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Mapping the CLEC12A expression on myeloid progenitors in normal bone marrow; implications for understanding CLEC12A‐related cancer stem cell biology

Abstract The C‐type lectin domain family 12, member A (CLEC12A) receptor has emerged as a leukaemia‐associated and cancer stem cell marker in myeloid malignancies. However, a detailed delineation of its expression in normal haematopoiesis is lacking. Here, we have characterized the expression pattern of CLEC12A on the earliest stem‐ and myeloid progenitor subsets in normal bone marrow. We demonstrate distinct CLEC12A expression in the classically defined myeloid progenitors, where on average 39.1% (95% CI [32.5;45.7]) of the common myeloid progenitors (CMPs) expressed CLEC12A, while for granulocyte‐macrophage progenitors and megakaryocyte‐erythroid progenitors (MEPs), the average percentages were 81.0% (95% CI [76.0;85.9]) and 11.9% (95% CI [9.3;14.6]), respectively. In line with the reduced CLEC12A expression on MEPs, functional assessment of purified CLEC12A+/− CMPs and MEPs in the colony‐forming unit assay demonstrated CLEC12A+ subsets to favour non‐erythroid colony growth. In conclusion, we provide evidence that the earliest CLEC12A+ cell in the haematopoietic tree is the classically defined CMP. Furthermore, we show that CLEC12A‐expressing CMPs and MEPs are functionally different than their negative counterparts. Importantly, these data can help determine which cells will be spared during CLEC12A‐targeted therapy, and we propose CLEC12A to be included in future studies of myeloid cancer stem cell biology.


| INTRODUCTION
The transmembrane receptor C-type lectin domain family 12, member A (CLEC12A) is emerging as a marker of blasts and leukaemic stem cells (LSC) in acute myeloid leukaemia (AML) and by inference as a useful tool in both the diagnostic and follow-up settings. [1][2][3][4][5] In addition, we have recently shown the relevance of this marker in the context of myelodysplastic syndrome (MDS). 6 In the setting of malignant haematology, one of the most appealing features of CLEC12A is its lack of expression on CD34 + CD38 À cells in normal bone mar-stem cells, 2 making it a potential treatment target. Recent ventures in this regard include the development of anti-CD3/anti-CLEC12A bispecific antibodies. [7][8][9] The upcoming roles of CLEC12A as a cancer stem cell marker and potential treatment target emphasize the need of knowing the details of its expression pattern on early stem and progenitor cells in healthy individuals.
Although the classical roadmap of haematopoiesis is constantly being revised, [10][11][12] it is nevertheless providing a powerful tool for understanding the production of mature circulating blood cells and continuously serves as a solid model for studying clonal evolution in the myeloid spectrum of haematological malignancies. [13][14][15][16] In this study, we opted to employ the immunophenotypic definition of the haematopoietic stem cell (HSC) as being Lin-CD34 + CD38 À CD90 + CD45RA À , [17][18][19] the multipotent progenitor (MPP) as being Lin-CD34 + CD38 À CD90 À CD45RA À20 and the human multilymphoid progenitor (MLP, also termed lymphoid-primed multipotent progenitor) as Lin-CD34 + CD38 À CD90 À /lowCD45RA + . 21 With respect to the Lin-CD34 + CD38 + myeloid progenitors, we distinguished the common myeloid progenitor (CMP), the granulocyte-macrophage progenitor (GMP) and the megakaryocyte-erythroid progenitor (MEP) subsets based on their CD123 and CD45RA expression as first described by Manz and colleagues. 22 Using these definitions, we have delineated the expression of CLEC12A on normal BM stem-and myeloid progenitor cells and evaluated the influence of this marker on the growth of purified CMPs, GMPs and MEPs in the colony-forming cell assay. Importantly, our results provide evidence that the earliest CLEC12A + cell in normal haematopoiesis is the classically defined CMP.  The gating strategy used for analysing the myeloid progenitor cells is depicted in Figure 1. In short, the lineage negative singlets were defined (not shown) and displayed in a CD34 vs. CD38 plot. Herein, the CD34 + CD38 + cell subset was defined ( Figure 1A) and the progenitors were further enriched by CD90 negativity ( Figure 1B). The CMPs were defined as being Lin-CD34 + CD38 + CD90 À CD123 + CD45RA À , GMPs as Lin-CD34 + CD38 + CD90 À CD123 + CD45RA + and MEP as Lin-CD34 + CD38 + CD90 À CD123 À CD45RA À ( Figure 1C). Next, these three myeloid progenitor subsets were further gated by their CLEC12A expression ( Figure 1D). The gating strategy used for analysing CLEC12A expression on HSCs, MPPs and MLPs in the Lin-CD34 + CD38 À subset is depicted in Figure S1. The number of analysed events and the corresponding percentages of CLEC12A + cells are given in Table S1.   data not shown).   Figure 2A). Notably, the CLEC12A + cells were distributed across the whole CMP and GMP compartments and extended into the MEP compartment ( Figure 2B). Taken together, while CLEC12A is a known marker of mature myeloid cells, 23,24 these data reveal that detectable expression of CLEC12A emerges at the level of the CMP, and is abundantly expressed on GMPs and to a lesser extent on the classically defined MEP.

