Proteins In Acute Myeloid Leukemia Research Articles

Targeting Fatty Acid Metabolism Abrogates the Differentiation Blockade in Pre-leukemic Cells.

Metabolism plays a key role in the maintenance of normal hematopoietic stem cells (HSCs) and in the development of leukemia. A better understanding of the metabolic characteristics and dependencies of pre-leukemic cells could help identify potential therapeutic targets to prevent leukemic transformation. As AML1-ETO, one of the most frequent fusion proteins in acute myeloid leukemia that is encoded by a RUNX1::RUNX1T1 fusion gene, is capable of generating pre-leukemic clones, here we used a conditional Runx1::Runx1t1 knock-in mouse model to evaluate pre-leukemic cell metabolism. AML1-ETO expression resulted in impaired hematopoietic reconstitution and increased self-renewal ability. Oxidative phosphorylation and glycolysis decreased significantly in these pre-leukemic cells accompanied by increased HSC quiescence and reduced cell cycling. Furthermore, HSCs expressing AML1-ETO exhibited an increased requirement for fatty acids through metabolic flux. Dietary lipid deprivation or loss of the fatty acid transporter FATP3 by targeted deletion using CRISPR/Cas9 partially restored differentiation. These findings reveal the unique metabolic profile of pre-leukemic cells and propose FATP3 as a potential target for disrupting leukemogenesis.

Cluster of Differentiation 38 Protein Significantly Adds to the Prognostic Value of Cytogenetic Risk in Patients with Acute Myeloid Leukemia.

There is some controversy regarding the clinical significance of cluster of differentiation 38 (CD38) protein in acute myeloid leukemia (AML). We aimed to study the prognostic value of CD38 expression in relation to the standard cytogenetic risk stratification in AML patients. In this prospective study, adult patients with AML were diagnosed based on the finding of ≥ 20% blasts in the bone marrow and the flow cytometry. The control group was selected from normal bone marrow samples examined at the same period in the lab. The CD38 expression levels were assessed by immunophenotyping the bone marrow samples of the patients prior to therapy, as well as those from the controls. The subjects were N = 52 newly diagnosed adult AML patients, while n = 25 sex-and-age matched normal volunteers served as the control. The CD38 expression among AML patients ranged from 3.6% to 79.9%, significantly higher levels than in the control population (p =0.001). Positive CD38 expression was higher in AML patients with favorable cytogenetics when compared to those with intermediate and poor cytogenetics (75.0%, 57.1%, and 68.2%, respectively) (p =0.007). Median CD38 expression in AML patients was higher in the favorable cytogenetic group and lower in intermediate and poor cytogenetic groups, though lacking in significance (p =0.578). Patients with poor cytogenetic risk were associated with significantly shorter median overall survival when compared to favorable and intermediate cytogenetic risk (p =0.010). The expression of CD38 significantly adds to the prognostic value of cytogenetic risk stratification at diagnosis of AML patients.

LYL1 facilitates AETFC assembly and gene activation by recruiting CARM1 in t(8;21) AML

Transcription factors (TFs) play critical roles in hematopoiesis, and their aberrant expression can lead to various types of leukemia. The t(8;21) leukemogenic fusion protein AML1-ETO (AE) is the most common fusion protein in acute myeloid leukemia and can enhance hematopoietic stem cell renewal while blocking differentiation. A key question in understanding AE-mediated leukemia is what determines the choice of AE to activate self-renewal genes or repress differentiation genes. Toward the resolution of this problem, we earlier showed that AE resides in the stable AETFC complex and that its components colocalize on up- or down-regulated target genes and are essential for leukemogenesis. In the current study, using biochemical and genomic approaches, we show that AE-containing complexes are heterogeneous, and that assembly of the larger AETFC (containing AE, CBFβ, HEB, E2A, LYL1, LMO2, and LDB1) requires LYL1. Furthermore, we provide strong evidence that the LYL1-containing AETFC preferentially binds to active enhancers and promotes AE-dependent gene activation. Moreover, we show that coactivator CARM1 interacts with AETFC and facilitates gene activation by AETFC. Collectively, this study describes a role of oncoprotein LYL1 in AETFC assembly and gene activation by recruiting CARM1 to chromatin for AML cell survival.

