Enhancement of hematopoietic recovery after radiation, chemotherapy, or hematopoietic stem cell (HSC) transplantation is clinically relevant. Dipeptidylpeptidase (DPP4) cleaves a wide variety of substrates, including the chemokine stromal cell-derived factor-1 (SDF-1). In the course of experiments showing that inhibition of DPP4 enhances SDF-1–mediated progenitor cell survival, ex vivo cytokine expansion and replating frequency, we unexpectedly found that DPP4 has a more general role in regulating colony-stimulating factor (CSF) activity. DPP4 cleaved within the N-termini of the CSFs granulocyte-macrophage (GM)-CSF, G-CSF, interleukin-3 (IL-3) and erythropoietin and decreased their activity. Dpp4 knockout or DPP4 inhibition enhanced CSF activities both in vitro and in vivo. The reduced activity of DPP4-truncated versus full-length human GM-CSF was mechanistically linked to effects on receptor-binding affinity, induction of GM-CSF receptor oligomerization and signaling capacity. Hematopoiesis in mice after radiation or chemotherapy was enhanced in Dpp4−/− mice or mice receiving an orally active DPP4 inhibitor. DPP4 inhibition enhanced engraftment in mice without compromising HSC function, suggesting the potential clinical utility of this approach.

Tyrosine kinase inhibitor (TKI) therapy for human cancers is not curative, with relapse due to the continuing presence of tumor cells, referred to as minimal residual disease (MRD) cells. MRD stem or progenitor cells survival in the absence of oncogenic kinase signaling, a phenomenon referred to as intrinsic resistance, depends on diverse growth factors. Here, we report that oncogenic kinase and growth factor signaling converge to induce the expression of the signaling proteins c-Fos and Dusp1. Genetic deletion of c-Fos and Dusp1 suppressed tumor growth in a BCR-ABL-induced mouse model of chronic myeloid leukemia (CML). Pharmacological inhibition of c-Fos, Dusp1 and BCR-ABL eradicated MRD in multiple in vivo models, as well as in primary CML patient xenotransplanted mice. Growth factor signaling also conferred TKI resistance and induced c-FOS and DUSP1 expression in tumor cells modeling other types of kinase-driven leukemias. Our data demonstrate that c-Fos and Dusp1 expression levels determine the threshold of TKI efficacy, such that growth factor-induced expression of c-Fos and Dusp1 confers intrinsic resistance to TKI therapy in a wide-ranging set of leukemias, and may represent a unifying Achilles heel of kinase-driven cancers.

Both membrane-proximal and truncation mutations in CSF3R have recently been reported to drive the onset of chronic neutrophilic leukemia (CNL). Here we show that although truncation mutation alone can not induce leukemia, both proximal and compound mutations (proximal and truncation mutations on same allele) are leukemogenic with a disease latency of 90 and 23 days, respectively. Comparative whole-genome expression profiling and biochemical experiments revealed that induced expression of Mapk adaptor protein Ksr1 and enhanced Mapk signaling are crucial to leukemogenesis by CSF3R proximal and compound mutants. Moreover, inhibition of Mek1/2 by trametinib alone is sufficient to suppress leukemia induced by both CSF3R proximal and ruxolitinib-resistant compound mutations. Together, these findings elucidate a Mapk-dependent mechanism of CSF3R-induced pathogenesis, and they establish the rationale for clinical evaluation of MEK1/2 inhibition in CNL.

