Developmental stage-specific effects of Pim-1 dysregulation on murine bone marrow B cell development

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). 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. 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
The steady-state production of B lineage lymphocytes in bone marrow is contingent on the developmental-stagespecific expression and combinatorial activities of multiple regulatory proteins including transcription factors, signaling molecules, microRNAs, and epigenetic modifiers. A critical early event in B cell development is lymphoid lineage specification within the multipotential progenitor (MPP) pool which we and others have shown is dependent, in part, on the homeodomain transcription factor Hoxa9 and signaling via the receptor tyrosine kinase Flt3 [1][2][3][4]. Germline knockout of Hoxa9 or Flt3-ligand largely phenocopy each other with respect to reductions in numbers of MPPs, the lymphoid progenitor subsets ALP and BLP, and B cell precursors in BM. Importantly, Hoxa9 and Flt3 signaling function synergistically to regulate a critical early checkpoint in lymphoid development, as combined loss of Hoxa9 and Flt3 signaling ablates the generation of lymphoid primed multipotential progenitors [4].
Pim-1 is a member of the Pim family of protooncogenes that encode a distinct class of serine/threonine kinases. Pim-1 was originally identified as a target for proviral activation in Moloney murine leukemia virus induced T cell lymphomas [5]. PIM proteins are widely expressed in most tissues and co-expression of family members has been found in various tissues [6]. The transcription of Pim family members is induced by a variety of cytokines and mitogens that transduce their primary signal through the JAK/STAT pathway [7]. A role for Pim-1 in B cell development was previously demonstrated through analysis of pim1-/-and Eμ-Pim1 transgenic mice [8,9]. Pim1-/-Pre-B cells exhibited impaired in vitro proliferation in response to IL-7 and stem cell factor (SCF) that was rescued by expression of a functional Pim-1 transgene [10]. In contrast, overexpression of Pim-1increased numbers of IL-7 + SCF responsive B cell colonies. These combined data provided the first evidence that Pim-1 was an important regulator of B lymphopoiesis in mice, and linked Pim-1 to the IL-7R signaling pathway.
Cytokine signaling plays an essential role in early lymphoid/B cell development. Threshold levels of Flt3 signaling are required for the proliferation, survival, and maintenance of MPPs competent to generate B cell precursors [1,11]. Flt3 signaling is mediated by the Ras and STAT5 pathways [12]. A dominant negative form of Ras was shown to phenocopy the B lineage developmental block in flt3-/-mice, impairing the proliferation of common lymphoid progenitors and Pre-Pro-B cells. The same study showed that Ras promoted STAT5-dependent Pro-B differentiation by enhancing expression of IL-7Rα [12]. Pim-1 is induced downstream of Jak2/STAT5 signaling and has also been implicated in playing a role in the proliferation and/or differentiation of myeloid progenitors [13][14][15]. Importantly, a role for Pim-1 in regulation of the early lymphoid/B cell progenitor pool, prior to expression of CD45R/B220, has not been reported.
Functional studies have confirmed a role for Pim-1 in regulating hematopoietic stem cell (HSC) proliferation and survival. HSCs from pim1-/-mice exhibited impaired repopulating capacity in competitive transplantation experiments [16]. In vitro assays revealed decreased cytokine mediated cell growth and differentiation of hematopoietic progenitors [7]. In contrast, overexpression of human Pim-1 driven by vav hematopoietic regulatory elements and SV40 showed enhanced hematopoietic progenitor function in vitro and in vivo [16]. The hematopoietic defects exhibited by pim1-/mice are strikingly similar to those in hoxa9-/-mice as loss of hoxa9 also impaired the proliferation and repopulating ability of HSCs [17]. Consistent with this observation, pim1 is a direct target of Hoxa9 [18]. Somatic ablation of pim1, hoxa9, or flt3 causes select reductions in hematopoietic progenitor subsets and B cell precursors. However, an obligate role for Pim-1 in regulation of lymphoid and/or early B cell development has not been investigated. In this study we evaluated the role of Pim-1 in murine lymphoid lineage specification and B cell development through comparative flow cytometric analysis of hoxa9-/-x Eμ-Pim1Tg, flt3l-/-x Eμ-Pim1Tg, Eμ-Pim1 transgenic, pim1-/-, hoxa9-/-, and flt3l-/-mice. Our experimental findings revealed that Pim-1 dysregulation has developmental-stage-specific effects on B lymphopoiesis and early myeloid, but not erythroid progenitors. Furthermore, we show that Pim1deficiency is not the basis of the lymphoid or early B cell developmental defects in hoxa9-/-or flt3l-/-mice.

