Cholesterol Rich-Domains Regulate VEGFR-1 (FLT-1) Expression and Signaling in Acute Leukemia Cells

Abstract Vascular endothelial growth factor receptors -1 (FLT-1) and -2 (KDR) are expressed by subsets of acute and chronic leukemias, where they signal in paracrine and/or autocrine manner to induce cell survival, proliferation and migration. We have previously shown that acute lymphocytic leukemia migration in response to VEGF via FLT-1 modulates the onset of extramedullary disease, and thus has clinical predictive value (Fragoso et al, Blood 2006). Acute leukemia cell (AML) migration, induced by PlGF/VEGF activation of FLT-1 results in the formation of actin membrane protrusions with concomitant increased ERK1/2 and P38 phosphorylation and activation of Rho-GTPases (Casalou et al, 2007). Since we have found an in vitro association of FLT-1 with caveolin-1, actin and HSp90, we hypothesised that cholesterol-rich domains might regulate FLT-1 mediated survival, proliferation or migration of acute myeloid (AML) and lymphoid (ALL) leukemias. First we found by FACS and RQ-PCR that FLT-1 expression is up-regulated by increased cholesterol/HDL levels in vitro. As shown by sucrose gradient fractionation and western blotting, PlGF/VEGF stimulation of AML cells results in re-localization of FLT-1 to cholesterol-rich domains. Accordingly, FLT-1 localization within cholesterol-rich domains is abrogated by exposing leukemia cells to b-methyl-cyclodextrin (MbCD) which removes intracellular cholesterol. Additionally, FLT-1 phosphorylation is abolished by treatment of AML cells with MbCD or Nystatin, an inhibitor of lipid raft endocytosis. Functionally, AML cells exposure to high levels of total cholesterol/HDL for 24 hours exerted a protective effect from actinomycin D-induced apoptosis and promoted PlGF/VEGF-induced AML migration in transwell migration assays. Together, these results show that on subsets of acute leukemias cholesterol/HDL cellular-content regulates FLT-1 expression and signalling, resulting in decreased apoptosis and induction of cell migration. In vivo, we show that cholesterol-rich diet significantly increases bone marrow VEGF levels in mice; inoculation of FLT-1 expressing acute leukemias into mice fed with cholesterolrich diet significantly accelerated disease progression and worsened disease outcome. Taken together, our data show the molecular basis by which cellular and systemic cholesterol regulates VEGF and VEGFR-1 signalling on subsets of acute leukemias, modulating cell migration and survival and thereby regulating disease progression.

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Cellular Cholesterol Modulates VEGFR-1 Function on Acute Leukemia Cells.

Blood ◽

10.1182/blood.v108.11.1847.1847 ◽

2006 ◽

Vol 108 (11) ◽

pp. 1847-1847

Author(s):

Rita Fragoso ◽

Cristina Casalou ◽

Sergio Dias

Keyword(s):

