Nucleolar phosphoprotein modifications as a marker of apoptosis induced by RITA treatment.

Reactivating p53 and Inducing Tumor Apoptosis (RITA) has been reported to increase the p53 activity and to trigger p53-dependent apoptosis in cancer cells with wild-type p53. Tumor suppressor p53 interacts with nucleolar phosphoproteins nucleophosmin (NPM) and nucleolin (NCL), which have crucial role in many cellular processes. Specific NPM mutations associated with acute myeloid leukemia (AML) cause aberrant localization of NPM and p53 in the cytoplasm with possible impact on the p53 function. We tested an effect of RITA on primary cells, and we found significant RITA-induced changes in NPM and NCL phosphorylation associated with apoptosis in cells of AML patients, but not that of healthy donors. Subsequent screening of several AML cell lines revealed heterogeneous response to RITA, and confirmed an association of the specific phosphorylation with apoptosis. While decreased NCL phosphorylation at Threonines T76 and T84 could be attributed to RITA-induced cell cycle arrest, enhanced NPM phosphorylation at Threonine T199 was not accompanied by the cell cycle changes and it correlated with sensitivity to RITA. Simultaneously, inverse changes occurred at Serine S4 of the NPM. These new findings of RITA mechanism of action could establish the NPM pT199/pS4 ratio as a marker for suitability of RITA treatment of AML cells.

Two-photon lifetime-based photoconversion of EGFP for 3D-photostimulation in FLIM.

Enhanced green fluorescence protein (EGFP) is a fluorescent tag commonly used in cellular and biomedical applications. Surprisingly, some interesting photochemical properties of EGFP have remained unexplored. Here we report on two-photon-induced photoconversion of EGFP, which can be permanently converted by intense IR irradiation to a form with a short fluorescence lifetime and spectrally conserved emission. Photoconverted EGFP thus can be distinguished from the unconverted tag by the time-resolved detection. Nonlinear dependence of the two-photon photoconversion efficiency on the light intensity allows for an accurate 3D localization of the photoconverted volume within cellular structures, which is especially useful for kinetic FLIM applications. For illustration, we used the two photon photoconversion of EGFP for measurements of redistribution kinetics of nucleophosmin and histone H2B in nuclei of live cells. Measurements revealed high mobility of fluorescently tagged histone H2B in the nucleoplasm and their redistribution between spatially separated nucleoli.

Cytoplasmic localization of Mdm2 in cells expressing mutated NPM is mediated by p53.

Specific C-terminal nucleophosmin (NPM) mutations are related to the acute myeloid leukaemia and cause mistargeting of mutated NPM (NPMmut) to the cytoplasm. Consequently, multiple NPM-interacting partners, e.g., the tumour suppressor p53, become also mislocalized. We found that ubiquitin ligase Mdm2 mislocalizes to the cytoplasm in the presence of NPMmut as well. Since p53 interacts with Mdm2, we searched for the NPMmut-p53-Mdm2 complex and interactions of its constituents in live cells and cell lysates using fluorescently tagged proteins, fluorescence lifetime imaging and immunoprecipitation. We proved existence of the ternary complex, which likely adopts a chain-like configuration. Interaction between Mdm2 and NPMmut was not detected, even under conditions of upregulated Mdm2 and p53 induced by Actinomycin D. We assume that p53 serves in the complex as a bridging link between Mdm2 and NPMmut. This conclusion was supported by disruption of the Mdm2-p53 interaction by Nutlin-3A, which resulted in relocalization of Mdm2 to the nucleus, while both NPMmut and p53 remained in the cytoplasm. Importantly, silencing of p53 also prevented mislocalization of Mdm2 in the presence of NPMmut.

NPM1 and DNMT3A mutations are associated with distinct blast immunophenotype in acute myeloid leukemia.

