non-APL Acute Myeloid Leukemia Cells Research Articles

Arsenic Trioxide Combine with G-CSF Triggers Distinct TP53 Mutations Acute Myeloid Leukemia Cells Ferroptosis through TP53-SLC7A11-GPX4 Pathway

Abstract ： Background ： Acute myeloid leukemia (AML) is a complex disease with diverse molecular subtypes. Approximately 8% of AML patients carry the TP53 mutation, which is associated with an aggressive disease course and dismal survival rates. Arsenic trioxide (ATO) has been remarkably effective in treating acute promyelocytic leukemia (APL), but its clinical activity in other AML subtypes remains limited. Recent evidence indicates that ATO demonstrates potential in rescuing multiple TP53 mutations effectively, offering promise as an individualized therapeutic strategy targeting mutant TP53 in leukemic cells. Our previous study showed that the upregulation of AQP9 by G-CSF enhanced the effect of ATO in non-APL AML cells. This demonstrates that ATO holds therapeutic promise in AML cases harboring distinct TP53 mutations. Methods ： We conducted a comprehensive analysis of TP53 mutations in the Analysis of Whole Genomes cohort, encompassing patients from the TCGA and OHSU databases. The frequency and distribution of TP53 mutations were determined. Median overall survival (OS) was calculated using the Kaplan-Meier method. To functionally assess the effect of TP53 mutation on AML phenotype, various cell lines harboring distinct TP53 mutation types were used for this study, including KASUMI-1 TP53 R248Q THP-1 TP53frameshift_delHL-60 TP53 deletionMOLM13 TP53 wild-type. The cellular lipid oxidation levels were measured by a C11 BODIPY flow cytometry assay and it was also observed by fluorescence microscopy. Additionally, the RNA and protein expressions of GPX4 and SLC7A11 were analyzed by real-time quantitative Polymerase Chain Reaction (QRT-PCR) and Western Blot. Results ： In the OHSU cohort and TCGALAML database, including 1005 newly diagnosed patients with 120 TP53 mutations, we found that missense mutations are the most common TP53 mutations and R248Q has the highest mutation frequency. Furthermore, TP53 mutationwas significantly associated with lower survival ( P <0.001). ATO potently induced cell death in each cell line. The KASUMI-1 R248Q cell line was the most sensitive to ATO (IC50=0.3607) and showed the most pronounced change in the IC50 of ATO with the addition of ferrostatin-1(Fer-1, ferroptosis inhibitor). ATO induced cell death was prevented by the addition of lipophilic antioxidant ferrostatin-1, supporting the hypothesis that ATO induced cell death occurred by ferroptosis. In KASUMI-1 R248Q cell line, ATO increases the lipid oxidation levels (observed by C11 BODIPY flow cytometry assay and fluorescence microscopy), MDA levels and decreases the expression of GPX4 and SLC7A11 (including RNA level and protein level). Whereas, in MOLM13 TP53 wildtype cell line, ATO increases the lipid oxidation levels but cannot prevented by Fer-1. THP-1 TP53 frameshift_del and HL-60 TP53 deletion only decrease the RNA expression of GPX4 and SLC7A11, nothing else changed in ferroptosis. These suggest that ATO has a more pronounced effect on missense mutations such as TP53 R248Q. In KASUMI-1 R248Q cell line, associated with G-CSF can further promote lipid oxidation levels (observed by C11 BODIPY flow cytometry assay and fluorescence microscopy) and further reduce the expression of GPX4 and SLC7A11. These results suggest that G-CSF promotes ATO-induced ferroptosis in KASUMI-1 R248Q cell line. Conclusion: We have demonstrated that ATO induces AML with distinct TP53 mutation cell death by ferroptosis resulting from depletion of GPX4 and SLC7A11. ATO associated with G-CSF may be able to be a new therapeutic strategy for the treatment of AML with distinct TP53 mutation.

Combined Application of Salinomycin and ATRA Induces Apoptosis and Differentiation of Acute Myeloid Leukemia Cells by Inhibiting WNT/β-Catenin Pathway.

All-trans retinoic acid (ATRA) is only effective in acute promyelocytic leukemia (APL), but not in other subtype of acute myeloid leukemia (AML). Salinomycin targets tumor cells rather than non-tumorigenic cells, and WNT/β-catenin pathway inhibition is one of the mechanisms of its anti-tumor activity. There is a crosstalk between RA and WNT/β-catenin pathway. Here, we investigate the effect of the combination of salinomycin and ATRA (S+RA) in non-APL AML cells. Apoptosis was evaluated by cell viability and Annexin-V assay. Cell differentiation was analyzed by CD11c expression and morphology. To explore the underlying mechanisms, Western blot analysis and mitochondrial transmembrane potentials (ΔΨm) were used. S+RA induced differentiation and apoptosis in AML cell lines and AML primary cells. S+RA inhibited the β-catenin signal pathway as determined by the decreased protein levels of β-catenin, the low-density lipoprotein receptor-related proteins 6 (LRP6), and its downstream proteins such as survivin, c-Myc, caspase-3/7, cdc25A and cyclinD1 and reduced phosphorylation level of GSK3β S9. S+RA also increased the protein levels of CCAAT/enhancer-binding proteins (C/EBPs) and PU.1 and collapsed Δψm. The above molecular and cellular changes induced by S+RA were inhibited by β-catenin specific activator and promoted by β-catenin specific inhibitor. S+RA induced differentiation by β-catenin-inhibition-mediated up-regulation of C/EBPs and PU.1 and suppression of c-Myc. S+RA triggered apoptosis through β-catenin-inhibition-regulated ΔΨm collapse and caspase-3/7 activation. Taken together, our findings may provide novel therapeutic strategies for AML patients by targeting the WNT/β-catenin pathway.

Dual inhibition of EZH2 and G9A/GLP histone methyltransferases by HKMTI-1-005 promotes differentiation of acute myeloid leukemia cells.

