Functional and molecular profiling of hematopoietic stem cells during regeneration.

Hematopoietic stem cells (HSCs) enable hematopoietic stem cell transplantation (HCT) through their ability to replenish the entire blood system. Proliferation of HSCs is linked to decreased reconstitution potential, and a precise regulation of actively dividing HSCs is thus essential to ensure long-term functionality. This regulation becomes important in the transplantation setting where HSCs undergo proliferation followed by a gradual transition to quiescence and homeostasis. Although mouse HSCs have been well studied under homeostatic conditions, the mechanisms regulating HSC activation under stress remain unclear. Here, we analyzed the different phases of regeneration after transplantation. We isolated bone marrow from mice at 8 time points after transplantation and examined the reconstitution dynamics and transcriptional profiles of stem and progenitor populations. We found that regenerating HSCs initially produced rapidly expanding progenitors and displayed distinct changes in fatty acid metabolism and glycolysis. Moreover, we observed molecular changes in cell cycle, MYC and mTOR signaling in both HSCs, and progenitor subsets. We used a decay rate model to fit the temporal transcription profiles of regenerating HSCs and identified genes with progressively decreased or increased expression after transplantation. These genes overlapped to a large extent with published gene sets associated with key aspects of HSC function, demonstrating the potential of this data set as a resource for identification of novel HSC regulators. Taken together, our study provides a detailed functional and molecular characterization of HSCs at different phases of regeneration and identifies a gene set associated with the transition from proliferation to quiescence.

GATA2 mitotic bookmarking is required for definitive haematopoiesis.

In mitosis, most transcription factors detach from chromatin, but some are retained and bookmark genomic sites. Mitotic bookmarking has been implicated in lineage inheritance, pluripotency and reprogramming. However, the biological significance of this mechanism in vivo remains unclear. Here, we address mitotic retention of the hemogenic factors GATA2, GFI1B and FOS during haematopoietic specification. We show that GATA2 remains bound to chromatin throughout mitosis, in contrast to GFI1B and FOS, via C-terminal zinc finger-mediated DNA binding. GATA2 bookmarks a subset of its interphase targets that are co-enriched for RUNX1 and other regulators of definitive haematopoiesis. Remarkably, homozygous mice harbouring the cyclin B1 mitosis degradation domain upstream Gata2 partially phenocopy knockout mice. Degradation of GATA2 at mitotic exit abolishes definitive haematopoiesis at aorta-gonad-mesonephros, placenta and foetal liver, but does not impair yolk sac haematopoiesis. Our findings implicate GATA2-mediated mitotic bookmarking as critical for definitive haematopoiesis and highlight a dependency on bookmarkers for lineage commitment.

Ciclopirox ethanolamine preserves the immature state of human HSCs by mediating intracellular iron content.

Culture conditions in which hematopoietic stem cells (HSCs) can be expanded for clinical benefit are highly sought after. To elucidate regulatory mechanisms governing the maintenance and propagation of human HSCs ex vivo, we screened libraries of annotated small molecules in human cord blood cells using an optimized assay for detection of functional HSCs during culture. We found that the antifungal agent ciclopirox ethanolamine (CPX) selectively supported immature CD34+CD90+ cells during culture and enhanced their long-term in vivo repopulation capacity. Purified HSCs treated with CPX showed a reduced cell division rate and an enrichment of HSC-specific gene expression patterns. Mechanistically, we found that the HSC stimulating effect of CPX was directly mediated by chelation of the intracellular iron pool, which in turn affected iron-dependent proteins and enzymes mediating cellular metabolism and respiration. Our findings unveil a significant impact of iron homeostasis in regulation of human HSCs, with important implications for both basic HSC biology and clinical hematology.

Engineered human Diamond-Blackfan anemia disease model confirms therapeutic effects of clinically applicable lentiviral vector at single-cell resolution.

Diamond-Blackfan anemia is a rare genetic bone marrow failure disorder which is usually caused by mutations in ribosomal protein genes. In the present study, we generated a traceable RPS19-deficient cell model using CRISPR-Cas9 and homology-directed repair to investigate the therapeutic effects of a clinically applicable lentiviral vector at single-cell resolution. We developed a gentle nanostraw delivery platform to edit the RPS19 gene in primary human cord bloodderived CD34+ hematopoietic stem and progenitor cells. The edited cells showed expected impaired erythroid differentiation phenotype, and a specific erythroid progenitor with abnormal cell cycle status accompanied by enrichment of TNFα/NF-κB and p53 signaling pathways was identified by single-cell RNA sequencing analysis. The therapeutic vector could rescue the abnormal erythropoiesis by activating cell cycle-related signaling pathways and promoted red blood cell production. Overall, these results establish nanostraws as a gentle option for CRISPR-Cas9- based gene editing in sensitive primary hematopoietic stem and progenitor cells, and provide support for future clinical investigations of the lentiviral gene therapy strategy.

