Dan A. Liebermann - US grants

The long term goal of the research is to better understand the molecular biology of normal cell development, malignancy and its suppression. Towards this goal, cDNA clones of differentiation primary response (MyD) genes, activated in the absence of protein synthesis following induction of myeloid cell terminal differentiation and growth arrest, have been isolated. The overall aim of the proposed studies is to gain a better understanding of the events in the signal transduction from the cell membrane to the nucleus which induce these differentiation primary response genes at the onset of terminal differentiation and the role these genes play in the regulated ordered expression of specific genes, resulting in the conversion of proliferating, nondifferentiated cells into nonproliferating, highly specialized cells. In these studies. In these studies the research exploits the attractive biological features of the myeloid cell system, where in addition to being able to manipulate and analyze the growth and differentiation of normal myeloid precursor cells in vitro, using physiological myelopoietic factors, several leukemic and nonleukemic myeloblast cells lines are available. These include: D+ leukemic precursors, where growth has been uncoupled from differentiation, yet can be induced to differentiate, thereby suppressing the leukemic phenotype, and nondifferentiating (D-) leukemic and nonleukemic factor dependent cell lines. Thus, one can study at the molecular-genetic level early events associated with normal myeloid cell differentiation, differentiation of D+ leukemic cells associated with growth arrest and suppression of the leukemic phenotype, and possible genetic lesions associated with nondifferentiating myeloid leukemic and factor dependent cell lines. cDNA clones of three differentiation primary response genes, MyD1,2, & 3, which appear novel (based on hybridization studies and homology searches of sequence databanks), have been selected for further structural and functional analysis. Specifically: 1) The full length sequence of the MyD cDNA clones will be determined to search for homologies with various functional domains, to be able to deduce the sequence of the encoded protein, and to be able to construct expression vectors. 2) The expression of the MyD genes will be analyzed during normal myelopoiesis, compared to in D+ leukemic myeloblasts following induction of terminal differentiation and suppression of their leukemic phenotype, and further compared to in nondifferentiating (D-) leukemic and factor dependent blasts. 3) The role of the MyD genes in myeloid differentiation, growth inhibition, and suppression of certain leukemic phenotypes will be determined, via: a. The use of MyD antisense oligonucleotides in the culture media to block MyD gene expression in normal and D+ leukemic myeloblasts to determine its effects on normal myeloid differentiation as well as differentiation and leukemia suppression of D+ leukemic phenotypes. b. Introduction of MyD genes, either expressed constitutively or under the control of an inducible promoter into D+ & D- leukemic blasts to determine whether any of the MyD genes may abrogate the need for an exogenous source of differentiation factor in D+ leukemic blast &/or may functionally complement genetic lesions of nondifferentiating (D)- leukemic and factor dependent phenotypes. 4) Ascertain the mode of activation of the MyD genes, which occurs in the absence of protein synthesis, by defining cis and trans acting control elements. First, the 5' regulatory region of a specific MyD gene will be isolated from available genomic libraries. Functional assays with a reporter gene whose expression is dependent on the MyD regulatory region will be done to identify, dissect and map positive and negative cis acting functional domains, whereas a combination of gel mobility shift assays and in-vitro footprinting will be used to define MyD gene trans acting transcription factors and their cognate binding sites.