Expression level and prognostic potential of beta-catenin-interacting protein in acute myeloid leukemia.

Inhibitor of beta-catenin and TCF (ICAT) is a key protein in the Wnt-β-catenin signaling pathway. However, its role in acute myeloid leukemia (AML) remains unknown. In this study, we evaluated its expression level as well as its prognostic value in AML patients. A total of 72 patients with AML and 30 control subjects were enrolled in this study during the period of January 2017 and December 2019 at Zhongshan Hospital of SunYat-sen University. ICAT and β-catenin expression levels in peripheral blood were determined via enzyme-linked immunosorbent assays. ICAT levels in AML patients were significantly lower and β-catenin levels were higher than those of the control group. After the first course of standard chemotherapy, the concentration of ICAT in the partial remission group (93.79 ng/mL) was significantly higher than that in the initial diagnosis group (49.38 ng/mL) and the no response group (39.94 ng/mL). AML subtypes had lower ICAT expression levels than controls, and ICAT levels were significantly correlated with body mass index, bone marrow/peripheral blood blast cell proportions, and white blood cell and red blood cell counts at initial diagnosis. Furthermore, low ICAT expression was found to be associated with poor disease-free survival and overall survival in AML. ICAT is closely associated with AML progression and can be used as an indicator to monitor AML treatment efficacy.

RUNX1T1, a potential prognostic marker in breast cancer, is co-ordinately expressed with ERα, and regulated by estrogen receptor signalling in breast cancer cells.

RUNX1T1 is extensively studied in the context of AML1-RUNX1T1 fusion protein in acute myeloid leukemia. Little is known about the function of RUNX1T1 itself, although data on its function and regulation have begun to emerge from clinical, and in vitro studies. It is a putative tumor suppressor, whose expression is altered in a variety of solid tumors. Recently, reduced expression of RUNX1T1 in triple-negative breast tumors, and its influence on prognosis was reported. The Kaplan-Meier Plotter online tool was used to study the relationship between RUNX1T1 expression and survival of breast cancer patients. High RUNX1T1 expression was associated with longer overall survival (OS), relapse-free survival (RFS) and distant metastasis free survival (DMFS). RUNX1T1 expression positively and negatively influenced OS of patients with ERα-positive and ERα-negative breast tumors, respectively. It was also associated with prolonged RFS, and DMFS in tamoxifen-treated patients. Expression of RUNX1T1 and ERα mRNA was analyzed in 40 breast tumor samples, and breast cancer cell lines using RT-PCR. TCGA-BRCA data was mined to study the relationship between RUNX1T1 and ERα mRNA expression. ERα-positive breast tumors showed significantly higher RUNX1T1 mRNA expressioncompared to ERα-negative tumors. RUNX1T1 mRNA expression was analyzed by qRT-PCR in MCF-7 or T47D cells, which were treated with 17β-estradiol, or the ERα agonist PPT, alone or in combination with 4-hydroxytamoxifen. Effect of ERα knockdown was also investigated. Results indicate that estrogen downmodulated RUNX1T1 mRNA expression via ERα. Higher expression of RUNX1T1 in breast tumors is associated with favourable prognosis. RUNX1T1 and ERα show co-ordinated expression in breast tumors, and breast cancer cell lines. Estrogen-ERα signalling downmodulates the expression of RUNX1T1 mRNA in ERα-positive breast cancer cells. In-depth investigations on the interaction between RUNX1T1 and ERα are warranted to unravel the role and relevance of RUNX1T1 in breast cancer.