Cell Migration ◽

Growth Factor ◽

Acute Leukemia ◽

Molecular Mechanisms ◽

Cholesterol Metabolism ◽

Leukemia Cell ◽

Protein Translation ◽

Leukemia Cells ◽

Cholesterol Levels

Abstract Vascular endothelial growth factor (VEGF) and its receptors play a crucial role in malignancy and in disease, regulating the survival, proliferation, and migration of several cell types, such as endothelium and also leukemia cells. Following our recent report on the role of VEGFR-1 (FLT-1) in ALL (Fragoso R et al, 2006), in the present study we analyzed the molecular mechanisms whereby it modulates acute leukemia cell migration in response to VEGF/Placental Growth Factor (PLGF). First, we observed the formation of cell protrusions on ALL cells after VEGF/PLGF stimulation, with evidence for polymerized actin and FLT-1 co-localization (as determined by phalloidin, immunofluorescence staining, and confocal microscopy). Western blot analysis revealed that PLGF/VEGF stimulation resulted in increased RhoA and Rac1 GTPases expression. Co-treatment with LY200942 significantly decreased RhoA and Rac1 induction and cell migration by PLGF/VEGF, demonstrating this effect is modulated via Pi3 kinase. Next, we investigated the mechanisms whereby FLT-1 and actin co-localize at the cell “leading edge” (protrusions), after VEGF/PLGF stimulation, and the relevance of such co-localization for cell migration. We addressed this question by impairing the formation of lipid rafts/caveolae using drugs that either sequester (nystatin) or deplete (methyl-β-ciclodextrin) total cholesterol. Accordingly, co-treatment of leukemia cells with nystatin or MβCD and PLGF/VEGF blocked cell migration, an effect that was associated with a decrease in FLT-1 polarization and co-localization with actin filaments. Instead, FLT-1 was now found mostly in the cell cytosol. Given that leukemia cells have an increased rate of cholesterol up-take we sought to understand if increased cholesterol levels affected FLT-1 function in leukemia cells. Cholesterol repletion in leukemia cells enhanced leukemia cells migration in response to VEGF/PlGF (about 3 folds). This significant increase was associated with an increase in FLT-1 protein expression that, very interestingly, was particularly concentrated intracellulary in the cytoplasm. At this time we are trying to understand if this increase in FLT-1 expression after cholesterol repletion is associated with increase protein translation or impairment in proteasome activity. Finally, our preliminary in vivo experiments using Nod-Scid mice subjected (n=3) or not (n=3) to high fat diet (that results in increased cholesterol levels in the BM and in the spleen), showed this metabolic condition worsens disease symptoms and significantly decreases mouse survival. These results reveal for the first time some of the molecular mechanisms involved in FLT-1-mediated leukemia migration, namely the involvement of cholesterol metabolism, which may be crucial for new therapeutics delineation.

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Incorporating Hemoglobin Levels to Map Leukostasis Risk in Acute Leukemia Using Microvasculature-on-Chip Technologies

Blood ◽

10.1182/blood-2020-137804 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 9-10

Author(s):

Jamie Oakley ◽

Evelyn K. Williams ◽

Christina Caruso ◽

Yumiko Sakurai ◽

Reginald Tran ◽

...

Keyword(s):

T Cell ◽

Acute Leukemia ◽

Blood Viscosity ◽

Leukemia Cell ◽

Leukemia Cells ◽

Cell Counts ◽

On Chip ◽

Wbc Count

