The Earliest T-Precursors in the Mouse Embryo Are Susceptible to Leukemic Transformation.

Acute lymphoblastic leukemia (ALL) is the most common malignancy in pediatric patients. About 10-15% of pediatric ALL belong to T-cell ALL (T-ALL), which is characterized by aggressive expansion of immature T-lymphoblasts and is categorized as high-risk leukemia. Leukemia initiating cells represent a reservoir that is responsible for the initiation and propagation of leukemia. Its perinatal origin has been suggested in some childhood acute B-lymphoblastic and myeloblastic leukemias. Therefore, we hypothesized that child T-ALL initiating cells also exist during the perinatal period. In this study, T-ALL potential of the hematopoietic precursors was found in the para-aortic splanchnopleura (P-Sp) region, but not in the extraembryonic yolk sac (YS) of the mouse embryo at embryonic day 9.5. We overexpressed the Notch intracellular domain (NICD) in the P-Sp and YS cells and transplanted them into lethally irradiated mice. NICD-overexpressing P-Sp cells rapidly developed T-ALL while YS cells failed to display leukemia propagation despite successful NICD induction. These results suggest a possible role of fetal-derived T-cell precursors as leukemia-initiating cells.

Systems Biology Approach Identifies Prognostic Signatures of Poor Overall Survival and Guides the Prioritization of Novel BET-CHK1 Combination Therapy for Osteosarcoma.

Osteosarcoma (OS) patients exhibit poor overall survival, partly due to copy number variations (CNVs) resulting in dysregulated gene expression and therapeutic resistance. To identify actionable prognostic signatures of poor overall survival, we employed a systems biology approach using public databases to integrate CNVs, gene expression, and survival outcomes in pediatric, adolescent, and young adult OS patients. Chromosome 8 was a hotspot for poor prognostic signatures. The MYC-RAD21 copy number gain (8q24) correlated with increased gene expression and poor overall survival in 90% of the patients (n = 85). MYC and RAD21 play a role in replication-stress, which is a therapeutically actionable network. We prioritized replication-stress regulators, bromodomain and extra-terminal proteins (BETs), and CHK1, in order to test the hypothesis that the inhibition of BET + CHK1 in MYC-RAD21+ pediatric OS models would be efficacious and safe. We demonstrate that MYC-RAD21+ pediatric OS cell lines were sensitive to the inhibition of BET (BETi) and CHK1 (CHK1i) at clinically achievable concentrations. While the potentiation of CHK1i-mediated effects by BETi was BET-BRD4-dependent, MYC expression was BET-BRD4-independent. In MYC-RAD21+ pediatric OS xenografts, BETi + CHK1i significantly decreased tumor growth, increased survival, and was well tolerated. Therefore, targeting replication stress is a promising strategy to pursue as a therapeutic option for this devastating disease.

The Role of MDM2 in Promoting Genome Stability versus Instability.

In cancer, the mouse double minute 2 (MDM2) is an oncoprotein that contributes to the promotion of cell growth, survival, invasion, and therapeutic resistance. The impact of MDM2 on cell survival versus cell death is complex and dependent on levels of MDM2 isoforms, p53 status, and cellular context. Extensive investigations have demonstrated that MDM2 protein-protein interactions with p53 and other p53 family members (p63 and p73) block their ability to function as transcription factors that regulate cell growth and survival. Upon genotoxic insults, a dynamic and intricately regulated DNA damage response circuitry is activated leading to release of p53 from MDM2 and activation of cell cycle arrest. What ensues following DNA damage, depends on the extent of DNA damage and if the cell has sufficient DNA repair capacity. The well-known auto-regulatory loop between p53-MDM2 provides an additional layer of control as the cell either repairs DNA damage and survives (i.e., MDM2 re-engages with p53), or undergoes cell death (i.e., MDM2 does not re-engage p53). Furthermore, the decision to live or die is also influenced by chromatin-localized MDM2 which directly interacts with the Mre11-Rad50-Nbs1 complex and inhibits DNA damage-sensing giving rise to the potential for increased genome instability and cellular transformation.

Ref-1/APE1 as a Transcriptional Regulator and Novel Therapeutic Target in Pediatric T-cell Leukemia.

