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The implementation of microarray (cDNA and oligonucleotide) technology is enabling investigators to assess gene expression at a tissue/cellular level by quantification of messenger RNA (mRNA) transcripts. Once assimilated, these data should provide gene-expression profiles that are unique to specific cells, their responses to specific stimuli, and also help unravel complex signalling pathways. With regards to a more holistic approach, comparisons between diseased and normal tissues could identify genes that are directly implicated in the disease pathology and hence identify targets for therapeutic intervention. However, as with all systems, there remains the possibility of generating false positives and, therefore, differential gene expression should be verified using other techniques such as real-time PCR with reverse-transcription or Northern blot analysis. Furthermore, certain genes can be upregulated without an increase in actual protein levels and, therefore, the use of tissue microarray technology allows high-throughput immunostaining to correlate protein and mRNA data.These technologies have recently been applied to dissect the molecular basis of the most common malignancy in men (with the exception of non-melanoma skin cancer) – prostate cancer. Dhanasekaran et al. 1xDelineation of prognostic biomarkers in prostate cancer. Dhanasekaran, S.M. et al. Nature. 2001; 412: 822–826Crossref | PubMed | Scopus (1213)See all References1 have used both cDNA and tissue microarrays to great effect in comparing and characterising gene-expression profiles in normal and neoplastic prostate specimens. Having generated a vast amount of data, the authors focused on the hepsin and PIM1 genes that encode a transmembrane protease and serine/threonine kinase respectively. Both proteins are overexpressed in prostate tumours and interestingly PIM1 appears to be co-regulated with the oncogene MYC, supporting the possibility of a synergistic oncogenic effect. Interestingly, immunostaining revealed that metastatic prostate samples expressed hepsin at levels in between malignant and benign prostate tumours. Men with elevated prostate-specific antigen (PSA) levels – a clinical marker for prostate cancer – are prone to die from metastases following radical prostatectomy. Extensive statistical analyses subsequently revealed that men conforming to this group have low or absent hepsin protein levels. The same trend was also demonstrated for PIM1 indicating that these two proteins can now be considered as additional clinical biomarkers for prostate cancer.In the past few months, there has been an array of similar studies reporting on the application of the expression array technology to study prostate cancer. Two such studies also identified hepsin as a useful biomarker 2xHuman prostate cancer and benign prostatic hyperplasia: molecular dissection by gene expression profiling. Luo, J. et al. Cancer Res. 2001; 61: 4683–4688PubMedSee all References, 3xExpression profiling reveals hepsin overexpression in prostate cancer. Magee, J.A. et al. Cancer Res. 2001; 61: 5692–5696PubMedSee all References. Further mining of all these microarray data sets will lead to the generation of a large panel of useful biomarkers to aid in the detection and prognosis of prostate cancer. In addition, they could also reveal new ‘molecular targets’ for potential therapeutic intervention towards improved treatment and management of this malignancy.

Mitogen-activated protein kinases (MAPKs), including c-Jun NH2-terminal kinases (JNKs) and extracellular signal-regulated kinases (ERKs), are important intermediates of siganal-transduction pathway from cell suraface to the nucleus in cell proliferation, differentiation, and apoptosis. Recent evidence shows that interleukin-113 (IL-113), a multifunctional cytokine, activates MAPKs in mesangial cells and fibroblasts. However, cellular response of gastric epithelial cells stimulated by IL-113 is not well known. In this study, to analyze the signal transduction pathways, we investigate whether IL-113 activates JNKs and ERKs in rat gastric epithelial ceils (RGM1) using in-gel kinase assays. RGM1 were cultured at 37°C in the optimal media (DMEM + Ham F12) with FCS finally concentrated at 20%. Identification of INKs and ERKs in RGM1 was performed by immunoprecipitation procedure. Recombinant mouse IL-113, at the final dose of 0, 0.1, 1, 10, 102, 103, or 5X103 pg/ml, was added to the culture media and incubated for 20 rain to investigate the dose-dependency of JNKs and ERKs activities in RGM1. IL-113 (100 pg/rnl) was added to the cuture media and incubated for 5, 10, 20, 30, 45, or 60 rain to investigate the time course of these activities. The activation of JNKs and ERKs in RGM1 were dose-dependent with maximal activity attained with 1000 pg/ml of IL-113, respectively. These two maximal activities occurred at 20 rain after stimulation of IL-113. Furthermore, pretreatment with neutralizing antibody against IL-113 inhibited IL-113-induced activation of JNKs and ERKs. The activation of JNKs, but not ERKs, was inhibited by 25% of the maximal levels by pretreatment with genistein, an inhibitor for receptor specific and cytoplasmic tyrosine kinase. On the other hand, Lavendustin A, an inhibitor for receptor specific tyrosine kinase, and GF109203X, an inhibitor for protein kinase C, affected neither activation of JNKs nor ERKs. Thus, the activation of JNKs in RGM1 by IL-113 might be in part mediated by cytoplasmic but not receptor specific tyrosine kinases. In contrast to the activation of JNKs, that of ERKs might not be mediated by neither cytoplasmic nor receptor specific tyrosine kinases. These results suggest that JNKs and ERKs in rat gastric epithelial ceils are activated by IL-l[3, possibly via the specific receptor for IL-[3. Activation of these two kinases may be mediated by some different signaling pathways.

