Oncogenic NRAS Primes Primary Acute Myeloid Leukemia Cells for Differentiation

RAS mutations are frequently found among acute myeloid leukemia patients (AML), generating a constitutively active signaling protein changing cellular proliferation, differentiation and apoptosis. We have previously shown that treatment of AML patients with high-dose cytarabine is preferentially beneficial for those harboring oncogenic RAS. On the basis of a murine AML cell culture model, we ascribed this effect to a RAS-driven, p53-dependent induction of differentiation. Hence, in this study we sought to confirm the correlation between RAS status and differentiation of primary blasts obtained from AML patients. The gene expression signature of AML blasts with oncogenic NRAS indeed corresponded to a more mature profile compared to blasts with wildtype RAS, as demonstrated by gene set enrichment analysis (GSEA) and real-time PCR analysis of myeloid ecotropic viral integration site 1 homolog (MEIS1) in a unique cohort of AML patients. In addition, in vitro cell culture experiments with established cell lines and a second set of primary AML cells showed that oncogenic NRAS mutations predisposed cells to cytarabine (AraC) driven differentiation. Taken together, our findings show that AML with inv(16) and NRAS mutation have a differentiation gene signature, supporting the notion that NRAS mutation may predispose leukemic cells to AraC induced differentiation. We therefore suggest that promotion of differentiation pathways by specific genetic alterations could explain the superior treatment outcome after therapy in some AML patient subgroups. Whether a differentiation gene expression status may generally predict for a superior treatment outcome in AML needs to be addressed in future studies.     

Exosomes Secreted by Apoptosis-Resistant Acute Myeloid Leukemia (AML) Blasts Harbor Regulatory Network Proteins Potentially Involved in Antagonism of Apoptosis

