PU.1 Is a Downstream Target of C/EBPα in Normal Hematopoiesis and Acute Myeloid Leukemia

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1353-1353
Author(s):  
Christian Bach ◽  
Philipp B. Staber ◽  
Min Ye ◽  
Pu Zhang ◽  
Alan D. Friedman ◽  
...  
Keyword(s):  
Gene Expression ◽  
Acute Myeloid Leukemia ◽  
Knockout Mice ◽  
Binding Sites ◽  
Myeloid Leukemia ◽  
Target Gene ◽  
Myeloid Differentiation ◽  
Downstream Target ◽  
Acute Myeloid

Abstract Abstract 1353 The transcription factors PU.1 and C/EBPα are key regulators of hematopoietic cell differentiation. Tight and coordinated regulation of these factors is essential for normal hematopoiesis and even moderate alterations can lead to acute myeloid leukemia (AML). Previous studies established that in PU.1 knockout mice myeloid differentiation is blocked at an earlier stage compared to C/EBPα knockouts, consistent with PU.1 acting upstream of C/EBPα during hematopoietic differentiation. Recently, however, we and others identified a PU.1 upstream regulatory element (URE) which contains potential C/EBP binding sites. C/EBPα binds to the PU.1 URE in vitro and in vivo. Furthermore, C/EBPα transactivated the PU.1 proximal promoter in a URE dependent manner. We, therefore, hypothesized that PU.1 is a target gene of C/EBPα in hematopoietic cells. To assess the role of PU.1 as a downstream target of C/EBPα in normal hematopoiesis we performed gene expression analysis in immature hematopoietic cells of conditional C/EBPα knockout mice (Mx1-Cre). Of note, we observed a strong reduction of PU.1 expression in hematopoietic stem cells (HSCs: CD150+CD48-LSK) after excision of C/EBPα, corroborating that PU.1 is a target of C/EBPα in murine HSCs in vivo. Moreover, lentiviral PU.1 expression alleviated the myeloid differentiation block of C/EBPα−/− KSL cells as evidenced by the differentiation to Gr-1 and Mac1 positive myeloid cells. Targeted deletion of the PU.1 URE reduces PU.1 expression and induces myeloid leukemia. Additionally, inactivation of C/EBPα by various mechanisms is a common observation in many AML subtypes. Therefore, we tested if dysregulation of C/EBPα is associated with decreased PU.1 expression. Gene expression studies in several human AML cell lines revealed a positive correlation between C/EBPα and PU.1 expression. Furthermore, we analyzed expression of C/EBPα and PU.1 in a well characterized cohort of 285 AML patients. Importantly, PU.1 expression was strongly reduced in cases with either C/EBPα mutations or C/EBPα promoter silencing compared to other AML subtypes. Taken together, our data support that PU.1 is a downstream target gene of C/EBPα in normal hematopoiesis as well as human leukemia. We currently develop a mouse model containing targeted mutations of three C/EBP binding sites in the PU.1 URE. This model will help to further pinpoint the functional impact of C/EBPα mediated regulation of PU.1 in different hematopoietic populations and to determine how this regulation may contribute to leukemia development in vivo. The first two authors contributed equally to this work. Disclosures: No relevant conflicts of interest to declare.

scholarly journals The transcriptional corepressor CBFA2T3 inhibits all-trans-retinoic acid–induced myeloid gene expression and differentiation in acute myeloid leukemia

2020 ◽  
Vol 295 (27) ◽  
pp. 8887-8900
Author(s):  
Nickolas Steinauer ◽  
Chun Guo ◽  
Jinsong Zhang
Keyword(s):  
Gene Expression ◽  
Acute Myeloid Leukemia ◽  
Retinoic Acid ◽  
Myeloid Leukemia ◽  
Target Gene ◽  
Retinoic Acid Receptor ◽  
Inhibitory Effect ◽  
Myeloid Differentiation ◽  
Transcriptional Corepressor ◽  
Acute Myeloid

