Elizabeth A. Eklund - US grants

Myeloid hematopoiesis involves the transcriptional regulation of a number of tissue specific genes. The gp91-phox gene is a marker of terminal differentiation in phagocytic leukocytes. This gene codes for the 91-kilodalton subunit of a unique b cytochrome involved in the NADPH- dependent respiratory burst oxidase. Respiratory burst activity is characteristic of mature phagocytes and is a marker for terminal hematopoieses in this lineage. The proximal promoter region of the gp91- phox gene is adequate to target reporter gene expression to a subset of monocyte/macrophages in transgenic mice. In the proposed experiments, the cis-elements of the gp91-phox promoter that target expression to monocyte/macrophages will be identified. This will be done employing site directed mutagenesis to regions of the proximal gp91-phox promoter that have sequence specific in vitro protein binding to ablate the protein binding site. In vitro protein binding assays will be done using nuclear extracts from terminally differentiated myeloid cell lines. Function of the cis-elements will be assayed by detection of reporter gene transcription in transgenic mice and terminally differentiated myeloid cell lines. DNA-binding proteins that interact with the identified cis-elements will be cloned using lambda gt 11 expression library screening or protein purification employing DNA-affinity chromatography. Full length proteins will be expressed in E coli to assay function in vitro.

Myeloid hematopoiesis involves the transcriptional regulation of a number of tissue specific genes. The gp91-phox gene is a marker of terminal differentiation in phagocytic leukocytes. This gene codes for the 91-kilodalton subunit of a unique b cytochrome involved in the NADPH- dependent respiratory burst oxidase. Respiratory burst activity is characteristic of mature phagocytes and is a marker for terminal hematopoieses in this lineage. The proximal promoter region of the gp91- phox gene is adequate to target reporter gene expression to a subset of monocyte/macrophages in transgenic mice. In the proposed experiments, the cis-elements of the gp91-phox promoter that target expression to monocyte/macrophages will be identified. This will be done employing site directed mutagenesis to regions of the proximal gp91-phox promoter that have sequence specific in vitro protein binding to ablate the protein binding site. In vitro protein binding assays will be done using nuclear extracts from terminally differentiated myeloid cell lines. Function of the cis-elements will be assayed by detection of reporter gene transcription in transgenic mice and terminally differentiated myeloid cell lines. DNA-binding proteins that interact with the identified cis-elements will be cloned using lambda gt 11 expression library screening or protein purification employing DNA-affinity chromatography. Full length proteins will be expressed in E coli to assay function in vitro.

DESCRIPTION (provided by applicant): The transcription factor HoxA10 is expressed early in myelopoiesis and is overexpressed in myeloid leukemia. These observations suggest that differentiation stage specific HoxA10 function may be involved in regulation of myelopoiesis. We noted that repressor elements in the promoters of the genes encoding the respiratory burst oxidase components gp91phox and p67phox (the CYBB and NCF2 genes) are similar to the derived DNA-binding consensus for HoxA10. We found that HoxA10 binds to these cis elements in EMSA with nuclear proteins from undifferentiated, but not WNy-differentiated, myeloid cells. Therefore, HoxA10 DNA-binding decreases as gp91phox and p67phox expression increases. Consistent with this, overexpression of HoxA10 represses gp91p0x and p67phox expression via these CYBB and NCF2 repressor elements. In vitro binding assays indicate that HoxA10 recruits transcriptional co-repressor proteins to the CYBB and NCF2 promoters. We found that IFNy-differentiation of myeloid cell lines results in HoxA10 tyrosine phosphorylation, which decreases HoxA10 DNA-binding affinity. Additionally, we found that SHP1 protein tyrosine phosphatase augments HoxA10 repression of the CYBB and NCF2 genes in undifferentiated myeloid cells. Therefore, we hypothesize that HoxA10 represses myeloid genes early in myelopoiesis and that HoxA10 DNA-binding is regulated by phosphorylation during differentiation. We will investigate this hypothesis by the following specific aims: Aim 1: Determine if HoxA10 repression requires recruitment of histone deacetylase activity to the CYBB and NCF2 genes. If so, does HoxA10 interact with co-repressor proteins directly, or via interaction with Pbxla? Aim 2: Determine if tyrosine phosphorylation during myeloid differentiation decreases the affinity of HoxA10 for the CYBB and NCF2 repressor elements. If so, does SHP1-PTP regulate HoxA10 phosphorylation.? Aim 3: Determine the roles of HoxA10 repression activity and tyrosine phosphorylation in leukemogenesis. These investigations will identify the molecular mechanisms of target gene regulation by HoxA10, which may suggest a role for HoxA10 in differentiation block in myeloid leukemia.

