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A. Liu et al.
ing differentiation of Mks, GATA1 also leads ultimately to cessation of cell proliferation.19, 37 This is consistent with our findings showing that IMiDs-induced loss of GATA1 expression in Mks not only inhibits maturation, but also leads to excessive proliferation of megakaryocytic progen- itors. GATA1 also regulates numerous CDKs such as cyclin D1 and CDK inhibitors.25 Moreover, overexpression of cyclin D1/CDK4 in GATA1-deficient Mks restored their growth and polyploidization.25 Correspondingly, we observed that downregulating GATA1 in Mks by IMiDs resulted in decreased cyclin D1 expression.
Our findings of the role of IKZF1 in regulating GATA1 expression are in accordance with results by Dijon et al., who reported that lentivirally induced Ik6 (a dominant negative isoform of IKZF1) overexpression resulted in decreased expression of GATA1.29 Cell-cycle regulation is an important mechanism governing the long-term self- renewal potential of HSCs.38 Therefore, long-term treat- ment of patients with IMiDs might induce a pool of con- stantly cycling progenitors, leading to premature exhaus- tion of the stem cell pool. Indeed, patients receiving long- term treatment with IMiDs very often exhibit a hypocel- lular bone marrow associated with cytopenias.39 Interestingly, Malinge and colleagues reported that IKZF1 knockout mice showed increased megakarayopoiesis. Consistent with this phenotype, studies using mice acute megakaryoblastic leukemia (AMKL) cell line 6133 showed that IKZF1 suppresses megakaryopoiesis by negatively regulating GATA1.40 In contrast, our experiments found that the knockdown of IKZF1 resulted in decreased GATA1 protein expression in human CD34+ cells. The mechanism of IKZF1 in regulating GATA1 in mice malig- nant cells may be different from that in human hematopoietic progenitor cells.
Interestingly, Kronke et al. reported that Lenalidomide induces ubiquitination and degradation of Casein kinase 1α (CK1α) in del(5q) MDS.41 Furthermore, in a murine model with conditional inactivation of Csnk1a1, Schneider et al. demonstrated that Csnk1a1 haploinsuffi- ciency induces hematopoietic stem cell expansion.42 This is in accordance with our data showing that both poma- lidomide and lenlidomide potently downregulate CK1α in hematopoietic progenitors, although pomalidomide exhibits a slightly lower efficiency compared to Lenalidomide (Online Supplementary Figure S1).
GATA1 overexpression allowed development of a more mature Mk phenotype. In addition, it preserved expres- sion of megakaryocyte-specific transcription factors such as NFE2 and ZFPM1 and of cell cycle regulators, including cyclin D1, despite IMiDs treatment, which further indi- cates that an IMiDs-induced decrease in GATA1 critically affects pathways involved in self-renewal, cell cycle regu- lation and lineage commitment of CD34+ cells. GATA1 mutations resulting in functional silencing have been found in patients with thrombocytopenia or megakary- ocytic acute leukemia.32,43 Therefore, our data suggest that the IMiDs-induced downregulation of IKZF1 and GATA1 favors myeloid lineage commitment and maintains a pool of immature cycling CD34+ cells without maturation, which may lead to stem cell exhaustion. Furthermore, downregulation of GATA1 results in an imbalance of key cellular and molecular regulators that block Mks from continued maturation, likely impeding platelet release and ultimately resulting in thrombocytopenia. sAt this moment, the clinical relevance of our in vitro findings is not entirely clear. However, prolonged treatment with IMiDs has been shown to be associated with an increased risk of MDS/AML and ALL, especially in patients treated with alkylating agents like Melphalan.44 It is therefore possible that the IMiDs-induced increased number of cycling CD34+ cells may enhance the probability of acquiring secondary DNA damage and leukemogenic events induced by other drugs. Hence, in vivo testing of various treatment combinations with IMiDs to explore and potentially predict leukemogenic effects of certain combinations is needed.
Acknowledgments
The authors would like to thank Mr. Dale Lewis at the University of Pittsburgh Cell Culture and Cytogenetics Facility for excellent FISH analysis. We would also like to thank Dr. Griffin P. Rodgers at the Molecular and Clinical Hematology Branch, National Heart, Lung, and Blood Institute, Bethesda, MD for providing the pLenti V5 GATA1 expression vectors and empty vectors.
Funding
This study was supported by a grant from the LLS and R01CA175313 (SL). MYM, CL, HM were supported in part by RO1 HL093716.
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