Dual targeting of p53 and c-MYC selectively eliminates leukaemic stem cells.

Chronic myeloid leukaemia (CML) arises after transformation of a haemopoietic stem cell (HSC) by the protein-tyrosine kinase BCR-ABL. Direct inhibition of BCR-ABL kinase has revolutionized disease management, but fails to eradicate leukaemic stem cells (LSCs), which maintain CML. LSCs are independent of BCR-ABL for survival, providing a rationale for identifying and targeting kinase-independent pathways. Here we show--using proteomics, transcriptomics and network analyses--that in human LSCs, aberrantly expressed proteins, in both imatinib-responder and non-responder patients, are modulated in concert with p53 (also known as TP53) and c-MYC regulation. Perturbation of both p53 and c-MYC, and not BCR-ABL itself, leads to synergistic cell kill, differentiation, and near elimination of transplantable human LSCs in mice, while sparing normal HSCs. This unbiased systems approach targeting connected nodes exemplifies a novel precision medicine strategy providing evidence that LSCs can be eradicated.

hsa-mir183/EGR1-mediated regulation of E2F1 is required for CML stem/progenitor cell survival.

Chronic myeloid leukemia (CML) stem/progenitor cells (SPCs) express a transcriptional program characteristic of proliferation, yet can achieve and maintain quiescence. Understanding the mechanisms by which leukemic SPCs maintain quiescence will help to clarify how they persist during long-term targeted treatment. We have identified a novel BCR-ABL1 protein kinase-dependent pathway mediated by the upregulation of hsa-mir183, the downregulation of its direct target early growth response 1 (EGR1), and, as a consequence, upregulation of E2F1. We show here that inhibition of hsa-mir183 reduced proliferation and impaired colony formation of CML SPCs. Downstream of this, inhibition of E2F1 also reduced proliferation of CML SPCs, leading to p53-mediated apoptosis. In addition, we demonstrate that E2F1 plays a pivotal role in regulating CML SPC proliferation status. Thus, for the first time, we highlight the mechanism of hsa-mir183/EGR1-mediated E2F1 regulation and demonstrate this axis as a novel, critical factor for CML SPC survival, offering new insights into leukemic stem cell eradication.

CXCR2 and CXCL4 regulate survival and self-renewal of hematopoietic stem/progenitor cells.

The regulation of hematopoietic stem cell (HSC) survival and self-renewal within the bone marrow (BM) niche is not well understood. We therefore investigated global transcriptomic profiling of normal human HSC/hematopoietic progenitor cells [HPCs], revealing that several chemokine ligands (CXCL1-4, CXCL6, CXCL10, CXCL11, and CXCL13) were upregulated in human quiescent CD34(+)Hoescht(-)Pyronin Y(-) and primitive CD34(+)38(-), as compared with proliferating CD34(+)Hoechst(+)Pyronin Y(+) and CD34(+)38(+) stem/progenitor cells. This suggested that chemokines might play an important role in the homeostasis of HSCs. In human CD34(+) hematopoietic cells, knockdown of CXCL4 or pharmacologic inhibition of the chemokine receptor CXCR2, significantly decreased cell viability and colony forming cell (CFC) potential. Studies on Cxcr2(-/-) mice demonstrated enhanced BM and spleen cellularity, with significantly increased numbers of HSCs, hematopoietic progenitor cell-1 (HPC-1), HPC-2, and Lin(-)Sca-1(+)c-Kit(+) subpopulations. Cxcr2(-/-) stem/progenitor cells showed reduced self-renewal capacity as measured in serial transplantation assays. Parallel studies on Cxcl4 demonstrated reduced numbers of CFC in primary and secondary assays following knockdown in murine c-Kit(+) cells, and Cxcl4(-/-) mice showed a decrease in HSC and reduced self-renewal capacity after secondary transplantation. These data demonstrate that the CXCR2 network and CXCL4 play a role in the maintenance of normal HSC/HPC cell fates, including survival and self-renewal.

Redirecting traffic using the XPO1 police.

