FIP1L1 Regulates Alternative Polyadenylation of Leukemia-Associated Genes in Acute Myeloid Leukemia

Abstract Alternative polyadenylation (APA) is a prevalent mechanism of post-transcriptional gene regulation. APA can impact the protein coding portion of a gene or the length of the 3' untranslated region (UTR), a crucial mRNA region mediating stability, localization, and translation efficiency. Global shifts in APA usage have been reported during development and in cancer transformation; however, the impact of APA in acute myeloid leukemia (AML) is currently unexplored. Therefore, we sought to address whether global shifts in APA patterns can be seen in leukemic blasts and if APA changes contribute to leukemogenesis. Here we report that leukemic blasts exhibit global transcript shortening that contributes to the upregulation of leukemia-associated genes: NRAS, BAALC, and MAPKAPK3. We also implicate FIP1L1 as an important mediator of APA alterations in AML. To identify whether global trends of APA are seen in leukemia patients, we performed a 3'RNA sequencing experiment comparing healthy CD34+ hematopoietic stem and progenitor cells (HSPCs) with CD34+ enriched leukemic blasts from primary patient samples. We observed a trend in whole transcript shortening, with an emphasis on 3'UTR shortening of affected genes. Importantly, genes with altered APA were among pathways associated with oncogenic transformation and specifically AML. We selected three genes for further functional validation (NRAS, BAALC, and MAPKAPK3) that exhibited a correlation of shortened 3'UTRs with higher mRNA expression. Using mRNA stability assays and luciferase assays, we confirmed that 3'UTR shortening contributes to the upregulation of these leukemia-associated genes in blood cells. To probe upstream regulators of APA alterations in leukemia, we performed overexpression and knockdown studies of APA machinery members that have altered expression in leukemia patients. We identified FIP1L1 as a novel regulator of APA in AML. Overexpression of FIP1L1 resulted in 3'UTR lengthening and downregulation of our three selected genes. Conversely, knockdown led to 3' UTR shortening and upregulation. Interestingly, altering FIP1L1 expression (both up and downregulation) is detrimental to leukemia cell lines, suggesting that modest changes in gene expression can have a dramatic impact on hematopoietic cell fitness. We conclude that APA changes represent another dysregulated layer of gene expression that can contribute to AML development. Current studies aimed at identifying the role of FIP1L1 in healthy HSPCs will elucidate the feasibility of FIP1L1 as a novel therapeutic target in AML. Disclosures No relevant conflicts of interest to declare.

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Importance of Altered Gene Expression of Metalloproteinases 2, 9, and 16 in Acute Myeloid Leukemia: Preliminary Study

Journal of Oncology ◽

10.1155/2021/6697975 ◽

2021 ◽

Vol 2021 ◽

pp. 1-8

Author(s):

Jacek Pietrzak ◽

Marek Mirowski ◽

Rafał Świechowski ◽

Damian Wodziński ◽

Agnieszka Wosiak ◽

...

Keyword(s):