Hyperleukocytosis, most commonly defined as a white blood cell (WBC) count > 100,000/μL, is an oncologic emergency in acute leukemia that can lead to leukostasis, which occurs when leukemia cells obstruct the microvasculature resulting in significant morbidity and mortality from neurologic (CNS hemorrhage, thrombosis) or pulmonary (respiratory distress, hypoxia) symptoms. The underlying mechanisms are poorly understood but are thought to be related to increased blood viscosity, secondary to high WBC count, leukemia cell aggregation, and the abnormal mechanical properties, size, and cell-cell interactions of leukemia cells. Leukapheresis is a commonly used therapy for rapid cytoreduction in symptomatic patients, but the procedure is not without risks. No existing methods reliably predict leukostasis or guide treatment including the commonly used WBC count, which only loosely correlates with leukostasis and does not accurately describe the blood viscosity at the microvascular level. Importantly, while hematocrit/hemoglobin levels (Hgb) are known to be major contributors to blood viscosity, they have not been systematically assessed in leukostasis risk, and Hgb often decreases as leukemic cell counts rise, complicating the issue. Incorporating Hgb levels may better predict leukostasis and assist physicians balancing the risk of hyperleukocytosis compared to the interventions themselves. To that end, we investigated how the differing presentations of acute leukemia lead to microvessel occlusion, thereby affecting effective blood viscosity at the microvascular level using "microvasculature-on-a-chip" devices that mimic the microvascular geometry (Figure 1) developed by our laboratory. This physiologically relevant microvascular model allows for in vitro investigation as in vivo studies are nearly impossible due to difficulty in visualizing and manipulating the animal microvasculature and cell counts. The devices were microfabricated using polydimethylsiloxane (PDMS). Acute T-cell lymphoblastic (Jurkat) and acute monocytic (THP-1) cell lines were maintained via standard cell culture conditions. Red cells from healthy donors were isolated and mixed with leukemia cells to achieve target Hgb and WBC levels. Various physiologic leukemia "mixtures" were then perfused under physiologic microcirculatory flow conditions through the microvascular device and microchannels occlusion was tracked via videomicroscopy (Figure 2). With T-cell leukemia, Hgb levels affected the risk of "in vitro leukostasis." Specifically, with severe anemia and WBC count less than the hyperleukocytosis range, time to microchannel occlusion was longer, and was more dependent on Hgb rather than WBC count. However, in cases with severe anemia and WBC counts > 100k/μL, WBC count exhibited a stronger effect on occlusion with little dependence on Hgb (Figure 3). At Hgb > 8g/dL, microchannel occlusion was dependent on WBC count regardless of hyperleukocytosis or not. In contrast, our data to date shows that with myeloid leukemia, in vitro leukostasis is not associated with Hgb levels, and is consistent with how myeloid leukemias in vivo cause leukostasis symptoms at lower WBC counts than lymphoid leukemias, not only due to size but also adhesive interactions. These data suggest when determining risk for leukostasis, WBC count should not be the sole determinant. Here we show Hgb levels affect microvascular blood viscosity and propensity for microvascular occlusion, but it appears to have a greater impact with T-cell leukemias versus myeloid leukemias (Figure 4). These studies indicate Hgb is an important clinical parameter for leukostasis risk in acute leukemia and will help inform guidelines for leukapheresis and even phlebotomy, a much simpler and safer procedure, to mitigate hyperviscosity in acute leukemia. These results can also impact decisions regarding the need for red blood cell transfusions, which iatrogenically increase blood viscosity. Studies incorporating patient myeloid and lymphoid leukemia cells and microvasculature-on-chip devices integrating live endothelium to assess leukemia cell adhesion are ongoing. Figure Disclosures Lam: Sanguina, Inc: Current equity holder in private company.

