Loss of the stress sensor GADD45A promotes stem cell activity and ferroptosis resistance in LGR4/HOXA9-dependent AML.

ABSTRACT: The overall prognosis of acute myeloid leukemia (AML) remains dismal, largely because of the inability of current therapies to kill leukemia stem cells (LSCs) with intrinsic resistance. Loss of the stress sensor growth arrest and DNA damage-inducible 45 alpha (GADD45A) is implicated in poor clinical outcomes, but its role in LSCs and AML pathogenesis is unknown. Here, we define GADD45A as a key downstream target of G protein-coupled receptor (LGR)4 pathway and discover a regulatory role for GADD45A loss in promoting leukemia-initiating activity and oxidative resistance in LGR4/HOXA9-dependent AML, a poor prognosis subset of leukemia. Knockout of GADD45A enhances AML progression in murine and patient-derived xenograft (PDX) mouse models. Deletion of GADD45A induces substantial mutations, increases LSC self-renewal and stemness in vivo, and reduces levels of reactive oxygen species (ROS), accompanied by a decreased response to ROS-associated genotoxic agents (eg, ferroptosis inducer RSL3) and acquisition of an increasingly aggressive phenotype on serial transplantation in mice. Our single-cell cellular indexing of transcriptomes and epitopes by sequencing analysis on patient-derived LSCs in PDX mice and subsequent functional studies in murine LSCs and primary AML patient cells show that loss of GADD45A is associated with resistance to ferroptosis (an iron-dependent oxidative cell death caused by ROS accumulation) through aberrant activation of antioxidant pathways related to iron and ROS detoxification, such as FTH1 and PRDX1, upregulation of which correlates with unfavorable outcomes in patients with AML. These results reveal a therapy resistance mechanism contributing to poor prognosis and support a role for GADD45A loss as a critical step for leukemia-initiating activity and as a target to overcome resistance in aggressive leukemia.

Gadd45 in Normal Hematopoiesis and Leukemia.

Gadd45a, Gadd45b, and Gadd45g have been implicated in cell cycle arrest, DNA repair, apoptosis, innate immunity, genomic stability, and modulation of normal blood cell development and leukemia. Each of the Gadd45 genes was shown to be regulated independently in myeloid cells in response to cytokine stimulation modulating blood cell survival and differentiation, including maintaining the quiescent stem cell pool. Gadd45a and Gadd45b were also shown to mediate the protective effects from UV in hematopoietic cells by separate signaling pathways involving either p38 activation or JNK inhibition. Furthermore, it was shown that gadd45a methylation in AML is predictive of poor survival. It was also shown that loss of Gadd45b accelerates the development of BCR-ABL driven CML in mice and leads to decreased median survival. The Gadd45b-deficient CML progenitors exhibited increased proliferation and decreased apoptosis, and this was associated with hyper-activation of c-Jun NH2-terminal kinase and Stat5. Moreover, loss of Gadd45a also accelerated the development of BCR-ABL driven CML, and this was associated with enhanced PI3K-AKT-mTOR-4E-BP1 signaling, upregulation of p30C/EBPα expression, and hyper-activation of p38 and Stat5. In human patients with chronic phase CML, gadd45a expression is up-regulated, whereas in accelerated and blast crisis phase patients, gadd45a is downregulated. Collectively, these results provide novel evidence that Gadd45a functions as a suppressor of BCR/ABL driven leukemia and may serve as a unique prognostic marker of CML progression. Thus Gadd45 proteins provide excellent targets for leukemia therapy.

Gadd45 in Senescence.