MAC-1 marks a quiescent and functionally superior HSC subset during regeneration.

Mouse hematopoietic stem cells (HSCs) have been extensively defined both molecularly and functionally at steady state, while regenerative stress induces immunophenotypical changes that limit high purity isolation and analysis. It is therefore important to identify markers that specifically label activated HSCs to gain further knowledge about their molecular and functional properties. Here, we assessed the expression of macrophage-1 antigen (MAC-1) on HSCs during regeneration following transplantation and observed a transient increase in MAC-1 expression during the early reconstitution phase. Serial transplantation experiments demonstrated that reconstitution potential was highly enriched in the MAC-1+ portion of the HSC pool. Moreover, in contrast to previous reports, we found that MAC-1 expression inversely correlates with cell cycling, and global transcriptome analysis showed that regenerating MAC-1+ HSCs share molecular features with stem cells with low mitotic history. Taken together, our results suggest that MAC-1 expression marks predominantly quiescent and functionally superior HSCs during early regeneration.

Schlafen2 is a regulator of quiescence in adult murine hematopoietic stem cells.

Even though hematopoietic stem cells (HSC) are characterized by their ability to self-renew and differentiate, they primarily reside in quiescence. Despite the immense importance of this quiescent state, its maintenance and regulation is still incompletely understood. Schlafen2 (Slfn2) is a cytoplasmic protein known to be involved in cell proliferation, differentiation, quiescence, interferon response, and regulation of the immune system. Interestingly, Slfn2 is highly expressed in primitive hematopoietic cells. In order to investigate the role of Slfn2 in the regulation of HSC we have studied HSC function in the elektra mouse model, where the elektra allele of the Slfn2 gene contains a point mutation causing loss of function of the Slfn2 protein. We found that homozygosity for the elektra allele caused a decrease of primitive hematopoietic compartments in murine bone marrow. We further found that transplantation of elektra bone marrow and purified HSC resulted in a significantly reduced regenerative capacity of HSC in competitive transplantation settings. Importantly, we found that a significantly higher fraction of elektra HSC (as compared to wild-type HSC) were actively cycling, suggesting that the mutation in Slfn2 increases HSC proliferation. This additionally caused an increased amount of apoptotic stem and progenitor cells. Taken together, our findings demonstrate that dysregulation of Slfn2 results in a functional deficiency of primitive hematopoietic cells, which is particularly reflected by a drastically impaired ability to reconstitute the hematopoietic system following transplantation and an increase in HSC proliferation. This study thus identifies Slfn2 as a novel and critical regulator of adult HSC and HSC quiescence.

Uncoupling key determinants of hematopoietic stem cell engraftment through cell-specific and temporally controlled recipient conditioning.

Hematopoietic stem cells (HSCs) are typically characterized by transplantation into irradiated hosts in a highly perturbed microenvironment. Here, we show that selective and temporally controlled depletion of resident HSCs through genetic deletion of Gata2 constitutes efficient recipient conditioning for transplantation without irradiation. Strikingly, we achieved robust engraftment of donor HSCs even when delaying Gata2 deletion until 4 weeks after transplantation, allowing homing and early localization to occur in a completely non-perturbed environment. When HSCs from the congenic strains Ly5.1 and Ly5.2 were competitively transplanted, we found that the more proliferative state of Ly5.2 HSCs was associated with superior long-term engraftment when using conditioning by standard irradiation, while higher CXCR4 expression and a better homing ability of Ly5.1 HSCs strongly favored the outcome in our inducible HSC depletion model. Thus, the mode and timing of recipient conditioning challenges distinct functional features of transplanted HSCs.

Histamine targets myeloid-derived suppressor cells and improves the anti-tumor efficacy of PD-1/PD-L1 checkpoint blockade.

