1986 — 1992 |
Andreeff, Michael W |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Function of Quiescent Cells in Pediatric Leukemia @ University of Texas Md Anderson Can Ctr
The proposed studies will further investigate the function of quiescent leukemic cells in non B - non T acute lymphoblastic leukemia in children and will develop means of recruiting them into the cell cycle. Multivariate analysis of DNS/RNA flow cytometric and clinical data has identified quiescent cells with low RNA content as resistant to early induction therapy. Correlation of WBC and cell kinetics showed that high WBC is associated with low RNA content (quiescence) of bone marrow blasts. These "G0-" cells will be further analyzed by multiparameter flow cytometric investigations of the nuclear antigens K167 and PCNA ("Cyclin") of chromatin structure in-situ and of a novel proliferation-related cell surface antigen (A18). The nuclear protein of the proto- oncogene p53 will be further studied by immunoprecipitation. P53 expression is cell cycle dependent and our preliminary data indicate that patients with high p53 expression have poor prognosis. A small population of aneuploid leukemic cells with restricted cell surface light chain expression has been identified in untreated patients. These cells appear to be quiescent and their relationship to the "G0" population will be further investigated in conjunction with the above proliferation markers. FACS-sorting of cells has identified these cells as having morphological features of more mature lymphocytes, consistent with the notion that a subpopulation of leukemic cells differentiates in-vivo. We will study the co-expression of these cells of light chains and of the CD19 antigen and then investigate if differentiated and/or quiescent cell can be diminished by an anti- CD19-ricin immunotoxin. The expression of the p-glycoprotein on proliferating, quiescent and differentiated leukemic cells will be studied using an novel antibody directed against a surface epitope of gp180. In-vitro studies have demonstrated the possibility of recruitment of leukemic cells from G0 to G1 by IL-2/OKT-3/PMA. BCGF and IL-6 will be studied in their ability to recruit aneuploid leukemic cells into the cell cycle and subsequent treatment with daunomycin, ARA-C and MTX will test the hypothesis that recruited cells have increased sensitivity to cytotoxic chemotherapy. If successful, these studies will introduce cytokines into chemotherapeutic regimen for the treatment of pediatric All and will hopefully improve the remission duration for children with poor risk and relapsed ALL.
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0.907 |
1992 — 1994 |
Andreeff, Michael W |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Cytokine/Chemotherapy Combinations in Myeloid Leukemia @ University of Texas Md Anderson Can Ctr
This grant proposal aims at investigating mechanisms of growth factor-induced cell cycle recruitment of leukemic cells, in vitro and in vivo and at developing optimized cytokine/chemotherapy regimens for patients with acute myeloblastic leukemia (AML). Rationale is based on the known cell cycle specificity of the main drugs used in the therapy of AML and MDS, i.e. ARA-C and anthracyclines, on prognostic models that recognize the negative impact of quiescent leukemic cells on therapeutic response and on the demonstrated recruitment of leukemic cells by granulocyte colony stimulating factor (G-CSF), granulocyte-macrophage colony stimulating factor (GM-CSF) and interleukin-3 (IL-3). We now propose to extend these studies to include the GM-CSF/IL-3 fusion molecule PIXY321, and the c-kit ligand stem cell factor (SCF). End points in these in vitro studies will be defined by multiparameter flow cytometry measurements of DNA, RNA, BUdR, IUdR and cell cycle-related proteins (c-myc, p53, PCNA) by results obtained in suspension cultures and in clonogenic assays. These studies of fresh leukemia samples (120 patients with newly diagnosed AML will provide detailed analysis of leukemia cell differentiation and proliferation, in particular of recruitment of Go cells into the cell cycle, and will identify the most active cytokine combinations. We will then expose the stimulated cells to ARA-C and to anthracyclines with the aim of maximizing cell kill by these agents. A newly developed FACS technique allows molecular analysis of gene expression in sorted hematopoietic cell populations of defined lineage and maturation, and will provide detailed analysis of lineage-specific gene regulation by cytokines. Fluorescence in-situ hybridization (FISH) using chromosome-specific probes will allow the crucial discrimination of cytokine effects on normal and leukemic cells. We already have evidence for selective cytokine effects on clonally abnormal leukemic cells, using the combined method of BUdR labelling and FISH analysis, and the proposed studies will allow us to optimize selective recruitment of leukemic cells. Clinical studies of IL-3 in AML will be hypothesis monitored by these new techniques and will allow us to test the that in vivo recruitment of hematopoietic cells is possible, and of therapeutic benefit.
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0.907 |
1993 — 1996 |
Andreeff, Michael W |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Fish Analysis of Lymphoma @ University of Texas Md Anderson Can Ctr
Follicular small cleaved cell lymphoma (FSCCL) is a low-grade lymphoma with indolent course with the characteristic chromosome translocation t(14; 18)(q32;q21) resulting in rearrangement of the bcl-2 proto-oncogene. Bcl-2 is over-expressed, abrogates apoptosis, and results in cell accumulation. Sixty-five percent (65%) of cases progress to large cell lymphoma within 10 years. Bcl-2 rearrangement or over-expression above may not be sufficient for lymphomagenesis, suggesting that other genetic events play a role in the development and progression of FSCCL. Karyotypic analysis of FSCCL is limited to metaphases of dividing cells and may not be representative for the cytogenetic abnormalities and for clonal heterogeneity, as these tumors exhibit a characteristically low growth fraction. Fluorescent in situ hybridization (FISH) allows the cytogenetic analysis of metaphase and of non-dividing interphase cells and is a powerful tool for the analysis of subpopulations of the malignant clone. FISH analysis has already demonstrated the emergence of subpopulations with trisomy and tetrasomy of chromosome 12. We propose to investigate prospectively samples from patients with FSCCL for emerging subpopulations with numerical abnormalities of chromosomes 3, 7, 12, 18, and y and for deletions such as 17p-, 6q-, and 1 p-. These changes will be correlated with progression of disease, relapse, or transformation. Samples also will be labelled with BUdR and the fraction of cells in 5- phase will be determined for each cytogenetically distinct subpopulation. Patients will be treated uniformly with standard therapeutic protocols. In addition, expression of bcl-2, as determined by Dr. T. McDonnell, will be correlated with cytogenetic results. Lymphoma cells from different sites in the same patient (lymph node, bone marrow, blood) will be compared to investigate possible associations between cytogenetic abnormalities and site of involvement. The novel technique of Comparative Genomic Hybridization (CGH) will be applied to the sequential study of FSCCL. DNA from involved lymph nodes will be comparatively hybridized to normal metaphase chromosomes with normal DNA and with DNA from subsequent samples from the same patient. We expect these studies to reveal new genetic abnormalities in FSCCL and to provide insight into the genetic changes underlying disease progression.