The long term goal of the research is to better understand the molecular biology of normal blood cell development, leukemogenicity & its suppression. Towards this end clones of myeloid differentiation primary response (MyD) genes, activated in the absence of protein synthesis upon induction of myeloid terminal differentiation, were isolated. Both normal myeloid precursor cells, & M1 myeloid leukemic myeloblasts are used. M1 cells proliferate autonomously and undergo terminal differentiation which culminates in programmed cell death, & loss of leukemogenicity when treated with the physiological inducers Interleukin- 6 (IL-6), leukemia inhibitory factor (LIF), or conditioned media of mouse lungs (containing both factors). Also available are M1myc/M1myb cell lines, where the genetic program of myeloid maturation has been disrupted at distinct developmental stages by deregulated c-myc/c-myb transgenes. Along the lines of the previous proposal it was shown that LIF/IL-6, trigger the same immediate early MyD response, including protein phosphorylation steps essential for MyD gene activation. Also, it was found that two novel MyD genes, MyD116 & MyD118, encode proteins stikingly similar to proteins encoded by two novel genes, gadd34 & gadd45, coordinately activated by growth arrest/DNA-damage (gadd) stimuli. MyD116 & gadd34 were found to be homologues of the same gene, whereas MyD1118 and gadd45 represent two separate closely related genes, both induced in response to growth arrest/DNA-damage stimuli. Evidence has been accumulating to suggest that MyD116, MyD118 & gadd45 play pivotal roles in growth arrest & apoptosis of M1 myeloid precursor cells. This proposal is aimed towards: 1. Analysis of the roles MyD116, MyD118, gadd45 play in growth arrest and apoptosis of myeloid cells. M1 & normal myeloblasts will be genetically manipulated to alter the expression of MyD116, MyD118 & gadd45 in order to study the roles these genes play in the control of myeloid cell growth arrest & programmed cell death, including how cell maturation is linked to growth arrest and apoptosis. Interactions between MyD116, MyD118, gadd45 and other positive & negative regulators, possible mechanisms of action, & the role of other players that participate in the regulation of myeloid cell growth suppression and apoptosis also will be studied. 2. Analysis of the molecular mechanisms utilized by hematopoietic differentiation inducers to activate MyD gene expression. Advantage will be taken of MyD cis-acting elements localized within the promoter region of a prototype MyD gene to clone & characterize pre-existing MyD trans-acting-factor(s), & analyze the molecular mechanisms employed by hematopoietic differentiation inducers to convert it to an active form that plays a role in promoting MyD gene transcription. Information should result which leads to increased understanding of terminal differentiation, including transduction of differentiation signals, control of growth arrest & programmed cell death, & how perturbing normal controls can block cell maturation & contribute to leukemogenesis, ultimately aiding in diagnosis, prognosis & eventual therapy.

The long term goal of the research is to better understand the molecular biology of normal blood cell development, leukemogenicity & its suppression. Towards this end clones of myeloid differentiation primary response (MyD) genes were isolated. Both normal progenitor enriched bone- marrow (BM) cells, whose differentiation capabilities are studied in vitro & in animal model systems in vivo, & hematopoietic cell lines are used. M1 myeloblastic leukemia cells proliferate autonomously & undergo terminal differentiation which culminates in programmed cell death, & loss of leukemogenicity when treated with the physiological inducers IL-6/LIF. Also available are M1myc/M1myb cell lines, where the genetic program of myeloid maturation has been disrupted at distinct developmental stages by deregulated c-myc/c-myb transgenes. The human myeloblastic leukemia HL60 cell line, which also proliferates autonomously, can be induced to differentiate to either macrophages or granulocytes, depending on the inducer, providing a good model to study cell lineage determination. Finally, the IL-3 dependent 32Dc13 myeloid cell line can be induced for granulocytic differentiation upon removal of IL-3 & addition of G-CSF. The research has shown that MyD genes which encode for transcription factors of the fos/jun family are positive regulators of terminal differentiation & apoptosis. Also, it was observed that the MyD transcription factor EGR-1 is essential for, restricts, & determines differentiation along the macrophage lineage. The proposed research plan entails deciphering how the transcription factors fos/jun and EGR-1 function as positive regulators of terminal differentiation, apoptosis & lineage specific development of hematopoietic cells, & what effect altering their function/expression may have in the progression of leukemias. The main thrust of the work involves genetic manipulation of leukemic & normal myeloid precursor cells to express either conditionally functional fos or EGR-1 transgenes (where function is dependent on beta-oestradiol in the culture medium and not on de novo synthesis of mRNA or protein), to clone fos/jun & EGR-1 target genes, & to assess their effect on terminal differentiation, apoptosis, lineage specific development & leukemogenicity in vitro & in vivo. How fos/jun interact with myc/myn & myb, negative regulators of myeloid differentiation which accelerate apoptosis, and the interactive effects of p53 and the Wilms tumor gene product (WT1) on the function of EGR-1, will be investigated at the level of normal & abnormal hematopoietic cell development. Finally, normal BM cells, as well as BM from fos-deficient or p53-deficient mice will be used to further assist in this research. These studies should lead to an increased understanding of the role of fos/jun & EGR-1 nuclear factors in terminal differentiation, apoptosis, and lineage specific differentiation, and how genetic lesions which effect their normal functions may contribute to leukemogenesis, ultimately aiding in diagnosis, prognosis and eventual therapy.