The increasing characterization of childhood acute lymphoblastic leukemia (ALL) has led to the identification of multiple molecular targets but has yet to translate into more effective targeted therapies, particularly for high-risk, relapsed T-cell ALL. Searching for master regulators controlling multiple signaling pathways in T-ALL, we investigated the multifunctional protein redox factor-1 (Ref-1/APE1), which acts as a signaling "node" by exerting redox regulatory control of transcription factors important in leukemia. Leukemia patients' transcriptome databases showed increased expression in T-ALL of Ref-1 and other genes of the Ref-1/SET interactome. Validation studies demonstrated that Ref-1 is expressed in high-risk leukemia T cells, including in patient biopsies. Ref-1 redox function is active in leukemia T cells, regulating the Ref-1 target NF-κB, and inhibited by the redox-selective Ref-1 inhibitor E3330. Ref-1 expression is not regulated by Notch signaling, but is upregulated by glucocorticoid treatment. E3330 disrupted Ref-1 redox activity in functional studies and resulted in marked inhibition of leukemia cell viability, including T-ALL lines representing different genotypes and risk groups. Potent leukemia cell inhibition was seen in primary cells from ALL patients, relapsed and glucocorticoid-resistant T-ALL cells, and cells from a murine model of Notch-induced leukemia. Ref-1 redox inhibition triggered leukemia cell apoptosis and downregulation of survival genes regulated by Ref-1 targets. For the first time, this work identifies Ref-1 as a novel molecular effector in T-ALL and demonstrates that Ref-1 redox inhibition results in potent inhibition of leukemia T cells, including relapsed T-ALL. These data also support E3330 as a specific Ref-1 small-molecule inhibitor for leukemia. Mol Cancer Ther; 16(7); 1401-11. ©2017 AACR.

Combination therapy in a xenograft model of glioblastoma: enhancement of the antitumor activity of temozolomide by an MDM2 antagonist.

OBJECTIVE Improvement in treatment outcome for patients with glioblastoma multiforme (GBM) requires a multifaceted approach due to dysregulation of numerous signaling pathways. The murine double minute 2 (MDM2) protein may fulfill this requirement because it is involved in the regulation of growth, survival, and invasion. The objective of this study was to investigate the impact of modulating MDM2 function in combination with front-line temozolomide (TMZ) therapy in GBM. METHODS The combination of TMZ with the MDM2 protein-protein interaction inhibitor nutlin3a was evaluated for effects on cell growth, p53 pathway activation, expression of DNA repair proteins, and invasive properties. In vivo efficacy was assessed in xenograft models of human GBM. RESULTS In combination, TMZ/nutlin3a was additive to synergistic in decreasing growth of wild-type p53 GBM cells. Pharmacodynamic studies demonstrated that inhibition of cell growth following exposure to TMZ/nutlin3a correlated with: 1) activation of the p53 pathway, 2) downregulation of DNA repair proteins, 3) persistence of DNA damage, and 4) decreased invasion. Pharmacokinetic studies indicated that nutlin3a was detected in human intracranial tumor xenografts. To assess therapeutic potential, efficacy studies were conducted in a xenograft model of intracranial GBM by using GBM cells derived from a recurrent wild-type p53 GBM that is highly TMZ resistant (GBM10). Three 5-day cycles of TMZ/nutlin3a resulted in a significant increase in the survival of mice with GBM10 intracranial tumors compared with single-agent therapy. CONCLUSIONS Modulation of MDM2/p53-associated signaling pathways is a novel approach for decreasing TMZ resistance in GBM. To the authors' knowledge, this is the first study in a humanized intracranial patient-derived xenograft model to demonstrate the efficacy of combining front-line TMZ therapy and an inhibitor of MDM2 protein-protein interactions.

Potentiation of Carboplatin-Mediated DNA Damage by the Mdm2 Modulator Nutlin-3a in a Humanized Orthotopic Breast-to-Lung Metastatic Model.

Triple-negative breast cancers (TNBC) are typically resistant to treatment, and strategies that build upon frontline therapy are needed. Targeting the murine double minute 2 (Mdm2) protein is an attractive approach, as Mdm2 levels are elevated in many therapy-refractive breast cancers. The Mdm2 protein-protein interaction inhibitor Nutlin-3a blocks the binding of Mdm2 to key signaling molecules such as p53 and p73α and can result in activation of cell death signaling pathways. In the present study, the therapeutic potential of carboplatin and Nutlin-3a to treat TNBC was investigated, as carboplatin is under evaluation in clinical trials for TNBC. In mutant p53 TMD231 TNBC cells, carboplatin and Nutlin-3a led to increased Mdm2 and was strongly synergistic in promoting cell death in vitro. Furthermore, sensitivity of TNBC cells to combination treatment was dependent on p73α. Following combination treatment, γH2AX increased and Mdm2 localized to a larger degree to chromatin compared with single-agent treatment, consistent with previous observations that Mdm2 binds to the Mre11/Rad50/Nbs1 complex associated with DNA and inhibits the DNA damage response. In vivo efficacy studies were conducted in the TMD231 orthotopic mammary fat pad model in NOD.Cg-Prkdc(scid)Il2rg(tm1Wjl)/SzJ (NSG) mice. Using an intermittent dosing schedule of combined carboplatin and Nutlin-3a, there was a significant reduction in primary tumor growth and lung metastases compared with vehicle and single-agent treatments. In addition, there was minimal toxicity to the bone marrow and normal tissues. These studies demonstrate that Mdm2 holds promise as a therapeutic target in combination with conventional therapy and may lead to new clinical therapies for TNBC.