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0.907 |
1995 — 2005 |
Andreeff, Michael W |
P01Activity Code Description: For the support of a broadly based, multidisciplinary, often long-term research program which has a specific major objective or a basic theme. A program project generally involves the organized efforts of relatively large groups, members of which are conducting research projects designed to elucidate the various aspects or components of this objective. Each research project is usually under the leadership of an established investigator. The grant can provide support for certain basic resources used by these groups in the program, including clinical components, the sharing of which facilitates the total research effort. A program project is directed toward a range of problems having a central research focus, in contrast to the usually narrower thrust of the traditional research project. Each project supported through this mechanism should contribute or be directly related to the common theme of the total research effort. These scientifically meritorious projects should demonstrate an essential element of unity and interdependence, i.e., a system of research activities and projects directed toward a well-defined research program goal. |
Therapy of Aml @ University of Texas Md Anderson Can Ctr
Acute myelogenous leukemia (AML) is a disease in which the accumulation of primitive nonfunctional precursor cells results in the death of 80% of patients due to bleeding and infection. Although allogeneic bone marrow transplantation is curative, only 25% of AML patients are eligible for this therapy, and only half of these survive long-term. Another 10-20% of patients are cured by combination chemotherapy. In order to develop new and improved directions of therapy for AML, we are proposing to use cell cycle regulatory molecules such as growth factors or differentiation induction agents to specifically sensitize the leukemia cells to phase-specific chemotherapy agents. These treatment programs will be linked to laboratory assays which will provide short-term molecular endpoints for the evaluation of response and minimal residual disease. The molecular changes found in these leukemia cells will be used as targets for therapy as well as for predictors of response, remission duration and survival. We will carry this analysis out in untreated patients and in patients who are in remission and relapse. We will classify patients as to whether they develop a proliferative response, a differentiation induction response, or an apoptotic response to the combination of chemotherapy plus a cell cycle regulatory molecule. We will compare in vitro with in vivo responses. We will compare responses to chemotherapy and a cell cycle regulatory molecule in vitro and in vivo with respect to chemotherapy metabolism, cell cycle, cytoplasmic signal transduction molecules, growth factor synthesis, intranuclear growth regulatory proteins (1)53, WAF- 1/CIP-1, and Rb), Bcl-2/Bax and other members of the apoptosis family, and apoptotic and non-apoptotic mechanisms of cell death. We will use PCR and FISH assays specific for each of the chromosomally defined subsets of AML to define response to therapy, response to cell cycle regulatory - molecules, and minimal residual disease. We will use the information derived from these assays to allocate patients to specific programs of therapy. We will use this information to build sequential therapeutic programs which integrate chemotherapy with cell cycle regulatory molecules, used singly and in combinations. In this way, we hope to be able to develop therapy which is targeted to the molecular and biological defects in AML, which is less toxic to normal tissues and provides more durable remissions in all subsets of AML in untreated patients, in relapsed patients, and in the minimal residual disease setting. Because the principles of therapy are relevant to the treatment of epithelial neoplasms as well, we expect the information to be derived in this program to provide direction for improvements in solid tumor therapy as well.
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0.907 |
1996 — 2001 |
Andreeff, Michael W |
P01Activity Code Description: For the support of a broadly based, multidisciplinary, often long-term research program which has a specific major objective or a basic theme. A program project generally involves the organized efforts of relatively large groups, members of which are conducting research projects designed to elucidate the various aspects or components of this objective. Each research project is usually under the leadership of an established investigator. The grant can provide support for certain basic resources used by these groups in the program, including clinical components, the sharing of which facilitates the total research effort. A program project is directed toward a range of problems having a central research focus, in contrast to the usually narrower thrust of the traditional research project. Each project supported through this mechanism should contribute or be directly related to the common theme of the total research effort. These scientifically meritorious projects should demonstrate an essential element of unity and interdependence, i.e., a system of research activities and projects directed toward a well-defined research program goal. |
Core--Fluorescence Activated Cell Sorting (Facs) @ University of Texas Md Anderson Can Ctr
The FACS Core facility provides cellular analysis to the investigators of this program project. The laboratory has provided and developed cutting edge techniques in single cell analysis. With the focus of this grant on apoptosis, the TdT-b-dUTP assay for apoptosis (TUNEL) was established and modified for multiparameter analysis in combination with DNA and BUdR measurements (cell cycle, ploidy), immunophenotype to analyze progenitor cells and lineage restricted cell populations, intracellular proteins related to apoptosis (bcl-2, p53, Rb), and cell surface antigens including fas and MDR1. Recently, binding of Annexin V to phosphatidyl serine (PS) was recognized as a test for changes in the membrane lipid structure that precedes changes detected by TUNEL in cells undergoing apoptosis. Quantitation of cellular antigens is now available allowing us to determine the Antibody Binding Capacity (ABC). Cell kinetic changes can be studied in patients with infusion of IUdR and BUdR and subsequent analysis by flow cytometry. Fluorescence in situ hybridization (FISH) has also been combined with the apoptosis-assays, allowing us to discriminate apoptosis in normal and leukemic cells. The number, phenotype and proliferation of residual leukemic cells (MRD) with abnormalities amenable to FISH analysis can be determined at levels of as few as 1 leukemic in 30,000 normal cells. This assay is predictive of remission duration and will be applied to patients after induction therapy and bone marrow transplantation.
