Angela Stoddart - Academia.edu (original) (raw)

Papers by Angela Stoddart

International Immunology, Oct 1, 1997

CD22 is a B cell-restricted glycoprotein involved in cell adhesion and signaling. Since CD22 is l... more CD22 is a B cell-restricted glycoprotein involved in cell adhesion and signaling. Since CD22 is likely to play an important role in interactions between B cells and other cells, and in regulating signaling thresholds, we characterized the expression of murine CD22 during different stages of B cell development. In contrast to previous reports, we show that CD22 is expressed on B cell progenitors prior to expression of IgM. IL-7-responsive B cell precursors from the fetal liver and early B lineage cells (B220 ⍣ IgM-) from the bone marrow both express a low density of surface CD22. The majority of the earliest B cell progenitors (B220 ⍣ IgM-CD43 ⍣) in the bone marrow, however, do not express CD22. As B cells mature, the density of CD22 molecules on the cell surface increases. B220 bright IgM ⍣ bone marrow cells express high levels of CD22, as do splenic B cells. The correlation of CD22 levels with B cell maturation is replicated in an in vitro culture system, which distinguishes stages of B cell development based on function. Following activation of mature resting splenic B cells with anti-µ mAb or lipopolysaccharide (LPS), levels of CD22 decrease. Finally, we show that the addition of anti-CD22 mAb augments the proliferative response of both anti-µ-and LPS-stimulated B cells, suggesting a role for CD22 in diverse signaling pathways.

Oncogene, Jun 27, 2011

The PICALM (CALM) gene, whose product is involved in clathrin-mediated endocytosis, has been iden... more The PICALM (CALM) gene, whose product is involved in clathrin-mediated endocytosis, has been identified in two recurring chromosomal translocations, involving either MLL or MLLT10 (AF10). We developed a mouse model of CALM-AF10 + leukemia to examine the hypothesis that disruption of endocytosis contributes to leukemogenesis. Exclusion of the C-terminal portion of CALM from the fusion protein, which is required for optimal binding to clathrin, resulted in the development of a myeloproliferative disease, while inclusion of this domain led to the development of acute myeloid leukemia and changes in gene expression of several cancer-related genes, notably Pim1 and Crebbp. Nonetheless, the development of leukemia could not be attributed directly to interference with endocytosis or consequential changes in proliferation and signaling. In leukemia cells, full-length CALM-AF10 localized to the nucleus with no consistent effect on growth factor endocyctosis, and suppressed H3K79 methylation regardless of the presence of clathrin. Using FRET analysis, we show that CALM-AF10 has a propensity to homooligomerize, raising the possibility that the function of endocytic proteins involved in chimeric fusions may be to provide dimerization properties, a recognized mechanism for unleashing oncogenic properties of chimeric transcription factors, rather than disrupting the internalization of growth factor receptors.

Blood, Jan 9, 2014

• Haploinsufficiency of Egr1, Apc, and Tp53 in mice cooperate to model the pathogenesis of the ea... more • Haploinsufficiency of Egr1, Apc, and Tp53 in mice cooperate to model the pathogenesis of the early stages of t-MN with a del(5q). • Exposure of an Apc haploinsufficient BM microenvironment to radiation and/or an alkylating agent accelerates disease development. Therapy-related myeloid neoplasms (t-MN) are a late complication of the successful use of cytotoxic therapy for patients with cancer. A heterozygous deletions of the long arm of chromosome 5 [del(5q)], observed in 40% of patients, is associated with prior exposure to alkylating agents, and a high frequency of TP53 loss or mutation. In previous studies, we demonstrated that haploinsufficiency of 2 del(5q) genes, Egr1, and Apc, individually play a role in the pathogenesis of hematologic disease in mice. We now show that loss of one copy of Egr1 or Tp53 in an Apc haploinsufficient background (Apc del/1) accelerated the development of a macrocytic anemia with monocytosis, early features of t-MN. The development of anemia was significantly accelerated by treatment of mice with the alkylating agent, N-ethyl-N-nitrosourea (ENU), regardless of the levels of expression of Egr1 and Tp53. Transplantation of either wild type; Egr1 1/2 ; Tp53 1/2 ; Apc del/1 ; or Egr1 1/2 , Apc del/1 bone marrow cells into lethally irradiated Apc del/1 recipients resulted in rapid development of anemia that was further accelerated by administration of ENU to recipients, demonstrating that the Apc del/1-induced anemia was cell extrinsic and potentiated by ENU mutagenesis. These data emphasize the synergistic role of cell intrinsic and cell extrinsic (microenvironment) factors in the pathogenesis of t-MN, and raise awareness of the deleterious effects of cytotoxic therapy on the stromal microenvironment.

Experimental Hematology, Nov 1, 2022

Mediterranean Journal of Hematology and Infectious Diseases, May 16, 2011

Haematologica, Feb 14, 2014

Chemico-Biological Interactions, Mar 1, 2010

Therapy-related myelodysplastic syndrome and acute myeloid leukemia (t-MDS/t-AML) are late compli... more Therapy-related myelodysplastic syndrome and acute myeloid leukemia (t-MDS/t-AML) are late complications of cytotoxic therapy used in the treatment of malignant diseases. The most common subtype of t-AML (~75% of cases) develops after exposure to alkylating agents, and is characterized by loss or deletion of chromosome 5 and/or 7 [−5/del(5q), −7/del(7q)], and a poor outcome (median survival 8 months). In the University of Chicago's series of 386 patients with t-MDS/t-AML, 79 (20%) patients had abnormalities of chromosome 5, 95 (25%) patients had abnormalities of chromosome 7, and 85 (22%) patients had abnormalities of both chromosomes 5 and 7. t-MDS/t-AML with a −5/del(5q) is associated with a complex karyotype, characterized by trisomy 8, as well as loss of 12p, 13q, 16q22, 17p (TP53 locus), chromosome 18, and 20q. In addition, this subtype of t-AML is characterized by a unique expression profile (higher expression of genes involved in cell cycle control (CCNA2, CCNE2, CDC2), checkpoints (BUB1), or growth (MYC), loss of expression of IRF8, and overexpression of FHL2. Haploinsufficiency of the RPS14, EGR1, APC, NPM1, and CTNNA1 genes on 5q has been implicated in the pathogenesis of MDS/AML. In previous studies, we determined that Egr1 acts by haploinsufficiency and cooperates with mutations induced by alkylating agents to induce myeloid leukemias in the mouse. To identify mutations that cooperate with Egr1 haploinsufficiency, we used retroviral insertional mutagenesis. To date, we have identified two common integration sites involving genes encoding transcription factors that play a critical role in hematopoiesis (Evi1 and Gfi1b loci). Of note is that the EVI1 transcription factor gene is deregulated in human AMLs, particularly those with −7, and abnormalities of 3q. Identifying the genetic pathways leading to t-AML will provide new insights into the underlying biology of this disease, and may facilitate the identification of new therapeutic targets.