DESCRIPTION (provided by applicant): Several lines of evidence suggest that the interferon consensus sequence binding protein (ICSBP or IRF8) functions as a myeloid leukemia tumor-suppressor. First, ICSBP-expression is decreased in bone marrow samples from subjects with myelodysplastic syndrome (MDS) and chronic myeloid leukemia (CML). Second, mice with targeted disruption of the IRF8-gene exhibit a CML-like myeloproliferative disorder which progresses to acute myeloid leukemia (AML) over time. These results suggest that ICSBP-deficiency alone is adequate to induce myeloproliferation, but additional mutations are necessary for progression to AML. However, initially identified target-genes did not suggest a mechanism by which ICSBP-deficiency predisposes to either of these events. For example, we found that ICSBP activates transcription of genes encoding the phagocyte NADPH-oxidase proteins, gp91PHOX and p67phox. Other investigators identified additional ICSBP-target-genes involved in phagocyte function. Therefore, ICSBP-deficiency decreases myeloid-specific gene transcription, which may contribute to differentiation block. However, ICSBP-deficient myeloid progenitor cells exhibit resistance to apoptosis, hypersensitivity to hematopoietic cytokines, and the tendency to accumulate additional genetic lesions. Genuine ICSBP-target-genes mediating these effects had not been identified. During the previous funding period, we pursued identification of such target-genes. Using chromatin immuno-precipitation and CpG island microarray screening, we identified ICSBP- target-genes which encode proteins involved in proliferation (Neurofibromin 1) and apoptosis (Nore1, Fap1 and a soluble guanylate cyclase component). Identification of these target-genes supports the hypothesis that ICSBP-deficiency is sufficient to induce a myeloproliferative disorder. We also identified target-genes involved in hematopoietic stem cell expansion via regulation of the Wnt/2catenin pathway (Gas2, Dapper2 and calpain2 and 12). Additionally, we identified an ICSBP-target-gene involved in a key DNA repair pathway in hematopoietic cells (Fanconi F). This is consistent with the hypothesis that abnormal target-gene expression ICSBP-deficient cells predisposes to acquisition of additional genetic mutations, leading to differentiation block and AML. We will pursue our hypotheses through the following specific aims;Aim 1: Determine if abnormal expression of apoptosis-related target-genes contributes to myeloproliferation in ICSBP-deficient hematopoiesis. Aim 2: Determine if abnormal expression of target-genes which regulate DNA-repair and hematopoietic stem cell expansion predisposes to AML in ICSBP-deficient hematopoiesis. Aim 3: Identify genetic lesions that cooperate with ICSBP-deficiency to lead to disease progression in myeloid malignancy. PUBLIC HEALTH RELEVANCE: Identifying ICSBP-target-genes may suggest common final pathways which is sufficient for myeloproliferation and necessary for susceptibility to myeloid blast crisis. Functional characterization of such a pathway has implications for identifying early markers of disease progression in human myeloid malignancy and rational targets for molecular therapeutic approaches to diseases such as CML and MDS.

