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Despite reaching remission with traditional chemotherapy, most adult patients with acute myeloid leukemia (AML) will relapse and die of their disease. Numerous studies have identified a rare subset of leukemia cells that evade traditional chemotherapy and are capable of self-renewal and initiating leukemia. These cells are thought to be responsible for relapse and are termed leukemia stem cells (LSCs). This article will review the current LSC translational research and focus on new approaches to detect LSC burden and its prognostic implications, as well as the identification and development of therapeutic agents active against LSCs., (Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.)

Ciclopirox, an antifungal agent commonly used for the dermatologic treatment of mycoses, has been shown recently to have antitumor properties. Although the exact mechanism of ciclopirox is unclear, its antitumor activity has been attributed to iron chelation and inhibition of the translation initiation factor eIF5A. In this study, we identify a novel function of ciclopirox in the inhibition of mTOR. As with other mTOR inhibitors, we show that ciclopirox significantly enhances the ability of the established preclinical antileukemia compound, parthenolide, to target acute myeloid leukemia. The combination of parthenolide and ciclopirox demonstrates greater toxicity against acute myeloid leukemia than treatment with either compound alone. We also demonstrate that the ability of ciclopirox to inhibit mTOR is specific to ciclopirox because neither iron chelators nor other eIF5A inhibitors affect mTOR activity, even at high doses. We have thus identified a novel function of ciclopirox that might be important for its antileukemic activity., (Copyright © 2013 ISEH - Society for Hematology and Stem Cells. Published by Elsevier Inc. All rights reserved.)

In the past year, there has been increasing attention towards understanding the clinical relevance of minimal residual disease (MRD) assessment. The monitoring of MRD levels at various stages of therapy has considerable potential to impact the guidance of treatment for AML patients and improve outcomes. Thus, efforts have increased to address important concerns regarding MRD measurements. These concerns include: (1) what should be monitored; (2) what methodologies should be used; (3) whether such methodologies are standardized across laboratories; (4) how prognostic levels are defined; (5) when MRD should be monitored; and (6) what treatment options are available for MRD positive patients. In this review, we will discuss the methodologies available for MRD and the studies available to date aiming to address the concerns around the use of MRD measurements for AML patients.

The highly dynamic membranous network of eukaryotic cells allows spatial organization of biochemical reactions to suit the complex metabolic needs of the cell. The unique lipid composition of organelle membranes in the face of dynamic membrane activities assumes that lipid gradients are constantly generated and maintained. Important advances have been made in identifying specialized membrane compartments and lipid transfer mechanisms that are critical for generating and maintaining lipid gradients. Remarkably, one class of minor phospholipids--the phosphoinositides--is emerging as important regulators of these processes. Here, we summarize several lines of research that have led to our current understanding of the connection between phosphoinositides and the transport of structural lipids and offer some thoughts on general principles possibly governing these processes., (Published by Elsevier Ltd.)

Objective: Chromosome 22q11 microdeletion syndrome (del22q11) is the most frequent microdeletion syndrome in humans, with an estimated incidence of 1/4000. It is recognized as a common identifiable cause of cleft palate. We characterized palatal abnormalities in a large cohort of Chilean patients with del22q11., Methods: Patients with the deletion were evaluated by geneticists and speech pathologists, including nasopharyngoscopy when indicated. Comparisons between groups with and without palatal abnormalities were performed using Fisher's exact test and Mann-Whitney U test., Results: Two hundred and one patients were included in the study. Palate abnormalities were present in 154 patients (76.6%). The most frequent finding was submucous cleft palate (both classic and occult forms) seen in 80 patients (39.8% of the total group). Overt cleft palate or cleft lip/palate was seen in 30 patients (14.9%). Patients without palate abnormalities had significantly greater frequency of congenital heart disease and higher mortality., Conclusions: Our data show a high frequency of palate abnormalities without significant association with congenital heart disease. The most common types of palate defects seen in this series are usually not evident on physical examination and thus require a high index of suspicion and active evaluation through nasopharyngoscopy., (Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.)