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0.907 |
1996 — 2001 |
Andreeff, Michael W |
P01Activity Code Description: For the support of a broadly based, multidisciplinary, often long-term research program which has a specific major objective or a basic theme. A program project generally involves the organized efforts of relatively large groups, members of which are conducting research projects designed to elucidate the various aspects or components of this objective. Each research project is usually under the leadership of an established investigator. The grant can provide support for certain basic resources used by these groups in the program, including clinical components, the sharing of which facilitates the total research effort. A program project is directed toward a range of problems having a central research focus, in contrast to the usually narrower thrust of the traditional research project. Each project supported through this mechanism should contribute or be directly related to the common theme of the total research effort. These scientifically meritorious projects should demonstrate an essential element of unity and interdependence, i.e., a system of research activities and projects directed toward a well-defined research program goal. |
Regulation of Proliferation and Apoptosis in Leukemia Progenitor Cells @ University of Texas Md Anderson Can Ctr
The main therapeutic challenge in the treatment of acute myelogenous leukemia (AML) is the refractory behavior of residual leukemic cells. The factors regulating the survival and subsequent clonal expansion of residual leukemic cells causing relapse remain to be elucidated and may be different from those determined in leukemia samples at diagnosis. This project is designed to investigate the dysregulated proliferation and apoptosis of leukemic progenitor cells which leads to resistance and subsequent expansion of the leukemic clone. We will determine the relationship between cell proliferation and cell death in normal and in AML progenitor cells at diagnosis, remission and relapse. To this end, we have developed new methods to determine gene expression (quantitative multiparametric FACS analysis of cellular proteins, and FACS/RT-PCR) and clonality (FACS/FISH) in very rare progenitor and minimal residual disease (MRD) cells. MRD was detectable in all patients studied in the first 4 years of this program project, but the mechanism of survival of residual leukemic cells in the bone marrow is still unknown and sensitive detection methods are necessary to evaluate them. We will characterize the proliferation of immunophenotypically characterized residual leukemic cells so that any differences found between normal diploid and genetically distinct neoplastic cells may later be exploited in the design of new therapeutic approaches. We will study the molecular determinants affecting survival and proliferation within progenitor cell compartments following induction and consolidation chemotherapy. By monitoring residual disease in AML patients, we will quantitate the level of MRD which requires therapeutic intervention. We will then test the effects of ATRA, G-CSF and Flt-3 ligand (already shown to regulate antiapoptotic genes) on chemotherapy- induced apoptosis in vitro and in vivo better. By understanding the differences which exist between leukemic and normal progenitors in their responses to stimuli of proliferation and apoptosis, using concomitant FISH with BUdR incorporation and Annexin V labeling of apoptotic cells, we will find ways to selectively sensitize leukemic cells to apoptosis by chemotherapeutic agents. Relapsed leukemia shows increased resistance to conventional chemotherapy because of the development of multidrug resistance and because residual leukemic cells have perhaps been selected for intrinsic resistance to the factors which normally regulate hematopoiesis. We will therefore determine the expression of the multidrug resistance gene (MDR-1) and the expression of molecules which regulate proliferation and apoptosis (Bcl-2 family proteins) in residual leukemic progenitors and at relapse. The respective contribution of the different mechanisms of resistance (proliferation, apoptosis, MDR) will be tested in appropriate mathematical models with the aim of identifying the key resistance factors and devising methods to sensitize and eradicate residual AML cells.
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0.907 |
1996 — 1997 |
Andreeff, Michael W |
P01Activity Code Description: For the support of a broadly based, multidisciplinary, often long-term research program which has a specific major objective or a basic theme. A program project generally involves the organized efforts of relatively large groups, members of which are conducting research projects designed to elucidate the various aspects or components of this objective. Each research project is usually under the leadership of an established investigator. The grant can provide support for certain basic resources used by these groups in the program, including clinical components, the sharing of which facilitates the total research effort. A program project is directed toward a range of problems having a central research focus, in contrast to the usually narrower thrust of the traditional research project. Each project supported through this mechanism should contribute or be directly related to the common theme of the total research effort. These scientifically meritorious projects should demonstrate an essential element of unity and interdependence, i.e., a system of research activities and projects directed toward a well-defined research program goal. |
Genetic Instability in Cml @ University of Texas Md Anderson Can Ctr
One of the hallmarks of CML is the continual evolution of the clinical behavior of the cells throughout the natural history of the disease from the indolent chronic phase to the accelerated and blastic phase in which the patients die of bleeding or infection. Moreover, patients are heterogeneous in phenotype at diagnosis and at each stage of the disease indicating that the genetic changes present at diagnosis vary among patients. Animal experiments also indicate that the bcr/abl gene interacts with other genes, and that once bcr/abl is introduced into cells, chromosomal changes evolve that mirror the changes seen in vivo in cells transduced with the v-abl oncogene. Reproducible chromosomal changes are also seen during the evolution of CML in man. In order to correlate the nature of these genetic changes with responsiveness to therapy, we have devised a novel method of following and characterizing the genetic changes in CML at the chromosomal level following remission induction, the method is sensitive to one leukemic in 10,000 normal cells, depends on FACS sorting based on immunophenotype followed by Fluorescent in-situ hybridization (FISH), and focuses on the following secondary chromosomal abnormalities in CML: duplication of the Philadelphia chromosome [t(9;22)], monosomy 7, trisomy 8, isochromosome 17 and loss of the y chromosome. FACS is used to isolate progenitor cells which are also labeled with BUdR to assess their proliferation. To further investigate genetic instability, we will apply a new technique to identify genetic changes: comparative genomic hybridization (CGH). This technique produces a map of DNA sequence copy numbers as a function of chromosomal location throughout the entire genome and allows the detection of genomic deletions, duplications and amplifications. It is based on the extraction, labeling and comparative hybridization of DNA from CML cells against normal DNA to metaphases from normal cells, and also allows comparison of DNA's from consecutive samples from the same patient. Samples from patients showing disease progression (with and without cytogenetic changes) will be hybridized in early and late stages of their disease and the sequences representing altered loci (amplified or deleted) will be further characterized. These studies should help in identifying the molecular basis for resistance, disease progression and relapse, and thereby help in establishing more effective forms of therapy.