All-trans-retinoic acid (ATRA)-based differentiation therapy of acute promyelocytic leukemia (APL) represents one of the most clinically effective examples of precision medicine and the first example of targeted oncoprotein degradation. The success of ATRA in APL, however, remains to be translated to non-APL acute myeloid leukemia (AML). We previously showed that aberrant histone modifications, including histone H3 lysine 4 (H3K4) and lysine 27 (H3K27) methylation, were associated with this lack of response and that epigenetic therapy with small molecule inhibitors of the H3K4 demethylase LSD1/KDM1A could reprogram AML cells to respond to ATRA. Serving as the enzymatic component of Polycomb Repressive Complex 2, EZH2/KMT6A methyltransferase plays a critical role in normal hematopoiesis by affecting the balance between self-renewal and differentiation. The canonical function of EZH2 is methylation of H3K27, although important non-canonical roles have recently been described. EZH2 mutation or deregulated expression has been conclusively demonstrated in the pathogenesis of AML and response to treatment, thus making it an attractive therapeutic target. In this study, we therefore investigated whether inhibition of EZH2 might also improve the response of non-APL AML cells to ATRA-based therapy. We focused on GSK-343, a pyridone-containing S-adenosyl-L-methionine cofactor-competitive EZH2 inhibitor that is representative of its class, and HKMTI-1-005, a substrate-competitive dual inhibitor targeting EZH2 and the closely related G9A/GLP H3K9 methyltransferases. We found that treatment with HKMTI-1-005 phenocopied EZH2 knockdown and was more effective in inducing differentiation than GSK-343, despite the efficacy of GSK-343 in terms of abolishing H3K27 trimethylation. Furthermore, transcriptomic analysis revealed that in contrast to treatment with GSK-343, HKMTI-1-005 upregulated the expression of differentiation pathway genes with and without ATRA, while downregulating genes associated with a hematopoietic stem cell phenotype. These results pointed to a non-canonical role for EZH2, which was supported by the finding that EZH2 associates with the master regulator of myeloid differentiation, RARα, in an ATRA-dependent manner that was enhanced by HKMTI-1-005, possibly playing a role in co-regulator complex exchange during transcriptional activation. In summary, our results strongly suggest that addition of HKMTI-1-005 to ATRA is a new therapeutic approach against AML that warrants further investigation.

P480: NKG2D-MEDIATED ANTI-TUMOR IMMUNITY CONTRIBUTES TO THE FAVORABLE PROGNOSIS IN APL

Background: Acute myeloid leukemia (AML) is an aggressive hematological malignancy with a poor prognosis. Acute promyelocytic leukemia (APL) is a unique subtype of AML, with PML-RARA fusion caused by t(15;17)(q22;q12) translocation, and is now considered a highly curable disease. Exploring the difference between APL and non-APL AML, may shed new insight on the treatment of AML. NKG2D is an activating receptor on NK cells that recognizes ligands on the surface of tumor cells and plays a crucial role in tumor cell killing. Prior studies have demonstrated that one mechanism of AML immune evasion is downregulation of NKG2D ligands (NKG2DL) or secretion of receptor-blocking soluble ligands. Whereas, reports on NKG2DL expression in APL are still lacking. In this study, we investigate the differences of NKG2DL expression on APL and non-APL cells. Aims: Exploring the difference between APL and non-APL AML, may shed new insight on the treatment of AML. Methods: Bone marrow samples were derived from de novo APL and non-APL AML patients, diagnosed according to French-American-British (FAB) criteria in The First Affiliated Hospital of Xi’an Jiao Tong University. Flow cytometry was performed with the anti-NKG2DL mAbs (MIC-A/B-PE, ULBP-1-APC, ULBP-2/5/6-PerCP). Leukemic blasts were identified using the CD45/SSC gating procedure. Non-APL AML patients were divided into high or low group based on the median expression levels of NKG2DL for survival analysis. Results: Totally 46 patients comprising of 23 APL and 23 non-APL AML were enrolled in this study. There was no significant difference in the baseline characteristics between the two groups. All non‐APL AML cells had low or no NKG2DL surface expression, while most APL cells expressed high NKG2DL levels. In particular, just two APL patients was negative for the NKG2DL but positive for CD34. The median percentage of NKG2DL among non-APL AML cells and APL cells were 1.2% vs 20.3% in MIC-A/B (P<0.01), 1.1% vs 54.8% in ULBP-1 (P=0.017), and 3.8% vs 36.35% in ULBP-2/5/6 (P<0.01). (Fig.1) 23 non-APL AML patients were enrolled for study inclusion; one patient dropped out due to cerebral infarction. The median follow-up time was 17.2 weeks. The high ULBP-2/5/6 group showed a superior overall survival rate compared to the low ULBP-2/5/6 group (P < 0.01), but there was no significant difference between high and low MIC-A/B groups (P = 0.31) or between the high and low ULBP-1 groups (P = 0.52). Image:Summary/Conclusion: NKG2DL was decreased significantly on the surface of non-APL AML cells compared with APL cells. These results may indicate that NKG2D-mediated anti-tumor immunity contributes to the favorable prognosis in APL, while immune escape is an underlying mechanism for unfavorable prognosis in non-APL AML. NKG2DL may be used as a potential biomarker to predict prognosis of patients with non-APL AML, and upregulation of its expression could improve the clinical efficacy. Further studies with larger sample size and longer follow-up are required to confirm our conclusion.

G-CSF upregulates the expression of aquaporin-9 through CEBPB to enhance the cytotoxic activity of arsenic trioxide to acute myeloid leukemia cells

