Contributions
Abstract: S1552
Type: Oral Presentation
Presentation during EHA23: On Sunday, June 17, 2018 from 08:30 - 08:45
Location: Room A2
Background
We have previously demonstrated that the transcriptional profile of diagnostic CD34+ cells from chronic phase chronic myeloid leukemia (CP-CML) patients exhibiting primary resistance to imatinib overlaps with that of patients with myeloid blast phase CML (BP-CML) (McWeeney et al. Blood 2010). These commonalities suggest that that primary TKI resistance and advanced disease are biologically related. Reduced expression of the hematopoietic cell cycle regulator, MS4A3, was identified as a principal component of the gene expression classifier predicting imatinib response.
Aims
The aim of this study was to identify the functional consequence of reduced MS4A3 expression in TKI resistance and blastic transformation of CML.
Methods
To assess the functional role of MS4A3 in CML and TKI response, we used ectopic MS4A3 expression and shRNA-mediated MS4A3 knockdown in CD34+ cells from BP-CML and CP-CML patients, respectively. The phenotypic consequence of altered MS4A3 expression was measured using colony formation assays, apoptosis assays, long-term culture-initiating cell assays, and xenografts into NSG recipient mice.
Results
Low MS4A3 correlated not only with primary TKI resistance, but also with shorter overall survival in CP-CML (n=35). Microarray (n=19 CP-CML; n=16 BP-CML), qRT-PCR (n=22 CP-CML; n=17 BP-CML), and immunoblot (n=3 CP-CML; n=3 BP-CML) analyses demonstrated that MS4A3 mRNA and protein levels are reduced in CD34+ progenitor cells from BP-CML versus CP-CML patients. Ectopic expression of MS4A3 in BP-CML CD34+ progenitors (n=5) markedly reduced colony formation in the presence and absence of imatinib, consistent with a tumor suppressor role for MS4A3 in CML. While re-expression of MS4A3 alone did not increase apoptosis compared to controls, imatinib-induced apoptosis in BP-CML CD34+ cells was increased by 62%, with no effect on normal CD34+ cord blood cells (n=3). Conversely, shRNA-mediated MS4A3 knockdown (shMS4A3) in CP-CML CD34+ cells (n=7) reduced the effects of imatinib in colony formation and apoptosis assays, with no effect on normal CD34+ progenitors (n=4). These data suggest that MS4A3 positively regulates patient survival and imatinib response in CML progenitor cells. To evaluate MS4A3 in the leukemic stem cell compartment, qRT-PCR revealed that MS4A3 mRNA levels are markedly higher in committed CD34+38+ progenitors compared to more primitive CD34+38- stem cells, suggesting a role for MS4A3 in differentiation. Flow cytometric analysis revealed that shMS4A3 in CP-CML CD34+ cells reduced CD11b+ cells by ~45% (n=3), which correlated with reduced cell surface expression of KIT, GM-CSFR, IL-3R, and FLT3R. To assess the function of MS4A3 in CML stem cells, we performed long-term culture-initiating cell (LTC-IC) assays and xenografts into NSG mice upon MS4A3 knockdown in CP-CML (n=3). shMS4A3 increased Ph+ LTC-IC colony formation in the presence absence of imatinib. Consistent with these data, shMS4A3 enhanced engraftment of CD34+CD45+GFP+ cells into the bone marrow of NSG recipient mice. DNA bisulfite conversion and patch PCR sequencing revealed that the MS4A3 promoter is highly methylated in CML compared to normal CD34+ cells, but could not explain downregulation in BP-CML or TKI resistance. Rather, downregulation in the latter is due to loss of the myeloid-specific transcription factor, C/EBPbeta.
Conclusion
Altogether, these data suggest that MS4A3 plays a key role in 1) BCR-ABL1 kinase-independent resistance, 2) progression of CML from the chronic to the blastic phase of disease, and 3) in primitive CML stem cells versus progenitors.