Myeloid-derived suppressor cells (MDSCs) are immature monocytes and granulocytes that impede immune-mediated clearance of malignant cells by multiple mechanisms, including the formation of immunosuppressive reactive oxygen species (ROS) via the myeloid cell NADPH oxidase (NOX2). Histamine dihydrochloride (HDC), a NOX2 inhibitor, exerts anti-cancer efficacy in experimental tumor models but the detailed mechanisms are insufficiently understood. To determine effects of HDC on the MDSC compartment we utilized three murine cancer models known to entail accumulation of MDSC, i.e. EL-4 lymphoma, MC-38 colorectal carcinoma, and 4T1 mammary carcinoma. In vivo treatment with HDC delayed EL-4 and 4T1 tumor growth and reduced the ROS formation by intratumoral MDSCs. HDC treatment of EL-4 bearing mice also reduced the accumulation of intratumoral MDSCs and reduced MDSC-induced suppression of T cells ex vivo. Experiments using GR1-depleted and Nox2 knock out mice supported that the anti-tumor efficacy of HDC required presence of NOX2+ GR1+ cells in vivo. In addition, treatment with HDC enhanced the anti-tumor efficacy of programmed cell death receptor 1 (PD-1) and PD-1 ligand checkpoint blockade in EL-4- and MC-38-bearing mice. Immunomodulatory effects of a HDC-containing regimen on MDSCs were further analyzed in a phase IV trial (Re:Mission Trial, ClinicalTrials.gov; NCT01347996) where patients with acute myeloid leukemia received HDC in conjunction with low-dose IL-2 (HDC/IL-2) for relapse prevention. Peripheral CD14+HLA-DR-/low MDSCs (M-MDSCs) were reduced during cycles of HDC/IL-2 therapy and a pronounced reduction of M-MDSCs during HDC/IL-2 treatment heralded favorable clinical outcome. We propose that anti-tumor properties of HDC may comprise the targeting of MDSCs.

Role of regulatory T cells in acute myeloid leukemia patients undergoing relapse-preventive immunotherapy.

Regulatory T cells (Tregs) have been proposed to dampen functions of anti-neoplastic immune cells and thus promote cancer progression. In a phase IV trial (Re:Mission Trial, NCT01347996, http://www.clinicaltrials.gov ) 84 patients (age 18-79) with acute myeloid leukemia (AML) in first complete remission (CR) received ten consecutive 3-week cycles of immunotherapy with histamine dihydrochloride (HDC) and low-dose interleukin-2 (IL-2) to prevent relapse of leukemia in the post-consolidation phase. This study aimed at defining the features, function and dynamics of Foxp3+CD25highCD4+ Tregs during immunotherapy and to determine the potential impact of Tregs on relapse risk and survival. We observed a pronounced increase in Treg counts in peripheral blood during initial cycles of HDC/IL-2. The accumulating Tregs resembled thymic-derived natural Tregs (nTregs), showed augmented expression of CTLA-4 and suppressed the cell cycle proliferation of conventional T cells ex vivo. Relapse of AML was not prognosticated by Treg counts at onset of treatment or after the first cycle of immunotherapy. However, the magnitude of Treg induction was diminished in subsequent treatment cycles. Exploratory analyses implied that a reduced expansion of Tregs in later treatment cycles and a short Treg telomere length were significantly associated with a favorable clinical outcome. Our results suggest that immunotherapy with HDC/IL-2 in AML entails induction of immunosuppressive Tregs that may be targeted for improved anti-leukemic efficiency.

Dynamics of myeloid cell populations during relapse-preventive immunotherapy in acute myeloid leukemia.

Relapse of leukemia in the postchemotherapy phase contributes to the poor prognosis and survival in patients with acute myeloid leukemia (AML). In an international phase IV trial (ClinicalTrials.gov; NCT01347996), 84 patients with AML in first complete remission who had not undergone transplantation received immunotherapy with histamine dihydrochloride (HDC) and low-dose IL-2 with the aim of preventing relapse. The dynamics of myeloid cell counts and expression of activation markers was assessed before and after cycles of immunotherapy and correlated with clinical outcome in terms of relapse risk and survival. During cycles, a pronounced increase in blood eosinophil counts was observed along with a reduction in monocyte and neutrophil counts. A strong reduction of blood monocyte counts during the first HDC/IL-2 treatment cycle predicted leukemia-free survival. The HDC component of the immunotherapy exerts agonist activity at histamine type 2 receptors (H2Rs) that are expressed by myeloid cells. It was observed that the density of H2 R expression in blood monocytes increased during cycles of immunotherapy and that high monocyte H2R expression implied reduced relapse risk and improved overall survival. Several other activation markers, including HLA-DR, CD86, and CD40, were induced in monocytes and dendritic cells during immunotherapy but did not predict clinical outcome. In addition, expression of HLA-ABC increased in all myeloid populations during therapy. A low expression of HLA-ABC was associated with reduced relapse risk. These results suggest that aspects of myeloid cell biology may impact clinical benefit of relapse-preventive immunotherapy in AML.