Gene Expression ◽

Acute Myeloid Leukemia ◽

Myeloid Leukemia ◽

Proteolytic Enzymes ◽

Regulation Of Gene Expression ◽

Expression Level ◽

Altered Expression ◽

Cytogenetic Changes ◽

The Impact ◽

Acute Myeloid

Acute myeloid leukemia is a group of hematological neoplasms characterized by a heterogeneous course and high mortality. The important factor in the neoplastic process is metalloproteinases, proteolytic enzymes capable of degrading various components of the extracellular matrix, which take an active part in modifying the functioning of the cell, including transformation to cancer cell. They interact with numerous signaling pathways responsible for the process of cell growth, proliferation, or apoptosis. In the present study, changes in the expression of MMP2, MMP9, and MMP16 genes between patients with AML and people without cancer were examined. The impact of cytogenetic changes in neoplastic cells on the expression level of MMP2, MMP9, and MMP16 was also assessed, as well as the impact of the altered expression on the effectiveness of the first cycle of remission-inducing therapy. To evaluate the expression of all studied genes MMP2, MMP9, and MMP16, SYBR Green-based real-time PCR method was used; the reference gene was GAPDH. For two investigated genes MMP2 and MMP16, the lower expression level was observed in patients with AML when compared to healthy people. The MMP9 gene expression level did not differ between patients with AML and healthy individuals which may indicate a different regulation of gene expression in acute myeloid leukemia. However, no correlation was observed between the genes’ expression of all tested metalloproteinases and the result of cytoreductive treatment or the presence of cytogenetic changes. The obtained results show that the expression of MMP2 and MMP16 genes is reduced while the expression of MMP9 is unchanged in patients with acute myeloid leukemia. This may indicate a different regulation of the expression of these genes, and possible disruptions in gene transcription or posttranscriptional mechanisms in the MMP2 and MMP16 genes, however, do not affect the level of MMP9 expression. Obtained results in AML patients are in contrary to various types of solid tumors where increased expression is usually observed.

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CD9, a potential leukemia stem cell marker, regulates drug resistance and leukemia development in acute myeloid leukemia

Stem Cell Research & Therapy ◽

10.1186/s13287-021-02155-6 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Yongliang Liu ◽

Guiqin Wang ◽

Jiasi Zhang ◽

Xue Chen ◽

Huailong Xu ◽

...

Keyword(s):

Gene Expression ◽

Stem Cells ◽

Drug Resistance ◽

Acute Myeloid Leukemia ◽

Myeloid Leukemia ◽

Stem Cell Marker ◽

Hematopoietic Stem ◽

Chemotherapy Drugs ◽

And Migration ◽

Acute Myeloid

Abstract Background Leukemia stem cells (LSCs) are responsible for the initiation, progression, and relapse of acute myeloid leukemia (AML). Therefore, a therapeutic strategy targeting LSCs is a potential approach to eradicate AML. In this study, we aimed to identify LSC-specific surface markers and uncover the underlying mechanism of AML LSCs. Methods Microarray gene expression data were used to investigate candidate AML-LSC-specific markers. CD9 expression in AML cell lines, patients with AML, and normal donors was evaluated by flow cytometry (FC). The biological characteristics of CD9-positive (CD9+) cells were analyzed by in vitro proliferation, chemotherapeutic drug resistance, migration, and in vivo xenotransplantation assays. The molecular mechanism involved in CD9+ cell function was investigated by gene expression profiling. The effects of alpha-2-macroglobulin (A2M) on CD9+ cells were analyzed with regard to proliferation, drug resistance, and migration. Results CD9, a cell surface protein, was specifically expressed on AML LSCs but barely detected on normal hematopoietic stem cells (HSCs). CD9+ cells exhibit more resistance to chemotherapy drugs and higher migration potential than do CD9-negative (CD9−) cells. More importantly, CD9+ cells possess the ability to reconstitute human AML in immunocompromised mice and promote leukemia growth, suggesting that CD9+ cells define the LSC population. Furthermore, we identified that A2M plays a crucial role in maintaining CD9+ LSC stemness. Knockdown of A2M impairs drug resistance and migration of CD9+ cells. Conclusion Our findings suggest that CD9 is a new biomarker of AML LSCs and is a promising therapeutic target.

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Alternative polyadenylation dysregulation contributes to the differentiation block of acute myeloid leukemia

Blood ◽

10.1182/blood.2020005693 ◽

2021 ◽

Author(s):

Amanda G Davis ◽

Daniel T. Johnson ◽

Dinghai Zheng ◽

Ruijia Wang ◽

Nathan D. Jayne ◽

...