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Altered Lipid and Mitochondrial Metabolism Are Viable Targets in Acute Leukemia,

Blood ◽

10.1182/blood.v118.21.3618.3618 ◽

2011 ◽

Vol 118 (21) ◽

pp. 3618-3618 ◽

Cited By ~ 1

Author(s):

Timothy Pardee ◽

Laura M DeFord-Watts ◽

Erica Peronto ◽

Denise A Levitan ◽

David Duane Hurd ◽

...

Keyword(s):

Fatty Acid ◽

Acute Leukemia ◽

Cell Lines ◽

Leukemia Cell ◽

K562 Cells ◽

Acid Synthesis ◽

Acute Leukemias ◽

Transfusion Independence

Abstract Abstract 3618 Acute leukemias are aggressive malignancies of the bone marrow. In adults these diseases disproportionally affect the elderly with a median age of onset of 72 for acute myeloid leukemia (AML) and 60 for acute lymphoblastic leukemia (ALL). Patients over the age of 60 have less than 10% survival at 5 years. This is the result of a combination of increased primary resistant disease and comorbidities that lead to treatment related mortality. Despite decades of research for the majority of AML and ALL patients, therapies have remained unchanged. There is a desperate need for novel agents with acceptable toxicities. Altered metabolism is a hallmark of cancer including the acute leukemias. Nearly all tumor cells will preferentially utilize anaerobic glycolysis even under aerobic conditions, a phenomenon known as the Warburg effect. Additionally, cancer cells can also increase lipid anabolism and upregulate fatty acid synthase (FASN). These alterations in metabolism represent a possible therapeutic target. The novel lipoate derivative CPI-613 is a first in class agent that targets a key mitochondrial enzyme involved in aberrant metabolism, pyruvate dehydrogenase complex (PDH). PDH is required for the conversion of pyruvate to acetyl-CoA, which then enters into the tricarboxylic acid cycle (TCA). TCA intermediates are utilized as biosynthetic precursors for the production of a variety of molecules, including fatty acids. To determine if leukemic blasts upregulate FASN we performed western blots on leukemic cell lines and primary patient samples and compared levels to normal hematopoietic precursor cells. We found all cell lines and many primary AML samples had increased levels of FASN when compared to controls implying an upregulation of fatty acid synthesis. Consistent with this, inhibition of fatty acid synthesis with the FASN inhibitor orlistat or acetyl-CoA carboxylase inhibitor TOFA resulted in loss of viability of HL60, Jurkat and K562 cells. To determine if PDH inhibition by CPI-613 has activity in the acute leukemias, we tested it against several human and murine acute leukemia cell lines in vitro and in vivo. CPI-613 was active against HL60, Jurkat and K562 cells with an average IC50 value of 14 μM (range 12.2 – 16.4). CPI-613 was found to be synergistic with doxorubicin with Combinatorial Index (CI) values between 0.478 and 0.765. Sensitivity to CPI-613 in a genetically defined murine AML cell line was increased with shRNA mediated knockdown of p53 or expression of MN1 despite their increased resistance to standard therapy. Intriguingly, in preliminary studies, CPI-613 appeared to be highly synergistic with the tyrosine kinase inhibitor nilotinib in Baf-3 cells transduced to express the p210 BCR-ABL kinase with CI values of 0.073–0.059. In vivo, CPI-613 was found to synergize with doxorubicin when these cells were injected into syngeneic Balb/c mice with an extension of median survival from 12 days with doxorubicin alone to 16 days with the combination of CPI-613 and doxorubicin (p=0.0001). In addition to these studies, CPI-631 is also the subject of a phase I clinical trial for patients with relapsed and refractory hematologic malignancies at the Wake Forest University Comprehensive Cancer Center. To date, ten patients have been treated. CPI-613 appears to be well tolerated with no adverse events > grade 1 attributed as probably associated with drug. Four patients had a diagnosis of relapsed or refractory AML. CPI-613 at the initial dosing level concurrent with hydroxyurea resulted in a transient reduction in peripheral blood blasts in the first AML patient. Treatment at the next dosing level resulted in a hematologic improvement leading to transfusion independence and transient decrease in the 7q minus clone in the second AML patient. He maintains transfusion independence after 7 cycles and continues on therapy to date. The third patient with refractory AML had an increase in neutrophil count but no reduction in peripheral blood blasts. Taken together, these data suggest that altered lipid and mitochondrial metabolism are viable targets in the acute leukemias. The novel agent CPI-613 has activity against several acute leukemia cell lines in vitro and in vivo and may have activity in patients with relapsed disease. The therapeutic index appears quite high with only minor toxicities seen. Disclosures: No relevant conflicts of interest to declare.