Chromosome 22q11 microdeletion syndrome has a wide range of clinical manifestations including congenital heart malformations, palatal defects, endocrine abnormalities, immunologic deficits, learning difficulties, and an increased predisposition to psychiatric disease. Short stature and poor weight gain in infancy are common findings and are usually seen in the absence of hormone deficiencies. An increased frequency of obesity has been observed in adolescents and adults. We generated gender-specific growth curves from 0 to 24 months of age, based on 479 length and 475 weight measurements from 138 Chilean patients with 22q11 deletion. Final adult height and weight on 25 individuals were analyzed. The 10th, 50th, and 90th centile-smoothed curves for infants were built using the LMS method and compared with World Health Organization Child Growth Standards. The 50th centile for length in the deleted patients was slightly lower than the 10th centile of WHO standards in boys and girls. The same was observed for weight, although a trend toward a gradual increase near 2 years of age was observed, particularly in boys. Average adult height was 152 cm (ranging from 143 to 162 cm) in females, corresponding to the 10th centiles of WHO standards, and 166 cm for males (160-172 cm), at the 20th centile of WHO standards. A third of the adult females and none of the males had body mass index (BMI) greater than 25. The curves should be useful to monitor growth in infants with 22q11 microdeletion syndrome., (Copyright © 2012 Wiley Periodicals, Inc.)

Unlabelled: Genetic lesions such as BCR-ABL1, E2A-PBX1, and MLL rearrangements (MLLr) are associated with unfavorable outcomes in adult B-cell precursor acute lymphoblastic leukemia (B-ALL). Leukemia oncoproteins may directly or indirectly disrupt cytosine methylation patterning to mediate the malignant phenotype. We postulated that DNA methylation signatures in these aggressive B-ALLs would point toward disease mechanisms and useful biomarkers and therapeutic targets. We therefore conducted DNA methylation and gene expression profiling on a cohort of 215 adult patients with B-ALL enrolled in a single phase III clinical trial (ECOG E2993) and normal control B cells. In BCR-ABL1-positive B-ALLs, aberrant cytosine methylation patterning centered around a cytokine network defined by hypomethylation and overexpression of IL2RA(CD25). The E2993 trial clinical data showed that CD25 expression was strongly associated with a poor outcome in patients with ALL regardless of BCR-ABL1 status, suggesting CD25 as a novel prognostic biomarker for risk stratification in B-ALLs. In E2A-PBX1-positive B-ALLs, aberrant DNA methylation patterning was strongly associated with direct fusion protein binding as shown by the E2A-PBX1 chromatin immunoprecipitation (ChIP) sequencing (ChIP-seq), suggesting that E2A-PBX1 fusion protein directly remodels the epigenome to impose an aggressive B-ALL phenotype. MLLr B-ALL featured prominent cytosine hypomethylation, which was linked with MLL fusion protein binding, H3K79 dimethylation, and transcriptional upregulation, affecting a set of known and newly identified MLL fusion direct targets with oncogenic activity such as FLT3 and BCL6. Notably, BCL6 blockade or loss of function suppressed proliferation and survival of MLLr leukemia cells, suggesting BCL6-targeted therapy as a new therapeutic strategy for MLLr B-ALLs., Significance: We conducted the first integrative epigenomic study in adult B-ALLs, as a correlative study to the ECOG E2993 phase III clinical trial. This study links for the first time the direct actions of oncogenic fusion proteins with disruption of epigenetic regulation mediated by cytosine methylation. We identify a novel clinically actionable biomarker in B-ALLs: IL2RA (CD25), which is linked with BCR-ABL1 and an inflammatory signaling network associated with chemotherapy resistance. We show that BCL6 is a novel MLL fusion protein target that is required to maintain the proliferation and survival of primary human adult MLLr cells and provide the basis for a clinical trial with BCL6 inhibitors for patients with MLLr., (©2012 AACR.)

Most cancers are characterized by multiple molecular alterations, but identification of the key proteins involved in these signaling pathways is currently beyond reach. We show that the inhibitor PU-H71 preferentially targets tumor-enriched Hsp90 complexes and affinity captures Hsp90-dependent oncogenic client proteins. We have used PU-H71 affinity capture to design a proteomic approach that, when combined with bioinformatic pathway analysis, identifies dysregulated signaling networks and key oncoproteins in chronic myeloid leukemia. The identified interactome overlaps with the well-characterized altered proteome in this cancer, indicating that this method can provide global insights into the biology of individual tumors, including primary patient specimens. In addition, we show that this approach can be used to identify previously uncharacterized oncoproteins and mechanisms, potentially leading to new targeted therapies. We further show that the abundance of the PU-H71-enriched Hsp90 species, which is not dictated by Hsp90 expression alone, is predictive of the cell's sensitivity to Hsp90 inhibition.