CBFA2/RUNX1 partner transcriptional co-repressor 3 (CBFA2T3, also known as MTG16 or ETO2) is a myeloid translocation gene family protein that functions as a master transcriptional corepressor in hematopoiesis. Recently, it has been shown that CBFA2T3 maintains leukemia stem cell gene expression and promotes relapse in acute myeloid leukemia (AML). However, a role for CBFA2T3 in myeloid differentiation of AML has not been reported. Here, we show that CBFA2T3 represses retinoic acid receptor (RAR) target gene expression and inhibits all-trans-retinoic acid (ATRA)-induced myeloid differentiation of AML cells. ChIP-Seq revealed that CBFA2T3 targets the RARα/RXRα cistrome in U937 AML cells, predominantly at myeloid-specific enhancers associated with terminal differentiation. CRISPR/Cas9-mediated abrogation of CBFA2T3 resulted in spontaneous and ATRA-induced activation of myeloid-specific genes in a manner correlated with myeloid differentiation. Importantly, these effects were reversed by CBFA2T3 re-expression. Mechanistic studies showed that CBFA2T3 inhibits RAR target gene transcription by acting at an early step to regulate histone acetyltransferase recruitment, histone acetylation, and chromatin accessibility at RARα target sites, independently of the downstream, RAR-mediated steps of transcription. Finally, we validated the inhibitory effect of CBFA2T3 on RAR in multiple AML subtypes and patient samples. To our knowledge, this is the first study to show that CBFA2T3 down-regulation is both necessary and sufficient for enhancing ATRA-induced myeloid gene expression and differentiation of AML cells. Our findings suggest that CBFA2T3 can serve as a potential target for enhancing AML responsiveness to ATRA differentiation therapies.

scholarly journals Dasatinib Inhibits FLT3/ITD and PTPN11mutated Acute Myeloid Leukemia Cells Overexpressing SRC Tyrosine Kinases

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 1451-1451
Author(s):  
Sigal Tavor ◽  
Tali Shalit ◽  
Noa Chapal Ilani ◽  
Yoni Moskovitz ◽  
Nir Livnat ◽  
...  
Keyword(s):  
Gene Expression ◽  
Acute Myeloid Leukemia ◽  
Board Of Directors ◽  
Myeloid Leukemia ◽  
Drug Sensitivity ◽  
Kinase Inhibitors ◽  
Advisory Committees ◽  
Acute Myeloid