Expression of apoptosis-regulating proteins (B-cell CLL/lymphoma 2 - BCL-2, Myeloid Cell Leukemia 1 - MCL-1, BCL-2 like 1 - BCL-X and BCL-2-associated X protein - BAX) in acute myeloid leukemia (AML) blasts at diagnosis is associated with disease-free survival. We previously found that the initially high apoptosis-resistance of AML cells decreased after therapy, while regaining high levels at relapse. Herein, we further explored this aspect of dynamic apoptosis regulation in AML. First, we showed that the intraindividual ex vivo apoptosis-related profiles of normal lymphocytes and AML blasts within the bone marrow of AML patients were highly correlated. The expression values of apoptosis-regulating proteins were far beyond healthy control lymphocytes, which implicates the influence of microenvironmental factors. Second, we demonstrated that apoptosis-resistant primary AML blasts, as opposed to apoptosis-sensitive cells, were able to up-regulate BCL-2 expression in sensitive AML blasts in contact cultures (p = 0.0067 and p = 1.0, respectively). Using secretome proteomics, we identified novel proteins possibly engaged in apoptosis regulation. Intriguingly, this analysis revealed that major functional protein clusters engaged in global gene regulation, including mRNA splicing, protein translation, and chromatin remodeling, were more abundant (p = 4.01E-06) in secretomes of apoptosis-resistant AML. These findings were confirmed by subsequent extracellular vesicle proteomics. Finally, confocal-microscopy-based colocalization studies show that splicing factors-containing vesicles secreted by high AAI cells are taken up by low AAI cells. The current results constitute the first comprehensive analysis of proteins released by apoptosis-resistant and sensitive primary AML cells. Together, the data point to vesicle-mediated release of global gene regulatory protein clusters as a plausible novel mechanism of induction of apoptosis resistance. Deciphering the modes of communication between apoptosis-resistant blasts may in perspective lead to the discovery of prognostic tools and development of novel therapeutic interventions, aimed at limiting or overcoming therapy resistance.Despite good remission rates observed in acute myeloid leukemia (AML) patients, the 5-year event-free survival rates reach only 35–40% in adults and 60–70% in children (1, 2). Apoptosis is one of the crucial mechanisms influencing survival of AML cells, and its deregulation can possibly lead to chemotherapy resistance and eventually relapse (3–5). The ability of cells to undergo apoptosis is largely defined by the relative expression of anti- (i.e. BCL-2, BCL-X long isoform - BCL-XL, or MCL-1) and proapoptotic (i.e. BAX, BH3 interacting domain death agonist - BID, caspases) proteins. Several studies have shown that the levels of BCL-2 and BCL-2/BAX ratio are a determinant of apoptosis-resistance in AML blasts and are associated with survival in AML patients (3, 6). We have previously demonstrated that the expression of several apoptosis-related proteins, such as BCL-2, BCL-XL, MCL-1, and BAX, can be reliably measured in AML samples by flow cytometry (6). These four quantitative parameters, which constitute an anti-apoptosis index (AAI)¹, have proven to be a reliable predictor of AML patients'' survival, with a high apoptosis-resistant profile (i.e. higher AAI) of diagnosis leukemic blasts being associated with shorter disease-free survival (7). Accordingly, AAI determined at the time of diagnosis also correlated with the frequency of minimal residual disease (MRD), which is a reflection of drug-resistant leukemic cells that have survived chemotherapy (7). MRD can be detected at a low frequency in bone marrow (BM) at the time of remission and is thought to contain the relapse-initiating cells (8–10). These observations imply that leukemic cells that harbor an apoptosis-resistant protein profile at diagnosis can better survive chemotherapy, thereby eventually causing a relapse. Consequently, we further hypothesized that the AAI of MRD cells would be either elevated or at least similar to the profile of leukemic cells at diagnosis. Surprisingly, in complete remission patients, the AAI decreased in the MRD situation compared with apoptosis-resistant profile as measured in leukemic blasts at diagnosis. The values of the AAI profile increased again at relapse, indicating apoptosis-resistance (11). Based on these unexpected findings, we hypothesized that expression of apoptosis-related proteins in AML blasts, and possibly also in bystander cells in the bone marrow, is regulated by extracellular factors present in the AML microenvironment.Tumor cell communication with its microenvironment is emerging as an important determinant playing multiple roles in cancer. In this respect, both soluble factors and extracellular vesicles (EVs), most notably exosomes, have been shown to influence cellular processes of malignant and normal cells in the tumor microenvironment (12–14). Apoptosis in the AML setting can be regulated by several cytokines as well as by EVs, which carry variable cargoes, including multiple proteins (15–18). In line with our hypothesis, apoptosis of BM cells was shown to be inhibited in the presence of secretome derived from AML blasts (19). These observations suggest that factors secreted by apoptosis-resistant leukemic blasts are likely to confer a drug resistance phenotype upon initially sensitive blasts. Therefore, the aim of our current study was to characterize the microenvironment produced by apoptosis-resistant AML blasts in terms of its capacity to influence apoptosis regulation in neighboring cells and protein content.     

miR-638 Regulates Differentiation and Proliferation in Leukemic Cells by Targeting Cyclin-dependent Kinase 2

MicroRNAs have been extensively studied as regulators of hematopoiesis and leukemogenesis. We identified miR-638 as a novel regulator in myeloid differentiation and proliferation of leukemic cells. We found that miR-638 was developmentally up-regulated in cells of myeloid but not lymphoid lineage. Furthermore, significant miR-638 down-regulation was observed in primary acute myeloid leukemia (AML) blasts, whereas miR-638 expression was dramatically up-regulated in primary AML blasts and leukemic cell lines undergoing forced myeloid differentiation. These observations suggest that miR-638 might play a role in myeloid differentiation, and its dysregulation may contribute to leukemogenesis. Indeed, ectopic expression of miR-638 promoted phorbol 12-myristate 13-acetate- or all-trans-retinoic acid-induced differentiation of leukemic cell lines and primary AML blasts, whereas miR-638 inhibition caused an opposite phenotype. Consistently, miR-638 overexpression induced G₁ cell cycle arrest and reduced colony formation in soft agar. Cyclin-dependent kinase 2 (CDK2) was found to be a target gene of miR-638. CDK2 inhibition phenotypically mimicked the overexpression of miR-638. Moreover, forced expression of CDK2 restored the proliferation and the colony-forming ability inhibited by miR-638. Our data suggest that miR-638 regulates proliferation and myeloid differentiation by targeting CDK2 and may serve as a novel target for leukemia therapy or marker for AML diagnosis and prognosis.     

