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N. Landberg et al.
Table 1. Cell surface expression of 17 candidate markers in CML cells as determined by flow cytometry.
Primary CMLa
Gene Symbol CD CD34+CD38low CD34+CD38+
CD36 CD36 ++ ++ LEPR CD295 + + TFRC CD71 ++ ++ ITGB3 CD61 ++
CD7 CD7 + FCGR2A CD32 +/- GP6 nc + IL12RB1 CD212
HMMR CD168
CML cell lines
KU812 K562 BV173 LAMA84
++ ++ +
++ - +/- + ++ ++ + ++ ++ ++ ++ ++ ++ - - ++ + - - + ++++++ ++
+ - - - +/- - - - - - - - - +/- - - +/- + - + - -+- + - +++++ ++
- - - - -
aTwo or more chronic myeloid leukemia (CML) patient samples were analyzed for each cell surface marker. ++: strong expression; +: moderate expression; +/−: expression on somecellsorweakexpression;−:noexpression;CD:clusterofdifferentiation;nc:notclustered;*:previouslydescribedtobeupregulatedonCD34+CD38low CMLcells;**:did not pass the thresholds used to identify differentially expressed cell surface markers in the RNA sequencing analysis.
ECE1
TNFRSF18
TYRO3
IL1RAP* nc
IL2RA* CD25 ++ - ++ + - + DPP4* CD26 ++ - - - - - NCAM1* CD56 + +/- - - - - CD93** CD93 ++ +/- ++++/--
nc CD357 nc
++ ++ ++ - ++ ++
450
detected or exhibited low expression on the cell surface of CD34+CD38low NBM cells, ITGB3 and TFRC showed a clearly detectable expression, albeit lower than in the corre- sponding CML cells (Figure 2A). Further, LEPR was not expressed on more mature CD34+CD38+ cells, whereas CD36 showed some expression in these cells (Online Supplementary Figure S3A). When examining specific sub- populations of normal hematopoietic cells, CD36 displayed a higher expression in more mature subsets (megakary- ocyte-erythroid progenitors [MEP], granulocyte- machrophage progenitors [GMP], and common myeloid progenitors [CMP]) as compared to more primitive subsets [HSCs, multipotent progenitors [MPP], and lymphoid- primed multipotent progenitors [LMPP]; Online Supplementary Figure S3B).
Notably, the expression of CD36 separated the CD34+CD38low CML cells into distinct CD36 positive and negative populations. The overexpression of CD36 in CD34+CD38low cells in relation to corresponding cells from healthy BM samples was confirmed in an independent cohort of 16 CML patients (P=0.0089, Figure 2B). Staining for LEPR resulted in a weak signal, but extended analyses of ten primary CML samples with a high leukemic burden in the CD34+CD38low compartment (as defined by 50-100% IL1RAP expression, mean 86% IL1RAP+ cells), confirmed a significantly higher LEPR expression compared with corre- sponding healthy cells (P=0.002), devoid of LEPR (Figure 2C). Moreover, all CML samples displayed higher mean flu- orescence intensity (MFI) for LEPR compared with paired isotype control stained samples, whereas the NBM samples did not (Online Supplementary Figure S4). Given the specific expression of LEPR in CML cells, we investigated if CML cells would respond to leptin.22 However, when assessing cell growth in vitro and colony forming capacity upon stim- ulation with leptin, no effects were observed (Online Supplementary Figure S5A-S5D). We conclude that CD36
and LEPR are specifically upregulated on the surface of primitive CML cells.
CD36 expression separates CD34+CD38lowIL1RAP+ CML cells into two distinct populations
We previously demonstrated that IL1RAP expression can be used to identify BCR/ABL1 positive cells within the CD34+CD38low compartment of BM cells from CML patients, with all cells in the IL1RAP positive fraction being BCR/ABL1 positive.11,13 Because CD36 was found to be expressed on a subpopulation of the CD34+CD38low CML cells, we investigated the co-expression of CD36 and IL1RAP. Although a significant correlation between CD36 and IL1RAP expression was observed (r=0.679, P=0.0048, Figure 3A), CD36 was distinctly expressed on a subset of the CD34+CD38lowIL1RAP+ cells (Figure 3B; Online Supplementary Figure S6). To investigate the co-expression of IL1RAP and CD36 in relation to the BCR/ABL1 status of the cells, we sorted cells based on IL1RAP and CD36 expression within the CD34+CD38low cell fraction from three CML patients. By fluorescence in situ hybridization (FISH) analy- ses, we found that on average 98% of CD34+CD38lowIL1RAP+CD36+ cells and 98% of CD34+CD38lowIL1RAP+CD36– cells were BCR/ABL1 posi- tive. By contrast, only 3% of the CD34+CD38lowIL1RAP–CD36– cells were BCR/ABL1 posi- tive (Figure 3C,D). Hence, CD36 divides the CD34+CD38lowIL1RAP+ compartment into a CD36 positive and a CD36 negative population that are both predomi- nantly BCR/ABL1 positive.
Primitive CML cells expressing CD36 are less sensitive to imatinib treatment
To delineate the difference between the CD36 positive and negative cell populations of primitive CML cells, we sorted CD34+CD38lowIL1RAP+CD36+ and CD34+ CD38low
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