The Oncogenic Fusion Protein CBFB-SMMHC Downregulates CD48 to Evade NK Cell Recognition

Chromosomal translocations are a common cytogenetic aberration in acute leukemia, which frequently generate fusion proteins with oncogenic properties. We have previously studied the immune evasion mechanisms of two common oncogenic fusion proteins in acute myeloid leukemia (AML), PML-RARA and AML1-ETO. We investigated, in particular, the activity of natural killer (NK) cells, which are part of the innate immune system and play a significant role in eliminating leukemic cells. We found that both oncogenic fusion proteins downregulate the expression of CD48, a ligand of the NK cell activating receptor 2B4, to escape NK cell-mediated recognition of the tumor cells.In the current study we tested the possibility that other oncogenic fusion proteins are also able to evade NK cell recognition. To this aim we cloned several leukemic fusion proteins into lentiviral vectors and expressed them in U937 cells, which are commonly used to express leukemic fusion proteins. The leukemic fusion proteins included MLL-AF4, NUP98-HOXA9, DEK-NUP98 and CBFB-SMMHC. By using flow cytometry, we examined the effect of these fusion proteins on the expression of several NK cell ligands. CBFB-SMMHC was the only fusion protein which downregulated the expression of CD48.Next, we tested the functional significance of the downregulation of CD48 by CBFB-SMMHC by performing cytotoxicity assays with NK cells. We first used the NK cell line YTS eco since the cytotoxicity of these cells is mainly dependent on the 2B4-CD48 interaction. We found that cells which express CBFB-SMMHC were killed significantly less than the control cells, and that this reduced killing was due to CD48 downregulation. We also tested the killing of CBFB-SMMHC expressing cells by primary bulk NK cells and found that cells which express this fusion protein were killed significantly less.Mechanistically we show that overexpression of CBFB-SMMHC reduces the mRNA levels of CD48. Additionally, we tested whether treatment with HDAC inhibitors (HDACi) can reverse the downregulation of CD48 by CBFB-SMMHC. We treated cells which express CBFB-SMMHC with various HDACi and examined the effect of this treatment on the expression of CD48. Two specific class I HDACi, mocetinostat and entinostat, upregulated the expression of CD48 in cells which express this fusion protein.Taken together we show that modulation of CD48 expression is an immune evasion mechanism, which is specific to core binding factor (CBF) proteins (AML1-ETO and CBFB-SMMHC) and PML-RARA. Although the three fusion proteins which we found to down-regulate the expression of CD48 are associated with better prognosis in AML, about 30% of CBF-AML patients eventually relapse. Based on our findings we suggest that down-regulation of CD48 by these fusion proteins, which leads to NK cell immune evasion, contributes to the persistence of residual disease in these subtypes of leukemia and eventually to a clinical relapse. Finally, we propose that NK cell based therapies (i.e., NK cell infusion) or class I HDACi could be potential adjunctive therapies for CBF-AML or APL. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Update on drug transporter proteins in acute myeloid leukemia: Pathological implication and clinical setting.

Acute myeloid leukemia (AML) is one of the most common hematological neoplasia causing death worldwide. The long-term overall survival is unsatisfactory due to many factors including older age, genetic heterogeneity and molecular characteristics comprising additional mutations, and resistance to chemotherapeutic drugs. The expression of ABCB1/P-glycoprotein, ABCC1/MRP1, ABCG2/BCRP and LRP transporter proteins is considered the major reason for multidrug resistance (MDR) in AML, however conflicting data have been reported. Here, we review the main issues about drug transporter proteins in AML clinical scenario, and highlight the clinicopathological significance of MDR phenotype associated with ABCB1 polymorphisms and FLT3 mutation.

Metformin exerts a synergistic effect with venetoclax by downregulating Mcl-1 protein in acute myeloid leukemia.