DESCRIPTION: For a number of years, the principal investigator's laboratory has been actively engaged in isolating potentially novel primary response genes induced during the differentiation of established myeloid cell lines (32D and M1). Several novel cDNAs, referred to as the myeloid differentiation primary response (MyD) genes, have been isolated. Two of these novel genes have been determined to be related to the Gadd family of genes involved in DNA excision repair, which are induced by genotoxic stress including alkylating agents and irradiation. MyD116 is the murine homologue of Gadd34, while MyD118 is related but distinct to Gadd45. A third member of the MyD118/Gadd45 family, termed CR6, has recently been cloned by other investigators. MyD116 and the MyD118 family of genes appear to play a role in the regulation of growth arrest and apoptosis. Physical interactions between MyD/Gadd proteins and proteins associated with DNA replication and cell cycle regulation have been demonstrated; MyD118 and Gadd45 also stimulate DNA repair in an in vitro assay. The focus of this revised renewal proposal is to understand at the molecular, genetic, and biochemical level how MyD116, MyD118, Gadd45, and CR6 influence growth arrest and apoptosis. The investigator now hypothesizes that MyD118 and Gadd45 positively regulate growth arrest and apoptosis, CR6 negatively regulates these pathways, and MyD116 may modulate these pathways in either a positive or negative fashion depending upon the biologic context. The specific aims of the proposal are to: 1) further characterize MyD116 and the MyD118 family of proteins by studying protein expression and post-translational modification in vivo as well as ascertaining if protein modification and cellular localization are cell cycle regulated; 2) perform genetic and functional analyses of these proteins during growth arrest and apoptosis. Functional studies will be performed by blocking or de-regulating expression using anti-sense technologies and then determining the phenotype in the 32D and M1 cell lines. It will be determined how these genes interact with other proteins, such as c-myc, c-myb, bcl2, and bax, in regulating growth arrest and apoptosis. The role of MyD/Gadd genes will also be addressed using a cell free apoptotic DNA degradation system; and 3) further study the mechanism of action and physical interaction of these genes with cell cycle genes. Additional interacting partners will be cloned and analyzed and the role of MyD/Gadd genes in DNA repair and DNA replication will be further studied.

DESCRIPTION (provided by applicant):Genotoxic stress is a common aspect of life that mammalian cells have to contend with, Paradoxically DNA-damage inducing agents, such as gamma-irradiation and alkylating agents, are also used in cancer therapy. It became evident that both the molecular basis for the initial increase in the susceptibility of malignant cells to anti-cancer agents, and the development of treatment resistance originate from genetic lesions that alter cell cycle arrest and apoptotic set points. Understanding the molecular-genetic pathways which mediate the response of mammalian cells to genotoxic and other types of environmental stress is, thus of high priority. In response to genotoxic stress mammalian cells have evolved an intricate defense mechanism, including activation of 01/S and G2/M cell cycle checkpoints &/or activation of a cell death program. How stress response pathways interact to signal cells to undergo either cell cycle arrest or programmed cell death is still not understood. Recently, the MyD1 18/CR6/GADD45 family of nuclear proteins [also termed GADD45f3, GADD45y, Gadd45cx] has been implicated in mediating the response of mammalian cells to genotoxic stress, either dependent or independent of p53. Evidence has accumulated that MyD1 18/CR6/GADD45 display a complex array of physical interactions with other proteins such as PCNA, p21, Cdc2, & MEKK4. To what extent the stress response function of each of the MyD1 18/CR6/GADD45 proteins is unique or overlaps with the functions of the other proteins, is unclear. Also, not understood is how the nature of the stress stimulus encountered, the cell type, its physiological state, and its genetic makeup, notably p53 status, modulate MyD1 18/CR6/GADD45 function to determine if the outcome will be cell cycle arrest, DNA repair and survival or apoptotic cell