Aberrant expression of functional BAFF-system receptors by malignant B-cell precursors impacts leukemia cell survival.

Despite exhibiting oncogenic events, patient's leukemia cells are responsive and dependent on signals from their malignant bone marrow (BM) microenvironment, which modulate their survival, cell cycle progression, trafficking and resistance to chemotherapy. Identification of the signaling pathways mediating this leukemia/microenvironment interplay is critical for the development of novel molecular targeted therapies.We observed that primary leukemia B-cell precursors aberrantly express receptors of the BAFF-system, BAFF-R, BCMA, and TACI. These receptors are functional as their ligation triggers activation of NF-κB, MAPK/JNK, and Akt signaling. Leukemia cells express surface BAFF and APRIL ligands, and soluble BAFF is significantly higher in leukemia patients in comparison to age-matched controls. Interestingly, leukemia cells also express surface APRIL, which seems to be encoded by APRIL-δ, a novel isoform that lacks the furin convertase domain. Importantly, we observed BM microenvironmental cells express the ligands BAFF and APRIL, including surface and secreted BAFF by BM endothelial cells. Functional studies showed that signals through BAFF-system receptors impact the survival and basal proliferation of leukemia B-cell precursors, and support the involvement of both homotypic and heterotypic mechanisms.This study shows an unforeseen role for the BAFF-system in the biology of precursor B-cell leukemia, and suggests that the target disruption of BAFF signals may constitute a valid strategy for the treatment of this cancer.

ClipR-59 interacts with Akt and regulates Akt cellular compartmentalization.

Akt is activated on the plasma membrane and its substrates are distributed throughout various cellular compartments. To phosphorylate its substrates, Akt needs to be recruited to specific intracellular compartments. Thus, regulation of Akt cellular compartmentalization constitutes an important mechanism to specify Akt signaling. Here, we report the identification of ClipR-59 as an Akt interaction protein. We show that the interaction of ClipR-59 with Akt is mediated by the CAP-Gly domain of ClipR-59 and kinase domain of Akt and is regulated by Akt phosphorylation. We demonstrate that ClipR-59 regulates the Akt membrane association through its interaction with Akt and membrane localization and, by modulating Akt cellular compartmentalization, differentially modulates phosphorylation of Akt substrates in adipocytes. Finally, we provide evidence that one of the Akt substrates whose phosphorylation is upregulated by ClipR-59 is AS160, a negative regulator of adipocyte glucose transport. Accordingly, ectopic expression of ClipR-59 enhances, whereas knockdown of ClipR-59 suppresses, adipocyte glucose transport. We suggest that ClipR-59 functions as a scaffold protein that interacts with phospho-Akt and recruits active Akt on the membrane and may play an important role in adipocyte glucose transport.

Insulin regulates TRB3 and other stress-responsive gene expression through induction of C/EBPbeta.

Pseudokinase TRB3 is an inducible gene whose expression is regulated by stress response and insulin and associated with insulin resistance and metabolic syndrome. In this report, we have investigated the mechanism under which insulin regulates TRB3 gene expression and demonstrated that insulin induces TRB3 expression via C/EBPbeta. We found that in Fao hepatoma and 3T3-L1 adipocytes, C/EBPbeta expression induced by insulin preceded that of TRB3 and that mutation of the C/EBPbeta binding site in TRB3 promoter abolished the responsiveness of the TRB3 gene to insulin. We further showed that ectopic expression of C/EBPbeta augmented, whereas knockdown of C/EBPbeta reduced, TRB3 expression induced by insulin. In addition, we presented data to show that insulin, through a similar mechanism under which insulin induces TRB3 expression, promotes the expression of genes such as ANAS, ATF3, BIP, and CHOP, which are typical stress-responsive genes. We also examined the impact of C/EBPbeta expression on Akt activation and found that inaction of C/EBPbeta not only augmented Akt activation but also obliterated the suppression of Akt activation due to prolonged insulin stimulation. We suggest, through induction of C/EBPbeta in hepatic cells and adipocytes, that insulin induces the expression of stress-responsive genes, which may represent a novel insulin action.

Ganglioside GM1 binding the N-terminus of amyloid precursor protein.

