Synergistic Cytarabine:Daunorubicin Ratios Delivered by CPX-351 to Human Leukemia Xenografts Is Associated with Liposome-Mediated Bone Marrow Drug Accumulation, Intracellular Delivery of Encapsulated Agents to Leukemia Cells, and Increased Efficacy.

Abstract CPX-351, a liposomal formulation of cytarabine:daunorubicin co-encapsulated at a synergistic 5:1 molar drug ratio, has been previously shown to be highly active in various preclinical leukemia models. In the present investigations, we utilized a bone marrow engrafted human lymphocytic leukemia xenograft model in order to elucidate the pharmacodynamic basis for its superior efficacy compared to un-encapsulated cytarabine:daunorubicin combinations. In normal bone marrow, the un-encapsulated cytarabine:daunorubicin saline-based cocktail and CPX-351 provided comparable cell suppression after intravenous treatment at maximum tolerated dose. In contrast, when these formulations were administered to tumor bearing mice, CPX-351 completely ablated leukemia cells from the bone marrow for multiple weeks whereas the saline-based drug cocktail induced only transient leukemia suppression. Examination of drug levels in the bone marrow compartment revealed that cytarabine:daunorubicin concentrations were significantly elevated for CPX-351. More striking was the observation that whereas bone marrow drug concentrations over time appeared comparable after each dose of the saline-based drug cocktail, the first dose of CPX-351 promoted elevated bone marrow drug accumulation for subsequent doses. Under these conditions, the cytarabine:daunorubicin ratio in bone marrow was maintained near the administered 5:1 molar ratio for CPX- 351 while the drug ratio changed more than 100-fold within 4 hours after administration of the saline-based drug cocktail. Confocal fluorescence microscopy of leukemia cells exposed to CPX-351 in vitro revealed that CPX-351 liposomes were taken up into cytoplasmic vacuoles and subsequently released their drug contents intracellularly. Taken together, these results indicate that the improved efficacy observed following CPX-351 administration in vivo is related to liposome-mediated prolonged and direct exposure of leukemia cells to synergistic drug ratios.

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cis-Retinoic acid stimulates the clonal growth of some myeloid leukemia cells in vitro

Blood ◽

10.1182/blood.v69.1.302.302 ◽

1987 ◽

Vol 69 (1) ◽

pp. 302-307 ◽

Cited By ~ 29

Author(s):

HJ Lawrence ◽

K Conner ◽

MA Kelly ◽

MR Haussler ◽

P Wallace ◽

...

Keyword(s):

Retinoic Acid ◽

T Lymphocytes ◽

Clonal Growth ◽

Colony Growth ◽

Leukemic Cells ◽

Leukemia Cells ◽

Human Leukemia ◽

Growth Responses ◽

Drug Concentrations

Abstract We studied the effects of cis-retinoic acid (cisRA) on the clonogenic growth of samples of leukemic cells from 35 patients with acute nonlymphocytic leukemia (ANLL). We observed significant inhibition of leukemic colony growth in 17 samples by 10(-7) to 10(-6)M cisRA. However, we found that retinoid exposure resulted in striking stimulation of clonal growth in ten samples at the same drug concentrations. With the exception of cases with promyelocytic features, there was no morphologic or functional evidence that cisRA induced the leukemic blasts to differentiate. Both inhibition and stimulation were dose-dependent and observable at pharmacologically achievable levels of cisRA. Leukemic cells with monocytic features more frequently demonstrated a stimulatory response than did those without monocytic features. Depletion of T lymphocytes and monocytes did not alter the type of growth response. Assays for cellular retinoic acid- binding protein (CRABP) were performed on five samples (two with inhibitory growth responses, two with stimulatory responses, and one with no growth) and failed to reveal detectable levels of CRABP in any case. The addition of cisRA to liquid suspensions of leukemic cells produced no significant change in the number of viable cells. We conclude that the effects of cisRA on leukemic colony growth are not cytotoxic and not mediated by T lymphocytes, monocytes, or CRABP. More importantly, cisRA appears to enhance the growth of certain human leukemia cells in vitro. Taking into account the increasing use of retinoids in clinical trials for patients with leukemia, the latter findings may represent a significant cautionary note.