Background: Recently, one of the specific BH3-mimetics, Venetoclax has been approved by FDA providing new options for newly diagnosed AML patient especially who are unfitted to receive conventional chemotherapy. Though the clinical success of venetoclax has been achieved in clinical outcomes such as complete remission (CR) and overall survival. Acquired resistance to ABT-199 which is induced by the regulation of apoptosis pathway is still an important clinical problem. To this end, the attempt to combine drugs which can reverse the compensatory regulation is urgent.Methods: In three AML cell lines (KG-1, Kasumi-1 and THP-1), the anti-AML effects of the combination of ABT-199 (Venetoclax) and metformin or the two drugs used alone were compared. CCK8 was used to evaluate the cell viability, and flow cytometry was used to estimate the rate of apoptosis, Western blot method was performed to detect apoptosis-related protein levels. In mice experiments, female BALB/c-nu nude mice were subcutaneously injected with THP-1 cells for subcutaneous tumor formation, and the combined effect of ABT-199 and metformin was tested. The evaluation indicators were tumor size, tumor weight, and Ki67 staining. Mouse body weight and HE staining were detected to evaluate liver damage and adverse drug reactions.Results: Both in vitro and in vivo experiments showed that compared with metformin or ABT-199 alone, the combined use of the two drugs exerts a synergistic effect on promoting apoptosis, thereby producing a strong anti-leukemia effect. Furthermore, after a short incubation time, ABT-199 swiftly increased the expression level of the anti-apoptotic protein Mcl-1, while the combined use of metformin and ABT-199 significantly reduced the level of Mcl-1. Notably, Metformin significantly downregulates the level of Mcl-1 protein by inhibiting its protein production. To less extent, metformin can also downregulate the expression of another anti-apoptotic protein, BCL-xl.Conclusion: Metformin downregulates the expression of anti-apoptotic proteins Mcl-1 and Bcl-xl by inhibiting protein production, and shows a synergistic anti-tumor effect with ABT-199 in acute myeloid leukemia.

Targeting Bcl-2 Proteins in Acute Myeloid Leukemia.

B cell lymphoma 2 (BCL-2) family proteins play an important role in intrinsic apoptosis. Overexpression of BCL-2 proteins in acute myeloid leukemia can circumvent resistance to apoptosis and chemotherapy. Considering this effect, the exploration of anti-apoptotic BCL-2 inhibitors is considered to have tremendous potential for the discovery of novel pharmacological modulators in cancer. This review outlines the impact of BCL-2 family proteins on intrinsic apoptosis and the development of acute myeloid leukemia (AML). Furthermore, we will also review the new combination therapy with venetoclax that overcomes resistance to venetoclax and discuss biomarkers of treatment response identified in early-phase clinical trials.

CDK6 is an essential direct target of NUP98 fusion proteins in acute myeloid leukemia

AbstractFusion proteins involving Nucleoporin 98 (NUP98) are recurrently found in acute myeloid leukemia (AML) and are associated with poor prognosis. Lack of mechanistic insight into NUP98-fusion–dependent oncogenic transformation has so far precluded the development of rational targeted therapies. We reasoned that different NUP98-fusion proteins deregulate a common set of transcriptional targets that might be exploitable for therapy. To decipher transcriptional programs controlled by diverse NUP98-fusion proteins, we developed mouse models for regulatable expression of NUP98/NSD1, NUP98/JARID1A, and NUP98/DDX10. By integrating chromatin occupancy profiles of NUP98-fusion proteins with transcriptome profiling upon acute fusion protein inactivation in vivo, we defined the core set of direct transcriptional targets of NUP98-fusion proteins. Among those, CDK6 was highly expressed in murine and human AML samples. Loss of CDK6 severely attenuated NUP98-fusion–driven leukemogenesis, and NUP98-fusion AML was sensitive to pharmacologic CDK6 inhibition in vitro and in vivo. These findings identify CDK6 as a conserved, critical direct target of NUP98-fusion proteins, proposing CDK4/CDK6 inhibitors as a new rational treatment option for AML patients with NUP98-fusions.