BackgroundArsenic trioxide (ATO) is highly effective in acute promyelocytic leukemia (APL) patients, but it fails to show satisfactory efficacy in other acute myeloid leukemia (AML) patients with non-APL subtypes. Different from the APL cells, most non-APL AML cells express low levels of the ATO transporter Aquaporin-9 (AQP9) protein, making them less sensitive to ATO treatment. Recently, we found that granulocyte colony stimulating factor (G-CSF) can upregulate the expression of AQP9. We hypothesized that the pretreatment with G-CSF may enhance the antitumor effect of ATO in non-APL AML cells. In addition, we aimed to elucidate the underlying mechanisms by which G-CSF upregulates the expression of AQP9.MethodsNon-APL AML cell lines including THP-1 and HL-60 were pretreated with or without G-CSF (100 ng/ml) for 24 h, followed by the treatment with ATO (2 μM) for 48 h. Cell morphology was observed under the microscope after Wright-Giemsa staining. Flow cytometry was performed to evaluate the cell apoptosis levels. The intracellular concentrations of ATO were determined by atomic fluorescence spectrometry. The mRNA and protein expression were respectively measured by quantitative reverse transcription PCR (RT-qPCR) and western blotting. Target genes were knocked down by transfection with small interfering RNA (siRNA), or overexpressed by transfection with overexpression plasmids. The cell line derived xenograft mouse model was established to confirm the results of the in vitro experiments.ResultsCompared with using ATO alone, the combination of G-CSF with ATO induced the cell apoptosis more dramatically. G-CSF upregulated the expression of AQP9 and enhanced the intracellular concentrations of ATO in AML cells. When AQP9 was overexpressed, it markedly enhanced the cytotoxic activity of ATO. On the other hand, when AQP9 was knocked down, it profoundly attenuated the combinational effect. Moreover, we found that the upregulation of AQP9 by G-CSF depends on the transcription factor CCAAT enhancer binding protein beta (CEBPB). We also demonstrated that the combination of G-CSF and ATO significantly inhibited tumor growth in the xenograft mouse model.ConclusionsThe combination of G-CSF and ATO may be a potential therapeutic strategy for AML patients.

Arsenic Trioxide Reprograms the Bone-Marrow Microenvironment to Sensitize Minimal Residual Disease in Acute Myeloid Leukemia

Eliminating minimal residual disease (MRD) in order to prevent relapse is an unmet need in acute myeloid leukemia (AML). One of the success stories is acute promyelocytic leukemia (APL), a unique subtype of AML. The use of arsenic trioxide (ATO) in combination with all-trans retinoic acid (ATRA) has essentially eliminated disease relapse (<1% ATRA+ATO vs. ~18% ATRA+ chemotherapy - NEJM (2013), 369(2), 111-121). This is even more impressive as neither agent alone results in significant cure of patients with APL. In fact, single agent ATRA induces remission without cure due to bone marrow stroma expression of CYP26 enzymes (PloS one (2015), 10(6), e0127790), which are induced by ATRA, inactivate retinoids and protect APL MRD (Leukemia (2020), 34(11), 3077-3081). While ATO may synergize with ATRA by restoring PML nuclear bodies (Nature medicine (2014), 20(2), 167-174) and rescuing RAR-α dependent transcription (Nature reviews. Cancer (2018), 18(2), 117-127) in the bulk of the APL tumor cells, it is unclear how these mechanisms contribute to elimination of MRD, particularly given the hypothesized lack of ATRA in the bone marrow niche. It was previously reported that combination of ATRA with ATO may be associated with high incidence of transaminitis and headaches (NEJM (2013), 369(2), 111-121). Since these may be clinical signs of ATRA toxicity, we investigated if addition of ATO changes the pharmacokinetics (PKs) of ATRA in patients with APL. We determined the area under the plasma concentration versus time curve (AUC) for ATRA in patients suspected of APL and treated with single agent ATRA (“preATO”) and in the same patients after the diagnoses of APL was confirmed and the patients received ATO (“postATO”). Even though ATRA should have an expected drop in AUC during the first 3-4 days of therapy (JPET (2017), 361(2), 246-258), addition of ATO resulted in improved ATRA PKs (Figure A). Since ATRA levels are mainly controlled by clearance via hepatic CYP26s, we tested if treatment with ATO changes the levels of CYP26 or its induction by ATRA. We found that exposure to ATO decreases both baseline and ATRA-induced upregulation of CYP26B1 in HepG2 cells (Figure B). Similarly, ATO decreases CYP26B1 levels in human bone marrow derived mesenchymal stroma cells (MSCs) at baseline (21%±7% of NO ATO control, n=3, p<0.01) and after ATRA-induced upregulation (66%±14% of ATRA only control, n=3, p<0.01). These changes resulted in improved ATRA PKs in vitro in the presence of bone marrow MSCs (Figure C). Next, we used NB4-EvAsR cells (NB4R), ATO-resistant APL cells to test if ATO-induced improvement of MSC-dependent ATRA PKs may contribute to elimination of APL MRD. We found that ATO sensitizes NB4R to ATRA in the presence of BM MSCs but not in stroma free conditions (Figure D). ATO had no effect on ATRA-induced differentiation of NB4R in the presence of CYP26B1 knockout stroma (this stroma offers no protection from ATRA) or if stroma expresses CYP26B1 under the control of a viral promoter and thus, resistant to ATO downregulation. This data suggest that ATO-induced changes of BM MSC expression of CYP26 may contribute to elimination of MRD in APL. Stromal CYP26 protects not only APL but also non-APL AML cells from retinoids (Leukemia (2020), 34(11), 3077-3081). Retinoids are synergistic with FLT3 inhibitors to eliminate mutant FLT3 AML cells (Blood (2016), 127(23), 2867-2878) and stromal CYP26 breaks this synergism (Blood (2015) 126 (23): 790). We tested if ATO sensitizes mutant FLT3 AML cells, MV4;11 to gilteritinib in the presence of BM MSCs. For this, we derived MV4.11 cells resistant to ATO (MV4.11R) via long term exposure to increasing concentration of ATO. As seen with NB4R, ATO treatment synergizes with gilteritinib to decrease the IC50 of gilteritinib against the clonogenic activity of MV4.11R in the presence of stroma but not in stroma free conditions (Figure E). In an effort to fully explore the translational potential of these findings, we are currently investigating the mechanism by which ATO changes CYP26 levels.Taken together these findings support a model in which ATO changes the bone marrow niche and it's ability to metabolize retinoids and thus, sensitizes MRD in AML to targeted therapy. [Display omitted] DisclosuresMathews: Christian Medical College: Patents & Royalties: US 2020/0345770 A1 - Pub.Date Nov.5, 2020; AML: Other: Co-Inventor. Ghiaur: Syros Pharmaceuticals: Consultancy; Menarini Richerche: Research Funding.

