Hematopoietic Stem Cells Are Not the Direct Target of Spontaneous Leukemic Transformation in p18 INK4C -Null Reconstituted Mice Youzhong Yuan, Hui Yu, Matthew J. Boyer, Xianmin Song, Shaonan Cao, Hongmei Shen, and Tao Cheng Department of Radiation Oncology, University of Pittsburgh School of Medicine and Stem Cell Biology Laboratory, University of Pittsburgh Cancer Institute, Pittsburgh, Pennsylvania Abstract Cell cycle inhibitors are important regulators in normal tissue regeneration and disruption of the regulators are involved in cancer development. Our recent study showed that the absence of the CDK inhibitor p18 INK4C (p18) enhances self-renewal of normal hematopoietic stem cell (HSC) in vivo , whereas previous studies by others showed an increased incidence of leukemogenesis in older p18-null mice. Here, we have examined potential leukemogenesis during experimentally induced regeneration of HSC in the absence of p18 in order to gauge the relation between these two processes. Reconstituted mice with p18-deficient HSCs under the condition of repetitive proliferative stress (serial transplantation) were followed for >3 years. T cell leukemia from the p18/ origin was recapitulated 24 months after secondary transplantation. However, no myeloid leukemia was found in the recipients. The T cell leukemia–initiating cells (mainly in a CD3 lo cell subset) did not share the same immunophenotype with normal HSCs and, in fact, the function of HSCs was significantly compromised with decreased abundance in the leukemic mice. Furthermore, we found that the p15 or p16 gene promoters were frequently methylated in the leukemic cells but not in HSCs. Our present study argues against the possibility of overgrowth of p18-null HSCs leading to a leukemic phenotype. The data also support the notion that p18 has an independent role in T cell maintenance such that CD3 + CD8 + cells, unlike HSCs, are more accessible to leukemogenic transformation after the loss of p18. (Cancer Res 2006; 66(1): 343-51) Introduction The hematopoietic stem cell (HSC) has defined therapeutic roles in clinical transplantation, but it might also directly or indirectly contribute to the development of leukemia due to its ability to self-renew and differentiate into multiple lineages over a lifetime (1, 2). The unique feature of HSC self-renewal must be physiologically balanced with cell differentiation or apoptosis. Imbalance of these processes may cause leukemogenesis, during which the leukemia-initiating cells (LICs) or leukemia stem cells (LSCs) must acquire a competitive self-renewal potential coupled with decreased cell death or a disrupted differentiation program to yield a leukemic phenotype (3). Therefore, it is vital that we gain a greater understanding of the relationship among these critical processes that underlie HSC kinetics in leukemogenesis as well as in normal hematopoietic regeneration. One of the fundamental mechanisms that coordinate these critical processes is cell cycle regulation. In mammalian cells, cell cycle progression is largely controlled at the G 1 phase (4). The G 1 phase is regulated by the sequential activation and inactivation of cyclin-dependent kinases (CDKs; refs. 5, 6). Two families of low molecular weight cyclin- dependent kinase inhibitors (CKIs), Cip/Kip and INK4, have been identified as capable of interacting with CDKs to suppress progression through G 1 . The Cip/Kip family, which includes p21 Cip1/Waf1 , p27 kip1 , and p57 Kip2 (p21, p27, and p57 hereafter), may interact with a broad range of cyclin-CDK complexes; whereas the INK4 family, which includes p16 INK4A , p15 INK4B , p18 INK4C , and p19 INK4D (p16, p15, p18, and p19 hereafter), specifically inhibit CDK4 and CDK6 kinases at early G 1 phase. Increasing lines of evidence suggest that CKIs may represent an interface between the cell cycle and upstream stem cell regulatory pathways (3). For instance, Bmi-1, a polycomb protein, critically regulates self-renewal of different adult stem cell populations through inhibition of p16 expression and its alternative reading frame (ARF; refs. 7–9). With knockout mouse models, previous work from several laboratories showed that p21 is crucial for the maintenance of stem cell quiescence in both hematopoietic and central nervous systems (10–14), whereas p27 more specifically inhibits the proliferation of early progenitor cells (10, 15, 16). Based on studies of the hematopoietic system, p18 seems to be an interesting and unique molecule because its absence results in the most significant enhancement of hematopoietic engraftment in mouse transplant models compared with the absence of other CKIs (p21, p27, and p16; refs. 10, 15, 17, 18). Loss of p18 increases in vivo self-renewing divisions of HSCs over a prolonged period of time and is able to compensate for the exhausting effect of irradiated hosts on transplanted HSCs (19), which implies that targeting of p18 may be used for therapeutic manipulations of human HSCs. However, p18-deficient mice exhibit predisposition to the development of both spontaneous and carcinogen-induced tumors in multiple organs (20–23). In hematopoietic and lymphoid systems, a small percentage (12%) of p18-null mice begin to develop T cell leukemia/lymphomas after reaching 1 year in age (21). Given the enhanced regeneration of HSC and the risks of T cell malignancy in the absence of p18, we sought to further define the relation between HSC self-renewal and potential leukemia development, and to explore the other molecular disruptions that, in addition to p18 deletion, lead to a leukemic phenotype. These issues Note: Y. Yuan and H. Yu contributed equally to this work. Requests for reprints: Tao Cheng, Office Suite 2.42e, Research Pavilion at The Hillman Cancer Center, University of Pittsburgh Cancer Institute, 5117 Center Avenue, Pittsburgh, PA 15213-1863. Phone: 412-623-3249; Fax: 412-623-7778; E-mail: chengt@ upmc.edu. I2006 American Association for Cancer Research. doi:10.1158/0008-5472.CAN-05-2945 www.aacrjournals.org 343 Cancer Res 2006; 66: (1). January 1, 2006 Research Article Research. on February 9, 2015. © 2006 American Association for Cancer cancerres.aacrjournals.org Downloaded from