Background: Recent advances in acute myeloid leukemia(AML) targeted therapy improve overall survival. While these targeted therapies can achieve prolonged remissions, most patients will eventually relapseunder therapy. Our recent studies suggest that relapse most often originates from several sub-clones of leukemic stem cells (LSCs), present before therapy initiation, and selected due to several resistance mechanisms. Eradication of these LSCs during treatment induction /remission could thus potentially prevent relapse. The overall goal of the current study was to identify drugs which can be safely administrated to patients at diagnosis and that will target LSCs. Since simultaneously testing multiple drugs in vivo is not feasible, we used an in vitrohigh throughput drug sensitivity assay to identify new targets in primary AML samples. Methods: Drug sensitivity and resistance testing (DSRT) was assessed in vitro (N=46 compounds) on primary AML samples from patients in complete remission (N=29). We performed whole exome sequencing and RNAseq on samples to identify correlations between molecular attributes and in vitro DSRT. Results:Unsupervised hierarchical clustering analysis of in vitro DSRT, measured by IC50, identified a subgroup of primary AML samples sensitive to various tyrosine kinase inhibitors (TKIs). In this subgroup, 52% (9/17) of AML samples displayed sensitivity to dasatinib (defined as a 10-fold decrease in IC50 compared to resistant samples). Dasatinib has broad TKI activity, and is safely administered in the treatment of leukemia. We therefore focused our analysis on predicting AML response to dasatinib, validating our results on the Beat AML cohort. Enrichment analysis of mutational variants in dasatinib-sensitive and resistant primary AML samples identified enrichment of FLT3/ITD (p=0.05) and PTPN11(p=0.05) mutations among dasatinib responders. Samples resistant to dasatinib were enriched with TP53 mutations (p=0.01). No global gene expression changes were observed between dasatinib-sensitive and resistant samples in our cohort, nor in the Beat AML cohort. Following this, we tested the differential expression of specific dasatinib-targeted genes between dasatinib-responding and resistant samples. No significant differences were identified. However, unsupervised hierarchical clustering of dasatinib targeted genes expression in our study and in the Beat AML cohort identified a subgroup of AML samples (enriched in dasatinib responders) that demonstrated overexpression of three SRC family tyrosine kinases:FGR, HCK and LYN as well as PTK6, CSK, GAK and EPHB2. Analysis of the PTPN11 mutant samples revealed that the IC50 for dasatinib in 23 carriers of the mutant PTPN11 was significantly lower compared to the IC50 of PTPN11 wild type samples (p=0.005). LYN was also upregulated (p<0.001) in the mutant samples. We therefore hypothesized that gene expression of dasatinib-targeted genes could be used as a predictive biomarker of dasatinib response among FLT3/ITD carriers. We found that among FLT3/ITD AML carriers in the Beat AML cohort LYN, HCK, CSK and EPHB2 were significantly over-expressed in the dasatinib responding samples (N=27) as compared to the dasatinib resistant samples (N=35). To predict response to dasatinib among FLT3/ITD carriers we used a decision tree classifier based on the expression levels of these four genes. Our prediction model yielded a sensitivity of 74% and specificity of 83% for differentiating dasatinib responders from non-responders with an AUC of 0.84. Based on our findings, we selected FLT3/ITD AML samples and injected them to NSG-SGM3 mice. We found that in a subset of these samples, dasatinib significantly inhibited LSCs engraftment. This subset of FLT3/ITD AML samples expressed higher levels of LYN, HCK,FGR and SRC as compared to the FLT3/ITD samples that were not sensitive to dasatinib therapy in vivo. In summary, we identified a subgroup of AML patients sensitive to dasatinib, based on mutational and expression profiles. Dasatinib has anti-leukemic effects on both blasts and LSCs. Further clinical studies are needed to demonstrate whether selection of tyrosine kinase inhibitors, based on specific biomarkers, could indeed prevent relapse. Disclosures Tavor: Novartis: Membership on an entity's Board of Directors or advisory committees; Abbvie: Membership on an entity's Board of Directors or advisory committees; Astellas: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees; BMS companies: Membership on an entity's Board of Directors or advisory committees.

scholarly journals Trib1 promotes acute myeloid leukemia progression by modulating the transcriptional programs of Hoxa9

Blood ◽  
2020 ◽  
Author(s):  
Seiko Yoshino ◽  
Takashi Yokoyama ◽  
Yoshitaka Sunami ◽  
Tomoko Takahara ◽  
Aya Nakamura ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Genetic Effect ◽  
Dependent Manner ◽  
Enhancer Activity ◽  
Downstream Target ◽  
Erk Phosphorylation ◽  
Acute Myeloid

The pseudokinase Trib1 functions as a myeloid oncogene that recruits the E3 ubiquitin ligase COP1 to C/EBPa and interacts with MEK1 to enhance ERK phosphorylation. Close genetic effect of Trib1 on Hoxa9 has been observed in myeloid leukemogenesis where Trib1 overexpression significantly accelerates Hoxa9-induced leukemia onset. However, the mechanism underlying how Trib1 functionally modulates Hoxa9 transcription activity is unclear. Herein, we provide evidence that Trib1 modulates Hoxa9-associated super-enhancers. ChIP-seq analysis identified increased histone H3K27Ac signals at super-enhancers of the Erg, Spns2, Rgl1, and Pik3cd loci, as well as increased mRNA expression of these genes. Modification of super-enhancer activity was mostly achieved via the degradation of C/EBPa p42 by Trib1, with a slight contribution from the MEK/ERK pathway. Silencing of Erg abrogated the growth advantage acquired by Trib1 overexpression, indicating that Erg is a critical downstream target of the Trib1/Hoxa9 axis. Moreover, treatment of acute myeloid leukemia (AML) cells with the BRD4 inhibitor JQ1 showed growth inhibition in a Trib1/Erg-dependent manner both in vitro and in vivo. Upregulation of ERG by TRIB1 was also observed in human AML cell lines, suggesting that Trib1 is a potential therapeutic target of Hoxa9-associated AML. Taken together, our study demonstrates a novel mechanism by which Trib1 modulates chromatin and Hoxa9-driven transcription in myeloid leukemogenesis.

