CREB: A Key Regulator of Normal and Neoplastic Hematopoiesis

The cAMP response element-binding protein (CREB) is a nuclear transcription factor downstream of cell surface receptors and mitogens that is critical for normal and neoplastic hematopoiesis. Previous work from our laboratory demonstrated that a majority of patients with acute myeloid leukemia (AML) and acute lymphoid leukemia (ALL) overexpress CREB in the bone marrow. To understand the role of CREB in leukemogenesis, we examined the biological effect of CREB overexpression on primary leukemia cells, leukemia cell lines, and CREB overexpressing transgenic mice. Our results demonstrated that CREB overexpression leads to an increase in cellular proliferation and survival. Furthermore, CREB transgenic mice develop a myeloproliferative disorder with aberrant myelopoiesis in both the bone marrow and spleen. Additional research from other groups has shown that the expression of the cAMP early inducible repressor (ICER), a CREB repressor, is also deregulated in leukemias. And, miR-34b, a microRNA that negative regulates CREB expression, is expressed at lower levels in myeloid leukemia cell lines compared to that of healthy bone marrow. Taken together, these data suggest that CREB plays a role in cellular transformation. The data also suggest that CREB-specific signaling pathways could possibly serve as potential targets for therapeutic intervention

CREB engages C/EBPδ to initiate leukemogenesis.

cAMP response element binding protein (CREB) is frequently overexpressed in acute myeloid leukemia (AML) and acts as a proto-oncogene; however, it is still debated whether such overactivation alone is able to induce leukemia as its pathogenetic downstream signaling is still unclear. We generated a zebrafish model overexpressing CREB in the myeloid lineage, which showed an aberrant regulation of primitive hematopoiesis, and in 79% of adult CREB-zebrafish a block of myeloid differentiation, triggering to a monocytic leukemia akin the human counterpart. Gene expression analysis of CREB-zebrafish revealed a signature of 20 differentially expressed human homologous CREB targets in common with pediatric AML. Among them, we demonstrated that CREB overexpression increased CCAAT-enhancer-binding protein-δ (C/EBPδ) levels to cause myeloid differentiation arrest, and the silencing of CREB-C/EBPδ axis restored myeloid terminal differentiation. Then, C/EBPδ overexpression was found to identify a subset of pediatric AML affected by a block of myeloid differentiation at monocytic stage who presented a significant higher relapse risk and the enrichment of aggressive signatures. Finally, this study unveils the aberrant activation of CREB-C/EBPδ axis concurring to AML onset by disrupting the myeloid cell differentiation process. We provide a novel in vivo model to perform high-throughput drug screening for AML cure improvement

MicroRNA profiling of paediatric AML with FLT-ITD or MLL-rearrangements: Expression signatures and in vitro modulation of miR-221-3p and miR-222-3p with BRD4/HATs inhibitors.

Novel therapeutic strategies are needed for paediatric patients affected by Acute Myeloid Leukaemia (AML), particularly for those at high-risk for relapse. MicroRNAs (miRs) have been extensively studied as biomarkers in cancer and haematological disorders, and their expression has been correlated to the presence of recurrent molecular abnormalities, expression of oncogenes, as well as to prognosis/clinical outcome. In the present study, expression signatures of different miRs related both to presence of myeloid/lymphoid or mixed-lineage leukaemia 1 and Fms like tyrosine kinase 3 internal tandem duplications rearrangements and to the clinical outcome of paediatric patients with AML were identified. Notably, miR-221-3p and miR-222-3p resulted as a possible relapse-risk related miR. Thus, miR-221-3p and miR-222-3p expression modulation was investigated by using a Bromodomain‑containing protein 4 (BRD4) inhibitor (JQ1) and a natural compound that acts as histone acetyl transferase inhibitor (curcumin), alone or in association, in order to decrease acetylation of histone tails and potentiate the effect of BRD4 inhibition. JQ1 modulates miR-221-3p and miR-222-3p expression in AML with a synergic effect when associated with curcumin. Moreover, changes were observed in the expression of CDKN1B, a known target of miR-221-3p and miR-222-3p, increase in apoptosis and downregulation of miR-221-3p and miR-222-3p expression in CD34+ AML primary cells. Altogether, these findings suggested that several miRs expression signatures at diagnosis may be used for risk stratification and as relapse prediction biomarkers in paediatric AML outlining that epigenetic drugs, could represent a novel therapeutic strategy for high-risk paediatric patients with AML. For these epigenetic drugs, additional research for enhancing activity, bioavailability and safety is needed