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Involvement of reactive oxygen species in adaphostin-induced cytotoxicity in human leukemia cells

Blood ◽

10.1182/blood-2003-02-0562 ◽

2003 ◽

Vol 102 (13) ◽

pp. 4512-4519 ◽

Cited By ~ 48

Author(s):

Joya Chandra ◽

Jennifer Hackbarth ◽

Son Le ◽

David Loegering ◽

Nancy Bone ◽

...

Keyword(s):

Cell Lines ◽

Buthionine Sulfoximine ◽

Lymphocytic Leukemia ◽

Myelogenous Leukemia ◽

Glutathione Depletion ◽

Leukemia Cells ◽

Clinical Samples ◽

Human Leukemia ◽

Lymphoid Leukemia

Abstract Adaphostin (NSC 680410), an analog of the tyrphostin AG957, was previously shown to induce Bcr/abl down-regulation followed by loss of clonogenic survival in chronic myelogenous leukemia (CML) cell lines and clinical samples. Adaphostin demonstrated selectivity for CML myeloid progenitors in vitro and remained active in K562 cells selected for imatinib mesylate resistance. In the present study, the mechanism of action of adaphostin was investigated in greater detail in vitro. Initial studies demonstrated that adaphostin induced apoptosis in a variety of Bcr/abl- cells, including acute myelogenous leukemia (AML) blasts and cell lines as well as chronic lymphocytic leukemia (CLL) samples. Further study demonstrated that adaphostin caused intracellular peroxide production followed by DNA strand breaks and, in cells containing wild-type p53, a typical DNA damage response consisting of p53 phosphorylation and up-regulation. Importantly, the antioxidant N-acetylcysteine (NAC) blunted these events, whereas glutathione depletion with buthionine sulfoximine (BSO) augmented them. Collectively, these results not only outline a mechanism by which adaphostin can damage both myeloid and lymphoid leukemia cells, but also indicate that this novel agent might have a broader spectrum of activity than originally envisioned. (Blood. 2003;102:4512-4519)

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Specific receptors for phorbol diesters on freshly isolated human myeloid and lymphoid leukemia cells: comparable binding characteristics despite different cellular responses

Blood ◽

10.1182/blood.v63.2.298.bloodjournal632298 ◽

1984 ◽

Vol 63 (2) ◽

pp. 298-304

Author(s):

BJ Goodwin ◽

JO Moore ◽

JB Weinberg

Keyword(s):