Blood, Nov 16, 2007

The clathrin assembly lymphoid myeloid leukemia gene, CALM, was first described as a translocatio... more The clathrin assembly lymphoid myeloid leukemia gene, CALM, was first described as a translocation partner for AF10 in morphologically distinct subsets of acute leukemia with a recurring 10;11 translocation. As the name implies, CALM is involved in the regulation of clathrin-mediated endocytosis. We hypothesize that expression of the CALM-AF10 protein disrupts endocytosis of growth receptors, leading to constitutive activation and, thereby, contributes to the development of leukemia. The CALM-AF10 fusion transcripts are characterized by two breakpoints in CALM at positions 1926 and 2091. Using the 293T human renal epithelial cell line as a model, we demonstrated that CALM1926-AF10 localized diffusely to the cytoplasm, whereas CALM2091-AF10 had a more prominent punctate appearance. This difference in distribution was likely attributable to protein domains in CALM rather than AF10, which is a transcription factor that localizes to the nucleus. The difference in localization extended to their effects on endocytosis. Whereas ∼4–8% of cells expressing truncated CALM2091, CALM1926, or AF10 displayed impaired transferrin uptake, this value was ∼60% in cells expressing CALM2091-AF10 and only ∼13% in cells expressing CALM1926-AF10. Next, we used a retroviral transduction-transplantation murine model to test the impact of CALM-AF10 expression on the development of leukemia. Mice transplanted with either CALM1926-AF10 or CALM2091-AF10 transduced fetal liver cells developed disease within a short period of time (∼8 to 21 weeks post-transplantation). Interestingly, expression of CALM1926-AF10 led to the development of a myeloproliferative disease (MPD)-like leukemia, with a predominance of mature neutrophils, whereas expression of CALM2091-AF10 led to the development of an acute myeloid leukemia (AML) with >30% immature myeloid blasts, with one exception. One mouse transplanted with progenitor cells expressing CALM2091-AF10 developed a MPD accompanied by a granulocytic sarcoma; however, examination of RNA from the spleen of this diseased mouse revealed that it had undergone a splicing event consistent with the size expected for the 1926 breakpoint. Both diseases were transplantable to secondary recipients confirming the leukemic nature of the CALM-AF10 expressing cells. Previous studies show that patients with the t(10;11) (p13;q14-21) develop either AML or T cell-acute lymphoblastic leukemia (T-ALL). Most of the patients with T-ALL express the CALM1926-AF10 fusion. Although there are differences in presentation between humans and mice, the hematological disease may be a reflection of the conditions of the mouse model. To identify possible secondary cooperating mutations, spectral karyotyping analysis of the mouse AMLs and MPDs was performed. However, to date we have not identified any significant abnormalities. We hypothesize that defective endocytosis synergizes with transcriptional deregulation, leading to an aggressive form of leukemia.

Blood cancer discovery, Apr 20, 2020

Therapy-related myeloid neoplasms (t-MN) comprise therapy-related acute myeloid leukemia (t-AML) ... more Therapy-related myeloid neoplasms (t-MN) comprise therapy-related acute myeloid leukemia (t-AML) and myelodysplastic syndrome (t-MDS), and are a late complication of cytotoxic therapy, chemotherapy and/or radiotherapy, used in the treatment of both malignant and nonmalignant diseases (1, 2). The genetic profile of t-MN is markedly skewed toward high-risk cytogenetic and molecular abnormalities, and complex karyotypes with deletions of the long arm of chromosome 5, del(5q), and TP53 mutation/loss are profoundly over-represented in t-MNs as compared with de novo counterparts (3, 4). A proximal minimally deleted region (MDR) in 5q31.2 (containing EGR1) was previously identified in t-MN, de novo AML and high-risk MDS, and a distal MDR in 5q33.1(containing mir145, RPS14, and CSNK1A1) was identified in MDS with an isolated del(5q), previously referred to as the 5q-syndrome (1, 5). However, the deletion of 5q in all but rare patients encompasses both MDRs, spanning 5q14-5q33, resulting in loss of one allele for hundreds of genes. Although challenging to identify involved genes, we previously chose to examine two well-characterized del(5q)

Blood, Jun 1, 2017

• Loss of 1 copy of Ctnnb1 (encoding b-catenin) in an Apc-haploinsufficient microenvironment prev... more • Loss of 1 copy of Ctnnb1 (encoding b-catenin) in an Apc-haploinsufficient microenvironment prevents the development of MDS. • Modulation of WNT signaling in the niche using pyrvinium inhibits the development of MDS in Apc-haploinsufficient mice. There is accumulating evidence that functional alteration(s) of the bone marrow (BM) microenvironment contribute to the development of some myeloid disorders, such as myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). In addition to a cellintrinsic role of WNT activation in leukemia stem cells, WNT activation in the BM niche is also thought to contribute to the pathogenesis of MDS and AML. We previously showed that the Apc-haploinsufficient mice (Apc del/1) model MDS induced by an aberrant BM microenvironment. We sought to determine whether Apc, a multifunctional protein and key negative regulator of the canonical b-catenin (Ctnnb1)/WNT-signaling pathway, mediates this disease through modulating WNT signaling, and whether inhibition of WNT signaling prevents the development of MDS in Apc del/1 mice. Here, we demonstrate that loss of 1 copy of Ctnnb1 is sufficient to prevent the development of MDS in Apc del/1 mice and that altered canonical WNT signaling in the microenvironment is responsible for the disease. Furthermore, the US Food and Drug Administration (FDA)-approved drug pyrvinium delays and/or inhibits disease in Apc del/1 mice, even when it is administered after the presentation of anemia. Other groups have observed increased nuclear CTNNB1 in stromal cells from a high frequency of MDS/AML patients, a finding that together with our results highlights a potential new strategy for treating some myeloid disorders.

Blood, Feb 13, 2014

• Egr1 haploinsufficiency in cooperation with reduced Tp53 activity accelerates the development o... more • Egr1 haploinsufficiency in cooperation with reduced Tp53 activity accelerates the development of hematologic disease in mice. • Loss of 1 copy of Egr1 and Apc in hematopoietic stem cells, in cooperation with Tp53 loss, results in myeloid neoplasms. An interstitial deletion of chromosome 5, del(5q), is the most common structural abnormality in primary myelodysplastic syndromes (MDS) and therapy-related myeloid neoplasms (t-MNs) after cytotoxic therapy. Loss of TP53 activity, through mutation or deletion, is highly associated with t-MNs with a del(5q). We previously demonstrated that haploinsufficiency of Egr1 and Apc, 2 genes lost in the 5q deletion, are key players in the progression of MDS with a del(5q). Using genetically engineered mice, we now show that reduction or loss of Tp53 expression, in combination with Egr1 haploinsufficiency, increased the rate of development of hematologic neoplasms and influenced the disease spectrum, but did not lead to overt myeloid leukemia, suggesting that altered function of additional gene(s) on 5q are likely required for myeloid leukemia development. Next, we demonstrated that cell intrinsic loss of Tp53 in hematopoietic stem and progenitor cells haploinsufficient for both Egr1 and Apc led to the development of acute myeloid leukemia (AML) in 17% of mice. The long latency (234-299 days) and clonal chromosomal abnormalities in the AMLs suggest that additional genetic changes may be required for full transformation. Thus, loss of Tp53 activity in cooperation with Egr1 and Apc haploinsufficiency creates an environment that is permissive for malignant transformation and the development of AML.

Molecular Biology of the Cell, May 1, 2005

B cell antigen receptor (BCR) association with lipid rafts, the actin cytoskeleton, and clathrin-... more B cell antigen receptor (BCR) association with lipid rafts, the actin cytoskeleton, and clathrin-coated pits influences B cell signaling and antigen presentation. Although all three cellular structures have been separately implicated in BCR internalization, the relationship between them has not been clearly defined. In this study, internalization pathways were characterized by specifically blocking each potential mechanism of internalization. BCR uptake was reduced by ϳ70% in B cells conditionally deficient in clathrin heavy chain expression. Actin or raft antagonists were both able to block the residual, clathrin-independent BCR internalization. These agents also affected clathrin-dependent internalization, indicating that clathrin-coated pits, in concert with mechanisms dependent on rafts and actin, mediate the majority of BCR internalization. Clustering G M1 gangliosides enhanced clathrin-independent BCR internalization, and this required actin. Thus, although rafts or actin independently did not mediate BCR internalization, they apparently cooperate to promote some internalization even in the absence of clathrin. Simultaneous inhibition of all BCR uptake pathways resulted in sustained tyrosine phosphorylation and activation of the extracellular signal-regulated kinase (ERK), strongly suggesting that downstream BCR signaling can occur without receptor translocation to endosomes and that internalization leads to signal attenuation.