Donor Cell Acute Myeloid Leukemia after Hematopoietic Stem Cell Transplantation for Chronic Granulomatous Disease: A Case Report and Literature Review

The patient reported here underwent hematopoietic stem cell transplantation (HSCT) due to chronic granulomatous disease (CGD) caused by biallelic mutations of the NCF1 gene. Two years later, he developed AML, which was unexpected and was recognized via sex-mismatched chromosomes as deriving from the donor cells; the patient was male, and the donor was his sister. Donor cell leukemia (DCL) is very rare, and it had never been reported in patients with CGD after HSCT. In the subsequent ten years, the AML relapsed three times and the patient underwent chemotherapy and three further HSCTs; donors were the same sister from the first HSCT, an unrelated donor, and his mother. The patient died during the third relapse. The DCL was characterized since onset by an acquired translocation between chromosomes 9 and 11, with a molecular rearrangement between the MLL and MLLT3 genes-a quite frequent cause of AML. In all of the relapses, the malignant clone had XX sex chromosomes and this rearrangement, thus indicating that it was always the original clone derived from the transplanted sister's cells. It exhibited the ability to remain quiescent in the BM during repeated chemotherapy courses, remission periods and HSCT. The leukemic clone then acquired different additional anomalies during the ten years of follow-up, with cytogenetic results characterized both by anomalies frequent in AML and by different, non-recurrent changes. This type of cytogenetic course is uncommon in AML

The lncRNA CASC15 regulates SOX4 expression in RUNX1-rearranged acute leukemia

Abstract Background Long non-coding RNAs (lncRNAs) play a variety of cellular roles, including regulation of transcription and translation, leading to alterations in gene expression. Some lncRNAs modulate the expression of chromosomally adjacent genes. Here, we assess the roles of the lncRNA CASC15 in regulation of a chromosomally nearby gene, SOX4, and its function in RUNX1/AML translocated leukemia. Results CASC15 is a conserved lncRNA that was upregulated in pediatric B-acute lymphoblastic leukemia (B-ALL) with t (12; 21) as well as pediatric acute myeloid leukemia (AML) with t (8; 21), both of which are associated with relatively better prognosis. Enforced expression of CASC15 led to a myeloid bias in development, and overall, decreased engraftment and colony formation. At the cellular level, CASC15 regulated cellular survival, proliferation, and the expression of its chromosomally adjacent gene, SOX4. Differentially regulated genes following CASC15 knockdown were enriched for predicted transcriptional targets of the Yin and Yang-1 (YY1) transcription factor. Interestingly, we found that CASC15 enhances YY1-mediated regulation of the SOX4 promoter. Conclusions Our findings represent the first characterization of this CASC15 in RUNX1-translocated leukemia, and point towards a mechanistic basis for its action

Prognostic impact of t(16;21)(p11;q22) and t(16;21)(q24;q22) in pediatric AML: A retrospective study by the I-BFM study group

To study the prognostic relevance of rare genetic aberrations in acute myeloid leukemia (AML), such as t(16:21), international collaboration is required. Two different types of t(16:21) translocations can be distinguished: t(16:21)(p11;q22), resulting in the FUS-ERG fusion gene; and t(16:21)(q24;q22), resulting in RUNX1-core binding factor (CBFA2T3). We collected data on clinical and biological characteristics of 54 pediatric AML cases with t(16:21) rearrangements from 14 international collaborative study groups participating in the international Berlin-Frankfurt-Miinster (I-BFM) AML study group. The AML-BFM cohort diagnosed between 1997 and 2013 was used as a reference cohort. RUNX1-CBFA2T3 (n = 23) had significantly lower median white blood cell count (12.5 x 10(9)/L, P = .03) compared with the reference cohort. FUS-ERG rearranged AML (n = 31) had no predominant French-American-British (FAB) type, whereas 76% of RUNX1-CBFA2T3 had an M1/M2 FAB type (M1, M2), significantly different from the reference cohort (P = .004). Four-year event-free survival (EFS) of patients with FUS-ERG was 7% (standard error [SE] = 5%), significantly lower compared with the reference cohort (51%, SE = 1%, P < .001). Four-year EFS of RUNX1-CBFA2T3 was 77% (SE = 8%, P = .06), significantly higher compared with the reference cohort. Cumulative incidence of relapse was 74% (SE = 8%) in FUS-ERG, 0% (SE = 0%) in RUNX1-CBFA2T3, compared with 32% (SE = 1%) in the reference cohort (P < .001). Multivariate analysis identified both FUS-ERG and RUNX1-CBFA2T3 as independent risk factors with hazard ratios of 1.9 (P < .0001) and 0.3 (P = .025), respectively. These results describe 2 clinically relevant distinct subtypes of pediatric AML. Similarly to other core-binding factor AMLs, patients with RUNX1-CBFA2T3 rearranged AML may benefit from stratification in the standard risk treatment, whereas patients with FUS-ERG rearranged AML should be considered high-risk

