Human stem cell factor-antibody [anti-SCF] enhances chemotherapy cytotoxicity in human CD34+ resistant myeloid leukaemia cells

Abstract

Acute myeloid leukaemia (AML) is a heterogenous malignant disease with diverse biological features in which disease progression at the level of CD34+ cells has a major impact on the resistance to chemotherapy and relapse. The AML blast cells in these elderly patients are often characterised by several unfavourable covariates that predict the poor treatment outcome, including high stem cell marker CD34 expression, minimally or undifferentiated features, high P-glycoprotein expression, high bcl-2/bax ratio, unfavourable karyotype and more frequent internal tandem duplications (ITDs) and mutations of class III receptor-type tyrosine kinase for key haematopoietic cytokines: Flt-3 (receptor for Flt-ligand), c-kit (receptor for stem cell factor) and fms (receptor for M-CSF). Testing the new and more specific molecular-targeted therapeutic approaches in CD34+ AML cells can provide the basis for a more effective combined molecular/chemotherapy regimen and may consequently improve the treatment outcome in elderly AML patients. Therefore, the present study was performed to evaluate whether stem cell factor-antibody (anti-SCF) can enhance the efficacy of the two main chemotherapeutic drugs used in AML therapy: cytarabine and daunorubicin at low doses in human-resistant CD34+ AML cells, in an attempt to identify a novel effective regimen with tolerable side-effects for elderly AML patients. The effect of anti-SCF on each of the two chemotherapeutic drugs-induced apoptosis and necrosis was investigated in KG1a human-resistant CD34+ AML cells expressing P-glycoprotein to determine its enhancing activity. Anti-SCF has significantly enhanced the low dose cytarabine- and daunorubicin-induced apoptosis + necrosis in KG1a CD34+ AML cells from 12.0 ± 1.7 to 40.9 ± 5.9% and from 16.3 ± 0.9 to 48.9 ± 1.0%, respectively, p < 0.01. It has also exerted its significant enhancement activity on the low dose cytarabine- and daunorubicin-induced apoptosis + necrosis in KG1a CD34+ AML cells in the presence of SCF, p < 0.05. Anti-SCF has significantly enhanced the low dose cytarabine- and daunorubicin-induced bcl-2 reduction in KG1a CD34+ AML cells from 26.7 ± 0.6 to 64.6 ± 1.0% and from 59.8 ± 3.1 to 80.1 ± 7.9%, respectively, p < 0.01. The addition of SCF has not altered the low dose cytarabine- and daunorubicin-induced bcl-2 reduction in KG1a CD34+ AML cells (Table 4). Anti-SCF has also significantly enhanced the low dose cytarabine- and daunorubicin-induced bcl-2 reduction in KG1a CD34+ AML cells in the presence of SCF, p < 0.05. The unique potent enhancing activity of anti-SCF on low dose chemotherapy-induced apoptosis and necrosis in extremely resistant AML cells suggest a novel promising role for the treatment of elderly AML patients. Further studies are warranted to evaluate a similar enhancing effect for anti-SCF in blast cells from elderly AML patients in primary cultures before its introduction in a pilot clinical study. In conclusion, the combination of anti-SCF and the low dose cytarabine provides a promising solution for the dilemma of therapy in elderly AML patients.

Introduction

Acute myeloid leukaemia (AML) is a heterogenous malignant disease with diverse biological features in which disease progression at the level of CD34+ cells has a major impact on resistance to chemotherapy and relapse [1]. Approximately 60% of the AML cases are in patients older than 60 years [2]. These elderly patients do not respond well to the current conventional chemotherapy used in younger AML patients due to the intrinsic resistant nature of their AML cells and/or their poor tolerance to the conventional chemotherapy regimens [3]. Very little progress has been made over the past 30 years in the treatment outcome of the AML patients older than 60 years and using chemotherapy.

The AML blast cells in these elderly patients are often characterised by several unfavourable covariates that predict poor treatment outcome, including high stem cell marker CD34 expression, minimally or undifferentiated features, high P-glycoprotein expression, high bcl-2/bax ratio, unfavourable karyotype and more frequent internal tandem duplications (ITDs) and mutations of the class III receptor-type tyrosine kinase for key haematopoietic cytokines: Flt-3 (receptor for Flt-ligand), c-kit (receptor for stem cell factor) and fms (receptor for M-CSF) [4], [5], [6], [7], [8], [9], [10], [11], [12], [13]. The frequent occurrence of ITDs and mutations in this receptor tyrosine kinase for Flt-3, c-kit and fms in AML cells were reported in 25–35%, 10–20% and 5–20% of the elderly patients, respectively [14]. Several tyrosine kinase inhibitors have been recently synthesised including imatinib (Gleevec or ST1571) as c-kit inhibitor, CEP-701 as flt-3 inhibitor and SU5416 as inhibitor for all the three, flt-3, c-kit and fms [14]. Their pilot phase I/II clinical trials as single agents in refractory AML patients revealed modest clinical activity in these patients and tolerable side-effects [15], [16], [17], [18], [19], [20].

Testing the new and more specific molecular targeted therapeutic approaches in CD34+ AML cells can provide the basis for a more effective combined molecular/chemotherapy regimen and may consequently improve the treatment outcome in elderly AML patients.

The stem cell factor (SCF) receptor: c-kit is expressed in 63% of the AML patients, two-third of which are also CD34+ and have lower complete remission rate and poor prognosis [21], [22]. Both c-kit and CD34 are overexpressed in AML blast cells compared to normal bone marrow [23] and associated with significant low complete remission and relapse-free survival rates among the AML patients [24], [25]. Also, c-kit exon 8 mutations seem to be a significant factor adversely affecting the relapse rate [26].

On the other hand, there is little information about the effect of the antibody against SCF (anti-SCF) on the chemotherapy cytotoxicity in AML cells. Therefore, the present study was performed to evaluate whether anti-SCF can enhance the efficacy of the two main chemotherapeutic drugs used in AML therapy: cytarabine and daunorubicin at low doses in human-resistant CD34+ AML cells in an attempt to identify a novel effective regimen with tolerable side-effects for elderly AML patients. The effect of anti-SCF on each of the two chemotherapeutic drugs-induced apoptosis and necrosis was investigated in KG1a human-resistant CD34+ AML cells expressing P-glycoprotein to determine its enhancing activity.