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The small-molecule compound AC-73 targeting CD147 inhibits leukemic cell proliferation, induces autophagy and increases the chemotherapeutic sensitivity of acute myeloid leukemia cells

CD147 is a transmembrane glycoprotein with multiple functions in human healthy tissues and diseases, in particular in cancer. Overexpression of CD147 correlates with biological functions that promote tumor progression and confers resistance to chemotherapeutic drugs. In contrast to solid tumors, the role of CD147 has not been extensively studied in leukemia. Understanding whether CD147 represents a new hematologic target and whether its inhibitor AC-73 may be used in leukemia therapy may reveal an alternative treatment strategy in patients with acute myeloid leukemia (AML). We analyzed CD147 expression and function in hematopoietic progenitor cells from normal cord blood, in several leukemic cell lines and in primary leukemic blasts obtained from patients with AML. We investigated the effects of AC-73, used alone or in combination with arabinosylcytosine (Ara-C) and arsenic trioxide (ATO), on leukemic cell proliferation. We demonstrated that CD147 overexpression promotes leukemic cell proliferation. We showed that AC-73 exhibits a potent growth inhibitory activity in leukemic cells, by inhibiting the ERK/STAT3 activation pathway and activating autophagy. We demonstrated that AC-73 exerts an anti-proliferative effect additive to chemotherapy by enhancing leukemic cell sensitivity to Ara-C-induced cytotoxicity or to ATO-induced autophagy. We also reported CD147 expression in the fraction of leukemic blasts expressing CD371, a marker of leukemic stem cells. Altogether, our study indicates CD147 as a novel potential target in the treatment of AML and AC-73 as an anti-proliferative drug and an inducer of autophagy in leukemic cells to use in combination with chemotherapeutic agents.

Ferrata Storti Foundation C D147 is a transmembrane glycoprotein with multiple functions in human healthy tissues and diseases, in particular in cancer. Overexpression of CD147 correlates with biological functions that promote tumor progression and confers resistance to chemotherapeutic drugs. In contrast to solid tumors, the role of CD147 has not been extensively studied in leukemia. Understanding whether CD147 represents a new hematologic target and whether its inhibitor AC-73 may be used in leukemia therapy may reveal an alternative treatment strategy in patients with acute myeloid leukemia (AML). We analyzed CD147 expression and function in hematopoietic progenitor cells from normal cord blood, in several leukemic cell lines and in primary leukemic blasts obtained from patients with AML. We investigated the effects of AC-73, used alone or in combination with arabinosylcytosine (Ara-C) and arsenic trioxide (ATO), on leukemic cell proliferation. We demonstrated that CD147 overexpression promotes leukemic cell proliferation. We showed that AC-73 exhibits a potent growth inhibitory activity in leukemic cells, by inhibiting the ERK/STAT3 activation pathway and activating autophagy. We demonstrated that AC-73 exerts an anti-proliferative effect additive to chemotherapy by enhancing leukemic cell sensitivity to Ara-C-induced cytotoxicity or to ATO-induced autophagy. We also reported CD147 expression in the fraction of leukemic blasts expressing CD371, a marker of leukemic stem cells. Altogether, our study indicates CD147 as a novel potential target in the treatment of AML and AC-73 as an anti-proliferative drug and an inducer of autophagy in leukemic cells to use in combination with chemotherapeutic agents.
The small-molecule compound AC-73 targeting CD147 inhibits leukemic cell proliferation, induces autophagy and increases the chemotherapeutic sensitivity of acute myeloid leukemia cells lar events by interacting with various binding partners, such as tumor-and inflammation-associated molecules including integrins, monocarboxylate transporters (MCTs), cyclophilins, caveolin-1, and E-selectin, explaining its significant role in the pathogenesis of several diseases. [3][4][5][6]10 CD147 overexpression and more recently its coexpression with MCTs 11,12 are regarded as unfavorable prognostic factors in cancers associated with hypoxia, a common feature of solid tumors, but also a major component of the bone marrow (BM) microenvironment, crucial in leukemia progression. 13,14 However, in contrast to solid tumors, the function of CD147 remains poorly defined in leukemia.
Recent studies have shown growing interest in the CD147 molecule in AML 15,16 and in some hematologic neoplasia, in particular in multiple myeloma (MM), where CD147 expression levels have a prognostic value and are required for the proliferation of MM cells. [17][18][19] Moreover, CD147 is over-expressed in erythroid cells of myelodysplastic syndrome (MDS) with 5q deletion. 18 Here, we show that CD147 is expressed in normal CD34 + hematopoietic progenitor cells (HPCs) and down-regulated during monocytic and granulocytic differentiation of HPCs. We then show that CD147 is over-expressed in blasts pertaining to different subtypes of AML and promotes leukemic cell proliferation. Interestingly, we report that CD147 is expressed at the level of CD34 + CD371 + AML cells, previously described for their leukemia-initiating properties. 20 Recently, the small-molecule AC-73 has been proposed as a specific inhibitor for CD147. 21 First, we checked that the response to AC-73 treatment is not involved in an offtarget mechanism in leukemic cells. Then, we analyzed the effects of CD147 inhibition by AC-73 in AML cell lines and in primary leukemic blasts. We found that AC-73 inhibits leukemic cell proliferation by suppressing the ERK/STAT3 activation pathway, known to play a role in AML cell proliferation and survival, 22 but also by activating autophagy, an essential phenomenon for hematopoietic stem cell (HSC) maintenance, resistance to stress, survival and differentiation, the machinery of which might be disrupted in AMLs. [23][24][25] Next, we analyzed whether AC-73 enhanced the sensitivity of leukemic cells to conventional chemotherapeutic agents. We used arabinosylcytosine (Ara-C), one of the most active cytotoxic agents in myeloid leukemia, and arsenic trioxide (ATO), an active anti-proliferative agent used in the treatment of patients with acute promyelocytic leukemia (APL) (AML-M3) 1,2,26 [although with low efficacy in AML lacking the t(15;17) translocation], and also an inducer of autophagy. 25,27 We found that AC-73 used in vitro in combination with Ara-C or ATO, increases the effects of these agents.
Altogether, our data suggest that CD147 plays a key role in leukemic cell proliferation and represents a potential therapeutic target in AML patients, and that AC-73 is a new promising inhibitor that could be used in combination with conventional chemotherapeutic agents as a novel treatment strategy in AML.

