634. The Role of Ink4a-Arf Locus in Control of Hematopoietic Stem Cell Proliferation

Abstract

The mechanisms that control hematopoietic stem cell (HSC) proliferation and self-renewal are incompletely understood, but are relevant for developing gene therapy strategies that target HSCs. Recent evidence shows that loss of Bmi-1, a polycomb transcriptional repressor of the Ink4a-Arf locus, results in progressive loss of HSCs in adult mice with subsequent failure of hematopoiesis. The Ink4a-Arf locus encodes two tumor suppressor proteins, p16Ink4a and p19Arf. Induced transcription of these genes results in cell cycle arrest in several model systems. Based on these facts, we have tested the hypothesis that these Ink4a-Arf gene products may play a role in limiting HSC self renewal during conditions of enhanced proliferation. We first tested whether p19Arf was induced in murine bone marrow cells that were cultured for 10–12 days in IL3, IL6, SCF, and 15% fetal calf serum, hypothesizing that the well described loss of HSCs under these conditions could be due to induction of p19Arf. Using cells from mice that had a GFP reporter gene inserted into the first exon of the p19Arf gene, we found that the p19Arf/GFP allele was significantly induced in the bulk cell population after culture, but not expressed in lineage negative, c-kit+, Sca1+ cells derived from uncultured bone marrow. Competitive repopulation studies were then performed using bone marrow from either p19Arf null mice, or from mice with a homozygous deletion that disrupt both p19Arf and p16Ink4a (Ink4a-Arf–/–). In both cases, there were no defects in repopulating activity when using either freshly isolated bone marrow cells, or cells grown in our 12 day cultures. These data indicate that the loss of HSCs with in vitro culture does not require either p19Arf or p16Ink4a. We next asked whether these genes play a role in the self-renewal of HSCs in vivo using serial bone marrow transplantation assay. Bone marrow cells from either strain or from wildtype controls were serially transplanted into lethally irradiated recipients in doses ranging from 1×105 to 1×106 cells per mouse. We are currently following the fourth group of transplant recipients. In the mice transplanted with bone marrow cells from Ink4a-Arf–/– donors, a survival advantage was noted relative to mice transplanted with strain-matched, wildtype controls. For instance, in secondary recipients receiving 1×105 bone marrow cells, 100% survival was noted in mice transplanted with Ink4a-Arf–/– cells after 10 weeks compared to 20% survival in mice receiving the same dose of wildtype cells. A similar survival advantage was also noted in groups given the lowest cell doses within the 3rd and 4th transplants. In parallel experiments using p19Arf null mice, there was no difference in survival relative to wildtype controls. Measurement of blood counts from recipients in all cohorts clearly showed that the deaths were due to hematopoietic failure. These results show that either both p16Ink4a and p19Arf can inhibit HSC self-renewal in a serial transplant setting, or that only p16Ink4a is necessary. Because animals transplanted with Ink4a-Arf–/– cells do die with hematopoietic failure, albeit later than wildtype controls, we conclude that mechanisms not associated with the Ink4a genes also play a role in limiting HSC self-renewal in vivo.