Identification of molecular mechanisms governing normal and malignant human blood stem cell self-renewal
Cell Biology
Final Report Abstract
This study aimed at gaining further insights into normal and malignant human blood stem cell biology in order to advance current knowledge and expand therapeutic options for hematopoietic stem cell (HSC) transplantation and leukemia patients. Since bona fide stem cell activity can only be read out only in long-term experiments (≧20wks) assaying repopulation and differentiation capacity, a high-throughput in vivo gain-of-function screen of 64 potential human stem cell self-renewal regulators was designed and carried out in a competitive fashion. Thereby the list could be narrowed down to 10 promising candidate genes that besides HOXA4 have not been attributed a functional role in blood stem cells. However, overexpressing these candidate genes in human cord blood stem and progenitor cells with subsequent xenotransplantation did not robustly result in the anticipated enhanced overall human cell engraftment. Hence, the major technical challenges faced in the course of this project were huge variability of biological replicates used for these long-term experiments on top of the access to the primary human cell material and cohorts of mice necessary for these kind of experiments crucial to determine self-renewal activity. Since most candidate genes are understudied in general, not only clues but also specific reagents (antibodies, inhibitors, etc.) are rare that are crucial to gain insight in the underlying molecular mechanism. Nonetheless, aware of these challenges already beforehand this project was never intended to be completed within the funding time as stated in the application. As a consequence, even more striking in the light of these challenges and inconsistencies across biological replicates was, that all candidate genes but one consistently induced a common phenotype of delayed repopulation that either did not alter or increased stem cell frequency when further analyzed focusing on the two candidates PHGDH and C3orf54/INKA1, respectively. The resulting follow-up functional and mechanistic studies with cord blood and in a primary human AML model imply that the induced delayed repopulation kinetics are a result of protracted stem cell latency either conferred by antioxidant protection or induction of stem cell quiescence via global epigenetic changes. Overall my work is leading to the uncovering of a novel stem cell property in xenograft based assay systems that might provide valuable insight into how normal stem cells are governed. Thereby, this study fulfilled its aim to identify previously unknown players in normal and leukemia stem cell (LSC) maintenance. Moreover, it also challenges the field with the observed latency phenomenon consistently induced by multiple candidate genes that are upregulated in LSC and hence, these kinetics can be understood as a phenocopy of disease latency in AML remission with relapse eventually initiating from a maintained leukemia stem cell. Consequently, this study sets the stage to investigate patterns of activation and latency of rare stem cells in normal hematopoiesis and leukemia and how these states are regulated. Identifying and dissecting those programs that not only distinguish HSC and LSC from their respective progenitor populations, but also those that are LSC specific will potentially define new targets for drugs affecting the vulnerabilities of LSC while sparing normal HSC. The novelty of the screen design, the candidate gene's previously unknown role in hematopoiesis, leukemogenesis and stem cells in general in addition to the latency phenomenon are the basis for publications currently in preparation.
Publications
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(2016) Deregulation of DUX4 and ERG in acute lymphoblastic leukemia. Nature Genetics. 48(12): 1481-1489
Zhang, Jinghui; Mccastlain, Kelly; Yoshihara, Hiroki; Xu, Beisi; Chang, Yunchao; Churchman, Michelle L.; Wu, Gang; Li, Yongjin; Wei, Lei; Iacobucci, Ilaria; Liu, Yu; Qu, Chunxu; Wen, Ji; Edmonson, Michael; Payne-Turner, Debbie; Kaufmann, Kerstin B.; Takay
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(2016) Distinct routes of lineage development reshape the human blood hierarchy across ontogeny. Science. 351(6269)
Notta, Faiyaz; Zandi, Sasan; Takayama, Naoya; Dobson, Stephanie; Gan, Olga I.; Wilson, Gavin; Kaufmann, Kerstin B.; McLeod, Jessica; Laurenti, Elisa; Dunant, Cyrille F.; McPherson, John D.; Stein, Lincoln D.; Dror, Yigal & Dick, John E.
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(2016) miR-126 Regulates Distinct Self-Renewal Outcomes in Normal and Malignant Hematopoietic Stem Cells. Cancer Cell. 29(2): 214-28
Lechman, Eric R.; Gentner, Bernhard; Ng, Stanley W.K.; Schoof, Erwin M.; van Galen, Peter; Kennedy, James A.; Nucera, Silvia; Ciceri, Fabio; Kaufmann, Kerstin B.; Takayama, Naoya; Dobson, Stephanie M.; Trotman-Grant, Aaron; Krivdova, Gabriela; Elzinga, Janneke; Mitchell, Amanda; Nilsson, Björn; Hermans, Karin G.; Eppert, Kolja; Marke, Rene; ... & Dick, John E.
