Supplementary Figures 1-7 from Gadd45a Functions as a Promoter or Suppressor of Breast Cancer Dependent on the Oncogenic Stress

Gadd45b is a member of Gadd45 stress sensor protein family that also includes Gadd45a & Gadd45g. To investigate the effect of Gadd45b in bcr-abl oncogene driven chronic myeloid leukemia (CML) development, syngeneic wild type lethally irradiated mice were reconstituted with either wild type or Gadd45b null myeloid progenitors transduced with a retroviral vector expressing BCR-ABL. Loss of Gadd45b was observed to accelerate BCR-ABL driven CML development with shortened median mouse survival time. BCR-ABL Gadd45b deficient CML progenitors exhibited increased proliferation and decreased apoptosis, associated with hyper-activation of c-Jun NH2-terminal kinase and Stat5. These results provide novel evidence that gadd45b, like gadd45a, functions as a suppressor of BCR-ABL driven leukemia, albeit via a different mechanism.

The Gadd45a stress sensor gene is a member in the Gadd45 family of genes that includes Gadd45b & Gadd45g. To investigate the effect of GADD45A in the development of CML, syngeneic wild type lethally irradiated mice were reconstituted with either wild type or Gadd45a null myeloid progenitors transduced with a retroviral vector expressing the 210-kD BCR-ABL fusion oncoprotein. Loss of Gadd45a was observed to accelerate BCR-ABL driven CML resulting in the development of a more aggressive disease, a significantly shortened median mice survival time, and increased BCR-ABL expressing leukemic stem/progenitor cells (GFP+Lin- cKit+Sca+). GADD45A deficient progenitors expressing BCR-ABL exhibited increased proliferation and decreased apoptosis relative to WT counterparts, which was associated with enhanced PI3K-AKT-mTOR-4E-BP1 signaling, upregulation of p30C/EBPa expression, and hyper-activation of p38 and Stat5. Furthermore, Gadd45a expression in samples obtained from CML patients was upregulated in more indolent chronic phase CML samples and down regulated in aggressive accelerated phase CML and blast crisis CML. These results provide novel evidence that Gadd45a functions as a suppressor of BCR/ABL driven leukemia and may provide a unique prognostic marker of CML progression.

There is substantial evidence that early growth response-1 (Egr1) gene, a zinc-finger transcription factor, behaves as a tumor suppressor in leukemia. This includes reports from this laboratory that constitutive Egr1 overrides leukemia conferred by deregulated c-Myc or E2F-1 in the M1 myeloid leukemic cell line by promoting differentiation. To investigate the effect of Egr1 on the initiation and progression of Chronic Myelogenous Leukemia (CML), lethally irradiated syngeneic wild type mice were reconstituted with bone marrow (BM) from either wild type or Egr1 null mice transduced with a 210-kD BCR-ABL-expressing MSCV-retrovirus (bone marrow transplantation {BMT}). Loss of Egr1 was observed to accelerate the development of BCR-ABL driven leukemia in recipient mice, resulting in the development of a more aggressive disease, a significantly shortened median survival time, and increased BCR-ABL expressing leukemic stem/progenitor cells (GFP+Lin-cKit+Sca+). Egr1 deficient progenitors expressing BCR-ABL exhibited decreased apoptosis, and increased cell viability and proliferation relative to WT counterparts. Secondary BMT of BCR-ABL BM revealed that loss of Egr1 resulted in enrichment of LSCs, consistent with shorter survival time and more aggressive disease of these mice compared to WT counterparts. Furthermore, serial re-plating colony assays indicated that loss of Egr1 increased self-renewal ability of BCR-ABL expressing BM. These novel findings on the tumor suppressor role of Egr1 in CML provide the impetus to study the effect of altering Egr1 expression in AML, where the overall five year survival rate remains low. The effect of loss of Egr1 in CML could reflect its established functions in normal hematopoiesis, maintaining quiescence of HSCs and driving terminal differentiation to the monocyte/macrophage lineage. Gain of function studies should validate these conclusions and provide further rationale for increased Egr1 as a therapeutic target in AML.