DESCRIPTION (provided by applicant): The Interferon Consensus Sequence Binding Protein (Icsbp or Irf8) is a transcription factor that functions as a leukemia-suppressor. We identified an Icsbp-target-gene set that is enriched for genes that control Fas and/or ?catenin activity. This is of interest, because decreased expression of Icsbp, Fas-resistance and increased ?catenin activity are associated with poor prognosis and disease progression in chronic myeloid leukemia (CML). Insensitivity of leukemia stem cells (LSC) to Fas-induced apoptosis is associated with development of drug resistance in CML, but does not correlate with decreased expression of Fas or FasL. Increased ?catenin activity precedes blast crisis (BC) in CML, but does not correlate with Wnt expression or CTNNB1 transcription. We determined that Icsbp represses the gene encoding Fap1; a Fas inhibitory protein. We found Icsbp/Fap1-dependent Fas-resistance in Bcr-abl+ cells. Fap1 also interacts with Apc, and we found Icsbp/Fap1/Gsk3b-dependent ?catenin stabilization in these cells. We identified GAS2 as another Icsbp- target-gene. Gas2 inhibits calpain; a protease with substrates that include ?catenin, Stat5 and Xiap. We found a Gas2/calpain-dependent increase in ?catenin in Bcr-abl+ or Icsbp-/- murine bone marrow cells. Stat5 and Xiap are also increased in Icsbp-/- cells in a Gas2/calpain-dependent manner. Xiap contribute to Fas- resistance, and we found that Stat5 represses the IRF8 promoter in a Gas2/calpain-dependent manner. Although tyrosine kinase inhibitors (TKI) induce remission in the majority of CML patients, an LSC sub- population persists during treatment, preventing cure with TKIs. The hypothesis of these studies is that Fap1, calpain and related pathways are rational therapeutic targets to abolish the persistent CML-LSC population; preventing emergence of overt drug-resistance and/or BC. This hypothesis will be pursued by 3 Aims; AIM 1: Define roles of calpain and Fap1 in development of TKI resistance in CML. The roles of Fap1 and calpain in Fas resistance will be studied in vitro using primary murine bone marrow cells and human CML bone marrow samples. The impact of targeting Fap1 or calpain on development of TKI resistance will be investigated in vivo in a murine CML bone marrow transplant model. AIM 2: Determine if cooperation between calpain and Fap1 facilitates BC in CML. Regulation of ?catenin activity by Fap1 and calpain in will be studied in vitro using murine bone marrow cells and human CML samples. The impact of targeting Fap1 or calpain on BC will be studied in vivo in a murine CML model. AIM 3: Investigate the impact of calpain activation on Icsbp-expression in CML. Regulation of Icsbp expression by Bcr-abl-dependent Stat5 activation will be studied in myeloid cell lines and primary murine bone marrow cells. Targeting calpain to increase Icsbp will be investigated in vivo in murine CML models. The goal of these studies is to identify molecular mechanisms that lead to LSC persistence, and therefore TKI resistance and/or BC in CML. Targeting these mechanisms might cure CML by abolishing the LSC.