In this issue of Blood, Walker et al investigate the preclinical potential of KPT-330, an exportin-1 (XPO1, also known as chromosome maintenance protein 1 [CRM1]) inhibitor, against both accelerated phase (AP) and blast crisis chronic myeloid leukemia (CML-BC) and against Philadelphia chromosome-positive (Ph1) acute lymphoblastic leukemia (ALL), all of which are diseases of significant unmet clinical need.1 The authors provide encouraging data from both a leukemic mouse model and a single CML-AP patient, corroborating mechanistic studies suggesting that KPT-330 efficacy relies on targeting abundantly expressed XPO1, followed by the reactivation of protein phosphatase 2A (PP2A).

A specific PTPRC/CD45 phosphorylation event governed by stem cell chemokine CXCL12 regulates primitive hematopoietic cell motility.

CXCL12 governs cellular motility, a process deregulated by hematopoietic stem cell oncogenes such as p210-BCR-ABL. A phosphoproteomics approach to the analysis of a hematopoietic progenitor cell line treated with CXCL12 and the Rac 1 and 2 inhibitor NSC23766 has been employed to objectively discover novel mechanisms for regulation of stem cells in normal and malignant hematopoiesis. The proteomic data sets identified new aspects of CXCL12-mediated signaling and novel features of stem cell regulation. We also identified a novel phosphorylation event in hematopoietic progenitor cells that correlated with motile response and governed by the chemotactic factor CXCL12. The novel phosphorylation site on PTPRC/CD45; a protein tyrosine phosphatase, was validated by raising an antibody to the site and also using a mass spectrometry absolute quantification strategy. Site directed mutagenesis and inhibitor studies demonstrated that this single phosphorylation site governs hematopoietic progenitor cell and lymphoid cell motility, lies downstream from Rac proteins and potentiates Src signaling. We have also demonstrated that PTPRC/CD45 is down-regulated in leukemogenic tyrosine kinase expressing cells. The use of discovery proteomics has enabled further understanding of the regulation of PTPRC/CD45 and its important role in cellular motility in progenitor cells.

Tandem SERS and MS/MS Profiling of Plasma Extracellular Vesicles for Early Ovarian Cancer Biomarker Discovery.

Extracellular vesicles (EVs) are vectors of biomolecular cargo that play essential roles in intercellular communication across a range of cells. Protein, lipid, and nucleic acid cargo harbored within EVs may serve as biomarkers at all stages of disease; however, the choice of methodology may challenge the specificity and reproducibility of discovery. To address these challenges, the integration of rigorous EV purification methods, cutting-edge spectroscopic technologies, and data analysis are critical to uncover diagnostic signatures of disease. Herein, we demonstrate an EV isolation and analysis pipeline using surface-enhanced Raman spectroscopy (SERS) and mass spectrometry (MS) techniques on plasma samples obtained from umbilical cord blood, healthy donor (HD) plasma, and plasma from women with early stage high-grade serous carcinoma (HGSC). Plasma EVs were purified by size exclusion chromatography and analyzed by surface-enhanced Raman spectroscopy (SERS), mass spectrometry (MS), and atomic force microscopy. After determining the fraction of highest EV purity, SERS and MS were used to characterize EVs from HDs, pooled donors with noncancerous gynecological ailments (n = 6), and donors with early stage [FIGO (I/II)] with HGSC. SERS spectra were subjected to different machine learning algorithms such as PCA, logistic regression, support vector machine, naïve Bayes, random forest, neural network, and k nearest neighbors to differentiate healthy, benign, and HGSC EVs. Collectively, we demonstrate a reproducible workflow with the potential to serve as a diagnostic platform for HGSC.

Quantitative proteomics analysis of BMS-214662 effects on CD34 positive cells from chronic myeloid leukaemia patients.