Protein-dependent Membrane Interaction of A Partially Disordered Protein Complex with Oleic Acid: Implications for Cancer Lipidomics.

Bovine α-lactalbumin (BLA) forms cytotoxic complexes with oleic acid (OA) that perturbs tumor cell membranes, but molecular determinants of these membrane-interactions remain poorly understood. Here, we aim to obtain molecular insights into the interaction of BLA/BLA-OA complex with model membranes. We characterized the folding state of BLA-OA complex using tryptophan fluorescence and resolved residue-specific interactions of BLA with OA using molecular dynamics simulation. We integrated membrane-binding data using a voltage-sensitive probe and molecular dynamics (MD) to demonstrate the preferential interaction of the BLA-OA complex with negatively charged membranes. We identified amino acid residues of BLA and BLA-OA complex as determinants of these membrane interactions using MD, functionally corroborated by uptake of the corresponding α-LA peptides across tumor cell membranes. The results suggest that the α-LA component of these cytotoxic complexes confers specificity for tumor cell membranes through protein interactions that are maintained even in the lipid complex, in the presence of OA.

Role of natural killer cell subsets and natural cytotoxicity receptors for the outcome of immunotherapy in acute myeloid leukemia.

In a phase IV trial, 84 patients (age 18-79) with acute myeloid leukemia (AML) in first complete remission (CR) received cycles of immunotherapy with histamine dihydrochloride (HDC) and low-dose human recombinant interleukin 2 (IL-2) for 18 months to prevent leukemic relapse. During cycles, the treatment resulted in expansion of CD56(bright) (CD3(-)/16(-)/56(bright)) and CD16(+) (CD3(-)/16(+)/56(+)) natural killer (NK) cells in the blood along with increased NK cell expression of the natural cytotoxicity receptors (NCRs) NKp30 and NKp46. Multivariate analyses correcting for age and risk group demonstrated that high CD56(bright) NK cell counts and high expression of NKp30 or NKp46 on CD16(+) NK cells independently predicted leukemia-free survival (LFS) and overall survival (OS). Our results suggest that the dynamics of NK cell subsets and their NCR expression may determine the efficiency of relapse-preventive immunotherapy in AML.

Dynamics of cytotoxic T cell subsets during immunotherapy predicts outcome in acute myeloid leukemia.

Preventing relapse after chemotherapy remains a challenge in acute myeloid leukemia (AML). Eighty-four non-transplanted AML patients in first complete remission received relapse-preventive immunotherapy with histamine dihydrochloride and low-dose interleukin-2 in an international phase IV trial (ClinicalTrials.gov; NCT01347996). Blood samples were drawn during cycles of immunotherapy and analyzed for CD8+ (cytotoxic) T cell phenotypes in blood. During the first cycle of therapy, a re-distribution of cytotoxic T cells was observed comprising a reduction of T effector memory cells and a concomitant increase of T effector cells. The dynamics of T cell subtypes during immunotherapy prognosticated relapse and survival, in particular among older patients and remained significantly predictive of clinical outcome after correction for potential confounders. Presence of CD8+ T cells with specificity for leukemia-associated antigens identified patients with low relapse risk. Our results point to novel aspects of T cell-mediated immunosurveillance in AML and provide conceivable biomarkers in relapse-preventive immunotherapy.

NK cell expression of natural cytotoxicity receptors may determine relapse risk in older AML patients undergoing immunotherapy for remission maintenance.

In a phase IV trial, eighty-four patients (age 18-79) with acute myeloid leukemia (AML) in first complete remission (CR) received cycles of immunotherapy with histamine dihydrochloride (HDC) and low-dose human recombinant interleukin-2 (IL-2) to prevent relapse in the post-consolidation phase. Aspects of natural killer (NK) cell biology were analyzed before and during immunotherapy with focus on outcome in older patients. In younger (<60 years old, n = 37) and older patients (>60 years old, n = 47), treatment with HDC/IL-2 resulted in an expansion of CD56(bright) and CD16+ NK cells in blood along with an increased NK cell expression of the natural cytotoxicity receptors (NCR) NKp30 and NKp46. In older patients, a high expression of NKp30 or NKp46 on CD16+ NK cells before and during therapy predicted leukemia-free and overall survival. These results suggest that NK cell functions determine relapse risk and survival in older AML patients and point to biomarkers of efficacy in protocols for remission maintenance.