death. Thus, the specific aims are: Aim I: Elucidate the physiological functions of MyD118 and CR6 (and Gadd4S when in combination with MyDI18) in normal development, growth control and the response to prototype stress agents. This will be done by analyzing the phenotype of mouse model systems deficient for either one or more of MyD1 18/CR6/GADD4S genes, and analyzing the phenotype of cells (primarily MEFs) obtained from such mice, untreated or following treatment with stress agents. AIM II: Dissect the role of MyD II 8/CR6/GADD45 interactions with cdc2/cyclinB 1, p21, PCNA and MEKK4 in cellular stress responses, including cell cycle arrest, DNA repair, cell survival & apoptosis. MEFs null for MyD1 l8/CR6/Gadd45 will be infected at high efficiency with retroviral vectors encoding for MyD1 18/CR6/GADD4S, either wt or interaction/function deficient, and the stress response will be analyzed. In vitro cell free systems for apoptosis and DNA repair, using null cell extracts spiked with recombinant wt or interaction/function deficient proteins, also will be used. AIM III: Understand how pS3 & p53 target genes; implicated in cell cycle control (p21, 14-3-3delta) or apoptosis (Bax), modulate MyDI18/CR6/GADD4S stress functions. MEFs, null for p53, p21, or Bax, and the HCT1 16 cells, null for 14-3-3s, will be infected with retroviral vectors encoding for MyD1 18/CR6/GADD45, and the response to stress will be analyzed. Following through on this research plan, should result in an increased understanding of negative growth control in response to genotoxic stress, how perturbing these controls may contribute to oncogenicity, and how treatment resistance in cancer therapy can arise.

DESCRIPTION (provided by applicant): The long-term goal of our research is to gain a better understanding of the molecular controls regulating homeostasis of hematopoietic cells. This research proposal focuses on the MyD118/Gadd45/CR6 gene family, which plays a role in negative growth control. The MyD 118/Gadd45/CR6 family members are rapidly induced by genotoxic agents, as well as by terminal differentiation and apoptotic cytokines, and the proteins encoded by these genes play pivotal roles in negative growth control. Examination of hematopoietic tissues in both MyD118 null and Gadd45 null mice revealed increased cellularity, enhanced survival and an increase in the proportion of immature cells. Deregulated expression of MyD 118/Gadd45/CR6 in myeloid cell lines appeared to accelerate the terminal differentiation program and its associated apoptosis. Our working hypothesis is that MyD 118/Gadd45/CR6 play a role in regulating homeostasis of hematopoietic tissues by modulating both the cell cycle and apoptosis; alterations in expression of MyD118/Gadd45/CR6 will modify cell cycle controls and survival, but also will manifest itself by changing the distribution of different lineages and stages of maturation of hematopoietic cells. The specific aims of this research proposal are: AIM 1: Analysis of loss of MyD118/Gadd45/CR6. Mouse strains that are null for MyD118 and Gadd45, and heterozygous CR6 null mice have been generated. The consequences of blocked expression of MyD118/Gadd45/CR6 within the hematopoietic compartment will be assessed, in vivo and in vitro, in both short term and long term cultures, as well as in response to TGFb, and to stress. The effect of loss of MyD118/Gadd45/CR6 expression on the ability to cooperate with oncogenes c-myc, c-myb, E2F-1 or ras, to alter growth and/or differentiation of primary BM will be determined. AIM 2: Analysis of deregulated expression of MyD118/Gadd45/CR6. Hematopoietic cell lines that express inducible deregulated MyD118/Gadd45/CR6 have been established to dissect the role of MyD118/Gadd45/CR6 in growth and differentiation, as well as in response to stress and TGFb. AIM 3: Analysis of the role for MyD11S/Gadd45/CR6 in apoptotic pathways, using cell free systems. Cell free systems will facilitate deciphering apoptosis, and provide an adjunct avenue of investigation to analyze the functions of MyD118, Gadd45, and CR6, as well as interacting proteins, where we can focus on apoptosis, segregated from cell cycle arrest. These investigations should contribute to a greater understanding of the genetic events involved in the pathogenesis of different leukemias and the response to chemo- and radiation therapy, ultimately aiding in diagnosis, prognosis and therapy.