All-trans retinoic acid induces differentiation in primary acute myeloid leukemia blasts carrying an inversion of chromosome 16

All-trans retinoic acid (ATRA)-based therapy for acute promyelocytic leukemia (APL), a subtype of acute myeloid leukemia (AML), is the most successful example of differentiation therapy. Although ATRA can induce differentiation in some non-APL AML cell lines and primary blasts, clinical results of adding ATRA to standard therapy in non-APL AML patients have been inconsistent, probably due to use of different regimens and lack of diagnostic tools for identifying which patients may be sensitive to ATRA. In this study, we exposed primary blasts obtained from non-APL AML patients to ATRA to test for differentiation potential in vitro. We observed increased expression of differentiation markers, indicating a response to ATRA, in four out of fifteen primary AML samples. Three samples in which CD11b increased in response to ATRA had an inversion of chromosome 16 as well as the CBFB-MYH11 fusion gene, and the fourth sample was from a patient with KMT2A-rearranged, therapy-related AML. In conclusion, we identified a subgroup of non-APL AML patients with inv(16) and CBFB-MYH11 as the most sensitive to ATRA-mediated differentiation in vitro, and our results can help identify patients who may benefit from ATRA treatment.

Reducing FASN expression sensitizes acute myeloid leukemia cells to differentiation therapy

Fatty acid synthase (FASN) is the only human lipogenic enzyme available for de novo fatty acid synthesis and is often highly expressed in cancer cells. We found that FASN mRNA levels were significantly higher in acute myeloid leukemia (AML) patients than in healthy granulocytes or CD34+ hematopoietic progenitors. Accordingly, FASN levels decreased during all-trans retinoic acid (ATRA)-mediated granulocytic differentiation of acute promyelocytic leukemia (APL) cells, partially via autophagic degradation. Furthermore, our data suggest that inhibition of FASN expression levels using RNAi or (-)-epigallocatechin-3-gallate (EGCG) accelerated the differentiation of APL cell lines and significantly re-sensitized ATRA refractory non-APL AML cells. FASN reduction promoted translocation of transcription factor EB (TFEB) to the nucleus, paralleled by activation of CLEAR network genes and lysosomal biogenesis. Together, our data demonstrate that inhibition of FASN expression in combination with ATRA treatment facilitates granulocytic differentiation of APL cells and may extend differentiation therapy to non-APL AML cells.

Inecalcitol and Decitabine Synergize to Inhibit Cell Proliferation and to Induce Differentiation of Human Acute Myeloid Leukemia Cell Lines

Decitabine (DAC) is a hypomethylating agent indicated as front-line therapy for de novo or secondary acute myeloid leukemia (AML) in newly diagnosed patients aged 65 years or older unfit for standard induction chemotherapy (Kantarjian et al., 2012, Malik & Cashen, 2014, Nieto et al., 2016, He et al., 2017). Its mechanism of action at the DNA level mostly results in inhibition of cell proliferation. Cellular differentiation can also be involved in some extent in a fraction of the leukemic cell population, as reported in initial pharmacological studies (Creusot et al., 1982; Pinto et al., 1984). Overall survival advantage is nevertheless limited to several more months and the next challenge is to combine DAC with other drugs to improve it further (Kubasch & Platzbecker, 2018).Inecalcitol (INE: 14epi-,19nor-,23yne-,1,25dihydroxy-cholecalciferol) is a vitamin D receptor agonist characterized by potent anti-proliferative and pro-differentiating general properties on cancer cells and by a low calcemic potential (Okamoto et al., 2012; Ma et al., 2013; Medioni et al. 2014), and especially on AML cell lines (AACR 2017, 2018). INE is currently being tested in combination with DAC in this category of elderly AML patients unfit for standard chemotherapy.The aim of the present report was to look for synergies in vitro between DAC and INE on four non-APL human AML cell lines (MOLM-13, U-937, THP-1, OCI-AML2) both on inhibition of proliferation and induction of differentiation. After 72 hours of incubation, cells were counted and labeled for CD11b and CD14 at the cell surface as biomarkers of monocytic/macrophagic differentiation. The range of DAC concentrations had to adapted to each cell line to avoid maximal cytotoxicity: 1.2 nM to 100 nM on MOLM-13, 3 nM to 250 nM on U-937 and THP-1, and 31 nM to 500 nM on OCI-AML2. The same range of 0.12 to 10 nM INE concentrations was tested on each cell line. Each concentration of INE was tested in combination with each concentration of DAC. Synergy was calculated as the excess over the highest single agent (HSA) using the open source Combenefit software (Di Veroli et al., 2016).The highest concentration of DAC alone (MOLM-13: 100 nM, U-937 and THP-1: 250 nM; OCI-AML2: 500 nM) induced a decrease in cell count of 30% of THP-1 cells, 50% of OCI-AML2 cells, 65% of U-937 cells and 80% of MOLM-13 cells. The highest concentration of INE alone (10 nM) induced a decrease in cell count of 20% of U-937 and THP-1 cells, 60% of OCI-AML2 cells and 70% of MOLM-13 cells. The antiproliferative effects of DAC and INE were at least additive in all combinations tested. Significant HSA synergy indexes were found for the decrease in cell number in all four cell lines, ranging from 12% to 23% depending on cell lines and combinations of concentrations.The highest concentration of DAC alone had no (U-937, THP-1) or limited activity (<+12% of labeled MOLM-13 or OCI-AML2 cells) to induce either CD11b or CD14 on the cell surface. By contrast, the highest concentration of INE alone (10 nM) stimulated the expression of CD11b and CD14 in up to 70% to 95% of the cells depending on the cell line (except the CD14 labeling of U-937 cells which remained < 8%). The respective EC50 of INE for CD11b and CD14 induction was 1 and 3 nM on THP-1 cells, 4 and 3 nM on MOLM-13 cells, 3 and 3 nM on OCI-AML2 cells and 1 nM on U-937 cells (50% not reached for CD14). There was no antagonistic effect of DAC towards the pro-differentiating properties of INE. A significant HSA synergy index in the 16% to 26% range was observed for both CD11b and CD14 in MOLM-13 cells and for CD14 in OCI-AML2 cells. A very high HSA synergy index of 75% was observed for the stimulation of CD14 in U-937 cells.In summary, DAC exerted more antiproliferative activity than INE which was more potent to induce monocytic/macrophagic differentiation of four non-APL human AML cell lines. The combination of DAC and INE systematically resulted in a synergy to inhibit cell proliferation, and the strong stimulation of cell differentiation induced by INE alone was in some cases boosted by DAC. These in vitro results provide the mechanistic basis for the potential interest of treating elderly AML patients with INE in addition to DAC in the ongoing double-blind placebo-controlled Phase II clinical trial (NCT02802267). DisclosuresMouly:Hybrigenics: Employment. Rousseau:Hybrigenics: Employment. Planquette:Hybrigenics: Employment, Equity Ownership, Patents & Royalties: inventor, but no royalties. Delansorne:Hybrigenics: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties: inventor, but no royalties.