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ANKHD1 Interacts with the Proapoptotic Protein SIVA and Plays a Role in the Proliferation and Stathmin Activation of Acute Leukemia Cells.

Blood ◽

10.1182/blood.v120.21.2419.2419 ◽

2012 ◽

Vol 120 (21) ◽

pp. 2419-2419

Author(s):

Joao Machado-Neto ◽

Mariana Lazarini ◽

Patricia Favaro ◽

Paula de Melo Campos ◽

Renata Scopim-Ribeiro ◽

...

Keyword(s):

Cell Migration ◽

Acute Leukemia ◽

Tumor Growth ◽

Chromosome Instability ◽

Leukemia Cells ◽

Alpha Tubulin ◽

Functional Studies

Abstract Abstract 2419 Introduction: Acute leukemia and solid tumors result from alterations in the essential pathways of cell physiology including apoptosis, proliferation and genome instability. In solid tumors, the proapoptotic SIVA protein modulates apoptosis, proliferation, migration and promotes Stathmin inhibition through phosphorylation. Stathmin regulates microtubules dynamics and its hyperactivity confers chromosome instability in leukemia cells. Using two-hybrid system assay, we have identified SIVA as a binding partner of ANKHD1, an ankyrin-repeat-containing protein. ANKHD1 is overexpressed in acute myeloid leukemia (AML) and acute lymphoblast leukemia (ALL) and has the potential role of regulating multiple cellular functions via their repeat motifs. We thus hypothesized that ANKHD1 and SIVA could be involved in leukemogenesis. We aimed to evaluate SIVA expression in normal and leukemia hematopoietic cells, to confirm the endogenous ANKHD1/SIVA association, and to investigate the functional role of both proteins in apoptosis, proliferation, migration, and Stathmin activation. Materials and Methods: Expression of SIVA was evaluated by qPCR in total bone marrow cells from 22 healthy donors, 42 AML and 21 ALL patients at diagnosis. All normal donors and patients provided informed written consent and the study was approved by the ethics committee of the Institution. Leukemia cell lines (Jurkat, Namalwa or U937 cells) were used for functional studies. Endogenous protein interaction was verified by immunopreciptation and cofocal microscopy. We stably knocked down the endogenous expression level of ANKHD1 or SIVA with specific shRNA-expressing lentiviral vector and in vitro apoptosis was examined by AnnexinV/PI, cell growth by MTT assay and colony formation, and migration by transwell assays. In addition, we investigated in vivo tumor growth; leukemia cells were implanted in the dorsal sub cutis of NOD/SCID mice and tumors were excised, measured and weighed after 15 days. Stathmin activation proteins (Stathmin, phospho-Stathmin, alpha tubulin and acetylated-alpha tubulin) and apoptotic proteins (BCL-XL, BAX, JNK and phospho-JNK) were evaluated by Western blot. Appropriated statistical analysis was performed. Results: SIVA expression was significantly decreased in AML and ALL cells compared with normal hematopoietic cells (P<0.05), a reverse pattern of ANKHD1 expression, when compared with published data. Immunopreciptation and confocal analyses confirmed that ANKHD1 and SIVA interact and co-localize in the cytoplasm of leukemia cells. Functional studies revealed that SIVA and ANKHD1 have antagonistic effects on migration, Stathmin activation, and in vivo tumor growth. SIVA silencing resulted in a significantly increased cell migration, Stathmin activation (decreased Stathmin phosphorylation), and augmented in vivo tumor growth (P<0.05). On the other hand, ANKHD1 silencing resulted in a significantly decreased cell migration, Stathmin inactivation (increased Stathmin phosphorylation and alpha tubulin acetylation), and reduced in vivo tumor growth (P<0.05). Regarding apoptosis and proliferation, SIVA knockdown resulted in a significant decrease in apoptosis response to UV and daunorubicin induction and a downregulation of proapoptotic proteins p-JNK and BAX, an upregulation of the antiapoptotic protein BCL-XL, but no modulation was observed in proliferation and clonal growth in vitro. In contrast, ANKHD1 knockdown resulted in a significant decrease of proliferation and clonogenicity (P<0.05), but no changes were observed in apoptosis in vitro. Conclusion: Our data indicate SIVA as a tumor suppressor gene in leukemia cells, and SIVA downmodulation may contribute to the apoptosis resistance and chromosome instability. ANKHD1 may be an oncogene, and the upregulation of this protein in leukemia cells might lead to increased proliferation and generate chromosomal instability through increased Stathmin activation. The results suggest that ANKHD1 inhibits SIVA and restoration of SIVA expression or inhibition of ANKHD1 may be an attractive approach in leukemia. Disclosures: No relevant conflicts of interest to declare.