ObjectiveThis study was aimed to evaluate whether maternal dried blood spots could be a potential source for the noninvasive fetal RHD genotyping, serving as a combined one-step test for both the First Trimester Screen and the fetal RHD genotyping.MethodBoth the maternal dried blood spots and the peripheral blood samples from 19 RhD-negative pregnant women were obtained during the First Trimester Screen. DNA was extracted and sequential real-time PCRs were performed to determine the fetal RHD genotypes. Fetal RhD serological types were obtained after delivery. This study was approved by the Institutional Review Board, and informed consents were obtained.ResultsA total of 19/19 fetal RHD genotyping with maternal DBS were consistent with the follow-up serological RhD test results after birth. Eleven were RhD positive, and eight were RhD negative (RHD deletion or RHD-CE-D=6, RHD pseudogene=1, RHDVI=1). Sensitivity=100%, specificity=100%, positive predictive value=100%, negative predictive value=100%. A total of 18/19 fetal gender were determined correctly with maternal DBS. One female fetus was falsely determined as male. Sensitivity=100%, specificity=91.6%, positive predictive value=87.5%, negative predictive value=100%.ConclusionMaternal dried blood spots, with the benefits of flexible sample transportation and processing, could be utilized for the noninvasive prenatal fetal RHD genotyping and potentially be incorporated into the routine First Trimester Screen. Larger scale study is in progress to implement fetal RHD genotyping in routine prenatal care. (c) 2017 John Wiley & Sons, Ltd.

The GADD45 family of proteins functions as stress sensors in response to various physiological and environmental stressors. Here we show that primary mouse embryo fibroblasts (MEFs) from Gadd45b null mice proliferate slowly, accumulate increased levels of DNA damage, and senesce prematurely. The impaired proliferation and increased senescence in Gadd45b null MEFs is partially reversed by culturing at physiological oxygen levels, indicating that Gadd45b deficiency leads to decreased ability to cope with oxidative stress. Interestingly, Gadd45b null MEFs arrest at the G2/M phase of cell cycle, in contrast to other senescent MEFs, which arrest at G1. FACS analysis of phospho-histone H3 staining showed that Gadd45b null MEFs are arrested in G2 phase rather than M phase. H2O2 and UV irradiation, known to increase oxidative stress, also triggered increased senescence in Gadd45b null MEFs compared to wild type MEFs. In vivo evidence for increased senescence in Gadd45b null mice includes the observation that embryos from Gadd45b null mice exhibit increased senescence staining compared to wild type embryos. Furthermore, it is shown that Gadd45b deficiency promotes senescence and aging phenotypes in mouse skin. Together, these results highlight a novel role for Gadd45b in stress-induced senescence and in tissue aging.

The transcription factor early growth response 1 (Egr-1) gene was identified as a macrophage differentiation primary response gene, shown to be essential for and to restrict differentiation along the macrophage lineage. There’s evidence consistent with Egr-1 behaving as a tumor suppressor of leukemia, both in vivo and in vitro, including (1) loss of Egr-1 associated with therapy derived MDS and AML; (2) deregulated Egr-1 overriding blocks in myeloid differentiation, and (3) haplo-insufficiency of Egr-1 in mice leading to increased development of myeloid disorders following treatment with the potent DNA alkylating agent, N- ethyl-nitrosourea (ENU). BCR-ABL driven leukemia (Chronic Myelogenous Leukemia [CML]) was chosen as a model system to investigate the role of Egr-1 as a tumor suppressor for leukemia. CML is a disease resulting from the neoplastic transformation of hematopoietic stem cells (HSC) with the BCR-ABL oncogene. To assess the effect of Egr-1 on BCR-ABL driven leukemia, lethally irradiated syngeneic wild type mice were reconstituted with bone marrow (BM) from either wild type or Egr-1 null mice transduced with a 210-kD BCR-ABL-expressing MSCV-GFP retrovirus (bone marrow transplantation {BMT}). It was observed that loss of Egr-1 accelerated the development of BCR-ABL driven leukemia in recipient mice. Analysis of hematopoietic organs is consistent with loss of Egr-1 accelerating development of CML but not altering the nature of the disease. Cells expressing BCR-ABL that are null for Egr-1 exhibit increased survival and proliferation, which can account for some of the above observations. Substantial evidence is available that loss of Egr1 increases the leukemia initiating cell population, which can account for the more rapid development of disease. An increased population of lineage negative BM cells was observed in Egr-1-/- BCR-ABL recipient mice compared to animals transplanted with WT BCR-ABL BM. Serial BMT has shown that Egr-1-/- BCR-ABL BM has an increased leukemic burden compared to the WT counterpart. Furthermore, data from serial colony transfer experiments confirmed that loss of Egr-1 increases leukemia initiating cells. Preliminary studies on the effect of gain of Egr-1 function are consistent with the tumor suppressor behavior of Egr-1. These data as well as analysis of human CML samples will be presented. Further investigation could result in novel targets for diagnosis, prognosis, and targeted therapeutics, including strategies for activating Egr-1 expression that can be used to treat CML, as well as other leukemic diseases. Disclosures No relevant conflicts of interest to declare.