Molecular dissection of valproic acid effects in acute myeloid leukemia identifies predictive networks

Histone deacetylase inhibitors (HDACIs) like valproic acid (VPA) display activity in leukemia models and induce tumor-selective cytotoxicity against acute myeloid leukemia (AML) blasts. As there are limited data on HDACIs effects, we aimed to dissect VPA effects in vitro using myeloid cell lines with the idea to integrate findings with in vivo data from AML patients treated with VPA additionally to intensive chemotherapy (n = 12). By gene expression profiling we identified an in vitro VPA response signature enriched for genes/pathways known to be implicated in cell cycle arrest, apoptosis, and DNA repair. Following VPA treatment in vivo, gene expression changes in AML patients showed concordant results with the in vitro VPA response despite concomitant intensive chemotherapy. Comparative miRNA profiling revealed VPA-associated miRNA expression changes likely contributing to a VPA-induced reversion of deregulated gene expression. In addition, we were able to define markers predicting VPA response in vivo such as CXCR4 and LBH. These could be validated in an independent cohort of VPA and intensive chemotherapy treated AML patients (n = 114) in which they were inversely correlated with relapse-free survival. In summary, our data provide new insights into the molecular mechanisms of VPA in myeloid blasts, which might be useful in further advancing HDAC inhibition based treatment approaches in AML.     

Distribution of the cytoskeletal protein,Nestin, in acute leukemia

Abstract

Nestin is a neuroepithelial stem cell marker that is expressed in some types of tumor cells. Recent reports suggest that Nestin may be closely related to malignant cell proliferation and migration. Acute leukemia (AL) is characterized by a lack of differentiation, which results in uncontrolled proliferation in the bone marrow and accumulation of immature cells. The expression and function of Nestin in AL is unclear. We investigated Nestin immunohistochemical patterns of 87 patients that included 47 cases of acute myeloid leukemia (AML) and 40 cases of acute lymphoblastic leukemia (ALL), and 20 patients in complete remission (CR) from AML or ALL. We also investigated the clinico-pathological features of 87 cases of AL and their CR and overall survival (OS). Nestin was expressed in leukemic blasts and mature granulocytic cells in most cases (39/47) of AML. Conversely, Nestin was expressed in mature granulocytic cells in fewer cases (6/40) of ALL, but not in blasts. Nestin expression appeared in leukemic blasts of AML, but not ALL. Nestin expression in AML blast cells was not associated with CR or OS. We provide evidence that Nestin is expressed in AL and might be a useful immunohistochemical marker for identifying AML and ALL.     

Calreticulin exposure on malignant blasts predicts a cellular anticancer immune response in patients with acute myeloid leukemia

Experiments performed in mice revealed that anthracyclines stimulate immunogenic cell death that is characterized by the pre-apoptotic exposure of calreticulin (CRT) on the surface of dying tumor cells. Here, we determined whether CRT exposure at the cell surface (ecto-CRT) occurs in human cancer in response to anthracyclines in vivo, focusing on acute myeloid leukemia (AML), which is currently treated with a combination of aracytine and anthracyclines. Most of the patients benefit from the induction chemotherapy but relapse within 1–12 months. In this study, we investigated ecto-CRT expression on malignant blasts before and after induction chemotherapy. We observed that leukemic cells from some patients exhibited ecto-CRT regardless of chemotherapy and that this parameter was not modulated by in vivo chemotherapy. Ecto-CRT correlated with the presence of phosphorylated eIF2α within the blasts, in line with the possibility that CRT exposure results from an endoplasmic reticulum stress response. Importantly, high levels of ecto-CRT on malignant myeloblasts positively correlated with the ability of autologous T cells to secrete interferon-γ on stimulation with blast-derived dendritic cell. We conclude that the presence of ecto-CRT on leukemia cells facilitates cellular anticancer immune responses in AML patients.     