Cell cultures
Human cord blood (CB) was obtained from healthy donors after informed consent. Leukemic blasts were isolated from BM obtained from patients with newly diagnosed AML, using Ficoll-Hypaque density gradient. Informed consent was obtained from patients in accordance with the Declaration of Helsinki. This study was approved by the local ethical committees of the Istituto Superiore di Sanità and the University of Tor Vergata, Rome, Italy.
Cord blood CD34 + HPC purification, unilineage monocytic (Mo) and granulocytic (G) differentiation and morphological analyses were performed, 28,29 as described in the Online Supplementary Methods.
Cell growth, cell cycle profile, viability and apoptosis analysis were performed, as described in the Online Supplementary Methods.
Flow cytometry, western blot and quantitative real-time RT-PCR analysis were as described in the Online Supplementary Methods.
Knockdown of CD147 expression by RNA interference, clonogenic assays and colony formation assays were performed, as described 30

in the Online Supplementary Methods.
Cyto-ID autophagy detection was performed using the Cyto-ID assay (Enzo Life Sciences ENZ-51031-K200) as described in the Online Supplementary Methods.  21 In leukemic cell lines, dose-response and time-course analysis were performed using AC-73 at 1.0, 2.5, 5.0 and 10 mM from 1 to 4 days of treatment; results were compared with 0.2% DMSOtreated leukemic cells, indicated as control leukemic cells. AC-73 was added in cultures every 2 days to maintain its activity. Combinations of treatment were performed using AC-73 (2.5 mM) alone over 24 hours and then by adding Ara-C (0.01; 0.1 and 1.0 µM) or ATO (0.1; 0.01 and 1.0 µM) for another 1 (for NB4 and NB4-R4 cells) or 2 (for U937, HL-60, MV4-11 and Kasumi-1 cells) days. Cell viability assays were performed to evaluate the effect of AC-73 alone or in combination on cell growth and viability of these cells.

Analysis of The Cancer Genome Atlas data
Datasets of The Cancer Genome Atlas (TCGA) Research Network 2008, were processed and obtained directly from the public access data portal (http://tcga-data.nci.nih.gov/).