The BCR-ABL fusion oncogene which encodes a fused deregulated tyrosine kinase causes chronic myelogenous leukemia (CML) in humans. Imatinib, a small molecule ABL kinase inhibitor has been highly effective in treating chronic phase (CP) CML patients. However, a substantial number of patients undergo relapse due to development of resistance to imatinib therapy that leads to blast crisis (BC-CML), which is invariably fatal within weeks to months. Additional genetic aberrations assist in progression and identification of key players that are responsible for transformation is of utmost importance from a therapeutic point of view. Growth arrest DNA damage 45a (Gadd45a) gene, a member in the gadd45 family of genes including Gadd45b & Gadd45g, was identified as a myeloid differentiation primary response gene. There is evidence consistent with it’s involvement in G2/M cell cycle arrest and apoptosis in response to multiple stressors, including genotoxic and oncogenic stress. To investigate the effect of Gadd45a in the development of CML, adaptive bone marrow transplantation experiments with either wild type or Gadd45a null myeloid progenitors expressing 210-kD BCR-ABL fusion oncoprotein revealed that loss of Gadd45a accelerated BCR-ABL driven CML resulting in the development of a more aggressive AML/BC like disease. Recent newly obtained data indicate that number of Gadd45a deficient Leukemic stem cells (LSC) harboring BCR-ABL increased as disease progressed confirming Gadd45a as a crucial tumor suppressor in CML. Recent data also indicate, that transformed Gadd45a deficient progenitors exhibit increased proliferation and decreased apoptosis, associated with enhanced PI3K-AKT-mTOR-4E-BP1 signaling and upregulated oncogenic p30C/EBPα. More importantly, newly obtained data indicate that Gadd45a transcript levels in peripheral blood of human blast crisis (BC-CML) samples was found to be reduced compared to accelerated phase (AP-CML), chronic phase (CP-CML) and normal controls, assessed by Quantitative real time PCR analysis. Collectivly these data strongly suggest that Gadd45a expression is a novel prognostic indicator of CML progression, implicating Gadd45a as a downregulated target of BCR-ABL associated with progression to more aggressive stages. To conclude, our findings provide novel evidence that Gadd45a functions as a suppressor of BCR/ABL driven myeloid leukemogenesis, & that suppresion of Gadd45a is associated with CML progression. These data provide the impetus to further elucidate the role Gadd45a plays in suppressing the development of CML, and explore how its loss contributes to the progression of CML to more aggressive leukemic phenotypes. Disclosures: No relevant conflicts of interest to declare.

Abstract Gadd45a, Gadd45b, and Gadd45g encode for small (18 kD), evolutionarily conserved proteins that play a pivotal role in diverse cellular functions such as cell cycle arrest, DNA repair, and are regulated by the nature of the stress stimulus encountered, its magnitude, and the cell type. Here, we show that Gadd45b negatively regulates stress-induced senescence. By using the 3T3 growth protocol to study tissue culture-induced senescence; we observed that Gadd45b KO MEFs display reduced growth rates at early passages (by passage 5) compared to wild type (WT) MEFs. Notably, this is in contrast with Gadd45a KO MEFs that show enhanced growth rate and escape senescence. Furthermore, senescence associated (SA) βgal staining showed increased blue stained cells at earlier passages in Gadd45b KO MEFs compared to WT MEFs. When MEFs were cultured at 3% oxygen, Gadd45b KO MEFs at 3% oxygen showed increased growth rate compared to MEFs at 21% oxygen, but growth was still retarded compared to WT MEFs at 21%, indicating that Gadd45b KO MEFs have increased sensitivity to oxidative stress leading to increased senescence. Additionally, we also observed that Gadd45b levels in WT MEFs increased with increasing passage number. Propidium Iodide staining and FACS analysis showed that Gadd45b KO MEFs arrest at the G2M phase of cell cycle. At passage 7, the majority (75%) of Gadd45b KO MEFs were observed to arrest at the G2M phase, whereas only 15% of WT MEFs arrested at G2M. This finding is in striking contrast to other senescent MEFs, which normally arrest at G1. Furthermore, FACS analysis of phospho-histone H3 (ser10) stained cells (mitotic) showed that, Gadd45b KO MEFs have less phospho-histone H3-positive cells than WT MEFs indicating that Gadd45b KO MEFs are arrested in G2 phase rather than M phase. Recent data also indicate that Gadd45b KO MEFs show increased phospho-histone H2Ax and phospho-p53 staining compared to WT MEFs, suggesting that loss of Gadd45b leads to increased DNA damage signaling. Interestingly, other stressors such as sub-lethal H2O2 as well as UV irradiation, known to increase oxidative stress, were observed to trigger increased premature senescence in Gadd45b KO MEFs compared to WT MEFs. Notably, staining embryos for SA βgal, we show that embryos from Gadd45b KO mice exhibit increased SA βgal staining compared to WT embryos, thus providing in vivo evidence for increased senescence in Gadd45b KO mice. Taken together, these data provide the impetus to further elucidate the role of Gadd45b in G2 arrest and premature senescence. Note: This abstract was not presented at the meeting. Citation Format: Andrew Magimaidas, Barbara Hoffman, Dan Liebermann. Gadd45b deficiency impairs G2/M cell cycle progression leading to premature senescence. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2252. doi:10.1158/1538-7445.AM2014-2252

