RelB and nuclear factor κB (NF-κB2) are the main effectors of NF-κB noncanonical signaling and play critical roles in many physiological processes. However, their role in hematopoietic stem/progenitor cell (HSPC) maintenance has not been characterized. To investigate this, we generated RelB/NF-κB2 double-knockout (dKO) mice and found that dKO HSPCs have profoundly impaired engraftment and self-renewal activity after transplantation into wild-type recipients. Transplantation of wild-type bone marrow cells into dKO mice to assess the role of the dKO microenvironment showed that wild-type HSPCs cycled more rapidly, were more abundant, and had developmental aberrancies: increased myeloid and decreased lymphoid lineages, similar to dKO HSPCs. Notably, when these wild-type cells were returned to normal hosts, these phenotypic changes were reversed, indicating a potent but transient phenotype conferred by the dKO microenvironment. However, dKO bone marrow stromal cell numbers were reduced, and bone-lining niche cells supported less HSPC expansion than controls. Furthermore, increased dKO HSPC proliferation was associated with impaired expression of niche adhesion molecules by bone-lining cells and increased inflammatory cytokine expression by bone marrow cells. Thus, RelB/NF-κB2 signaling positively and intrinsically regulates HSPC self-renewal and maintains stromal/osteoblastic niches and negatively and extrinsically regulates HSPC expansion and lineage commitment through the marrow microenvironment. STEM CELLS 2012; 30:709-718.

Pancytopenia is a major cause of morbidity in acute myeloid leukemia (AML), yet its cause is unclear. Normal osteoblastic cells have been shown to support hematopoiesis. To define effects of leukemia on osteoblastic cells, we employed an immunocompetent murine model of AML. Leukemic mice had inhibition of osteoblastic cells, with decreased serum levels of the bone formation marker osteocalcin. Osteoprogenitor cells and endosteal-lining osteopontin+ cells were reduced, and osteocalcin mRNA in CD45- marrow cells was diminished. This resulted in severe loss of mineralized bone. Osteoclasts were only transiently increased without significant increases in bone resorption, and their inhibition only partially rescued leukemia-induced bone loss. In vitro data suggested a leukemia-derived secreted factor inhibited osteoblastic cells. Since the chemokine CCL-3 was recently reported to inhibit osteoblastic function in myeloma, we tested its expression in our model, and in AML patients. Consistent with its potential novel role in leukemic-dependent bone loss, CCL-3 mRNA was significantly increased in malignant marrow cells from leukemic mice and from samples from AML patients. Based on these results, we propose that therapeutic mitigation of leukemia-induced uncoupling of osteoblastic and osteoclastic cells may represent a novel approach to promote normal hematopoiesis in patients with myeloid neoplasms.

Acute myelogenous leukemia (AML) is typically a disease of stem progenitor cell origin. Interestingly, the leukemic stem cell (LSC) shares many characteristics with normal hematopoietic stem cells (HSCs) including the ability to self-renew and a predominantly G(0) cell-cycle status. Thus, although conventional chemotherapy regimens often ablate actively cycling leukemic blast cells, the primitive LSC population is likely to be drug-resistant. Moreover, given the quiescent nature of LSCs, current drugs may not effectively distinguish between malignant stem cells and normal HSCs. Nonetheless, based on recent studies of LSC molecular biology, we hypothesized that certain unique properties of leukemic cells could be exploited to induce apoptosis in the LSC population while sparing normal stem cells. In this report we describe a strategy using treatment of primary AML cells with the proteasome inhibitor carbobenzoxyl-l-leucyl-l-leucyl-l-leucinal (MG-132) and the anthracycline idarubicin. Comparison of normal and leukemic specimens using in vitro culture and in vivo xenotransplantation assays shows that the combination of these two agents induces rapid and extensive apoptosis of the LSC population while leaving normal HSCs viable. Molecular genetic studies using a dominant-negative allele of inhibitor of nuclear factor kappaB (IkappaBalpha) demonstrate that inhibition of nuclear factor kappaB (NF-kappaB) contributes to apoptosis induction. In addition, gene-expression analyses suggest that activation of p53-regulated genes are also involved in LSC apoptosis. Collectively, these findings demonstrate that malignant stem cells can be preferentially targeted for ablation. Further, the data begin to elucidate the molecular mechanisms that underlie LSC-specific apoptosis and suggest new directions for AML therapy.