Although neurotrophic factors have long been recognized as potent agents for protecting against neuronal degeneration, clinical success in treating Parkinson’s disease and other neurodegenerative disorders has been hindered by difficulties in delivery of trophic factors across the blood brain barrier (BBB). Bone marrow hematopoietic stem cell-based gene therapy is emerging as a promising tool for overcoming drug delivery problems, as myeloid cells can cross the BBB and are recruited in large numbers to sites of neurodegeneration, where they become activated microglia that can secrete trophic factors. We tested the efficacy of bone marrow-derived microglial delivery of neurturin (NTN) in protecting dopaminergic neurons against neurotoxin-induced death in mice. Bone marrow cells were transduced ex vivo with lentivirus expressing the NTN gene driven by a synthetic macrophage-specific promoter. Infected bone marrow cells were then collected and transplanted into recipient animals. Eight weeks after transplantation, the mice were injected with the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropuridine (MPTP) for seven days to induce dopaminergic neurodegeneration. Microglia-mediated NTN delivery dramatically ameliorated MPTP-induced degeneration of tyrosine hydroxylase (TH)-positive neurons of the substantia nigra and their terminals in the striatum. Microglia-mediated NTN delivery also induced significant recovery of synaptic marker staining in the striatum of MPTP-treated animals. Functionally, NTN treatment restored MPTP-induced decline in general activity, rearing behavior, and food intake. Thus, bone marrow-derived microglia can serve as cellular vehicles for sustained delivery of neurotrophic factors capable of mitigating dopaminergic injury.

Since cord blood (CB) has become a commonly used source of transplantable hematopoietic stem (HSC) and hematopoietic progenitor cells (HPC), there has been a need to overcome the limited HSC and HPC numbers available to transplant from a single CB, especially for adult recipients. Our laboratory previously demonstrated that Rheb2 overexpression significantly impaired the repopulating ability of HSC. Since overexpression of Rheb2 leads to increased signaling through mTOR, we examined the effect of the mTOR inhibitor rapamycin ex vivo on cytokine expanded CD34 + CB cells for the engraftment of these cells in non-obese diabetic, severe combined immunodeficient, IL-2 receptor γ chain null (NSG) mice. We observed significant enhancement in engraftment of the CB treated ex vivo with cytokines in the presence of rapamycin prior to transplant, effects seen in primary as well as secondary transplants. These pre-clinical results suggest a positive role for rapamycin during ex vivo culture of CB SCID repopulating cells/HSC.

The contribution of specific cell types to the production of cytokines that regulate hematopoiesis is still not well defined. IntroductionT cells are an important source of cytokines that regulate hematopoietic progenitor cell (HPC) homeostasis. Mice deficient in T cells have altered myeloid cell development that is rescued by the transfer of CD4 ϩ T cells. 1 Moreover, mice that are deficient in STAT4 or STAT6, transcription factors that respectively promote Th1 and Th2 development, have altered HPC homeostasis due in part to the effects of IL-12-priming Th1 development and the secretion of Oncostatin M. 2,3 The ability of other Th subsets to regulate this process has not been examined.STAT3 promotes the development of Th17 cells, 4,5 Tfh cells, 6 and Th2 cells, 7 by activating genes encoding subset-specific cytokines and transcription factors. 7,8 Although the requirement for STAT3 in T cells to regulate HPC homeostasis has not been examined, one of the genes regulated and bound by STAT3 is Il21. 9 IL-21 is produced by multiple T-cell subsets and has been reported to promote hematopoiesis. [10][11][12][13] In this report, we examine the requirement for STAT3-dependent Th subsets in regulating HPC homeostasis. Methods MiceC57BL/6 Stat3 fl/fl mice 14 with a CD4-Cre (Stat3 CD4-/-) transgene and Cre-negative littermates, wild-type mice purchased from Harlan Laboratories, Il17 Ϫ/Ϫ mice provided by Dr Alison Finnegan (Rush University, Chicago, IL) with agreement from Dr Yoichiro Iwakura (University of Tokyo, Tokyo, Japan), and Rorc gfp/gfp mice purchased from The Jackson Laboratory were maintained in specific pathogen-free conditions and experiments were approved by the Indiana University Institutional Animal Care and Use Committee. In vitro T-cell differentiation and analysis of phospho-STAT proteinsIn vitro differentiation, analysis of cytokine production, retroviral transduction, and analysis of phospho-STAT proteins were performed as described. 7,15 Assay of hematopoietic progenitor numbers and cyclingNumbers of granulocyte-macrophage (CFU-GM), erythroid (BFU-E) and multipotential (CFU-GEMM) progenitor cells, and cycling of progenitor cells using high specific activity tritiated thymidine, were assessed as previously described. 2,16,17 Results and discussionTo test the requirement for STAT3-dependent T-cell function in regulating HPC homeostasis, we isolated bone marrow and spleen from mice carrying a floxed Stat3 allele and expressing Cre controlled by a CD4 promoter (noted as Stat3 CD4Ϫ/Ϫ ) and from littermates that did not have the Cre-transgene (noted as wild-type). CD4-Cre expression results in conditional deletion of Stat3 in T cells but not HPC. There were no differences in the number of nucleated bone marrow or spleen cells, and no difference in apoptosis of Lineage Ϫ Sca-1 ϩ c-Kit ϩ cells that are highly enriched for HPC between mice with wild-type or STAT3-deficient T cells (data not shown). However, there was a significant decrease in absolute numbers of CFU-GM, BFU-E and CFU-GEMM in bone marrow and ...