Background Gadd45a is a member of the Gadd45 family of genes that are known stress sensors. Gadd45a has been shown to serve as an effector in oncogenic stress in breast carcinogenesis in murine models. The present study was aimed at clarifying the expression of Gadd45a in human breast cancer and its correlation with clinicopathologic features. Methods The expression levels of Gadd45a in breast tissue samples of female breast surgery cases were examined by immunohistochemistry (IHC) using a Gadd45a antibody. Percent staining was determined and statistical analyses were applied to determine prognostic correlations. Results 56 female breast surgery cases were studied: Normal (11), Luminal A (9), Luminal B (11), HER2+ (10), Triple Negative (15). There was a highly significant difference in percent Gadd45a staining between groups [Mean]: Normal 16.3%; Luminal A 65.3%; Luminal B 80.7%; HER2+ 40.5%; TN 32%, P < 0.001, ANOVA. Gadd45a IHC levels for Normal cases found 82% negative/low. Luminal A breast cancer cases were found to be 67% high. Luminal B breast cancers were 100% high. Her2+ cases were 50% negative/low. Triple Negative cases were 67% negative/low. This difference in distribution of Gadd45a levels across breast cancer receptor subtypes was significant, P = 0.0009. Conclusions Gadd45a levels are significantly associated with hormone receptor status in human breast cancer. Normal breast tissue displays low Gadd45a levels. High Gadd45a levels are associated with Luminal A and Luminal B subtypes. Absence of hormone receptors in Triple Negative subtype is associated with Negative/Low levels of Gadd45a. Further studies are indicated to elucidate the role of Gadd45a in breast cancer as a potential prognosticator or target for treatment.

Emerging evidence indicates that the Gadd45 family of genes play a unique and critical role as sensors of stress, including genotoxic, physiological and oncogenic stress. The stress response Gadd45 fa

To determine fetal gender, RhD genotyping and fetal DNA quantification during first screen using peripheral maternal dry blood spot. Fetal DNA was extracted from both Guthrie card and peripheral blood samples of RhD-negative pregnant in the first trimester. Probe specific Realtime PCRs were used to detect fetal gender gene (male -DSY14, female - S05, S06 and S10A) and Exon 4-6 of the RhD gene. Confirmation was obtained after delivery. Both Simplex (S) and Duplex (D) realtime PCRs were performed for quantification of fetal DNA. RhD amplification of exon 4, 5 or 6 was determined 8/13 and 5/13 fetuses to be Rh-positive or Rh-negative, respectively. One Rh-negative baby was incorrectly diagnosed as positive. Except for one, sex determination was correct in all cases. Quantification studies using S PCR and ratio of expressional levels of fetal gender genes over the β-actin levels detected fetal DYS14 gene: positive, ratio= 3.45814893 from mononuclear layer and 0.00445611 from Guthrie card. Using D PCR and ratio of expressional levels of fetal gender genes (female) and RhD gene over the predetermined copy number gene levels detected fetal female polymorphism gene S06: positive, copy number= 1.254070363 and RhD gene exon4 copy number= 6.601552872, exon5 copy number= 9.003016417, exon6 copy number=11.21182. Our study revealed that fetal DNA from maternal dry blood samples placed on Guthrie card during the first trimester screen were reliable sources for fetal sex and fetal RhD determination as well as fetal DNA quantification. Although this method is prospective for convenient testing, fetal DNA trace from previous pregnancy may impair the accuracy of the results. Fetal DNA quantification has the potential in clinical use not only for accurate fetal genotyping but also for numerous applications such as predicting anomalies & pregnancy complications in the current pregnancy, early in the first trimester.