| Statistical analyses
3.2 | Different clonogenic potential in CLEC12A positive and negative myeloid progenitors Next, the clonogenic potential of CLEC12A positive and negative myeloid progenitors was evaluated in the colony-forming cell assay (  Figure 3). Being the most immature of the three cell types, the CMPs gave rise to both myeloid, erythroid and CFU-GEMM colonies in both the CLEC12A + and CLEC12A À subsets. The total number of colonies was significantly higher in the seeded CLEC12A À CMPs (P = .02). Moreover, significantly fewer erythroid colonies were observed in the seeded CLEC12A + CMP subset (P = .002), showing that CLEC12A positivity favours a non-erythroid lineage.
For both CLEC12A +/À GMPs, the clonogenic growth was almost exclusively of myeloid origin and no CFU-GEMMs were evident (Table 2), which is in accordance with the original definition of this cell subset. 22 We found no difference neither in the overall clonogenic potential of the CLEC12A + and CLEC12A À GMPs (P = . and for CD34-AML even more mature progenitors/precursors. 15,28 In addition, in low-risk myelodysplastic syndrome (MDS), the cancer stem cells have been shown to reside in the HSC compartment, 13 although studies on high-risk MDS indicate the MDS-propagating cell to arise in the MPP or GMP compartment, much resembling AML. 29,30 In this context, the likely usefulness of CLEC12A as a marker of the leukaemic blasts and/or stem cells could be linked to the fact that the level of differentiation arrest occurs in a cell type naturally expressing this marker. By inference-regarding CLEC12A as a marker of myeloid progenitors and their downstream progeny-the presence of CLEC12A on AML blasts and stem cells could indicate a myeloid progenitor/precursor disease, while CLEC12A À AML could correspond to a more immature stem cell disease originating from a cell at the level of the CLEC12A À CMP or upstream. In accordance with this, CLEC12A expression on AML blasts is highly correlated with CD34 negativity, 1 which is often associated with a more mature subtype of AML. 15 Whether CLEC12A expression on leukaemic blasts has prognostic impact in CD34 + AML remains to be elucidated. Future studies are warranted to explore this in detail and also to examine whether the aberrant expression of CLEC12A on CD34 + CD38 À cells shown in some cases of AML 2,4 and MDS 6 is due to an up-regulation of CLEC12A in these early stem/progenitor cells or a down-regulation of CD38 in the leukaemic CLEC12A + cells.
Our data impact on the notion of CLEC12A as a druggable pro- with RNA sequencing of 63 single pre-granulocyte-macrophage cells, and interestingly, CLEC12A turned up as a potential subpopulation classifier being one of the 55 genes showing differential expression by cluster analysis and proved to be mutually exclusive with GATA1. 37 In this study, the GATA1 + (and thereby F I G U R E 3 Colony-forming cell assays on CLEC12A +/À CMPs, GMPs and MEPs from 6 normal donors. Asterisk (*) denotes mean number of colonies from plates seeded in doublets.
CLEC12A À ) cells gave rise to mast cells and eosinophils, while the GATA1 À (CLEC12A + ) cells gave rise to monocytes and neutrophils -cells that are also known to express CLEC12A in humans. 23,24,37 Interestingly, by adding the surface markers CD71 and BAH-1 to the established sorting schemes of myeloid progenitors, a study by Notta and colleagues found the originally defined CMPs to be quite heterogeneous and to consist of unipotent myeloid and erythroid progenitors with very sparse megakaryocyte activity. 11 In line with this, our data indicate that adding CLEC12A as a deselection marker in the sorting scheme of MEPs could purify the early erythroid subset further.
In conclusion, we here show the differentiated expression of CLEC12A at the level of myeloid progenitors and provide evidence that the earliest CLEC12A + cell in the haematopoietic tree is the classically defined CMP. While this knowledge is readily translational in the setting of CLEC12A-targeted treatment, we also suggest that the marker is continuously explored in basic research studying the early myeloid differentiation.