Elucidating the Non-Catalytic Function of Fatty Acid Synthase and Its Autophagy-Dependent Degradation in Acute Myelocytic Leukemia Differentiation Therapy

Acute myeloid leukemia (AML) is characterized by a block in myeloid differentiation and an aberrant cell proliferation of leukemia blasts. While other AML therapies cure only 30% of the patients, acute promyelocytic leukemia (APL) is successfully treated by differentiation therapy using all-trans retinoic acid (ATRA) in combination with chemotherapy or arsenic trioxide. A better understanding of the molecular mechanisms underlying ATRA therapy in APL may offer new perspectives in the treatment of additional AML subtypes.Fatty acid synthase (FASN) is the only human lipogenic enzyme able of de novo fatty acid synthesis. Its expression is very low in healthy adult tissues, whereas increased expression is commonly found in a variety of cancerous tissues. Therefore, FASN represents an attractive potential drug target.To investigate if a similar expression pattern is found in normal versus malignant myeloid cells, we determined FASN expression in fresh leukemic blast cells from untreated AML patients obtained at the Inselspital Bern (Switzerland) (n=68) and expression data available online from the Bloodspot server (n=203). In both AML cohorts, we found that FASN expression is significantly higher in AML patients than in healthy granulocytes (p<0.05). Accordingly, FASN protein levels are reduced in NB4 and HT93 APL cells upon ATRA-induced granulocyte differentiation. Additionally, silencing FASN in NB4 cells led to an acceleration of differentiation upon ATRA treatment. Surprisingly, solely inhibiting the catalytic function of FASN using C75 or Orlistat did not affect granulocyte differentiation nor FASN expression. To validate that FASN levels and not activity attenuate granulocyte differentiation, we took advantage of the green tea polyphenol epigallocatechin-3-gallate (EGCG) that reduces FASN protein expression. Indeed, similar to knocking down FASN, EGCG treatment of NB4 cells resulted in increased differentiation paralleled by decreased FASN protein levels.We and others demonstrated the importance of autophagy for successful ATRA-induced APL differentiation. Autophagy is an intracellular degradation system that ensures a dynamic recycling of cytoplasmic contents. This process is regulated by the so-called autophagy related genes or ATGs. ATG1, or ULK1, is a key autophagy gene of the initiation complex that is inhibited by mTOR-mediated phosphorylation leading to reduced autophagy activity.FASN expression correlates with mTOR activity in several solid cancers, therefore we hypothesized that FASN activates mTOR resulting in decreased autophagy in AML cells. Indeed, lowering FASN protein expression by either shRNA or EGCG resulted in increased autophagy together with a decreased mTOR-mediated phosphorylation of ULK1.Next, we aimed at understanding how FASN expression is regulated during ATRA treatment. We found a negative correlation between KLF5 and FASN expression in both AML patient cohorts (r=-0.3) and 4 potential KLF binding sites on the FASN promoter, suggesting that KLF5 inhibits FASN transcription.However, since the protein half-life of FASN is around 24 hours, transcriptional inhibition is not sufficient to fully explain the reduced protein levels upon ATRA treatment. We therefore tested if FASN protein itself is degraded via autophagy. Co-treatment of NB4 cells with ATRA and the lysosome inhibitor Bafilomycin A1 led to a dose dependent accumulation of FASN protein suggesting that FASN is an autophagy substrate during ATRA treatment. Furthermore, using mCherry-LC3B expressing NB4 cells, we were able to show FASN and LC3B co-localization by fluorescence microscopy.Finally, we asked whether inhibiting FASN expression levels would increase ATRA sensitivity in non-APL AML cells. To this end, we treated KG-1, OCI-AML2 and MOLM-13 AML cells with EGCG in combination with ATRA. Importantly, combining ATRA with EGCG resulted in increased differentiation of these non-APL cells paralleled by reduced FASN levels, decreased mTOR activity and ULK1 phosphorylation.Together, our data suggest that EGCG treatment is beneficial for differentiation therapy of non-APL AML cells by activating autophagy via degradation of FASN and thereby inactivating mTOR. Furthermore, high FASN expression in AML is likely due to low expression of its positive transcriptional regulator KLF5 and low autophagy activity. DisclosuresNo relevant conflicts of interest to declare.

The Genes for Tissue Factor F3 and Nuclear Receptor 4A Are Down-Regulated in Early Death Acute Promyelocytic Leukemia Patients