Blood, Dec 3, 2015

A del(5q) is frequently noted in MDS, AML, and therapy-related myeloid neoplasms (t-MN) following... more A del(5q) is frequently noted in MDS, AML, and therapy-related myeloid neoplasms (t-MN) following alkylating agent therapy. Mutation/loss of the TP53 is gene found in 80% of t-MNs with a del(5q). Recent studies suggest that TP53 mutations are found at a low frequency in hematopoietic stem/progenitor cells (HSPCs) in adults (Xie et al., Nat Med 20:1472, 2014; Genovese et al., NEJM 371:2477, 2014; Jaiswal et al., NEJM 371:2488, 2014), and chemotherapy confers a selective growth advantage to these rare clones (Wong et al., Nature 518:552, 2015). We previously established a mouse model for t-MN with a del(5q) and showed that haploinsufficiency of two del(5q) genes, Egr1 and Apc, cooperate with loss of function of the Trp53 (p53) gene to induce myeloid neoplasms in mice. Specifically, transplantation of Egr1+/-, Apcdel/+ bone marrow (BM) cells transduced with p53 shRNAs into wild type (WT) recipients resulted in the development of a transplantable AML, characterized by a complex karyotype and genetic instability, in 17% of mice. There is growing evidence that microenvironment perturbations play a major role in the malignant process; however, the effect of cytotoxic therapy on HSPCs as well as the BM niche is not well understood. Using our mouse model of t-MN with a del(5q), we explored the effects of ENU, an alkylating agent, on both HSPCs and the BM microenvironment by exposing both donor and recipient mice to ENU (Panel 1 in the figure). In mice transplanted with Egr1+/-, Apcdel/+, p53 shRNA HSPCs, exposure to ENU strikingly decreased survival (median survival: 200d vs. not reached) and increased the incidence of AML or MDS with multilineage dysplasia (73% vs. 17%). In the absence of p53 knockdown (i.e., control shRNA), mice survived longer (370d vs. 200d, P = 0.0014); however, 100% of mice developed MDS with dyserythropoiesis. None developed AML, suggesting that loss of p53 function is critical for leukemic transformation (Panel 2). Loss of both del(5q) genes, EGR1 and APC, was necessary to develop AML. Compared to mice transplanted with Egr1+/-, Apcdel/+, p53 shRNA HSPCs, mice transplanted with Egr1+/-, p53 shRNA HSPCs survived longer (369 d vs. 200 d, P = 0.0117) and only 40% of mice developed MDS with dysgranulopoiesis and/or dyserythropoiesis (Panel 3). None developed AML. Thus, severity of disease increases with loss of more than one del(5q) gene. Finally, to determine the separate effects of alklating agent therapy on HSPCs vs. the niche, we treated either the recipient or donor mice with ENU. Whereas ENU exposure to both donor and recipient resulted in a profound expansion of p53 shRNA+ cells and the development of MDS/AML in 73% of mice, ENU exposure of either donor or recipient led to only modest expansion of p53 shRNA+ cells and none of the mice developed MDS or AML. This suggests that the clonal expansion of cells with loss of multiple 5q genes and p53 is likely promoted by cytotoxic exposure to the cells themselves, as well as exposure to the surrounding niche cells. t-MN patients with a del(5q) typically present with trilineage dysplasia implicating all three hematopoietic cell lineages (erythroid, myeloid, and megakaryocytic) in the dysplastic process. Our mouse models shed light on some of the key genes on 5q, as well as the environmental exposure, that contributes to trilineage dysplasia in patients. Finally, our data suggests that t-MN is a "disease of the…

Blood, Nov 16, 2012

Abstract 117 Heterozygous deletions of the long arm of chromosome 5 are among the most common abn... more Abstract 117 Heterozygous deletions of the long arm of chromosome 5 are among the most common abnormalities in de novo (∼15% of patients) and therapy-related myeloid neoplasms (t-MN) (∼40% of patients). Two minimally deleted segments have been identified - the minimally deleted segment within 5q31.2 is associated with de novo AML and t-MNs, whereas the other spans 5q33.1 and is associated with MDS with an isolated del(5q). Current studies support a haploinsufficiency model, in which loss of a single allele of more than one gene on 5q contributes to the development of myeloid neoplasms. Using mouse models, we previously showed that haploinsufficiency of Egr1 (5q31.2) or Apc (5q22-frequently deleted in t-MN) independently recapitulates some features of human myelodysplastic syndromes (MDS). To test the hypothesis that reduced levels of EGR1 and APC cooperate in the pathogenesis of MDS/AML, we generated mice expressing a single allele of Egr1 and Apc: Mx1-Cre+Apcfl/+Egr1+/−(Apcdel/+Egr1+/−). At 2 mos of age, we induced deletion of a single allele of Apc by injection of 3 doses of pI-pC. Survival curves clearly show that Egr1 and Apc haploinsufficiency cooperate in the development of disease with a median survival of 129 days for Apcdel/+Egr1+/− mice and 296 days for Apcdel/+mice (P<0.0001). Although disease latency was significantly shorter for Apcdel/+Egr1+/− mice, their phenotype was similar to Apcdel/+ mice, with only two exceptions. For both cohorts, mice typically developed splenomegaly and a lethal macrocytic anemia with monocytosis. Anemic mice had an increased proportion of CD71+Ter119+ erythroblasts, indicating a block in erythroid development between the early and late basophilic erythroblast stage. Two mice displayed anemia and leukocytosis (WBC 51–72 k/mL) with an increased proportion of Mac1+ cells in the spleen and Kit+ cells in the bone marrow (1 mouse). As anticipated, mice with wild type levels of Apc (Mx1-Cre-Apcfl/+) or with loss of one allele of Egr1 showed no signs of anemia. Mutations in TP53 are commonly found in t-MNs with a del(5q) and loss of Tp53 in mouse models has been shown to promote AML by enabling aberrant self renewal. To test the hypothesis that loss of TP53 may adversely advance disease development, we crossed Tp53+/− to Egr1+/− and Apcdel/+ mice. Similar to Apcdel/+Egr1+/− mice, Apcdel/+Tp53+/− mice rapidly developed macrocytic anemia with a median survival of 144 days, suggesting that partial loss of TP53 function accelerates the Apcdel/+ -induced macrocytic anemia. Triple heterozygous mice (Apcdel/+Tp53+/−Egr1+/−) had a median survival of 178 days, but survival was not statistically different than Apcdel/+Egr1+/− mice (P=0.35) suggesting that Egr1 and Tp53 loss play redundant roles in the development of disease in Apcdel/+ mice. Thus, in the context of Apc haploinsufficiency, loss of Egr1 or Tp53 function promotes erythroid failure. These results are in contrast to the setting of ribosomal protein haploinsufficiency, as is the case in MDS with an isolated del(5q), where induction of TP53 is essential for erythroid failure. It has been proposed that inactivation of TP53 (through additional TP53 mutations) would be required for progression to AML, in the setting of a 5q deletion. To this end we transduced Egr1+/−Apcdel/+ bone marrow cells with a Tp53-specific shRNA, known to reduce Tp53 transcripts by ∼90%, and transplanted them into lethally irradiated C57BL/6 mice. Although penetrance of disease was low, 2 out of 13 mice (15%) developed an aggressive AML, as compared to 0 of 12 mice transplanted with Egr1+/−Apcdel/+ cells transduced with control shRNA. These data suggest that EGR1 and APC haploinsufficiency cooperate in the development of myeloid disorders, characterized by ineffective erythropoiesis, and that further mutations, such as that achieved by complete inactivation of TP53, are required for progression to AML. Disclosures: No relevant conflicts of interest to declare.