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0.907 |
1998 — 2002 |
Andreeff, Michael W |
P30Activity Code Description: To support shared resources and facilities for categorical research by a number of investigators from different disciplines who provide a multidisciplinary approach to a joint research effort or from the same discipline who focus on a common research problem. The core grant is integrated with the center's component projects or program projects, though funded independently from them. This support, by providing more accessible resources, is expected to assure a greater productivity than from the separate projects and program projects. |
Core--Automated Cytometry &Cell Sorter Laboratory/Confocal Microscopy @ University of Texas Md Anderson Can Ctr
The Automated Cytometry and Cell Sorter Laboratory/Confocal Microscopy and Image Analysis Facility provides cellular analysis to investigators with peer-reviewed grants at M.D. Anderson Cancer Center. The facility develops and provides cutting-edge techniques in single-cell analysis. Cell phenotyping, proliferation, and apoptosis assays have been established and modified as needed for multi-parameter analysis. Immunophenotypic analysis was combined with assays of intracellular proteins related to apoptosis (bcl-2, BAG-1, Bcl-Xl, p53, Rb, and fas), proliferation and membrane lipid asymmetry. Quantitation of cellular antigens allow determination of antibody binding capacity per cell. Very rare events and progenitor cell subpopulations have been detected and isolated by three-laser excitation/eight-parameter fluorescence-activated cell sorting (FACS) for subsequent analysis by molecular cytogenetics and other molecular techniques. Acquisition of a high-speed cell sorter and a laser-scanning microscope will provide state-of-the-art isolation and analysis. Laser confocal microscopy has been used extensively, and so the acquisition of a second instrument is necessary. By using a charge coupled-device (CCD)- based image analysis system, a method for image capture in perfect registration was developed and has been used extensively for molecular cytogenetics fluorescence in situ hybridization (FISH) and comparative genomic hybridization (CGH). FISH has also been combined with the apoptosis assay to detect apoptosis in normal and leukemic cells. The number, phenotype, and proliferation of minimal residual disease cells with abnormalities amenable to FISH analysis can be determined at levels of as few as one malignant cell in 30,000 normal cells. Methods to detect transgene expression in cells have been established using beta- galactosidase (beta-gal), nerve growth factor receptor (NGF-R), and green fluorescent protein. The Facility has served 26 investigators with peer- reviewed grants who used the laser confocal microscope for 1,797 and the FACS facility for 1,970 hours last year, a 33% increase over the last 2 years. The Core has continuously developed new methodology to suit the evolving needs of its users.
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0.907 |
1998 — 2002 |
Andreeff, Michael W |
P01Activity Code Description: For the support of a broadly based, multidisciplinary, often long-term research program which has a specific major objective or a basic theme. A program project generally involves the organized efforts of relatively large groups, members of which are conducting research projects designed to elucidate the various aspects or components of this objective. Each research project is usually under the leadership of an established investigator. The grant can provide support for certain basic resources used by these groups in the program, including clinical components, the sharing of which facilitates the total research effort. A program project is directed toward a range of problems having a central research focus, in contrast to the usually narrower thrust of the traditional research project. Each project supported through this mechanism should contribute or be directly related to the common theme of the total research effort. These scientifically meritorious projects should demonstrate an essential element of unity and interdependence, i.e., a system of research activities and projects directed toward a well-defined research program goal. |
Core--Cell Analysis and Facs @ University of Texas Md Anderson Can Ctr
The cell analysis and FACS Core facility provides genotypic and phenotypic analysis and separated cell populations to the investigators of this program. The laboratory has provided and developed cutting edge techniques in single cell analysis. For the quantitation of apoptotic cells, the Tdt- b-dUTP assay (TUNEL) was established and modified for multi-parameter analysis in combination with DNA and BUdR measurements (cell cycle ploidy). Also, immunophenotypic analysis of progenitor cells and lineage restricted cell populations, analysis of intracellular proteins related to apoptosis (bcl-2, bax, p53), and of cell surface antigens including fas and MDR1 is provided. Recently, binding of Annexin V to phosphatidyl serine (PS) was recognized as a test for changes in the membrane lipid structure that precedes changes detected by TUNEL in cells initiating apoptosis. Quantitation of cellular antigens allows us to determine the Antibody Binding Capacity (ABC). Retrovirally-transduced cells will be analyzed for expression of transgene (NGF receptor) and FACS-sorted CD34 cells will be separated for subsequent analysis by MACS, and CD34 subpopulations by MACS/FACS. Cell kinetic changes can be studied in vitro and the number of actual cell divisions can be determined using PKH26, with cells undergoing none or up to ten divisions being separated for subsequent molecular analysis. Fluorescence in situ hybridization (FISH) to determine the number of t(9,22) containing interphase cells has been established. The combination of FISH and TUNEL or PS/Annexin V assays allows us to discriminate apoptosis in normal and leukemic cells. A novel assay has been developed to determine bcr-abl transcripts in situ by RT- PCR. This assay will be compared to hypermetaphase FISH and competitive RT-PCR for the detection of low levels of residual leukemic cells. Finally, progenitor cell compartments (CD34 and "SP" cells) will be analyzed for the presence of Ph cells, based on our observation that normal but not CML cells express MDR1 and that fas (CD95) is expressed more in CML than in normal progenitor cells.