Gadd45a, Gadd45b, and Gadd45g have been implicated in cell cycle arrest, DNA repair, apoptosis, innate immunity, genomic stability, and more recently in senescence. Evidence has accumulated that Gadd45a deficiency results in escape of mouse embryo fibroblasts from senescence, whereas Gadd45b deficiency promotes premature senescence and skin aging. Moreover, recently Gadd45b deficiency was found to promote senescence and attenuate liver fibrosis, whereas Gadd45a was observed to exert a protective effect against hepatic fibrosis. These findings indicate that the Gadd45 stress response proteins play important roles in modulating cellular responses to senescence. Thus, exploring how Gadd45 proteins modulate cellular senescence has the potential to provide new and innovative tools to treat cancer as well as liver disease.

The ERK2-DBP domain opposes pathogenesis of a mouse JAK2V617F-driven myeloproliferative neoplasm.

Although Ras/mitogen-activated protein kinase (MAPK) signaling is activated in most human cancers, attempts to target this pathway using kinase-active site inhibitors have not typically led to durable clinical benefit. To address this shortcoming, we sought to test the feasibility of an alternative targeting strategy, focused on the ERK2 substrate binding domains, D and DEF binding pocket (DBP). Disabling the ERK2-DBP domain in mice caused baseline erythrocytosis. Consequently, we investigated the role of the ERK2-D and -DBP domains in disease, using a JAK2-dependent model of polycythemia vera (PV). Of note, inactivation of the ERK2-DBP domain promoted the progression of disease from PV to myelofibrosis, suggesting that the ERK2-DBP domain normally opposes progression. ERK2-DBP inactivation also prevented oncogenic JAK2 kinase (JAK2V617F) from promoting oncogene-induced senescence in vitro. The ERK2-DBP mutation attenuated JAK2-mediated oncogene-induced senescence by preventing the physical interaction of ERK2 with the transcription factor Egr1. Because inactivation of the ERK2-DBP created a functional ERK2 kinase limited to binding substrates through its D domain, these data suggested that the D domain substrates were responsible for promoting oncogene-induced progenitor growth and tumor progression and that pharmacologic targeting of the ERK2-D domain may attenuate cancer cell growth. Indeed, pharmacologic agents targeting the ERK2-D domain were effective in attenuating the growth of JAK2-dependent myeloproliferative neoplasm cell lines. Taken together, these data indicate that the ERK-D and -DBP domains can play distinct roles in the progression of neoplasms and that the D domain has the potential to be a potent therapeutic target in Ras/MAPK-dependent cancers.

Inhibiting calpain 1 and 2 in cyclin G associated kinase-knockout mice mitigates podocyte injury.

Evidence for reduced expression of cyclin G associated kinase (GAK) in glomeruli of patients with chronic kidney disease was observed in the Nephroseq human database, and GAK was found to be associated with the decline in kidney function. To examine the role of GAK, a protein that functions to uncoat clathrin during endocytosis, we generated podocyte-specific Gak-knockout mice (Gak-KO), which developed progressive proteinuria and kidney failure with global glomerulosclerosis. We isolated glomeruli from the mice carrying the mutation to perform messenger RNA profiling and unearthed evidence for dysregulated podocyte calpain protease activity as an important contributor to progressive podocyte damage. Treatment with calpain inhibitor III specifically inhibited calpain-1/-2 activities, mitigated the degree of proteinuria and glomerulosclerosis, and led to a striking increase in survival in the Gak-KO mice. Podocyte-specific deletion of Capns1, essential for calpain-1 and calpain-2 activities, also improved proteinuria and glomerulosclerosis in Gak-KO mice. Increased podocyte calpain activity-mediated proteolysis of IκBα resulted in increased NF-κB p65-induced expression of growth arrest and DNA-damage-inducible 45 beta in the Gak-KO mice. Our results suggest that loss of podocyte-associated Gak induces glomerular injury secondary to calcium dysregulation and aberrant calpain activation, which when inhibited, can provide a protective role.

Targeting RSPO3-LGR4 Signaling for Leukemia Stem Cell Eradication in Acute Myeloid Leukemia.

