Brd4 regulates the expression of essential autophagy genes and Keap1 in AML cells.

We have recently reported that activation of Brd4 is associated with the presence of autophagy in NPMc+ and MLL AML cells. In order to determine the mechanisms underlying this relationship, we have examined the role of Brd4 in regulating the expression of several genes that are central to the process of autophagy. We found that Brd4 binds to the promoters of ATG 3, 7 and CEBPß, and expression of these genes is markedly reduced by inhibitors of Brd4, as well as by Brd4-shRNA and depletion of CEBPß. Inhibitors of Brd4 also dramatically suppress the transcription of Keap1, thereby increasing the expression of anti-oxidant genes through the Nrf2 pathway and reducing the cytotoxicity induced by Brd4 inhibitors. Elimination of ATG3 or KEAP1 expression using CRISPR-cas9 mediated genomic editing markedly reduced autophagy. We conclude that Brd4 plays a significant role in autophagy activation through the direct transcriptional regulation of genes essential for it, as well as through the Keap1-Nrf2 axis in NPMc+ and MLL-fusion AML cells.

Autophagy mediates proteolysis of NPM1 and HEXIM1 and sensitivity to BET inhibition in AML cells.

The mechanisms underlying activation of the BET pathway in AML cells remain poorly understood. We have discovered that autophagy is activated in acute leukemia cells expressing mutant nucleophosmin 1 (NPMc+) or MLL-fusion proteins. Autophagy activation results in the degradation of NPM1 and HEXIM1, two negative regulators of BET pathway activation. Inhibition of autophagy with pharmacologic inhibitors or through knocking down autophagy-related gene 5 (Atg5) expression increases the expression of both NPM1 and HEXIM1. The Brd4 inhibitors JQ1 and I-BET-151 also inhibit autophagy and increase NPM1 and HEXIM1 expression. We conclude that the degradation of NPM1 and HEXIM1 through autophagy in certain AML subsets contributes to the activation of the BET pathway in these cells.

Expression and Role of the ErbB3-Binding Protein 1 in Acute Myelogenous Leukemic Cells.

PURPOSE: The ErbB3-binding protein 1 (Ebp1) has been implicated in diverse cancers as having either oncogenic or tumor suppressor activities. The present study was undertaken to determine the effects of Ebp1 expression in AML cells and to determine the mechanisms by which Ebp1 promotes cell proliferation in these cells. EXPERIMENTAL DESIGN: The expression of Ebp1 was studied in mononuclear cells obtained from the peripheral blood of 54 patients with AML by Western blot analysis. The effects of Ebp1 expression on proliferating cell nuclear antigen (PCNA) expression and cell proliferation was measured using Western blot analysis, immunoprecipitation, in vitro ubiquitination, and colony-forming assays. The role of Ebp1 in promoting rRNA synthesis and cell proliferation was evaluated by measuring the level of pre-rRNA and the recruitment of Pol I to rDNA. RESULTS: Ebp1 is highly expressed in acute myelogenous leukemia (AML) cells and regulates the level of ribosomal RNA (rRNA) synthesis by binding to RNA Polymerase I (Pol I) and enhancing the formation of the Pol I initiation complex. Ebp1 also increases the stability of PCNA protein by preventing its interaction with Mdm2, for which it is a substrate. CONCLUSIONS: These results demonstrate an important role of Ebp1 in promoting cell proliferation in AML cells through the regulation of both rRNA synthesis and PCNA expression. Clin Cancer Res; 22(13); 3320-7. ©2016 AACR.

Selective Toxicity of Investigational Ixazomib for Human Leukemia Cells Expressing Mutant Cytoplasmic NPM1: Role of Reactive Oxygen Species.

