A Novel Role of Pim Serine/Threonine Kinases in Hematopoiesis: Pim 1 Kinase Increases Hematopoietic Stem Cell Population

Pim kinases are a small family of constitutively active, highly conserved serine/threonine kinases, and have 3 members (Pim1, 2 and 3). Pim kinase is considered to be oncogenic because ∼5-10% of murine Pim1 transgenic (Tg) mice driven by the Eμ immunoglobulin promoter developed lymphoid malignancies (van Lohuizen, M. et al, 1989, Cell 56:673-682). However, the roles of Pim kinases in the development of myeloid malignancies and in the regulation of hematopoietic stem cells (HSCs) are not completely understood. In the current study, we used Tg and knockout (KO) mouse models to characterize the roles of Pim1 kinase in myeloid oncogenesis and/or hematopoiesis. The regulation and the downstream events of Pim1 kinase in HSCs were also investigated. We used the vav hematopoietic regulatory elements to drive human PIM1 gene expression in our Tg mouse model. Vav regulatory elements have been shown to promote hematopoietic- specific expression and induce myeloid malignancies (Lin, Y.W. et al, 2005. Blood 106:287-295).

We generated PIM1 Tg mice bearing human PIM1 kinase under the vav-regulatory elements (vav-hPIM1). Pim1, 2, 3 single, double and triple KO mice were also available to us and were used in this study. The percentage and the absolute number of c-Kit⁺Sca-1⁺Lin⁻ (LSK) hematopoietic stem/progenitor cells (HSPCs) in bone marrow and in spleen were measured by flow cytometry. The frequency of hematopoietic progenitor cells was measured by colony-forming unit assays (CFU-GM, BFU-E and CFU-GEMM). The capacity of HSPCs to protect and reconstitute lethally irradiated recipient mice was determined using a bone marrow transplantation model. The regulation of PIM1 expression in HSPCs was determined by quantitative RT-PCR following exposure to various cytokines including IL-3, IL-6, stem cell factor, TPO, and Flt3 ligand. Finally, HSPCs were transduced with lentiviral vector expressing PIM1 and its downstream gene targets were measured by quantitative RT-PCR.

1). Vav-hPIM1 Tg mice developed lymphoid, but not myeloid, malignancies. In our vav-hPIM 1 Tg mice, human PIM1 kinase was expressed in all hematopoietic tissues including bone marrow, spleen and thymus, but not in non-hematopoietic tissues. Approximately10% of vav-hPIM 1 Tg mice developed lymphoid leukemia/lymphoma. Interestingly, no myeloid malignancies were observed in vav-hPIM 1 Tg mice. 2). Vav-hPIM1Tg mice had increased HSPC population. In those vav-hPIM1 Tg mice that were free of leukemia/lymphoma, a > 2 fold increase in the number of LSK HSPCs were noted when compared to age-matched, wild-type littermates (p <0.05). Vav-hPIM 1 Tg mice also exhibited enlarged spleen, higher CXCR4 surface expression and increased frequencies of CFUs, compared to littermate controls. Additionally, lethally irradiated mice transplanted with splenocytes from vav-hPIM1 Tg mice had better survival compared to those transplanted with splenocytes from wild-type control mice. 3). Pim1/2 KO mice had decreased HSPC population. When compared to wild-type littermate controls, Pim1/2 KO mice exhibited lower number of LSK HSPCs, lower CXCR4 surface expression, and reduced frequencies of CFUs. Furthermore, lethally irradiated mice transplanted with marrow cells from Pim1/2 KO mice had much slower hematopoietic recovery, compared to those receiving marrow from normal control mice. 4). The expression of Pim1 kinase in HSCs was up-regulated by cytokine stimulation, in particular, IL-3, IL-6, and stem cell factor, suggesting that cytokines in marrow niche microenvironment could affect Pim 1 expression in HSCs. 5). Finally, we identified a gene family that was up-regulated when Pim1 kinase was over-expressed in HSCs, illustrating the mechanism through which Pim1 enhances hematopoiesis.

Our current studies demonstrate a novel role of Pim serine/threonine kinase in hematopoiesis. Our data also provide a novel molecular mechanism in the regulation of self-renewal, proliferation, and expansion of HSCs.

We thank Dr. Jerry M. Adams, Walter and Eliza Hall Institute of Medical Research, Australia for the kind gift of vav-hCD4 vector, and Dr. Peter D. Aplan, NCI, Bethesda MD for advice on developing the construct for generating Tg mouse. This work is partly supported by MUSC Hollings Cancer Center Startup Fund, Hollings Cancer Center ACS IRG, and ASCO Conquer Cancer Foundation Career Development Award.

No relevant conflicts of interest to declare.