Specific Binding ◽

Blast Crisis ◽

Cellular Responses ◽

Lymphocytic Leukemia ◽

Leukemia Cells ◽

Human Leukemia ◽

Lymphoid Leukemia ◽

Binding Characteristics ◽

Specific Receptors

Freshly isolated human leukemia cells have been shown in the past to display varying in vitro responses to phorbol diesters, depending on their cell type. Specific receptors for the phorbol diesters have been demonstrated on numerous different cells. This study was designed to characterize the receptors for phorbol diesters on leukemia cells freshly isolated from patients with different kinds of leukemia and to determine if differences in binding characteristics for tritium-labeled phorbol 12,13-dibutyrate (3H-PDBu) accounted for the different cellular responses elicited in vitro by phorbol diesters. Cells from 26 patients with different kinds of leukemia were studied. PDBu or phorbol 12- myristate 13-acetate (PMA) caused cells from patients with acute myeloblastic leukemia (AML), acute promyelocytic (APML), acute myelomonocytic (AMML), acute monocytic (AMoL), acute erythroleukemia (AEL), chronic myelocytic leukemia (CML) in blast crisis (myeloid), acute undifferentiated leukemia (AUL), and hairy cell leukemia (HCL) (n = 15) to adhere to plastic and spread. However, they caused no adherence or spreading and only slight aggregation of cells from patients with acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL), or CML-blast crisis (lymphoid) (n = 11). All leukemia cells studied, irrespective of cellular type, displayed specific receptors for 3H-PDBu. The time courses for binding by all leukemia types were similar, with peak binding at 5–10 min at 37 degrees C and 120 min at 4 degrees C. The binding affinities were similar for patients with ALL (96 +/- 32 nM, n = 4), CLL (126 +/- 32 nM, n = 6), and acute nonlymphoid leukemia (73 +/- 14 nM, n = 11). Likewise, the numbers of specific binding sites/cell were comparable for the patients with ALL (6.2 +/- 1.3 X 10(5) sites/cell, n = 4), CLL (5.0 +/- 2.0 X 10(5) sites/cell, n = 6), and acute nonlymphoid leukemia (4.4 +/- 1.9 X 10(5) sites/cell, n = 11). Thus, the differing responses to phorbol diesters of various types of freshly isolated leukemia cells appear to be due to differences other than initial ligand-receptor binding.

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Adipocytes Secrete Factors That Cause Leukemia Drug Resistance.

Blood ◽

10.1182/blood.v112.11.1346.1346 ◽

2008 ◽

Vol 112 (11) ◽

pp. 1346-1346

Author(s):

James W Behan ◽

Jason P Yun ◽

Marina P Proektor ◽

Ehsan A Ehsanipour ◽

Anna Butturini ◽

...

Keyword(s):

Bone Marrow ◽

Chemotherapy Resistance ◽

Leukemia Cell ◽

Conditioned Media ◽

Leukemia Cells ◽

Cell Contact ◽

Human Leukemia ◽

Bone Marrow Niche

Abstract We have previously shown that obesity is an independent predictor of leukemia relapse in children. We have also shown that obese mice transplanted with syngeneic leukemia cells have poorer survival after chemotherapy, even when they are dosed proportional to body weight. Since interactions between leukemia cells and cells of the bone marrow niche are considered important for chemotherapy resistance and relapse, and adipocytes can comprise ~50% of the bone marrow niche, we developed in vivo and in vitro models to investigate the role of adipocytes in the leukemia microenvironment. Obese C57Bl/6J mice were transplanted with GFP+ murine preB cell ALL (“8093”) cells and then treated with vincristine (0.5 mg/kg/week × 3 weeks). At the time of relapse, we found that GFP+ leukemia cells persisted in the fat pads of the mice. We then developed an in vitro co-culture system in which human or murine leukemia cells were cultured together with adipocytes (differentiated 3T3-L1s). Undifferentiated 3T3-L1 cells, which are fibroblastic in nature, were used as a control. In this model, adipocytes severely diminished the anti-leukemic effect of all chemotherapeutics tested against murine 8093 cells, including vincristine, dexamethasone, nilotinib, daunorubicin, and L-asparaginase. Adipocytes also protected murine T-cell ALL and human SD-1, RCH-ACV, and BV173 cells from vincristine and daunorubicin. Adipocyte protection of leukemia cells occurred independent of cell contact. Further experiments demonstrated that media conditioned by adipocytes was able to protect 8093 cells from a 3-day exposure to 25 nM dexamethasone (viable cells were at 40±12% of their plated value in regular media, 66±17% in fibroblast-conditioned media, and 109±24% in adipocyte-conditioned media, p<0.05). Surprisingly, adipocyte-conditioned media did not protect leukemia cells from daunorubicin. However, media conditioned by the presence of both adipocytes and leukemia cells simultaneously conferred a high degree of resistance to the leukemia cells (n=3, p<0.05 compared to all other media types). In summary, adipose tissue is a reservoir for relapsed leukemia cells in vivo. Adipocytes engender protection from multiple chemotherapies in murine and human leukemia cell lines. Adipocytes secrete factor(s) that confer dexamethasone and daunorubicin resistance to leukemia cells, though for the latter drug it appears that a two-way communication between leukemia and adipocytes may be necessary for this protection. Figure Figure