Signals driving aberrant self-renewal in the heterogeneous leukemia stem cell (LSC) pool determine aggressiveness of acute myeloid leukemia (AML). We report that a positive modulator of canonical WNT signaling pathway, RSPO-LGR4, upregulates key self-renewal genes and is essential for LSC self-renewal in a subset of AML. RSPO2/3 serve as stem cell growth factors to block differentiation and promote proliferation of primary AML patient blasts. RSPO receptor, LGR4, is epigenetically upregulated and works through cooperation with HOXA9, a poor prognostic predictor. Blocking the RSPO3-LGR4 interaction by clinical-grade anti-RSPO3 antibody (OMP-131R10/rosmantuzumab) impairs self-renewal and induces differentiation in AML patient-derived xenografts but does not affect normal hematopoietic stem cells, providing a therapeutic opportunity for HOXA9-dependent leukemia.

Loss of Gadd45b accelerates BCR-ABL-driven CML.

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.

Loss of Egr1, a human del5q gene, accelerates BCR-ABL driven chronic myelogenous leukemia.

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.

First trimester noninvasive fetal RHD genotyping using maternal dried blood spots.

OBJECTIVE: This 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. METHOD: Both 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. RESULTS: A 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%. CONCLUSION: Maternal 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. © 2017 John Wiley & Sons, Ltd.

Gadd45a deficiency accelerates BCR-ABL driven chronic myelogenous leukemia.

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/EBPα 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.

Gadd45b deficiency promotes premature senescence and skin aging.

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.

Gadd45 in modulation of solid tumors and leukemia.

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.

Gadd45a levels in human breast cancer are hormone receptor dependent.

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.

Preeclampsia-associated stresses activate Gadd45a signaling and sFlt-1 in placental explants.

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.

Genetic deletion of Gadd45b, a regulator of active DNA demethylation, enhances long-term memory and synaptic plasticity.

Dynamic epigenetic mechanisms including histone and DNA modifications regulate animal behavior and memory. While numerous enzymes regulating these mechanisms have been linked to memory formation, the regulation of active DNA demethylation (i.e., cytosine-5 demethylation) has only recently been investigated. New discoveries aim toward the Growth arrest and DNA damage-inducible 45 (Gadd45) family, particularly Gadd45b, in activity-dependent demethylation in the adult CNS. This study found memory-associated expression of gadd45b in the hippocampus and characterized the behavioral phenotype of gadd45b(-/-) mice. Results indicate normal baseline behaviors and initial learning but enhanced persisting memory in mutants in tasks of motor performance, aversive conditioning and spatial navigation. Furthermore, we showed facilitation of hippocampal long-term potentiation in mutants. These results implicate Gadd45b as a learning-induced gene and a regulator of memory formation and are consistent with its potential role in active DNA demethylation in memory.

Gadd45b knockout mice exhibit selective deficits in hippocampus-dependent long-term memory.

Growth arrest and DNA damage-inducible ß (Gadd45b) has been shown to be involved in DNA demethylation and may be important for cognitive processes. Gadd45b is abnormally expressed in subjects with autism and psychosis, two disorders associated with cognitive deficits. Furthermore, several high-throughput screens have identified Gadd45b as a candidate plasticity-related gene. However, a direct demonstration of a link between Gadd45b and memory has not been established. The current studies first determined whether expression of the Gadd45 family of genes was affected by contextual fear conditioning. Gadd45b, and to a lesser extent Gadd45g, were up-regulated in the hippocampus following contextual fear conditioning, whereas Gadd45a was not. Next, Gadd45b knockout mice were tested for contextual and cued fear conditioning. Gadd45b knockout mice exhibited a significant deficit in long-term contextual fear conditioning; however, they displayed normal levels of short-term contextual fear conditioning. No differences between Gadd45b knockout and wild-type mice were observed in cued fear conditioning. Because cued fear conditioning is hippocampus independent, while contextual fear conditioning is hippocampus dependent, the current studies suggest that Gadd45b may be important for long-term hippocampus-dependent memory storage. Therefore, Gadd45b may be a novel therapeutic target for the cognitive deficits associated with many neurodevelopmental, neurological, and psychiatric disorders.