Pharmacological Targeting of BET Bromodomain Proteins in Acute Myeloid Leukemia and Malignant Lymphomas: From Molecular Characterization to Clinical Applications.

Alterations in protein-protein and DNA-protein interactions and abnormal chromatin remodeling are a major cause of uncontrolled gene transcription and constitutive activation of critical signaling pathways in cancer cells. Multiple epigenetic regulators are known to be deregulated in several hematologic neoplasms, by somatic mutation, amplification, or deletion, allowing the identification of specific epigenetic signatures, but at the same time providing new therapeutic opportunities. While these vulnerabilities have been traditionally addressed by hypomethylating agents or histone deacetylase inhibitors, pharmacological targeting of bromodomain-containing proteins has recently emerged as a promising approach in a number of lymphoid and myeloid malignancies. Indeed, preclinical and clinical studies highlight the relevance of targeting the bromodomain and extra-terminal (BET) family as an efficient strategy of target transcription irrespective of the presence of epigenetic mutations. Here we will summarize the main advances achieved in the last decade regarding the preclinical and clinical evaluation of BET bromodomain inhibitors in hematologic cancers, either as monotherapies or in combinations with standard and/or experimental agents. A mention will finally be given to the new concept of the protein degrader, and the perspective it holds for the design of bromodomain-based therapies.

The Role of Forkhead Box Proteins in Acute Myeloid Leukemia.

Forkhead box (FOX) proteins are a group of transcriptional factors implicated in different cellular functions such as differentiation, proliferation and senescence. A growing number of studies have focused on the relationship between FOX proteins and cancers, particularly hematological neoplasms such as acute myeloid leukemia (AML). FOX proteins are widely involved in AML biology, including leukemogenesis, relapse and drug sensitivity. Here we explore the role of FOX transcription factors in the major AML entities, according to “The 2016 revision to the World Health Organization classification of myeloid neoplasms and acute leukemia”, and in the context of the most recurrent gene mutations identified in this heterogeneous disease. Moreover, we report the new evidences about the role of FOX proteins in drug sensitivity, mechanisms of chemoresistance, and possible targeting for personalized therapies.

Exploiting Protein Corona around Gold Nanoparticles Conjugated to p53 Activating Peptides To Increase the Level of Stable p53 Proteins in Cells.

Therapeutic peptides suffer from major drawbacks such as peptide degradation in vivo due to proteolysis. Gold nanoparticles (AuNPs) are an effective carrier for therapeutic peptides that improve their stability in vivo, while also enabling nonspecific adsorption of complementary proteins to enhance their effectiveness. Using p53 peptide as a model known to disrupt the intracellular MDM2-p53 protein-protein interaction which tags the endogenous p53 proteins for degradation, we conjugated p53 peptides to AuNPs (AuNP-p53) and examined the functionality of AuNP-p53 to release the endogenous p53 proteins from being tagged for degradation, thereby increasing the level of stable p53 proteins in acute myeloid leukemia 2 (AML2) cells. We found that AuNPs did not just protect conjugated p53 peptides from trypsin degradation, but also helped to recruit 56.5% and 26.4% of total MDM2 and p53 proteins in the cells to form a protein corona around AuNP-p53. The proximity of MDM2/p53 complexes and p53 peptide on the surface of AuNP-p53 facilitated the action of p53 peptides to cause a sustained elevation of the p53 level in AML2 cells up to 6 h, which was not possible with free p53 peptide alone at the same concentration. Even a 20-fold higher concentration of free p53 peptide caused only a short-lived elevated p53 level of 1 h. The outcome of this study highlights the utility of combining conjugated ligands and complementary protein adsorption on nanoparticles to improve the biological functionality of the therapeutic ligands.