Introduction: Acute promyelocytic leukemia (APL) patients are successfully treated via differentiation therapy with all-trans retinoic acid (ATRA) and arsenic trioxide (ATO). Attempts to apply ATRA-based differentiation therapy to non-APL acute myeloid leukemia (AML) patients have not been effective so far (Johnson & Redner, 2015). Furthermore, 10-30% of the APL patients suffer an early death (ED) within 30 days, due to hemorrhages, infections, differentiation syndrome and thrombosis. Different risk factors have been identified, but the underlying mechanisms and successful treatment of ED in APL patients still have not been established (Kwaan et al., 2014, Lehmann et al., 2017). Hence, it is highly important to identify novel risk factors to understand the mechanistic processes in APL ED patients.Methods: To identify target genes, 50 bp single-read RNA sequencing (RNAseq) of 6 ED and 9 APL controls (cohort #1; n=15) and 4 ED and 5 APL controls (cohort #2; n=9) was executed. Samples were taken from patients with confirmed diagnosis of APL before beginning of treatment. Cohort #1 consisted of 15 patients: median age 60 years (range 37-73 years), 10 high-risk patients and 5 low/intermediate-risk patients according to Sanz score. Patients of cohort #1 were treated according to the AIDA2000 protocol of the Study Alliance Leukemia (SAL) study group. Cohort #2 consisted of 9 patients: median age 55 years (range 36-79 years), 4 high-risk patients and 5 low/intermediate-risk patients. Most patients of cohort 2 were treated according to the AIDA2000 (SAL) or APL0406 protocol. Gene validation was performed via RT-qPCR. PML-RARα variants were determined by Mentype AMLplexQS (Biotype). Survival and RT-qPCR analyses were performed with 64 non-APL AML patients´ samples treated within the clinical trial of the AMLSG HD98B study (Schlenk et al., 2004). In this trial, all patients received ICE (idarubicin, cytarabine, etoposide) chemotherapy. The patients were randomly assigned to ATRA or no ATRA. In our cohort, 26 patients had received ICE-ATRA, whereas 38 only ICE. Amaxa nucleofector technology was used for overexpression of PML-RARα bcr1 or bcr3 variant in U937 cells. NB4 or AML cells treated with 1 µM ATRA up to 72 h or 1-2 µM ATO up to 48 h were analyzed via flow cytometry, CellTiter-Glo viability assay, RT-qPCR or Western blot.Results: RNAseq of cohorts 1 and 2 showed F3 (Tissue Factor) and members of nuclear receptor 4A family, NR4A1/2/3, significantly downregulated in ED compared to control APL cases. GSEA analysis further identified a gene family including F3 and members of nuclear receptor 4A family, NR4A1/2/3, co-regulated upon leukotriene and thrombin treatment. Downregulation of F3 was further validated by RT-qPCR. Analysis of PML-RARα variants in APL control and ED cases (n=38, Chi-square p < 0.041) showed a significant enrichment of the short variant bcr3 in ED APL. Artificial overexpression of the short bcr3 and long bcr1 PML/RARα variant in U937 further revealed a correlation between bcr3 and downregulation of NR4A2/3. Moreover, treatment of the APL NB4 cell line with ATRA or ATO induced further downregulation of F3 but upregulation of NR4A2/3 transcripts. ATRA treatment induced the same effects on F3 and NR4A3 protein levels, while ATO led not only to a decrease of F3 but also NR4A3 protein levels.We next sought to address the role of F3 and NR4A in non-APL AML. In 5 non-APL AML cell lines high F3 transcript and protein levels were positively correlated with a better response to ATRA treatment in vitro. Consistently, analyzing samples taken from the AMLSG HD98B trial, AML patients with high F3 but also NR4A3 transcript levels treated with ICE chemotherapy showed a significantly prolonged overall survival upon additional ATRA treatment in vivo (Log-rank p < 0.008).Conclusions: Expression of F3 and NR4A1/2/3 is downregulated in APL ED and decreased expression of NR4A2/3 is associated with short PML-RARα variant bcr3, which is significantly enriched in ED APL. Since NR4A members are associated with coagulation and inflammation, they may be important F3-related factors, contributing to the bcr3 ED APL phenotype. As ATRA and ATO treatment is known to further inhibit F3 expression, alternative therapies not inhibiting F3 expression could be worthwhile in APL ED patients. Moreover, F3 and NR4A3 expression levels seem to be highly relevant as marker for the prediction of ATRA response in non-APL AML patients. [Display omitted] DisclosuresPlatzbecker:Celgene: Research Funding. Thiede:Novartis: Honoraria, Research Funding; AgenDix: Other: Ownership. Schlenk:Pfizer: Research Funding, Speakers Bureau.

KMT2E-Mediated Epigenetic Reprogramming Promotes the Sensitivity to All-Trans Retinoic Acid and Increases the Granulocytic Differentiation in AML Cells

Background: The histone-lysine N-methyltransferase 2E (KMT2E) is a member of the KMT2 family that regulates terminal myeloid differentiation and hematopoietic stem cell self-renewal. However, unlike the other members of KMT2 family, KMT2E does not exhibit the intrinsic histone methyl transferase activity, but has been shown to indirectly interact with chromatin by regulating SET7/9 expression and directing the tri-methylation state of the histone H3K4 (H3K4me3). We previously reported that the low expression of KMT2E predicts poorer prognosis in acute promyelocytic leukemia (APL) and that the overexpression of KMT2E contributed to granulocytic differentiation of APL cells in response to all-trans retinoic acid (ATRA). ATRA as a single agent is not effective in cell differentiation of non-APL acute myeloid leukemia (AML) but changes in H3K4 methylation promotes ATRA sensitivity and upregulates myeloid-differentiation-associated genes in AML cells. Therefore, to better understand the role of KMT2E in leukemogenesis and to test whether ATRA was capable to epigenetically mediate the KMT2E function in non-APL AML blasts, we investigated the effect of KMT2E overexpression in the cell viability, tumor growth and signaling response to ATRA in vitro and in vivo.Methods: qPCR analyzes revealed that the AML cell lines OCI-AML3, U937 and THP-1 express low levels of KMT2E in relation to the APL cell line NB4 and then, they were selected for further experiments. OCIAML3, THP1, and U937 were transduced with the pMEG or the pMEG-KMT2E vector, and U937 cells were also submitted to KMT2E gene silencing by a specific shRNA. The transduced cells were subjected to ATRA (1µM) treatment to evaluate proliferation (Ki-67), clonogenicity, cell cycle and apoptosis (annexin V). For dose-response curves (MTT), cells were administrated graduated ATRA concentrations and the IC50 values were calculated. ATRA-induced granulocytic differentiation was determined by immunophenotyping analysis of myeloid markers: CD11b, CD11c, CD14, CD16, CD64, and HLA-DR. Genes (SETD7/9) and protein (H3K4me3) expressions were established by qPCR and Western blot analysis respectively. 8-10 week-old NSG mice received a dorsal s.c. injection of 5 x 106 U937 cells (transduced with empty/KMT2E/shKMT2E) and 7 days after initial tumor growth treatment was initiated by intraperitoneal injection of the vehicle (n=26) or ATRA (5 mg/kg, n=30) for another 7 days. Tumor volume (V) was obtained using the formula (V=W2 x L x 0.52), where W represents the smaller and L the large diameter. The tumor apoptotic index was determined by TUNEL assay.Results:KMT2E overexpression significantly increased cell proliferation and the proportion of G2/M cells in all cell lines (all P<.01) but no differences in clonogenicity were observed. The IC50 value of ATRA for U937 was of 23 µM for cells transduced with empty vector and 11 µM for pMEG-KMT2E cells, while for THP1 empty/KMT2E cells received a dose of 9.93 µM and 1.68 µM respectively. ATRA increased apoptosis in a time-dependent manner in U937 and THP1 cells (all P<.05), but not in OCIAML3 p-MEG-KMT2E cells. Furthermore, ATRA-treated U937 and THP1 cells presented G0/G1-cell cycle arrest (all P<.05) after 72 hours, while no effect was observed in OCI-AML3. ATRA-induced granulocytic differentiation increased the CD11b/CD14 expression in U937 and THP1-KMT2E cells, within 72 hours of treatment compared to controls (all P<.05). ATRA treatment enhanced expression of H3K4me3 in a time-dependent manner which was correlated with enhanced transcript levels of SETD7/9 (all P<.05).In NSG mice, ATRA significantly reduced the tumor burden in U937- KMT2E cells and increased the apoptotic index (P<.01) after 7 days of treatment, albeit the high tumor burden in KMT2E cells treated with the vehicle. As a result the mean±SD of tumor volume for empty/KMT2E cells was 1296±95.6 mm3 and 445±59.6 mm3, respectively (P=.001). Finally, U937 shKMT2E indicated no difference in ATRA sensitivity and tumor volume (840±54.8 mm3 and 550±32.1 mm3 for empty/shKMT2E cells, respectively (P>.05).Conclusion: Our results demonstrated that KMT2E increases the cellular proliferation and ATRA-induced granulocytic differentiation in non-APL AML cell lines. Furthermore, ATRA triggered epigenetic reprogramming resulting in increased levels of H3K4me3, which redirected the biological function of KMT2E. DisclosuresNo relevant conflicts of interest to declare.

