The cancer-testis antigen NXF2 is activated by the hypomethylating agent decitabine in acute leukemia cells in vitro and in vivo

Abstract Introduction Epigenetic therapies with azanucleoside DNA hypomethylating agents, alone or in combination with histone deacetylase inhibitors (HDACi), show clinical activity in myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML), particularly when given at non-cytotoxic doses. They are able to reactivate epigenetically silenced genes including, among others, a number of highly immunogenic proteins dubbed Cancer/testis antigens (CTAs), predominantly the CTAs located on the X chromosome. We have previously shown that decitabine can induce expression of several CTAs, including MAGEB2 and NY-ESO-1, in myeloid cells in vitro and thereby trigger an immune response (Almstedt et al., Leuk. Res. 2010). Induction of a CTA-specific cytotoxic T cell response in vivo was reported also in AML patients treated with azacitidine and sodium valproate (VPA) and correlated with clinical response (Goodyear et al., Blood 2010). To the best of our knowledge, no data have yet been reported on the effect of combination treatment with decitabine and panobinostat or sodium valproate (VPA) on CTA reactivation in myeloid leukemia. Aim We hypothesized that by combining decitabine with HDACi we could further enhance expression of CTAs in myeloid leukemia cells and thereby boost recognition of the malignant cells by the cytotoxic T lymphocytes. Methods The myeloid cell lines U937 and Kasumi-1 were treated with decitabine alone or in combination with the HDACi VPA or panobinostat applied at non-toxic concentrations (>80% cell viability). Expression of CTAs was analyzed by RT-qPCR and Western blot after 48 hours of HDACi treatment. DNA methylation of NY-ESO-1 and MAGEB2 promoter regions was quantified by pyrosequencing. Bone marrow mononuclear cells from 19 AML patients (treated with or without VPA as add-on to decitabine in the ongoing randomized phase II DECIDER clinical trial, NCT00867672) were collected before and on day 15 of treatment, in some patients also after 2 treatment cycles. CTA mRNA expression and promoter DNA methylation were quantified as described above. Results VPA or panobinostat alone did not induce MAGEB2 or NY-ESO-1 expression in vitro. However the pretreatment of cells with decitabine prior to addition of either HDACi resulted in a synergistic dose-dependent reactivation of MAGEB2 and NY-ESO-1 on the mRNA level (confirmed for the latter on the protein level). Pyrosequencing analysis of the heavily methylated NY-ESO-1 and MAGEB2 promoters revealed, as expected, no methylation changes upon HDACi treatment, but a dose-dependent hypomethylation upon decitabine. In recently initiated in vivo studies (DECIDER trial), until now cells from 19 AML patients receiving epigenetic treatment were sequentially analyzed. Induction of MAGEB2 mRNA was observed in 9 patients (from absent to a median of 0.002 relative to GAPDH, range 0.0004-0.043), with concomitant DNA hypomethylation of the MAGEB2 promoter from median 83% pretreatment methylation (range 63%-90%) to 63% posttreatment (range 44%-74%). In 5 patients modest hypomethylation without changes in MAGEB2 expression was observed (from median pretreatment values of 89% [72%-92%] to 82% [58%-87%] posttreatment). Another 5 patients disclosed neither hypomethylation nor reexpression of MAGEB2 (results as yet blinded to treatment arm and clinical response). Conclusions Combined epigenetic treatment with the hypomethylating agent decitabine and the HDACi VPA or panobinostat synergistically induced a dose-dependent reactivation of the CTAs MAGEB2 and NY-ESO-1 in vitro, accompanied by promoter hypomethylation. First translational results of the DECIDER AML trial also indicate in vivo effects of the epigenetic treatment on CTA induction. The unmasking of CTAs to the immune system by epigenetically active drugs can increase anti-tumor immune responses, and thus has clear implications for future clinical trials combining epigenetic therapy and specific immunotherapy in myeloid neoplasia. Disclosures: No relevant conflicts of interest to declare.