Discovery of the selective and efficacious inhibitors of FLT3 mutations

Fms-like tyrosine kinase 3 (FLT3) is among the most frequently mutated protein in acute myeloid leukemia (AML), which has been confirmed as an important drug target for AML chemotherapy. Starting from the lead compound LT-106-175, a series of 1-H-pyrazole-3-carboxamide derivatives were synthesized to improve the FLT3 inhibitory potency and selectivity. Among them, compound 50 was identified as a highly potent and selective FLT3 inhibitor (IC50 = 0.213 nM), which showed equal activities against various mutants of FLT3 including FLT3 (ITD)-D835V and FLT3 (ITD)-F691L that is resistant to quizartinib. Compound 50 also exhibited efficacy against the human AML cell line MV4-11 (IC50 = 16.1 nM) harboring FLT3-ITD mutants. Inversely, compound 50 displayed no cytotoxicity to FLT3-independent cells, and the biochemical analyses showed that its effects were related to the inhibition of FLT3 signal pathways. Additionally, compound 50 induced apoptosis in MV4-11 cell as demonstrated by flow cytometry. Moreover, compound 50 showed enhanced metabolic stability. Altogether, it was concluded that compound 50 could be a promising FLT3 inhibitor for further developing therapeutic remedy of AML.

Structural investigation of nucleophosmin interaction with the tumor suppressor Fbw7\u03b3

Nucleophosmin (NPM1) is a multifunctional nucleolar protein implicated in ribogenesis, centrosome duplication, cell cycle control, regulation of DNA repair and apoptotic response to stress stimuli. The majority of these functions are played through the interactions with a variety of protein partners. NPM1 is frequently overexpressed in solid tumors of different histological origin. Furthermore NPM1 is the most frequently mutated protein in acute myeloid leukemia (AML) patients. Mutations map to the C-terminal domain and lead to the aberrant and stable localization of the protein in the cytoplasm of leukemic blasts. Among NPM1 protein partners, a pivotal role is played by the tumor suppressor Fbw7γ, an E3-ubiquitin ligase that degrades oncoproteins like c-MYC, cyclin E, Notch and c-jun. In AML with NPM1 mutations, Fbw7γ is degraded following its abnormal cytosolic delocalization by mutated NPM1. This mechanism also applies to other tumor suppressors and it has been suggested that it may play a key role in leukemogenesis. Here we analyse the interaction between NPM1 and Fbw7γ, by identifying the protein surfaces implicated in recognition and key aminoacids involved. Based on the results of computational methods, we propose a structural model for the interaction, which is substantiated by experimental findings on several site-directed mutants. We also extend the analysis to two other NPM1 partners (HIV Tat and CENP-W) and conclude that NPM1 uses the same molecular surface as a platform for recognizing different protein partners. We suggest that this region of NPM1 may be targeted for cancer treatment.

Helicase-like transcription factor is a RUNX1 target whose downregulation promotes genomic instability and correlates with complex cytogenetic features in acute myeloid leukemia.

Helicase-like transcription factor is a SWI/SNF chromatin remodeling factor involved in various biological processes. However, little is known about its role in hematopoiesis. In this study, we measured helicase-like transcription factor mRNA expression in the bone marrow of 204 adult patients with de novo acute myeloid leukemia. Patients were dichotomized into low and high expression groups at the median level for clinicopathological correlations. Helicase-like transcription factor levels were dramatically reduced in the low expression patient group compared to those in the normal controls (n=40) (P<0.0001). Low helicase-like transcription factor expression correlated positively with French-American-British M4/M5 subtypes (P<0.0001) and complex cytogenetic abnormalities (P=0.02 for ≥3 abnormalities;P=0.004 for ≥5 abnormalities) but negatively with CEBPA double mutations (P=0.012). Also, low expression correlated with poorer overall (P=0.005) and event-free (P=0.006) survival in the intermediate-risk cytogenetic subgroup. Consistent with the more aggressive disease associated with low expression, helicase-like transcription factor knockdown in leukemic cells promoted proliferation and chromosomal instability that was accompanied by downregulation of mitotic regulators and impaired DNA damage response. The significance of helicase-like transcription factor in genome maintenance was further indicated by its markedly elevated expression in normal human CD34(+)hematopoietic stem cells. We further demonstrated that helicase-like transcription factor was a RUNX1 target and transcriptionally repressed by RUNX1-ETO and site-specific DNA methylation through a duplicated RUNX1 binding site in its promoter. Taken together, our findings provide new mechanistic insights on genomic instability linked to helicase-like transcription factor deregulation, and strongly suggest a tumor suppressor function of the SWI/SNF protein in acute myeloid leukemia.