Chronic myeloid leukaemia (CML) arises in a haemopoietic stem cell and is driven by the Bcr-Abl oncoprotein. Abl kinase inhibitors (protein tyrosine kinase inhibitors) represent standard treatment for CML and induce remission in the majority of patients with early disease, however these drugs do not target leukaemic stem cells (LSCs) effectively, thus preventing cure. Previously, we identified the farnesyl transferase inhibitor BMS-214662 as a selective inducer of apoptosis in LSCs of CML patients relative to normal controls; however, the mechanism underlying LSC-specific apoptosis remains unclear. To identify pathways involved in the favourable effects of BMS-214662 in CML, we employed a proteomic approach (based on iTRAQ) to analyse changes in protein expression in response to drug treatment in the nuclear and cytoplasmic fractions of CD34(+) CML cells. The study identified 88 proteins as altered after drug treatment, which included proteins known to be involved in nucleic acid metabolism, oncogenesis, developmental processes and intracellular protein trafficking. We found that expression of Ebp1, a negative regulator of proliferation, was upregulated in the nucleus of BMS-214662-treated cells. Furthermore, proteins showing altered levels in the cytosol, such as histones, were predominantly derived from the nucleus and BMS-214662 affected expression levels of nuclear pore complex proteins. Validation of key facets of these observations suggests that drug-induced alterations in protein localisation, potentially via loss of nuclear membrane integrity, contributes to the LSC specificity of BMS-214662, possibly via Ran proteins as targets.

Mitochondrial hyperfusion via metabolic sensing of regulatory amino acids.

The relationship between nutrient starvation and mitochondrial dynamics is poorly understood. We find that cells facing amino acid starvation display clear mitochondrial fusion as a means to evade mitophagy. Surprisingly, further supplementation of glutamine (Q), leucine (L), and arginine (R) did not reverse, but produced stronger mitochondrial hyperfusion. Interestingly, the hyperfusion response to Q + L + R was dependent upon mitochondrial fusion proteins Mfn1 and Opa1 but was independent of MTORC1. Metabolite profiling indicates that Q + L + R addback replenishes amino acid and nucleotide pools. Inhibition of fumarate hydratase, glutaminolysis, or inosine monophosphate dehydrogenase all block Q + L + R-dependent mitochondrial hyperfusion, which suggests critical roles for the tricarboxylic acid (TCA) cycle and purine biosynthesis in this response. Metabolic tracer analyses further support the idea that supplemented Q promotes purine biosynthesis by serving as a donor of amine groups. We thus describe a metabolic mechanism for direct sensing of cellular amino acids to control mitochondrial fusion and cell fate.

Extracellular vesicles tell all: How vesicle-mediated cellular communication shapes hematopoietic stem cell biology with increasing age.

Extracellular vesicles (EVs) are small lipid bilayer particles containing biologically important cargo and impart regulatory changes in target cells. Despite the importance of EVs in cellular communication, there remains a gap in our understanding of how EVs influence HSC fate and, in turn, how aging and longevity are affected. This review summarizes the current literature dealing with how age-altered intercellular communication mediated by EVs influences HSC biology.

BRD4-mediated repression of p53 is a target for combination therapy in AML.

Acute myeloid leukemia (AML) is a typically lethal molecularly heterogeneous disease, with few broad-spectrum therapeutic targets. Unusually, most AML retain wild-type TP53, encoding the pro-apoptotic tumor suppressor p53. MDM2 inhibitors (MDM2i), which activate wild-type p53, and BET inhibitors (BETi), targeting the BET-family co-activator BRD4, both show encouraging pre-clinical activity, but limited clinical activity as single agents. Here, we report enhanced toxicity of combined MDM2i and BETi towards AML cell lines, primary human blasts and mouse models, resulting from BETi's ability to evict an unexpected repressive form of BRD4 from p53 target genes, and hence potentiate MDM2i-induced p53 activation. These results indicate that wild-type TP53 and a transcriptional repressor function of BRD4 together represent a potential broad-spectrum synthetic therapeutic vulnerability for AML.

Blood extracellular vesicles from healthy individuals regulate hematopoietic stem cells as humans age.

Hematopoietic stem cells (HSCs) maintain balanced blood cell production in a process called hematopoiesis. As humans age, their HSCs acquire mutations that allow some HSCs to disproportionately contribute to normal blood production. This process, known as age-related clonal hematopoiesis, predisposes certain individuals to cancer, cardiovascular and pulmonary pathologies. There is a growing body of evidence suggesting that factors outside cells, such as extracellular vesicles (EVs), contribute to the disruption of stem cell homeostasis during aging. We have characterized blood EVs from humans and determined that they are remarkably consistent with respect to size, concentration, and total protein content, across healthy subjects aged 20-85 years. When analyzing EV protein composition from mass spectroscopy data, our machine-learning-based algorithms are able to distinguish EV proteins based on age and suggest that different cell types dominantly produce EVs released into the blood, which change over time. Importantly, our data show blood EVs from middle and older age groups (>40 years) significantly stimulate HSCs in contrast to untreated and EVs sourced from young subjects. Our study establishes for the first time that although EV particle size, concentration, and total protein content remain relatively consistent over an adult lifespan in humans, EV content evolves during aging and potentially influences HSC regulation.