BAG1: The Guardian of Anti-Apoptotic Proteins in Acute Myeloid Leukemia

BCL2 associated Athano-Gene 1 (BAG1) is a multifunctional protein that has been described to be involved in different cell processes linked to cell survival. It has been reported as deregulated in diverse cancer types. Here, BAG1 protein was found highly expressed in children with acute myeloid leukemia at diagnosis, and in a cohort of leukemic cell lines. A silencing approach was used for determining BAG1's role in AML, finding that its down-regulation decreased expression of BCL2, BCL-XL, MCL1, and phospho-ERK1/2, all proteins able to sustain leukemia, without affecting the pro-apoptotic protein BAX. BAG1 down-regulation was also found to increase expression of BAG3, whose similar activity was able to compensate the loss of function of BAG1. BAG1/BAG3 co-silencing caused an enhanced cell predisposition to death in cell lines and also in primary AML cultures, affecting the same proteins. Cell death was CASPASE-3 dependent, was accompanied by PARP cleavage and documented by an increased release of pro-apoptotic molecules Smac/DIABLO and Cytochrome c. BAG1 was found to directly maintain BCL2 and to protect MCL1 from proteasomal degradation by controlling USP9X expression, which appeared to be its novel target. Finally, BAG1 was found able to affect leukemia cell fate by influencing the expression of anti-apoptotic proteins crucial for AML maintenance

Characterization and role of the transcription factor cAMP response element binding protein (CREB) in childhood leukemia