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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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XAGE-1b Cancer/Testis Antigen Is a Potential Target for Immunotherapy in Prostate Cancer

Urologia Internationalis ◽

10.1159/000363333 ◽

2015 ◽

Vol 94 (3) ◽

pp. 354-362 ◽

Cited By ~ 1

Author(s):

Chong Xie ◽

Guomin Wang

Keyword(s):

Prostate Cancer ◽

Gene Transfection ◽

Suppressor Cell ◽

Eukaryotic Expression ◽

Cancer Testis Antigen ◽

Recombinant Vector ◽

Cell Expression ◽

Cancer Testis

Introduction: Gene-modified cell vaccines are now considered to be the best way to achieve immunotherapy for a variety of cancers including prostate cancer (PCa). XAGE-1b is a member of the cancer/testis antigen family which has demonstrated strong immunogenicity. We investigated whether XAGE-1b is an ideal target for PCa immunotherapy. Materials and Methods: The recombinant eukaryotic expression vector pDisplay-XAGE-1b was constructed. Then the recombinant vector was transfected into Myc-CaP cells and its immunogenicity in vitro was studied. After transfection, the Myc-CaP-XAGE-1b cells were injected into FVB mice subcutaneously. Tumor growth was periodically observed and the anti-tumor effect and mechanism in vivo were further studied. Results: The recombinant vector was correctly constructed by DNA sequencing and restriction endonuclease digestion. Myc-CaP cells were successfully transfected with XAGE-1b gene by immunofluorescence staining and Western blot. The transfected cells exhibited increased IFN-γ secretion, decreased IL-6 secretion and enhanced killing activity. Tumor grew slower in XAGE-1b-modified FVB mice than in wild-type FVB mice. High dendritic cell expression and low myeloid-derived suppressor cell expression were observed in tumor tissues expressed with XAGE-1b. Conclusions: XAGE-1b gene transfection could significantly enhance the immunogenicity of Myc-CaP cells. Therefore, XAGE-1b may be an attractive target for antigen-specific immunotherapy in PCa.

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A CRISPR/Cas9 Library Screen in Patients' Leukemia Cells In Vivo

Blood ◽

10.1182/blood-2019-124288 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 3945-3945

Author(s):

Ehsan Bahrami ◽

Martin Becker ◽

Anna-Katharina Wirth ◽

Jan Phillip Schmid ◽

Tobias Herold ◽

...

Keyword(s):

Acute Leukemia ◽

Lymphoblastic Leukemia ◽

Drop Out ◽

Leukemia Cells ◽

Functional Genomic ◽

Cell Homing ◽

Nsg Mice ◽

Crispr Screen

Background: Functional genomic screens elegantly increase our understanding of biology of leukemias. So far, CRISPR/Cas9 screens are widely performed in cell lines and in genetically engineered mouse models, in vitro and in vivo; here, we extended their use to patient-derived leukemia cells in vivo. Methods Serially transplantable patient-derived xenograft (PDX) models were generated from children and adults with acute lymphoblastic leukemia (ALL). Cas9 was stably expressed in PDX ALL cells using a split form of Cas9 assembled by inteins, facilitating lentiviral-mediated gene delivery. Customized sgRNA library was generated using golden gate cloning, at 5 sgRNAs per target gene. The sgRNA vector additionally expressed a fluorochrome marker and a tag, for sequential magnetic-activated cell sorting (MACS) and flow cytometry (FACS) enrichment of sgRNA transduced PDX cells. Highly enriched Cas9/sgRNA double transgenic cells were transplanted into NSG mice and animals sacrificed after different periods of time. Cells were re-isolated from bone marrow, purified and subjected to PCR-based amplification of sgRNA library followed by next generation sequencing. Differential sgRNA distributions were analysed using a MAGeCK pipeline. Results We aimed to establish a comprehensive CRISPR screen pipeline allowing functional genomic screens in patients' acute leukemia cells. We investigated surface molecules required for cell homing and growth in mice, using a distinct customized sgRNA library. Quality controls of the sgRNA plasmid pool as well as transgenic PDX input samples verified standard distribution of all sgRNAs. As knockout was required at the time point of transplantation, conditions for prolonged culture of PDX ALL cells in vitro were optimized. Before injection into NSG mice, transduced PDX ALL cells were successfully enriched to above 95% using MACS and FACS. Over time in vivo, deep sequencing of re-isolated PDX cells revealed unchanged distribution of control sgRNAs, but strong loss of sgRNAs targeting CXCR4 and ITGB1, suggesting that CXCR4 and ITGB1 might be required for PDX ALL cell homing and engraftment. To validate the findings of drop-out CRISPR screen, we analyzed single sgRNAs targeting CXCR4 and ITGB1 in PDX cells. Competitive in vivo assays monitored by recombinant fluorochrome markers showed that the cells with CXCR4 or ITGB1 knockout had a significant disadvantage in vivo with respect to homing and growth in mice, compared to the control population. Taken together, we established a comprehensive workflow for CRISPR screen in PDX model of ALL in vivo. Our data identify and validate that CXCR4 and ITGB1 are required for homing and growth of PDX ALL cells in mice. Conclusion We show that CRISPR/Cas9 functional genetic screens are feasible in PDX acute leukemia models in vivo and report the first such screen, as far as to our knowledge. Extending CRISPR/Cas9 screens to patients' cells will greatly facilitate our understanding of individual leukemia biology and therapeutic targets in the future. Disclosures Becker: AVA Lifescience GmbH: Consultancy.