PubMed, Jun 15, 1998

The proliferation, survival, and differentiation of B cell progenitors in primary hematopoietic t... more The proliferation, survival, and differentiation of B cell progenitors in primary hematopoietic tissues depends on extracellular signals produced by stromal cells within the microenvironment. IL-7 is a stromal-derived growth factor that plays a crucial role in B lineage development. We have shown that in the presence of IL-7, pro-B cells proliferate and differentiate to a stage in which they are responsive to stromal cells and LPS, leading to terminally differentiated IgM-secreting plasma cells. In this report, we examine in detail the role of stromal cells in the transition from the IL-7-responsive pro-B cell stage to the mature LPS-responsive B cell stage. We demonstrate that this transition fails to occur, even in the presence of stromal cells and LPS, if constant exposure to IL-7 is maintained. The transition from the large pro-B cell stage to the small cmu+ pre-B cell stage occurs independent of stromal cells. Moreover, the "stromal cell-dependent" maturation that occurs subsequent to the expression of surface IgM leading to responsiveness to B cell mitogens can also be accomplished in the absence of stromal cells if pre-B cells are cultured in proximity to each other or at high cell concentrations. Together these results suggest that stromal cells mediate B cell differentiation by providing the necessary growth requirements (i.e., IL-7) to sustain the development of pre-B cells. The progeny of these pre-B cells can then differentiate through as yet unidentified homotypic interactions, leading to the production of LPS-responsive B cells.

Journal of Biological Chemistry, Sep 1, 1999

The proliferation, survival and differentiation of B cell precursors depends on extracellular sig... more The proliferation, survival and differentiation of B cell precursors depends on extracellular signals provided by cells within the microenvironment. Cell-bound and secreted molecules direct B lineage progression and regulate the selection of clones from which the immune repertoire emerges. In fact, a myriad of signals derived from B cell progenitors themselves and the microenvironment, in which they develop, cooperatively regulate the progression of progenitors along the B lineage pathway. In this thesis, I describe several new molecular interactions that had previously not been considered to play a role in the early B cell differentiation process. I first characterize the cloning of a gene encoding a sialic acid specific 9-0-acetylesterase that was isolated by differential display analysis of a preBCR-proB cell line and a preBC~* preB cell line. Differential expression is confirmed by the analysis of several B cell lines and primary B lineage cells. Furthermore, the isolation of various cDNA clones with differing 5' sequences suggests an additional level of transcriptional regulation. This finding directs my studies to an examination of the B lineage-specific, sialic acid-binding lectin CD22, whose interactions are modified by 9-0-acetylation. In contrast to previous reports, I show that surface CD22 is expressed at an early stage of development. The study of CD22, whose ligands are also expressed at an early stage of development, finally leads to an analysis of the role of homotypic B cell precursor interactions during B cell development. Using an in vibo assay system I demonstrate that interactions between B cell precursors themselves promote their further development to a mature B cell stage. That preB-preB interactions promote or regulate the critical preBCR-driven signal is suggested by the dramatic inhibition of maturation observed upon blocking p heavy chain cell surface interactions. In summary, these results suggest two novel means of regulating the B cell differentiation process: the regulation of early CD22 interactions through 9-0-acetylation and the generation of differentiation signals through homotypic B cell precursor interactions.

Blood, Nov 15, 2013

Therapy-related myeloid neoplasm (t-MN) is a distinctive clinical syndrome occurring after treatm... more Therapy-related myeloid neoplasm (t-MN) is a distinctive clinical syndrome occurring after treatment with chemotherapy and/or radiotherapy, typically for a primary malignant disease. Loss of the long arm of chromosome 5, del(5q), is the most common recurring cytogenetic abnormality and is observed in 40% of t-MN patients, as well as 10-15% of patients with primary MDS or AML de novo. These deletions typically encompass over 70Mb [spanning 5q14-q33] and numerous genes, making the identification of relevant del(5q) genes very challenging. In previous studies, we identified a commonly deleted region within 5q and identified Egr1 as a del(5q) haploinsufficient myeloid leukemia gene. Using Egr1+/- mice, we previously showed that Egr1 cooperates with mutations, induced by the alkylating agent, ENU, to induce a myeloproliferative disorder with ineffective erythropoiesis (MPD). However, loss of Egr1 on its own was not sufficient for the development of MPD. To identify cooperating mutations in MPDs in Egr1 haploinsufficient mice, we conducted a retroviral insertional mutagenesis (RIM) screen. Egr1 WT (n=61) and Egr1+/- (n=77) neonates were injected with the MOL4070LTR retrovirus. Although the overall survival of MOL4070LTR-treated Egr1+/- and WT controls was similar, Egr1+/- mice developed MPD or AML with a shorter latency and at a higher overall frequency than WT littermate controls. Forty-six percent of WT mice developed myeloid disease versus 61% for Egr1+/- mice, with a median survival of 474 d for Egr1 WT and 389 d for Egr1+/- mice (p=0.03). We mapped the retroviral integration sites in myeloid neoplasms from 29 WT and 46 Egr1+/- mice using barcoded splinkerette PCR and Illumina high-throughput sequencing. To identify and analyze the statistically significant common insertion sites (CISs) we used the TAPDANCE software developed by A. Sarver. In total, 159 CISs were identified in WT mice and 365 CISs were identified in Egr1+/- mice. Several of these CIS-associated genes, such as Sox4, Pim1 and Myb have been previously identified in other genetic screens according to the Retroviral Tagged Cancer Gene Database (RTCGD). As the main goal of this study is to identify mutations that cooperate with Egr1 haploinsufficiency, we were particularly interested in CISs that were identified exclusively or more frequently in Egr1+/- mice with myeloid neoplasms. The TAPDANCE software automatically identifies associations between phenotypes and CISs using Fisher’s exact test with multiple testing correction. Using this analysis, we identified six CISs that were statistically associated with myeloid neoplasms in Egr1+/- mice, but not WT mice. The candidate cancer genes proximal to these proviral insertions included Evi1, Gfi1, Evi5, and Cd47. Analysis of transcript levels revealed elevated expression of Evi1, but not Gfi1, Evi5, or Cd47 in myeloid leukemias with proviral insertions proximal to these genes. Of interest was a CIS associated with Egr1+/- mice that mapped to a region of mouse chromosome 18 that is syntenic to human 5q31.2, proximal to the Dnajc18, Ecscr, Tmem173, Cxxc5 and Psd2 genes and adjacent to the commonly deleted region. Moreover, an analysis of co-occurring CISs revealed that this CIS co-occurred with a CIS that mapped to a region of mouse chromosome 13 that is syntenic to human 5q31.1, also deleted in t-MN patients and proximal to the Tifab, and H2afy genes. Of the genes in these two CISs, CXXC5, TMEM173, TIFAB, and H2AFY each show significantly decreased expression in bone marrow cells from t-MN del(5q) patients, consistent with haploinsufficiency. Identification of the relevant del(5q) genes in t-MN continues to be a challenge for developing therapeutic targets. Loss of expression of the tumor suppressor gene, EGR1, which is expressed at haploinsufficient levels in t-MN patients with a del(5q), on its own is not sufficient for the development of myeloid leukemia. Here we performed a forward genetic screen with Egr1+/- mice and have identified several candidate del(5q) genes, including CXXC5, TMEM173, TIFAB, and H2AFY, that should now be evaluated as candidate genes that cooperate with EGR1 haploinsufficiency in the pathogenesis of t-MN. The identification of aberrant pathways resulting from haploinsufficiency of EGR1 in cooperation with these del(5q) genes may potentially lead to the new therapeutic targets for t-MNs with chromosome 5 abnormalities. Disclosures: Largaespada: Discovery Genomics, Inc: Consultancy, Share Holder Other; NeoClone Biotechnology, Inc: Consultancy, Share Holder, Share Holder Other.