Keyword(s):

Acute Myeloid Leukemia ◽

Myeloid Leukemia ◽

Alternative Polyadenylation ◽

Hematopoietic Stem ◽

Global Trends ◽

Protein Levels ◽

Mtorc1 Signaling ◽

Differentiation Block ◽

Acute Myeloid

Post-transcriptional regulation has emerged as a driver for leukemia development and an avenue for therapeutic targeting. Among post-transcriptional processes, alternative polyadenylation (APA) is globally dysregulated across cancer types. However, limited studies have focused on the prevalence and role of APA in myeloid leukemia. Furthermore, it is poorly understood how altered poly(A) site (PAS) usage of individual genes contributes to malignancy or whether targeting global APA patterns might alter oncogenic potential. In this study, we examined global APA dysregulation in acute myeloid leukemia (AML) patients by performing 3' Region Extraction And Deep Sequencing (3'READS) on a subset of AML patient samples along with healthy hematopoietic stem and progenitor cells (HSPCs) and by analyzing publicly available data from a broad AML patient cohort. We show that patient cells exhibit global 3' untranslated region (UTR) shortening and coding sequence (CDS) lengthening due to differences in PAS usage. Among APA regulators, expression of FIP1L1, one of the core cleavage and polyadenylation factors, correlated with the degree of APA dysregulation in our 3'READS dataset. Targeting global APA by FIP1L1 knockdown reversed the global trends seen in patients. Importantly, FIP1L1 knockdown induced differentiation of t(8;21) cells by promoting 3'UTR lengthening and downregulation of the fusion oncoprotein AML1-ETO. In non-t(8;21) cells, FIP1L1 knockdown also promoted differentiation by attenuating mTORC1 signaling and reducing MYC protein levels. Our study provides mechanistic insights into the role of APA in AML pathogenesis and indicates that targeting global APA patterns can overcome the differentiation block of AML patients.

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Collaboration of the Meningioma 1 (MN1) Oncogene with MLL-Fusions in Pediatric Leukemia

Blood ◽

10.1182/blood.v112.11.3786.3786 ◽

2008 ◽

Vol 112 (11) ◽

pp. 3786-3786

Author(s):

Ting Liu ◽

Dragana Jankovic ◽

Laurent Brault ◽

Sabine Ehret ◽

Vincenzo Rossi ◽

...

Keyword(s):

Gene Expression ◽

Acute Myeloid Leukemia ◽

Myeloid Leukemia ◽

Lymphoblastic Leukemia ◽

Leukemia Cell ◽

Leukemic Cells ◽

Human Leukemia ◽

Fusion Genes ◽

Pediatric Leukemia ◽

Acute Myeloid

Abstract Expression of meningioma 1 (MN1) has been proposed to be a negative prognostic marker in adult acute myeloid leukemia (AML). In pediatric leukemia, we found overexpression of MN1 in 53 of 88 cases: whereas no MN1 expression was detected in T-cell acute lymphoblastic leukemia (T-ALL), significant amounts of MN1 were found in immature B-cell ALL and most cases of infant leukemia. Interestingly, 17 of 19 cases harboring fusion genes involving the mixed-lineage leukemia (MLL-X) gene showed elevated MN1 expression. Lentiviral siRNA mediated MN1 knock-down resulted in cell cycle arrest and impaired clonogenic growth of 3 MLL-X-positive human leukemia cell lines overexpressing MN1 (THP-1, RS4;11, MOLM-13). In a mouse model of MLL-ENL-induced leukemia we found MN1 to be overexpressed as a consequence of provirus integration. Strikingly co-expression of MN1 with MLL-ENL resulted in significantly reduced latency for induction of an AML phenotype in mice suggesting functional cooperation. Immunophenotyping and secondary transplant experiments suggested that MN1 overexpression seems to expand the L-GMP cell population targeted by the MLL-ENL fusion. Gene expression profiling allowed defining a number of potential MN1 hematopoietic targets. Upregulation of CD34, FLT3, HLF, or DLK1 was validated in bone marrow transiently overexpressing MN1, in MN1-induced mouse acute myeloid leukemia, as well as in pediatric leukemias with elevated MN1 levels. Our work shows that MN1 is overexpressed in a significant fraction of pediatric acute leukemia, is essential for growth of leukemic cells, and that MN1 can act as a cooperating oncogene with MLL-ENL most probably through modification of a distinct gene expression program that leads to expansion of a leukemic progenitor population targeted by MLL-fusion genes.

