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I. Spinello et al.
line with the data observed from fresh samples obtained from AML-M3 patients shown in Figure 2A, while signif- icantly lower levels were observed in AMLs blasts mutat- ed for NPM1, DNMT3A or FLT3 (Figure 7A). By analyzing CD147 levels in all AMLs that we have stratified into three risk groups according to the European LeukemiaNet (ENL) risk classification, we found that: 1) the lower CD147 levels were observed in poor- and intermediate- risk AMLs; 2) surprisingly, the highest CD147 levels were observed in AMLs with favorable risk profile, mainly due to the presence in this group of AML-M3 (Figure 7B). However, it is important to point out that approximately 20% of poor- and intermediate-risk AMLs also display elevated CD147 expression (Figure 7B). Because AML-M3 have the best prognosis among all AML subtypes for the efficiency of target therapy based on ATRA and ATO,26 those patients with favorable risk and high CD147 levels may represent a bias for our analysis. Therefore, we eval- uated a possible correlation between CD147 expression level and overall survival of AML patients, excluding AML-M3 patients, and according to CD147 levels classi- fied as: low (CD147<70); medium (CD147; range, 70- 120); high (CD147 >120) (Figure 7C). In these non-M3 patients who died within 60 months after diagnosis, ear- lier death (within 20 months) was observed more fre- quently in cases with high CD147 levels, as compared with (non-M3)-AML subgroups with low-medium CD147 levels (P=0.003 and P=0.0003, respectively) (Figure 7D). These three (non-M3)-AML subgroups had comparable age and white blood count (WBC) at diagno- sis (Figure 7D, right panels). Our data indicate that, excluding AML-M3, high CD147 levels are correlated with early death of AML patients.
Discussion
CD147 represents a prognostic marker in several solid tumors and in hematologic malignancies.3,6-9,15-19 However, the biological function of CD147 and its potential role as a marker in leukemia remain poorly defined. Apart from the erythrocyte lineage, the expression, regulation and function of CD147 in normal and leukemic hematopoietic cells have not been extensively studied.35 CD147, previ-
ously identified as a carrier molecule for the blood group antigen OKa,36 has been involved in the recirculation of mature erythrocytes from the spleen into the general cir- culation.37 In our study, we show that CD147 is important for the proliferation of normal CD34+ HPCs and its expression is down-regulated during Mo and G differenti- ation. Notably, we found that CD147 is over-expressed in all leukemic cell lines and in the large majority of primary leukemic blasts analyzed, as compared to normal CD34+ HPCs. However, CD147 expression significantly decreas- es during Mo and G differentiation of leukemic cells, thus mimicking what occurs in Mo and G lineage cells. By using AC-73 to inhibit CD147 function in leukemic cell lines and in primary AML blasts, we demonstrated that CD147 overexpression promotes leukemic cell prolifera- tion. Interestingly, we observed that AC-73 treatment inhibits leukemic cell proliferation by suppressing the acti- vation of the ERK/STAT3 signaling pathway, in line with previous studies.5,21 As constitutive STAT3 activity has been described to promote AML cell proliferation and sur- vival,22,38 targeting key tyrosine kinases upstream of STAT3 has been proposed as a strategy to treat AML.38-40 However, due to its role in stem cell renewal, the potential risk of systemic STAT3 inhibition could be a deregulation of hematopoiesis.41 Here, we found that CD147 is expressed in normal HPCs and also showed that a low dose of AC-73 induces only a slight inhibition of cell pro- liferation, without affecting differentiation of CD34+ HPCs. Importantly, we found that AC-73 does not cause cell death via apoptosis or cell cycle arrest, but induces autophagy in leukemic cells, as described in previous stud- ies carried out in AML cells treated with ATO as an induc- er of autophagy,23 or with other novel targeted drugs.23,27,42- 44 In some cases, autophagy accounted for a non-apoptotic decrease in cell viability.42,43
To further validate its potential therapeutic activity, we used AC-73 in combination with conventional anti- leukemia treatment, and showed that the anti-prolifera- tive effect of AC-73 on leukemic cells enhanced the sensi- tivity of leukemic cells to chemotherapeutic treatments such as Ara-C or ATO, which could be consequently used at lower concentration. Our findings are in line with recent studies showing that other compounds, such as chi- damide that down-regulates the JAK2/STAT3 signaling,45
Table 1. Analysis of cell surface antigen expression in primary acute myeloid leukemia samples.
Patient n.
1
2 3 4 5 6 7 8 9
10
FAB subtype
M1
M1 M1 M2 M2 M3 M3 M5 M5a
M5
CD147 CD34 (%) (%)
63.1 21.2
CD371 CD11b CD14 (%) (%) (%)
80.3 31 1
93.7
44.4
95
92.5
63.7
97.9
71.5
96.6
91.3
92.4 98.6 11.1 39.5 0.2 12.5 5 8.3
88.6 64.5 88.3 94 31 95.9 90.9 93.2
ND ND 28 2.3 51 78 18.5
4 ND 3.2 0 60 21
33.6 8.8 69 17
73 ND
Flow cytometry analysis of CD147,CD34,CD371 and CD11b,CD14 cell surface antigen expression in 10 primary acute myeloid leukemia (AML) samples,obtained from patients with AML pertaining to different French-American-British (FAB) classification subtypes. Results are shown as percentage (%) of expression. ND: not determined.
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haematologica | 2019; 104(5)