scholarly journals The Transcription Factor PU.1 Controls a Reversible Differentiation Program in Acute Myeloid Leukemia

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 3930-3930
Author(s):  
Mark D McKenzie ◽  
Margherita Ghisi ◽  
Luisa Cimmino ◽  
Michael Erlichster ◽  
Ethan P Oxley ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Transcription Factor ◽  
Myeloid Leukemia ◽  
Conflicts Of Interest ◽  
Significant Loss ◽  
Polymorphonuclear Neutrophil ◽  
Myeloid Differentiation ◽  
Myeloid Lineage ◽  
Acute Myeloid

Abstract Background: Acute myeloid leukemia (AML) is an aggressive malignancy characterized by clonal expansion of transformed myeloid precursors that fail to differentiate into mature cells. Since myeloid lineage maturation curbs self-renewal and is considered irreversible, engaging this process in AML is an attractive therapeutic strategy. Results: Normal myeloid differentiation requires the transcription factor PU.1 (SPI1), which is functionally compromised in several AML subtypes and is directly inhibited by the recurrent fusion oncoproteins AML1-ETO and PML-RARA. To examine the importance of PU.1 suppression in AML maintenance in vivo, we have combined RNAi-mediated PU.1 inhibition with p53 deficiency to drive highly aggressive AML in mice. Using these models we find that restoring endogenous PU.1 activity in established AML in vivo is sufficient to trigger robust transcriptional, immunophenotypic, and morphological differentiation of leukemic blasts, yielding polymorphonuclear, neutrophil-like cells. Maturation of AML is associated with significant loss of cell viability and yields sustained disease clearance in vivo. Although PU.1 restoration is potently anti-leukemic, remarkably we find that subsequent suppression of PU.1 in mature neutrophil-like cells reverts them to a transformed state within several days. While mature AML-derived cells are slower to form blast colonies in methylcellulose cultures, their clonogenic frequency is only reduced four-fold relative to AML blasts suggesting highly efficient de-differentiation. Conclusions: These results demonstrate that triggering myeloid differentiation can effectively resolve a p53-deficient model of treatment resistant AML, but also identify a previously unrecognised ability of AML cells to bidirectionally transition between transformed and differentiated states based on the activity of a single transcription factor. Our findings challenge the concept of 'terminal differentiation' in AML and highlight the importance of therapeutically eradicating leukemia cells at all stages of myeloid lineage maturation. Disclosures No relevant conflicts of interest to declare.

scholarly journals KDM7A Is Targeted By MiR-155 in Acute Myeloid Leukemia and Impacts Differentiation

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 3641-3641 ◽  
Author(s):  
Maya D. Hughes ◽  
Valerie A. Morris ◽  
Carrie Cummings ◽  
Soheil Meshinchi ◽  
Vivian G. Oehler
Keyword(s):  
Gene Expression ◽  
Cell Death ◽  
Acute Myeloid Leukemia ◽  
Cell Differentiation ◽  
Binding Sites ◽  
Myeloid Leukemia ◽  
Target Genes ◽  
K562 Cells ◽  
Erythroid Differentiation ◽  
Acute Myeloid