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A Novel Three-Dimensional Co-Culture System to Study Leukemia in the Bone Marrow Microenvironment

Blood ◽

10.1182/blood.v126.23.1864.1864 ◽

2015 ◽

Vol 126 (23) ◽

pp. 1864-1864 ◽

Cited By ~ 1

Author(s):

Zeena Salman ◽

Juan Carlos Balandrán-Juárez ◽

Rosana Pelayo ◽

Monica L. Guzman

Keyword(s):

Bone Marrow ◽

Culture System ◽

Lymphoblastic Leukemia ◽

Cell Monolayer ◽

Leukemia Cell ◽

Leukemia Cells ◽

Human Leukemia ◽

Bone Marrow Niche ◽

3 Dimensional

Abstract The need for novel therapies in acute leukemia has been motivated by sub-optimal 5-year survival rates of 25.4% in acute myeloid leukemia (AML) and approximately 70% in acute lymphoblastic leukemia (ALL). While these rates are higher in the pediatric population, novel approaches are necessary in all age groups to improve outcomes. Pre-clinical studies of novel therapeutics using in vitro and in vivo methods remain suboptimal with frequent lack of correlation with clinical outcomes at the bedside. Recent evidence has shown that human leukemia xenografts into immunodeficient mice yield variable results, indicating that treatment using these methods is not replicable. When using in vitro cell culture methods, the well-documented protective effects of the bone marrow (BM) microenvironment (BMME) on leukemia are not mimicked. Furthermore, these techniques cannot be used to investigate the effects of novel agents on leukemia stem cells (LSC) and their mobilization, which is important in the ablation of leukemia. Thus, we explored a novel 3-dimensional co-culture system to study the effects of drugs on leukemia cells in the presence of stroma in an environment more similar to that of human leukemia in the BMME. We generated a 3-dimensional (3D) spheroid co-culture system using human stromal cell line (HS-5) cells or human mesenchymal stromal cells (hBMSC) from primary AML or ALL BM. To evaluate the dynamics of the 3D system, we labeled the stroma cells with GFP and the leukemia cells with mCherry. We observed rapid homing to the center of the 3D stroma. We evaluated ROS levels, proliferation status, hypoxia and expression of key niche proteins such as CXCL12 in leukemia cells found outside and inside the 3D system. These methods were compared to similar treatments in leukemia cell monolayer culture and 2-dimensional co-culture systems. We treated this system with various drugs such as cytarabine, doxorubicin, TG02 (a multi-kinase inhibitor with LSC mobilization effects), and plerixafor; we then harvested cells from the outer and inner layers and evaluated these separately by multi-parameter flow cytometry for viability and mobilization of LSCs in relation to the stroma and xenotransplant assays. Our studies reveal that the 3D culture system has lower ROS internally, suggesting a similarly hypoxic environment to BMME. Our studies also reveal that, when treated with cytarabine, AML cells closest to the stromal center of the spheroid remain protected, with higher viability compared to those farther from stroma, and even more so than leukemia cells in a 2-D bilayer with stroma or in a monolayer. A lower CXCL12 level was also observed in the stroma of leukemic BM compared to healthy BM within the co-culture system. This culture method possesses many of the characteristics of leukemia cells within the bone marrow niche and should be considered for future in vitro pre-clinical drug testing to model the tumor within its microenvironment. Disclosures No relevant conflicts of interest to declare.