Session topic: 7. Chronic myeloid leukemia – Biology & Translational Research
Keyword(s): Chronic myeloid leukemia, Cytokine, Stem and progenitor cell
Abstract: S1552
Type: Oral Presentation
Presentation during EHA23: On Sunday, June 17, 2018 from 08:30 - 08:45
Location: Room A2
Background
We have previously demonstrated that the transcriptional profile of diagnostic CD34+ cells from chronic phase chronic myeloid leukemia (CP-CML) patients exhibiting primary resistance to imatinib overlaps with that of patients with myeloid blast phase CML (BP-CML) (McWeeney et al. Blood 2010). These commonalities suggest that that primary TKI resistance and advanced disease are biologically related. Reduced expression of the hematopoietic cell cycle regulator, MS4A3, was identified as a principal component of the gene expression classifier predicting imatinib response.
Aims
The aim of this study was to identify the functional consequence of reduced MS4A3 expression in TKI resistance and blastic transformation of CML.
Methods
To assess the functional role of MS4A3 in CML and TKI response, we used ectopic MS4A3 expression and shRNA-mediated MS4A3 knockdown in CD34+ cells from BP-CML and CP-CML patients, respectively. The phenotypic consequence of altered MS4A3 expression was measured using colony formation assays, apoptosis assays, long-term culture-initiating cell assays, and xenografts into NSG recipient mice.
Results
Low MS4A3 correlated not only with primary TKI resistance, but also with shorter overall survival in CP-CML (n=35). Microarray (n=19 CP-CML; n=16 BP-CML), qRT-PCR (n=22 CP-CML; n=17 BP-CML), and immunoblot (n=3 CP-CML; n=3 BP-CML) analyses demonstrated that MS4A3 mRNA and protein levels are reduced in CD34+ progenitor cells from BP-CML versus CP-CML patients. Ectopic expression of MS4A3 in BP-CML CD34+ progenitors (n=5) markedly reduced colony formation in the presence and absence of imatinib, consistent with a tumor suppressor role for MS4A3 in CML. While re-expression of MS4A3 alone did not increase apoptosis compared to controls, imatinib-induced apoptosis in BP-CML CD34+ cells was increased by 62%, with no effect on normal CD34+ cord blood cells (n=3). Conversely, shRNA-mediated MS4A3 knockdown (shMS4A3) in CP-CML CD34+ cells (n=7) reduced the effects of imatinib in colony formation and apoptosis assays, with no effect on normal CD34+ progenitors (n=4). These data suggest that MS4A3 positively regulates patient survival and imatinib response in CML progenitor cells. To evaluate MS4A3 in the leukemic stem cell compartment, qRT-PCR revealed that MS4A3 mRNA levels are markedly higher in committed CD34+38+ progenitors compared to more primitive CD34+38- stem cells, suggesting a role for MS4A3 in differentiation. Flow cytometric analysis revealed that shMS4A3 in CP-CML CD34+ cells reduced CD11b+ cells by ~45% (n=3), which correlated with reduced cell surface expression of KIT, GM-CSFR, IL-3R, and FLT3R. To assess the function of MS4A3 in CML stem cells, we performed long-term culture-initiating cell (LTC-IC) assays and xenografts into NSG mice upon MS4A3 knockdown in CP-CML (n=3). shMS4A3 increased Ph+ LTC-IC colony formation in the presence absence of imatinib. Consistent with these data, shMS4A3 enhanced engraftment of CD34+CD45+GFP+ cells into the bone marrow of NSG recipient mice. DNA bisulfite conversion and patch PCR sequencing revealed that the MS4A3 promoter is highly methylated in CML compared to normal CD34+ cells, but could not explain downregulation in BP-CML or TKI resistance. Rather, downregulation in the latter is due to loss of the myeloid-specific transcription factor, C/EBPbeta.
Conclusion
Altogether, these data suggest that MS4A3 plays a key role in 1) BCR-ABL1 kinase-independent resistance, 2) progression of CML from the chronic to the blastic phase of disease, and 3) in primitive CML stem cells versus progenitors.
Session topic: 7. Chronic myeloid leukemia – Biology & Translational Research
Keyword(s): Chronic myeloid leukemia, Cytokine, Stem and progenitor cell