PURPOSE: This study was performed to determine whether the investigational proteasome inhibitor ixazomib demonstrated selective antineoplastic activity against acute myelogenous leukemia cells expressing a mutated nucleophosmin-1 gene and to gain a better understanding of its mechanisms of action. EXPERIMENTAL DESIGN: The cytotoxic effects of ixazomib treatment were analyzed in human acute myelogenous leukemia (AML) cell lines and primary AML samples expressing wild-type or mutated NPM1 (NPMc(+)). The potential roles of oxidative stress in mediating cytotoxic activity were determined using flow cytometry, enzyme-based assays, and Western blots. RESULTS: Apoptosis induced by ixazomib was abrogated by knockdown of NPM1/NPMc(+)expression using an inducible shRNA construct and enhanced by NPMc(+)overexpression. Cytotoxicity was associated with superoxide generation and was reduced by the addition of the antioxidant N-acetylcysteine. AML cells expressing NPMc(+)had significantly reduced levels of intracellular glutathione and NADPH associated with reduced antioxidant responses to drug treatment. Treatment of 3 patients with relapsed NPMc(+)AML resulted in an antileukemic effect in 1 patient as demonstrated by a marked reduction of leukemic blasts in the peripheral blood. Efficacy was associated with superoxide generation, reduced glutathione levels, and reduced mRNA and protein expression of antioxidant effectors in responding cells. CONCLUSIONS: In this study, a direct association was observed between NPMc(+)expression in AML, reduced antioxidant responses, and enhanced sensitivity to an oral proteasome inhibitor that induces oxidative stress. These data suggest that intracellular determinants of antioxidant responses may be good predictors of therapeutic response to ixazomib.

Reversibility of Defective Hematopoiesis Caused by Telomere Shortening in Telomerase Knockout Mice.

Telomere shortening is common in bone marrow failure syndromes such as dyskeratosis congenita (DC), aplastic anemia (AA) and myelodysplastic syndromes (MDS). However, improved knowledge of the lineage-specific consequences of telomere erosion and restoration of telomere length in hematopoietic progenitors is required to advance therapeutic approaches. We have employed a reversible murine model of telomerase deficiency to compare the dependence of erythroid and myeloid lineage differentiation on telomerase activity. Fifth generation Tert-/- (G5 Tert-/-) mice with shortened telomeres have significant anemia, decreased erythroblasts and reduced hematopoietic stem cell (HSC) populations associated with neutrophilia and increased myelopoiesis. Intracellular multiparameter analysis by mass cytometry showed significantly reduced cell proliferation and increased sensitivity to activation of DNA damage checkpoints in erythroid progenitors and in erythroid-biased CD150hi HSC, but not in myeloid progenitors. Strikingly, Cre-inducible reactivation of telomerase activity restored hematopoietic stem and progenitor cell (HSPC) proliferation, normalized the DNA damage response, and improved red cell production and hemoglobin levels. These data establish a direct link between the loss of TERT activity, telomere shortening and defective erythropoiesis and suggest that novel strategies to restore telomerase function may have an important role in the treatment of the resulting anemia.

The effect of an intense mentoring program on junior investigators' preparation for a patient-oriented clinical research career.

PROBLEM: There is a recognized need to translate scientific discoveries to patient-oriented clinical research (POCR). Several obstacles interfere with the successful recruitment and retention of physicians for POCR careers. APPROACH: The American Society of Hematology developed a yearlong educational and mentoring experience, the Clinical Research Training Institute (CRTI), for early-career physician-scientists from multiple institutions throughout the United States and Canada pursuing POCR careers. Several academic outcome measures of the 140 participants in the first seven years (2003-2010) of CRTI were evaluated by reviewing former trainee participants' curriculum vitae and survey responses. OUTCOMES: Ethnic, racial, and gender diversity of CRTI trainees was reflective of the proportions represented across U.S. hematology/oncology fellowship programs. Eighty-six percent (109/126) of trainees reported success establishing a POCR study; nearly half (62/126) had primarily research-focused jobs. Former CRTI trainees received at least 262 external grant awards and published 1,035 peer-reviewed manuscripts, 173 chapters, and 115 review articles. NEXT STEPS: Because mentorship is key to developing a successful career, the CRTI program is being modified to enhance longitudinal mentorship by CRTI faculty mentors and mentors at trainees' home institutions, as well as to encourage the establishment of collaborations and the potential for research project success. Efforts to make the CRTI experience available to more phy sicians, include more CRTI graduates as faculty, and increase participation by hematologists from backgrounds under represented in medicine are under way.

Regulation of ribosomal RNA synthesis in T cells: requirement for GTP and Ebp1.