Biological Analysis of Human CML Stem Cells; Xenograft Model of Chronic Phase Human Chronic Myeloid Leukemia.

Xenograft mouse models have been instrumental in expanding our knowledge of hematopoiesis and can provide a functional description of stem cells that possess engrafting potential. Here we describe methodology outlining one way of analyzing human malignant cells that are able to engraft immune compromised mice. Using models such as these will allow researchers to gain valuable insight into the primitive leukemic subtypes that evade current therapy regimes and are critical to understand, in order to eradicate malignancy.

Improved retention of idarubicin after intravenous injection obtained for cholesterol-free liposomes.

To date there has been a focus on the application of sterically stabilized liposomes, composed of saturated diacylphospholipid, polyethylene glycol (PEG) conjugated lipids (5-10 mole%) and cholesterol (CH) (>30 mole%), for the systemic delivery of drugs. However, we are now exploring the utility of liposome formulations composed of diacylphospholipid conjugated PEG mixtures prepared in the absence of added cholesterol, with the primary objective of developing formulations that retain encapsulated drug better than comparable formulations prepared with cholesterol. In this report the stability of cholesterol-free distearoylphosphatidylcholine (DSPC):distearoylphosphatidylethanolamine (DSPE)-PEG(2000) (95:5 mol/mol) liposomes was characterized in comparison to cholesterol-containing formulations DSPC:CH (55:45 mol/mol) and DSPC:CH:DSPE-PEG(2000) (50:45:5 mol/mol/mol), in vivo. Circulation longevity of these formulations was determined in consideration of variables that included varying phospholipid acyl chain length, PEG content and molecular weight. The application of cholesterol-free liposomes as carriers for the hydrophobic anthracycline antibiotic, idarubicin (IDA), was assessed. IDA was encapsulated using a transmembrane pH gradient driven process. To determine stability in vivo, pharmacokinetic studies were performed using 'empty' and drug-loaded [(3)H]cholesteryl hexadecyl ether radiolabeled liposomes administered intravenously to Balb/c mice. Inclusion of 5 mole% of DSPE-PEG(2000) or 45 mole% cholesterol to DSPC liposomes increased the mean plasma area under the curve (AUC(0-24h)) 19-fold and 10-fold, respectively. Cryo-transmission electron micrographs of IDA loaded liposomes indicated that the drug formed a precipitate within liposomes. The mean AUC(0-4h) for free IDA was 0.030 micromole h/ml as compared to 1.38 micromole h/ml determined for the DSPC:DSPE-PEG(2000) formulation, a 45-fold increase, demonstrating that IDA was retained better in cholesterol-free compared to cholesterol-containing liposomes.

The liposomal formulation of doxorubicin.