The immune system is important for elimination of residual leukemic cells during acute myeloid leukemia (AML) therapy. Anti-leukemia immune response can be inhibited by various mechanisms leading to immune evasion and disease relapse. Selected markers of immune escape were analyzed on AML cells from leukapheresis at diagnosis (N = 53). Hierarchical clustering of AML immunophenotypes yielded distinct genetic clusters. In the absence of DNMT3A mutation, NPM1 mutation was associated with decreased HLA expression and low levels of other markers (CLIP, PD-L1, TIM-3). Analysis of an independent cohort confirmed decreased levels of HLA transcripts in patients with NPM1 mutation. Samples with combined NPM1 and DNMT3A mutations had high CLIP surface amount suggesting reduced antigen presentation. TIM-3 transcript correlated not only with TIM-3 surface protein but also with CLIP and PD-L1. In our cohort, high levels of TIM-3/PD-L1/CLIP were associated with lower survival. Our results suggest that AML genotype is related to blast immunophenotype, and that high TIM-3 transcript levels in AML blasts could be a marker of immune escape. Cellular pathways regulating resistance to the immune system might contribute to the predicted response to standard therapy of patients in specific AML subgroups and should be targeted to improve AML treatment.

AML-Related NPM Mutations Drive p53 Delocalization into the Cytoplasm with Possible Impact on p53-Dependent Stress Response.

Nucleophosmin (NPM) interaction with tumor suppressor p53 is a part of a complex interaction network and considerably affects cellular stress response. The impact of NPM1 mutations on its interaction with p53 has not been investigated yet, although consequences of NPMmut-induced p53 export to the cytoplasm are important for understanding the oncogenic potential of these mutations. We investigated p53-NPM interaction in live HEK-293T cells by FLIM-FRET and in cell lysates by immunoprecipitation. eGFP lifetime-photoconversion was used to follow redistribution dynamics of NPMmut and p53 in Selinexor-treated cells. We confirmed the p53-NPMwt interaction in intact cells and newly documented that this interaction is not compromised by the NPM mutation causing displacement of p53 to the cytoplasm. Moreover, the interaction was not abolished for non-oligomerizing NPM variants with truncated oligomerization domain, suggesting that oligomerization is not essential for interaction of NPM forms with p53. Inhibition of the nuclear exporter XPO1 by Selinexor caused expected nuclear relocalization of both NPMmut and p53. However, significantly different return rates of these proteins indicate nontrivial mechanism of p53 and NPMmut cellular trafficking. The altered p53 regulation in cells expressing NPMmut offers improved understanding to help investigational strategies targeting these mutations.

NSC348884 cytotoxicity is not mediated by inhibition of nucleophosmin oligomerization.

Nucleophosmin (NPM) mutations causing its export from the nucleoli to the cytoplasm are frequent in acute myeloid leukemia (AML). Due to heterooligomerization of wild type NPM with the AML-related mutant, the wild-type becomes misplaced from the nucleoli and its functions are significantly altered. Dissociation of NPM heterooligomers may thus restore the proper localization and function of wild-type NPM. NSC348884 is supposed to act as a potent inhibitor of NPM oligomerization. The effect of NSC348884 on the NPM oligomerization was thoroughly examined by fluorescence lifetime imaging with utilization of FRET and by a set of immunoprecipitation and electrophoretic methods. Leukemia-derived cell lines and primary AML cells as well as cells transfected with fluorescently labeled NPM forms were investigated. Our results clearly demonstrate that NSC348884 does not inhibit formation of NPM oligomers neither in vivo nor in vitro. Instead, we document that NSC348884 cytotoxicity is rather associated with modified cell adhesion signaling. The cytotoxic mechanism of NSC348884 has therefore to be reconsidered.

Group I p21-activated kinases in leukemia cell adhesion to fibronectin.

P21-activated kinases (PAK) regulate processes associated with cytoskeleton dynamics. PAK expression in leukemia cells was measured on protein and mRNA levels. In functional assays, we analyzed the effect of PAK inhibitors IPA-3 and FRAX597 on cell adhesivity and viability. PAK2 was dominant in cell lines, whereas primary cells also expressed comparable amount of PAK1 transcription isoforms: PAK1-full and PAK1Δ15. PAK1Δ15 and PAK2 levels correlated with surface density of integrins ß1 and αVß3. PAK1-full, but not PAK2, was present in membrane protrusions. IPA-3, which prevents PAK activation, induced cell contraction in semi-adherent HEL cells only. FRAX597, which inhibits PAK kinase activity, increased cell-surface contact area in all leukemia cells. Both inhibitors reduced the stability of cell attachment and induced cell death.

Exosomes released by imatinib­resistant K562 cells contain specific membrane markers, IFITM3, CD146 and CD36 and increase the survival of imatinib­sensitive cells in the presence of imatinib.

