CD157 REGULATES THE SENSITIVITY OF ACUTE MYELOID LEUKEMIA CELLS TO CHEMOTHERAPY BY MODULATING THE APOPTOTIC RESPONSE

INTRODUCTION

Figure 2. CD157 ligation promotes survival in OCI-AML3 cells and

triggers the transduction of intracellular signals

CONCLUSIONS

Figure 1. Impact of CD157 ligation on primary AML cells viability

Figure 3. Effect of CD157 knockdown in AML cells on survival and

sensitivity to AraC

)

CD157 REGULATES THE SENSITIVITY OF ACUTE MYELOID LEUKEMIA CELLS

TO CHEMOTHERAPY BY MODULATING THE APOPTOTIC RESPONSE

Y. Yakymiv

_{, S. Augeri}

_{, G. Fissolo}

_{, C. Bracci}

_{, S. Peola}

_{, S. D’Ardia}

_{, S. Aydin}

_{, M. Massaia}

_,

E. Ortolan

_{and A. Funaro}

1_{Laboratory of Immunogenetics, Department of Medical Sciences, University of Torino, Italy.} 2_{Department of Hematology and Oncology, A.O.U Città della Salute e}

della Scienza, Torino, Italy. 3_{Laboratory of Blood Tumor Immunology, Department of Molecular Biotechnology and Health Sciences, University of Torino, Italy;}

Hematology Division, AO S Croce e Carle, Cuneo, Italy.

Figure 4. Effect of S63845 Mcl-1 inhibitor on CD157-positive cells as a

single agent and when combined with AraC

Acute myeloid leukemia (AML) is a heterogeneous malignancy characterized by expansion of immature myeloid cells. Patients survival is low due to the occurrence of chemoresistance and tumor relapse. However, mechanisms underlying drug resistance in AML patients are still largely unknown. Therefore, to clarify the molecular basis of drug resistance and to identify novel markers with potential clinical utility represent an urgent need. CD157, an adhesion molecule expressed by myelomonocytic cells, bone marrow stromal cells and selected epithelial cancers, was found expressed in more than 90% of AML both at diagnosis and after relapse, although at variable levels. Moreover, CD157 is involved in the protection of selected tumor cells from apoptosis and from platinum-induced cytotoxicity.

In this study we investigated i) the role of CD157 in drug response, and ii) explored the intracellular pathways underlying the functional properties of CD157 in AML cells.

In order to evaluate the functional role of CD157 in AML, blasts were isolated from bone marrow of newly diagnosed AML patients and were treated with an agonistic α-CD157 (SY11B5) antibody for 24h. Ligation of α-CD157 resulted in protection of AML cells from spontaneous cell death induced by in-vitro culture, compared to the isotype-matched control (Fig. 1 A), and this effect was time- and dose-dependent (Fig. 1 B,C). Moreover, ligation of CD157 was able to improve survival of leukemic blasts exposed to Cytarabine (AraC) treatment (Fig. 1 D,E).

U937, THP1 (with high levels of CD157 expression) and OCI-AML3 (with intermediate levels of CD157) cell lines were chosen as in-vitro models to evaluate the functional role of CD157 in AML (Fig. 2 A). To reproduce spontaneous apoptosis occurring in fresh AML blasts when kept in culture, we maintained OCI-AML3 cells in serum-free medium. Under nutrient deprivation, OCI-AML3 treated with α-CD157 mAb for 24h showed a significantly increased cell survival (Fig. 2 B). Moreover, ligation of CD157 for 24h increased the phosphorylation of mTOR, FAK, AKT and ERK, which mediate pro-survival signals, and enhanced the expression of Bcl2 anti-apoptotic protein in both fresh AML cells and in OCI-AML3 cells (Fig. 2 C,D)

We selected CD157-positive U937, THP1 and OCI-AML3 cell lines for gene knockdown to investigate the effect of loss of CD157. Results showed that loss of CD157 increased the sensitivity of THP1 (Fig. 3 A) and OCI-AML3 (Fig. 3 B) cells to nutrient deprivation, compared to the CD157-positive counterpart. Moreover, CD157-negative U937 cells (U937/shCD157) proved significantly more sensitive to AraC treatment than CD157-positive U937 cells (Fig. 3 C). In keeping with this observation, CD157-CD157-positive cells treated with AraC showed a remarkable increase of AKT protein phosphorylation (Fig. 3 D), and decreased cleavage of Bax pro-apoptotic protein, counterbalanced by reduced degradation of anti-apoptotic Mcl-1 protein (Fig. 3 E). These findings suggest that the modulation of these intracellular pathways could be implicated in the pro-survival effects mediated by CD157.

Based on the previous observations, we investigated the effect of S63845 (a specific inhibitor of Mcl-1 protein) used alone or in combination with chemotherapy. At molecular level, treatment with S63845 induced a stronger increase of Mcl-1 protein expression and half life extension in CD157-positive cells than in CD157-negative cells, with the activation of BAX/BAK-dependent mitochondrial apoptotic pathway (Fig. 4 A). Treatment of CD157-positive cells with the S63845 inhibitor in combination with AraC was able to restore the sensitivity of CD157-positive cells to chemotherapy (Fig. 4 B). By means of combination index (CI) method of Chou-Talalay (Chou and Talalay 1983) we observed a strong synergism (CI<0.5) between AraC and S63845 in CD157-positive cells, keeping a constant 1:100 S63845:AraC ratio (CI<1, CI=1, and CI>1 indicates synergism, addictive effect and antagonism, respectively) (Fig. 4 C).

 CD157 contributes to primary AML cells and cell lines survival and resistance to AraC by induction of intracellular signals converging on the PI3K/Akt/Bcl2 pathway.

 CD157 knockdown enhances AML cells sensitivity to nutrient deprivation and AraC toxicity.

 CD157-positive U937 cells are less sensitive than CD157-negative cells to AraC treatment and upregulate anti-apoptotic Mcl-1 protein

 S63845 Mcl-1 inhibitor is able to restore the sensitivity of CD157-positive cells to AraC, moreover, combined use of S63845 and AraC has a strong synergic effect

 Our results demonstrate an essential role of CD157 as a regulator of survival, stress resistance and drug response in AML, and suggest a potential clinical utility of CD157 as biomarker to select AML patient that could benefit from treatment combining AraC with Mcl-1 inhibitors.