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MicroRNA-29a Is An Oncogene in Acute Myeloid Leukemia

Blood ◽

10.1182/blood.v112.11.683.683 ◽

2008 ◽

Vol 112 (11) ◽

pp. 683-683

Author(s):

Christopher Y. Park ◽

Yoon-Chi Han ◽

Govind Bhagat ◽

Jian-Bing Fan ◽

Irving L Weissman ◽

...

Keyword(s):

Gene Expression ◽

Cell Cycle ◽

Stem Cells ◽

Acute Myeloid Leukemia ◽

Hematopoietic Stem Cells ◽

Mirna Expression ◽

Myeloid Leukemia ◽

Hematopoietic Stem ◽

Acute Myeloid

Abstract microRNAs (miRNAs) are short, non-protein encoding RNAs that bind to the 3′UTR’s of target mRNAs and negatively regulate gene expression by facilitating mRNA degradation or translational inhibition. Aberrant miRNA expression is well-documented in both solid and hematopoietic malignancies, and a number of recent miRNA profiling studies have identified miRNAs associated with specific human acute myeloid leukemia (AML) cytogenetic groups as well as miRNAs that may prognosticate clinical outcomes in AML patients. Unfortunately, these studies do not directly address the functional role of miRNAs in AML. In fact, there is no direct functional evidence that miRNAs are required for AML development or maintenance. Herein, we report on our recent efforts to elucidate the role of miRNAs in AML stem cells. miRNA expression profiling of AML stem cells and their normal counterparts, hematopoietic stem cells (HSC) and committed progenitors, reveals that miR-29a is highly expressed in human hematopoietic stem cells (HSC) and human AML relative to normal committed progenitors. Ectopic expression of miR-29a in mouse HSC/progenitors is sufficient to induce a myeloproliferative disorder (MPD) that progresses to AML. During the MPD phase of the disease, miR-29a alters the composition of committed myeloid progenitors, significantly expedites cell cycle progression, and promotes proliferation of hematopoietic progenitors at the level of the multipotent progenitor (MPP). These changes are manifested pathologically by marked granulocytic and megakaryocytic hyperplasia with hepatosplenomegaly. Mice with miR-29a-induced MPD uniformly progress to an AML that contains a leukemia stem cell (LSC) population that can serially transplant disease with as few as 20 purified LSC. Gene expression analysis reveals multiple tumor suppressors and cell cycle regulators downregulated in miR-29a expressing cells compared to wild type. We have demonstrated that one of these genes, Hbp1, is a bona fide miR-29a target, but knockdown of Hbp1 in vivo does not recapitulate the miR-29a phenotype. These data indicate that additional genes are required for miR-29a’s leukemogenic activity. In summary, our data demonstrate that miR-29a regulates early events in normal hematopoiesis and promotes myeloid differentiation and expansion. Moreover, they establish that misexpression of a single miRNA is sufficient to drive leukemogenesis, suggesting that therapeutic targeting of miRNAs may be an effective means of treating myeloid leukemias.

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Characterization of Extramedullary Acute Myeloid Leukemia – Results of the AML96 Trial

Blood ◽

10.1182/blood.v116.21.2154.2154 ◽

2010 ◽

Vol 116 (21) ◽

pp. 2154-2154

Author(s):

Friedrich Stölzel ◽

Christoph Röllig ◽

Michael Kramer ◽

Brigitte Mohr ◽

Uta Oelschlägel ◽

...