Mice
Wildtype C57Bl/6 mice were generated from our breeding colony. Pim1-/-and Eμ − Pim1 transgenic mice have been previously described [10]. Pim1-/-mice were provided by Andrew S. Kraft and Eμ − Pim1 transgenic mice were provided by Jung-Hyun Park and permission for both obtained from A. Berns. All mice evaluated in this study were 8-12 weeks of age. C57Bl/6, EμPim-1Tg, hoxa9-/-x Eμ-Pim1Tg, and flt3l-/-x Eμ-Pim1Tg mice were bred in the Mayo Clinic animal facility, and all animal experiments detailed in this study were conducted under guidelines established and approved by the Mayo Clinic Institutional Animal Care and Use Committee. PCR, using DNA isolated from tail clips, was performed using previously published primers to confirm the genotype of compound mice [19,20].

Statistics
Statistical significance was determined using the Student-t test. Data are reported as standard error of the mean (SEM) and p-values less than 0.05 were considered significant.

Impact of Pim1-deficiency versus Eμ − transgene driven expression of Pim-1 on BM lymphoid progenitors and BCPs
A role for Pim-1 in B cell development was previously demonstrated from analysis of pim1-/-and Eμ-Pim1 transgenic mice [10]. Our findings that Eμ transgene driven expression of Pim-1 abrogated lymphoid/early B cell development supported further evaluation of the effects of dysregulated expression of Pim-1 on development of this lineage. BM cells from WT, pim1-/-, and Eμ-Pim1 transgenic mice were stained with combinations of antibodies to resolve ALP, BLP, Pre-Pro-B, IgM − BCP, and IgM + B cells. Comparison of BM mononuclear cell counts revealed no significant difference between WT and Eμ-Pim1 transgenic mice, but a statistically significant reduction in pim1-/-mice (5.2×10 7 ± 0.33, n = 3; p = 0.0055) compared to WT (7.9 ×10 7 ± 1.3, n = 10). No statistically significant alterations in absolute numbers of ALP, BLP, or Pre-Pro-B were identified between WT and pim1-/-mice (Fig. 3a). The inverse was found in Eμ-Pim1 transgenic mice wherein absolute numbers of phenotypic ALP, BLP, and Pre-Pro-B cells were significantly reduced compared to WT (Fig. 3a). Finally, while numbers of IgM − BCP and IgM + B cells in Eμ-Pim1 transgenic mice were comparable to WT (Fig. 3a), we found that IgM − , but not IgM + , BCPs were reduced in pim1-/-mice [10]. CD19 + IgM − BCPs constitute Pro-B and Pre-B cells. To gain further insight into the role of Pim-1 in hematopoietic development and B cell differentiation, we evaluated pim-1 transcript levels in purified bone marrow progenitor subsets. As shown in Fig. 3b, abundant pim-1 transcript expression was high exclusively in Pre-B cells across the B lineage developmental spectrum. Taken together, these data suggest that elevated levels of Pim-1 are inhibitory for lymphoid/B cell development prior to commitment to the B cell fate.
On the other hand, loss of Pim-1 decreased IgM − Pro-B/ Pre-B cells, suggesting an essential role at this stage.