Abstract Acute myeloid leukemia (AML) is a heterogeneous disease that develops secondary to the acquisition of mutations that disrupt cell differentiation, proliferation and survival. MicroRNAs (miRNAs or miRs) are short non-coding RNA molecules that modulate post-transcriptional gene expression by either cleaving or repressing translation of target mRNA transcripts. Differential expression of miRNAs has been identified in AML and noted to correlate with specific disease characteristics, cytogenetic abnormalities and prognosis. MiR-155 expression is upregulated in both adult and pediatric patients with cytogenetically normal AML (CN-AML) and correlates with adverse clinical outcomes. Specifically, we have shown that high miR-155 expression is associated with an increased incidence of induction chemotherapy failure and inferior overall and event free survival. However, how miR-155 up-regulation contributes mechanistically to adverse clinical outcomes is poorly understood. In prior work, we correlated the expression of predicted or validated miR-155 target genes with miR-155 expression in a gene expression profiling (GEP) dataset of pediatric AML samples. We identified 22 candidates with inversely correlated expression by GEP for further validation in diagnostic bone marrow specimens from children with the highest miR-155 expression levels (n=9) vs. children with the lowest miR-155 expression levels (n=9). Although the expression of miR-155 inversely correlated with 9 target genes, only expression of the putative target KDM7A demonstrated a statistically significant difference in expression between low and high miR-155 expressing cases (p = 0.03). KDM7A is a lysine-specific histone demethylase enzyme that may play a role in regulating differentiation by impacting transcriptional elongation. Computational software programs, i.e. TargetScan, identified two predicted miR-155 binding sites in the KDM7A 3'UTR. To evaluate whether miR-155 directly binds to the KDM7A 3'UTR, we cloned two regions of the KDM7A 3'UTR containing predicted miR-155 binding sequences into luciferase reporter vectors and then mutated the binding sites by site-directed DNA mutagenesis. We validated that both predicted binding sites in KDM7A 3'UTR were direct miR-155 targets using HEK293T cells. Next, we examined the impact of miR-155 overexpression in K562 cells, an acute leukemia cell line that express very low levels of endogenous miR-155, and can be differentiated along the erythroid lineage after hemin exposure. KDM7A RNA expression was decreased 16-fold in miR-155 versus control lentivirally transduced K562 cells as detected by qPCR. KDM7A protein expression was also decreased in miR-155 versus control expressing K562 cells as measured via Western blot. These data demonstrate that KDM7A is a previously uncharacterized target of miR-155. Next, we explored the effect of differential KDM7A expression on cell differentiation, and cell death and apoptosis after exposure to daunorubicin chemotherapy. For this work we used GFP-labeled miR-155 and YFP-labeled KDM7A lentiviral constructs and labeled control constructs. To examine differentiation we used benzidine staining of hemin-exposed K562 cells transduced with empty control vector (ECV), miR-155, KDM7A, or both constructs. The lowest percentage of benzidine staining, consistent with limited erythroid differentiation, was seen in K562 cells with miR-155 overexpression compared to ECV (28.2% vs. 39.8% positive). This effect on blocked erythroid differentiation was fully reversed with overexpression of KDM7A in miR-155 overexpressing cells (41.4% positive). Confirming these observations, we also observed decreased benzidine staining in hemin exposed K562 cells that were transduced with KDM7A shRNA versus control (28.9% versus 42.7%). Together, these data support that KDM7A plays a role in cell differentiation that is in part controlled by miR-155 expression. Preliminary data also support that re-expression of KDM7A in miR-155 overexpressing cells promotes cell death after exposure to daunorubicin. Further work is ongoing. In conclusion, we have identified a new target of miR-155, KDM7A. Our data suggest that KDM7A plays a role in cell differentiation and that decreased KDM7A expression in AML cells that overexpress miR-155 contributes to blocked differentiation, and may also contribute to resistance to chemotherapy. Disclosures No relevant conflicts of interest to declare.

scholarly journals Thyrointegrin αVβ3 Antagonist: Implications in Acute Myeloid Leukemia

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 4434-4434
Author(s):  
Noureldien Darwish ◽  
Gennadi V. Glinsky ◽  
Shaker A Mousa
Keyword(s):  
Gene Expression ◽  
Bone Marrow ◽  
Acute Myeloid Leukemia ◽  
Anticancer Activity ◽  
Myeloid Leukemia ◽  
Leukemic Cells ◽  
Biological Functions ◽  
Regulated Gene Expression ◽  
Acute Myeloid