[unreadable] DESCRIPTION (provided by applicant): Gadd45 genes (a, b, g) are stress sensors that modulate the response of cells to genotoxic/physiological stress, & modulate tumor formation. Gadd45 proteins interact with other stress response proteins, including PCNA, p21, Cdc2/CyclinB1, MEKK4 & p38 kinase. To what extent the functions of Gadd45 proteins overlap, & how the nature of stress stimuli dictate Gadd45 functions to signal cell survival or cell death is unclear. The hypothesis tested is that the nature/magnitude of stress dictates which partners Gadd45 proteins will associate with to signal cell survival or cell death. In response to low stress Gadd45 proteins may interact with p21, cdc2/cyclinB1 & PCNA to activate cell cycle arrest and DNA repair to promote cell survival, whereas in response to high stress, including cellular aging & activated oncogenes, Gadd45 proteins may interact with stress kinases (MEKK4, p38, JNK) to promote apoptosis or senescence. It is surmised that stress sensing functions of Gadd45 proteins play a role in modulating tumor formation. Mice deficient for one or more gadd45 genes & Gadd45 mutant proteins deficient in binding to particular partners were generated to test the hypothesis. Aim 1 will assess the role of Gadd45 & interacting partners in the response of cells to varying doses of genotoxic stress. The effect of Gadd45 deficiencies on cell cycle arrest, survival or apoptosis in gadd45 KO & WT cells in vitro & in vivo, following exposure to low/high levels of genotoxic stress will be tested. Also, the role of Gadd45/partner protein interactions will be explored by testing the ability of transduced wt/mutant gadd45 genes to rescue wt phenotypes. Aim 2 will assess role of Gadd45 & partners in the response of MEFs to physiological/oncogenic stress. Gadd45a-/- & gadd45g-/- MEFs were found to escape replicative & oncogene mediated senescence, whereas gadd45b-/- MEFs lose viability. Gadd45a-/- & gadd45g-/- MEFs were also found to be susceptible to ras transformation, whereas gadd45a/gadd45g double KO MEFs were susceptible for transformation by ras or myc. The role Gadd45 & interacting partners play in senescence, survival & susceptibility to transformation will be explored. Aim 3 will assess function of gadd45 genes as modulators of tumor development. Breast cancer prone MMTV-ras & MMTV-myc mice wt or null for gadd45a were generated. Gadd45a deficiency accelerated MMTV-ras tumor formation, yet retarded MMTV-myc carcinogenesis, indicating that gadd45a functions as tumor promoter/suppressor depending on the oncogene. Experiments are targeted at assessing the role stress response functions of gadd45 genes play in differentially modulating ras/myc driven breast tumorigenesis. Data obtained will be of great importance to better understand the role of stress sensors in tumorigenesis, & how treatment resistance in cancer therapy can arise & be abrogated. [unreadable] [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): Gadd45 proteins are key players in cellular stress responses. These proteins interact with cell cycle & stress response proteins, including PCNA, p21, Cdc2/cyclinB1, MEKK4 & p38 kinase. Taking advantage of the gadd45 null mice several important observations have been made. This includes evidence that gadd45 genes play an important role in regulating the response of hematopoietic cells to genotoxic & physiological stress, including acute stimulation by hematopoietic cytokines, myeloablating agents & inflammatory substances. The unifying hypothesis is that Gadd45 proteins are modulators of interrelated hematopoietic stress signaling pathways in response to genotoxic & physiological stimuli, and have distinct roles in sub-compartments of the hematopoietic cascade; in addition, Gadd45 functions are manifested by binding to partner proteins implicated in stress signaling. Gadd45a and Gadd45b play pro-survival roles, protecting myeloid cells from genotoxic stress induced cell death, including ultraviolet-radiation (UV), VP-16 & daunorubicin (DNR). Aim 1 is targeted at elucidating signaling pathways via which Gadd45 proteins protect myeloid cells from genotoxic stress. Evidence was obtained that