Keyword(s):

Bone Marrow ◽

Acute Myeloid Leukemia ◽

Induction Chemotherapy ◽

Myeloid Leukemia ◽

The Body ◽

Hematopoietic Stem ◽

Npm1 Mutation ◽

Mutation Status ◽

The Impact ◽

Acute Myeloid

Abstract Abstract 2154 Background: Myeloid Sarcoma (MS) is defined as an extramedullary mass composed of myeloid blasts occurring at an anatomical site other than the bone marrow. Furthermore, the term extramedullary manifestation (EM) is applied if it accompanies overt acute myeloid leukemia (AML) and represents non-effacing tissue infiltration. EM is reported to correspond often to the skin but can affect almost every site of the body. The prognosis of MS or EM has been discussed controversially in the past. EM at diagnosis of AML is generally thought to be a rare event. However, data defining the prevalence of EM at diagnosis of AML and its prognostic value are missing. The aim of this analysis was to provide data for estimating the prevalence of EM at diagnosis of AML and to determine its relevance by including clinical and laboratory data from patients being treated in the prospective AML96 trial of the Study Alliance Leukemia (SAL) study group. Patients and Methods: A total of 326 patients with AML (age 17 – 83 years) and EM were treated within the AML96 trial with a median follow up of 8.8 years (95% CI, 8.4 to 9.3 years). All patients received double induction chemotherapy. Consolidation therapy contained high-dose cytosine arabinoside and for patients ≤ 60 years of age the option of autologous or allogeneic hematopoietic stem cell transplantation (HSCT). Logistic regression analyses were used to identify prognostic variables for CR rates. The method of Kaplan-Meier was used to estimate OS and EFS. Confidence interval (CI) estimation for the survival curves was based on the cumulative hazard function using the Greenwood's formula for the SE estimation. Survival distributions were compared using the log rank test. Results: 17% of the AML patients entered into the AML96 trial were diagnosed with EM. In 313 of the 326 patients (96%) EM was evident at diagnosis. The majority of patients with EM were diagnosed with de novo AML (84%, n=273), whereas gingival infiltration (51%, n=166) displayed the main EM of AML with CNS involvement being less common (4%, n=14). The majority of patients had a cytogenetic intermediate risk profile (71%, n=221) with a total of 172 patients (56%) harboring a normal karyotype. Patients with EM had a statistically significant lower median CD34-positivity of bone marrow blasts, higher percentage of FAB subtypes M4 and M5, higher WBC counts and LDH at diagnosis and higher percentage of NPM1 mutations compared to those patients without EM (all p<.001). When comparing achievement of CR between patients with EM to patients without EM, no statistical difference between these two groups was observed. Analysis according to the NPM1/FLT3-ITD mutation status revealed highest 5-year-OS (37%, 95% CI: .24 - .508) and 5-year-EFS (36%, 95% CI: .224 - .448) in the NPM1-mut/FLT3-wt group and lowest 5-year-OS (12%, 95% CI: 0 - .261) and 5-year-EFS (4%, 95% CI: 0 - .124) in the NPM1-wt/FLT3-ITD group, p=.007 and p=.001, respectively. Of the 49 relapsed patients with EM who had a NPM1-mutation at diagnosis 48 deceased despite of intensified relapse therapies. Conclusions: This analysis represents the largest study so far investigating the impact of EM AML. Patients with EM AML have distinct differences from AML patients without EM regarding their clinical and molecular characteristics at diagnosis. However these differences do not translate into differences in response to induction chemotherapy. Compared to patients without EM, survival analysis revealed differences according to the NPM1/FLT3-ITD mutation status which is also described for patients without EM AML. However, the prognosis for patients with EM who harbor a mutated NPM1 the prognosis at relapse seems to be dismal. Disclosures: No relevant conflicts of interest to declare.