Mycophenolic acid (MPA) is the active metabolite of mycophenolate mofetil, an effective immunosuppressive drug. Both MPA and mycophenolate mofetil are highly specific inhibitors of guanine nucleotide synthesis and of T-cell activation. However, the mechanism by which guanine nucleotide depletion suppresses T-cell activation is unknown. Depletion of GTP inhibits ribosomal RNA synthesis in T cells by inhibiting transcription initiation factor I (TIF-IA), a GTP-binding protein that recruits RNA polymerase I to the ribosomal DNA promoter. TIF-IA-GTP binds the ErbB3-binding protein 1, and together they enhance the transcription of proliferating cell nuclear antigen (PCNA). GTP binding by TIF-IA and ErbB3-binding protein 1 phosphorylation by protein kinase C δ are both required for optimal PCNA expression. The protein kinase C inhibitor sotrastaurin markedly potentiates the inhibition of ribosomal RNA synthesis, PCNA expression, and T-cell activation induced by MPA, suggesting that the combination of the two agents are more highly effective than either alone in inducing immunosuppression.

Regulation of ribosomal gene expression in cancer.

The ability of a cell to undergo malignant transformation is both associated with and dependent on a concomitant increase in protein synthesis due to increased cell division rates and biosynthetic activities. Protein synthesis, in turn, depends upon the synthesis of ribosomes and thus ultimately on the transcription of ribosomal RNA by RNA polymerase I that occurs in the nucleolus. Enlargement of nucleoli has long been considered a hallmark of the malignant cell, but it is only recently that the rate of synthesis of rRNA in the nucleolus has been recognized as both a critical regulator of cellular proliferation and a potential target for therapeutic intervention. As might be expected, the factors regulating rRNA synthesis are both numerous and complex. It is the objective of this review to highlight recent advances in understanding how rRNA synthesis is perturbed in transformed mammalian cells and to consider the impact of these findings on the development of new approaches to the treatment of malignancies. In-depth analysis of the process of rRNA transcription itself may be found in several recently published reviews (Drygin et al., 2010, Annu Rev Pharmacol Toxicol 50:131-156; Bywater et al., 2013,Cancer Cell 22: 51-65; Hein et al., 2013,Trends Mol Med 19:643-654).

Age and genetics: how do prognostic factors at diagnosis explain disparities in acute myeloid leukemia?

OBJECTIVES: Survival disparities in acute myeloid leukemia (AML) among blacks and Hispanics have been described but not studied extensively in adults. Although younger age and cytogenetic profiles of t(8;21) and acute promyelocytic leukemia (APL) subtypes of AML are associated with improved survival, these factors have not been investigated by race. The purpose is to evaluate whether the observed survival differences for blacks and Hispanics with AML are attributable to older age at diagnosis or lower rates of favorable cytogenetic profiles at diagnosis. The hypothesis is that survival disparities for blacks and Hispanics with AML will be explained by older age at diagnosis and lower rates of favorable cytogenetics. METHODS: Patients with AML were identified in the Surveillance Epidemiology and End Results database (1999 to 2008). Kaplan-Meier (KM) survival curves predicted survival by race/ethnicity, stratified by age. Cox proportional hazard models estimated mortality by race with adjustment for age, sex, year of diagnosis, t(8;21), and APL subtypes. RESULTS: A total of 25,692 patients were included. Blacks and Hispanics were diagnosed at younger ages (younger than 61 y), and had higher rates of t(8;21) and APL compared with non-Hispanic whites (NHWs). The overall KM curve shows that NHWs had a worse survival compared with other races/ethnicities. However, when KM curves were stratified by age, blacks and Hispanics had worse survival in younger age categories (younger than 61 y). In multivariable models, black race was associated with an increased risk of death compared with NHWs (HR, 1.10; 95% CI, 1.04-1.16). Adjustment for t(8;21) and APL subtypes did not attenuate the disparity. CONCLUSIONS: Despite younger age and higher prevalence of favorable cytogenetics at diagnosis, blacks and Hispanics have an increased mortality from AML compared with other racial/ethnic groups. Future studies should investigate other factors that may influence outcomes among minority populations.

Interaction of TIF-90 and filamin A in the regulation of rRNA synthesis in leukemic cells.