Statistical analysis
Student t-test was applied to assess statistical significance of differences between multiple/group of experiments. Data were analyzed using GraphPad Prism software. For univariate survival analysis, Kaplan-Meier plots with a log-rank test were presented using the overall survival data of AML patients from the TGCA. Additional information is provided in the Online Supplementary Appendix.

CD147 is directly involved in hematopoietic progenitor cell proliferation
We analyzed CD147 expression at both mRNA and protein level in CD34 + HPCs and during Mo and G proliferation and differentiation of these cells. CD147 is well expressed in CD34 + HPCs and its level of expression decreases during Mo and G differentiation and maturation of these cells ( Figure 1A-C and Online Supplementary Figure   S1A and B). Western blot analysis shows that CD147 protein is highly glycosylated (HG-CD147 40-60 kDa) in CD34 + HPCs and at all stages of Mo and G cell differentiation ( Figure 1B), indicating the presence of CD147 protein in a stable and biologically active conformation mainly translocated to plasma membrane, 5 as also shown by flow cytometry analysis ( Figure 1C and Online Supplementary Figure S1B). To analyze the role of CD147 in HPCs, we performed siRNA-mediated CD147 knockdown experiments in CD34 + HPCs. We transiently trans-CD147 as potential therapeutic target in AML haematologica | 2019; 104(5) 975 fected HPCs with specific-CD147 interfering small RNAs (CD147-siRNA) or with a non-targeting control siRNA (c-siRNA). Silencing of CD147 mRNA decreased CD147 protein levels in transfected (CD147-siRNA)-HPCs, as compared to (c-siRNA)-HPCs of control ( Figure 1D); then, transfected-HPCs were grown under culture conditions, to allow their selective G or Mo proliferation and differentiation. We observed that the decreased expression of CD147 impaired the proliferation of both G and Mo differentiating (CD147-siRNA)-HPCs, as compared to control (c-siRNA)-HPCs ( Figure 1E), without any significant modulation of G or Mo specific antigen expression on these cells (data not shown) and without any significant induction of apoptosis (data not shown). Clonogenic G and Mo progenitor assays performed with transfected-HPCs, showed a significant decrease in the number of CFU-GM and CFU-M colonies obtained from (CD147-siRNA)-HPCs, compared with the number of colonies obtained from (c-siRNA)-HPCs ( Figure 1F). Altogether, our data indicate that CD147 plays a role in HPC proliferation and clonogenic activity.

Over-expressed in AML, CD147 is down-regulated during differentiation of leukemic cells
By analyzing CD147 mRNA expression levels in primary leukemic blast cells obtained from 48 patients with different subtypes of AML, 29 we found that CD147 is over-expressed in all AML subtypes, in particular in the AML-M3 subtype, as compared to normal CD34 + HPCs ( Then, we also examined CD147 expression in several AML cell lines induced to terminal differentiation. All leukemic cell lines analyzed over-expressed the fully glycosylated mature CD147 protein, biologically active ( Figure 2B, HG-CD147), like also its mRNA, as compared to normal CD34 + HPCs ( Figure 2C). We found that CD147 expression is down-regulated during vitamin D3-induced Mo differentiation of U937 cells and ATRA-induced G differentiation of HL-60 and NB4 cell lines, at both mRNA and protein levels during terminal differentiation ( Figure  2D-F and Online Supplementary Figure S1C-H), similarly to Mo and G terminal differentiation of normal HPCs ( Figure  1A-C). However, CD147 expression is not affected by ATRA treatment of the NB4-R4 cell line ( Figure 2G and Online Supplementary Figure S1I and L), indicating that induction of cell differentiation is required for CD147 downregulation.