A number of mink cell focus-forming (MCF) proviruses was molecularly cloned from mouse lymphoma DNA. From each clone, flanking probes were prepared to detect common integration regions in other MuLV-induced lymphomas. One clone frequently revealed variations in the molecular structure of the corresponding region (Pim-1) in other lymphomas. The results show the following. Changes in the Pim region are seen in 24 out of 93 lymphomas tested. Over 50% of the early T-cell lymphomas show integration in the Pim-1 region. The alterations are seen in different mouse strains and with various MuLVs. The observed variations are caused by the integration of predominantly MCF genomes. All integrations occur in a region spanning less than 20 kb and are associated with the transcriptional activation of a distinct region within the Pim-1 domain. The activated region does not show any homology with 13 known and three putative oncogenes.

T-cell acute lymphoblastic leukemia (T-ALL) and T-cell acute lymphoblastic lymphoma (T-LBL) are aggressive hematological malignancies that are currently treated with high-dose chemotherapy. Over the last several years, the search toward novel and less-toxic therapeutic strategies for T-ALL/T-LBL patients has largely focused on the identification of cell-intrinsic properties of the tumor cell. However, non–cell-autonomous activation of specific oncogenic pathways might also offer opportunities that could be exploited at the therapeutic level. In line with this, we here show that endogenous interleukin 7 (IL7) can increase the expression of the oncogenic kinase proviral integration site for Moloney-murine leukemia 1 (PIM1) in CD127+ T-ALL/T-LBL, thereby rendering these tumor cells sensitive to in vivo PIM inhibition. In addition, using different CD127+ T-ALL/T-LBL xenograft models, we also reveal that residual tumor cells, which remain present after short-term in vivo chemotherapy, display consistent upregulation of PIM1 as compared with bulk nontreated tumor cells. Notably, this effect was transient as increased PIM1 levels were not observed in reestablished disease after abrogation of the initial chemotherapy. Furthermore, we uncover that this phenomenon is, at least in part, mediated by the ability of glucocorticoids to cause transcriptional upregulation of IL7RA in T-ALL/T-LBL patient-derived xenograft (PDX) cells, ultimately resulting in non–cell-autonomous PIM1 upregulation by endogenous IL7. Finally, we confirm in vivo that chemotherapy in combination with a pan-PIM inhibitor can improve leukemia survival in a PDX model of CD127+ T-ALL. Altogether, our work reveals that IL7 and glucocorticoids coordinately drive aberrant activation of PIM1 and suggests that IL7-responsive CD127+ T-ALL and T-LBL patients could benefit from PIM inhibition during induction chemotherapy.

Constitutive expression of the Pim-1 kinase prolongs survival of cytokine-deprived FDCP1 cells, partly via maintenance of Bcl-2 expression. Here, we show that Pim-1 colocalizes and physically interacts with the pro-apoptotic Bad protein and phosphorylates it in vitro on serine 112, which is a gatekeeper site for its inactivation. Furthermore, wild-type Pim-1, but not a kinase-deficient mutant, enhances phosphorylation of this site in FDCP1 cells and protects cells from the pro-apoptotic effects of Bad. Our results suggest that phosphorylation of Bad by Pim-1 is one of several mechanisms via which the Pim-1 kinase can enhance Bcl-2 activity and promote cell survival.

