The biology of hematopoietic stem cells (HSCs) has predominantly been studied under transplantation conditions 1 , 2 . Particularly challenging has been the study of dynamic HSC behaviors given that live animal HSC visualization in the native niche still represents an elusive goal in the field. Here, we describe a dual genetic strategy in mice that restricts reporter labeling to a subset of the most quiescent long-term HSCs (LT-HSCs) and that is compatible with current intravital imaging approaches in the calvarial bone marrow (BM) 3 – 5 . We find that this subset of LT-HSCs resides in close proximity to both sinusoidal blood vessels and the endosteal surface. In contrast, multipotent progenitor cells (MPPs) display a broader distance distribution from the endosteum and are more likely to be associated with transition zone vessels. LT-HSCs are not found in BM niches with the deepest hypoxia and instead are found in similar hypoxic environments as MPPs. In vivo time-lapse imaging reveals that LT-HSCs display limited motility at steady-state. Following activation, LT-HSCs display heterogenous responses, with some cells becoming highly motile and a fraction of HSCs expanding clonally within spatially restricted domains. These domains have defined characteristics, as HSC expansion is found almost exclusively in a subset of BM cavities exhibiting bone-remodeling activities. In contrast, cavities with low bone-resorbing activities do not harbor expanding HSCs. These findings point to a new degree of heterogeneity within the BM microenvironment, imposed by the stages of bone turnover. Overall, our approach enables direct visualization of HSC behaviors and dissection of heterogeneity in HSC niches.

Axin negatively regulates the Wnt pathway during axis formation and plays a central role in cell growth control and tumorigenesis. We found that Axin also serves as a scaffold protein for mitogen-activated protein kinase activation and further determined the structural requirement for this activation. Overexpression of Axin in 293T cells leads to differential activation of mitogen-activated protein kinases, with robust induction for c-Jun NH 2 -terminal kinase (JNK)/stress-activated protein kinase, moderate induction for p38, and negligible induction for extracellular signal-regulated kinase. Axin forms a complex with MEKK1 through a novel domain that we term MEKK1-interacting domain. MKK4 and MKK7, which act downstream of MEKK1, are also involved in Axin-mediated JNK activation. Domains essential in Wnt signaling, i.e. binding sites for adenomatous polyposis coli, glycogen synthase kinase-3␤, and ␤-catenin, are not required for JNK activation, suggesting distinct domain utilization between the Wnt pathway and JNK signal transduction. Dimerization/oligomerization of Axin through its C terminus is required for JNK activation, although MEKK1 is capable of binding C terminus-deleted monomeric Axin. Furthermore, Axin without the MEKK1-interacting domain has a dominant-negative effect on JNK activation by wild-type Axin. Our results suggest that Axin, in addition to its function in the Wnt pathway, may play a dual role in cells through its activation of JNK/stress-activated protein kinase signaling cascade.