Insulin-like Growth Factor Binding Protein 7 Activates the Retinoid Acid Differentiation Pathway in Acute Myeloid Leukemia Cells

Acute myeloid leukemia (AML) is characterized by the accumulation of malignant blasts with impaired transcriptional differentiation programs. Despite important advances in AML therapy, the five-year overall survival rate of AML patients remains a disappointing 30-40%. This poor prognosis is mainly caused by survival of chemotherapy resistant leukemic cells, named leukemic stem cells (LSC), re-initiating relapse. However, for AML patients with PML-RARA positive acute promyelocytic leukemia (APL), treatment with all trans retinoic acid (ATRA) results in cure rates of >90%. Upon ATRA treatment, APL cells can restore transcription leading to granulocytic differentiation, and in combination with arsenic trioxide APL cells go into apoptosis. While the success of ATRA treatment has been demonstrated for APL patients, so far it has not proved effective for non-APL AML patients.Previously, we demonstrated that insulin-like growth factor binding protein 7 (IGFBP7), a negative regulator of IGF1 receptor (IGF1R) activity, induces apoptosis of AML cells and sensitizes AML cells to chemotherapy-induced cell death. Since it has been shown that IGF1R inhibitors can eliminate therapy-resistant cells by modifying their chromatin state, we hypothesized that IGFBP7 may also have the capacity to modify an epigenetic state and unlock the ATRA-driven differentiation response. To this end, we generated APL cell lines with down- or upregulated IGFBP7 levels and demonstrated that knockdown of IGFBP7 in NB4 cells blocked ATRA-induced differentiation, whereas overexpression of IGFBP7 led to an 8-fold increase in differentiation in the presence of low concentrations of ATRA, together suggesting a role for IGFBP7 in ATRA-induced differentiation in APL cells.Strategies to increase efficacy of ATRA-based therapy might also improve treatment outcomes for non-APL AML, and therefore we investigated the potential of IGFBP7 to induce susceptibility for ATRA-driven differentiation in this group. ATRA and IGFBP7 treatment of non-APL AML cell lines and primary AML cells derived from patients at diagnosis demonstrated an enhanced efficiency of the combination therapy to induce differentiation of myeloid CD45dimCD33+ AML cells (2.5-fold increase in CD11b-expression) and/or to reduce viability of AML CD45dim cells as compared to single treatments (from 30-39% to 70% reduction upon IGFBP7, ATRA or combination therapy, respectively), in 50% of tested primary AML samples, while this combination therapy did not influence normal hematopoietic cell survival. Remarkably, ATRA-IGFBP7 treatment diminished the in vivo engraftment potential of primary AML cells as compared to treatment with either drug alone in NSG mice (from 28% and 52% to 17% engraftment upon ATRA, IGFBP7 or combination therapy, respectively). Re-transplantation of human AML derived from first transplanted mice into secondary recipients demonstrated that the ATRA-IGFBP7 combination treatment also eliminated LSC more effectively (1.4-fold reduction). Together these data suggest that IGFBP7 enhances sensitivity of AML (stem) cells to ATRA, and is able to induce a transcriptional program sensitizing AML cells for ATRA-induced differentiation and cell death.To identify factors responsible for IGFBP7-induced ATRA sensitivity, we performed gene expression profiling of primary AML samples treated with IGFBP7, and identified growth factor independent protein 1 (GFI1) as one of the top down-regulated genes upon IGFBP7 stimulation. As overexpression of GFI1 in non-APL AML patient samples resulted in a >2.5-fold reduction in IGFBP7-induced susceptibility to ATRA-driven differentiation, low GFI1 expression is suggested to be associated with susceptibility to ATRA in AML cells.In conclusion, our results indicate that treatment of AML patient with a combination of ATRA and IGFBP7 might be successful in preventing relapse and improving AML patient survival, which has to be confirmed in further clinical studies. DisclosuresOssenkoppele:Karyopharm: Consultancy, Research Funding; Novartis: Consultancy, Honoraria, Research Funding; Jazz: Consultancy, Honoraria; Genentech: Consultancy, Honoraria; Roche: Consultancy, Honoraria; BMS: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria; Genmab: Research Funding; Celgene: Honoraria, Research Funding; Johnson & Johnson: Consultancy, Honoraria, Research Funding.