Summary The Janus kinase (JAK)/signal transducer and activator of transcription (STAT) pathway is involved in many cellular processes, including cell growth and differentiation, immune functions and cancer. It is activated by various cytokines, growth factors, and protein tyrosine kinases (PTKs) and regulates the transcription of many genes. Of the four JAK isoforms and seven STAT isoforms known, JAK2 and STAT3 are highly expressed in the brain where they are present in the postsynaptic density (PSD). Here, we demonstrate a new neuronal function for the JAK/STAT pathway. Using a variety of complementary approaches, we show that the JAK/STAT pathway plays an essential role in the induction of NMDA-receptor dependent long-term depression (NMDAR-LTD) in the hippocampus. Therefore, in addition to established roles in cytokine signaling, the JAK/STAT pathway is involved in synaptic plasticity in the brain., Highlights ► Identification of a specific role of the JAK/STAT pathway in synaptic plasticity ► Activation of JAK2 and STAT3 is required for NMDAR-LTD ► Activation of STAT3 in dendrites and nucleus during NMDAR-LTD ► Nuclear translocation of STAT3 is not required for NMDAR-LTD, The authors demonstrate a new neuronal function for the JAK/STAT pathway in the induction of NMDA-receptor-dependent long-term depression (NMDAR-LTD) in the hippocampus.

A 53-yr-old man with a rare form of partial α 1 antitrypsin deficiency, PiM 1 /PiM duarte , died of endstage cirrhosis. Typical cytoplasmic α 1 -antitrypsin globules were present in the hepatocyte cytoplasm-Initial protease inhibitor phenotyping on the patient was reported as normal PiM 1 in more than one laboratory. This case emphasizes the diagnostic importance of α 1 -antitrypsin and illustrates the point that protease inhibitor phenotyping without family genotyping may be misleading in heterozygous patients with liver disease.

Transgenic mice bearing the pim-1 gene supplemented with an upstream immunoglobulin enhancer and a downstream murine leukemia virus long terminal repeat express pim-1 mRNA at high levels in both B and T cells. Between 5% and 10% of the pim-1 transgenic mice develop clonal T cell lymphomas before 7 months of age, whereas none of the age-matched control mice do, providing direct evidence for the oncogenic potential of pim-1. Histological examination and FACS analysis revealed no abnormalities in hematopoietic tissues of disease-free pim-1 transgenic mice. When newborn pim-1 transgenic mice are infected with MuLV, T cell lymphomas develop much faster (latency 7-8 weeks) than in nontransgenic mice (latency 22 weeks). In all these T cell lymphomas either c-myc or N-myc was activated by proviral insertion, suggesting strong cooperation between pim-1 and myc in lymphomagenesis.

We have isolated a cDNA clone of a new member of protein serine/threonine kinases, MPK38, from a cDNA library constructed from the murine teratocarcinoma PCC4 cell line by the polymerase chain reaction. MPK38 was transcribed as an approx. 2.5 kb mRNA encoding for a protein of 643 amino acids. N-terminus of MPK38 contains the kinase catalytic domain which exhibits approximately 60% protein sequence identity with the SNF1 serine/threonine kinase family. The MPK38 cDNA directs the in vitro translation of two protein species of approx. 70 and approx. 50 kDa, which appear to result from an internal initiation of translation. MPK38 was predominantly expressed in thymus and spleen, but was not detectable in kidney, liver, and muscle in the adult tissues. In addition, MPK38 was apparently expressed in T lineage cells and a macrophage/monocyte cell, but was not detectable in a B cell line and an embryonic cell line. However, a low level of MPK38 transcript was detectable in a mast cell line after a longer exposure. Furthermore, MPK38 gene product showed the kinase activity which was assessed by immune complex kinase assay. Thus, MPK38 gene product seems to play an important role in signal transduction of certain lineages of hematopoietic cells.