International Immunology, Oct 1, 1997

CD22 is a B cell-restricted glycoprotein involved in cell adhesion and signaling. Since CD22 is l... more CD22 is a B cell-restricted glycoprotein involved in cell adhesion and signaling. Since CD22 is likely to play an important role in interactions between B cells and other cells, and in regulating signaling thresholds, we characterized the expression of murine CD22 during different stages of B cell development. In contrast to previous reports, we show that CD22 is expressed on B cell progenitors prior to expression of IgM. IL-7-responsive B cell precursors from the fetal liver and early B lineage cells (B220 ⍣ IgM-) from the bone marrow both express a low density of surface CD22. The majority of the earliest B cell progenitors (B220 ⍣ IgM-CD43 ⍣) in the bone marrow, however, do not express CD22. As B cells mature, the density of CD22 molecules on the cell surface increases. B220 bright IgM ⍣ bone marrow cells express high levels of CD22, as do splenic B cells. The correlation of CD22 levels with B cell maturation is replicated in an in vitro culture system, which distinguishes stages of B cell development based on function. Following activation of mature resting splenic B cells with anti-µ mAb or lipopolysaccharide (LPS), levels of CD22 decrease. Finally, we show that the addition of anti-CD22 mAb augments the proliferative response of both anti-µ-and LPS-stimulated B cells, suggesting a role for CD22 in diverse signaling pathways.

Oncogene, Jun 27, 2011

The PICALM (CALM) gene, whose product is involved in clathrin-mediated endocytosis, has been iden... more The PICALM (CALM) gene, whose product is involved in clathrin-mediated endocytosis, has been identified in two recurring chromosomal translocations, involving either MLL or MLLT10 (AF10). We developed a mouse model of CALM-AF10 + leukemia to examine the hypothesis that disruption of endocytosis contributes to leukemogenesis. Exclusion of the C-terminal portion of CALM from the fusion protein, which is required for optimal binding to clathrin, resulted in the development of a myeloproliferative disease, while inclusion of this domain led to the development of acute myeloid leukemia and changes in gene expression of several cancer-related genes, notably Pim1 and Crebbp. Nonetheless, the development of leukemia could not be attributed directly to interference with endocytosis or consequential changes in proliferation and signaling. In leukemia cells, full-length CALM-AF10 localized to the nucleus with no consistent effect on growth factor endocyctosis, and suppressed H3K79 methylation regardless of the presence of clathrin. Using FRET analysis, we show that CALM-AF10 has a propensity to homooligomerize, raising the possibility that the function of endocytic proteins involved in chimeric fusions may be to provide dimerization properties, a recognized mechanism for unleashing oncogenic properties of chimeric transcription factors, rather than disrupting the internalization of growth factor receptors.

Blood, Jan 9, 2014

• Haploinsufficiency of Egr1, Apc, and Tp53 in mice cooperate to model the pathogenesis of the ea... more • Haploinsufficiency of Egr1, Apc, and Tp53 in mice cooperate to model the pathogenesis of the early stages of t-MN with a del(5q). • Exposure of an Apc haploinsufficient BM microenvironment to radiation and/or an alkylating agent accelerates disease development. Therapy-related myeloid neoplasms (t-MN) are a late complication of the successful use of cytotoxic therapy for patients with cancer. A heterozygous deletions of the long arm of chromosome 5 [del(5q)], observed in 40% of patients, is associated with prior exposure to alkylating agents, and a high frequency of TP53 loss or mutation. In previous studies, we demonstrated that haploinsufficiency of 2 del(5q) genes, Egr1, and Apc, individually play a role in the pathogenesis of hematologic disease in mice. We now show that loss of one copy of Egr1 or Tp53 in an Apc haploinsufficient background (Apc del/1) accelerated the development of a macrocytic anemia with monocytosis, early features of t-MN. The development of anemia was significantly accelerated by treatment of mice with the alkylating agent, N-ethyl-N-nitrosourea (ENU), regardless of the levels of expression of Egr1 and Tp53. Transplantation of either wild type; Egr1 1/2 ; Tp53 1/2 ; Apc del/1 ; or Egr1 1/2 , Apc del/1 bone marrow cells into lethally irradiated Apc del/1 recipients resulted in rapid development of anemia that was further accelerated by administration of ENU to recipients, demonstrating that the Apc del/1-induced anemia was cell extrinsic and potentiated by ENU mutagenesis. These data emphasize the synergistic role of cell intrinsic and cell extrinsic (microenvironment) factors in the pathogenesis of t-MN, and raise awareness of the deleterious effects of cytotoxic therapy on the stromal microenvironment.

Experimental Hematology, Nov 1, 2022

Mediterranean Journal of Hematology and Infectious Diseases, May 16, 2011

Haematologica, Feb 14, 2014

Chemico-Biological Interactions, Mar 1, 2010

Therapy-related myelodysplastic syndrome and acute myeloid leukemia (t-MDS/t-AML) are late compli... more Therapy-related myelodysplastic syndrome and acute myeloid leukemia (t-MDS/t-AML) are late complications of cytotoxic therapy used in the treatment of malignant diseases. The most common subtype of t-AML (~75% of cases) develops after exposure to alkylating agents, and is characterized by loss or deletion of chromosome 5 and/or 7 [−5/del(5q), −7/del(7q)], and a poor outcome (median survival 8 months). In the University of Chicago's series of 386 patients with t-MDS/t-AML, 79 (20%) patients had abnormalities of chromosome 5, 95 (25%) patients had abnormalities of chromosome 7, and 85 (22%) patients had abnormalities of both chromosomes 5 and 7. t-MDS/t-AML with a −5/del(5q) is associated with a complex karyotype, characterized by trisomy 8, as well as loss of 12p, 13q, 16q22, 17p (TP53 locus), chromosome 18, and 20q. In addition, this subtype of t-AML is characterized by a unique expression profile (higher expression of genes involved in cell cycle control (CCNA2, CCNE2, CDC2), checkpoints (BUB1), or growth (MYC), loss of expression of IRF8, and overexpression of FHL2. Haploinsufficiency of the RPS14, EGR1, APC, NPM1, and CTNNA1 genes on 5q has been implicated in the pathogenesis of MDS/AML. In previous studies, we determined that Egr1 acts by haploinsufficiency and cooperates with mutations induced by alkylating agents to induce myeloid leukemias in the mouse. To identify mutations that cooperate with Egr1 haploinsufficiency, we used retroviral insertional mutagenesis. To date, we have identified two common integration sites involving genes encoding transcription factors that play a critical role in hematopoiesis (Evi1 and Gfi1b loci). Of note is that the EVI1 transcription factor gene is deregulated in human AMLs, particularly those with −7, and abnormalities of 3q. Identifying the genetic pathways leading to t-AML will provide new insights into the underlying biology of this disease, and may facilitate the identification of new therapeutic targets.