Fluorescent ligands for the heat shock protein 90 (Hsp90) were synthesized containing either fluorescein isothiocyanate (FITC), 4-nitrobenzo[1,2,5]oxadiazole (NBD) or the red shifted dye sulforhodamine 101 (Texas Red) conjugated to PU-H71. Two of the compounds, PU-H71-FITC2 (9) and PU-H71-NBD1 (8), were shown to be suitable for fluorescence-activated flow cytometry and fluorescence microscopy. Thus these molecules serve as useful probes for studying Hsp90 in heterogeneous live cell populations., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

4-Benzyl-2-methyl-1,2,4-thiadiazolidine-3,5-dione (TDZD-8) was previously identified as an antileukemic agent exhibiting no evident toxicity toward normal hematopoietic cells. An SAR study has been carried out to examine the effect of varying the C-2 and C-4- substituents on the thiadiazolidinone ring of TDZD-8 on antileukemic activity. These studies resulted in the identification of more druglike analogs that exhibited comparable potency to TDZD-8 in killing acute myelogenous leukemia (AML) cells in culture. Surprisingly, the cell death kinetics induced by several of these novel analogs on MV-411 cells were extremely fast, with commitment to death occurring within 30 min. At a concentration of 10 μM, 3f (LD(50)=3.5 μM) completely eradicated cell viability of MV-411 cells within 2h, while analog 3e (LD(50)=2.0 μM) decimated cell viability within 30 min at a concentration of 10 μM and effectively abolished cell viability at 5 μM within 1-2h., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

A number of compounds from different chemical classes are known to bind competitively to the ATP-pocket of Hsp90 and inhibit its chaperone function. The natural product geldanamycin was the first reported inhibitor of Hsp90 and since then synthetic inhibitors from purine, isoxazole and indazol-4-one chemical classes have been discovered and are currently or soon to be in clinical trials for the treatment of cancer. In spite of a similar binding mode to Hsp90, distinct biological profiles were demonstrated among these molecules, both in vitro and in vivo. To better understand the molecular basis for these dissimilarities, we report here the synthesis of chemical tools for three Hsp90 inhibitor classes. These agents will be useful for probing tumor-by-tumor the Hsp90 complexes isolated by specific inhibitors. Such information will lead to better understanding of tumor specific molecular markers to aid in their clinical development. It will also help to elucidate the molecular basis for the biological differences observed among Hsp90 inhibitors., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

We have previously shown that the plant-derived compound parthenolide (PTL) can impair the survival and leukemogenic activity of primary human acute myeloid leukemia (AML) stem cells. However, despite the activity of this agent, PTL also induces cellular protective responses that likely function to reduce its overall cytotoxicity. Thus, we sought to identify pharmacologic agents that enhance the antileukemic potential of PTL. Toward this goal, we used the gene expression signature of PTL to identify compounds that inhibit cytoprotective responses by performing chemical genomic screening of the Connectivity Map database. This screen identified compounds acting along the phosphatidylinositol 3-kinase and mammalian target of rapamycin pathways. Compared with single agent treatment, exposure of AML cells to the combination of PTL and phosphatidylinositol 3-kinase/mammalian target of rapamycin inhibitors significantly decreased viability of AML cells and reduced tumor burden in vitro and in murine xenotransplantation models. Taken together, our data show that rational drug combinations can be identified using chemical genomic screening strategies and that inhibition of cytoprotective functions can enhance the eradication of primary human AML cells.