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0.907 |
1998 — 2000 |
Andreeff, Michael W |
R03Activity Code Description: To provide research support specifically limited in time and amount for studies in categorical program areas. Small grants provide flexibility for initiating studies which are generally for preliminary short-term projects and are non-renewable. |
Bcl2 and P53 in Cyto Protec of Aml Cells From Apoptosis @ University of Texas Md Anderson Can Ctr
DESCRIPTION The applicant wishes to test the hypotheses that hemopoietic cytokines exert their anti-apoptotic effects by inactivating p53 activity and thus lowering Bax levels in leukemic cells. Apoptosis will be induced in myeloid cell lines M1 and M07e by expressing the temperature sensitive (ts) mutant of p53 and by deprivation of serum in the culture medium in primary AML cells. Various cytokines (GM-CSF, G-CSF, IL6) and flt-3 ligand will either be used alone or in different combinations. Expression of Bcl-2, Bax, Bcl-X, and X will be measured in leukemic cells subjected to apoptotic stress. Changes in expression of these molecules will be correlated with anti-apoptotic effects. Since apoptosis is asynchronous in nature, the activity of the Bcl-2 family members will be separately measured in apoptotic and viable cells that have been sorted by FACS. To prove that a given molecule is important for apoptosis in leukemic cells, antisense oligonucleotides will be used to block their activity. Inactivation of p53 will be measured by testing p53 DNA binding activity with gel shift assay and the ability to activate transcription from Bax promoter driven reporter gene constructs.
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0.907 |
2002 — 2004 |
Andreeff, Michael W |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Anti-Leukemic Activity of the Novel Triterpeniod Cddo @ University of Texas Md Anderson Can Ctr
DESCRIPTION (PROVIDED BY APPLICANT): Treatment results for acute myeloid leukemia's (AML) have not changed for over two decades, except for the improved response rates and survival of patients with acute promyelocytic leukemia treated with all-transretinoic acid. 2-Cyano-3,12-Dioxoolean-1,9-Dien-28-Oic Acid (CDDO) is a novel triterpenoid with unique properties: it induces differentiation, inhibits cell growth and induces apoptosis in leukemia cell lines and in primary samples from patients with AML and blast transformation of CML. CDDO ligates and transcaptivates the nuclear transcription factor peroxisome proliferator-activated receptor gamma (PPARgamma), which forms heterodimers with the retinoid X receptor (RXR). We here propose to extend our initial studies on the efficacy and mechanisms of CDDO activity in AML, with the goal of developing CDDO as a drug for the treatment of hematological malignancies. First, we will further investigate the growth-inhibitory effects of CDDO on primary AML and normal hematopoietic progenitors in suspension and clonogenic assay systems. Second, we will define the effects of CDDO on the transcriptional function of PPARgamma, which we found to be highly expressed in AML. Third, we will elucidate mechanisms of apoptotic cell death and growth arrest downstream from CDDO-induced PPARgamma ligation. Preliminary data demonstrate that CDDO induces loss of mitochondrial membrane potential and activation of caspases. Finally, we will conduct in vivo experiments in the NOD/Scid model of AML. The long-term goal of our proposed mechanistic and efficacy studies is to determine the potential of CDDO as a novel anti-leukemia and anti-tumor agent.
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0.907 |
2008 — 2012 |
Andreeff, Michael |
P50Activity Code Description: To support any part of the full range of research and development from very basic to clinical; may involve ancillary supportive activities such as protracted patient care necessary to the primary research or R&D effort. The spectrum of activities comprises a multidisciplinary attack on a specific disease entity or biomedical problem area. These grants differ from program project grants in that they are usually developed in response to an announcement of the programmatic needs of an Institute or Division and subsequently receive continuous attention from its staff. Centers may also serve as regional or national resources for special research purposes. |
P53 Activation as Novel Therapeutic Stratgey For Acute Myelogenous Leukemia @ University of Tx Md Anderson Can Ctr
The main therapeutic challenge in the treatment of myeloid leukemias is the development of strategies that maximize the induction of leukemia cell apoptosis before resistance to chemotherapy develops. p53 is the master switch that determines whether a stressed cell undergoes apoptosis, thus acting as a tumor suppressor. p53 mutations lead to inactivation of this suppressor function. Mouse Double Minute 2 (HDM2) and its homolog HDMX can also inactivate p53 activity: while HDM2 is an ubiquitin ligase that mediates degradation of p53 by ubiquitin-mediated proteolysis, HDMX inhibits the transcriptional activity of p53. p53 mutations leading to p53 inactivation are rare in newly diagnosed and relapsed AML. There is a reported loss of p53 function through over-expression of HDM2 in approximately 50% of AML cases; expression of HDMX have not been investigated in AML. Restoration of p53 activity by inhibiting HDM2/p53 interaction utilizing non-genotoxic small molecule inhibitors (Nutlin 3a, Ml 63) induces apoptosis in AML cells with unmutated p53. While these HDM2 inhibitors dramatically increase p53 levels that initiate transcription of p53 targets, transcription-independent direct interactions of p53 with Bcl-2 family members also occur. Furthermore, Chemotherapeutic agents such as cytarabine and daunorubicin syngergize with BH3 mimetics, and with MARK inhibitors, which inhibit induction of anti-apoptotic p21 and regulate the subcellular distribution of p53. Thus, we propose to investigate the molecular and clinical consequences of a clinical trial with small molecule inhibitors of HDM2 (Nutlin 3a, MI-63) and to develop a better understanding of the mechanisms regulating p53 activation and the observed synergism with chemotherapy. If successful, these studies will provide rationale for a novel therapeutic strategy in AML based on the non-genotoxic activation of p53 signaling. Specific Aim 1: Identify the molecular determinants of apoptosis induced by non-genotoxic small molecule inhibitors of HDM2 (Nutlin 3a, Ml 63) in leukemia cell lines, primary leukemia cells and stem cells. Specific Aim 2 Determine mechanisms by which HDM2 inhibition synergizes with chemotherapy (Ara- C, Doxorubicin) in AML. Specific Aim 3 Conduct first-in-man Phase I trial of HDM2 inhibitors (Nutlin 3a analog R7112, Ml 63) in AML Lay Language Summary: AML is a largely incurable malignancy of bone marrow stem cells. A gene termed p53, a master regulator of cell survival and death, is frequently dysregulated in cancers and leukemias. While cancers have a high frequency of p53 mutations, leukemias do not. This finding opens a window to activate the function of this gene by releasing p53 from its inhibitor, the HDM2 protein, by a novel class of drugs with great therapeutic promise. This proposal lays the groundwork for an entirely novel concept for the treatment of AML.