Blood, Nov 16, 2007

The clathrin assembly lymphoid myeloid leukemia gene, CALM, was first described as a translocatio... more The clathrin assembly lymphoid myeloid leukemia gene, CALM, was first described as a translocation partner for AF10 in morphologically distinct subsets of acute leukemia with a recurring 10;11 translocation. As the name implies, CALM is involved in the regulation of clathrin-mediated endocytosis. We hypothesize that expression of the CALM-AF10 protein disrupts endocytosis of growth receptors, leading to constitutive activation and, thereby, contributes to the development of leukemia. The CALM-AF10 fusion transcripts are characterized by two breakpoints in CALM at positions 1926 and 2091. Using the 293T human renal epithelial cell line as a model, we demonstrated that CALM1926-AF10 localized diffusely to the cytoplasm, whereas CALM2091-AF10 had a more prominent punctate appearance. This difference in distribution was likely attributable to protein domains in CALM rather than AF10, which is a transcription factor that localizes to the nucleus. The difference in localization extended to their effects on endocytosis. Whereas ∼4–8% of cells expressing truncated CALM2091, CALM1926, or AF10 displayed impaired transferrin uptake, this value was ∼60% in cells expressing CALM2091-AF10 and only ∼13% in cells expressing CALM1926-AF10. Next, we used a retroviral transduction-transplantation murine model to test the impact of CALM-AF10 expression on the development of leukemia. Mice transplanted with either CALM1926-AF10 or CALM2091-AF10 transduced fetal liver cells developed disease within a short period of time (∼8 to 21 weeks post-transplantation). Interestingly, expression of CALM1926-AF10 led to the development of a myeloproliferative disease (MPD)-like leukemia, with a predominance of mature neutrophils, whereas expression of CALM2091-AF10 led to the development of an acute myeloid leukemia (AML) with >30% immature myeloid blasts, with one exception. One mouse transplanted with progenitor cells expressing CALM2091-AF10 developed a MPD accompanied by a granulocytic sarcoma; however, examination of RNA from the spleen of this diseased mouse revealed that it had undergone a splicing event consistent with the size expected for the 1926 breakpoint. Both diseases were transplantable to secondary recipients confirming the leukemic nature of the CALM-AF10 expressing cells. Previous studies show that patients with the t(10;11) (p13;q14-21) develop either AML or T cell-acute lymphoblastic leukemia (T-ALL). Most of the patients with T-ALL express the CALM1926-AF10 fusion. Although there are differences in presentation between humans and mice, the hematological disease may be a reflection of the conditions of the mouse model. To identify possible secondary cooperating mutations, spectral karyotyping analysis of the mouse AMLs and MPDs was performed. However, to date we have not identified any significant abnormalities. We hypothesize that defective endocytosis synergizes with transcriptional deregulation, leading to an aggressive form of leukemia.

Blood cancer discovery, Apr 20, 2020

Therapy-related myeloid neoplasms (t-MN) comprise therapy-related acute myeloid leukemia (t-AML) ... more Therapy-related myeloid neoplasms (t-MN) comprise therapy-related acute myeloid leukemia (t-AML) and myelodysplastic syndrome (t-MDS), and are a late complication of cytotoxic therapy, chemotherapy and/or radiotherapy, used in the treatment of both malignant and nonmalignant diseases (1, 2). The genetic profile of t-MN is markedly skewed toward high-risk cytogenetic and molecular abnormalities, and complex karyotypes with deletions of the long arm of chromosome 5, del(5q), and TP53 mutation/loss are profoundly over-represented in t-MNs as compared with de novo counterparts (3, 4). A proximal minimally deleted region (MDR) in 5q31.2 (containing EGR1) was previously identified in t-MN, de novo AML and high-risk MDS, and a distal MDR in 5q33.1(containing mir145, RPS14, and CSNK1A1) was identified in MDS with an isolated del(5q), previously referred to as the 5q-syndrome (1, 5). However, the deletion of 5q in all but rare patients encompasses both MDRs, spanning 5q14-5q33, resulting in loss of one allele for hundreds of genes. Although challenging to identify involved genes, we previously chose to examine two well-characterized del(5q)

Blood, Jun 1, 2017

• Loss of 1 copy of Ctnnb1 (encoding b-catenin) in an Apc-haploinsufficient microenvironment prev... more • Loss of 1 copy of Ctnnb1 (encoding b-catenin) in an Apc-haploinsufficient microenvironment prevents the development of MDS. • Modulation of WNT signaling in the niche using pyrvinium inhibits the development of MDS in Apc-haploinsufficient mice. There is accumulating evidence that functional alteration(s) of the bone marrow (BM) microenvironment contribute to the development of some myeloid disorders, such as myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). In addition to a cellintrinsic role of WNT activation in leukemia stem cells, WNT activation in the BM niche is also thought to contribute to the pathogenesis of MDS and AML. We previously showed that the Apc-haploinsufficient mice (Apc del/1) model MDS induced by an aberrant BM microenvironment. We sought to determine whether Apc, a multifunctional protein and key negative regulator of the canonical b-catenin (Ctnnb1)/WNT-signaling pathway, mediates this disease through modulating WNT signaling, and whether inhibition of WNT signaling prevents the development of MDS in Apc del/1 mice. Here, we demonstrate that loss of 1 copy of Ctnnb1 is sufficient to prevent the development of MDS in Apc del/1 mice and that altered canonical WNT signaling in the microenvironment is responsible for the disease. Furthermore, the US Food and Drug Administration (FDA)-approved drug pyrvinium delays and/or inhibits disease in Apc del/1 mice, even when it is administered after the presentation of anemia. Other groups have observed increased nuclear CTNNB1 in stromal cells from a high frequency of MDS/AML patients, a finding that together with our results highlights a potential new strategy for treating some myeloid disorders.

Blood, Feb 13, 2014

• Egr1 haploinsufficiency in cooperation with reduced Tp53 activity accelerates the development o... more • Egr1 haploinsufficiency in cooperation with reduced Tp53 activity accelerates the development of hematologic disease in mice. • Loss of 1 copy of Egr1 and Apc in hematopoietic stem cells, in cooperation with Tp53 loss, results in myeloid neoplasms. An interstitial deletion of chromosome 5, del(5q), is the most common structural abnormality in primary myelodysplastic syndromes (MDS) and therapy-related myeloid neoplasms (t-MNs) after cytotoxic therapy. Loss of TP53 activity, through mutation or deletion, is highly associated with t-MNs with a del(5q). We previously demonstrated that haploinsufficiency of Egr1 and Apc, 2 genes lost in the 5q deletion, are key players in the progression of MDS with a del(5q). Using genetically engineered mice, we now show that reduction or loss of Tp53 expression, in combination with Egr1 haploinsufficiency, increased the rate of development of hematologic neoplasms and influenced the disease spectrum, but did not lead to overt myeloid leukemia, suggesting that altered function of additional gene(s) on 5q are likely required for myeloid leukemia development. Next, we demonstrated that cell intrinsic loss of Tp53 in hematopoietic stem and progenitor cells haploinsufficient for both Egr1 and Apc led to the development of acute myeloid leukemia (AML) in 17% of mice. The long latency (234-299 days) and clonal chromosomal abnormalities in the AMLs suggest that additional genetic changes may be required for full transformation. Thus, loss of Tp53 activity in cooperation with Egr1 and Apc haploinsufficiency creates an environment that is permissive for malignant transformation and the development of AML.

Molecular Biology of the Cell, May 1, 2005

B cell antigen receptor (BCR) association with lipid rafts, the actin cytoskeleton, and clathrin-... more B cell antigen receptor (BCR) association with lipid rafts, the actin cytoskeleton, and clathrin-coated pits influences B cell signaling and antigen presentation. Although all three cellular structures have been separately implicated in BCR internalization, the relationship between them has not been clearly defined. In this study, internalization pathways were characterized by specifically blocking each potential mechanism of internalization. BCR uptake was reduced by ϳ70% in B cells conditionally deficient in clathrin heavy chain expression. Actin or raft antagonists were both able to block the residual, clathrin-independent BCR internalization. These agents also affected clathrin-dependent internalization, indicating that clathrin-coated pits, in concert with mechanisms dependent on rafts and actin, mediate the majority of BCR internalization. Clustering G M1 gangliosides enhanced clathrin-independent BCR internalization, and this required actin. Thus, although rafts or actin independently did not mediate BCR internalization, they apparently cooperate to promote some internalization even in the absence of clathrin. Simultaneous inhibition of all BCR uptake pathways resulted in sustained tyrosine phosphorylation and activation of the extracellular signal-regulated kinase (ERK), strongly suggesting that downstream BCR signaling can occur without receptor translocation to endosomes and that internalization leads to signal attenuation.