Decreased expression of DNAM-1 on NK cells from acute myeloid leukemia patients

This study tested the hypothesis that the expression of CD112 and CD155 (DNAM-1 ligands) on leukemic blasts induces a decreased expression of the activating receptor DNAM-1 on natural killer (NK) cells from acute myeloid leukemia (AML) patients. DNAM-1 is a co-receptor involved in the activation of NK cell cytotoxicity after its interaction with its ligands CD112 and CD155 on target cells. Here we study the expression of DNAM-1 on NK cells and DNAM-1 ligands on blasts from AML patients stratified by age. The results demonstrate that NK cells from AML patients younger than 65 years have a reduced expression of DNAM-1 compared with age-matched controls. The analysis of DNAM-1 ligands showed a high expression of CD112 and CD155 on leukemic blasts. An inverse correlation between CD112 expression on leukemic blasts and DNAM-1 expression on NK cells was found. Furthermore, downregulation of DNAM-1 was induced on healthy donors' NK cells after in vitro culture with leukemic blasts expressing DNAM-1 ligands. In conclusion, these results support the hypothesis that receptor-ligand crosslinking downregulates DNAM-1 expression on NK cells from patients <65 years of age. Considering the relevance of DNAM-1 in NK recognition and killing of leukemic cells, the reduced expression of this receptor on NK cells from AML patients can represent an additional mechanism of tumor escape.     

Upregulation of microRNA-375 is associated with poor prognosis in pediatric acute myeloid leukemia

A genome-wide serum miRNA expression analysis previously showed the upregulation of microRNA-375 (miR-375) in acute myeloid leukemia (AML) patients compared with healthy controls. The aim of this study was to investigate the expression patterns and the prognostic relevance of miR-375 in pediatric AML. Expression levels of miR-375 in bone marrow mononuclear cells were detected by real-time quantitative PCR in a cohort of 106 patients with newly diagnosed pediatric AML. Expression levels of miR-375 in the bone marrow of pediatric AML patients were significantly higher than those in normal controls (P < 0.001). Then, miR-375 upregulation occurred more frequently in French–American–British classification subtype M7 than in other subtypes (P < 0.001). Regarding to cytogenetic risk, the expression levels of miR-375 in pediatric AML patients with unfavorable karyotypes were dramatically higher than those in intermediate and favorable groups (P = 0.002). Moreover, high miR-375 expression was significantly associated with shorter relapse-free survival (P < 0.001) and overall survival (P < 0.001) in pediatric AML patients. Multivariate analysis further identified miR-375 expression and cytogenetics risk as independent prognostic factors for both relapse-free survival and overall survival. In particular, the prognostic relevance of miR-375 expression was more obvious in the subgroup of patients with intermediate-risk cytogenetics. Our findings suggest for the first time that the upregulation of miR-375 may be one of the molecular mechanisms involved in the development and progression of pediatric AML. Since its correlation with poor relapse-free survival and overall survival, miR-375 may be a novel biomarker to improve the management of pediatric AML patients.     

Functional Pathway Analysis Using SCNP of FLT3 Receptor Pathway Deregulation in AML Provides Prognostic Information Independent from Mutational Status

FMS-like tyrosine kinase 3 receptor (FLT3) internal tandem duplication (ITD) mutations result in constitutive activation of this receptor and have been shown to increase the risk of relapse in patients with acute myeloid leukemia (AML); however, substantial heterogeneity in clinical outcomes still exists within both the ITD mutated and unmutated AML subgroups, suggesting alternative mechanisms of disease relapse not accounted by FLT3 mutational status. Single cell network profiling (SCNP) is a multiparametric flow cytometry based assay that simultaneously measures, in a quantitative fashion and at the single cell level, both extracellular surface marker levels and changes in intracellular signaling proteins in response to extracellular modulators. We previously reported an initial characterization of FLT3 ITD-mediated signaling using SCNP. Herein SCNP was applied sequentially to two separate cohorts of samples collected from elderly AML patients at diagnosis. In the first (training) study, AML samples carrying unmutated, wild-type FLT3 (FLT3 WT) displayed a wide range of induced signaling, with a fraction having signaling profiles comparable to FLT3 ITD AML samples. Conversely, the FLT3 ITD AML samples displayed more homogeneous induced signaling, with the exception of patients with low (<40%) mutational load, which had profiles comparable to FLT3 WT AML samples. This observation was then confirmed in an independent (verification) cohort. Data from the second cohort were also used to assess the association between SCNP data and disease-free survival (DFS) in the context of FLT3 and nucleophosmin (NPM1) mutational status among patients who achieved complete remission (CR) to induction chemotherapy. The combination of SCNP read outs together with FLT3 and NPM1 molecular status improved the DFS prediction accuracy of the latter. Taken together, these results emphasize the value of comprehensive functional assessment of biologically relevant signaling pathways in AML as a basis for the development of highly predictive tests for guidance of post-remission therapy.     