Progress in molecularly targeted therapies for acute myeloid leukemia

Genetic abnormalities including specific point mutations have recently been confirmed by applying comprehensive genome sequencing analyses. Molecular targeting therapies, which focus on the mutated proteins and over-expressed proteins in acute myeloid leukemia (AML) cells, are now being developed in clinical studies and/or based on in vitro analyses. This manuscript summarizes the genetic abnormalities in AML cells and some of the current molecular targeting therapies including FLT3 inhibitors (e.g. AC220; Quizartinib), Polo like kinase 1 (PLK1) inhibitors (e.g. BI-6727; Volasertib), IDH2 inhibitors (e.g. AG-221), and XPO1 inhibitors (e.g. KPT-330; Selinexor).

Acute Myeloid Leukemia Cells Acquire Chemo-Resistance By Inducing Osteoblast Differentiation in Mesenchymal Stem Cells through up-Regulation of RUNX2

The leukemia bone marrow micro-environment (BME) is comprised of the endosteal and vascular niches, provides vital support for cellular growth and conveys drug resistance to leukemia cells. Several reports suggest that mesenchymal stem/stromal cells (MSCs) present in the bone marrow niche induce cell survival and anti-apoptotic proteins in acute myeloid leukemia (AML) cells and protect them from chemotherapy. The mechanisms underlying BME-mediated chemo-resistance however have not been fully elucidated. Here, we hypothesize that AML cells induce functional changes and prime MSCs to protect leukemia cells from chemotherapy. To test our hypothesis, we have compared age matched (between 40-60 years) bone marrow derived MSCs from AML patients (AML-MSC, n=10) and normal (N-MSC, n=10) individuals and analyzed their proliferation, cell surface phenotype, multi-lineage differentiation and chemo-protection potential. AML-MSCs are phenotypically different, with their polygonal morphology and larger cell size compared to N-MSCs which are elongated and spindle shaped appearance. The average cell doubling time of AML-MSCs is 52±8hrs compared to 34±6hours for N-MSCs during their exponential growth phase (p<0.01). Cell surface phenotyping by flow cytometry revealed that most of the markers known to be expressed on N-MSCs including CD105, CD90, CD73, CD51, CD44, SUSD2, CD106, CD140b, CD140a, CD106 and CD271 were also expressed on AML-MSCs at similar levels. Interestingly, tissue non-specific alkaline phosphatase (TNAP, clone W8B2), a cell surface protein highly expressed in naïve-MSCs and osteoblast progenitors (Battula VL et al., Haematologica, 2009) was 10-14 fold higher in AML- as compared to N-MSCs. Since TNAP is also a osteoblast specific marker, we compared osteoblast differentiation potential of N- vs AML-MSCs. Surprisingly, a dramatic increase in alkaline phosphatase activity (by BCIP/NBT substrate) was observed in AML-MSCs even without induction of osteoblast differentiation. mRNA analysis by qRT-PCR revealed that osteoblast specific genes including osteopontin, TNAP, osteocalcin, and osterix were 5-10 fold up-regulated in AML-MSCs compared to N-MSCs before induction. In N-MSCs, the expression of these markers was induced only under osteoblast differentiation conditions. These data indicate that AML-MSCs are primed to differentiate into-osteoblasts. Adipocyte differentiation was assessed by Oil-Red O staining for lipid droplets and revealed a > 95% reduction (p<0.0001) in the number mature adipocytes in AML-MSCs compared to N-MSCs suggesting that AML-MSCs lack the ability to differentiate into adipocytes. To understand the mechanism inducing osteogenic specific differentiation of AML-MSCs, we performed mRNA expression analysis of genes that regulate this process. We found RUNX2, a transcription factor that induces osteogenic but inhibits adipogenic differentiation, was 4-5 fold increased in AML-MSCs compared to N-MSCs. To validate these observations, we co-cultured N-MSCs in the presence or absence of OCI-AML3 cells for 3-5 days and FACS sorted the MSCs for gene expression analysis. We observed a 3-4 fold up-regulation of TNAP protein expression by flow cytometry and 4-6 fold up-regulation of osteoblast specific markers including osteopontin, alkaline phosphatase and osterix in MSCs co-cultured with OCI-AML3 cells. In addition, RUNX2 was up-regulated in MSCs when co-cultured with OCI-AML3 cells. These data suggest that AML cells induce osteogenic differentiation in BM-MSCs by up-regulation of RUNX2. To identify the clinical significance of these observations, we examined the ability of AML- and N-MSCs to protect AML cells from chemotherapy. Co-culture of OCI-AML3 cells with either AML- or N-MSCs and treatment with Cytarabine revealed a 15±4.5% increase in the number of live leukemia cells when co-cultured with AML-MSCs compared to N-MSCs. These data indicate that AML-MSCs protect leukemia cells better from chemotherapy than normal MSCs. In conclusion, AML cells induce osteogenic differentiation in MSCs through up-regulation of the RUNX2 transcription factor. Increased chemo-protection of AML cells by AML-MSCs suggests a prominent role of these cells in AML relapse. Targeting RUNX2 and thereby inhibition of osteoblast differentiation of MSCs may provide enhanced treatment options for AML therapy. DisclosuresNo relevant conflicts of interest to declare.