Abstract Leukemic cells are able to receive and send several signals within bone marrow niche that play an important role in their survival. One of the important crosstalk is the interaction between the bone marrow microenvironment proteins (vitronectin, fibronectin, fibrinogen, and ostepontin) and thyrointegrin αVβ3 on leukemic cells, generating ligand-specific outside-in signals that are relevant to a variety of cell functions, including gene transcription, cell division, cell attachment, and motility Our previous experiment using in vivo AML animal models with primary AML cells and cell lines have shown significant reduction of leukemic cell burden 74% and >95% (P<0.0001), respectively, after daily subcutaneous treatment with thyrointegrin αvβ3 antagonist fb-PMT (Ki 0.23 nM) at 3 and 10 mg/kg, for 3-4 weeks. In this study we focused on evaluations of the molecular effects of fb-PMT in leukemic cells. Acute myeloid leukemia cell lines (K562-Luc and KG1a cells) were cultured in 50 cm² cell culture flasks with 10 mL phenol red free RPMI media containing 10% fetal bovine albumin. The leukemic cells were treated (at 50% confluence) with 30 µM fb-PMT for 48 hours. Total RNA was immediately isolated from harvested cells using Triazole and used for microarray analysis. Overall, there were 370 significantly down-regulated gene expression records and 273 significantly up-regulated gene expression records, expression of which were changed at least 1.5-fold in fb-PMT-treated human leukemic cells. Significant examples of the fb-PMT-induced gene expression signatures (GES) of pathway's interference include SNAI, MYC, HIF1A, TWIST1, and TFAP2C (P<0.05). Notably, inference of potential contribution to the fb-PMT anticancer activity of the interference with these pathways seems highly congruent with their known biological functions such as cell cycle control (MYC), survival and maintenance of stem cells (HIF1A, TFAP2C), and essential features of the malignant phenotype (TWIST1, SNAI) (Figure 1). Consistently, examples of the fb-PMT-induced GES of transcriptional pathway's activation include RB1, IRF9, MAML1, RAP1A, and GATA4 pathways (P<0.05), known biological functions of which appear highly consistent with the hypothesis that activation of these pathways might contribute to fb-PMT anticancer activity. Finally, we found that fb-PMT interfered with estrogen signaling in human AML cells. The fb-PMT was associated with decreased phosphorylation and nuclear enrichment of Erα (Figure 1). Collectively, our in vivo study and genomic data have shown the key role thyrointegrin αvβ3 in leukemogenesis. The thyrointegrin αvβ3 antagonist fb-PMT demonstrated potent anticancer actions on human AML through the molecular interference mechanism with multiple signaling pathways supporting growth and survival of leukemic cells Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

scholarly journals Differentiation Therapy with Novel Epigenetic Inhibitors in Acute Myeloid Leukemia

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 3762-3762
Author(s):  
Edurne San Jose ◽  
Naroa Gimenez-Camino ◽  
Obdulia Rabal ◽  
Estibaliz Miranda ◽  
Leire Garate ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Small Molecules ◽  
Myeloid Leukemia ◽  
Binding Proteins ◽  
Therapeutic Agents ◽  
Myeloid Differentiation ◽  
Differentiation Therapy ◽  
Acute Myeloid