Recent studies have described malignant stem cells as central to the initiation, growth, and potential relapse of acute and chronic myelogenous leukemia (AML and CML). Because of their important role in pathogenesis, rare and biologically distinct leukemia stem cells (LSCs) represent a critical target for therapeutic intervention. However, to date, very few agents have been shown to directly target the LSC population. The present studies demonstrate that parthenolide (PTL), a naturally occurring small molecule, induces robust apoptosis in primary human AML cells and blast crisis CML (bcCML) cells while sparing normal hematopoietic cells. Furthermore, analysis of progenitor cells using in vitro colony assays, as well as stem cells using the nonobese diabetic/severe combined immunodeficient (NOD/SCID) xenograft model, show that PTL also preferentially targets AML progenitor and stem cell populations. Notably, in comparison to the standard chemotherapy drug cytosine arabinoside (Ara-C), PTL is much more specific to leukemia cells. The molecular mechanism of PTL-mediated apoptosis is strongly associated with inhibition of nuclear factor kappa B (NF-kappaB), proapoptotic activation of p53, and increased reactive oxygen species (ROS). On the basis of these findings, we propose that the activity of PTL triggers LSC-specific apoptosis and as such represents a potentially important new class of drugs for LSC-targeted therapy.

The investigation and study of cancer stem cells (CSCs) have received enormous attention over the past 5 to 10 years but remain topics of considerable controversy. Opinions about the validity of the CSC hypothesis, the biological properties of CSCs, and the relevance of CSCs to cancer therapy differ widely. In the following commentary, we discuss the nature of the debate, the parameters by which CSCs can or cannot be defined, and the identification of new potential therapeutic targets elucidated by considering cancer as a problem in stem cell biology.

Major strides have been made in our understanding of the molecular basis of adult and pediatric leukemias. More than one hundred disease alleles have been identified and characterized in cell culture and murine models of leukemia. In some instances, molecularly targeted therapies have been developed based on these insights that are currently in clinical trials, such as small molecule inhibitors of FLT3. In addition, it has recently been appreciated that, as with normal hematopoiesis, there is a hierarchical organization among leukemic cells that includes a rare population of leukemic stem cells that have properties of self-renewal. Understanding the characteristics of these leukemic stem cells may provide new insights into leukemia therapies that target self-renewal pathways. In Section I, Dr. Craig Jordan reviews the data that supports the existence of a "leukemia stem cell." He provides an overview of the functional properties of leukemic stem cells, their relationship to hematopoietic stem cells, and the relevance of leukemic stem cells in other human malignancies including solid tumors. He briefly discusses what is known of the pathways that regulate properties of self-renewal. Dr. Gary Gilliland provides an overview of the genetics of adult leukemias in Section II and ongoing genome-wide strategies for discovery of new disease alleles. He describes the clinical and therapeutic implications of these findings and provides examples of bench-to-bedside translation of molecularly targeted therapies for AML, including the use of FLT3 inhibitors. In Section III, Dr. Carolyn Felix reviews recent advances in our understanding of the genetics and therapy of pediatric leukemias. She provides an overview of leukemias that are common in pediatric malignancies but rarely observed in adults, including the TEL-AML1 (ETV6-RUNX1) fusion associated with pediatric B-cell ALL, the OTT-MAL fusion associated with infant megakaryoblastic leukemia, PTPN11 mutations in juvenile myelomonocytic leukemia, and MLL fusion genes in leukemogenesis, among others.

Leukemia stem cells (LSCs) are thought to play a central role in the pathogenesis of acute leukemia and likely contribute to both disease initiation and relapse. Therefore, identification of agents that target LSCs is an important consideration for the development of new therapies. To this end, we have previously demonstrated that the naturally-occurring compound parthenolide (PTL) can induce death of human LSCs in vitro, while sparing normal hematopoietic cells. However, PTL has relatively poor pharmacological properties that limit its potential clinical use. Consequently, we generated a family of PTL analogs designed to improve solubility and bioavailability. These studies identified an analog, dimethylamino-parthenolide (DMAPT), which induces rapid death of primary human LSC from both myeloid and lymphoid leukemias, and is also highly cytotoxic to bulk leukemic cell populations. Molecular studies indicate the prevalent activities of DMAPT include induction of oxidative stress responses, inhibition of NF-kB, and activation of p53. The compound has approximately 70% oral bioavailability and pharmacological studies using both mouse xenograft models and spontaneous acute canine leukemias demonstrate in vivo bioactivity as determined by functional assays and multiple biomarkers. Therefore, based on the collective preclinical data, we propose that the novel compound DMAPT has the potential to target human LSCs in vivo.