Chronic myeloid leukemia (CML) is a malignant hematopoietic disorder distinguished by the presence of a BCR­ABL1 fused oncogene with constitutive kinase activity. Targeted CML therapy by specific tyrosine kinase inhibitors (TKIs) leads to a marked improvement in the survival of the patients and their quality of life. However, the development of resistance to TKIs remains a critical issue for a subset of patients. The most common cause of resistance are numerous point mutations in the BCR­ABL1 gene, followed by less common mutations and multiple mutation-independent mechanisms. Recently, exosomes, which are extracellular vesicles excreted from normal and tumor cells, have been associated with drug resistance and cancer progression. The aim of the present study was to characterize the exosomes released by imatinib­resistant K562 (K562IR) cells. The K562IR­derived exosomes were internalized by imatinib­sensitive K562 cells, which thereby increased their survival in the presence of 2 µM imatinib. The exosomal cargo was subsequently analyzed to identify resistance­associated markers using a deep label­free quantification proteomic analysis. There were >3,000 exosomal proteins identified of which, 35 were found to be differentially expressed. From this, a total of 3, namely the membrane proteins, interferon­induced transmembrane protein 3, CD146 and CD36, were markedly upregulated in the exosomes derived from the K562IR cells, and exhibited surface localization. The upregulation of these proteins was verified in the K562IR exosomes, and also in the K562IR cells. Using flow cytometric analysis, it was possible to further demonstrate the potential of CD146 as a cell surface marker associated with imatinib resistance in K562 cells. Taken together, these results suggested that exosomes and their respective candidate surface proteins could be potential diagnostic markers of TKI drug resistance in CML therapy.

PAK1, PAK1Δ15, and PAK2: similarities, differences and mutual interactions.

P21-activated kinases (PAK) are key effectors of the small GTPases Rac1 and Cdc42, as well as of Src family kinases. In particular, PAK1 has several well-documented roles, both kinase-dependent and kinase-independent, in cancer-related processes, such as cell proliferation, adhesion, and migration. However, PAK1 properties and functions have not been attributed to individual PAK1 isoforms: besides the full-length kinase (PAK1-full), a splicing variant lacking the exon 15 (PAK1Δ15) is annotated in protein databases. In addition, it is not clear if PAK1 and PAK2 are functionally overlapping. Using fluorescently tagged forms of human PAK1-full, PAK1Δ15, and PAK2, we analyzed their intracellular localization and mutual interactions. Effects of PAK inhibition (IPA-3, FRAX597) or depletion (siRNA) on cell-surface adhesion were monitored by real-time microimpedance measurement. Both PAK1Δ15 and PAK2, but not PAK1-full, were enriched in focal adhesions, indicating that the C-terminus might be important for PAK intracellular localization. Using coimmunoprecipitation, we documented direct interactions among the studied PAK group I members: PAK1 and PAK2 form homodimers, but all possible heterocomplexes were also detected. Interaction of PAK1Δ15 or PAK2 with PAK1-full was associated with extensive PAK1Δ15/PAK2 cleavage. The impedance measurements indicate, that PAK2 depletion slows down cell attachment to a surface, and that PAK1-full is involved in cell spreading. Altogether, our data suggest a complex interplay among different PAK group I members, which have non-redundant functions.

High PD-L1 Expression Predicts for Worse Outcome of Leukemia Patients with Concomitant NPM1 and FLT3 Mutations.

Compared to solid tumors, the role of PD-L1 in hematological malignancies is less explored, and the knowledge in this area is mostly limited to lymphomas. However, several studies indicated that PD-L1 is also overexpressed in myeloid malignancies. Successful treatment of the acute myeloid leukemia (AML) is likely associated with elimination of the residual disease by the immune system, and possible involvement of PD-L1 in this process remains to be elucidated. We analyzed PD-L1 expression on AML primary cells by flow cytometry and, in parallel, transcript levels were determined for the transcription variants v1 and v2. The ratio of v1/v2 cDNA correlated with the surface protein amount, and high v1/v2 levels were associated with worse overall survival (p = 0.0045). The prognostic impact of PD-L1 was limited to AML with mutated nucleophosmin and concomitant internal tandem duplications in the FLT3 gene (p less than 0.0001 for this particular AML subgroup).

Nucleophosmin in leukemia: Consequences of anchor loss.