Acute myeloid leukemia (AML) is a malignant disease characterized by uncontrolled proliferation, differentiation arrest and accumulation of immature myeloid progenitors. Despite recent developments and the approval of new therapeutic agents in the last few years, long term survival of AML, particularly in elderly patients remains an unmet medical need.The use of all-trans retinoic acid (ATRA) in Acute Promyelocytic Leukemia has proven that differentiation therapy may significantly change the survival of AML patients, however the success in APL has not been translated to other groups of AML. Therefore, the identification of new therapeutic agents that may induce the differentiation of AML blasts represents an attractive new target. Furthermore, it is well known that epigenetic alterations have an important role in the development and maintenance of cancer and AML in particular. Thus, our aim was to develop new small molecules targeting epigenetic modifying enzymes like DNA methyltransferases (DNMT), histone methyltransferases or histone deacetylase (HDAC) with the aim of inducing differentiation in AML. We performed a screening of over 50 small molecules synthesized by our group. The design was performed in-house using a knowledge and structure based strategy and the read out of the screening was based on changes in expression of CD11b (a well described marker of myeloid differentiation) after in vitro treatment of AML cells lines. Interestingly, we found several compounds with high capacity to promote the differentiation of leukemic cells in AML cells lines at low non-cytotoxic doses, selecting CM-444 and CM-1758 as our lead compounds (Figure 1a).A complete biochemical characterization showed that both compounds are specific pan-HDACs inhibitors (HDACi). CM-444 and CM-1758 induced in vitro cell differentiation in all subtypes of AML, independently of the AML genetic subgroups or the presence of mutations, which was significantly more pronounced that differentiation induced by reference compounds such as Panobinostat, Vorinostat, Entinostat, Tubastatin or Quisinostat, previously described HDACi. CM-444 and CM-1758 also induced in vivo differentiation in xenogeneic models of AML. AML differentiation was associated with induction of CD11b, downregulation of c-MYC, overexpression of transcription factors that govern the myeloid differentiation and morphologic changes. In addition, these compounds promoted in vitro differentiation of patient-derived AML blasts. The complete transcriptome analysis by RNA-Seq carried out in AML cell lines after CM-444, CM-1758, Panobinostat or Vorinostat treatment showed changes in genes implicated in differentiation, but without explaining the differences among the different HDACi. Analysis of the complete acetylome and proteome before and after treatment with CM-444 and CM-1758 in comparison with other HDACi showed differential acetylation of non-histone proteins included in the GO categories of Zn binding proteins and nucleic acid binding proteins (Figure 1b). Most of these proteins are epigenetic enzymes and have been related to AML and myeloid differentiation, such as MLL2, EP300 or BRD4. In summary, we have developed and characterized novel epigenetic small molecules with a high in vitro and in vivo capacity of differentiating AML cells. These compounds might be an effective differentiation-based therapy to be tested in AML. Besides, the mechanism of differentiation of these compounds is due, at least in part, to the acetylation of non-histone epigenetic proteins, which are key in the myeloid differentiation. Disclosures Paiva: Celgene, Janssen, Sanofi and Takeda: Consultancy; Amgen, Bristol-Myers Squibb, Celgene, Janssen, Merck, Novartis, Roche and Sanofi: Honoraria, Membership on an entity's Board of Directors or advisory committees. San-Miguel:Amgen, Bristol-Myers Squibb, Celgene, Janssen, MSD, Novartis, Roche, Sanofi, and Takeda: Consultancy, Honoraria.

scholarly journals PP2A is a therapeutically targetable driver of cell fate decisions via a c-Myc/p21 axis in Acute Myeloid Leukemia

Blood ◽  
2021 ◽  
Author(s):  
Swagata Goswami ◽  
Rajeswaran Mani ◽  
Jessica Nunes ◽  
Chi-ling Chiang ◽  
Kevan Zapolnik ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Cell Fate ◽  
Myeloid Leukemia ◽  
Myeloid Differentiation ◽  
Cell Fate Decisions ◽  
Phosphatase 2A ◽  
Differentiation Block ◽  
Pp2a Activation ◽  
Acute Myeloid

Dysregulated cellular differentiation is a hallmark of acute leukemogenesis. Phosphatases are widely suppressed in cancers but have not been traditionally associated with differentiation. Herein, we identified that the silencing of Protein Phosphatase 2A (PP2A) directly contributes to differentiation block in acute myeloid leukemia (AML). Gene expression and mass cytometric profiling reveal that PP2A activation modulates cell cycle and transcriptional regulators that program terminal myeloid differentiation. Using a novel pharmacological agent OSU-2S in parallel with genetic approaches, we discovered that PP2A enforces c-Myc and p21 dependent terminal differentiation, proliferation arrest and apoptosis in AML. Finally, we demonstrate that PP2A activation decreases leukemia initiating stem cells, increases leukemic blast maturation, and improves overall survival in murine Tet2-/-Flt3ITD/WT and human AML models in-vivo. Our findings identify the PP2A/c-Myc/p21 axis as a critical regulator of the differentiation/proliferation switch in AML that can be therapeutically targeted in malignancies with dysregulated maturation fate.

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