Interactions between the nuclear factor (NF)-κB inhibitor parthenolide and the pan-histone deacetylase inhibitors (HDACIs) vorinostat and LBH589 were investigated in human acute myeloid leukaemia (AML) cells, including primary AML blasts. Co-administration of parthenolide blocked HDACI-mediated phosphorylation/activation of IKK and RelA/p65 in association with increased JNK1 activation in various AML cell types. These events were accompanied by an increase in apoptosis in multiple AML cell lines (e.g. U937, HL-60, NB4, MV-4-11, and MOLM-13). Significantly, parthenolide also increased HDACI-mediated cell death in haematopoietic cells transduced with the MLL-MLLT1 fusion gene, which exhibit certain leukaemia-initiating cell characteristics, as well as primary AML blasts. Exposure to parthenolide/HDACI regimens clearly inhibited the growth of AML-colony-forming units but was relatively sparing toward normal haematopoietic progenitors. Notably, blockade of c-Jun N-terminal kinase (JNK) signalling by either pharmacological inhibitors or genetic means (e.g. dominant-negative JNK1 or JNK1 shRNA) diminished parthenolide/HDACI-mediated lethality. Moreover, dominant-negative MKK7, but not dominant-negative MKK4/SEK1, blocked JNK1 activation and apoptosis induced by parthenolide/HDACI regimens. Together, these findings indicate that parthenolide potentiates HDACI lethality in human AML cells through a process involving NF-κB inhibition and subsequent MKK7-dependent activation of the SAPK/JNK pathway. They also raise the possibility that this strategy may target leukaemic progenitor cells., (© 2010 Blackwell Publishing Ltd.)

Background: There has been much interest in targeting intracellular redox pathways as a therapeutic approach for cancer. Given recent data to suggest that the redox status of extracellular protein thiol groups (i.e. exofacial thiols) effects cell behavior, we hypothesized that redox active anti-cancer agents would modulate exofacial protein thiols., Methodology/principal Findings: To test this hypothesis, we used the sesquiterpene lactone parthenolide, a known anti-cancer agent. Using flow cytometry, and western blotting to label free thiols with Alexa Fluor 633 C(5) maleimide dye and N-(biotinoyl)-N-(iodoacetyl) ethylendiamine (BIAM), respectively, we show that parthenolide decreases the level of free exofacial thiols on Granta mantle lymphoma cells. In addition, we used immuno-precipitation techniques to identify the central redox regulator thioredoxin, as one of the surface protein thiol targets modified by parthenolide. To examine the functional role of parthenolide induced surface protein thiol modification, we pretreated Granta cells with cell impermeable glutathione (GSH), prior to exposure to parthenolide, and showed that GSH pretreatment; (a) inhibited the interaction of parthenolide with exofacial thiols; (b) inhibited parthenolide mediated activation of JNK and inhibition of NFkappaB, two well established mechanisms of parthenolide activity and; (c) blocked the cytotoxic activity of parthenolide. That GSH had no effect on the parthenolide induced generation of intracellular reactive oxygen species supports the fact that GSH had no effect on intracellular redox. Together these data support the likelihood that GSH inhibits the effect of parthenolide on JNK, NFkappaB and cell death through its direct inhibition of parthenolide's modulation of exofacial thiols., Conclusions/significance: Based on these data, we postulate that one component of parthenolide's anti-lymphoma activity derives from its ability to modify the redox state of critical exofacial thiols. Further, we propose that cancer cell exofacial thiols may be important and novel targets for therapy.

IREM-1 is an inhibitory cell surface receptor with an unknown function and is expressed on myeloid cell lineages, including cell lines derived from acute myeloid leukemia (AML) patients. We have generated a series of monoclonal antibodies (mAbs) against the extracellular domain of IREM-1 and further assessed its expression in normal and AML cells. IREM-1 was restricted to cells from myeloid origin and extensive expression analysis in primary cells obtained from AML patients showed IREM-1 expression in leukemic blasts of 72% (39/54) of samples. We therefore searched for specific IREM-1 mAbs with activity in functional complement-dependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC). Lead mAbs against IREM-1 showed specific cytotoxic activity against a variety of AML-derived cell lines and freshly isolated blasts from AML patients. Internalization of mAbs upon IREM-1 binding was also shown. In vivo anticancer activity of lead mAbs was observed in an established HL-60 xenograft model with a tumor growth delay of up to 40% and in a model using primary human AML cells, where treatment with anti-IREM-1 mAb resulted in a significant reduction of engrafted human cells. These results demonstrate IREM-1 as a potential novel target for immunotherapy of AML.