Abstract 3946 Poster Board III-882 INTRODUCTION The Pim kinases are a family of serine/threonine kinases composed by three members: Pim1, Pim2 and Pim3, involved in the phosphorylation and regulation of several proteins that are essential for cell cycle progression, metabolism or apoptosis (BAD, p21, p27KIP, AKT, Mdm2 and cMyc, among them). Overexpression, translocation or amplification of Pim family have been described in many human cancers, including B-cell Non Hodgkin's Lymphoma, Multiple Myeloma, Prostate cancer and Pancreatic cancer. In addition, 50% of patients diagnosed with diffuse large B-cell Lymphoma (DLBCL) present somatic mutations in Pim1. Despite of its important role in cancer progression, very few chemical inhibitors have been described in the literature, being effective all of them in the high micromolar range. PURPOSE Validating PIM as a rational therapeutic target in B-cell lymphoma, developing tools for patient stratification and pharmacodynamic studies on PIM inhibition. MATERIAL AND METHODS Gene expression profiling and Copy Number data were obtained from a series of 94 B-cell Non-Hodgkin Lymphoma patients (DLBCL, FL, MALT, MCL and NMZL). The effect of Pim inhibition was checked on cell lines by using a novel specific inhibitor for the Pim family (ETP-39010). Newly produced antibodies and RT-PCR primers and protocols were standarized. RESULTS Gene expression data revealed high Pim isoforms expression in a subset of patients with Mantle cell lymphoma (MCL), and Diffuse Large B-cell lymphoma (DLBLC)-ABC type. CGH analysis focused on chromosomal regions containing Pim family and its main regulatory upstream pathway (JAK/STAT) was performed. Heterozygous gains of Pim1 (6p21.2) and Pim3 (22q13.33) were identified in 13.6% of DLBCL patients and in 4.2% of MCL. Alterations in JAK/STAT pathway were also detected in 59.1% of DLBCL patients, and 37.5% of MCL patients presented any alteration in JAK/STAT pathway, being frequent losses of JAK2 chromosomal region. Analysis of additional pathways involved in the up-stream regulation of Pim family disclosed heterozygous gains of PIK3C3 in 40.9% of DLBCL patients, and gains of PIK3CA in 45.9% of MCL patients. Lymphoma cell lines (15) derived from both MCL (9) and ABC-DLBLC (6) subtype, have been analyzed by qRT-PCR and Western-blot, showing variable expression levels of Pim1, Pim2 and Pim3. IC50 obtained for the ETP-39010 compound is in the low micromolar range for the MCL (0.7-8.7 micromolar) and DLBCL-ABC (0.8-10.3 micromolar) cell lines. Since Pim kinase family phosphorilate multiple sites of Bad and AKT, we have checked the inhibition of its phosphorilation as molecular biomarkers for the ETP-39010 effect. Our data show an inhibition of at least 20% of pBad (S112) and almost a complete inhibition of pAKT (S473) 4h after treatment. In addition, cell cycle arrest at G1 and induction of apoptosis were observed 24h after the treatment. CONCLUSION Pim family genes are a rational therapeutic target in MCL and DLBCL-ABC lymphoma subtypes. Stratification and pharmacodynamic markers have been developed for PIM inhibition using a novel specific inhibitor compound -ETP-39010-. Disclosures: No relevant conflicts of interest to declare.

Objectives Dysregulated signal transduction and activator of transcription-3 (STAT3) signalling in CD4+ T cells has been proposed as an early pathophysiological event in RA. We sought further evidence for this observation, and to determine its clinical relevance. Methods Microarray technology was used to measure gene expression in purified peripheral blood CD4+ T cells from treatment-naïve RA patients and disease controls newly recruited from an early arthritis clinic. Analysis focused on 12 previously proposed transcripts, and concurrent STAT3 pathway activation was determined in the same cells by flow cytometry. A pooled analysis of previous and current gene expression findings incorporated detailed clinical parameters and employed multivariate analysis. Results In an independent cohort of 161 patients, expression of 11 of 12 proposed signature genes differed significantly between RA patients and controls, robustly validating the earlier findings. Differential regulation was most pronounced for the STAT3 target genes PIM1, BCL3 and SOCS3 (>1.3-fold difference; P < 0.005), each of whose expression correlated strongly with paired intracellular phospho-STAT3. In a meta-analysis of 279 patients the same three genes accounted for the majority of the signature’s ability to discriminate RA patients, which was found to be independent of age, joint involvement or acute phase response. Conclusion The STAT3-mediated dysregulation of BCL3, SOCS3 and PIM1 in circulating CD4+ T cells is a discriminatory feature of early RA that occurs independently of acute phase response. The mechanistic and functional implications of this observation at a cellular level warrant clarification.

