Search

Systemic mastocytosis (SM) is a myeloid neoplasm involving uncommitted and mast cell (MC) committed hematopoietic progenitors. In most SM patients, the transforming c-KIT point mutation D816V is detectable. However, so far, little is known about the molecular mechanisms underlying KIT D816V-induced transformation. We examined the expression of two Bcl-2 family members, i.e. anti-apoptotic Mcl-1 and the pro-apoptotic tumor suppressor Bim, in the KIT D816V+ human mast cell line HMC-1. As assessed by RT-PCR, immunocytochemistry, and Western blotting, these cells were found to express Mcl-1 mRNA and the Mcl-1 protein, whereas only trace amounts of Bim were detectable in HMC-1 cells. Exposure of HMC-1 cells to the KIT D816V-targeting drug PKC412 resulted in a substantial increase in expression of Bim and in a marked decrease in expression of Mcl-1. Correspondingly, in Ba/F3 cells with doxycycline-inducible expression of KIT D816V (Ton.Kit.D816V), exposure to doxycycline (induction of KIT D816V) resulted in a significant decrease in expression of Bim, and PKC412 was found to counteract the KIT D816V-induced downregulation in Bim in these cells. Transfection of HMC-1 cells with Mcl-1-specific antisense oligonucleotides (ASO) or an Mcl-1-specific siRNA resulted in reduced survival and in an increased percentage of apoptotic cells compared to cells transfected with scramble control oligonucleotides. In consecutive experiments, combinations of PKC412 and Mcl-1 ASO were applied using varying drug concentrations. In these experiments, both compounds were found to synergize with each other in producing growth inhibition and apoptosis in HMC-1 cells. By contrast, no synergism was observed with combinations of Mcl-1 ASO and imatinib or with Mcl-1 ASO and 2CdA. In summary, our data show that the Bcl-2 family members Mcl-1 and Bim are differentially expressed in neoplastic MC, and that targeting of Mcl-1 is associated with reduced survival of HMC-1 cells. In addition, our data show that Mcl-1 ASO synergize with PKC412 in producing growth inhibition in neoplastic MC. These data support the concept that optimal growth inhibition of neoplastic MC in aggressive variants of SM may be achieved with combinations of targeted drugs.

The cell surface antigen Siglec-3 = CD33 is becoming increasingly important as target of therapy in acute myeloid leukemia (AML). In particular, a conjugate consisting of the humanized CD33 antibody P67.6 (gemtuzumab) and the cytostatic drug calicheamicin has been developed for clinical use and was found to work as an effective antileukemic agent (Mylotarg®) in patients with CD33+ AML. In normal myelopoiesis, expression of CD33 is restricted to advanced stages of differentiation, whereas primitive stem cells do not express CD33 (Siglec-3). In line with this notion, CD33-targeting therapy is a non-myeloablative approach. In the present study, we asked whether leukemic stem cells in patients with AML express CD33. For this purpose, a multicolor-staining technique was applied in eleven patients with AML. Leukemic stem cells were defined as CD34+/CD38−/CD123+ cells. In all patients in whom the majority of myeloblasts expressed CD33 (=CD33+ AML, n=8), the AML progenitor cells reacted with the CD33 antibody P67.6. Repopulation experiments utilizing NOD/SCID mice confirmed that the AML stem cells in these patients reside within the CD33+ subpopulation of leukemic cells. Moreover, AML stem cells (CD34+/CD38−/CD123+ cells) highly purified (>98% purity) from patients with (CD33+) AML by cell sorting, were found to express CD33 mRNA in RT-PCR analyses. To demonstrate that AML stem cells can also reside within the CD33-negative fraction of the AML clone, we purified CD33-negative cells in a patient with AML in whom a majority of leukemic stem cells were found to lack CD33. In this particular patient, the CD33-negative cells were found to repopulate NOD/SCID mice with leukemias in the same way as the entire leukemic clone did. The CD33 antigen was neither detectable on CD34+/CD38− cells in the normal bone marrow nor on leukemic stem cells in patients with CD33-negative AML. In summary, our data show that leukemic stem cells in patients with CD33+ AML frequently express the target receptor CD33. This observation is in favor of novel treatment concepts employing CD33-targeting antibodies (Mylotarg®) in acute myeloid leukemia.

The BCR-ABL tyrosine kinase inhibitor STI571 (Imatinib) has successfully been introduced in the treatment of patients with CML. However, despite encouraging initial data and high expectations long term results are not available yet, and recent data suggest that resistance against STI571 can occur. Therefore, current studies focus on novel potential drug targets in CML cells. Recently, several anti-apoptotic members of the Bcl-2-family including Mcl-1 have been implicated in the regulation of survival of BCR/ABL+ cell lines and therefore proposed as potential targets. We have examined expression of Mcl-1 in primary CML cells and various BCR/ABL-transformed cell lines. Independent of the phase of disease, isolated primary CML cells expressed Mcl-1 mRNA and the Mcl-1 protein in a constitutive manner. The BCR/ABL-inhibitor STI571 decreased the expression of Mcl-1 in these cells. Correspondingly, BCR/ABL enhanced Mcl-1 promoter activity, Mcl-1 mRNA, and Mcl-1 protein in Ba/F3 cells. BCR/ABL-dependent expression of Mcl-1 in Ba/F3 cells was counteracted by the MEK-inhibitor PD98059, but not by the PI3-kinase inhibitor LY294002. Identical results were obtained for constitutive expression of Mcl-1 in primary CML cells and the CML-derived cell lines K562 and KU812. To investigate the role of Mcl-1 as a survival-related target gene in CML cells, Mcl-1 siRNA or an Mcl-1 antisense oligonucleotide (ASO) were applied. Both the ASO and the siRNA-induced downregulation of Mcl-1 were found to be associated with a substantial decrease in viability of K562 cells. Moreover, the Mcl-1 ASO was found to cooperate with STI571 in producing growth-inhibition in both STI571-sensitive and STI571-resistant K562 cells. Together, our data identify Mcl-1 as a survival factor and novel target in CML. Whether this concept is of clinical significance remains to be determined in forthcoming clinical trials.