Abstract
In chronic lymphocytic leukemia (CLL) the proliferation rate and resistance to drug-induced apoptosis are recognized as important factors in the outcome of treatment. In this study, we assess the activity and the mechanism of action of the prototype cell division cycle kinase 7 (Cdc7) inhibitor, PHA-767491, which inhibits the initiation of DNA replication but also has cyclin-dependent kinase 9 (Cdk9) inhibitory activity. We have studied the effects of this dual Cdc7/Cdk9 inhibitor in both quiescent CLL cells and CLL cells that have been induced to proliferate using a cellular coculture system that mimics the lymph node microenvironment. We find that this compound, originally developed as a DNA replication inhibitor, is particularly active in promoting mitochondrial dependent apoptosis in quiescent CLL cells purified from peripheral blood of patients regardless of recognized risk factors. In this setting, apoptosis is preceded by a decrease in the levels of Mcl-1 protein and transcript possibly due to inhibition of Cdk9. Following stimulation by CD154 and interleukin-4, CLL cells become highly chemoresistant, reenter into the cell cycle, reexpress Cdc7 kinase, a key molecular switch for the initiation of DNA replication, replicate their DNA, and undergo cell division. In this context, treatment with PHA-767491 abolished DNA synthesis by inhibiting Cdc7 but is less effective in triggering cell death, although Mcl-1 protein is no longer detectable. Thus, dual Cdc7/Cdk9 inhibition has the potential to target both the quiescent and actively proliferating CLL populations through two distinct mechanisms and may be a new therapeutic strategy in CLL.
Highlights
Chronic lymphocytic leukemia (CLL) is the commonest leukemia in the Western world
Peripheral blood CLL cells are sensitive to the cell division cycle kinase 7 (Cdc7)/cyclin-dependent kinase 9 (Cdk9) inhibitor PHA-767491
Peripheral blood CLL cells do not proliferate [14] but surprisingly, when challenged with PHA-767491 (Fig. 1A and Supplementary Fig. S1), originally developed as a DNA replication inhibitor [27], we observed a concentration and time-dependent induction of apoptosis measured by phosphatidylserine exposure (Supplementary Fig. S2)
Summary
Chronic lymphocytic leukemia (CLL) is the commonest leukemia in the Western world. Despite advances in treatment, CLL remains an incurable disease. CLL cells in the peripheral blood represent a population of nondividing tumor cells that display high chemosensitivity; in this setting the antiapoptotic protein myeloid cell leukemia sequence 1 (Mcl-1) seems to be a critical survival factor [3,4,5,6,7]. Instead CLL cells that reside in secondary lymphoid organs and the bone marrow display high chemoresistance and proliferative capacity [8, 9]. A subpopulation of CLL cells expressing the cell cycle markers Ki67 and cyclin D1 can be identified in pseudofollicles or proliferation centers [10] In this environment, the interactions between leukemic and accessory cells, such as monocyte-derived nurse-like cells, CD4þ CD154þ T cells and mesenchymal stromal cells, provide essential signals to maintain CLL survival and growth [11]. Proliferation centers may harbor dividing and resistant leukemic cells, which sustain clonal maintenance, growth, and genetic diversification and may represent a relevant chemotherapeutic target to limit tumor burden and clonal evolution
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