Protein receptor-independent plasma membrane remodeling by HAMLET: a tumoricidal protein-lipid complex.

A central tenet of signal transduction in eukaryotic cells is that extra-cellular ligands activate specific cell surface receptors, which orchestrate downstream responses. This ''protein-centric" view is increasingly challenged by evidence for the involvement of specialized membrane domains in signal transduction. Here, we propose that membrane perturbation may serve as an alternative mechanism to activate a conserved cell-death program in cancer cells. This view emerges from the extraordinary manner in which HAMLET (Human Alpha-lactalbumin Made LEthal to Tumor cells) kills a wide range of tumor cells in vitro and demonstrates therapeutic efficacy and selectivity in cancer models and clinical studies. We identify a ''receptor independent" transformation of vesicular motifs in model membranes, which is paralleled by gross remodeling of tumor cell membranes. Furthermore, we find that HAMLET accumulates within these de novo membrane conformations and define membrane blebs as cellular compartments for direct interactions of HAMLET with essential target proteins such as the Ras family of GTPases. Finally, we demonstrate lower sensitivity of healthy cell membranes to HAMLET challenge. These features suggest that HAMLET-induced curvature-dependent membrane conformations serve as surrogate receptors for initiating signal transduction cascades, ultimately leading to cell death.

Spontaneous colitis in Muc2-deficient mice reflects clinical and cellular features of active ulcerative colitis.

BACKGROUND: The colonic mucus layer plays a critical role in intestinal homeostasis by limiting contact between luminal bacteria and the mucosal immune system. A defective mucus barrier in animal models allows bacterial contact with the intestinal epithelium and results in spontaneous colitis. A defective mucus barrier is also a key feature of active ulcerative colitis (UC). Alterations in the immune compartment due to intestinal bacterial breach in mice lacking the colon mucus barrier have not been characterized and correlated to active UC. AIMS: To characterize alterations in the immune compartment due to intestinal bacterial breach in Muc2-/- mice, which lack the colon mucus barrier, and correlate the findings to active UC. METHODS: Bacterial contact with colon epithelium and penetration into colon tissue was examined in Muc2-/- mice and colon biopsies from patients with active UC using fluorescence microscopy and qPCR. Neutrophils, lymphocytes, CD103+ dendritic cell subsets and macrophages in colon from Muc2-/- mice and biopsies from UC patients were quantitated by flow cytometry. RESULTS: Inflamed UC patients and Muc2-/- mice had bacteria in contact with the colon epithelium. Bacterial rRNA was present in colonic mucosa in humans and Muc2-/- mice and in the draining lymph nodes of mice. Inflamed Muc2-/- mice and UC patients had elevated colon neutrophils, T cells and macrophages while a reduced frequency of CD103+ DCs was present in the inflamed colon of both mice and humans. CONCLUSIONS: The parallel features of the colon immune cell compartment in Muc2-/- mice and UC patients supports the usefulness of this model to understand the early phase of spontaneous colitis and will provide insight into novel strategies to treat UC.

Lipids as tumoricidal components of human α-lactalbumin made lethal to tumor cells (HAMLET): unique and shared effects on signaling and death.

Long-chain fatty acids are internalized by receptor-mediated mechanisms or receptor-independent diffusion across cytoplasmic membranes and are utilized as nutrients, building blocks, and signaling intermediates. Here we describe how the association of long-chain fatty acids to a partially unfolded, extracellular protein can alter the presentation to target cells and cellular effects. HAMLET (human α-lactalbumin made lethal to tumor cells) is a tumoricidal complex of partially unfolded α-lactalbumin and oleic acid (OA). As OA lacks independent tumoricidal activity at concentrations equimolar to HAMLET, the contribution of the lipid has been debated. We show by natural abundance (13)C NMR that the lipid in HAMLET is deprotonated and by chromatography that oleate rather than oleic acid is the relevant HAMLET constituent. Compared with HAMLET, oleate (175 µm) showed weak effects on ion fluxes and gene expression. Unlike HAMLET, which causes metabolic paralysis, fatty acid metabolites were less strongly altered. The functional overlap increased with higher oleate concentrations (500 µm). Cellular responses to OA were weak or absent, suggesting that deprotonation favors cellular interactions of fatty acids. Fatty acids may thus exert some of their essential effects on host cells when in the deprotonated state and when presented in the context of a partially unfolded protein.