AC-73 inhibits leukemic cell proliferation by blocking ERK/STAT3 signaling and induces autophagy
To investigate whether a high level of CD147 can promote cell proliferation, we used AC-73 to inhibit CD147 function in normal and leukemic cells.
First, we examined the effect of AC-73 treatment on G and Mo differentiation of normal HPCs. AC-73 used at 5 µM moderately decreased cell growth without affecting cell cycling or differentiation of HPCs ( Figure 3A-C). Morphology analysis of HPCs at different days of culture supported these observations, showing that AC-73-treated cells regularly progress along G and Mo differentiation and do not show any significant morphology abnormality or delay in differentiation ( Figure 3D).
We analyzed the effects of chronic AC-73 administra-tion on leukemic cell growth, apoptosis and viability of AML cell lines. Leukemic cells were treated from 1 to 3 days, with various AC-73 doses (2.5, 5 and 10 mM) to determine their sensitivity to AC-73 treatment. We observed a time-and dose-dependent effect of AC-73 on leukemic cell growth, with growth inhibition of all leukemic cell lines treated ( Figure 4A and Online Supplementary Figure S2B). We could not detect any effect of AC-73 on cell growth and apoptosis of the Chinese Hamster Ovary (CHO) cells (data not shown) used as a CD147-negative cell line (Online Supplementary Figure S2A, left panel). NB4 and NB4-R4 cell lines were more sensitive to AC-73 treatment than U937 and HL-60 cell lines. Indeed, a higher significant decrease in cell growth ( Figure 4A Figure S2F).
Altogether, despite the different sensitivities to AC-73 manifested by the different leukemic cell lines, probably related to the specific molecular alteration, such as PML/RARA, FLT3-ITD or RUNX1/RUNX1T1, of these cell lines, AC-73 exhibits potent growth inhibition and cytotoxic activity on leukemic cells only at high doses. Because low doses of AC-73 inhibit leukemic cell proliferation, but do not cause cell death via apoptosis or cell cycle arrest, we investigated the possibility that AC-73 treatment induces autophagy in leukemic cells. First, we assessed the effect of AC-73 on the level of the autophagic indicator LC3 by western blotting in U937 and NB4 cells. Our results showed a dose-dependent effect of AC-73 on the increase of LC3-II/LC3-I ratio in leukemic cells as compared to control (-) cells ( Figure 4E), indicating induction of autophagy. 31 Then we monitored autophagy flux by flow cytometry analysis in live leukemic cells treated for 72 hours (h) by AC-73 compared to control cells. Our data demonstrated that AC-73 induces dose-dependent autophagy ( Figure 4F) in CD147-expressing leukemic cells ( Figure 4G), but not in CHO cells (Online Supplementary Figure S2A, right panels), again indicating that the biological effects induced by AC-73 require CD147 expression on target cells. Considering that AC-73 suppresses CD147/ERK1/2/STAT3/MMP-2 pathways in hepatocellu-lar carcinoma cells, and that ERK/STAT3 signaling plays a significant role in promoting AML cell proliferation, survival and autophagy, 22,23,32,33 we also investigated the effect of AC-73 on ERK1/2 and STAT3 activation in leukemic cells. In line with previous studies, 31,32 western blot analysis showed that ERK and STAT3 are constitutively phosphorylated in all leukemic cell lines [Online Supplementary Figure S3, lanes (-)]. We then found that AC-73 (5 mM, for 3 days) notably decreases both pERK and pSTAT3 (S727) levels without affecting the levels of total ERK and STAT3, in leukemic cells (lanes 5 mM) (Online Supplementary Figure  S3, lower panels).
Altogether, our data demonstrate that AC-73 inhibits leukemic cell proliferation, in part by suppressing the ERK/STAT3 activation pathway in these cells, in part by activating a non-apoptotic, autophagic form of cell death, which may account for the efficacy of AC-73 in leukemic cells.