The transcription initiation factor I (TIF-IA) is an important regulator of the synthesis of ribosomal RNA (rRNA) through its facilitation of the recruitment of RNA polymerase I (Pol I) to the ribosomal DNA promoter. Activation of the phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt) pathway, which occurs commonly in acute myelogenous leukemia, enhances rRNA synthesis through TIF-IA stabilization and phosphorylation. We have discovered that TIF-IA coexists with a splicing isoform, TIF-90, which is expressed preferentially in the nucleolus and at higher levels in proliferating and transformed hematopoietic cells. TIF-90 interacts directly with Pol I to increase rRNA synthesis as a consequence of Akt activation. Furthermore, TIF-90 binds preferentially to a 90-kDa cleavage product of the actin binding protein filamin A (FLNA) that inhibits rRNA synthesis. Increased expression of TIF-90 overcomes the inhibitory effect of this cleavage product and stimulates rRNA synthesis. Because activated Akt also reduces FLNA cleavage, these results indicate that activated Akt and TIF-90 function in parallel to increase rRNA synthesis and, as a consequence, cell proliferation in leukemic cells. These results provide evidence that the direct targeting of Akt would be an effective therapy in acute leukemias in which Akt is activated.

Akt activation enhances ribosomal RNA synthesis through casein kinase II and TIF-IA.

Transcription initiation factor I (TIF-IA) plays an essential role in regulating ribosomal RNA (rRNA) synthesis by tethering RNA polymerase I (Pol I) to the rDNA promoter. We have found that activated Akt enhances rRNA synthesis through the phosphorylation of casein kinase IIα (CK2α) on a threonine residue near its N terminus. CK2 in turn phosphorylates TIF-IA, thereby increasing rDNA transcription. Activated Akt also stabilizes TIF-IA, induces its translocation to the nucleolus, and enhances its interaction with Pol I. Treatment with AZD8055, an inhibitor of both Akt and mammalian target of rapamycin phosphorylation, but not with rapamycin, disrupts Akt-mediated TIF-IA stability, translocation, and activity. These data support a model in which activated Akt enhances rRNA synthesis both by preventing TIF-IA degradation and phosphorylating CK2α, which in turn phosphorylates TIF-IA. This model provides an explanation for the ability of activated Akt to promote cell proliferation and, potentially, transformation.

Sequential azacitidine plus lenalidomide combination for elderly patients with untreated acute myeloid leukemia.

There are limited treatment options for older patients with acute myeloid leukemia and prognosis of these patients remains poor, thereby warranting development of novel therapies. We evaluated the efficacy and safety of azacitidine in combination with lenalidomide as front-line therapy for older patients with acute myeloid leukemia. Patients ≥ 60 years of age with untreated acute myeloid leukemia received azacitidine 75 mg/m2 for 7 days followed by escalating doses of lenalidomide daily for 21 days starting on day 8 of each cycle every 6 weeks. Patients received continued therapy until disease progression, unacceptable toxicity, or completion of 12 cycles. Forty-two patients (median age, 74 years) were enrolled with equal distribution according to European LeukemiaNet risk. The overall response rate was 40% (rate of complete remission with or without complete recovery of blood counts = 28%). The median time to complete remission with or without complete recovery of blood counts was 12 weeks, and duration of this status was 28 weeks (range, 4 - >104 weeks). Therapy-related acute myeloid leukemia and a high score on the Hematopoietic Cell Transplantation Comorbidity Index were negative predictors of response. Early death was noted in 17% of patients. Grades ≥ 3 toxicities were uncommon and most adverse events were gastrointestinal, fatigue and myelosuppression. In conclusion, a sequential combination of azacitidine plus lenalidomide has clinical activity in older patients with acute myeloid leukemia, and further studies of this combination are underway.

Reduced rRNA expression and increased rDNA promoter methylation in CD34+ cells of patients with myelodysplastic syndromes.