Doxorubicin is the best known and most widely used member of the anthracycline antibiotic group of anticancer agents. It was first introduced in the 1970s, and since that time has become one of the most commonly used drugs for the treatment of both hematological and solid tumors. The therapy-limiting toxicity for this drug is cardiomyopathy, which may lead to congestive heart failure and death. Approximately 2% of patients who have received a cumulative (lifetime) doxorubicin dose of 450-500 mg?m(2) will experience this condition. An approach to ameliorating doxorubicin-related toxicity is to use drug carriers, which engender a change in the pharmacological distribution of the drug, resulting in reduced drug levels in the heart. Examples of these carrier systems include lipid-based (liposome) formulations that effect a beneficial change in doxorubicin biodistribution, with two formulations approved for clinical use. Drug approval was based, in part, on data suggesting that beneficial changes in doxorubicin occurred in the absence of decreased therapeutic activity. Preclinical (animal) and clinical (human) studies showing that liposomes can preferentially accumulate in tumors have provided a rationale for improved activity. Liposomes represent ideal drug delivery systems, as the microvasculature in tumors is typically discontinuous, having pore sizes (100-780 nm) large enough for liposomes to move from the blood compartment into the extravascular space surrounding the tumor cells. Liposomes, in the size range of 100-200 nm readily extravasate within the site of tumor growth to provide locally concentrated drug delivery, a primary role of liposomal formulation. Although other liposomal drugs have been prepared and characterized due to the potential for liposomes to improve antitumor potency of the encapsulated drug, the studies on liposomal doxorubicin have been developed primarily to address issues of acute and chronic toxicity that occur as a consequence of using this drug. It is important to recognize that research programs directed toward the development of liposomal doxorubicin occurred concurrently with synthetic chemistry programs attempting to introduce safer and more effective anthracycline analogues. Although many of these drugs are approved for use, and preliminary liposomal formulations of these analogues have been prepared, doxorubicin continues to be a mainstay of drug cocktails used in the management of most solid tumors. It will be of great interest to observe how the approved formulations of liposomal doxorubicin are integrated into combination regimes for treatment of cancer. In the meantime, we have learned a great deal about liposomes as drug carriers from over 20 years of research on different liposomal doxorubicin formulations, the very first of which were identified in the late 1970s. This chapter will discuss the various methods for encapsulation of doxorubicin into liposomes, as well as some of the important interactions between the formulation components of the drug and how this may impact the biological activity of the associated drug. This review of methodology, in turn, will highlight research activities that are being pursued to achieve better performance parameters for liposomal formulations of doxorubicin, as well as other anticancer agents being considered for use with lipid-based carriers.

Encapsulation of doxorubicin into thermosensitive liposomes via complexation with the transition metal manganese.

In the present study, doxorubicin was encapsulated into two thermosensitive liposome formulations which were composed of DPPC/MSPC/DSPE-PEG(2000) (90/10/4 mole ratio) or DPPC/DSPE-PEG(2000) (95/5 mole ratio). Doxorubicin loading was achieved through the use of a pH gradient or a novel procedure that involved doxorubicin complexation with manganese. Regardless of the initial drug-to-lipid ratios (D:L), the final D:L reached a maximum of 0.05 (w/w) when doxorubicin was encapsulated via a pH gradient for both thermosensitive liposome formulations. In contrast, the final maximum D:L achieved through manganese complexation was 0.2 (w/w), and this loading method did not affect temperature-induced drug release, with 85% of drug released from MSPC-containing liposomes within 10 min at 42 degrees C but <5% released over 60 min at 37 degrees C. When the thermosensitive liposomes prepared via the two different loading methods were injected into mice, similar plasma elimination profiles were observed. Cryo-transmission electron microscopy analysis indicated the presence of doxorubicin fiber bundles in liposomes loaded via pH gradient, compared to a stippled and diffuse morphology in those loaded via manganese complexation. To investigate the effect of intraliposomal pH on drug precipitate morphology, the A23187 ionophore (mediates Mn(2+)/H(+) exchange) was added to liposomes loaded with doxorubicin-manganese complex, and the stippled and diffuse appearance could be converted to one exhibiting fiber bundles after acidification of the liposome core. This suggests that the formation of doxorubicin-manganese complex is favored when the intraliposomal pH is >6.5. During the conversion to the fiber bundle morphology, no doxorubicin release was observed when A23187 was added to liposomes exhibiting a 0.05 (w/w), whereas a significant release was noted when the initial D:L was 0.2 (w/w). Following acidification of the liposomal interior and establishment of an apparent new D:L equilibrium, the measured D:L ratio was 0.05 (w/w). In conclusion, the manganese complexation loading method increased the encapsulation efficiency of doxorubicin in thermosensitive liposomes with no major impact on temperature-triggered drug release or pharmacokinetics.

In vitro and in vivo characterization of doxorubicin and vincristine coencapsulated within liposomes through use of transition metal ion complexation and pH gradient loading.