AC-73 has an additive anti-proliferative effect on chemotherapeutic treatment of leukemic cells and increases ATO-induced autophagy in leukemic cells
To evaluate the effect of AC-73 in combination with standard drugs used for AML treatment, AML cell lines CD147 as potential therapeutic target in AML haematologica | 2019; 104(5) 977 and primary AML blasts were initially treated for 24 h with a low concentration (2.5 mM) of AC-73 and then cotreated for 48 h with Ara-C or ATO at different concentrations (0.01-1 mM). Cell viability assays were performed to assess leukemic cell survival and sensitivity to AC-73 treatment used in combination with Ara-C or ATO, as compared to single AC-73, Ara-C, or ATO treatment ( Figure 5A and B and Online Supplementary Figure S4). We observed a significant decrease in leukemic cell viability of all leukemic cell lines after AC-73 treatment in combination with Ara-C or ATO, as compared to AC-73, Ara-C or ATO used alone ( Figure 5A and B and Online Supplementary Figure S4). Furthermore, we identified three subgroups of leukemic cells according to their Ara-C drugsensitivity: high-sensitivity lines (U937 and HL-60); intermediate-sensitivity lines (NB4 and NB4-R4); low-sensitivity lines (MV4-11 and Kasumi-1). AC-73 potentiates the sensitivity to Ara-C treatment of all leukemic cells, even those belonging to the low-sensitivity subgroup ( Figure  5A and B and Online Supplementary Figure S4). The antiproliferative effect of AC-73 enhances sensitivity to ATO treatment of both M3 leukemic cells, such as NB4 and NB4-R4 ( Figure 5B and Online Supplementary Figure S4B), and non-M3 leukemic cells, such as U937, HL-60, Kasumi-1 and MV4-11 cells ( Figure 5A and Online Supplementary Figure S4A, C and D).
Then, we showed that when used in combination with Ara-C and ATO, AC-73 treatment inhibits ERK and STAT3 activation in leukemic cells ( Figure 5C).
Because ATO is also an inducer of autophagy, 25 we analyzed the autophagy flux in both M3 and non-M3 leukemic cells treated by ATO or AC-73 used alone or in combination, as compared to control cells. Our data showed that AC-73 and ATO are both inducers of autophagy and the autophagy flux is significantly increased in M3 (NB4) and non-M3 (U937) leukemic cells I. Spinello et al. 978 haematologica | 2019; 104(5)  Figure  5D and E). Altogether, our data demonstrate that AC-73 is a potent, novel anti-proliferative molecule that enhances the sensitivity of leukemic cells to conventional chemotherapeutic agents, but also increases ATO-induced autophagy in M3 and non-M3 leukemic cells.

AC-73 increases the sensitivity of primary acute myeloid leukemia blasts co-expressing CD147 and CD371 to chemotherapy
We evaluated the effects of AC-73 used alone and in combination with Ara-C or ATO on primary AML blasts. First, we analyzed CD147 protein expression in samples obtained from different subtypes of AML and we controlled the expression of CD34, CD38 and CD371 mark-CD147 as potential therapeutic target in AML haematologica | 2019; 104(5) 979  34 We confirmed that CD147 is expressed at high levels in all AML subtypes (M1, M2, M3, M5 and M5a) analyzed (Table 1). We also report for the first time that CD147 is co-expressed with CD34, and particularly with CD371, in these cells (Table 1 and Figure 6A). It is interesting to note that in 8 non-M3 AMLs analyzed for CD147 and CD371 expression, there was a strong positive correlation between CD147 and CD371 expression (P<0.001) ( Table 1). These data suggest the existence of CD34 + /CD147 + and CD371 + /CD147 + subpopulations in AMLs that may have functional properties of leukemic stem cells (LSCs). Then, we investigated the effects of AC-73 used alone or combined with Ara-C and ATO at low concentration in primary AML blasts, and particularly in M2, M3 and M5 AML cells. We found that AC-73 used alone has no significant effect on AML cell apoptosis, unless used at high concentration (10 mM) for AML (7) Figure 6B). However, AC-73 induces autophagy by increasing the LC3-II/LC3-I ratio in leukemic blasts, as compared to control (-) leukemic cells, even at 2.5 or 5 mM ( Figure 6C). However, combination of low-dose AC-73 (2.5 mM) and ATO or Ara-C (0.1 and 1 mM) more efficiently decreased leukemic blast viability, compared with single AC-73, ATO or Ara treatment ( Figure 6D). Overall, our data indicate that AC-73 activates a non-apoptotic autophagic form of cell death in AML blasts and increases the sensitivity of these cells to Ara-C or ATO agents.