The stress response gadd45 gene family participates in cell cycle control, cell survival, apoptosis, maintenance of genomic stability, DNA repair, and active DNA demethylation, in response to environmental and physiological stress including oncogenic stress. Given these diverse functions, it is anticipated that gadd45 genes can influence the initiation and progression of malignancy and the response to different treatments. This chapter will provide an overview of how the different members of the gadd45 gene family are expressed in different tumors and leukemia, how this may impact on progression of disease, and what happens when expression is manipulated. Studies from human tumor/leukemia samples, cell lines, and animal models are included in this review. An overriding theme is that each of the gadd45 genes has both tumor suppressor and tumor promoter functions, dependent on the tissue/cell type and transforming event.

The transcription factor early growth response 1 (Egr-1) gene was identified as a macrophage differentiation primary response gene, shown to be essential for and to restrict differentiation along the macrophage lineage. There’s evidence consistent with Egr-1 behaving as a tumor suppressor of leukemia, both in vivo and in vitro, including (1) loss of Egr-1 associated with treatment derived AMLs; (2) deregulated Egr-1 overriding blocks in myeloid differentiation, and (3) haplo-insufficiency of Egr-1 in mice leading to increased development of myeloid disorders following treatment with the potent DNA alkylating agent, N- ethyl-nitrosourea (ENU). BCR-ABL driven leukemia (Chronic Myelogenous Leukemia [CML]) was chosen as a model system to investigate the role of Egr-1 as a tumor suppressor for different leukemias. CML is a disease resulting from the neoplastic transformation of hematopoietic stem cells (HSC) with the BCR-ABL oncogene. The BCR-ABL protein is a constitutively active tyrosine kinase, which promotes cell survival and proliferation by means of diverse intracellular signaling pathways, thereby being the culprit for malignant transformation. Although the Tyrosine Kinase Inhibitor (TKI) imatinib mesylate (Gleevec, Novartis) is effectively used on CML patients, resistance to imatinib has been described. Thus there is a high priority to enhance our understanding of how BCR/ABL subverts normal hematopoiesis and to identify novel targets for therapy. It was observed that Egr-1 expression is reduced in bone marrow (BM) of CML patients, and its expression is further reduced in more advanced stages of CML. Consistent with this data, Egr-1 expression is reduced in BCR-ABL-expressing murine BM. The tumor suppressor role of Egr-1 in CML was validated using mouse models. Lethally irradiated syngeneic wild type mice were reconstituted with bone marrow (BM) from either wild type or Egr-1 null mice transduced with a 210-kD BCR-ABL-expressing MSCV-retrovirus (bone marrow transplantation {BMT}). Loss of Egr-1 accelerated the development of BCR-ABL driven leukemia in recipient mice. Furthermore, no statistically significant difference in the percentage of stem cells (Lin-Sca+c-Kit+, LSK) was observed between Egr-1 WT and Egr-1-/- BM. Thus, the BM stem cell compartment of the Egr-1-/- mice does not offer a quantitative advantage to justify the faster development of leukemia compared to Egr-1 WT mice. An increased population of lineage negative BM cells was observed in Egr-1-/- BCR-ABL recipients when compared to animals transplanted with WT BCR-ABL BM, consistent with more rapid development of disease. Preliminary results from serial BMT has shown that Egr-1-/- BCR-ABL BM has an increased leukemic burden when compared to the WT counterpart. Data from serial colony transfer and studies using spleens from diseased mice as well as BCR-ABL-expressing BM will be presented. These data could result in novel targets for diagnosis, prognosis, and targeted therapeutics, including strategies for activating Egr-1 expression, that can be used to treat CML, as well as other leukemic diseases. Disclosures: No relevant conflicts of interest to declare.

Accumulating evidence suggests that placental stresses during pregnancy can play an important role in the pathogenesis of preeclampsia. A common signal pathway that senses and converts placental stresses into intracellular stress response may be contributing to this pathology. Based on our previous findings, we extended our investigation to establish that Gadd45a stress signaling regulates sFlt-1 levels, particularly in placenta, when exposed to various preeclampsia-associated stresses including AT-1 receptor agonist (Angiotensin II), hypoxia, and inflammatory cytokines. Using a placental explant model, we found that Gadd45a was induced in response to all the preeclampsia stresses stated above. Although stress induced Gadd45a was associated with the activation of its downstream effectors phospho-p38 and phospho-JNK, the subsequent regulation of sFlt-1 levels occurred through either one of these effectors, but not both. These observations indicate that Gadd45a signaling may work as a hub connecting placental stresses and the pathogenesis of preeclampsia. It also provides evidence to justify testing the role of Gadd45 in the etiology of preeclampsia using in vivo mouse (i.e., Gadd45a null mice) models. J. Cell. Physiol. 228: 362370, 2013. (c) 2012 Wiley Periodicals, Inc.