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Identification of GPAT1 As a Novel Therapeutic Target for Acute Leukemia By Inhibiting Leukemia Specific Metabolism

Blood ◽

10.1182/blood-2019-125661 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 1384-1384

Author(s):

Hidetoshi Irifune ◽

Yu Kochi ◽

Masayasu Hayashi ◽

Yoshikane Kikushige ◽

Toshihiro Miyamoto ◽

...

Keyword(s):

Acute Leukemia ◽

Cell Lines ◽

Mass Spectrometer ◽

Mitochondrial Function ◽

Therapeutic Target ◽

Leukemia Cell ◽

Leukemia Cells ◽

Leukemia Cell Lines

With the development of mass spectrometer technology, recent studies revealed the critical roles of cancer-specific metabolism for tumor propagation in several types of cancers. In leukemia, many studies have been conducted to elucidate a leukemia-specific metabolism, and several effective treatments such as IDH1/2 inhibitors targeting acute myeloid leukemia (AML) with IDH1/2 mutation have been developed. To identify the new metabolic pathways on which acute leukemia cells depend, we purified water-soluble metabolites from CD34+ hematopoietic stem and progenitor cells (HSPCs) of healthy donors, AML and acute lymphoblastic leukemia (ALL) patients, and we comprehensively measured 116 metabolites using mass spectrometer analysis. From this experiment, we found that the cellular content of glycerol 3-phosphate (G3P) in CD34+ AML and ALL cells was lower than that of normal CD34+ HSPCs. G3P is an intermediate metabolite in the glycolysis metabolic pathway and is utilized as a substrate for phospholipids synthesis. The initial and rate-limiting step of phospholipids synthesis is the synthesis of lysophosphatidic acid (LPA) from G3P and acyl-CoA mediated by glycerol 3-phosphate acyltransferases (GPATs). Since CD34+ acute leukemia cells contained significantly lower level of G3P, we hypothesized that leukemia cells actively consumed G3P and synthesized LPA by GPATs. GPATs are classified into four isoforms based on intracellular localization and substrate preference. GPAT1 and GPAT2 are mitochondrial GPATs that are localized to the mitochondrial outer membrane, but on the other hand, GPAT3 and GPAT4 are microsomal GPATs that are localized to the endoplasmic reticulum membrane, each encoded by independent genes. GPAT1 is identified as an essential gene for the growth of leukemia cells by RNAi screen analysis in the public database (DepMap). We found that CD34+ immature AML cells exhibited higher GPAT1 expression as compared to CD34- more differentiated AML cells and normal T cells. GPAT1 knockdown inhibited the proliferation of several acute leukemia cell lines including THP-1 and Kasumi-1 in vitro and in vivo. Moreover, a mitochondrial GPATs specific inhibitor (FSG67), which was originally developed as a drug to treat obesity and diabetes, suppressed the growth of the leukemia cell lines through the induction of G1 cell cycle arrest. Growth inhibition was rescued by exogenous administration of LPA, suggesting that the synthetic activity mediated by mitochondrial GPATs should be required for acute leukemia growth. Furthermore, FSG67 induced the apoptosis of leukemia cells derived from AML and ALL patients without affecting normal CD34+ HSPCs at least in vitro. We also confirmed that the injection of FSG67 resulted in the suppression of AML and ALL propagation in vivo using patient-derived xenograft models (see figure). GPAT1 regulates the mitochondrial function by producing LPA which is an essential metabolite for maintaining mitochondrial fusion. Actually, the amount of LPA was decreased in GPAT1 knockdown acute leukemia cells. We next examined mitochondrial energy production by extracellular flux assay, and found that GPAT1 knockdown as well as FSG67 significantly suppressed oxygen consumption rate of acute leukemia cells. Consistent with the impaired mitochondrial function, FSG67 suppressed the mitochondrial membrane potential, indicating that GPAT1 should play a pivotal role in maintaining leukemia-specific mitochondrial function. These results collectively suggest that the synthesis of LPA from G3P catalyzed by GPAT1 has a critical role in propagation of acute leukemia cells irrespective of their lineage origin. Thus, GPAT1 is a novel and common therapeutic target for human acute leukemia through suppressing leukemia-specific mitochondrial function. Figure Disclosures Akashi: Celgene, Kyowa Kirin, Astellas, Shionogi, Asahi Kasei, Chugai, Bristol-Myers Squibb: Research Funding; Sumitomo Dainippon, Kyowa Kirin: Consultancy.

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VEGFR-1 (FLT-1) activation modulates acute lymphoblastic leukemia localization and survival within the bone marrow, determining the onset of extramedullary disease

Blood ◽

10.1182/blood-2005-06-2530 ◽

2006 ◽

Vol 107 (4) ◽

pp. 1608-1616 ◽

Cited By ~ 68

Author(s):

Rita Fragoso ◽

Teresa Pereira ◽

Yan Wu ◽

Zhenping Zhu ◽

José Cabeçadas ◽

...

Keyword(s):

Bone Marrow ◽

Cell Migration ◽

Acute Lymphoblastic Leukemia ◽

Acute Leukemia ◽

Actin Polymerization ◽

Lymphoblastic Leukemia ◽

Leukemia Cells ◽

Acute Leukemias ◽

Extramedullary Disease

The presence of persistent circulating leukemia cells, or engrafted into extramedullary tissues, is a bad prognostic factor for patients with acute leukemia. However, little is known about the mechanisms that regulate the exit of leukemia cells from the bone marrow (BM) microenvironment. We reveal that vascular endothelial growth factor receptor 1 (FLT-1) modulates acute leukemia distribution within the BM, along VEGF and PlGF gradients, regulating leukemia survival and exit into the peripheral circulation. FLT-1 activation on acute lymphoblastic leukemia (ALL) cells results in cell migration and proliferation in vitro, whereas in vivo FLT-1-overexpressing cells accumulate in the BM epiphysis of nonobese diabetic-severe combined immunodeficient (NOD-SCID) recipients and are detected in circulation 2 weeks after inoculation. In turn, FLT-1 neutralization affects leukemia localization (now in the BM diaphysis), increases leukemia apoptosis, and impedes the exit of ALL cells, prolonging the survival of inoculated mice. We demonstrate further that FLT-1-induced cell migration involves actin polymerization and lipid raft formation. Taken together, we show that FLT-1 regulates the BM localization of ALL cells, determining their survival and exit into the circulation and ultimately the survival of inoculated recipients. FLT-1 targeting on subsets of acute leukemias may delay the onset of extramedullary disease, which may be advantageous in combinatorial therapeutic settings.

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In Vivo RNA Interference Screening Identifies a Leukemia-Specific Dependence on Integrin Beta 3 Signaling

Blood ◽

10.1182/blood.v118.21.758.758 ◽

2011 ◽

Vol 118 (21) ◽

pp. 758-758

Author(s):

◽

Fatima Al-Shahrour ◽

Kimberly A. Hartwell ◽

Lisa P Chu ◽

Jaras Marcus ◽

...