PURPOSE: There is an opportunity to augment the therapeutic potential of drug combinations through use of drug delivery technology. This report summarizes data obtained using a novel liposomal formulation with coencapsulated doxorubicin and vincristine. The rationale for selecting these drugs is due in part to the fact that liposomal formulations of doxorubicin and vincristine are being separately evaluated as components of drug combinations. EXPERIMENTAL DESIGN: Doxorubicin and vincristine were coencapsulated into liposomes using two distinct methods of drug loading. A manganese-based drug loading procedure, which relies on drug complexation with a transition metal, was used to encapsulate doxorubicin. Subsequently the ionophore A23187 was added to induce formation of a pH gradient, which promoted vincristine encapsulation. RESULTS: Plasma elimination studies in mice indicated that the drug:drug ratio before injection [4:1 doxorubicin:vincristine (wt:wt ratio)] changed to 20:1 at the 24-h time point, indicative of more rapid release of vincristine from the liposomes than doxorubicin. Efficacy studies completed in MDA MB-435/LCC6 tumor-bearing mice suggested that at the maximum tolerated dose, the coencapsulated formulation was therapeutically no better than liposomal vincristine. This result was explained in part by in vitro cytotoxicity studies evaluating doxorubicin and vincristine combinations analyzed using the Chou and Talalay median effect principle. These data clearly indicated that simultaneous addition of vincristine and doxorubicin resulted in pronounced antagonism. CONCLUSION: These results emphasize that in vitro drug combination screens can be used to predict whether a coformulated drug combination will act in an antagonistic or synergistic manner.

An evaluation of transmembrane ion gradient-mediated encapsulation of topotecan within liposomes.

Topotecan can be encapsulated in liposomes, however little is known about the role encapsulated counter ions play in drug loading efficiency and drug release. Using 1,2-distearoyl-sn-glycero-3 phosphatidylcholine and cholesterol liposomes (55:45 mole ratio), encapsulation was achieved using manganese ion gradients (MnSO(4) or MnCl(2)), with the addition of A23187, a divalent cation/proton exchanger, to maintain a pH gradient. This methodology was compared to procedures where the pH gradient was generated by use of encapsulated (NH(4))(2)SO(4) or citrate (300 mM, pH 3.5). All methods facilitated topotecan encapsulation. Liposomes prepared in the presence of the citrate and MnCl(2) (+A23187) exhibited reduced loading capacities. Liposomes prepared in the presence of (NH(4))(2)SO(4) and MnSO(4) (+A23187) could be used to generate liposomes exhibiting a drug-to-lipid ratio of 0.3 (wt/wt) with an encapsulation efficiency of >90%. In vitro drug release data suggested that the (NH(4))(2)SO(4) and MnSO(4) (+A23187) formulations released drug at a reduced rate. For these formulations, the drug release rates decreased as the drug-to-lipid ratio (wt/wt) increased from 0.1 to 0.2. Cryo-electron micrographs indicated that encapsulated topotecan precipitated as linear particles within liposomes. The stability of topotecan loaded liposomes appeared to be dependent on the presence of both a pH gradient and encapsulated sulfate.

Arachidonate 15-lipoxygenase is required for chronic myeloid leukemia stem cell survival.

Cancer stem cells (CSCs) are responsible for the initiation and maintenance of some types of cancer, suggesting that inhibition of these cells may limit disease progression and relapse. Unfortunately, few CSC-specific genes have been identified. Here, we determined that the gene encoding arachidonate 15-lipoxygenase (Alox15/15-LO) is essential for the survival of leukemia stem cells (LSCs) in a murine model of BCR-ABL-induced chronic myeloid leukemia (CML). In the absence of Alox15, BCR-ABL was unable to induce CML in mice. Furthermore, Alox15 deletion impaired LSC function by affecting cell division and apoptosis, leading to an eventual depletion of LSCs. Moreover, chemical inhibition of 15-LO function impaired LSC function and attenuated CML in mice. The defective CML phenotype in Alox15-deficient animals was rescued by depleting the gene encoding P-selectin, which is upregulated in Alox15-deficient animals. Both deletion and overexpression of P-selectin affected the survival of LSCs. In human CML cell lines and CD34+ cells, knockdown of Alox15 or inhibition of 15-LO dramatically reduced survival. Loss of Alox15 altered expression of PTEN, PI3K/AKT, and the transcription factor ICSBP, which are known mediators of cancer pathogenesis. These results suggest that ALOX15 has potential as a therapeutic target for eradicating LSCs in CML.