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TIM-3 Is a Promising Target to Eradicate Acute Myeloid Leukemia Stem Cells Sparing Normal Hematopoietic Stem Cells

Blood ◽

10.1182/blood.v118.21.559.559 ◽

2011 ◽

Vol 118 (21) ◽

pp. 559-559

Author(s):

Toshihiro Miyamoto ◽

Yoshikane Kikushige ◽

Takahiro Shima ◽

Koichi Akashi

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Acute Myeloid Leukemia ◽

Hematopoietic Stem Cells ◽

Myeloid Leukemia ◽

Leukemia Cell ◽

Cytotoxic Activities ◽

Antibody Treatment ◽

Hematopoietic Stem ◽

Acute Myeloid

Abstract Abstract 559 Acute myeloid leukemia (AML) originates from self-renewing leukemic stem cells (LSCs), an ultimate therapeutic target for permanent cure. To selectively kill AML LSCs sparing normal hematopoietic stem cells (HSCs), one of the most practical approaches is to target the AML LSCs-specific surface or functionally indispensable molecules. Based on differential transcriptome analysis of prospectively-purified CD34+CD38− LSCs from AML patient samples and normal HSCs, we found that T-cell immunoglobulin mucin-3 (TIM-3) was highly expressed in AML LSCs but not in normal HSCs (Kikushige et al., Cell Stem Cell, 2010). In normal hematopoiesis, TIM-3 is mainly expressed in mature monocytes and a fraction of NK cells, but not in granulocytes, T cells or B cells. In the bone marrow, TIM-3 is expressed only in a fraction of granulocyte/macrophage progenitors (GMPs) at a low level, but not in HSCs, common myeloid progenitors, or megakaryocyte/erythrocyte progenitors. In contrast, in human AML, TIM-3 was expressed on cell surface of the vast majority of CD34+CD38− LSCs and CD34+CD38+ leukemic progenitors in AML of most FAB types, except for acute promyelocytic leukemia (M3). FACS-sorted TIM-3+ but not TIM-3− AML cells reconstituted human AML in the immunodeficient mice, indicating that the TIM-3+ population contains most of functional LSCs. To selectively eradicate TIM-3-expressing AML LSCs, we established an anti-human TIM-3 mouse IgG2a antibody, ATIK2a, possessing antibody-dependent cellular cytotoxic and complement-dependent cytotoxic activities in leukemia cell lines transfected with TIM-3. We first tested the effect of ATIK2a treatment on reconstitution of normal HSCs in a xenograft model. ATIK2a was intraperitoneally injected to the mice once a week after 12 hours of transplantation of human CD34+ cells. Injection of ATIK2a did not affect reconstitution of normal human hematopoiesis except removing TIM-3-expressing mature monocytes. In contrast, injection of TIM-3 to the mice transplanted with human AML samples markedly reduced leukemic repopulation. In some mice transplanted with AML bone marrow, only normal hematopoiesis was reconstituted after anti-TIM-3 antibody treatment, suggesting that the antibody selectively killed AML cells, sparing residual normal HSCs. To further test the inhibitory effect of ATIK2a on established human AML, eight weeks after transplantation of human AML cells, engraftment of human AML cells was confirmed by blood sampling and thereafter ATIK2a was injected to these mice. In all cases tested, ATIK2a treatment significantly reduced human TIM-3+ AML fraction as well as the CD34+CD38− LSCs fraction. In addition, to verify the anti-AML LSCs effect of ATIK2a treatment, human CD45+AML cells from the primary recipients were re-transplanted into secondary recipients. All mice transplanted from primary recipients treated with control IgG developed AML, whereas none of mice transplanted with cells from ATIK2a-treated primary recipients developed AML, suggesting that functional LSCs were effectively eliminated by ATIK2a treatment in primary recipients. Thus, TIM-3 is a promising surface molecule to target AML LSCs. Our experiments strongly suggest that targeting this molecule by monoclonal antibody treatment is a practical approach to eradicate human AML. Disclosures: No relevant conflicts of interest to declare.

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Inhibition of HDAC8 Reactivates p53 and Abrogates Leukemia Stem Cell Activity in CBFβ-SMMHC Associated Acute Myeloid Leukemia

Blood ◽

10.1182/blood.v124.21.363.363 ◽

2014 ◽

Vol 124 (21) ◽

pp. 363-363

Author(s):

Jing Qi ◽

Qi Cai ◽

Sandeep Singh ◽

Ling Li ◽

Hongjun Liu ◽

...