The eukaryotic translation initiation factor 4E (eIF4E) acts as both a key translation factor and as a promoter of nucleocytoplasmic transport of specific transcripts. Traditionally, its transformation capacity in vivo is attributed to its role in translation initiation in the cytoplasm. Here, we demonstrate that elevated eIF4E impedes granulocytic and monocytic differentiation. Our subsequent mutagenesis studies indicate that this block is a result of dysregulated eIF4E-dependent mRNA transport. These studies indicate that the RNA transport function of eIF4E could contribute to leukemogenesis. We extended our studies to provide the first evidence that the nuclear transport function of eIF4E contributes to human malignancy, specifically in a subset of acute and chronic myelogenous leukemia patients. We observe an increase in eIF4E-dependent cyclin D1 mRNA transport and a concomitant increase in cyclin D1 protein levels. The aberrant nuclear function of eIF4E is due to abnormally large eIF4E bodies and the loss of regulation by the proline-rich homeodomain PRH. We developed a novel tool to modulate this transport activity. The introduction of IkappaB, the repressor of NF-kappaB, leads to suppression of eIF4E, elevation of PRH, reorganization of eIF4E nuclear bodies, and subsequent downregulation of eIF4E-dependent mRNA transport. Thus, our findings indicate that this nuclear function of eIF4E can contribute to leukemogenesis by promoting growth and by impeding differentiation.

Human cytomegalovirus (HCMV) resides latently in hematopoietic cells of the bone marrow. Although viral genomes can be found in CD14+ monocytes and CD34+ progenitor cells, the primary reservoir for latent cytomegalovirus is unknown. We analyzed human hematopoietic subpopulations infected in vitro with a recombinant virus that expresses a green fluorescent protein marker gene. Although many hematopoietic cell subsets were infected in vitro, CD14+ monocytes and various CD34+ subpopulations were infected with the greatest efficiency. We have developed an in vitro system in which to study HCMV infection and latency in CD34+ cells cultured with irradiated stromal cells. Marker gene expression was substantially reduced by 4 days postinfection, and infectious virus was not made during the culture period. However, viral DNA sequences were maintained in infected CD34+ cells for >20 days in culture, and, importantly, virus replication could be reactivated by coculture with human fibroblasts. Using an HCMV gene array, we examined HCMV gene expression in CD34+ cells. The pattern of viral gene expression was distinct from that observed during productive or nonproductive infections. Some of these expressed viral genes may function in latency and are targets for further analysis. Altered gene expression in hematopoietic progenitors may be indicative of the nature and outcome of HCMV infection.

Myeloid leukemia arises from leukemia stem cells (LSC), which are resistant to standard chemotherapy agents and likely to be a major cause of drug resistant disease and relapse. To investigate the in vivo properties of LSC, we developed a mouse model in which the biological features of human LSC are closely mimicked. Primitive normal hematopoietic cells were modified to express the BCR/ABL and Nup98/HoxA9 translocation products and a distinct LSC population, with the aberrant immunophenotype of lineage-, Kit(+/-), Flt3+, Sca+, CD34+, and CD150-, was identified. In vivo studies were then performed to assess the response of LSC to therapeutic insult. Treatment of animals with the ABL kinase inhibitor imatinib mesylate induced specific modulation of blasts and progenitor cells but not stem cell populations, thereby recapitulating events inferred to occur in human CML patients. In addition, challenge of leukemic mice with total body irradiation was selectively toxic to normal HSC, suggesting that LSC are resistant to apoptosis and/or senescence in vivo. Taken together, the system provides a powerful means by which the in vivo behavior of LSC vs. HSC can be characterized and candidate treatment regimens can be optimized for maximal specificity towards primitive leukemia cells.

The cellular reservoir for latent human cytomegalovirus (HCMV) in the hematopoietic compartment, and the mechanisms governing a latent infection and reactivation from latency are unknown. Previous work has demonstrated that HCMV infects CD34+ progenitors and expresses a limited subset of viral genes. The outcome of HCMV infection may depend on the cell subpopulations infected within the heterogeneous CD34+ compartment. We compared HCMV infection in well-defined CD34+ cell subpopulations. HCMV infection inhibited hematopoietic colony formation from CD34+/CD38- but not CD34+/c-kit+ cells. CD34+/CD38- cells transiently expressed a large subset of HCMV genes that were not expressed in CD34+/c-kit+ cells or cells expressing more mature cell surface phenotypes. Although viral genomes were present in infected cells, viral gene expression was undetectable by 10 days after infection. Importantly, viral replication could be reactivated by coculture with permissive fibroblasts only from the CD34+/CD38- population. Strikingly, a subpopulation of CD34+/CD38- cells expressing a stem cell phenotype (lineage-/Thy-1+) supported a productive HCMV infection. These studies demonstrate that the outcome of HCMV infection in the hematopoietic compartment is dependent on the nature of the cell subpopulations infected and that CD34+/CD38- cells support an HCMV infection with the hallmarks of latency.