A series of aminoparthenolide analogs (6-37) were synthesized and evaluated for their anti-leukemic activity. Eight compounds exhibited good anti-leukemic activity with LD(50)'s in the low microM range (1.5-3.0microM). Compounds 16, 24 and 30 were the most potent compounds in the series, causing greater than 90% cell death at 10microM concentration against primary AML cells in culture, with LD(50) values of 1.7, 1.8 and 1.6microM.

JunB is important in maintaining normal hematopoietic stem cell functions, but the mechanisms underlying its activity are not well understood. In the current issue of Cancer Cell, a study by Santaguida et al. provides new insights into JunB's function and the genesis of myeloid disease.

Increasing evidence indicates that malignant stem cells are important for the pathogenesis of acute myelogenous leukemia (AML) and represent a reservoir of cells that drive the development of AML and relapse. Therefore, new treatment regimens are necessary to prevent relapse and improve therapeutic outcomes. Previous studies have shown that the sesquiterpene lactone, parthenolide (PTL), ablates bulk, progenitor, and stem AML cells while causing no appreciable toxicity to normal hematopoietic cells. Thus, PTL must evoke cellular responses capable of mediating AML selective cell death. Given recent advances in chemical genomics such as gene expression-based high-throughput screening (GE-HTS) and the Connectivity Map, we hypothesized that the gene expression signature resulting from treatment of primary AML with PTL could be used to search for similar signatures in publicly available gene expression profiles deposited into the Gene Expression Omnibus (GEO). We therefore devised a broad in silico screen of the GEO database using the PTL gene expression signature as a template and discovered 2 new agents, celastrol and 4-hydroxy-2-nonenal, that effectively eradicate AML at the bulk, progenitor, and stem cell level. These findings suggest the use of multicenter collections of high-throughput data to facilitate discovery of leukemia drugs and drug targets.

In this study, we characterized nuclear factor kappaB (NF-kappaB) subunit DNA binding in chronic lymphocytic leukemia (CLL) samples and demonstrated heterogeneity in basal and inducible NF-kappaB. However, all cases showed higher basal NF-kappaB than normal B cells. Subunit analysis revealed DNA binding of p50, Rel A, and c-Rel in primary CLL cells, and Rel A DNA binding was associated with in vitro survival (P = .01) with high white cell count (P = .01) and shorter lymphocyte doubling time (P = .01). NF-kappaB induction after in vitro stimulation with anti-IgM was associated with increased in vitro survival (P < .001) and expression of the signaling molecule ZAP-70 (P = .003). Prompted by these data, we evaluated the novel parthenolide analog, LC-1, in 54 CLL patient samples. LC-1 induced apoptosis in all the samples tested with a mean LD(50) of 2.8 microM after 24 hours; normal B and T cells were significantly more resistant to its apoptotic effects (P < .001). Apoptosis was preceded by a marked loss of NF-kappaB DNA binding and sensitivity to LC-1 correlated with basal Rel A DNA binding (P = .03, r(2) = 0.15). Furthermore, Rel A DNA binding was inversely correlated with sensitivity to fludarabine (P = .001, r(2) = 0.3), implicating Rel A in fludarabine resistance. Taken together, these data indicate that Rel A represents an excellent therapeutic target for this incurable disease.

Leukemia is thought to arise from malignant stem cells, which have been described for acute and chronic myeloid leukemia (AML and CML) and for acute lymphoblastic leukemia (ALL). Leukemia stem cells (LSCs) are relatively resistant to current chemotherapy and likely contribute to disease relapse and progression. Consequently, the identification of drugs that can efficiently eradicate LSCs is an important priority. In the present study, we investigated the antileukemia activity of the compound TDZD-8. Analysis of primary AML, blast crisis CML (bcCML), ALL, and chronic lymphoblastic leukemia (CLL) specimens showed rapid induction of cell death upon treatment with TDZD-8. In addition, for myeloid leukemias, cytotoxicity was observed for phenotypically primitive cells, in vitro colony-forming progenitors, and LSCs as defined by xenotransplantation assays. In contrast, no significant toxicity was observed for normal hematopoietic stem and progenitor cells. Notably, cell death was frequently evident within 2 hours or less of TDZD-8 exposure. Cellular and molecular studies indicate that the mechanism by which TDZD-8 induces cell death involves rapid loss of membrane integrity, depletion of free thiols, and inhibition of both the PKC and FLT3 signaling pathways. We conclude that TDZD-8 uses a unique and previously unknown mechanism to rapidly target leukemia cells, including malignant stem and progenitor populations.