Keyword(s):

Bone Marrow ◽

Acute Myeloid Leukemia ◽

Myeloid Leukemia ◽

Bone Marrow Cells ◽

Conflicts Of Interest ◽

Cell Activity ◽

Disease Free Survival ◽

Hematopoietic Stem ◽

The Impact ◽

Acute Myeloid

Abstract The inv(16)-created CBFβ-SMMHC fusion protein inhibits differentiation of hematopoietic stem and progenitor cells (HSPCs) and creates pre-leukemic populations predisposed to acute myeloid leukemia (AML) transformation. However, the molecular mechanism underlying the leukemogenic function of CBFβ-SMMHC has been elusive. Given the low TP53 mutation rate in AML, alternative mechanisms disrupting p53 function are expected. We showed thatCBFβ-SMMHC impairs p53 acetylation and p53 target gene activation through formation of an aberrant protein complex with p53 and HDAC8 (Blood, 120: A772; 122(21): 224). We now show that CBFβ-SMMHC binds to p53 and HDAC8 independently through distinct regions and that HDAC8 mediates the deacetylation of p53 associated with CBFβ-SMMHC. In addition, we generated mice carrying a floxed Hdac8 (Hdac8f) allele and crossed with Cbfb56M/+/Mx1-Cre (Kuo YH et al, Cancer Cell 2006). Deletion of Hdac8 signifiacntly (p<0.0001) reduced the incidence of AML and prolonged disease-free survival. Pharmacologic inhibition of HDAC8 activity with HDAC8-selective inhibitors (HDAC8i) reactivates p53 and selectively induces apoptosis of inv(16)+ AML CD34+ cells while sparing normal HSPCs. To test the effect of HDAC8i on LSC engraftment and leukemia-initiating capacity, we generated Cbfb56M/+/Mx1-Cre mice with a Cre-reporter line expressing tdTomato fluorescence protein following Cre-mediated recombination. AML cells (dTomato+/cKit+) treated with HDAC8i (22d) ex vivo showed reduced engraftment (p=0.025) and enhanced survival (p=0.025) in transplanted mice. To examine whether HDAC8i 22d treatment affects the engraftment capacity on surviving cells, we transplanted equal number (2 x 106) of AML cells treated with either 22d or vehicle in another cohort of mice (n=4). We show that HDAC8i 22d treatment reduced the engraftment of dTomato+/cKit+ AML cells and enhanced survival, suggesting that the engraftment capacity is altered in addition to reducing AML cell survival. We next performed preclinical studies to determine the efficacy of in vivo administration of HDAC8i 22d. AML transplanted mice were randomized into two groups, one group treated with vehicle and the other treated with HDAC8i 22d for 2 weeks. Flow cytometry analysis revealed significantly reduced frequency (p=0.0097) and number (p=0.0101) of dTomato+/cKit+ AML cells in the bone marrow and spleen of 22d treated mice compared to vehicle treated group. To further assess the impact on LSC activity, we transplanted bone marrow cells from these treated mice into secondary recipients and analyzed for AML engraftment. Significant reduction in the frequency (p<0.0001) and the number (p=0.0006) of dTomato+/cKit+ AML cells was observed in the bone marrow and spleen. Furthermore, HDAC8i 22d treated transplants showed no signs of leukemia while vehicle treated transplants are moribund with aggressive AML. These results indicate that HDAC8 inhibition by 22d treatment effectively eliminates engraftment and leukemia-initiating capacity of AML LSCs. In conclusion, our studies identify a novel post-translational p53-inactivating mechanism and demonstrate selective HDAC8 inhibition as a promising approach to target inv(16)+ AML LSCs. Disclosures No relevant conflicts of interest to declare.

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Different regulation of PARP1, PARP2, PARP3 and TRPM2 genes expression in acute myeloid leukemia cells

10.21203/rs.2.19004/v1 ◽

2019 ◽

Author(s):

Paulina Gil-Kulik ◽

Ewa Dudzińska ◽

Elżbieta Radzikowska-Büchner ◽

Joanna Wawer ◽

Mariusz Jojczuk ◽

...