MicroRNA Patterns Associated with Clinical Prognostic Parameters and CNS Relapse Prediction in Pediatric Acute Leukemia

Background

Recent reports have indicated that microRNAs (miRNAs) play a critical role in malignancies, and regulations in the progress of adult leukemia. The role of miRNAs in pediatric leukemia still needs to be established. The purpose of this study was to investigate the aberrantly expressed miRNAs in pediatric acute leukemia and demonstrate miRNA patterns that are pediatric-specific and prognostic parameter-associated.

Methodology/Principal Findings

A total of 111 pediatric bone marrow samples, including 99 patients and 12 normal donors, were enrolled in this study. Of those samples, 36 patients and 7 normal samples were used as a test cohort for the evaluation of miRNA profiling; 63 pediatric patients and 5 normal donors were used as a validation cohort to confirm the miRNA differential expression. Pediatric ALL- and AML-specific microRNA expression patterns were identified in this study. The most highly expressed miRNAs in pediatric ALL were miR-34a, miR-128a, miR-128b, and miR-146a, while the highly expressed miRNAs in pediatric AML were miR-100, miR-125b, miR-335, miR-146a, and miR-99a, which are significantly different from those reported for adult CLL and AML. miR-125b and miR-126 may serve as favorable prognosticators for M3 and M2 patients, respectively. Importantly, we identified a “miRNA cascade” associated with central nervous system (CNS) relapse in ALL. Additionally, miRNA patterns associated with prednisone response, specific risk group, and relapse of ALL were also identified.

Conclusions/Significance

There are existing pediatric-associated and prognostic parameter-associated miRNAs that are independent of cell lineage and could provide therapeutic direction for individual risk-adapted therapy for pediatric leukemia patients.     

Identification of differentially methylated markers among cytogenetic risk groups of acute myeloid leukemia

Aberrant DNA methylation is known to occur in cancer, including hematological malignancies such as acute myeloid leukemia (AML). However, less is known about whether specific methylation profiles characterize specific subcategories of AML. We examined this issue by using comprehensive high-throughput array-based relative methylation analysis (CHARM) to compare methylation profiles among patients in different AML cytogenetic risk groups. We found distinct profiles in each group, with the high-risk group showing overall increased methylation compared with low- and mid-risk groups. The differentially methylated regions (DMRs) distinguishing cytogenetic risk groups of AML were enriched in the CpG island shores. Specific risk-group associated DMRs were located near genes previously known to play a role in AML or other malignancies, such as MN1, UHRF1, HOXB3, and HOXB4, as well as TRIM71, the function of which in cancer is not well characterized. These findings were verified by quantitative bisulfite pyrosequencing and by comparison with results available at the TCGA cancer genome browser. To explore the potential biological significance of the observed methylation changes, we correlated our findings with gene expression data available through the TCGA database. The results showed that decreased methylation at HOXB3 and HOXB4 was associated with increased gene expression of both HOXB genes specific to the mid-risk AML, while increased DNA methylation at DCC distinctive to the high-risk AML was associated with increased gene expression. Our results suggest that the differential impact of cytogenetic changes on AML prognosis may, in part, be mediated by changes in methylation.     