Gadd45a and Gadd45b modulate innate immune functions of granulocytes and macrophages by differential regulation of p38 and JNK signaling.

Gadd45 proteins function as stress sensors in response to various physiological and environmental stressors, interacting with other cellular proteins implicated in cellular stress responses, including p38 and JNK. This study shows that mice lacking either Gadd45a or Gadd45b are defective in the recruitment of granulocytes and macrophages to the intra-peritoneal cavity following intra-peritoneal administration of the bacterial cell wall pathogen-associated molecular pattern lipopolysaccharide (LPS). Bone marrow derived granulocytes and macrophages lacking either Gadd45a or Gadd45b are shown to be impaired in their chemotactic response to LPS, as well as other inflammatory stimuli such as N-formyl-methionine-leucine-phenylalanine and IL-8. Evidence was obtained also implicating Gadd45a and Gadd45b in other myeloid innate immune functions, including reactive oxygen species production, phagocytosis, and adhesion. Gadd45a and Gadd45b activation of p38 kinase was implicated in the response of granulocytes to LPS mediated chemotaxis, whereas Gadd45a and Gadd45b curtailment of JNK activation was linked to chemotaxis of macrophages in response to LPS. Collectively, these data highlight a novel role for both Gadd45a and Gadd45b in myeloid innate immune functions by differential modulation of p38 and JNK signaling in granulocytes compared to macrophages.

Gadd45 stress sensors in malignancy and leukemia.

Gadd45 proteins, including Gadd45a, Gadd45b, and Gadd45g, have been implicated in stress signaling in response to physiological and environmental stress, including oncogenic stress, which can result in cell cycle arrest, DNA repair, cell survival, senescence, and apoptosis. The function of Gadd45 as a stress sensor is mediated via a complex interplay of physical interactions with other cellular proteins implicated in cell cycle regulation and the response of cells to stress, notably PCNA, p21, cdc2/cyclinB1, and the p38 and JNK stress response kinases. Altered expression of Gadd45 has been observed in multiple types of solid tumors as well as in hematopoietic malignancies. Using genetically engineered mouse models and bone-marrow transplantation, evidence has been obtained indicating that Gadd45 proteins can function to either promote or suppress tumor development and leukemia; this is dependent on the molecular nature of the activated oncogene and the cell type, via engagement of different signaling pathways.

Gadd45ß is an inducible coactivator of transcription that facilitates rapid liver growth in mice.

The growth arrest and DNA damage-inducible 45 (Gadd45) proteins act in many cellular processes. In the liver, Gadd45b (encoding Gadd45ß) is the gene most strongly induced early during both compensatory regeneration and drug-induced hyperplasia. The latter response is associated with the dramatic and rapid hepatocyte growth that follows administration of the xenobiotic TCPOBOP (1,4-bis[2-(3,5)-dichoropyridyloxy] benzene), a ligand of the nuclear receptor constitutive androstane receptor (CAR). Here, we have shown that Gadd45b-/- mice have intact proliferative responses following administration of a single dose of TCPOBOP, but marked growth delays. Moreover, early transcriptional stimulation of CAR target genes was weaker in Gadd45b-/- mice than in wild-type animals, and more genes were downregulated. Gadd45ß was then found to have a direct role in transcription by physically binding to CAR, and TCPOBOP treatment caused both proteins to localize to a regulatory element for the CAR target gene cytochrome P450 2b10 (Cyp2b10). Further analysis defined separate Gadd45ß domains that mediated binding to CAR and transcriptional activation. Although baseline hepatic expression of Gadd45b was broadly comparable to that of other coactivators, its 140-fold stimulation by TCPOBOP was striking and unique. The induction of Gadd45ß is therefore a response that facilitates increased transcription, allowing rapid expansion of liver mass for protection against xenobiotic insults.