Keyword(s):

Gene Expression ◽

Bone Marrow ◽

Acute Myeloid Leukemia ◽

Peripheral Blood ◽

Myeloid Leukemia ◽

Mrna Level ◽

Hematopoietic Stem ◽

Genes Expression ◽

Acute Myeloid Leukemia Cells ◽

Acute Myeloid

Abstract Acute myeloid leukemia (AML) is a heterogenic lethal disorder characterized by the accumulation of abnormal myeloid progenitor cells in the bone marrow, which results in hematopoietic failure. Despite various efforts in detection and treatment, many patients with AML die of this cancer. That is why it is important to develop novel therapeutic options, employing strategic target genes involved in apoptosis and tumor progression. The aim of the study was to evaluate PARP1, PARP2, PARP3, and TRPM2 gene expression at the mRNA level in the cells of the hematopoietic system of the bone marrow in patients with acute myeloid leukemia, bone marrow collected from healthy patients, peripheral blood of healthy individuals, and hematopoietic stem cells from the peripheral blood after mobilization.Results: The results found that the bone marrow cells of patients with acute myeloid leukemia (AML) show over expression of PARP1 and PARP2 genes and decreased TRPM2 gene expression. In the hematopoietic stem cells derived from the normal marrow and peripheral blood after mobilization, the opposite situation was observed, i.e. TRPM2 gene showed increased expression while PARP1 and PARP2 gene expression was reduced. We observed the positive correlations between PARP1, PARP2, PARP3, and TRPM2 genes expression in the group of mature mononuclear cells derived from the peripheral blood and in the group of bone marrow-derived cells. In AML cells significant correlations were not observed between the expression of the examined genes.Conclusions: Our research suggests that in physiological conditions in the cells of the hematopoietic system there is mutual positive regulation of PARP1, PARP2, PARP3, and TRPM2 genes expression. PARP1, PARP2, and TRPM2 genes at mRNA level deregulate in acute myeloid leukemia cells.

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Variants ofDNMT3Acause transcript-specific DNA methylation patterns and affect hematopoiesis

Life Science Alliance ◽

10.26508/lsa.201800153 ◽

2018 ◽

Vol 1 (6) ◽

pp. e201800153 ◽

Cited By ~ 7

Author(s):

Tanja Božić ◽

Joana Frobel ◽

Annamarija Raic ◽

Fabio Ticconi ◽

Chao-Chung Kuo ◽

...

Keyword(s):

Gene Expression ◽

Dna Methylation ◽

Acute Myeloid Leukemia ◽

Myeloid Leukemia ◽

Dna Methyltransferase ◽

De Novo ◽

Hematopoietic Differentiation ◽

Hematopoietic Stem ◽

Acute Myeloid

De novo DNA methyltransferase 3A (DNMT3A) plays pivotal roles in hematopoietic differentiation. In this study, we followed the hypothesis that alternative splicing ofDNMT3Ahas characteristic epigenetic and functional sequels. SpecificDNMT3Atranscripts were either down-regulated or overexpressed in human hematopoietic stem and progenitor cells, and this resulted in complementary and transcript-specific DNA methylation and gene expression changes. Functional analysis indicated that, particularly, transcript 2 (coding for DNMT3A2) activates proliferation and induces loss of a primitive immunophenotype, whereas transcript 4 interferes with colony formation of the erythroid lineage. Notably, in acute myeloid leukemia expression of transcript 2 correlates with its in vitro DNA methylation and gene expression signatures and is associated with overall survival, indicating thatDNMT3Avariants also affect malignancies. Our results demonstrate that specificDNMT3Avariants have a distinct epigenetic and functional impact. Particularly, DNMT3A2 triggers hematopoietic differentiation and the corresponding signatures are reflected in acute myeloid leukemia.

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