The Mds1 and Evi1 complex locus (Mecom) gives rise to several alternative transcripts implicated in leukemogenesis. However, the contribution that Mecom-derived gene products make to normal hematopoiesis remains largely unexplored. To investigate the role of the upstream transcription start site of Mecom in adult hematopoiesis, we created a mouse model with a lacZ knock-in at this site, termed ME m1 , which eliminates Mds1-Evi1 (ME), the longer, PR-domaincontaining isoform produced by the gene (also known as PRDM3). ␤-galactosidasemarking studies revealed that, within hematopoietic cells, ME is exclusively expressed in the stem cell compartment. ME deficiency leads to a reduction in the number of HSCs and a complete loss of long-term repopulation capacity, whereas the stem cell compartment is shifted from quiescence to active cycling. Genetic exploration of the relative roles of endogenous ME and EVI1 isoforms revealed that ME preferentially rescues long-term HSC defects. RNA-seq analysis in Lin ؊ Sca-1 ؉ cKit ؉ cells (LSKs) of ME m1 documents near complete silencing of Cdkn1c, encoding negative cell-cycle regulator p57-Kip2. Reintroduction of ME into ME m1 LSKs leads to normalization of both p57-Kip2 expression and growth control. Our results clearly demonstrate a critical role of PR-domain-containing ME in linking p57-kip2 regulation to long-term HSC function. (Blood. 2011;118(14):3853-3861) IntroductionMaintenance of normal hematopoiesis depends on balanced selfrenewal of HSCs and differentiation of blood cell progenitors within a supportive BM environment. Defects in stem cell function can result in a wide range of phenotypes, including cytopenias, dysplasias, and leukemias. HSCs can be functionally divided into those that can reconstitute long-term (Ͼ 16 weeks) and those with short-term repopulating ability (3-6 weeks). 1 It was inferred early on from studies with 5-fluorouracil (5-FU) that the most primitive, long-term reconstituting cells within the marrow are largely quiescent, whereas the short-term repopulating cells are actively cycling. 2,3 Further studies showed that a second injection of 5-FU 2 days after the first can kill off long-term repopulating cells, 4 indicating that the stem cell pool enters the cell cycle after depletion of cycling progenitors by the first 5-FU injection. 5 More recent studies indicate that immunophenotypic HSCs, Lin Ϫ Sca-1 ϩ cKit ϩ cells (LSKs), can be separated into actively dividing HSCs and dormant HSCs, with the latter being in a quiescent state but able to be recruited to the active state during stress. 6 HSCs with the ability to long-term engraft irradiated recipients reside in the dormant pool. These are uniquely marked by the cell-surface phenotype CD150 ϩ /CD48 Ϫ LSKs or by CD135 Ϫ /CD34 Ϫ LSKs. There is good evidence that the relative quiescence of HSCs is due to negative regulation of the cell cycle, 7 metabolic activity, 8,9 and differentiation 10 ; pathways involved in aging, 11,12 response to oxidative stress, 13 and apoptosis 14 play important roles as we...

Expression of the EVI1 proto-oncogene is deregulated by chromosomal translocations in some cases of acute myeloid leukemia (AML) and is associated with poor clinical outcome. Here, through transcriptomic and metabolomic profiling of hematopoietic cells, we reveal that EVI1 overexpression alters cellular metabolism. A pooled shRNA screen identified the ATP-buffering, mitochondrial creatine kinase CKMT1 as a metabolic dependency in EVI1-positive AML. EVI1 promotes CKMT1 expression by repressing the myeloid differentiation regulator RUNX1. Suppression of arginine-creatine metabolism by CKMT1-directed shRNAs or by the small molecule cyclocreatine selectively decreased the viability, promoted cell cycle arrest and apoptosis of human EVI1-positive AML cells, and prolonged survival in human orthotopic and mouse primary AML models. CKMT1 inhibition alters mitochondrial respiration and ATP production, an effect that is abrogated by phospho-creatine-mediated reactivation of the arginine-creatine pathway. Targeting CKMT1 is a promising therapeutic strategy for this EVI1-driven AML subtype that is highly resistant to current treatment regimens.

Axin is a multifunctional protein, regulating Wnt signaling and the c-Jun N-terminal/stress-activated protein kinase (JNK/SAPK) pathway as well as tumorigenesis. In the present study, we found that Axin interacts with three SUMO-1 (small ubiquitin-related modifier) conjugating enzymes 3 (E3), PIAS1, PIASx␤, and PIASy. The extreme C-terminal six amino acid residues of Axin are critical for the Axin/E3 interaction as deletion of the six residues (Axin⌬C6) completely abolished the ability of Axin to interact with E3 enzymes. Axin⌬C6 also failed to activate JNK, although it was intact in both its interaction with MEKK1 and homodimerization. Consistent with the presence of a doublet of the KV(E/D) sumoylation consensus motif at the C-terminal end (KVEKVD), we found that Axin is heavily sumoylated. Deletion of the C-terminal six amino acids drastically reduced sumoylation, indicating that the C-terminal six amino acids stretch is the main sumoylation site for Axin. Sumoylation-defective mutants failed to activate JNK but effectively destabilized ␤-catenin and attenuated LEF1 transcriptional activity. In addition, we show that dominant negative Axin mutants blocked PIAS-mediated JNK activation, in accordance with the requirement of sumoylation for Axin-mediated JNK activation.Taken together, we demonstrate that sumoylation plays a role for Axin to function in the JNK pathway.