Secreted amyloid precursor protein (APPs) plays a role in several neuronal functions, including the promotion of synaptogenesis, neurite outgrowth and neuroprotection. Previous study has demonstrated that ganglioside GM1 inhibits the secretion of APPs; however the underlying mechanism remains unknown. Here we reported that GM1 can bind cellular full length APP and APPs secreted from APP(695) stably-transfected SH-SY5Y cells. To characterize the GM1-APP interaction further, we expressed and purified recombinant fragments of the N-terminal APP. Immunoprecipitation experiments revealed that GM1 was able to bind the recombinant APP(18-81) fragment. Moreover, the synthetic peptide APP(52-81) could inhibit the binding. Therefore, the binding site for GM1 appears to be located within residues 52-81 of APP. Furthermore, we found that only GM1, but not GD1a, GT1b and ceramide, binds APP-N-terminus, indicating that the specific binding depends on the sugar moiety of GM1. Fluorescent studies revealed a decrease in the intrinsic fluorescence intensity of the APP(52-81) peptide in phosphatidylcholine (PC)/GM1 vesicles. By using FTIR techniques, we found that the major secondary structure of the APP(52-81) peptide was altered in PC/GM1 vesicles. Our results demonstrate that GM1 binds the N-terminus of APP and induces a conformational change. These findings suggest that secreted APP is decreased by membrane GM1 binding to its precursor protein and provide a possible molecular mechanism to explain the involvement of GM1 in APP proteolysis and pathogenesis of Alzheimer's disease.

COP1 functions as a FoxO1 ubiquitin E3 ligase to regulate FoxO1-mediated gene expression.

COP1 is a Ring-Finger E3 ubiquitin ligase that is involved in plant development, mammalian cell survival, growth, and metabolism. Here we report that COP1, whose expression is enhanced by insulin, regulates FoxO1 protein stability. We found that in Fao hepatoma cells, ectopic expression of COP1 decreased, whereas knockdown of COP1 expression increased the level of endogenous FoxO1 protein without impacting other factors such as C/EBPalpha and CREB (cAMP-response element-binding protein). We further showed that COP1 binds FoxO1, enhances its ubiquitination, and promotes its degradation via the ubiquitin-proteasome pathway. To determine the biological significance of COP1-mediated FoxO1 protein degradation, we have examined the impact of COP1 on FoxO1-mediated gene expression and found that COP1 suppressed FoxO1 reporter gene as well as FoxO1 target genes such as glucose-6-phosphatase and phosphoenolpyruvate carboxykinase, two key targets for FoxO1 in the regulation of gluconeogenesis, with corresponding changes of hepatic glucose production in Fao cells. We suggest that by functioning as a FoxO1 E3 ligase, COP1 may play a role in the regulation of hepatic glucose metabolism.

PI3K activates negative and positive signals to regulate TRB3 expression in hepatic cells.

TRB3 is a pseudokinase whose expression is regulated during stress response and changing of nutrient status. TRB3 negatively regulates Akt activation and noticeably, TRB3 expression is induced by insulin. Here, we sought to determine the dynamic relationship between TRB3 expression and Akt activation. We find that insulin induces TRB3 expression in cell type dependent manner such that in hepatic cells and adipocytes but not Beta cells and muscle cells. In Fao hepatoma cells, induction of TRB3 expression by insulin restrains Akt activation and renders Akt refractory to further activation. In addition, we have also analyzed the roles of PI3K and its downstream kinases Akt and atypical PKC in TRB3 expression. Induction of TRB3 expression by insulin requires PI3K. However, inactivation of Akt enhances TRB3 expression whereas inhibition of PKCzeta expression impairs TRB3 expression induced by insulin. Our data demonstrated that PI3K conveys both negative and positive signals to TRB3 expression. We suggest that insulin-induced TRB3 expression functions as an indicator how multiple insulin-induced signal transduction pathways are balanced.

Differential activation of CREB by Akt1 and Akt2.

Members of Akt family are highly conserved protein kinase and yet, they show clearly distinct in vivo functions. Here, we have examined the abilities of Akt1 and Akt2 to activate CREB. We found that, in contrast to Akt1 that induces CREB phosphorylation at Ser-133 and CREB target gene expression, Akt2 was unable to induce CREB phosphorylation at Ser-133 in vivo and CREB target gene expression. This difference is specific to CREB as both Akt1 and Akt2 similarly inhibits FoxO1 mediated gene expression. We further showed that the regulatory domain of Akt plays a critical role to confer Akt substrate specificity as substitution of regulatory domain of Akt1 with that of Akt2 abolished the ability of Akt1 to activate CREB. We suggest that the regulatory domain of Akts contributes to the functional difference between Akt1 and Akt2.