Nucleophosmin (NPM), one of the most abundant nucleolar proteins, has crucial functions in ribosome biogenesis, cell cycle control, and DNA-damage repair. In human cells, NPM occurs mainly in oligomers. It functions as a chaperone, undergoes numerous interactions and forms part of many protein complexes. Although NPM role in carcinogenesis is not fully elucidated, a variety of tumor suppressor as well as oncogenic activities were described. NPM is overexpressed, fused with other proteins, or mutated in various tumor types. In the acute myeloid leukemia (AML), characteristic mutations in NPM1 gene, leading to modification of NPM C-terminus, are the most frequent genetic aberration. Although multiple mutation types of NPM are found in AML, they are all characterized by aberrant cytoplasmic localization of the mutated protein. In this review, current knowledge of the structure and function of NPM is presented in relation to its interaction network, in particular to the interaction with other nucleolar proteins and with proteins active in apoptosis. Possible molecular mechanisms of NPM mutation-driven leukemogenesis and NPM therapeutic targeting are discussed. Finally, recent findings concerning the immunogenicity of the mutated NPM and specific immunological features of AML patients with NPM mutation are summarized.

Lifetime-based photoconversion of EGFP as a tool for FLIM.

BACKGROUND: EGFP is a fluorescent tag extensively used in biological and biomedical research. Over the years many researches have gathered collections of cell lines bearing specific EGFP-tagged proteins. Despite its popularity some photochemical properties of EGFP remain undocumented and unused. We report on so far unexplored lifetime photoconversion of EGFP usable in FLIM. METHODS: Fluorescence lifetime imaging and spectral FLIM has been used for characterization of the EGFP photoconversion and protein tracking. RESULT: Our data suggest that EGFP can be permanently photoconverted to a short-fluorescence-lifetime form (PC-EGFP) by intense blue irradiation. PC-EGFP cannot be reverted back by 405 nm light and exhibits the same spectral emission properties with blue-shifted absorption compared to the unconverted EGFP. Fluorescence of PC-EGFP is pH-independent and the photoconversion efficiency decreases with the solvent viscosity. Utilization of the EGFP photoconversion was demonstrated by tracking of a nucleophosmin mutant in live HEK-293 T cells during its cytoplasm-nuclear relocalization induced by Leptomycin B. CONCLUSIONS: Besides potential FLIM artifacts caused by an unintended EGFP photoconversion, the controlled photoconversion turns EGFP to an excellent tool for kinetic FLIM applications. Since the photoconversion occurs in the lifetime domain, PC-EGFP can be easily distinguished from the unconverted tag by time-resolved detection while all other spectral channels stay free for multicolor labeling. GENERAL SIGNIFICANCE: The reported lifetime photoconversion lines up EGFP with other photoconvertible fluorescent proteins with special advantage for fluorescence lifetime imaging where lifetime-photoconvertible labels are scarce.

Association of HLA class I type with prevalence and outcome of patients with acute myeloid leukemia and mutated nucleophosmin.

Acute myeloid leukemia with mutated nucleophosmin (NPMc+ AML) forms a distinct AML subgroup with better prognosis which can potentially be associated with immune response against the mutated nucleophosmin (NPM). As the T-cell-mediated immunity involves antigen presentation on HLA class I molecules, we hypothesized that individuals with suitable HLA type could be less prone to develop NPMc+ AML. We compared HLA class I distribution in NPMc+ AML patient cohort (398 patients from 5 centers) with the HLA allele frequencies of the healthy population and found HLA-A*02, B*07, B*40 and C*07 underrepresented in the NPMc+ AML group. Presence of B*07 or C*07:01 antigen was associated with better survival in patients without concomitant FLT3 internal tandem duplication. Candidate NPM-derived immunopeptides were found for B*40 and B*07 using prediction software tools. Our findings suggest that a T-cell-mediated immune response could actually explain better prognosis of NPMc+ patients and provide a rationale for attempts to explore the importance of immunosuppressive mechanisms in this AML subgroup.

AML-associated mutation of nucleophosmin compromises its interaction with nucleolin.

C-terminal mutations of the nucleolar protein nucleophosmin (NPM) are the most frequent genetic aberration detected in acute myeloid leukemia (AML) with normal karyotype. The mutations cause aberrant cytoplasmic localization of NPM and lead to loss of functions associated with NPM nucleolar localization, e.g. in ribosome biogenesis or DNA-damage repair. NPM has many interaction partners and some of them were proved to interact also with the mutated form (NPMmut) and due to this interaction thereby to be withdrawn from their site of action. We analyzed the impact of the mutation on NPM interaction with nucleolin (NCL) which is also prevalently localized into the nucleolus and cooperates with wild-type NPM (NPMwt) in many cellular processes. We revealed that the NCL-NPM complex formation is completely abolished by the mutation and that the presence/absence of the interaction is not affected by drugs causing genotoxic stress or differentiation. Deregulation resulting from changes of NCL/NPMwt ratio may contribute to leukemogenesis.