Keyword(s):

Rna Interference ◽

Cell Growth ◽

Leukemia Cell ◽

Leukemia Cells ◽

Growth And Survival ◽

Integrin Beta 3 ◽

Shrna Screen ◽

Primary Leukemia

Abstract Abstract 758 Primary leukemia stem cells (LSCs) reside in an in vivo microenvironment that supports the growth and survival of malignant cells. Despite the increasing understanding of the importance of niche interactions and primary cell biology in leukemia, many studies continue to focus on cell autonomous processes in artificial model systems. The majority of strategies to-date that attempt to define therapeutic targets in leukemia have relied on screening cell lines in culture; new strategies should incorporate the use of primary disease within a physiologic niche. Using a primary murine MLL-AF9 acute myeloid leukemia (AML) model highly enriched for LSCs, we performed an in vivo short hairpin RNA (shRNA) screen to identify novel genes that are essential for leukemia growth and survival. LSCs infected with pools of shRNA lentivirus were transplanted and grown in recipient mice for 2 weeks, after which bone marrow and spleen cells were isolated. Massively parallel sequencing of infected LSCs isolated before and after transplant was used to quantify the changes in shRNA representation over time. Our in vivo screens were highly sensitive, robust, and reproducible and identified a number of positive controls including genes required for MLL-AF9 transformation (Ctnnb1, Mef2c, Ccna1), genes universally required for cell survival (Ube2j2, Utp18), and genes required in other AML models (Myb, Pbx1, Hmgb3). In our primary and validation screens, multiple shRNAs targeting Integrin Beta 3 (Itgb3) were consistently depleted by more than 20-fold over two weeks in vivo. Follow up studies using RNA interference (RNAi) and Itgb3−/− mice identified Itgb3 as essential for murine leukemia cells growth and transformation in vivo, and loss of Itgb3 conferred a statistically significant survival advantage to recipient mice. Importantly, neither Itgb3 knockdown or genetic loss impaired normal hematopoietic stem and progenitor cell (HSPC) function in 16 week multilineage reconstitution assays. We further identified Itgav as the heterodimeric partner of Itgb3 in our model, and found that knockdown of Itgav inhibited leukemia cell growth in vivo. Consistent the therapeutic aims or our study, flow cytometry on primary human AML samples revealed ITGAV/ITGB3 heterodimer expression. To functionally assess the importance of gene expression in a human system, we performed another RNAi screen on M9 leukemia cells, primary human cord blood CD34+ cells transduced with MLL-ENL that are capable of growing in vitro or in a xenotransplant model in vivo. We found that ITGB3 loss inhibited M9 cell growth in vivo, but not in vitro, consistent with the importance of ITGB3 in a physiologic microenvironment. We explored the signaling pathways downstream of Itgb3 using an additional in vivo, unbiased shRNA screen and identified Syk as a critical mediator of Itgb3 activity in leukemia. Syk knockdown by RNAi inhibited leukemia cell growth in vivo; downregulation of Itgb3 expression resulted in decreased levels of Syk phosphorylation; and expression of an activated form of Syk, TEL-SYK, rescued the effects of Itgb3 knockdown on leukemia cell growth in vivo. To understand cellular processes controlled by Itgb3, we performed gene expression studies and found that, in leukemia cells, Itgb3 knockdown induced differentiation and inhibited multiple previously published LSC transcriptional programs. We confirmed these results using primary leukemia cell histology and a model system of leukemia differentiation. Finally, addition of a small molecule Syk inhibitor, R406, to primary cells co-cultured with bone marrow stroma caused a dose-dependent decrease in leukemia cell growth. Our results establish the significance of the Itgb3 signaling pathway, including Syk, as a potential therapeutic target in AML, and demonstrate the utility of in vivo RNA interference screens. Disclosures: Armstrong: Epizyme: Consultancy.

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ATR inhibition induces synthetic lethality and overcomes chemoresistance in TP53- or ATM-defective chronic lymphocytic leukemia cells

Blood ◽

10.1182/blood-2015-05-644872 ◽

2016 ◽

Vol 127 (5) ◽

pp. 582-595 ◽

Cited By ~ 104

Author(s):

Marwan Kwok ◽

Nicholas Davies ◽

Angelo Agathanggelou ◽

Edward Smith ◽

Ceri Oldreive ◽

...