Myelodysplastic syndromes (MDS) are clonal disorders of hematopoietic stem cells characterized by ineffective hematopoiesis. The DNA-hypomethylating agents 5-azacytidine and 5-aza-2'-deoxycytidine are effective treatments for patients with MDS, increasing the time to progression to acute myelogenous leukemia and improving overall response rates. Although genome-wide increases in DNA methylation have been documented in BM cells from MDS patients, the methylation signatures of specific gene promoters have not been correlated with the clinical response to these therapies. Recently, attention has been drawn to the potential etiologic role of decreased expression of specific ribosomal proteins in MDS and in other BM failure states. Therefore, we investigated whether rRNA expression is dysregulated in MDS. We found significantly decreased rRNA expression and increased rDNA promoter methylation in CD34(+) hematopoietic progenitor cells from the majority of MDS patients compared with normal controls. Treatment of myeloid cell lines with 5-aza-2'-deoxycytidine resulted in a significant decrease in the methylation of the rDNA promoter and an increase in rRNA levels. These observations suggest that an increase in rDNA promoter methylation can result in decreased rRNA synthesis that may contribute to defective hematopoiesis and BM failure in some patients with MDS.

Cyclopentenyl cytosine induces senescence in breast cancer cells through the nucleolar stress response and activation of p53.

The induction of senescence has emerged as a potentially important contributor to the effects of chemotherapeutic agents against tumors. We have demonstrated that depletion of CTP induced by cyclopentenyl cytosine (CPEC; NSC 375575), a specific inhibitor of the enzyme CTP synthetase, induces irreversible growth arrest and senescence characterized by altered morphology and expression of senescence-associated ß-galactosidase activity in MCF-7 breast cancer cells expressing wild-type p53. In contrast, differentiation in the absence of senescence resulted from CPEC treatment in MDA-MB-231 breast cancer cells that express a mutated p53. Both senescence of MCF-7 cells and differentiation of MDA-MB-231 cells were prevented by repletion of CTP through the cytidine salvage pathway. Senescence in MCF-7 cells was associated with a G(2)- and S-phase arrest, whereas differentiation in MDA-MB-231 cells was associated with arrest in G(1) phase at 5 days. Mechanistic studies revealed that CTP depletion induced a rapid translocation of nucleolar proteins, including nucleostemin and nucleolin into the nucleoplasm. This nucleolar stress response resulted in a sustained elevation of p53 and the p53 target genes, p21 and Mdm2, in cells with wild-type p53. Furthermore, short interfering RNA-induced knockdown of p53 in MCF-7 cells treated with CPEC prevented cellular senescence and increased apoptotic cell death. We conclude that CTP depletion and the resulting nucleolar stress response results in a senescence-like growth arrest through activation of p53, whereas cells with mutated p53 undergo differentiation or apoptotic cell death.

Reactive oxygen species regulate nucleostemin oligomerization and protein degradation.

Nucleostemin (NS) is a nucleolar-nucleoplasmic shuttle protein that regulates cell proliferation, binds p53 and Mdm2, and is highly expressed in tumor cells. We have identified NS as a target of oxidative regulation in transformed hematopoietic cells. NS oligomerization occurs in HL-60 leukemic cells and Raji B lymphoblasts that express high levels of c-Myc and have high intrinsic levels of reactive oxygen species (ROS); reducing agents dissociate NS into monomers and dimers. Exposure of U2OS osteosarcoma cells with low levels of intrinsic ROS to hydrogen peroxide (H(2)O(2)) induces thiol-reversible disulfide bond-mediated oligomerization of NS. Increased exposure to H(2)O(2) impairs NS degradation, immobilizes the protein within the nucleolus, and results in detergent-insoluble NS. The regulation of NS by ROS was validated in a murine lymphoma tumor model in which c-Myc is overexpressed and in CD34+ cells from patients with chronic myelogenous leukemia in blast crisis. In both instances, increased ROS levels were associated with markedly increased expression of NS protein and thiol-reversible oligomerization. Site-directed mutagenesis of critical cysteine-containing regions of nucleostemin altered both its intracellular localization and its stability. MG132, a potent proteasome inhibitor and activator of ROS, markedly decreased degradation and increased nucleolar retention of NS mutants, whereas N-acetyl-L-cysteine largely prevented the effects of MG132. These results indicate that NS is a highly redox-sensitive protein. Increased intracellular ROS levels, such as those that result from oncogenic transformation in hematopoietic malignancies, regulate the ability of NS to oligomerize, prevent its degradation, and may alter its ability to regulate cell proliferation.

Synthesis of 2'-deoxy-2'-[18F]fluoro-9-ß-D-arabinofuranosylguanine: a novel agent for imaging T-cell activation with PET.