DESCRIPTION (provided by applicant): The Fanconi pathway repairs collapsed and stalled DNA-replication forks through homologous recombination repair (HRR) and translesional synthesis (TLS); thereby maintaining genomic integrity during S phase of the cell cycle. Congenital absence of any Fanconi protein results in Fanconi Anemia (FA); a disorder that is characterized during early stages by bone marrow failure (BMF). BMF is hypothesized to occur as unrepaired DNA damage triggers apoptosis in FA hematopoietic stem cells (HSC) and progenitor cells. Patients who survive the BMF stage of FA have a tendency to develop bone marrow dysplasia with clonal progression. This is hypothesized to be caused by accumulating mutations that induce resistance to cell cycle checkpoints and/or the apoptotic response to DNA-damage. FA patients exhibit steady state granulocytopenia and susceptibility to infection. Our studies suggest that impaired emergency granulopoiesis (EG) during infectious challenge also contributes to immuno-deficiency in FA. Under normal circumstances, EG-related cytokines stimulate immediate granulocyte release from the bone marrow, followed by expansion of HSC and granulocyte/monocyte progenitors (GMP). This proliferative phase involves S phase-shortening. We found that expression of Fanconi C and F increased in primary murine GMP treated with IL1? and other cytokines that mediate the EG response. And, we determined that FancC deficient mice are unable to mount an in vivo EG-response. Instead, we found that repeated episodes of EG-stimulation result in pancytopenia, BMF, and death in the majority of FancC-/- mice. We also found that the adverse effects of EG stimulation in FancC-/- mice are blocked by an IL1-R antagonist. We hypothesize that repeated, failed episodes of emergency granulopoiesis accelerate bone marrow failure in FA. And, that unsuccessful EG episodes provide opportunity for mutations that result in clonal progression. This hypothesis will be pursued through three aims: AIM 1: Define mechanisms of Fanconi pathway activation and DNA-repair during EG. Wt, FancC- deficient, or FancA-deficient murine bone marrow cells will be treated with EG-related cytokines and analyzed for Fanconi pathway activation and DNA-repair. In vivo studies will be performed with various EG stimuli. AIM 2: Identify molecular mechanisms involved in EG-related bone marrow failure in FA. We will also use the models described above to determine if EG-related cytokines induce apoptosis in Fanc-deficient bone marrow in association with cell cycle checkpoint activation. AIM 3: Determine if multiple failed episodes of EG facilitate clonal progression in FA: We will use these models to determine if blocking specific cytokines prevents clonal progression during repeated EG episodes. The goal of these studies is to define the role of ineffective EG episodes in BMF and clonal progression in FA. These studies may suggest therapeutic approaches that could be rapidly translated to the clinic.

DESCRIPTION (provided by applicant): This application is a renewal request for funding of the Training Program in Oncogenesis and Developmental Biology (T32 CA080621) at the Robert H. Lurie Comprehensive Cancer Center of Northwestern University. Over the past 10 years, this Training Program has been preparing pre-doctoral students and post-doctoral trainees to work at the interface between cancer and developmental biology through laboratory research, formal course work and participation in seminars, journal clubs and group meetings. The hypothesis of this Training Program is that an understanding of key processes in developmental biology informs studies of oncogenesis. Trainees will use their understanding of normal development to identify lesions that are functionally significant for oncogenic transformation. The intent is to train a cohort of investigators who ultimately use this informatio for therapeutically targeting the cancer stem cell. In the first ten years of funding, the Training Program in Oncogenesis and Developmental Biology effectively trained 15 pre-doctoral students and 23 post-doctoral fellows; 14 remain in training. Of the trainees who have completed the Training Program, 9 have obtained faculty appointments, 9 have obtained relevant positions in industry, and over 25% have successfully competed for national funding. In this renewal application, we propose expansion of the Training Program to include a required career development curriculum and multi-disciplinary training in cutting edge technologies that are relevant to cancer research. We also further define the required and recommended courses in cancer and developmental biology that are specific to the trainee level. To facilitate identification and support of the highest quality of trainees, we have expanded the Northwestern University based selection and evaluation committee. We have also added an External Advisory Committee that includes individuals with involvement in high caliber T32 programs to further assist us in achieving this goal. In the current renewal application, we request funding for two pre-doctoral students and four post- doctoral fellows per year for a period of five years. This wil enable us to continue focused training in Oncogenesis and Developmental Biology.

