients with major molecular remission or complete molecular remission remains unresolved. Our data indicate that the frequency of BCRABL+CD34+CD38 − cells was 1–2% in the blood of patients in remission.1 This is with agreement with clinical data showing that ~ 60% of patients relapse after TKI discontinuation.2 A subset of patients will achieve deep molecular remission, and in these patients TKI treatment will be discontinued. Forty to fifty percent of these patients will maintain treatment-free remission; however, BCR-ABL+ cells are still detectable.3 We believe that heterogeneity of leukemia stem cell (LSC) potential and immune/microenvironmental regulation allow these cells to be present even without the recurrence of leukemia. Co-culture with BM mesenchymal cells (MSC) enhances survival and maintenance of CML stem cells as assessed by in vitro analysis of CML CD34+CD38 − cells or by engraftment of CML CD34+ cells in NSG mice.4 We found that the N-cadherin receptor plays an important role in MSC-mediated protection of CML stem cells from TKI. N-cadherin-mediated adhesion to MSC was associated with increased cytoplasmic N-cadherin–β-catenin complex formation, as well as enhanced β-catenin nuclear translocation and transcriptional activity. Increased exogenous Wnt-mediated β-catenin signaling played an important role in MSC-mediated protection of CML progenitors from TKI treatment. These results reveal novel mechanisms of CML LSC resistance to TKI treatment, and suggest new targets for treatment. We have also used an SCL-tTA /TRE-BCR-ABL transgenic mouse model based on targeted Bcr-Abl expression in murine hematopoietic stem cells (HSC) via a Tet-regulated SCL promoter to study CML stem cell regulation and targeting. In this model, withdrawal of tetracycline leads to BCR-ABL expression in murine HSC and development of a CML-like disorder with leukocytosis and splenomegaly.5 We have used this model to characterize the main features of the LSC. Long-term hematopoietic stem cell (LT-HSC) numbers were markedly reduced in the BM of BCR-ABL mice, but were increased in the spleen compared with controls. Long-term engrafting and leukemia-initiating capacity is restricted to BCR-ABL cells with LT-HSC phenotype. Cell cycling of BCR-ABL LT-HSC was increased compared to control mice. We observed that both reduced homing to the BM and enhanced egress from the BM to the spleen contribute to altered LT-HSC localization in BCR-ABL mice. However, we also found that there are altered chemokine and cytokine levels in the BM of BCR-ABL mice compared to control mice, and we confirmed that altered chemokine and cytokine expression in CML BM may persist after TKI treatment.6 CML LT-HSC demonstrated reduced homing and
retention in the bone marrow (BM), related to decreased CXCL12 expression in CML BM, resulting from increased G-CSF production by leukemia cells. In transplantation experiments, we observed that CML stem cells had a competitive advantage in the CML BM microenvironment in comparison to normal LT-HSC. Leukemia-induced abnormalities in cytokine expression selectively suppressed normal stem cell growth and enhanced LSC growth in CML BM. This observation was confirmed in an in vitro setting, where CML stem cells had a growth advantage over the normal HSC upon growth in CML versus normal conditioned medium. Our data indicated that IL-1α, IL-1β, TNF-α, MIP-1α and MIP-1β contributed to enhanced growth of leukemic compared to normal stem cells. We are currently assessing whether inhibition of the major pro-inflammatory cytokine IL-1 can lead to reduced leukemia development and prolonged survival of CML mice, as well as target primary CML CD34+ CD38 − CD90+ cells. Our data show that maintenance of HSC depends on interaction with their niche, and these interactions become abnormal in the pathophysiology of leukemia and contribute to persistence of leukemic stem cells in the niche. Interestingly, in limiting dilution transplantation experiments, we have observed that Bcr-Abl+ LT-HSC have a heterogeneous leukemogenic capacity. In gene expression profiling we identified MPL as one of the genes that are differentially expressed in LT-HSC isolated from leukemic and non-leukemic mice. We are currently assessing the role of MPL in the leukemogenic potential of Bcr-Abl+ LT-HSC, and as a potential target for LSC-directed therapeutics. CONFLICT OF INTEREST The author declares no conflict of interest.
ACKNOWLEDGEMENTS The symposium and publication of this supplement were sponsored by the Division of Hematology/Oncology at the Warren Alpert Medical School of Brown University and NIH Center of Biomedical Research Excellence (COBRE) for Stem Cells Biology at Rhode Island Hospital.
REFERENCES 1 Chu S, McDonald T, Lin A, Chakraborty S, Huang Q, Snyder DS et al. Persistence of leukemia stem cells in chronic myelogenous leukemia patients in prolonged remission with imatinib treatment. Blood 2011; 118: 5565–5572. 2 Mahon FX, Rea D, Guilhot J, Guilhot F, Huguet F, Nicolini F et al. Discontinuation of imatinib in patients with chronic myeloid leukaemia who have maintained complete molecular remission for at least 2 years: the prospective, multicentre Stop Imatinib (STIM) trial. Lancet Oncol 2010; 11: 1029–1035.
Division of Hematopoietic Stem Cell and Leukemia Research, City of Hope National Medical Center, Duarte, CA, USA. Correspondence: Dr R Bhatia, Division of Hematopoietic Stem Cell and Leukemia Research, City of Hope National Medical Center, Duarte, CA 91010, USA. E-mail: [email protected]
Microenvironmental regulation of CML cells R Bhatia
S2 3 Ross DM, Branford S, Seymour JF, Schwarer AP, Arthur C, Yeung DT et al. Safety and efficacy of imatinib cessation for CML patients with stable undetectable minimal residual disease: results from the TWISTER study. Blood 2013; 122: 515–522. 4 Zhang B, Li M, McDonald T, Holyoake TL, Moon RT, Campana D et al. Microenvironmental protection of CML stem and progenitor cells from tyrosine kinase inhibitors through N-cadherin and Wnt-beta-catenin signaling. Blood 2013; 121: 1824–1838.
Leukemia Supplements
5 Koschmieder S, Gottgens B, Zhang P, Iwasaki-Arai J, Akashi K, Kutok JL et al. Inducible chronic phase of myeloid leukemia with expansion of hematopoietic stem cells in a transgenic model of BCR-ABL leukemogenesis. Blood 2005; 105: 324–334. 6 Zhang B, Ho YW, Huang Q, Maeda T, Lin A, Lee SU et al. Altered microenvironmental regulation of leukemic and normal stem cells in chronic myelogenous leukemia. Cancer Cell 2012; 21: 577–592.