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Specific receptors for phorbol diesters on freshly isolated human myeloid and lymphoid leukemia cells: comparable binding characteristics despite different cellular responses

Blood ◽

10.1182/blood.v63.2.298.298 ◽

1984 ◽

Vol 63 (2) ◽

pp. 298-304 ◽

Cited By ~ 2

Author(s):

BJ Goodwin ◽

JO Moore ◽

JB Weinberg

Keyword(s):

Specific Binding ◽

Blast Crisis ◽

Cellular Responses ◽

Lymphocytic Leukemia ◽

Leukemia Cells ◽

Human Leukemia ◽

Lymphoid Leukemia ◽

Binding Characteristics ◽

Specific Receptors

Abstract Freshly isolated human leukemia cells have been shown in the past to display varying in vitro responses to phorbol diesters, depending on their cell type. Specific receptors for the phorbol diesters have been demonstrated on numerous different cells. This study was designed to characterize the receptors for phorbol diesters on leukemia cells freshly isolated from patients with different kinds of leukemia and to determine if differences in binding characteristics for tritium-labeled phorbol 12,13-dibutyrate (3H-PDBu) accounted for the different cellular responses elicited in vitro by phorbol diesters. Cells from 26 patients with different kinds of leukemia were studied. PDBu or phorbol 12- myristate 13-acetate (PMA) caused cells from patients with acute myeloblastic leukemia (AML), acute promyelocytic (APML), acute myelomonocytic (AMML), acute monocytic (AMoL), acute erythroleukemia (AEL), chronic myelocytic leukemia (CML) in blast crisis (myeloid), acute undifferentiated leukemia (AUL), and hairy cell leukemia (HCL) (n = 15) to adhere to plastic and spread. However, they caused no adherence or spreading and only slight aggregation of cells from patients with acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL), or CML-blast crisis (lymphoid) (n = 11). All leukemia cells studied, irrespective of cellular type, displayed specific receptors for 3H-PDBu. The time courses for binding by all leukemia types were similar, with peak binding at 5–10 min at 37 degrees C and 120 min at 4 degrees C. The binding affinities were similar for patients with ALL (96 +/- 32 nM, n = 4), CLL (126 +/- 32 nM, n = 6), and acute nonlymphoid leukemia (73 +/- 14 nM, n = 11). Likewise, the numbers of specific binding sites/cell were comparable for the patients with ALL (6.2 +/- 1.3 X 10(5) sites/cell, n = 4), CLL (5.0 +/- 2.0 X 10(5) sites/cell, n = 6), and acute nonlymphoid leukemia (4.4 +/- 1.9 X 10(5) sites/cell, n = 11). Thus, the differing responses to phorbol diesters of various types of freshly isolated leukemia cells appear to be due to differences other than initial ligand-receptor binding.

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cis-Retinoic acid stimulates the clonal growth of some myeloid leukemia cells in vitro

Blood ◽

10.1182/blood.v69.1.302.bloodjournal691302 ◽

1987 ◽

Vol 69 (1) ◽

pp. 302-307

Author(s):

HJ Lawrence ◽

K Conner ◽

MA Kelly ◽

MR Haussler ◽

P Wallace ◽

...

Keyword(s):