? DESCRIPTION (provided by applicant): Triad1 is an E3 ubiquitin ligase that impairs proliferation of bone marrow progenitor cells and increases in expression during granulopoiesis. We found that Triad1 enhances ubiquitin (Ub) mediated degradation of Fgf- R1, Flt3 and av integrin in myeloid cells. Fgf-R1 and Flt3 activate phospho-inositol-3-kinase; resulting in stabilization of ßcatenin and expression of ßcatenin-target-genes involved in proliferation/survival. Syk is activated by avß3 integrin, resulting in Pak1-dependent proliferatin and PLC?-dependent survival. Triad1 also inhibits Ub of p53 by Mdm2. We hypothesize that increasing Triad1 expression during granulopoiesis favors degradation of Fgf-R1, Flt3 and avß3 integrin, but stabilizes p53; decreasing proliferation and enhancing sensitivity to apoptosis. This identifies a possible leukemia suppressor function for Triad1, since impaired activity would sustain Fgf-R1, Flt3 and avß3 signaling and destabilize p53. Consistent with this, Triad1 is specifically decreased in subsets of acute myeloid leukemia (AML) with MLL-translocations (i.e. 11q23-AML) or activating FLT3 mutation. Our studies identified a mechanism for this. 11q23-AML is characterized by increased expression of a set of HOX genes, including HoxA9 and A10. We found that HoxA9 and A10 regulate transcription of ARIH2 (encoding Triad1) in a manner that requires cytokine-induced tyrosine phosphorylation of the Hox proteins. We found that constitutive activation of Shp2-PTP blocks ARIH2 transcription by preventing Hox phosphorylation. Interestingly, FLT3 mutations are frequent in Hox-over- expressing AML and activate Shp2. A myeloproliferative neoplasm (MPN) develops in mice transplanted with bone marrow expressing MLL1 fusion proteins or overexpressing HoxA9 or A10. This MPN evolves to AML over time, suggesting that Hox-overexpression is inadequate for AML in the absence of cooperating mutations. We find constitutive Shp2-activation performs this function. We hypothesize Triad1 is a leukemia suppressor that decreases proliferation/survival of cytokine-stimulated progenitor cells, and that impaired Triad1-activity facilitates disease progression/drug resistance in Hox-overexpressing AML. We will pursue this via 3 aims: Aim 1: Identify Triad1-regulated events that have functional implications for leukemia. The influence of Triad1 on Ub/degradation of Fgf-R1, avß3, Flt3 and p53 will be investigated in vitro and in vivo. Aim 2: Determine if Triad1 is a leukemia suppressor in AML with Hox-overexpression and Shp2 activation. The contributions of Triad1-expression and Shp2-activation to drug resistance/disease progression will be explored in studies with murine AML models and human AML bone marrow samples. Aim 3: Define targetable mechanisms of leukemia suppression by Triad1. Contributions of Fgf-R1, Flt3, av integrin and p53 to leukemogenesis and drug resistance will be explored in vivo in murine models. Hox-overexpressing AML has poor prognosis and is treatment refractory. Clarifying cooperating lesions and down-stream events may suggest therapeutic targets for this subset of individuals.

ABSTRACT ? HEMATOLOGIC MALIGNANCIES The Hematologic Malignancies (HM) Program of the Lurie Cancer Center (LCC) combines the talents and expertise of outstanding basic scientists, nationally recognized clinical researchers, and translational investigators. The goal of the program is to leverage discoveries from member laboratories into clinical interventions for the treatment of malignant hematologic diseases. This goal is pursued through two program aims: a) Identify key pathways that regulate hematopoiesis and lymphopoiesis and determine the functional significance of their alteration in hematologic malignancies, and b) Define molecular therapeutic targets and evaluate the efficacy of novel agents in clinical trials for hematologic malignancies. Disease specific areas of focus for program members include acute and chronic myeloid leukemia, myeloproliferative neoplasms (MPNs), acute and chronic lymphoid leukemia, lymphomas, and multiple myeloma. HM members have made significant contributions to our understanding of molecular and cellular drivers of hematologic malignancies; specifically, in the areas of epigenetic modification, cellular signaling and gene expression. Translation of these results to clinical trials is facilitated by an extensive network of collaborative interactions between laboratory-based faculty and clinical investigators in the program. During the current funding period, such intra-programmatic interactions resulted in a number of therapeutic clinical trials that were developed from work in investigator laboratories. This multidisciplinary inter-departmental program has 32 members from 10 departments in 3 schools. During the current funding period, program members published 520 papers that were relevant to malignant hematologic diseases. 131 (25%) of these publications represented intra-programmatic collaborations, 91 (18%) inter- programmatic collaborations, and 68% involved inter-institutional collaborations. 143 (28%) were high impact (impact factor >9). Peer reviewed funding for program members totaled $5,190,595 (direct) with $2,117,851 (direct) from the NCI, and $3,072,744 (direct) from other NIH institutes and other sources. Program Leader Elizabeth Eklund, MD and co-leader Leo Gordon, MD have complementary expertise in the areas of laboratory investigation, translational research and clinical trials for hematologic malignancies. They work together to foster collaborative interactions between program members through regularly occurring clinical and research oriented conferences, retreats and other program activities.!