PURPOSE: 9-(ß-D-Arabinofuranosyl)guanine (AraG) is a guanosine analog that has a proven efficacy in the treatment of T-cell lymphoblastic disease. To test the possibility of using a radiofluorinated AraG as an imaging agent, we have synthesized 2'-deoxy-2'-[(18)F]fluoro-9-ß-D-arabinofuranosylguanine ([(18)F]F-AraG) and investigated its uptake in T cells. PROCEDURE: We have synthesized [(18)F]F-AraG via a direct fluorination of 2-N-acetyl-6-O-((4-nitrophenyl)ethyl)-9-(3',5'-di-O-trityl-2'-O-trifyl-ß-D-ribofuranosyl)guanine with [(18)F]KF/K.2.2.2 in DMSO at 85°C for 45 min. [(18)F]F-AraG uptake in both a CCRF-CEM leukemia cell line (unactivated) and activated primary thymocytes was evaluated. RESULTS: We have successfully prepared [(18)F]F-AraG in 7-10% radiochemical yield (decay corrected) with a specific activity of 0.8-1.3 Ci/µmol. Preliminary cell uptake experiments showed that both a CCRF-CEM leukemia cell line and activated primary thymocytes take up the [(18)F]F-AraG. CONCLUSION: For the first time to the best of our knowledge, [(18)F]F-AraG has been successfully synthesized by direct fluorination of an appropriate precursor of a guanosine nucleoside. This approach maybe also useful for the synthesis of other important positron emission tomography (PET) probes such as [(18)F]FEAU, [(18)F]FMAU, and [(18)F]FBAU which are currently synthesized by multiple steps and involve lengthy purification. The cell uptake studies support future studies to investigate the use of [(18)F]F-AraG as a PET imaging agent of T cells.

Depletion of guanine nucleotides leads to the Mdm2-dependent proteasomal degradation of nucleostemin.

Nucleostemin is a positive regulator of cell proliferation and is highly expressed in a variety of stem cells, tumors, and tumor cell lines. The protein shuttles between the nucleolus and the nucleus in a GTP-dependent fashion. Selective depletion of intracellular guanine nucleotides by AVN-944, an inhibitor of the de novo purine synthetic enzyme, IMP dehydrogenase, leads to the rapid disappearance of nucleostemin protein in tumor cell lines, an effect that does not occur with two other nucleolar proteins, nucleophosmin or nucleolin. Endogenous nucleostemin protein is completely stabilized by MG132, an inhibitor of the 26S proteasome, as are the levels of expressed enhanced green fluorescent protein-tagged nucleostemin, both wild-type protein and protein containing mutations at the G(1) GTP binding site. Nutlin-3a, a small molecule that disrupts the binding of the E3 ubiquitin ligase, Mdm2, to p53, stabilizes nucleostemin protein in the face of guanine nucleotide depletion, as does siRNA-mediated knockdown of Mdm2 expression and overexpression of a dominant-negative form of Mdm2. Neither Doxorubicin nor Actinomycin D, which cause the release of nucleostemin from the nucleolus, results in nucleostemin degradation. We conclude that nucleostemin is a target for Mdm2-mediated ubiquitination and degradation when not bound to GTP. Because this effect does not occur with other chemotherapeutic agents, the induction of nucleostemin protein degradation in tumor cells by IMP dehydrogenase inhibition or by other small molecules that disrupt GTP binding may offer a new approach to the treatment of certain neoplastic diseases.

Guanine nucleotide depletion mediates translocation of nucleolar proteins, including RNA helicase A (DHX-9).

DHX-9, a member of the DEXH family of RNA helicases, unwinds dsRNA/dsDNA by ATP or GTP-dependent hydrolysis. We asked whether DHX-9 played a role in the GTP depletion-induced inhibition of rRNA synthesis and/or nucleolar disruption. MPA, a specific inhibitor of inosine monophosphate dehydrogenase (IMPDH), induced a rapid translocation of DHX-9 from the nucleolus to the nucleus. EGFP-tagged DHX-9 mutated at the GTP binding site also localized to the nucleus. However, knockdown of DHX-9 by siRNA did not inhibit the rRNA synthesis or cause the nucleolar disruption. Thus, DHX-9 translocation found with IMPDH inhibition does not mediate the inhibition of rRNA synthesis.