Blood, Dec 3, 2015

A del(5q) is frequently noted in MDS, AML, and therapy-related myeloid neoplasms (t-MN) following... more A del(5q) is frequently noted in MDS, AML, and therapy-related myeloid neoplasms (t-MN) following alkylating agent therapy. Mutation/loss of the TP53 is gene found in 80% of t-MNs with a del(5q). Recent studies suggest that TP53 mutations are found at a low frequency in hematopoietic stem/progenitor cells (HSPCs) in adults (Xie et al., Nat Med 20:1472, 2014; Genovese et al., NEJM 371:2477, 2014; Jaiswal et al., NEJM 371:2488, 2014), and chemotherapy confers a selective growth advantage to these rare clones (Wong et al., Nature 518:552, 2015). We previously established a mouse model for t-MN with a del(5q) and showed that haploinsufficiency of two del(5q) genes, Egr1 and Apc, cooperate with loss of function of the Trp53 (p53) gene to induce myeloid neoplasms in mice. Specifically, transplantation of Egr1+/-, Apcdel/+ bone marrow (BM) cells transduced with p53 shRNAs into wild type (WT) recipients resulted in the development of a transplantable AML, characterized by a complex karyotype and genetic instability, in 17% of mice. There is growing evidence that microenvironment perturbations play a major role in the malignant process; however, the effect of cytotoxic therapy on HSPCs as well as the BM niche is not well understood. Using our mouse model of t-MN with a del(5q), we explored the effects of ENU, an alkylating agent, on both HSPCs and the BM microenvironment by exposing both donor and recipient mice to ENU (Panel 1 in the figure). In mice transplanted with Egr1+/-, Apcdel/+, p53 shRNA HSPCs, exposure to ENU strikingly decreased survival (median survival: 200d vs. not reached) and increased the incidence of AML or MDS with multilineage dysplasia (73% vs. 17%). In the absence of p53 knockdown (i.e., control shRNA), mice survived longer (370d vs. 200d, P = 0.0014); however, 100% of mice developed MDS with dyserythropoiesis. None developed AML, suggesting that loss of p53 function is critical for leukemic transformation (Panel 2). Loss of both del(5q) genes, EGR1 and APC, was necessary to develop AML. Compared to mice transplanted with Egr1+/-, Apcdel/+, p53 shRNA HSPCs, mice transplanted with Egr1+/-, p53 shRNA HSPCs survived longer (369 d vs. 200 d, P = 0.0117) and only 40% of mice developed MDS with dysgranulopoiesis and/or dyserythropoiesis (Panel 3). None developed AML. Thus, severity of disease increases with loss of more than one del(5q) gene. Finally, to determine the separate effects of alklating agent therapy on HSPCs vs. the niche, we treated either the recipient or donor mice with ENU. Whereas ENU exposure to both donor and recipient resulted in a profound expansion of p53 shRNA+ cells and the development of MDS/AML in 73% of mice, ENU exposure of either donor or recipient led to only modest expansion of p53 shRNA+ cells and none of the mice developed MDS or AML. This suggests that the clonal expansion of cells with loss of multiple 5q genes and p53 is likely promoted by cytotoxic exposure to the cells themselves, as well as exposure to the surrounding niche cells. t-MN patients with a del(5q) typically present with trilineage dysplasia implicating all three hematopoietic cell lineages (erythroid, myeloid, and megakaryocytic) in the dysplastic process. Our mouse models shed light on some of the key genes on 5q, as well as the environmental exposure, that contributes to trilineage dysplasia in patients. Finally, our data suggests that t-MN is a "disease of the…

Blood, Nov 16, 2012

Abstract 117 Heterozygous deletions of the long arm of chromosome 5 are among the most common abn... more Abstract 117 Heterozygous deletions of the long arm of chromosome 5 are among the most common abnormalities in de novo (∼15% of patients) and therapy-related myeloid neoplasms (t-MN) (∼40% of patients). Two minimally deleted segments have been identified - the minimally deleted segment within 5q31.2 is associated with de novo AML and t-MNs, whereas the other spans 5q33.1 and is associated with MDS with an isolated del(5q). Current studies support a haploinsufficiency model, in which loss of a single allele of more than one gene on 5q contributes to the development of myeloid neoplasms. Using mouse models, we previously showed that haploinsufficiency of Egr1 (5q31.2) or Apc (5q22-frequently deleted in t-MN) independently recapitulates some features of human myelodysplastic syndromes (MDS). To test the hypothesis that reduced levels of EGR1 and APC cooperate in the pathogenesis of MDS/AML, we generated mice expressing a single allele of Egr1 and Apc: Mx1-Cre+Apcfl/+Egr1+/−(Apcdel/+Egr1+/−). At 2 mos of age, we induced deletion of a single allele of Apc by injection of 3 doses of pI-pC. Survival curves clearly show that Egr1 and Apc haploinsufficiency cooperate in the development of disease with a median survival of 129 days for Apcdel/+Egr1+/− mice and 296 days for Apcdel/+mice (P<0.0001). Although disease latency was significantly shorter for Apcdel/+Egr1+/− mice, their phenotype was similar to Apcdel/+ mice, with only two exceptions. For both cohorts, mice typically developed splenomegaly and a lethal macrocytic anemia with monocytosis. Anemic mice had an increased proportion of CD71+Ter119+ erythroblasts, indicating a block in erythroid development between the early and late basophilic erythroblast stage. Two mice displayed anemia and leukocytosis (WBC 51–72 k/mL) with an increased proportion of Mac1+ cells in the spleen and Kit+ cells in the bone marrow (1 mouse). As anticipated, mice with wild type levels of Apc (Mx1-Cre-Apcfl/+) or with loss of one allele of Egr1 showed no signs of anemia. Mutations in TP53 are commonly found in t-MNs with a del(5q) and loss of Tp53 in mouse models has been shown to promote AML by enabling aberrant self renewal. To test the hypothesis that loss of TP53 may adversely advance disease development, we crossed Tp53+/− to Egr1+/− and Apcdel/+ mice. Similar to Apcdel/+Egr1+/− mice, Apcdel/+Tp53+/− mice rapidly developed macrocytic anemia with a median survival of 144 days, suggesting that partial loss of TP53 function accelerates the Apcdel/+ -induced macrocytic anemia. Triple heterozygous mice (Apcdel/+Tp53+/−Egr1+/−) had a median survival of 178 days, but survival was not statistically different than Apcdel/+Egr1+/− mice (P=0.35) suggesting that Egr1 and Tp53 loss play redundant roles in the development of disease in Apcdel/+ mice. Thus, in the context of Apc haploinsufficiency, loss of Egr1 or Tp53 function promotes erythroid failure. These results are in contrast to the setting of ribosomal protein haploinsufficiency, as is the case in MDS with an isolated del(5q), where induction of TP53 is essential for erythroid failure. It has been proposed that inactivation of TP53 (through additional TP53 mutations) would be required for progression to AML, in the setting of a 5q deletion. To this end we transduced Egr1+/−Apcdel/+ bone marrow cells with a Tp53-specific shRNA, known to reduce Tp53 transcripts by ∼90%, and transplanted them into lethally irradiated C57BL/6 mice. Although penetrance of disease was low, 2 out of 13 mice (15%) developed an aggressive AML, as compared to 0 of 12 mice transplanted with Egr1+/−Apcdel/+ cells transduced with control shRNA. These data suggest that EGR1 and APC haploinsufficiency cooperate in the development of myeloid disorders, characterized by ineffective erythropoiesis, and that further mutations, such as that achieved by complete inactivation of TP53, are required for progression to AML. Disclosures: No relevant conflicts of interest to declare.