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0.93 |
2008 — 2012 |
Andreeff, Michael |
P30Activity Code Description: To support shared resources and facilities for categorical research by a number of investigators from different disciplines who provide a multidisciplinary approach to a joint research effort or from the same discipline who focus on a common research problem. The core grant is integrated with the center's component projects or program projects, though funded independently from them. This support, by providing more accessible resources, is expected to assure a greater productivity than from the separate projects and program projects. |
Flow Cytometry @ University of Tx Md Anderson Can Ctr
The Flow Cytometry and Cellular Imaging Core Facility (FCCICF) provides cellular analysis to investigators with peer-reviewed grants. The FCCICF has two sites, on the north campus and on the recently-opened south campus. It occupies 1900 sq. ft. and is directed by Dr. Michael Andreeff. The FCCICF develops and provides techniques for single-cell analysis. Cell phenotyping, proliferation, signaling and apoptosis assays have been established and modified for multiparametric analysis. Immunophenotypic analysis was combined with assays of intracellular proteins related to apoptosis (Bcl-2, Bcl-XL, BAG-1, p53, Rb, caspase activation, mitochondria! membrane potential and Fas), cell signaling (MARK), proliferation (Ki67, cyclins, BrDU, PCNA, DNA) and cell division history (CFSE). Quantitation of cellular antigens allows determination of the antibodybinding capacity per cell. Rare events and progenitor/stem cell subpopulations can be detected and isolated by three-laser excitation/eight-parameter fluorescence-activated cell sorting (FACS) for subsequent analysis by molecular cytogenetic and other molecular techniques including quantitation of intracellular PKCoc, Bax, Bcl-2, ERK, pERK, XIAP by laser scanning cytometry. FISH has been combined with apoptosis assays to discriminate apoptosis in normal and malignant cells. The number, phenotype and proliferation of minimal residual disease cells can be determined at levels of one malignant in 30,000 normal cells. Methods for detection of transgene expression in cells are in place using p-galactosidase (p-gal), nerve growth factor receptor (NGF-R), and green fluorescent protein (EGFP). Acquisition of 3 new FACS Aria cell sorters and a four-laser flow cytometer (B&D LSRII) has upgraded the facility to provide state-of-the-art isolation and analysis. Laser confocal microscopy has been used extensively and was upgraded by acquisition of an Olympus FV-500 multi-user instrument and a DSU spinning disc confocal system. High-impact studies in cancer prevention, growth factor signaling in breast cancer, apoptosis regulation in leukemias and multistep tumorigenesis all utilized confocal microscopy. The FCCICF now utilizes 17 major instrument systems. The Core supports numerous R01, P01, R21, and SPORE grants. Since the previous review, the number of users has increased 145% (169 investigators with peer-reviewed grants from 19 different programs). 67% of users have peer-reviewed funding. During the previous funding period, 11,668 hours of service was provided. Use has tripled to greater than 7,000 hours estimated for 2007. Future plans are focused on the continued development and application of cutting-edge technologies in cell analysis.