Monitoring of nucleophosmin oligomerization in live cells.

Oligomerization plays a crucial role in the function of nucleophosmin (NPM), an abundant nucleolar phosphoprotein. Two dual-color methods based on modern fluorescence confocal microscopy are applied for tracking NPM aggregates in live cells: cross-correlation Number and Brightness analysis (ccN&B) combined with pulsed interleaved excitation (PIE) and fluorescence-lifetime imaging microscopy (FLIM) utilizing resonance energy transfer (FRET). HEK-293T cells were transfected with mixture of plasmids designed for tagging with fluorescent proteins so that the cells express mixed population of NPM labeled either with eGFP or mRFP1. We observe joint oligomers formed from the fluorescently labeled NPM. Having validated the in vivo methods, we study an effect of substitutions in cysteine 21 (Cys21) of the NPM N-terminus on the oligomerization to demonstrate applicability of the methods. Inhibitory effect of mutations of the Cys21 to nonpolar Ala or to aromatic Phe on the oligomerization was reported in literature using in vitro semi-native electrophoresis. However, we do not detect any break-up of the joint NPM oligomers due to the Cys21 mutations in live cells. In vivo microscopy observations are supported by an in vitro method, the GFP-Trap immunoprecipitation assay. Our results therefore show importance of utilizing several methods for detection of biologically relevant protein aggregates. In vivo monitoring of the NPM oligomerization, a potential cancer therapy target, by the presented methods offers a new way to monitor effects of drugs that are tested as NPM oligomerization inhibitors directly in live cells.

Localization of AML-related nucleophosmin mutant depends on its subtype and is highly affected by its interaction with wild-type NPM.

Mutations of the gene for nucleophosmin (NPM1) are the most frequent genetic aberration in patients with acute myeloid leukemia (AML). The mechanism of leukemic transformation in this leukemia subtype is not fully understood, but aberrant cytoplasmic localization of mutated NPM (NPMmut) is widely considered as an important factor for leukemia manifestation. We analyzed the subcellular localization of three types of NPM with a C-terminal mutation (A, B and E). Genes for the individual NPM forms were fused with a gene for one of fluorescent protein variants in plasmids, which were transfected into three cell lines with different endogenous NPM expression. Subcellular localization of the fluorescent protein-labeled NPM was further correlated with the relative expression of all NPM forms. We confirmed a high cytoplasmic expression of NPMmutA and NPMmutB whereas a substantial fraction of NPMmutE was found to be localized in nucleoli. Moreover, we revealed that the localization of fluorescently labeled NPM is affected by the interaction between various forms of the protein.

Correlation of PD-L1 Surface Expression on Leukemia Cells with the Ratio of PD-L1 mRNA Variants and with Electrophoretic Mobility.

The expression on the surface of tumor cells of ligands for the PD-1 inhibitory receptor prevents the antitumor immune response and is considered to be a negative prognostic factor in a variety of solid tumors as well as in hematologic malignancies. To determine if it were possible to analyze PD-L1 with PCR-based methods, we assessed the expression of PD-L1 in primary samples from patients with acute myeloid leukemia, in healthy donors, and in a panel of cell lines, by means of flow cytometry, RT-PCR, and Western blotting. Although the surface density of the protein was not correlated with the amount of expressed full-length mRNA, we found a statistically significant positive correlation between PD-L1 surface density and the ratio of two transcript variants (variant 1/variant 2). Our PCR-based method allows for retrospective examination of PD-L1 surface expression from frozen cDNA samples, without the need for a reference gene. Our results also suggest that variant 2, which is produced by alternative splicing, negatively regulates PD-L1 protein expression on the cell surface. In addition, PD-L1 exposition on the cell surface is clearly associated with a shift of electrophoretic mobility, observed on Western blots. This finding can explain the relatively large variability in PD-L1 apparent molecular weight reported in the literature and offers an alternate means for the assessment of PD-L1 surface expression. Cancer Immunol Res; 4(10); 815-9. ©2016 AACR.