A unifying mechanism for cancer cell death through ion channel activation by HAMLET.

Ion channels and ion fluxes control many aspects of tissue homeostasis. During oncogenic transformation, critical ion channel functions may be perturbed but conserved tumor specific ion fluxes remain to be defined. Here we used the tumoricidal protein-lipid complex HAMLET as a probe to identify ion fluxes involved in tumor cell death. We show that HAMLET activates a non-selective cation current, which reached a magnitude of 2.74±0.88 nA within 1.43±0.13 min from HAMLET application. Rapid ion fluxes were essential for HAMLET-induced carcinoma cell death as inhibitors (amiloride, BaCl2), preventing the changes in free cellular Na(+) and K(+) concentrations also prevented essential steps accompanying carcinoma cell death, including changes in morphology, uptake, global transcription, and MAP kinase activation. Through global transcriptional analysis and phosphorylation arrays, a strong ion flux dependent p38 MAPK response was detected and inhibition of p38 signaling delayed HAMLET-induced death. Healthy, differentiated cells were resistant to HAMLET challenge, which was accompanied by innate immunity rather than p38-activation. The results suggest, for the first time, a unifying mechanism for the initiation of HAMLET's broad and rapid lethal effect on tumor cells. These findings are particularly significant in view of HAMLET's documented therapeutic efficacy in human studies and animal models. The results also suggest that HAMLET offers a two-tiered therapeutic approach, killing cancer cells while stimulating an innate immune response in surrounding healthy tissues.

Everything you always wanted to know about CADY-mediated siRNA delivery* (* but afraid to ask).

Although siRNA consist in very promising therapeutics, their clinical development is limited by several biological barriers including low cellular permeability, poor stability and lack of tissue specificity. Therefore the Achilles' heel for siRNA-based therapy is directly related to the lack of efficient system to promote their delivery. During the last two decades, cell-penetrating peptides (CPPs) have been widely developed to enhance the cellular delivery of therapeutics. In this context we have elaborated a new strategy based on self-assembling peptide-based nanoparticles. The CADY peptide is a 20-residue secondary amphipathic peptide which is able to spontaneously self associate with siRNA with a strong affinity, by combining both electrostatic and hydrophobic interactions, to form stable nanoparticles. Investigations of both physico-chemical properties and cellular siRNA delivery revealed that the CADY/siRNA complexes were able to enter a wide variety of cell lines by a mechanism independent of any endocytotic pathway. In addition a deeper understanding of the self assembly of CADY molecules around siRNA leads to a "raspberry"-like nanoparticle architecture which provides new perspectives for the CADY/siRNA formulations. Finally the robustness of the biological response infers that peptide-based nanoparticle technology holds a strong promise for therapeutic applications. The present review deals with most of the biophysical characteristics as well as the cellular mechanism and cellular applications of CADY/siRNA nanoparticles.

HAMLET: functional properties and therapeutic potential.

Human α-lactalbumin made lethal to tumor cells (HAMLET) is the first member in a new family of protein-lipid complexes that kills tumor cells with high selectivity. The protein component of HAMLET is α-lactalbumin, which in its native state acts as a substrate specifier in the lactose synthase complex, thereby defining a function essential for the survival of lactating mammals. In addition, α-lactalbumin acquires tumoricidal activity after partial unfolding and binding to oleic acid. The lipid cofactor serves the dual role as a stabilizer of the altered fold of the protein and a coactivator of specific steps in tumor cell death. HAMLET is broadly tumoricidal, suggesting that the complex identifies conserved death pathways suitable for targeting by novel therapies. Sensitivity to HAMLET is defined by oncogene expression including Ras and c-Myc and by glycolytic enzymes. Cellular targets are located in the cytoplasmic membrane, cytoskeleton, mitochondria, proteasomes, lysosomes and nuclei, and specific signaling pathways are rapidly activated, first by interactions of HAMLET with the cell membrane and subsequently after HAMLET internalization. Therapeutic effects of HAMLET have been demonstrated in human skin papillomas and bladder cancers, and HAMLET limits the progression of human glioblastomas, with no evidence of toxicity for normal brain or bladder tissue. These findings open up new avenues for cancer therapy and the understanding of conserved death responses in tumor cells.