Objective-Cell cycle checkpoints guarantee movement through the cell cycle. Mitotic arrest deficiency 2 (Mad2), a mitotic checkpoint protein, appears crucial for generating the wait anaphase signal to prevent onset of anaphase. We evaluated effects of Mad2 haploinsufficiency on hematopoietic stem (HSC) and progenitor (HPC) function in response to stress.Materials and Methods-We studied effects of Mad2 +/− on in vivo recovery of bone marrow HPC from cytotoxic effects and also effects of cytostatic agents on HPC growth in vitro using Mad2 +/− mice.Results-Mad2 +/− HPCs were protected from cytotoxic effects in vivo of a cell cycle specific agent, Ara-C, events consistent with Mad2 +/− HPCs being in a slow or noncycling state, but not from recovery of functional HPC after treatment with non-cycle specific cyclophosphamide or sub-lethal irradiation. There were no differences in phenotyped HSCs in Mad2 +/− & Mad2 +/+ mice, information confirmed by no changes in short or long term repopulating HSC assay. To better understand Mad2 +/− HPC function, E3330, a cytostatic agent, was used to assess redox function of Ape1/Ref-1; colony growth was examined under 5% and 20% O 2 tension. Mad2 +/− HPCs were less responsive to E3330 than Mad2 +/+ HPCs, and E3330 was more effective under lowered O 2 tension. Mad2 +/− HPCs were not enhanced at lowered oxygen, as were Mad2 +/+ HPCs.Conclusions-Our studies have unexpectedly found that Mad2 haploinsufficiency is protective in the presence of a cycle specific DNA synthesis agent in vivo, and Ape1/Ref-1 inhibitor in vitro.

Previously we reported that the spindle assembly checkpoint (SAC), which is coupled in somatic cells, is uncoupled from apoptosis-initiation in mouse and human ESCs. This condition allows ESCs to tolerate and proliferate as polyploidy/aneuploid cells. Proper function of the SAC is vital to prevent polyploidy/aneuploidy during ex-vivo HSC expansion. Here we address, for the first time, whether hematopoietic stem cells are more like ESC or somatic cells with respect to SAC-apoptosis coupling. Using multiparametric permeablized cell flow-cytometric analysis to identify and analyze the mouse sca 1+/c-kit+/lin− (LSK) population, we found the mitotic spindle checkpoint to be functional in primary murine LSK cells, a population enriched in primitive hematopoietic stem/progenitor cells, after prolonged activation of the SAC by microtubule-depolymerizing agents such as nocodazole. HSCs can efficiently initiate apoptosis after activation of the SAC in LSK cells as indicated by increased hypopdiploidy and increased levels of activated caspase-3 suggesting that HSCs behave more like somatic cells instead of ESCs with respect to this important cell cycle checkpoint. We conclude that mouse HSCs are not subject to the same kinds of chromosomal-instability as are ESCs, knowledge that might aid in optimizing in-vitro culture and expansion of human bone marrow or cord blood HSC for clinical applications.