The Fanconi DNA repair pathway is required for rescue of stalled or collapsed replication forks. Fanconi Anemia (FA) is caused by inherited mutation of Fanconi genes. FA patients develop bone marrow failure (BMF) in childhood, with survivors frequently developing clonal progression. We identified a role for emergency (stress) granulopoiesis (EG) in BMF and clonal progression in FA. EG is an episodic process for granulocyte production in response to infectious challenge. During EG, S phase is shortened and FancC and F expression increase. Unlike wild type mice, Fancc-/- mice did not develop granulocytosis upon stimulation of EG. Repeated EG challenge in Fancc-/- mice induced either BMF, with apoptosis of HSC and progenitors, or clonal progression. Treatment of Fancc-/- mice with an IL1-R antagonist protected them from these adverse consequences. IL1? is an essential cytokine for EG; inducing myeloid lineage commitment, and G-CSF expression. During the S phase, Atr activates p53 and apoptosis of cells with unrepaired replication fork damage. In Fancc-/- mice, Tp53-haplo- insufficiency rescued granulocytosis during EG; delaying BMF but accelerating clonal progression. In Fancc-/- mice, increasing activity of Atr/p53 occurred with each unsuccessful EG episode; associated with BMF. In contrast, Atr/p53 activity decreased with consecutive, successful EG cycles in Fancc-/-Tp53+/- mice. We hypothesize unsuccessful PMN production in FA during EG prevents activation of unknown negative regulatory pathways; sustaining cell cycle checkpoint activity and HSC/GMP expansion signals. This induces BMF and accumulation of mutations that lead to clonal progression. We will pursue this through three aims: Aim 1: Define molecular triggers for termination of emergency granulopoiesis and the role of this process in BMF in FA. We will investigate contribution of PMN density to apoptosis and BMF during unsuccessful EG in Fancc-/- mice. The impact of PMN bone marrow density on known EG-related pathways will be determined in Wt vs Fancc-/- mice, and novel pathways identified in non-biased studies. Aim 2: Identify events associated with emergency granulopoiesis-induced clonal progression in FA. We will define events involved in clonal progression in Fancc-/- mice by studying leukemia suppressor pathways that mediate EG termination and by non-biased approaches. Results will be compared to gene expression profiles in CD34+ bone marrow cells from human Fanconi Anemia to identify potential translational targets. Aim 3: Investigate potential translational targets to delay BMF or clonal progression in FA. We will determine the impact of novel pathways that are activated during EG on BMF and/or clonal progression in murine genetic models. Relevant intermediates with small molecule inhibitors will be tested in pre-clinical studies. The goal of these studies is to define molecular mechanisms for BMF and/or clonal progression during recurrent, unsuccessful EG attempts in FA. This may suggest therapeutic approaches to decrease morbidity due to anemia and infection, or bridge patients to definitive treatments such as stem cell/bone marrow transplant.