Low-Dose Actinomycin-D Induces Redistribution of Wild-Type and Mutated Nucleophosmin Followed by Cell Death in Leukemic Cells.

Specific mutations involving C-terminal part of the nucleolar protein nucleophosmin (NPM) are associated with better outcome of acute myeloid leukemia (AML) therapy, possibly due to aberrant cytoplasmic NPM localization facilitating induction of anti-NPM immune response. Actinomycin D (actD) is known to induce nucleolar stress leading to redistribution of many nucleolar proteins, including NPM. We analyzed the distribution of both wild-type and mutated NPM (NPMmut) in human cell lines, before and after low-dose actD treatment, in living cells expressing exogenous fluorescently labeled proteins as well as using immunofluorescence staining of endogenous proteins in fixed cells. The wild-type NPM form is prevalently nucleolar in intact cells and relocalizes mainly to the nucleoplasm following actD addition. The mutated NPM form is found both in the nucleoli and in the cytoplasm of untreated cells. ActD treatment leads to a marked increase in NPMmut amount in the nucleoplasm while a mild decrease is observed in the cytoplasm. Cell death was induced by low-dose actD in all the studied leukemic cell lines with different p53 and NPM status. In cells expressing the tumor suppresor p53 (CML-T1, OCI-AML3), cell cycle arrest in G1/G0 phase was followed by p53-dependent apoptosis while in p53-null HL60 cells, transient G2/M-phase arrest was followed by cell necrosis. We conclude that although actD does not increase NPM concentration in the cytoplasm, it could improve the effect of standard chemotherapy in leukemias through more general mechanisms.

Altered HLA Class I Profile Associated with Type A/D Nucleophosmin Mutation Points to Possible Anti-Nucleophosmin Immune Response in Acute Myeloid Leukemia.

Nucleophosmin 1 (NPM1) mutations are frequently found in patients with acute myeloid leukemia (AML) and the newly generated sequences were suggested to induce immune response contributing to the relatively favorable outcome of patients in this AML subset. We hypothesized that if an efficient immune response against mutated nucleophosmin can be induced in vivo, the individuals expressing HLA alleles suitable for presenting NPM-derived peptides should be less prone to developing AML associated with NPM1 mutation. We thus compared HLA class I frequencies in a cohort of patients with mutated NPM1 (63 patients, NPMc+), a cohort of patients with wild-type NPM1 (94 patients, NPMwt) and in normal individuals (large datasets available from Allele Frequency Net Database). Several HLA allelic groups were found to be depleted in NPMc+ patients, but not in NPMwt compared to the normal distribution. The decrease was statistically significant for HLA B(*)07, B(*)18, and B(*)40. Furthermore, statistically significant advantage in the overall survival was found for patients with mutated NPM1 expressing at least one of the depleted allelic groups. The majority of the depleted alleles were predicted to bind potent NPM-derived immunopeptides and, importantly, these peptides were often located in the unmutated part of the protein. Our analysis suggests that individuals expressing specific HLA allelic groups are disposed to develop an efficient anti-AML immune response thanks to aberrant cytoplasmic localization of the mutated NPM protein.

Decitabine and SAHA-induced apoptosis is accompanied by survivin downregulation and potentiated by ATRA in p53-deficient cells.

While p53-dependent apoptosis is triggered by combination of methyltransferase inhibitor decitabine (DAC) and histone deacetylase inhibitor suberoylanilide hydroxamic acid (SAHA) in leukemic cell line CML-T1, reactive oxygen species (ROS) generation as well as survivin and Bcl-2 deregulation participated in DAC + SAHA-induced apoptosis in p53-deficient HL-60 cell line. Moreover, decrease of survivin expression level is accompanied by its delocalization from centromere-related position in mitotic cells suggesting that both antiapoptotic and cell cycle regulation roles of survivin are affected by DAC + SAHA action. Addition of subtoxic concentration of all-trans-retinoic acid (ATRA) increases the efficiency of DAC + SAHA combination on viability, apoptosis induction, and ROS generation in HL-60 cells but has no effect in CML-T1 cell line. Peripheral blood lymphocytes from healthy donors showed no damage induced by DAC + SAHA + ATRA combination. Therefore, combination of ATRA with DAC and SAHA represents promising tool for therapy of leukemic disease with nonfunctional p53 signalization.