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Abstract 2746: Combined inhibition of Wee1 and Poly(ADP Ribose)Polymerase 1/2 synergistically inhibits acute leukemia cells by enhancing DNA damage and inducing apoptosis in vitro and in vivo

10.1158/1538-7445.am2016-2746 ◽

2016 ◽

Author(s):

Tamara B. Garcia ◽

Jonathan C. Snedeker ◽

Christopher C. Porter

Keyword(s):

Dna Damage ◽

Acute Leukemia ◽

Leukemia Cells

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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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DNA-PK inhibitor peposertib enhances p53-dependent cytotoxicity of DNA double-strand break inducing therapy in acute leukemia

Scientific Reports ◽

10.1038/s41598-021-90500-3 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Eric Haines ◽

Yuki Nishida ◽

Michael I. Carr ◽

Rafael Heinz Montoya ◽

Lauren B. Ostermann ◽

...

Keyword(s):

Acute Leukemia ◽

Topoisomerase Ii ◽

Therapeutic Potential ◽

P53 Protein ◽

Fixed Ratio ◽

Myeloid Cell ◽

Leukemia Cells ◽

Topoisomerase Ii Inhibitors

AbstractPeposertib (M3814) is a potent and selective DNA-PK inhibitor in early clinical development. It effectively blocks non-homologous end-joining repair of DNA double-strand breaks (DSB) and strongly potentiates the antitumor effect of ionizing radiation (IR) and topoisomerase II inhibitors. By suppressing DNA-PK catalytic activity in the presence of DNA DSB, M3814 potentiates ATM/p53 signaling leading to enhanced p53-dependent antitumor activity in tumor cells. Here, we investigated the therapeutic potential of M3814 in combination with DSB-inducing agents in leukemia cells and a patient-derived tumor. We show that in the presence of IR or topoisomerase II inhibitors, M3814 boosts the ATM/p53 response in acute leukemia cells leading to the elevation of p53 protein levels as well as its transcriptional activity. M3814 synergistically sensitized p53 wild-type, but not p53-deficient, AML cells to killing by DSB-inducing agents via p53-dependent apoptosis involving both intrinsic and extrinsic effector pathways. The antileukemic effect was further potentiated by enhancing daunorubicin-induced myeloid cell differentiation. Further, combined with the fixed-ratio liposomal formulation of daunorubicin and cytarabine, CPX-351, M3814 enhanced the efficacy against leukemia cells in vitro and in vivo without increasing hematopoietic toxicity, suggesting that DNA-PK inhibition could offer a novel clinical strategy for harnessing the anticancer potential of p53 in AML therapy.

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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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The Effects of Anti-High Molecular Weight-Melanoma Associated Antigen (HMW-MAA) Monoclonal Antibodies (mAb) Against 11q23 Positive Acute Leukemia Cells.