Retinoic Acid ◽

T Lymphocytes ◽

Clonal Growth ◽

Colony Growth ◽

Leukemic Cells ◽

Leukemia Cells ◽

Human Leukemia ◽

Growth Responses ◽

Drug Concentrations

We studied the effects of cis-retinoic acid (cisRA) on the clonogenic growth of samples of leukemic cells from 35 patients with acute nonlymphocytic leukemia (ANLL). We observed significant inhibition of leukemic colony growth in 17 samples by 10(-7) to 10(-6)M cisRA. However, we found that retinoid exposure resulted in striking stimulation of clonal growth in ten samples at the same drug concentrations. With the exception of cases with promyelocytic features, there was no morphologic or functional evidence that cisRA induced the leukemic blasts to differentiate. Both inhibition and stimulation were dose-dependent and observable at pharmacologically achievable levels of cisRA. Leukemic cells with monocytic features more frequently demonstrated a stimulatory response than did those without monocytic features. Depletion of T lymphocytes and monocytes did not alter the type of growth response. Assays for cellular retinoic acid- binding protein (CRABP) were performed on five samples (two with inhibitory growth responses, two with stimulatory responses, and one with no growth) and failed to reveal detectable levels of CRABP in any case. The addition of cisRA to liquid suspensions of leukemic cells produced no significant change in the number of viable cells. We conclude that the effects of cisRA on leukemic colony growth are not cytotoxic and not mediated by T lymphocytes, monocytes, or CRABP. More importantly, cisRA appears to enhance the growth of certain human leukemia cells in vitro. Taking into account the increasing use of retinoids in clinical trials for patients with leukemia, the latter findings may represent a significant cautionary note.

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Function of Growth Factor Independence 1 (GFI1) in the Polarization AML -Associated Stroma

Blood ◽

10.1182/blood.v124.21.4366.4366 ◽

2014 ◽

Vol 124 (21) ◽

pp. 4366-4366

Author(s):

Lacramioara Botezatu ◽

Yahya S. A. Al-Matary ◽

Bertram Opalka ◽

Jan Dürig ◽

Thomas Schroeder ◽

...

Keyword(s):

Bone Marrow ◽

Solid Tumors ◽

Tumor Cells ◽

Conflicts Of Interest ◽

Leukemic Cells ◽

Leukemia Cells ◽

Human Leukemia ◽

Deficient Mice

Abstract The growth of various solid tumors, lymphoma and leukemias is not only the result of cell-specific changes at the genetic and epigenetic level, but is also affected by the surrounding stroma and the cells therein. It has been shown that tumor cells induce surrounding immune cells to express various cytokines and other factors, which promote further growth and spread of tumor cells. Most studies have been conducted with solid tumors, however, not much is known about the role of stroma cells with regard to haematopoietic tumors. In addition, our knowledge is limited with regard to the cell intrinsic factors governing the polarization of stroma cells. We intended to study the role of stroma cells in acute myeloid leukemia (AML), a malignant disease of the myeloid lineage and in myelodysplastic syndrome (MDS), a disease which is characterized by disturbed function of the bone marrow, which can often progress to AML. We focused our studies on two different components of the stroma: mesenchymal stroma cells (MSC) and monocytes. Bone marrow derived MSCs and monocytes originating from AML patients better supported the growth of human leukemia cells in vitrothan MSCs and monocytes from control healthy persons. Interestingly, after achieving a remission, MSCs and monocytes from the cured patients did not any more support the growth of leukemia cells to the same extent as monocytes and MSCs from leukemic patients. To better understand the mechanism behind this observation, we used different murine MDS and AML models. Similar to the finding in human patients, bone marrow derived MSCs and monocytes from MDS and AML mice better supported the growth of leukemic cells in vitro than monocytes and MDSc from healthy mice. In addition, we observed a higher number of non-malignant MSCs and monocytes in the bone marrow of leukemic mice than in the bone marrow of healthy mice. Furthermore, the higher the number of monocytes was in the bone marrow of the mice, the more aggressive was the course of the leukemia. On a molecular level we found that the transcription factor Gfi1 is 3-4 fold upregulated in the MSCs and monocytes of leukemic mice. To verify whether Gfi1 is indeed required for the polarization of MSCs and monocytes we used leukemic Gfi1-deficient mice. In these mice the accumulation of monocytes was less pronounced than in the bone marrow of Gfi1 wildtype mice. In addition MSCs and monocytes from Gfi1-deficient mice did not support to the same extent the growth of leukemic cell lines in vitro, as did the monocytes and MSCs of Gfi1 wildtype leukemic mice. Thus, we have first indications that MSCs and monocytes play an important role in the support of leukemic cells and that Gfi1 is involved in the polarization of these cells and thus Gfi1 could serve as an additional approach to treat leukemia and MDS. Disclosures No relevant conflicts of interest to declare.

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