VSTM-v1, a potential myeloid differentiation antigen that is downregulated in bone marrow cells from myeloid leukemia patients.

Leukocyte differentiation antigens often represent important markers for the diagnosis, classification, prognosis, and therapeutic targeting of myeloid leukemia. Herein, we report a potential leukocyte differentiation antigen gene VSTM1 (V-set and transmembrane domain-containing 1) that was downregulated in bone marrow cells from leukemia patients and exhibited a higher degree of promoter methylation. The expression level of its predominant encoded product, VSTM1-v1, was positively correlated with myeloid cell maturation state. Restoration of VSTM1-v1 expression inhibited myeloid leukemia cells' growth. Therefore, VSTM1-v1 might represent an important myeloid leukocyte differentiation antigen and provide a potential target for the diagnosis and treatment of leukemia.

To the Editor

Acute myeloid leukemia (AML) and chronic myeloid leukemia (CML) are myeloid blood cell malignancies that show great heterogeneity. VSTM1 (V-set and transmembrane domain-containing 1) encodes a potential leukocyte differentiation antigen that is highly expressed in myeloid cells, but silenced in multiple leukemia cell lines [1]. To determine whether it plays a role in leukemogenesis, we characterized its expression pattern and function in bone marrow cells from AML/CML patients and myeloid leukemia cell lines.

We measured VSTM1 expression in leukemia cell lines and bone marrow biopsies from leukemia patients using qRT-PCR. VSTM1 was downregulated or silenced in all cell lines tested (Additional file 1: Table S1). Compared to healthy donors (HDs), VSTM1 was downregulated in AML (Table 1). Additionally, in CML-AP/BC (accelerated phase/blast crisis), which clinically behaves like AML, VSTM1 expression levels were much lower than those in CML-CP (chronic phase, P = 0.003, Table 1). Analogous protein expression differences were found by Western blotting. Compared to HD bone marrow, VSTM1 was similarly expressed in CML-CP patients, whereas it was barely detectable in untreated AML patients. However, in AML patients who achieved complete remission, VSTM1 expression was completely restored (Additional file 2: Figure S1). Higher levels of VSTM1 promoter methylation in bone marrow cells from AML patients compared to those from HDs were observed, which might contribute to its downregulation (Additional file 3: Figure S2).

Table 1

The expression level of in bone marrow cells from leukemia patients and healthy donors

Groups	MIC	Sample size	Mean ratio, VSTM1 / ABL	P value a
HD		36	17.358 ± 17.904
Untreated AML		145	4.333 ± 7.895	<0.001
	M1	3	0.374 ± 0.619	n.a.
	M2	72	5.292 ± 10.171	<0.001
	M3	29	2.884 ± 3.960	<0.001
	M4	24	4.814 ± 5.293	0.001
	M5	14	3.052 ± 4.867	<0.001
	M6	3	1.417 ± 2.205	n.a.
Untreated ALL		40	0.381 ± 0.755	<0.001
Untreated CML		57	5.479 ± 8.266	<0.001
	CP	38	7.743 ± 9.312	0.001
	AP/BC	19	0.950 ± 1.367	<0.001

MIC morphological, immunological, and cytogenetic classifications, n.a. not available due to a small sample size.

aThe P value was calculated using Wilcoxon signed ranks test as compared to the HD group.

VSTM1-v1 is the most abundantly expressed gene product encoded by VSTM1 [2] and is an ITIM-bearing immune receptor that negatively regulates neutrophil activity [3-5]. We used flow cytometry to divide bone marrow cells into subpopulations based on CD45 levels and SSC [6], and found that VSTM1-v1 was highly expressed in mature granulocytes and monocytes from HDs (94.01 ± 6.80% positive with a mean fluorescence intensity (MFI) of 167.61 ± 90.95), but was much less abundant in naive cells (37.14 ± 14.47% with a MFI of 46.70 ± 23.51, n = 27; P <0.001). Moreover, the percentage of VSTM1-v1-positive cells among naive cells from AML patients (19.60 ± 21.09%, n = 52, including 4 M0/M1, 22 M2, 4 M3, 14 M4, and 8 M5) was even lower than that in HDs (P = 0.047). Therefore, we speculated that VSTM1-v1 expression might be associated with the maturity of myeloid cells. A combination of CD34/CD117/CD13/CD16 staining showed that VSTM1-v1 expression in myeloid cells was positively correlated with cell maturation state. Differences between any two continuous stages were significant (n = 11; P < 0.001; Table 2 and Additional file 4: Figure S3). Using CD16 and CD14 as phenotypic markers for mature granulocytes and monocytes, respectively, we found a similar result (Additional file 1: Table S2). This correlation was subsequently confirmed by increased VSTM1 expression in bone marrow cells from APL patients and in NB4 cells after ATRA treatment in vitro (Additional file 5: Figure S4). These findings provide a potential reason why VSTM1 expression levels were reduced so markedly in AML and CML-AP/BC patients.

Table 2

The expression of VSTM1-v1 at various stages of myelocytic differentiation

	Myeloblasts	Promyelocytes	Myelocytes	Metamyelocytes
CD markers	CD34+	CD34−, CD117+, CD16−	CD34−, CD117−, CD16−, CD13+	CD16+
VSTM1-v1-positive cells (%)	5.69 ± 2.69	11.48 ± 4.58	35.02 ± 11.58	61.63 ± 8.53
P valuea	<0.001	<0.001	<0.001

aThe P value was calculated using a Wilcoxon signed ranks test as compared with the next stage.

Similar to its function in Jurkat cells [1], restoration of VSTM1-v1 expression in the myeloid leukemia cell lines K562 and MEG-01 also inhibited cell growth (Additional file 6: Figure S5). Moreover, when searching for clinical features that could be related to VSTM1 expression in AML patients, we detected a higher expression level of VSTM1 in AML1-ETO-positive patients (Additional file 1: Table S3 and Additional file 7: Figure S6). This chimeric oncogene in AML is often associated with a relatively favorable prognosis [7,8]. Considering the inhibitory effect of VSTM1-v1 on leukemia cell growth, we can hypothesize that VSTM1-v1 might enhance the cytotoxic effects of chemotherapeutics in patients with this genetic abnormality.

In conclusion, our findings suggest that VSTM1-v1 might be an important myeloid leukocyte differentiation antigen. Our elucidation of its expression pattern throughout myeloid cell differentiation and its effect on leukemia cell growth could help to establish it as a novel target for the development of diagnostics and treatments for myeloid leukemia.