Impact of Pim1-deficiency versus Eμ − transgene driven expression of Pim-1 on HSC, MPP, and erythroid/myeloid progenitors The Eμ enhancer is active very early in hematopoiesis, within LSK + MPPs [11,24]. A previous study reported increased numbers of LSK + cells in vav-Pim1Tg mice, but a similar analysis has not been reported for Eμ-Pim1Tg mice [16]. To begin, we compared frequencies of LSK + cells. In contrast to results reported for vav-Pim1Tg mice, frequencies and numbers of total LSK + cells were significantly reduced in Eμ-Pim1Tg mice ( Fig. 4a-b and data not shown). Next we compared frequencies and numbers of LSK + CD150 + CD34 − HSC and LSK + CD150 − Flt3 + MPP in Eμ-Pim1Tg mice. Absolute numbers of HSC and MPP were significantly reduced in Eμ-Pim1Tg mice (Fig. 4c-d). However, the reduction was attributed to the total decrease in LSK + cells as percentages of CD150 + CD34 − HSC within the LSK + compartment were increased in Eμ-Pim1Tg mice (6.23 ± 0.990 vs. 4.05 ± 1.07 %, p = 0.014, in Eμ-Pim1Tg vs. WT, respectively). The same was true for the reduction in absolute numbers of MPP as percentages CD150 − Flt3 + within the LSK + compartment was not significantly reduced (73.7 ± 4.53 vs. 80.1 ± 3.72 %, p = 0.064, in Eμ-Pim1Tg vs. WT, respectively). The reduction in LSK + cells in Eμ-Pim1Tg mice could influence numbers of erythroid/myeloid progenitors. Frequencies of Lin − ckit hi Sca1 − cells were not altered in Eμ-Pim1Tg mice (Fig. 4a). However, significant alterations in frequencies and absolute numbers of CMP and GMP, but not MEP, were documented Eμ-Pim1Tg mice (Fig. 4a,e-m). No significant alterations in frequencies or numbers of LSK + , HSC, MPP, CMP, MEP, or GMP were observed in pim1-/-mice, compared to WT (Fig. 4). Taken together, these data show that Eμ-Pim1Tg driven expression of Pim-1, but not Pim1-deficiency, reduces the myeloid but not the erythroid/megakaryocytic progenitor pools.

Loss of Pim-1 does not phenocopy hoxa9deficiency on the lymphoid/early B cell progenitor pools
Pim-1 is a Hoxa9 target gene and forced expression of Pim-1 rescued the in vitro proliferation of hoxa9-/-BM progenitors [18]. We reasoned that if Pim1-deficiency is a b

Discussion
Previous studies have implicated a role for Pim-1 in regulation of B cell development in BM and pim-1 is a Hoxa9 target gene [10,18]. Furthermore, hoxa9-and pim1-deficient mice share similar hematopoietic phenotypes and forced expression of Pim-1 rescued the in vitro proliferation defect in hoxa9-/-BM progenitors [17,18]. To determine if forced expression of Pim-1 was sufficient to restore the lymphoid progenitor/B cell precursor defect in hoxa9-/-or flt3l-/-mice, we generated hoxa9-/-Eμ-Pim1Tg mice and flt3l-/-Eμ-Pim1Tg mice. Unexpectedly, Eμ driven expression of Pim-1 exacerbated the deficiencies in ALP and BLP in flt3l-/-, but not hoxa9-/-mice. The detrimental effect in flt3l-/-Eμ-Pim1Tg mice was specific to the ALP and BLP stages of B cell differentiation, as the Eμ-Pim1Tg did not further exacerbate the B cell deficiencies in flt3l-/-mice from the Pre-Pro-B through the IgM + stages. The negative effect of Eμ-Pim1Tg expression was not restricted to the ALP and BLP compartments. We previously showed that Eμ is active from a very early stage in hematopoiesis and here we also show that Eμ-Pim1Tg expression caused significant reductions in numbers of HSCs, MPPs, CMPs, and GMPs suggesting that Pim-1 expression levels must be carefully controlled during early hematopoietic differentiation [11,24]. In contrast, loss of Pim-1, which we hypothesized might have deleterious consequences on the early lymphoid/B cell progenitor pools, was dispensable for hematopoiesis with the exception of IgM − BCPs [10]. Consistent, with our findings, realtime PCR of pim-1 transcription across the hematopoietic spectrum, showed significant levels limited to BCPs at the Pre-B cell stage. Finally, through direct comparative hematopoietic progenitor subset analysis of hoxa9-/-, (See figure on previous page.) Fig. 4 Impact of Eμ-Pim1Tg or Pim1-deficiency on primitive hematopoietic and myeloid-erythroid progenitor subsets in bone marrow. a Flow cytometry profile and gating strategy for HSC, Flt3+ MPP, CMP, GMP, and MEP. Bar graphs depict absolute numbers or frequencies of the various progenitor