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0.93 |
2009 — 2012 |
Andreeff, Michael |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Plerixafor/G-Csf With Sorafenib For Acute Myelogenous Leukemia With Flt3-Itd Muta @ University of Tx Md Anderson Can Ctr
DESCRIPTION (provided by applicant): The prognosis of patients with relapsed AML harboring Fms-like tyrosine kinase 3 gene (FLT3) mutations is extremely poor. Studies have demonstrated that microenvironment/leukemia interactions play a major role in the chemoresistance of leukemic stem cells residing in bone marrow niches and that the SDF-1a/CXCR4 axis is a key regulator of this interaction. The applicant states they have recently discovered that Sorafenib, an agent approved by the FDA for the treatment of renal cell and hepatocellular carcinoma, is a superb inhibitor of FLT3-ITD (internal tandem duplication) signaling in AML (IC502nM) with high clinical activity in Phase 1 studies as a single agent, and in combination with Idarubicin and Ara-C3. Sorafenib has shown greater clinical activity in early studies than PKC-412 or CEP-701, probably because of lower protein binding. A study by Dr. Small from Johns Hopkins has shown complete inhibition of FLT3-ITD phosphorylation by serum from patients treated with Sorafenib at MD Anderson Cancer Center. This effect was not consistent with other inhibitors. In the applicant's Phase 1 study, Sorafenib alone eradicated leukemic cells from circulation and showed a 55% reduction of bone marrow blasts. High CXCR4 levels have been associated with poor prognosis, and FLT3 mutations have been reported to highly upregulate CXCR4, thus anchoring leukemic cells/stem cells firmly in the bone marrow microenvironment. These findings provide the rationale for the currently proposed studies. The applicant has recently reported that in preclinical leukemia studies, inhibition of CXCR4 with an analogue of the first clinically available and recently FDA approved CXCR4 inhibitor (AMD3100, Plerixafar) resulted in mobilization of leukemic cells into the circulation and sensitization to the pro-apoptotic effects of the FLT3 inhibitor Sorafenib. G-CSF is now known to cleave SDF-1 and has been shown to enhance the effect of CXCR4 blockade. The applicant and others have used G-CSF for priming AML to chemotherapy, and it has been widely used for the treatment of relapsed AML in the FLAG protocol. In recent studies of stem cell mobilization, G-CSF was found to greatly enhance the ability of CXCR4 inhibitor AMD3100 to mobilize hematopoietic stem cells. AMD3100 has been extensively used, in combination with G-CSF, for the mobilization of normal hematopoietic stem cells into the circulation and was recently approved by the FDA for this indication. AML patients in remission who were treated with AMD3100/G-CSF had massive egress of AML cells into the circulation, providing first proof of principle in leukemia patients. In addition, preferential mobilization of AML over normal cells has been found, further supporting the clinical development of this therapeutic concept. Of note, Sorafenib is not toxic to normal hematopoietic cells. Based on these findings, the investigator proposes to test the hypothesis that mobilization of leukemic stem cells by disrupting the SDF-1a/CXCR4 axis by AMD3100/G-CSF will result in improved anti-leukemia activity of Sorafenib in AML patients with mutated FLT3. CXCR4 inhibitor AMD3100, G-CSF and Sorafenib will be administered sequentially to patients with advanced myeloid leukemia.
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2013 |
Andreeff, Michael |
P50Activity Code Description: To support any part of the full range of research and development from very basic to clinical; may involve ancillary supportive activities such as protracted patient care necessary to the primary research or R&D effort. The spectrum of activities comprises a multidisciplinary attack on a specific disease entity or biomedical problem area. These grants differ from program project grants in that they are usually developed in response to an announcement of the programmatic needs of an Institute or Division and subsequently receive continuous attention from its staff. Centers may also serve as regional or national resources for special research purposes. |
P53 Activation as Novel Therapeutic Strategy For Acute Myelogenous Leukemia @ University of Tx Md Anderson Can Ctr
The primary goal of this project is to improve treatment outcomes and cure rates of patients with acute myelogenous leukemia (AML) by developing novel, non-genotoxic therapeutic strategies that maximize the induction of leukemia cell apoptosis. TP53 is the master regulator of apoptosis that is frequently inactivated by overexpression of MDM2. Restoration of p53 activity by inhibition of MDM2-p53 interaction with non-genotoxic small molecule inhibitors (Nutlin-3a, RG7112, MI-63) dramatically increases cellular p53 levels and induces apoptosis. During the past funding period, we have generated pre-clinical and clinical evidence to support this concept. While much p53 in AML is localized in the cytoplasm, only nuclear p53 can function as transcription factor. Exportini (CRM1) is the major nuclear transporter of p53. Preliminary data suggest that CRM1 overexpression is associated with poor prognosis in AML. SINEs (selective inhibitors of nuclear export) are new, potent, irreversible and selective small molecule inhibitors of CRM1 [5, 6]. Our overall hypothesis is that nuclear retention of p53 by CRMI inhibition and non- genotoxic activation of p53 by inhibition of MDM2 will induce/enhance apoptosis in AML. in addition, we hypothesize that p53 is an important determinant of microenvironmental function. In Aim 1 we will test the hypothesis that blockade of p53 nuclear export by CRMI inhibition in AML enhances apoptosis induced by M0M2 inhibition. We reported that AML cells express p53 predominantly in the cytoplasm and hypothesize that CRMI inhibition results in nuclear accumulation and activity of p53, thereby enhancing p53-mediated transcription- dependent apoptosis. SINEs have minimal toxicities in normal human cells, including hematopoietic cells in vitro and in vivo. Our preliminary data show that SINEs induce cell death in AML in a p53-dependent manner. We will investigate if nuclear retention of p53 by CRMI inhibition synergizes with accumulation of p53 by MDM2 inhibition to induce apoptosis in AML. In Aim 2 we will investigate the role of p53 activation by MDM2 and CRMI inhibition in the bone marrow microenvironment. Bone marrow stromal cells protect AML cells from various anti-leukemic agents, but the MDM2 inhibitor Nutlin-3a or SINE KPT-185 kill AML cells even in the presence of
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0.93 |
2019 |
Andreeff, Michael Ishizawa, Jo |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Ph1/2 Study of the Imipridone Onc201 For Treatment of Aml Ind125,203 (12/23/2014) @ University of Tx Md Anderson Can Ctr