The survival of chronic lymphocytic leukemia (CLL) cells depends on their interactions with microenvironment components, such as stromal and T cells. Lymph node microenvironment provides protective signals that enable the formation of proliferation centers and favor resistance to conventional chemotherapeutics. One of the key molecules engaged in the communication of CLL cells with their microenvironment is C-X-C chemokine receptor type 4 (CXCR4). The surface expression of CXCR4 is regulated by PIM1 (provirus integration site for Moloney murine leukemia virus) kinase. PIM 1-3 kinases are overexpressed in CLL cells and recent data suggest that targeting PIMs might be a rational therapeutic approach in this type of leukemia. Herein, we assessed associations of PIM kinase expression with clinical characteristics of CLL patients and investigated the consequences of PIM kinase inhibition for cell survival and CXCR4 - dependent signal transduction and migration of primary CLL cells. In primary CLL cells from peripheral blood, PIM2 transcript abundance was higher than PIM1 and PIM3 (p Taken together, our data demonstrate that CXCR4/SDF1 signal in chronic lymphocytic leukemia cells is transduced through mTOR pathway and that CXCR4 - triggered mTOR activity is modulated by PIM kinases. Pan-PIM inhibitor SEL24-B489 decreased CXCR4 surface expression and SDF-1 - triggered mTOR activity. Finally, SEL24-B489 decreased protective effects of tumor microenvironment and induced CLL cells apoptosis even in the presence of stromal cells. Since overexpression of PIM kinases might be associated with adverse clinical characteristics at diagnosis, PIM inhibition might be a rational therapeutic strategy in CLL. Disclosures Czardybon: Selvita S.A.: Employment. Galezowski:Selvita S.A.: Employment. Windak:Selvita S.A.: Employment. Brzozka:Selvita S.A.: Employment. Juszczynski:Selvita S.A.: Other: member of Selvita Scientific Advisory Board.

Background Receptor tyrosine kinase, c-Kit (CD117) plays a pivotal role in the maintenance and expansion of hematopoietic stem/progenitor cells (HSPCs). Additionally, over-expression and/or mutational activation of c-Kit have been implicated in numerous malignant diseases including acute myeloid leukemia. However, the translational regulation of c-Kit expression remains largely unknown. Methods and results We demonstrated that loss of Pim1 led to specific down-regulation of c-Kit expression in HSPCs of Pim1−/− mice and Pim1−/−2−/−3−/− triple knockout (TKO) mice, and resulted in attenuated ERK and STAT3 signaling in response to stimulation with stem cell factor. Transduction of c-Kit restored the defects in colony forming capacity seen in HSPCs from Pim1−/− and TKO mice. Pharmacologic inhibition and genetic modification studies using human megakaryoblastic leukemia cells confirmed the regulation of c-Kit expression by Pim1 kinase: i.e., Pim1-specific shRNA knockdown down-regulated the expression of c-Kit whereas overexpression of Pim1 up-regulated the expression of c-Kit. Mechanistically, inhibition or knockout of Pim1 kinase did not affect the transcription of c-Kit gene. Pim1 kinase enhanced c-Kit 35S methionine labeling and increased the incorporation of c-Kit mRNAs into the polysomes and monosomes, demonstrating that Pim1 kinase regulates c-Kit expression at the translational level. Conclusions Our study provides the first evidence that Pim1 regulates c-Kit gene translation and has important implications in hematopoietic stem cell transplantation and cancer treatment.

Background The serine threonine kinase Pim-1 has documented roles in hematopoietic progenitor and B cell precursor proliferation and survival. Pim-1 is a molecular target of the transcription factor Hoxa9. Previous studies showed that Pim-1 deficiency phenocopied the hematopoietic progenitor defect in hoxa9-/- mice and forced expression of Pim-1 normalized the in vitro proliferation defect inherent to hoxa9-/- hematopoietic progenitors. Pim-1 is induced by cytokine signaling, including the early lymphoid/B lineage regulators Flt3 and IL-7, and expression levels were shown to influence the size of the B cell compartment in bone marrow (BM). Results In this study, we sought to determine if transgenic expression of Pim-1, driven by the immunoglobulin enhancer, Eμ, was sufficient to rescue the lymphoid/B cell precursor defect in hoxa9 or flt3-ligand (flt3l) deficient mice. Unexpectedly, expression of Eμ − Pim1 exacerbated lymphoid progenitor deficiencies in flt3l-/-, and to a lesser extent, hoxa9-/- mice. Furthermore, Eμ − Pim1 expression alone reduced early myeloid and lymphoid, but not erythroid, progenitors. In contrast, Pim-1 deficiency had no significant effect on early lymphoid/B cell development through the Pre-Pro-B cell stage, but caused a significant reduction in IgM− B cell precursors. Importantly, loss of Pim-1 did not phenocopy hoxa9- or flt3l-deficiency on the lymphoid/early B cell progenitor pools. Conclusions These experimental findings demonstrate that Pim-1 overexpression has developmental-stage-specific effects on B lymphopoiesis and myelopoiesis. Importantly, these suggest that Pim-1 deficiency does not contribute significantly to the early lymphoid/B cell developmental deficiency in hoxa9-/- or flt3l-/- mice.