subsets. b Percentage of LSK+ in total BM. c-d Absolute number of HSC or MPP in BM. e-g Frequencies of CMP, MEP, or GMP within the Lin-ckithi Sca1-subset. h-j Absolute numbers of CMP, MEP, or GMP in BM. k-m Frequencies of CMP, MEP, or GMP in BM. Data represents the mean ± SEM of 3-10 mice per group. * denotes p < 0.05 and ** denotes p < 0.005 Fig. 5 Comparison of Hoxa9-, Fl3tl-, and Pim1-deficiencies on hematopoietic progenitor subsets and Pre-Pro-B cells. Bar graphs depict absolute numbers of the various progenitor subsets. Data represents the mean ± SEM of 3-4 mice per single knockout and 10 WT mice. * denotes p < 0.05 and ** denotes p < 0.005 flt3l-/-, and pim1-/-mice, we suggest that loss of Pim-1 is unlikely to contribute significantly to the hematopoietic progenitor defects in either hoxa9-/-or flt3l-/-mice. Taken together, these comprehensive flow cytometric analyses demonstrate very select lineage and developmentalstage-specific consequences of dysregulated expression of Pim-1 expression on BM hematopoietic progenitor and B lineage subsets. The serine threonine phosphokinase Pim-1 is highly expressed in hematopoietic cells in mouse and man [20,25]. The pim-1 gene was originally discovered as a common insertion site in MoMuLV-induced T cell lymphomas and later found in B-cell lymphomas and erythroleukemias. Overexpression of Pim-1 driven by the Eμ enhancer was shown to lead to a low incidence of T cell lymphomas and increased sensitivity to chemically induced T cell transformation. In the B lineage, overexpression of Eμ − Pim-1 facilitated establishment of B cell progenitor cell lines blocked at the Pre-B cell stage of differentiation [10]. The same study reported that marrows with the highest Pim-1 expression had a reduction in M-CSF responsive cells, and suggested the result could be due to impaired macrophage differentiation or on a macrophage progenitor. Our immunophenotypic analysis of Eμ-Pim1 transgenic mice provides insight into the reductions in M-CSF responsive cells as we identified significant reductions in frequencies and absolute numbers of CMP and GMP.
PIM kinases are short-lived proteins induced at the level of transcription upon mitogenic stimulation [26]. They do not have regulatory domains and are constitutively active when expressed. Under physiologic conditions, their short half-life limits their activity [27]. Many cytokines that induce pim gene transcription act through the JAK/STAT pathway. The pim1 promoter has binding sites for STAT3 and STAT5 [28,29]. Pim-1 protein then, in turn, functions to inhibit the JAK/STAT pathway by binding and activating SOCS proteins [30]. SOCS proteins support cytokine signaling through the Ras/MAPK pathway by binding the Ras inhibitor RasGAP [30]. Interestingly, dysregulated Ras/SOCS function mediated by overexpression of Pim-1 provides a plausible cellular mechanism for the impaired lymphoid/early B lineage development exhibited by Eμ − Pim1Tg mice. Ras proteins are involved in regulating proliferation and differentiation of various cell types in a tissue specific manner. Pertinent to this study, mice expressing a dominant negative form of human H-Ras have a severe block in B cell development at the Pre-Pro-B to Pro-B transition [31]. Li, et al., previously showed that a Flt3/Ras dependent signal governs lymphoid progenitor (ALP + BLP) and Pre-Pro-B proliferation [12]. In addition, they showed that Flt3/Ras suppresses expression of socs2 and socs3. They concluded that Flt3 activation of Ras primes B cell development by inducing a state of STAT5 responsiveness, a key event downstream of IL-7R signaling in lymphoid progenitors leading to induction of the B cell fate specification and commitment factor EBF1. Sustained expression of Pim-1 would be predicted to stabilize SOCS expression, impairing STAT5-mediated induction of ebf1, suppressing EBF1directed B cell differentiation. Indeed, this hypothetical molecular explanation is consistent with our findings that the Flt3 dependent ALP and BLP stages are exquisitely sensitive to dysregulated Pim-1 in flt3l-/-mice. It also suggests that it is unlikely that elevated levels of Pim-1 are inhibitory for lymphoid/B cell development by accelerating B cell differentiation, given the critical role for STAT5/ EBF1 in this process.