Keyword(s):

Chronic Lymphocytic Leukemia ◽

Synthetic Lethality ◽

Lymphocytic Leukemia ◽

Leukemia Cells ◽

Selective Cytotoxicity ◽

Key Points ◽

Synthetically Lethal

Key PointsATR inhibition is synthetically lethal to TP53- or ATM-defective CLL cells. ATR targeting induces selective cytotoxicity and chemosensitization in TP53- or ATM-defective CLL cells in vitro and in vivo.

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Nurture versus Nature: The Microenvironment in Chronic Lymphocytic Leukemia

Hematology ◽

10.1182/asheducation-2011.1.96 ◽

2011 ◽

Vol 2011 (1) ◽

pp. 96-103 ◽

Cited By ~ 101

Author(s):

Jan A. Burger

Keyword(s):

Chronic Lymphocytic Leukemia ◽

Tyrosine Kinase ◽

B Cell ◽

Disease Progression ◽

Drug Testing ◽

Cell Receptor ◽

Lymphocytic Leukemia ◽

In Vivo Models

Abstract Intrinsic factors such as genetic lesions, anti-apoptotic proteins, and aberrant signaling networks within leukemia cells have long been the main focus of chronic lymphocytic leukemia (CLL) research. However, over the past decade, it became increasingly clear that external signals from the leukemia microenvironment make pivotal contributions to disease progression in CLL and other B-cell malignancies. Consequently, increasing emphasis is now placed on exploring and targeting the CLL microenvironment. This review highlights critical cellular and molecular pathways of CLL-microenvironment cross-talk. In vitro and in vivo models for studying the CLL microenvironment are discussed, along with their use in searching for therapeutic targets and in drug testing. Clinically, CXCR4 antagonists and small-molecule antagonists of B cell receptor (BCR)-associated kinases (spleen tyrosine kinase [Syk], Bruton's tyrosine kinase [Btk], and PI3Kδ) are the most advanced drugs for targeting specific interactions between CLL cells and the miocroenvironment. Preclinical and first clinical evidence suggests that high-risk CLL patients can particularly benefit from these alternative agents. These findings indicate that interplay between leukemia-inherent and environmental factors, nature and nurture determines disease progression in CLL.

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Chronic lymphocytic leukemia of Eμ-TCL1 transgenic mice undergoes rapid cell turnover that can be offset by extrinsic CD257 to accelerate disease progression

Blood ◽

10.1182/blood-2009-06-230169 ◽

2009 ◽

Vol 114 (20) ◽

pp. 4469-4476 ◽

Cited By ~ 36

Author(s):

Thomas Enzler ◽

Arnon P. Kater ◽

Weizhou Zhang ◽

George F. Widhopf ◽

Han-Yu Chuang ◽

...

Keyword(s):

Chronic Lymphocytic Leukemia ◽

Disease Progression ◽

Leukemia Cell ◽

Lymphocytic Leukemia ◽

Leukemia Cells ◽

Cell Turnover ◽

Proliferating Cells ◽

Rapid Reduction ◽

Disease Evolution ◽

Dying Cells

AbstractResults of heavy-water labeling studies have challenged the notion that chronic lymphocytic leukemia (CLL) represents an accumulation of noncycling B cells. We examined leukemia cell turnover in Eμ-TCL1 transgenic (TCL1-Tg) mice, which develop a CLL-like disease at 8 to 12 months of age. We found that leukemia cells in these mice not only had higher proportions of proliferating cells but also apoptotic cells than did nonleukemic lymphocytes. We crossed TCL1-Tg with BAFF-Tg mice, which express high levels of CD257. TCL1×BAFF-Tg mice developed CLL-like disease at a significantly younger age and had more rapid disease progression and shorter survival than TCL1-Tg mice. Leukemia cells of TCL1×BAFF-Tg mice had similar proportions of proliferating cells, but fewer proportions of dying cells, than did the CLL cells of TCL1-Tg mice. Moreover, leukemia cells from either TCL1×BAFF-Tg or TCL1-Tg mice produced more aggressive disease when transferred into BAFF-Tg mice than into wild-type (WT) mice. Neutralization of CD257 resulted in rapid reduction in circulating leukemia cells. These results indicate that the leukemia cells of TCL1-Tg mice undergo high levels of spontaneous apoptosis that is offset by relatively high rates of leukemia cell proliferation, which might allow for acquisition of mutations that contribute to disease evolution.

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