Gadd45a & Gadd45b modulate survival & differentiation of myeloid progenitors in response to acute stimulation with differentiating cytokines. Experiments proposed in Aim 2 are targeted at assessing how Gadd45 proteins interact with partners to regulate the program of terminal differentiation following acute stimulation with differentiating cytokines, conditions that mimic what occurs during inflammatory stress. Preliminary evidence shows that Gadd45b and Gadd45a also modulate the function of hematopoietic stem cells (HSC). Aim 3 will determine what role(s) these Gadd45 proteins play in modulating HSC functions. This research plan should result in increased understanding of stress responses of hematopoietic cells, & set the stage to evaluate, in clinically relevant settings, the impact the status of Gadd45 proteins has on the efficacy of chemotherapeutic agents & anti inflammatory drugs. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Gadd45 family of genes (Gadd45a, b, g) encode for cellular proteins rapidly induced by multiple stressors, including genotoxic & oncogenic stress. The unique role of Gadd45 proteins as sensors of oncogenes has been born out by several novel cancer mouse models in this laboratory, indicating that, dependent on the activated oncogene, gadd45 can function as either tumor promoter or suppressor by tethering distinct signaling pathways. Gadd45 has been identified as a mediator of oncogenic ras signaling. Ras mutations occur frequently in hematopoietic malignancies, including in AML, MPD and MDS. Recent evidence indicates that oncogenic N-RAS, K-RAS, and H- RAS exhibit different leukemogenic potentials in mice, suggesting that myeloid leukemogenesis by oncogenic RAS involves unique RAS signaling networks that need to be determined. On the other hand, BCR-ABL (BA) is known as the most common translocation in the myeloproliferative (MPD) disorder chronic myelogenous leukemia (CML) where an activated BA kinase oncoprotein impacts on cell proliferation and survival signaling pathways including Ras, PI3K, JAK-STAT, and PDk2-NFkB. The complex nature of these signaling pathways in the pathogenesis of CML is not fully understood. Recently Gadd45a expression was documented to be altered in a subset of AML patients. Our preliminary data indicate that Gadd45a behaves as an oncogene in context of N-RAS driven leukemia whereas both Gadd45a & b function as tumor suppressors in context of BA-driven leukemia. Also, Gadd45a,b expression was observed to be altered in human CML correlating with disease progression. The role of Gadd45 proteins as oncogenic stress sensors that modulate oncogene driven leukemias has not been studied, and understanding the role of these novel modulators in the molecular pathology of RAS and BCR-ABL is important. To this end, two Specific Aims are delineated: Aim 1 is targeted at assessing how Gaddd45a modulates RAS driven leukemogenicity. Sub-aim 1A will ascertain the effect of loss of Gadd45a on RAS-driven leukemic transformation in vivo~ Sub-aim 1B is targeted at studying the effect of loss of Gadd45a in BM expressing oncogenic RAS in vitro~ finally Sub-aim 1C will analyze human AML/MDS for activated RAS, alterations in Gadd45a and genes/pathways regulated by altered Gadd45a function. Aim 2 is targeted at exploring how Gaddd45a,b tumor suppressor functions impact on BA-driven leukemia and signaling. Sub-aim 2A will ascertain the effect of loss of Gadd45a & b function on BA-driven leukemia and Imatinib treatment~ Sub-aim 2B will explore how loss of Gadd45a and Gadd45b impact on BA oncogenic potential and imatinib treatment in vitro~ Sub-aim 2C will ascertain how gain of function of Gadd45a or b in primary BM impacts on BA oncogenic effect and imatinib treatment~ and, finally, Sub-aim 2D will analyze human CML for alterations in Gadd45a and Gadd45b and genes/pathways differentially regulated by altered function of Gaddd45a & Gadd45b. Knowledge gained from the proposed research should influence understanding of leukemogensis as well as add to the understanding of the role stress response genes play in other cancers, and contribute to the development of new/improved modalities for treatment of cancer.