Pim kinases act as oncogenes by promoting cell cycle progression by phosphorylating and down-regulating the cyclin dependent kinase inhibitor p27Kip1. Pim kinases also have the ability to suppress p27kip1 transcription through phosphorylation and inactivation of forkhead transcription factors, including FoxO1 and FoxO3 [32]. Pim-1 inactivation of FoxO1 would have deleterious effects on B cell fate determination as FoxO1 is an integral member of a global network of transcription factors, including E2A and EBF1 [33]. Importantly, FoxO1-deficient mice also have an arrest in B cell development at the CLP stage, specifically at the BLP stage, and phenocopy EBF1-deficiency [34]. Importantly, FoxO1-deficient BLPs have increased transcripts for socs2, reminiscent of Pim-1 overexpression [30]. Based on our immunophenotyping results, together with previous phenotypic, molecular, and cellular data in the literature database, we hypothesize that dyregulated expression on Pim-1, driven by Eμ, impairs lymphoid/early B cell differentiation through upregulation of SOCS proteins and inactivation of FoxO1. Upregulation of SOCS proteins inhibits STAT5 mediated B cell differentiation leading to upregulation of EBF1. Inactivation of FoxO1 impairs the global transcriptional network orchestrated by E2A, EBF1, and FoxO1 that directs the early program of B lineage gene expression, requisite for B cell differentiation. In contrast, loss of Pim-1, would be predicted to suppress SOCS protein expression and stabilize FoxO1, favoring B cell developmental potential, providing an explanation for the slight, but not statistically significant, increase in BLP and Pre-Pro-B cells we observed in pim1-/-mice. We note that additional studies are required to determine if this molecular explanation is valid.
The goal of this project was to investigate the role of Pim-1 in early lymphoid/B cell development. However, we also found reductions in numbers of HSC, MPP, CMP, and GMP in Eμ − Pim1Tg mice. An, et al., reported an increase in frequencies and numbers of LSK + cells in vav-Pim1Tg mice, whereas we report decreased frequencies and numbers of LSK + cells in Eμ − Pim1Tg mice [16]. The overall decrease in LSK + cells was the basis of the decreased numbers of HSCs and MPPs, as frequencies of CD150 + CD34 − immunophenotypic HSC within the LSK + compartment were actually increased in Eμ − Pim1Tg mice. We believe it unlikely that Eμ − Pim1Tg expression has any functional consequence on HSC biology for two reasons. First, we previously showed that Eμ driven reporter expression is confined to CD27 + MPPs within the LSK + compartment and HSCs are CD27 − [11,24,35]. Second, the frequencies of CD150 + CD34 − HSCs were substantially increased in Eμ − Pim1Tg mice.
The cellular and/or molecular basis of the inhibitory consequence of Eμ − Pim1Tg expression on numbers of Flt3 + MPPs in noteworthy, as Flt3 + MPPs are the precursors of CMP and CLP and these hematopoietic subsets were the most sensitive to Eμ − Pim1Tg expression. Eμ − Pim1Tg expression did not impact Flt3 + CMP or CLP by altering the expression levels of Flt3. However, Flt3 + CMP and CLP were particularly sensitive to Eμ − Pim1Tg expression. These findings suggest that overexpression of Pim-1 might negatively impact signaling networks downstream of Flt3 stimulation in Flt3 + progenitor subsets, impairing either their proliferation or survival. Regardless of the mechanism, differentiating CMP and CLP overcome the inhibitory effects and their downstream progeny are refractory to sustained expression of Pim-1. Overexpression of Pim-1 in myeloid progenitors is likely limited by inactivation of Eμ due to lack of expression of critical activators necessary to maintain enhancer activity. It is interesting to note that Pim-1 driven by vav regulatory elements was low in mature myeloid cells [16].
Finally, although our flow cytometry analysis revealed that Pim1-deficiency does not provide a molecular explanation for the lymphoid/B cell deficiency in hoxa9-/-mice, pim1-/-and hoxa9-/-mice do share similar phenotypic and functional HSC defects. These observations and reported findings underscore developmental stage specific requirements for Hoxa9 and Pim-1 in hematopoietic development.

Conclusions
The goal of this study was to determine the requirement for Pim-1 in early lymphoid/B cell development. We show that controlled expression of Pim-1 is important in lymphoid/B cell development. High levels of Pim-1 are inhibitory while Pim-1 is dispensable for establishment of lymphoid progenitor and Pre-Pro-B cell pools.