Investigation of KLF5 Function in Normal Hematopoiesis.

Abstract 2313

Krüppel-like factor 5 (KLF5) is a member of a highly conserved group of transcription factors (the KLF family) that have regulatory roles in development and in the growth and differentiation of many adult tissues. In humans, KLF5 is located at 13q21–22, which is frequently lost in multiple tumour types, including tumours of the breast, cervix, endometrium, ovary and prostate where it is associated with loss of KLF5 expression, however little is known about the potential role of Klf5 in the hematopoietic system. We initially identified that Klf5 mRNA is up-regulated during myeloid differentiation of murine myeloid cell lines (J Leukoc Biol, 80: 433), consistent with published microarray data in human and mouse primary haemopoietic system. Functionally we have shown that knockdown of Klf5 gene expression reduces granulocyte differentiation in response to cytokine simulation (Leuk Res, 2012. 36: 110).

Here we show that retroviral over-expression of Klf5 in the murine myeloid FDB1 cell line induces growth arrest associated with differentiation and increased apoptosis. Consistent with this, enforced expression of Klf5 in primary mouse bone marrow progenitors inhibited cell growth in liquid culture, with a reduction in c-Kit+ cells co-incident with an increase in differentiated myeloid cells at day 3 post-transduction. In colony forming assays, Klf5-expressing cells had significantly reduced myeloid colony forming ability in response to a combination of IL-3, IL-6 and SCF as well as M-CSF and G-CSF alone.

To extend this functional analysis of Klf5 we have generated an in vivo gene-ablation model. As non-conditional Klf5 knockout (KO) die at embryonic day 8.5 we have generated pan-hematopoietic Klf5 conditional gene KO mice by using the Cre/LoxP recombination system with vav-cre transgenic mice. For this we first generated Klf5 floxed mice (Klf5^fl/fl) allowing specific deletion of exon 2, which is the largest exon of Klf5 and encodes the majority of the protein.

Klf5^fl/fl, Vav-cre^+/− mice were born at the expected Mendelian ratios and showed no difference in overall weight or growth compared to heterozygote Klf5 KO or floxed littermates. We analysed the Klf5^fl/fl, Vav-cre^+/− mice (n=10) and Klf5^fl/fl mice (n=9) at 12 weeks of age for defects in steady state hematopoiesis. Interestingly, our preliminary data shows that the spleen weight of Klf5 KO animals (Klf5^fl/fl, Vav-cre^+/−) is significantly higher compared to Klf5^fl/fl mice (mean 0.097 vs 0.070 gram respectively, p<0.01). Peripheral blood (PB) analysis showed that Klf5 KO animals (Klf5^fl/fl, Vav-cre^+/−) displayed a significant reduction in total white blood cell (WBC) count compared to Klf5^fl/fl mice (mean 4.25×10⁹ vs 5.37×10⁹ cells/L respectively, p<0.05). In addition, Klf5^fl/fl, Vav-cre^+/− mice displayed significantly lower values for red blood cell count (RBC), hemoglobin and hematocrit (p <0.05). To further characterise the changes in blood cell populations, we used flow cytometry with a range of different specific cell surface antibody markers. From this we observed a significant increase in CD45⁺/GR-1⁺ and CD3e⁺ cells in PB of Klf5^fl/fl, Vav-cre^+/−mice (p <0.05).

Overall our data in the Klf5 conditional KO model are consistent with Klf5 playing a role in growth and differentiation in the myeloid lineage, as suggested by our in vitro data, and further suggests that Klf5 may have roles in multiple haematopoietic lineages. We will present further data from this model including flow cytometric analysis assessing the stem and progenitor cell compartments from bone marrow, as well as clonal assays to enumerate different haemopoietic progenitors and to assess changes in self renewal capacity of cells.