Background Progression of prostate cancer from benign local tumors to metastatic carcinomas is a multistep process. Here we have investigated the signaling pathways that support migration and invasion of prostate cancer cells, focusing on the role of the NFATC1 transcription factor and its post-translational modifications. We have previously identified NFATC1 as a substrate for the PIM1 kinase and shown that PIM1-dependent phosphorylation increases NFATC1 activity without affecting its subcellular localization. Both PIM kinases and NFATC1 have been reported to promote cancer cell migration, invasion and angiogenesis, but it has remained unclear whether the effects of NFATC1 are phosphorylation-dependent and which downstream targets are involved. Methods We used mass spectrometry to identify PIM1 phosphorylation target sites in NFATC1, and analysed their functional roles in three prostate cancer cell lines by comparing phosphodeficient mutants to wild-type NFATC1. We used luciferase assays to determine effects of phosphorylation on NFAT-dependent transcriptional activity, and migration and invasion assays to evaluate effects on cell motility. We also performed a microarray analysis to identify novel PIM1/NFATC1 targets, and validated one of them with both cellular expression analyses and in silico in clinical prostate cancer data sets. Results Here we have identified ten PIM1 target sites in NFATC1 and found that prevention of their phosphorylation significantly decreases the transcriptional activity as well as the pro-migratory and pro-invasive effects of NFATC1 in prostate cancer cells. We observed that also PIM2 and PIM3 can phosphorylate NFATC1, and identified several novel putative PIM1/NFATC1 target genes. These include the ITGA5 integrin, which is differentially expressed in the presence of wild-type versus phosphorylation-deficient NFATC1, and which is coexpressed with PIM1 and NFATC1 in clinical prostate cancer specimens. Conclusions Based on our data, phosphorylation of PIM1 target sites stimulates NFATC1 activity and enhances its ability to promote prostate cancer cell migration and invasion. Therefore, inhibition of the interplay between PIM kinases and NFATC1 may have therapeutic implications for patients with metastatic forms of cancer. Graphical abstract

Background Pim (p roviral i nsertion in m urine lymphoma) kinases are a small family of constitutively active, highly conservative serine/threonine oncogenic kinases and have 3 members: Pim1, Pim2, and Pim3. Pim kinases are also implicated in the regulation of B- and T- cell responses to cytokines and hematopoietic growth factors. The roles of Pim kinases in the regulation of primitive hematopoietic stem cells (HSCs) are largely unknown. Methods In the current study, Pim1−/−2−/−3−/− triple knockout (TKO) mice were used to determine the role of Pim kinases in hematopoiesis. Peripheral blood hematological parameters were measured in Pim TKO mice and age-matched wild-type (WT) controls. Primary, secondary, and competitive transplantations were performed to assay the long-term repopulating HSCs in Pim TKO mice. In vivo BrdU incorporation assay and ex vivo Ki67 staining and caspase 3 labeling were performed to evaluate the proliferation and apoptosis of HSCs in Pim TKO mice. Results Compared to age-matched WT controls, Pim TKO mice had lower peripheral blood platelet count and exhibited erythrocyte hypochromic microcytosis. The bone marrow cells from Pim TKO mice demonstrated decreased hematopoietic progenitor colony-forming ability. Importantly, Pim TKO bone marrow cells had significantly impaired capacity in rescuing lethally irradiated mice and reconstituting hematopoiesis in primary, secondary and competitive transplant models. In vivo BrdU incorporation in long-term HSCs was reduced in Pim TKO mice. Finally, cultured HSCs from Pim TKO mice showed reduced proliferation evaluated by Ki67 staining and higher rate of apoptosis via caspase 3 activation. Conclusions Pim kinases are not only essential in the hematopoietic lineage cell development, but also important in HSC expansion, self-renewal, and long-term repopulation.