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 pharmacologic 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 LSCs 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-kappaB, and activation of p53. The compound has approximately 70% oral bioavailability, and pharmacologic 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.

We previously demonstrated that activation of the Parathyroid Hormone Receptor (PTH1R) in osteoblastic cells increases the Notch ligand Jagged1 and expands hematopoietic stem cells (HSC) through Notch signaling. However, regulation of Jagged1 by PTH in osteoblasts is poorly understood. The present study demonstrates that PTH treatment increases Jagged1 levels in a subpopulation of osteoblastic cells in vivo and in UMR106 osteoblastic cells in vitro. Since PTH(1-34) activates both Adenylate Cyclase/Protein Kinase A (AC/PKA) and Protein Kinase C (PKC) downstream of the PTH1R in osteoblastic cells, we independently determined the effect of either pathway on Jagged1. Activation of AC with Forskolin or PKA with PTH(1-31) or cell-permeable cAMP analogues increased osteoblastic Jagged1. This PTH-dependent Jagged1 increase was blocked by H89 and PKI, specific PKA inhibitors. In contrast, PKC activation with phorbol ester (PMA) or PTH(13-34) did not stimulate Jagged1 expression, and PTH-dependent Jagged1 stimulation was not blocked by Gö6976, a conventional PKC inhibitor. Therefore, PTH treatment stimulates osteoblastic Jagged1 mainly through the AC/PKA signaling pathway downstream of the PTH1R. Since Jagged1/Notch signaling has been implicated not only in stromal-HSC interactions but also in osteoblastic differentiation, Jagged1 may play a critical role in mediating the PTH-dependent expansion of HSC, as well as the anabolic effect of PTH in bone.

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 eukaryotic translation initiation factor 4E (eIF4E) alters gene expression on multiple levels. In the cytoplasm, eIF4E acts in the rate-limiting step of translation initiation. In the nucleus, eIF4E facilitates nuclear export of a subset of mRNAs. Both of these functions contribute to eIF4E's ability to oncogenically transform cells. We report here that the homeodomain protein, HOXA9, is a positive regulator of eIF4E. HOXA9 stimulates eIF4E-dependent export of cyclin D1 and ornithine decarboxylase (ODC) mRNAs in the nucleus, as well as increases the translation efficiency of ODC mRNA in the cytoplasm. These activities depend on direct interactions of HOXA9 with eIF4E and are independent of the role of HOXA9 in transcription. At the biochemical level, HOXA9 mediates these effects by competing with factors that repress eIF4E function, in particular the proline-rich homeodomain PRH/Hex. This competitive mechanism of eIF4E regulation is disrupted in a subset of leukemias, where HOXA9 displaces PRH from eIF4E, thereby contributing to eIF4E's dysregulation. In regard to these results and our previous finding that approximately 200 homeodomain proteins contain eIF4E binding sites, we propose that homeodomain modulation of eIF4E activity is a novel means through which this family of proteins implements their effects on growth and development.

Stem cells are an integral component of normal mammalian physiology and have been intensively studied in many systems. Intriguingly, substantial evidence indicates that stem cells also play an important role in the initiation and pathogenesis of at least some cancers. In particular, myeloid leukemias have been extensively characterized with regard to stem and progenitor cell involvement. Thus, as a focal point for both scientific and therapeutic endeavors, leukemic stem cells (LSC) represent a critical area of investigation. LSC appear to retain many characteristics of normal hematopoietic stem cells (HSC) as evidenced by a hierarchical developmental pattern, a mostly quiescent cell cycle profile, and an immunophenotype very similar to HSC. Consequently, defining unique properties of LSC remains a high priority in order to elucidate the molecular mechanisms driving stem cell transformation, and for developing therapeutic strategies that specifically target the LSC population. In this review, we discuss emerging concepts in the field and describe how various molecular and cellular characteristics of leukemia cells might be exploited as a means to preferentially ablate malignant stem cells.