Project Summary/Abstract Survival rates (app. 30 %) of acute myeloid leukemia (AML) have not been improved over 4 decades, except in some specialized instances. The long term aim of this study is to increase the cure rates of AML through clinical implementation of targeting a new cellular survival mechanism, i.e. mitochondrial (mt) unfolded protein response (mtUPR). We are proposing to conduct a clinical Phase 1/2 trial of ONC201, a first-in-class imipridone and to confirm and further investigate the underlying novel mechanism of action (MOA). We discovered in extensive preclinical studies that ONC201 induces apoptosis in AML but not in normal cells. Importantly, ONC201 has great efficacy in p53-mutated AML, the most chemotherapy-resistant subset, as well as in p53 wild-type AML. Our preclinical studies further demonstrate that ONC201 eliminates functionally- defined leukemia stem cells in patient-derived xenografts. Early trials initiated at MD Anderson show excellent tolerability of ONC201, micromolar plasma concentrations, and early clinical responses. We previously reported that ONC201 induces apoptosis mediated by the transcription factor ATF4, a hallmark of integrated stress response (ISR). However, ONC201 did not induce all characteristic molecular changes associated with classical ISRs (e.g., ER stress), suggesting an atypical MOA to induce ATF4. As break through progress reported in this re-submission, we have discovered that ONC201 directly binds and activates the mitochondrial protease, ClpP, resulting in selective mitochondrial proteolysis. The resultant reduction of mt protein pools induces so-called mt protein folding stress (mtPFS) and the protective transcriptional response against mtPFS termed mt unfolded protein response (mtUPR). Importantly, ATF4 is known to be induced through mtUPR, connecting our previous findings on ATF4 in a way different from classical ISRs. We here hypothesize that AML progenitor and stem cells are more susceptible to mtPFS than normal cells, and that ONC201 is targeting a novel point of vulnerability in AML pathobiology. The proposed clinical trial in leukemia provides a unique opportunity to thoroughly investigate this hypothesis. We will conduct a Phase 1/2 study of ONC201 in AML (Aim 1), and evaluate the underlying MOA (Aim 2). The Phase 1 trial will determine the safety and preliminary efficacy of ONC201 and Phase 2 the overall response rate. Changes in ATF4, mtUPR effector proteins, mt function and biogenesis in AML cells will be investigated using standard immunoblot and PCR methods as well as novel tools including CyTOF (single cell proteomics). We will also determine if ClpP, ATF4 and mtUPR effector proteins are potential biomarkers of clinical response to ONC201. Changes in clonal architecture will be monitored by flow cytometry and single-cell DNA sequencing. Genome-wide RNAseq will also be performed to further elucidate MOA and potential resistance. We expect these studies, which are at the cutting edge of our evolving knowledge of mitochondrial pathophysiology, to be developed into a highly effective and novel concept for the treatment of AML.
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0.93 |
2019 — 2021 |
Andreeff, Michael |
P30Activity Code Description: To support shared resources and facilities for categorical research by a number of investigators from different disciplines who provide a multidisciplinary approach to a joint research effort or from the same discipline who focus on a common research problem. The core grant is integrated with the center's component projects or program projects, though funded independently from them. This support, by providing more accessible resources, is expected to assure a greater productivity than from the separate projects and program projects. |
07 Flow Cytometry and Cellular Imaging Facility @ University of Tx Md Anderson Can Ctr
PROJECT SUMMARY: FLOW CYTOMETRY AND CELLULAR IMAGING FACILITY (FCCIF) The Flow Cytometry and Cellular Imaging Facility (FCCIF) was established to provide access to state-of-the-art equipment for single-cell analysis and isolation, and to provide expertise in cell sorting, analytical flow cytometry, cellular imaging, and custom monoclonal antibody-fluorochrome conjugations. Core personnel are available to design and execute experiments and analyze the results using a variety of commercially available or Core- designed custom software packages. The Core is comprised of 2 independently managed sites: one on the North Campus (NC) and the other on the South Campus (SC). Dr. Michael Andreeff is the director of the FCCIF; Dr. Jared K. Burks is co-director of the NC site, and Dr. Karen Clise-Dwyer is co-director of the SC laboratory. The FCCIF-NC specializes in fluorescence-activated cell sorting, sorting, mass cytometry (CyTOF -cytometry by time of flight), and cellular imaging. A major investment has been made in mass cytometry; the Core is now part of The University of Texas System Proteomics Network and has 2 CyTOFs and a Hyperion mass cytometer funded by both MD Anderson Cancer Center and The University of Texas System. The FCCIF-SC specializes in highly multi-parametric fluorescence-based analytical flow cytometry and cell sorting, and offers imaging flow cytometry using an Amnis ImageStream system. Over the past 6 years, the institution has provided $695,000 in renovations and $4,512,156 in funds for capital equipment. The FCCIF now uses 27 major instrument systems (NC: 14; SC: 13) and over the past 6 years has provided service to 404 cancer center members in support of all 16 CCSG programs as well as numerous MD Anderson Moon Shot programs and platforms, P01s, R01s, and P50 SPOREs. Cancer center members with peer-reviewed funding account for 92% of use, and 28% ($467,180) of the total Core budget is requested from the CCSG. In the current grant cycle, the FCCIF has provided 95,375 hours of service, and grant Yr42 had a 35% increase in use over that in Yr37 and a 63% increase over the average in the previous grant period. Since the previous grant period, the FCCIF has supported 589 publications, with 421 (71%) appearing in journals with IF >5 and 167 (28%) in journals with IF >10, including Science, Nature, Nat Med, Nat Immunol, Cell, Immunity, Cancer Cell, and Cell Stem Cell. The FCCIF specific aims are: Aim 1) Collaboration: To provide the MD Anderson research community with unparalleled expertise to address important research hypotheses using robust, state-of-the-art flow cytometry and cellular imaging techniques; Aim 2) Innovation: To develop and validate new methods in the fields of cytometry, cell sorting, and cellular imaging; and Aim 3) Education: To educate users in applicable methodology and best practices in data collection and analysis to facilitate research rigor and reproducibility.
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