Blood ◽

10.1182/blood.v108.11.4550.4550 ◽

2006 ◽

Vol 108 (11) ◽

pp. 4550-4550

Author(s):

Allison S. Drake ◽

Brady T. Michael ◽

Xin Hui Wang ◽

Sheila J.N. Sait ◽

Justin C. Earp ◽

...

Keyword(s):

Acute Leukemia ◽

Lymphoblastic Leukemia ◽

Scid Mice ◽

Leukemic Cells ◽

Leukemia Cells ◽

High Dose ◽

Tumor Inhibition ◽

Tyrosine Kinase 2

Abstract Acute leukemia with 11q23 aberrations is associated with especially poor outcome to standard treatment. HMW-MAA is a membrane bound proteoglycan that has been targeted in the treatment of malignant melanoma. It is widely distributed on 11q23-positive leukemic cells and is not expressed on normal hematopoietic cells. Finally, HMW-MAA is believed to function by signaling through the focal adhesion kinase (Fak)/proline-rich tyrosine kinase 2 (Pyk2) pathways and by supporting angiogenesis via its expression on pericytes. We hypothesized that inhibition of HMW-MAA with mAb directed against specific HMW-MAA determinants would effectively limit the growth of 11q23-positive acute leukemia cells in vitro and in vivo, by affecting Fak/Pyk2 pathways. We examined an 11q23-positive acute myeloid leukemia (AML) cell line (ML-2) and leukemia cells from two patients (one AML, one acute lymphoblastic leukemia) with 11q23 aberrations. Our results demonstrate that ML-2 cells expressed only the VT68.2 and 225.28 determinants, while primary acute leukemia cells expressed VT68.2, 225.28, and 763.74 determinants. Treatment of ML-2 cells with mAb against VT68.2 and 225.28 in vitro did not affect overall proliferation but downregulated phosphorylated (p) Pyk2 expression as determined by Western blotting. Further, anti-HMW-MAA mAb treatment enhanced the anti-proliferative effect of cytarabine (y=0.7875) in vitro. Based on these results, we then evaluated the dose-response relationships and toxicities of anti-HMW-MAA mAb alone and in combination with cytarabine in subcutaneous ML-2 xenografts grown in SCID mice. A trend towards tumor inhibition was noted following high dose (1 mg/kg 2x/week) anti-225 mAb therapy but was not seen at lower (0.25 and 0.5 mg/kg) anti-225 mAb doses. Mice demonstrated no side effects or weight loss as a result of anti-225 therapy. No downregulation of pPyk2 in tumor cells was observed following anti-225 therapy at any dose. Furthermore, combination anti-225.28 HMW-MAA mAb and cytarabine treatment (at increasing cytarabine doses) did not result in improved tumor inhibition as compared to cytarabine alone (p>0.05). We then evaluated the effects of HMW-MAA anti-225.28 and anti-763.74 mAb alone or together for treatment of NOD/SCID mice engrafted with primary 11q23-positive leukemia patient cells. Combination anti-HMW-MAA mAb therapy resulted in no prolongation in survival of engrafted mice as compared to control treated animals. We were able to confirm the presence of high levels of circulating HMW-MAA mAb in the serum of animals 4 days following mAb injection. In addition, explanted ML-2 cells harvested following xenograft growth still maintained expression of the HMW-MAA determinants. We conclude that naïve anti-HMW-MAA mAb neither caused a statistically significant inhibitory effect nor enhanced the effect of cytarabine on 11q23-positive acute leukemia cells in vivo. Future studies using radiolabelled or toxin-conjugated antibodies may enhance the anti-tumor effects of targeted HMW-MAA immunotherapy for 11q23-positive acute leukemia.

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

Blood ◽

10.1182/blood.v112.11.5315.5315 ◽

2008 ◽

Vol 112 (11) ◽

pp. 5315-5315

Author(s):

Cristina Casalou ◽

Ana Gomes ◽

Tania Carvalho ◽

Sergio Dias

Keyword(s):

Cell Migration ◽

Acute Leukemia ◽

Disease Progression ◽

Rho Gtpases ◽

Leukemia Cell ◽

Lymphocytic Leukemia ◽

Leukemia Cells ◽

Acute Leukemias

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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