Background: Malignant stem cells have been identified in acute myelogenous leukemia, chronic myeloid leukemia, and some types of acute lymphoblastic leukemia. Like normal stem cells, these leukemic stem cells (LSCs) are able to self-renew, differentiate, and proliferate extensively. Evidence suggests that LSCs are critical for the initiation and perpetuation of leukemic disease., Methods: We reviewed the literature describing the characteristic features of LSCs in various leukemias and the novel molecular approaches being used to specifically ablate the LSC population., Results: Studies have demonstrated the potential importance of ablating LSCs when treating leukemia. The unique characteristics of LSCs that differentiate them from their normal counterparts can be exploited to specifically target the malignant population., Conclusions: Current therapeutic strategies may not effectively ablate the LSC, leaving the potential for disease progression or relapse. A better understanding of LSC cell and molecular biology will allow the design of more effective therapies.

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.

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.

Human acute myelogenous leukemia (AML) is thought to arise from a rare population of malignant stem cells. Cells of this nature, herein referred to as leukemic stem cells (LSCs), have been documented for nearly all AML subtypes and appear to fulfill the criteria for stem cells in that they are self-renewing and give rise to the cells found in many leukemic populations. Because these cells are likely to be critical for the genesis and perpetuation of leukemic disease, the present studies sought to characterize unique molecular properties of the LSC population, with particular emphasis on the transcription factor, nuclear factor-kappaB (NF-kappaB). Previous experiments have shown that unstimulated human CD34(+) progenitor cells do not express NF-kappaB. In contrast, primary AML CD34(+) cells display readily detectable NF-kappaB activity as assessed by electrophoretic mobility shift assay and gene expression studies. Furthermore, detailed analyses of enriched AML stem cells (CD34(+)/CD38(-)/CD123(+)) indicate that NF-kappaB is also active in the LSC population. Given the expression of NF-kappaB in leukemic, but not normal primitive cells, the hypothesis that inhibition of NF-kappaB might induce leukemia-specific apoptosis was tested by treating primary cells with the proteasome inhibitor MG-132, a well-known inhibitor of NF-kappaB. Leukemic CD34(+)/CD38(-) cells displayed a rapid induction of cell death in response to MG-132, whereas normal CD34(+)/CD38(-) cells showed little if any effect. Taken together, these data indicate that primitive AML cells aberrantly express NF-kappaB and that the presence of this factor may provide unique opportunities to preferentially ablate LSCs.

Previous studies indicate that human acute myelogenous leukemia (AML) arises from a rare population of leukemic stem cells. Cells of this nature can initiate and maintain leukemic cell growth in both long-term cultures and nonobese diabetic/severe combined immune-deficient mice. To characterize the biology of primitive AML cells, gene expression screens were performed with 7 primary AML and 3 normal specimens. For each sample, stem cell populations (CD34(+)/CD38(-)) were isolated and used to synthesize radiolabeled complementary DNA (cDNA). AML vs normal probes were then hybridized to cDNA arrays containing genes related to cancer and apoptosis. Of approximately 1400 genes analyzed, 2 tumor-suppressor genes were identified that were overexpressed in all 7 of the AML CD34(+)/CD38(-) cell populations: death-associated protein kinase and interferon regulatory factor 1. Expression of each gene was confirmed by reverse-transcription polymerase chain reaction and immunoblot analysis. It is proposed that tumor-suppressor proteins play a role in the biology of primitive AML cells. (Blood. 2001;97:2177-2179)

Prematurity and fetal death are common complications in patients with cholestasis of pregnancy. Both conditions appear to be associated with abnormal patterns of uterine activity. We studied the oxytocin-induced contractile activity in uterine strips taken from patients with cholestasis of pregnancy (n = 6) and from women with normal pregnancy (n = 6). Contractile activity of the myometrium in response to oxytocin was significantly higher in patients with cholestasis of pregnancy than in normally pregnant patients, at doses of 10(-6), 10(-4), and 10(-2) M. We found that there is a greater maximal response to oxytocin in strips of myometrium from patients with cholestasis of pregnancy than from normally pregnant patients.