PubMed, Jun 15, 1998

The proliferation, survival, and differentiation of B cell progenitors in primary hematopoietic t... more The proliferation, survival, and differentiation of B cell progenitors in primary hematopoietic tissues depends on extracellular signals produced by stromal cells within the microenvironment. IL-7 is a stromal-derived growth factor that plays a crucial role in B lineage development. We have shown that in the presence of IL-7, pro-B cells proliferate and differentiate to a stage in which they are responsive to stromal cells and LPS, leading to terminally differentiated IgM-secreting plasma cells. In this report, we examine in detail the role of stromal cells in the transition from the IL-7-responsive pro-B cell stage to the mature LPS-responsive B cell stage. We demonstrate that this transition fails to occur, even in the presence of stromal cells and LPS, if constant exposure to IL-7 is maintained. The transition from the large pro-B cell stage to the small cmu+ pre-B cell stage occurs independent of stromal cells. Moreover, the "stromal cell-dependent" maturation that occurs subsequent to the expression of surface IgM leading to responsiveness to B cell mitogens can also be accomplished in the absence of stromal cells if pre-B cells are cultured in proximity to each other or at high cell concentrations. Together these results suggest that stromal cells mediate B cell differentiation by providing the necessary growth requirements (i.e., IL-7) to sustain the development of pre-B cells. The progeny of these pre-B cells can then differentiate through as yet unidentified homotypic interactions, leading to the production of LPS-responsive B cells.

Journal of Biological Chemistry, Sep 1, 1999

The proliferation, survival and differentiation of B cell precursors depends on extracellular sig... more The proliferation, survival and differentiation of B cell precursors depends on extracellular signals provided by cells within the microenvironment. Cell-bound and secreted molecules direct B lineage progression and regulate the selection of clones from which the immune repertoire emerges. In fact, a myriad of signals derived from B cell progenitors themselves and the microenvironment, in which they develop, cooperatively regulate the progression of progenitors along the B lineage pathway. In this thesis, I describe several new molecular interactions that had previously not been considered to play a role in the early B cell differentiation process. I first characterize the cloning of a gene encoding a sialic acid specific 9-0-acetylesterase that was isolated by differential display analysis of a preBCR-proB cell line and a preBC~* preB cell line. Differential expression is confirmed by the analysis of several B cell lines and primary B lineage cells. Furthermore, the isolation of various cDNA clones with differing 5' sequences suggests an additional level of transcriptional regulation. This finding directs my studies to an examination of the B lineage-specific, sialic acid-binding lectin CD22, whose interactions are modified by 9-0-acetylation. In contrast to previous reports, I show that surface CD22 is expressed at an early stage of development. The study of CD22, whose ligands are also expressed at an early stage of development, finally leads to an analysis of the role of homotypic B cell precursor interactions during B cell development. Using an in vibo assay system I demonstrate that interactions between B cell precursors themselves promote their further development to a mature B cell stage. That preB-preB interactions promote or regulate the critical preBCR-driven signal is suggested by the dramatic inhibition of maturation observed upon blocking p heavy chain cell surface interactions. In summary, these results suggest two novel means of regulating the B cell differentiation process: the regulation of early CD22 interactions through 9-0-acetylation and the generation of differentiation signals through homotypic B cell precursor interactions.

Blood, Nov 15, 2013

Therapy-related myeloid neoplasm (t-MN) is a distinctive clinical syndrome occurring after treatm... more Therapy-related myeloid neoplasm (t-MN) is a distinctive clinical syndrome occurring after treatment with chemotherapy and/or radiotherapy, typically for a primary malignant disease. Loss of the long arm of chromosome 5, del(5q), is the most common recurring cytogenetic abnormality and is observed in 40% of t-MN patients, as well as 10-15% of patients with primary MDS or AML de novo. These deletions typically encompass over 70Mb [spanning 5q14-q33] and numerous genes, making the identification of relevant del(5q) genes very challenging. In previous studies, we identified a commonly deleted region within 5q and identified Egr1 as a del(5q) haploinsufficient myeloid leukemia gene. Using Egr1+/- mice, we previously showed that Egr1 cooperates with mutations, induced by the alkylating agent, ENU, to induce a myeloproliferative disorder with ineffective erythropoiesis (MPD). However, loss of Egr1 on its own was not sufficient for the development of MPD. To identify cooperating mutations in MPDs in Egr1 haploinsufficient mice, we conducted a retroviral insertional mutagenesis (RIM) screen. Egr1 WT (n=61) and Egr1+/- (n=77) neonates were injected with the MOL4070LTR retrovirus. Although the overall survival of MOL4070LTR-treated Egr1+/- and WT controls was similar, Egr1+/- mice developed MPD or AML with a shorter latency and at a higher overall frequency than WT littermate controls. Forty-six percent of WT mice developed myeloid disease versus 61% for Egr1+/- mice, with a median survival of 474 d for Egr1 WT and 389 d for Egr1+/- mice (p=0.03). We mapped the retroviral integration sites in myeloid neoplasms from 29 WT and 46 Egr1+/- mice using barcoded splinkerette PCR and Illumina high-throughput sequencing. To identify and analyze the statistically significant common insertion sites (CISs) we used the TAPDANCE software developed by A. Sarver. In total, 159 CISs were identified in WT mice and 365 CISs were identified in Egr1+/- mice. Several of these CIS-associated genes, such as Sox4, Pim1 and Myb have been previously identified in other genetic screens according to the Retroviral Tagged Cancer Gene Database (RTCGD). As the main goal of this study is to identify mutations that cooperate with Egr1 haploinsufficiency, we were particularly interested in CISs that were identified exclusively or more frequently in Egr1+/- mice with myeloid neoplasms. The TAPDANCE software automatically identifies associations between phenotypes and CISs using Fisher’s exact test with multiple testing correction. Using this analysis, we identified six CISs that were statistically associated with myeloid neoplasms in Egr1+/- mice, but not WT mice. The candidate cancer genes proximal to these proviral insertions included Evi1, Gfi1, Evi5, and Cd47. Analysis of transcript levels revealed elevated expression of Evi1, but not Gfi1, Evi5, or Cd47 in myeloid leukemias with proviral insertions proximal to these genes. Of interest was a CIS associated with Egr1+/- mice that mapped to a region of mouse chromosome 18 that is syntenic to human 5q31.2, proximal to the Dnajc18, Ecscr, Tmem173, Cxxc5 and Psd2 genes and adjacent to the commonly deleted region. Moreover, an analysis of co-occurring CISs revealed that this CIS co-occurred with a CIS that mapped to a region of mouse chromosome 13 that is syntenic to human 5q31.1, also deleted in t-MN patients and proximal to the Tifab, and H2afy genes. Of the genes in these two CISs, CXXC5, TMEM173, TIFAB, and H2AFY each show significantly decreased expression in bone marrow cells from t-MN del(5q) patients, consistent with haploinsufficiency. Identification of the relevant del(5q) genes in t-MN continues to be a challenge for developing therapeutic targets. Loss of expression of the tumor suppressor gene, EGR1, which is expressed at haploinsufficient levels in t-MN patients with a del(5q), on its own is not sufficient for the development of myeloid leukemia. Here we performed a forward genetic screen with Egr1+/- mice and have identified several candidate del(5q) genes, including CXXC5, TMEM173, TIFAB, and H2AFY, that should now be evaluated as candidate genes that cooperate with EGR1 haploinsufficiency in the pathogenesis of t-MN. The identification of aberrant pathways resulting from haploinsufficiency of EGR1 in cooperation with these del(5q) genes may potentially lead to the new therapeutic targets for t-MNs with chromosome 5 abnormalities. Disclosures: Largaespada: Discovery Genomics, Inc: Consultancy, Share Holder Other; NeoClone Biotechnology, Inc: Consultancy, Share Holder, Share Holder Other.