Erratum: WTAP is a novel oncogenic protein in acute myeloid leukemia

Correction to: Leukemia (2014) 28, 1171–1174; doi:10.1038/leu.2014.16 Since the publication of this article, the authors have identified an error concerning one of the author names. D Proia should be listed as DA Proia. The corrected list is shown above. The Authors would like to apologise for any inconvenience this may have caused.

KAT6A, a Chromatin Modifier from the 8p11-p12 Amplicon is a Candidate Oncogene in Luminal Breast Cancer

The chromosome 8p11-p12 amplicon is present in 12% to 15% of breast cancers, resulting in an increase in copy number and expression of several chromatin modifiers in these tumors, including KAT6A. Previous analyses in SUM-52 breast cancer cells showed amplification and overexpression of KAT6A, and subsequent RNAi screening identified KAT6A as a potential driving oncogene. KAT6A is a histone acetyltransferase previously identified as a fusion partner with CREB binding protein in acute myeloid leukemia. Knockdown of KAT6A in SUM-52 cells, a luminal breast cancer cell line harboring the amplicon, resulted in reduced growth rate compared to non-silencing controls and profound loss of clonogenic capacity both in mono-layer and in soft agar. The normal cell line MCF10A, however, did not exhibit slower growth with knockdown of KAT6A. SUM-52 cells with KAT6A knockdown formed fewer mammospheres in culture compared to controls, suggesting a possible role for KAT6A in self-renewal. Previous data from our laboratory identified FGFR2 as a driving oncogene in SUM-52 cells. The colony forming efficiency of SUM-52 KAT6A knockdown cells in the presence of FGFR inhibition was significantly reduced compared to cells with KAT6A knockdown only. These data suggest that KAT6A may be a novel oncogene in breast cancers bearing the 8p11-p12 amplicon. While there are other putative oncogenes in the amplicon, the identification of KAT6A as a driving oncogene suggests that chromatin-modifying enzymes are a key class of oncogenes in these cancers, and play an important role in the selection of this amplicon in luminal B breast cancers.