The t(15;17)-Associated Fusion Protein PML/RAR Upregulates c-Kit Which Contributes to the Induction of the Leukemic Phenotype.

The pathogenesis of acute myeloid leukemia (AML) is strictly related to a block of terminal differentiation combined with an increased proliferation of hematopoietic progenitors (HP) in the bone marrow (BM). Furthermore an aberrant self-renewal of leukemic stem cells seems to be obligatory for the establishment of the leukemic clone in the BM. The block of differentiation is due to a deregulated function of differentiation-relevant transcription factors, whereas the proliferation is induced by aberrantly activated signaling pathways related to growth factor-dependent receptor kinases such as Flt3 or c-Kit (CD117). The aberrant self renewal can be attributed to specific pathways such as the Wnt-signaling, which play an important role also in the maintenance of the normal hematopoietic stem cell (HSC). The acute promyelocytic leukemia (APL) is characterized by the t(15;17) which leads to the expression of the PML/RAR fusion protein in the leukemic cells. PML/RAR mediates the APL phenotype given by a differentiation block at the promyelocytic level and an increased self renewal of the APL stem cells by the activation of the Wnt-signaling. The differentiation block, but not the aberrant self renewal, can be overcome by treatment with ATRA resulting ina high percentage of complete remissions. Nevertheless, if ATRA is used as a monotherapy a relapse is inevitable. APL blasts are frequently positive for constitutive activating Flt3 mutations and are constantly c-Kit-positive. Given the fact that c-Kit is a stem cell marker, the expression of c-Kit has to be considered aberrant and not related to the differentiation stage of the promyelocytes in APL.

Therefore we investigated first the relationship between PML/RAR and the aberrant expression of c-Kit and then the role of aberrantly activated c-Kit for the pathogenesis of APL by studying its influence on the biology of early HSC. Here we report that i.) in contrast to the t(8;21)-associated AML-1/ETO, PML/RAR activated the c-Kit promotor in HP; ii.) the inhibition of the endogenous c-Kit kinase activity by imatinib or by AZD2171 abrogated the aberrant “replating efficiency“ of PML/RAR-positive HSC; iii.) activated c-Kit (c-Kit-D814H) accelerated cell cycle progression of Sca1+/lin- HSC; iv.) activated c-Kit blocked the differentiation of Sca1+/lin- HSC and increased their “replating efficiency“; v.) “colony forming unit-spleen“ (CFU-S) as well as “competitive repopulation“ assays“ (CRA) revealed that c-Kit-D814H strongly increased the “self renewal“-potential of Sca1+/lin- HSC; vi) c-Kit-D814H augmented the migration of Sca1+/lin- HSC into a 3D stromal spheroid model based on M2-10B4 cells, but did not have any influence on their adhesion (flow chamber on TNFalpha-stimulated HUVEC cells) as well as on their chemotaxis (SDF-1 gradient in transwell assays); vii.) c-Kit-D814H further increased the aberrant replating efficiency of PML/RAR- as well as of AML-1/ETO-positive HSC.

Taken together our results strongly indicate not only that c-Kit importantly contributes to the leukemogenesis of APL, but that PML/RAR has a dual effect on c-Kit - the amplification of its expression at the promotor level as well as driving its expression in cells which normally do not have this proliferation stimulus. Thus it seems that there is a sort of positive feedback between PML/RAR and c-Kit which establishes c-Kit as a valuable therapeutic target for the treatment of APL-patients.