We investigated the transcription factor cyclic adenosine monophosphate response-element binding protein (CREB) in childhood acute leukemia. We analyzed CREB expression in series of bone marrow samples from children with acute lymphoblast (ALL) or myeloid leukemia (AML) at diagnosis and during remission therapy. We documented that CREB protein was significantly (p<0.001) over expressed in 84% of patients with ALL and 66% of those with AML. CREB protein was always present in the active phosphorylated form, and transcriptional active wholly at diagnosis of leukemia. In order to find the causes of CREB abnormalities we looked at CREB mRNA which was not amplified and at gene sequencing analyses that revealed a wild type genome in leukemic samples. cAMP concentration was then measured but not significant differences in diagnosis samples respect to controls were found. We described an inverse correlation between CREB and its inducible cAMP early repressor (ICER) mRNA expression and decided to considere this phenomenon to be better elucidated. ICER and CREB are both transcriptional activator and repressor, respectively, of the cAMP-mediated signaling pathway. The research continued by looking at the possible function of restored ICER expression in leukemia cell lines. ICER exogenously expressed in HL60 was demonstrated to decrease CREB protein level and induce a lowered clonogenic potential in vitro. ICER was demonstrated to repress a series of genes involved in cell cycle, proliferation and MAPK signaling. Its binding to CRE promoters confirmed its role in controlling gene expression of the main cellular pathways. HL60 cell line expressing ICER was then evaluated in their ability to repopulate non-obese diabetic-severe combined immunodeficient mice demonstrating a lower extramedullary sites invasion and bone marrow angiogenesis. So, its potential effects on tumor progression was documented in vivo. We then tried to give a reason of ICER lower expression found in HL60 and demonstrated that it was subjected to degradation through a constitutively active form of the extracellular signal-regulated protein kinase (ERK1/2) maintained by CREB, which drives it to the proteasome. Considerable attention was focused on the role played by ICER in the control of genes related to apoptosis and MAPK signaling and chemotherapics were used as stimuli for cell death. We found that a series of ICER’s target cyclins, phosphatases DUSP1/4, kinases and oncogenes were strongly downregulated after chemotherapic treatment, supporting ICER restored expression to confer an enhanced sensibility to drugs to leukemic cells. Cell cycle was impaired and apoptosis significantly increased. Addressing the mechanism by which exogenous ICER improves apoptosis, we found that the simple inhibition of DUSP1/4 phosphatases expression triggers the activation of the p38 apoptotic pathway and the disruption of ERK, AKT and RAS survival signaling. This is a process caspases dependent and abrogated by p38 specific inhibitor. We reveal that ICER induces CREB proteasome degradation supporting apoptotic signaling and a lowered tumorigenicity. ICER is here proposed to be able to switch leukemia cell fate from survival to apoptosis by counteracting CREB transcriptional activity and modifing its protein stability. At this point, we revealed that CREB was overexpressed in leukemia, but the underlying mechanism remained unknown. MicroRNAs , that act as negative regulators of gene expression principally through translational repression, were then investigated for the mediation of high CREB protein levels. A series of miRNAs which target CREB were identified. RQ-PCR revealed that miR-34b was expressed significantly less in myeloid cell lines, previously known for high CREB protein levels. Exogenous miR-34b expression was induced, and results revealed a direct interaction with CREB in vitro. MiR-34b restored expression caused cell cycle abnormalities, reduced anchorage independent growth, and altered CREB target gene expression, suggesting its suppressor potential. CREB target proteins (BCL-2, Cyclins A1, B1, D, NfKB, JAK1, STAT3), as well as many downstream protein kinases and cell survival signaling pathways (AKT/mTOR, ERK) usually elicited by CREB, were observed to have decreased. The miR-34b/34c promoter was demonstrated to be methylated finding this epigenetic regulation able to control the observed lower miR-34b expression levels in order to maintain the CREB protein overexpressed. In addition, the inverse correlation between miR-34b and CREB expression was found in a cohort of pediatric patients at diagnosis of acute myeloid leukemia, supporting this relationship also in vivo. Finally, the research defines CREB as an oncogene and ICER as a tumor suppressor in leukemia. Unbalanced CREB/ICER expression needs to be considered a pathogenetic feature in leukemogenesis. Our results identify a targets and pathways to be considered for future therapeutic approaches. The direct miR-34b targeting of CREB provides new information about myeloid transformation.In questo dottorato di ricerca è stato studiato il fattore di trascrizione CREB che dirige l’espressione genica regolata per lo più dall’ AMPciclico. CREB è uno dei fattori di trascrizione più conosciuti, ma ancora poco è noto del suo ruolo fisiologico e oncologico nel tessuto ematopoietico. L’espressione proteica di CREB è stata definita in linee cellulari leucemiche e in una ampia coorte di pazienti all’esordio di leucemia acuta linfoide (LLA) o leucemia acuta mieloide (LMA). I dati sono stati confrontati con quelli ottenuti dall’analisi di prelievi raccolti durante il follow up in documentata remissione di malattia e donatori sani. I risultati hanno evidenziato una elevata espressione proteica di CREB nell’84% delle ALL e nel 66% delle LAM (p<0.001). Il legame specifico di CREB con i siti di riconoscimento