Analysis of CD147 expression in acute myeloid leukemia based on TCGA dataset
By providing selected genetic and clinical data from 200 AML patients, useful for prognosis and diagnosis, the TCGA dataset offered a unique opportunity to explore a possible relationship between the recurrent gene mutations observed in AML and the level of CD147 expression. TCGA data set analysis indicates that CD147 level is particularly elevated in AML-M3, those AML subtypes bearing PML-RARA fusion gene 1,26 ( Figure Figure 2A, while significantly lower levels were observed in AMLs blasts mutated 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 intermediaterisk 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 evaluated a possible correlation between CD147 expression level and overall survival of AML patients, excluding AML-M3 patients, and according to CD147 levels classified 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, earlier death (within 20 months) was observed more frequently 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 diagnosis ( 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][7][8][9][15][16][17][18][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 circulation. 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 differentiation. 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 decreases 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 proliferation. Interestingly, we observed that AC-73 treatment inhibits leukemic cell proliferation by suppressing the activation 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 survival, 22,38 targeting key tyrosine kinases upstream of STAT3 has been proposed as a strategy to treat AML. [38][39][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 proliferation, 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 studies carried out in AML cells treated with ATO as an inducer of autophagy, 23 or with other novel targeted drugs. 23,27,[42][43][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 antileukemia treatment, and showed that the anti-proliferative effect of AC-73 on leukemic cells enhanced the sensitivity 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 chidamide that down-regulates the JAK2/STAT3 signaling, 45 inhibit the viability of AML cells and synergize with cytarabine in inhibiting AML cell viability. 46 Importantly, we also showed that AC-73 increases ATO-induced autophagy in both M3 and non-M3 leukemic cells, indicating a possible synergistic anti-tumor interaction of ATO with an inhibitor of CD147, both agents being able to induce autophagy. The analysis of CD147 expression in AML subsets presented several interesting findings. First, CD147 is overexpressed in all French-American-British classification AML subtypes, and particularly in AML-M3; a finding confirmed through the analysis of primary AML samples. This finding was also corroborated by the analysis of the TCGA data set. Moreover, about 20% of non-M3 AMLs displayed elevated CD147 levels. Although the overall long-term survival of these patients (defined as highlyexpressing CD147) was similar to the other two non-M3 AML groups (defined as middle-and low-expressing CD147), death occurs earlier in patients showing high expression of CD147. This observation supports the negative prognostic role of high CD147 expression levels in AMLs, as observed also in other tumors, including some hematologic neoplasia. 3,19 Other recurrent AML mutations, 1,2 such as NPM1, FLT3-ITD and DNMT3A, were not associated to particularly elevated levels of CD147. Therefore, AC-73 used in combination with Ara-C or ATO may have clinical potential implications in treatment of AML patients expressing CD147. Future toxicological and pharmacodynamic studies in suitable animal models will be required for a preclinical evaluation of the possible impact of AC-73 as an anti-leukemic drug.
Our study also reports for the first time that CD147 is expressed in a sub-fraction CD34 + CD371 + of AML cells that is able to engraft immunodeficient mice, 20 CD34 being predominantly regarded as a marker of hematopoietic stem cells (HSC) and HPCs, and CD371 as a marker of CD147 as potential therapeutic target in AML haematologica | 2019; 104(5) 983 gene expression and stratified according to their CD147 mRNA levels in 3 groups: low (CD147<70); medium (CD147, range, 70-120); high (CD147 >120). (D) Kaplan-Meier survival analysis in the new group of (non-M3)-AML patients deceased within 50-60 months years after diagnosis, indicates that the kinetics of death is more rapid (within 20 months) among CD147 high patients, as compared with those with low (P<0.01) or medium (P<0.001) CD147 levels; P-values calculated by log-rank test. The histograms show that white blood count (WBC) number, age at diagnosis and proportion of patients with poor cytogenetics were comparable in the 3 (non-M3)-AML subgroups subdivided according to CD147 expression level.

A B C D
High vs. medium P=0.0003 High vs. low P=0.003 LSCs and a novel prognostic predictor in AML. 20,34,47 Interestingly, in non-M3 AMLs a strong positive correlation was observed between CD147 and CD371 expression. In this context, while a dedicated future study would shed light on the functional properties of LSCs of the fractions of (CD34 + CD147 + ) and (CD34 + CD371 + CD147 + ) AML cells, our data suggest that CD147 is expressed in LSCs and may be a potential new prognostic marker in AML and a potential therapeutic target. Taken together, our study demonstrates a pivotal role for CD147 in leukemic cell proliferation and indicates CD147 as a potential therapeutic target of AC-73 in AML.
Thus, targeting CD147 by using AC-73 or derivate molecules to induce autophagy or to inhibit the growth of leukemic clones, including LSCs, may be an effective option to improve leukemia treatment.