Proteinase-Activated Receptor 1 (PAR1) Regulates Leukemic Stem Cell Functions

External signals that are mediated by specific receptors determine stem cell fate. The thrombin receptor PAR1 plays an important role in haemostasis, thrombosis and vascular biology, but also in tumor biology and angiogenesis. Its expression and function in hematopoietic stem cells is largely unknown. Here, we analyzed expression and function of PAR1 in primary hematopoietic cells and their leukemic counterparts. AML patients'' blast cells expressed much lower levels of PAR1 mRNA and protein than CD34⁺ progenitor cells. Constitutive Par1-deficiency in adult mice did not affect engraftment or stem cell potential of hematopoietic cells. To model an AML with Par1-deficiency, we retrovirally introduced the oncogene MLL-AF9 in wild type and Par1^−/− hematopoietic progenitor cells. Par1-deficiency did not alter initial leukemia development. However, the loss of Par1 enhanced leukemic stem cell function in vitro and in vivo. Re-expression of PAR1 in Par1^−/− leukemic stem cells delayed leukemogenesis in vivo. These data indicate that Par1 contributes to leukemic stem cell maintenance.     

Shift of aberrant antigen expression at relapse or at treatment failure in acute leukemia

The flow cytometric detection of aberrant antigen expression is one method proposed for the quantification of minimal residual disease (MRD) in acute leukemias. The present study was designed to investigate the stability of the aberrant antigen expression at relapse or at treatment failure of initial chemotherapy. For this purpose, multiparameter immunophenotyping with a panel of 15 monoclonal antibodies was used at diagnosis as well as at relapse (43 patients with overall 65 aberrations) and at treatment failure (35 patients with overall 66 aberrations). There was a significant decrease in the percentage of the initially described aberrant antigen expression on leukemia blasts at relapse (P = 0.001; n = 65) as well as at treatment failure (P = 0.0001; n = 66) considering all aberrations in the whole leukemia population. Concerning only patients with acute myelogenous leukemia (AML), significant decreases in the aberrant expression could be detected at relapse (P = 0.031; n = 42) and at treatment failure (P = 0.0001; n = 52). The changes in patients with acute lymphoblastic leukemia (ALL) were significant only at relapse (P = 0.006; n = 23). Initially, the most informative aberration was not detectable in four patients at relapse and in seven patients at treatment failure. A decrease of under 50% of the initial value was observed in another 8 patients at relapse and in 10 patients at treatment failure. In further studies assessing the detection of aberrant antigen expression for MRD, quantification of the relapses should be explicitly analyzed regarding the persistence of the initially described aberrant antigen expression.     

Epigenetic Priming of AML Blasts for All-trans Retinoic Acid-Induced Differentiation by the HDAC Class-I Selective Inhibitor Entinostat

All-trans retinoic acid (ATRA) has only limited single agent activity in AML without the PML-RARα fusion (non-M3 AML). In search of a sensitizing strategy to overcome this relative ATRA resistance, we investigated the potency of the HDAC class-I selective inhibitor entinostat in AML cell lines Kasumi-1 and HL-60 and primary AML blasts. Entinostat alone induced robust differentiation of both cell lines, which was enhanced by the combination with ATRA. This “priming” effect on ATRA-induced differentiation was at least equivalent to that achieved with the DNA hypomethylating agent decitabine, and could overall be recapitulated in primary AML blasts treated ex vivo. Moreover, entinostat treatment established the activating chromatin marks acH3, acH3K9, acH4 and H3K4me3 at the promoter of the RARβ2 gene, an essential mediator of retinoic acid (RA) signaling in different solid tumor models. Similarly, RARβ2 promoter hypermethylation (which in primary blasts from 90 AML/MDS patients was surprisingly infrequent) could be partially reversed by decitabine in the two cell lines. Re-induction of the epigenetically silenced RARβ2 gene was achieved only when entinostat or decitabine were given prior to ATRA treatment. Thus in this model, reactivation of RARβ2 was not necessarily required for the differentiation effect, and pharmacological RARβ2 promoter demethylation may be a bystander phenomenon rather than an essential prerequisite for the cellular effects of decitabine when combined with ATRA. In conclusion, as a “priming” agent for non-M3 AML blasts to the differentiation-inducing effects of ATRA, entinostat is at least as active as decitabine, and both act in part independently from RARβ2. Further investigation of this treatment combination in non-M3 AML patients is therefore warranted, independently of RARβ2 gene silencing by DNA methylation.