cAMP-Responsive-Element (CRE, 5’TGACGTCA3’) sui promotori si è dimostrato avvenire esclusivamente nei prelievi alla diagnosi di leucemia dove dunque l’overespressione di CREB sosteneva la possibilità di una sua attività trascrizionale anomala. Per capire le cause di questo fenomeno sono stati misurati i livelli di CREB mRNA che non erano significativamente differenti negli esordi rispetto ai controlli utilizzati. E’ stato condotto il sequenziamento del gene CREB, e dei domini di interazione tra CREB la CREB binding protein (CBP) in quanto necessario alla attivazione trascrizionale, che però non ha rivelato nessuna mutazione tale da giustificare l’overespressione di CREB. Infine è stata misurata la quantità di cAMP che non è risultata significativamente diversa nei campioni all’esordio rispetto ai controlli. Allo stesso fine, è stata valutata l’espressione del cAMP inducible early repressor (ICER). ICER si è dimostrato essere sotto espresso all’esordio di leucemia e upregolato nei prelievi in remissione e nei donatori sani, contrariamenti ai livelli espressione di CREB documentati nei medesimi campioni. La ricerca è proseguita con lo studio di ICER, che è un fattore di trascrizione noto riconoscere CRE nei promotori con la funzione di reprimerne la trascrizione. ICER dunque ha il ruolo di modulare l’attività di CREB controllando l’espressione genica in diversi tessuti. Essendo ICER sotto espresso nel tessuto leucemico è stato da noi costruito un modello in vitro reintroducendo una esogena espressione di ICER in linee cellulari. E’ stato dimostrato che l’espressione esogena di ICER induceva una diminuzione della proteina CREB e una alterazione di una serie di geni target. I promotori di alcuni geni, scelti per svolgere attività cellulari primarie quali la proliferazione e la sopravvivenza, sono stati dimostrati infatti essere occupati da ICER invece che da CREB. Le attività cellulari delle HL60 risultavano fortemente modificate: la capacità di formare colonie dei blasti esprimenti ICER in vitro era fortemente ridotta, supportando l’ipotesi che ICER potesse svolgere il ruolo di onco-soppressore. E’ stato così utilizzato un test di ripopolamento in vivo in topi NOD-SCID con le HL60 stabilizzate con ICER e HL60 stabilizzate con il vettore vuoto. La linea leucemica si è dimostrata meno tumorigenica se esprimente ICER, la disseminazione dei blasti nel sangue periferico e nella milza era molto rallentata e l’angiogenesi ridotta. L’identificazione di quali geni ICER fosse in grado di reprimere e quindi contrastare il fenotipo leucemico sono stati studiati. I dati di espressione genica hanno dimostrato che ICER reprimeva per lo più geni del ciclo cellulare, dell’apoptosi e del pathway delle MAPK. Con l’utilizzo di chemioterapici come stimoli apoptotici è stato dimostrato che alcune cicline, le fosfatasi DUSP1/4, alcune MAP chinasi e importanti oncogeni, tutte proteine target di ICER, erano fortemente represse nelle cellule leucemiche se esprimenti ICER esogeno, rispetto alle stesse cellule non trattate con farmaci. Il trattamento inoltre induceva un blocco del ciclo cellulare e una aumentata apoptosi dimostrando che ICER conferiva una maggior sensibilità ai farmaci. In particolare,è stato dimostrato che il pathway pro-apoptotico diretto dalla proteina di stress p38 si manteneva attivo grazie alla repressione di DUSP1/4 indotta da ICER, fornendo così il pathway diretto da ICER e nuovi target terapeutici. Infine, sono state indagate le cause della bassa espressione di ICER all’esordio di malattia. E’ stato dimostrato che il pathway legato alla kinase ERK1/2, fortemente mantenuto anche dagli elevati livelli di CREB, permetteva a ERK1/2 di fosforilare ICER e portarlo alla degradazione via proteasoma. Inoltre, è stato dimostrato che una volta reintrodotta l’espressione di ICER in HL60, ICER dimerizzava con CREB portando quest’ultimo alla degradazione via proteasoma, spiegando dunque i bassi livelli di CREB nel nostro modello in vitro. Infine la ricerca si è concentrata sul cercare di determinare le cause della iper espressione di CREB nella leucemia. L’osservazione che i livelli proteici di CREB erano alterati, ma non i livelli di mRNA, ha condotto lo studio verso il ruolo delle modifiche post-trascrizionali. L’ipotesi che CREB rimanesse ad alti livelli per anomalie di traduzione ha spinto l’analisi verso la ricerca di possibili microRNAs. E’ stato condotto uno studio di 5 micrornas su una serie di linee cellulari mieloidi che ha permesso di puntare su miR-34b in quanto la sua espressione era fortemente abbassata nelle linee leucemiche in concomitanza agli elevati livelli di CREB. MiR-34b è stato così re-inserito nelle linee come oligonucleotide esogeno ed è stato dimostrato che provocava un drastico abbassamento dei livelli di CREB, con conseguente diminuzione dell’espressione genica e proteica dei target di CREB. La vitalità cellulare inoltre era compromessa, compresa la capacità di formare colonie. Il fatto che miR-34b si mantenesse sotto espresso nella leucemia è stato dimostrato dipendere dalla metilazione del suo promotore. Alle analisi in vitro, sono state affiancate le analisi di espressione di miR-34b in una serie di pazienti all’esordio di LAM, dimostrando che anche in vivo miR-34b è fortemente sotttoespresso e CREB upregolato. Infine, la ricerca ha caratterizzato il gene CREB come un oncogene e ICER come un oncosopressore nella leucemia mieloide. La modulazione della loro espressione risulta di fondamentale importanza nel mantenimento del tumore. La caratterizzazione di nuovi geni target e pathways cellulari implicati nella leucemia mieloide aprono nuove prospettive di studio e di possibilità terapeutiche.La caratterizzazione di CREB come target di miR-34b e la metilazione del promotore come evento regolatore dei livelli di proteina nella leucemia, confermano l’importanza degli eventi epigenetici nella leucemogenesi