Targeting the SUMO Pathway Primes All-trans Retinoic Acid–Induced Differentiation of Nonpromyelocytic Acute Myeloid Leukemias

Differentiation therapies using all-trans retinoic acid (ATRA) are highly efficient at treating acute promyelocytic leukemia (APL), a subtype of acute myeloid leukemia (AML). However, their efficacy, if any, is limited in the case of non-APL AML. We report here that inhibition of SUMOylation, a posttranslational modification related to ubiquitination, restores the prodifferentiation and antiproliferative activities of retinoids in non-APL AML. Controlled inhibition of SUMOylation with the pharmacologic inhibitors 2-D08 or anacardic acid, or via overexpression of SENP deSUMOylases, enhanced the ATRA-induced expression of key genes involved in differentiation, proliferation, and apoptosis in non-APL AML cells. This activated ATRA-induced terminal myeloid differentiation and reduced cell proliferation and viability, including in AML cells resistant to chemotherapeutic drugs. Conversely, enhancement of SUMOylation via overexpression of the SUMO-conjugating enzyme Ubc9 dampened expression of ATRA-responsive genes and prevented differentiation. Thus, inhibition of the SUMO pathway is a promising strategy to sensitize patients with non-APL AML to retinoids and improve the treatment of this poor-prognosis cancer.Significance: SUMOylation silences key ATRA-responsive genes in nonpromyelocytic acute myeloid leukemias. Cancer Res; 78(10); 2601-13. ©2018 AACR.

Reprogramming acute myeloid leukemia into sensitivity for retinoic-acid-driven differentiation.

The success of all-trans retinoic acid (ATRA) therapy for acute promyelocytic leukemia (APL) provides a rationale for using retinoic acid (RA)-based therapy for other subtypes of acute myeloid leukemia (AML). Recently, several studies showed that ATRA may drive leukemic cells efficiently into differentiation and/or apoptosis in a subset of AML patients with an NPM1 mutation, a FLT3-ITD, an IDH1 mutation, and patients overexpressing EVI-1. Because not all patients within these molecular subgroups respond to ATRA and clinical trials that tested ATRA response in non-APL AML patients have had disappointing results, the identification of additional biomarkers may help to identify patients who strongly respond to ATRA-based therapy. Searching for response biomarkers might also reveal novel RA-based combination therapies with an efficient differentiation/apoptosis-inducing effect in non-APL AML patients. Preliminary studies suggest that the epigenetic or transcriptional state of leukemia cells determines their susceptibility to ATRA. We hypothesize that reprogramming by inhibitors of epigenetic-modifying enzymes or by modulation of microRNA expression might sensitize non-APL AML cells for RA-based therapy. AML relapse is caused by a subpopulation of leukemia cells, named leukemic stem cells (LSCs), which are in a different epigenetic state than the total bulk of the AML. The survival of LSCs after therapy is the main cause ofthe poor prognosis of AML patients, and novel differentiation therapies should drive these LSCs into maturity. In this review, we summarize the current knowledge on the epigenetic aspects of susceptibility to RA-induced differentiation in APL and non-APL AML.

Sorafenib Inhibition of Mcl-1 Accelerates ATRA-Induced Apoptosis in Differentiation-Responsive AML Cells.

All trans-retinoic acid (ATRA) is successful in treating acute promyelocytic leukemia (APL) by inducing terminal differentiation-mediated cell death, but it has limited activity in non-APL acute myeloid leukemia (AML). We aim to improve ATRA therapy of AML by enhancing apoptosis through repression of the antiapoptotic proteins Bcl-2 and Mcl-1. APL and AML cell lines, as well as primary AML samples, were used to explore the mechanisms regulating differentiation and apoptosis during ATRA treatment. Stable transfection and gene silencing with siRNA were used to identify the key factors that inhibit apoptosis during induction of differentiation and drugs that accelerate apoptosis. In differentiation-responsive AML cells, ATRA treatment induces long-lasting repression of Bcl-2 while first upmodulating and then reducing the Mcl-1 level. The Mcl-1 level appears to serve as a gatekeeper between differentiation and apoptosis. During differentiation induction, activation of MEK/ERK and PI3K/Akt pathways by ATRA leads to activation of p90RSK and inactivation of glycogen synthase kinase 3β (GSK3β), which increase Mcl-1 levels by increasing its translation and stability. Sorafenib blocks ATRA-induced Mcl-1 increase by reversing p90RSK activation and GSK3β inactivation, maintains the repressed Bcl-2 level, and enhances ATRA induced apoptosis in non-APL AML cell lines and in primary AML cells. Inhibition of Mcl-1 is required for apoptosis induction in ATRA differentiation-responsive AML cells. ATRA and sorafenib can be developed as a novel drug combination therapy for AML patients because this drug combination augments apoptosis by inhibiting Bcl-2 and Mcl-1.

The mechanism of synergistic effects of arsenic trioxide and rapamycin in acute myeloid leukemia cell lines lacking typical t(15;17) translocation.

Arsenic trioxide (ATO) has potent clinical activity in the treatment of patients with acute promyelocytic leukemia (APL), but is much less efficacious in acute myeloid leukemia (AML) lacking t(15;17) translocation. Recent studies have indicated that the addition of mammalian target of rapamycin (mTOR) inhibitors may increase the sensitivity of malignant cells to ATO. The aim of the present study was to test for possible synergistic effects of ATO and rapamycin at therapeutically achievable doses in non-APL AML cells. In HL-60 and U937 cell lines, the inhibitory effects of low concentrations of ATO and rapamycin were synergistic and more pronounced in U937 cells. The combination of drugs increased apoptosis in HL-60 cells and increased the percentage of cells in G(0)/G(1) phase in both cell lines. In U937 cells, rapamycin alone increased the activity of mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) and the addition of ATO decreased the level of phosphorylated ERK, Ser473 phosphorylated Akt and anti-apoptotic Mcl-1 protein. Primary AML cells show high sensitivity to growth-inhibitory effects of rapamycin alone or in combination with ATO. The results of the present study reveal the mechanism of the synergistic effects of two drugs at therapeutically achievable doses in non-APL AML cells.

Abstract 4239: Differentiation and apoptosis induced by all-trans retinoic acid is associated with downregulation of peroxiredoxin 1 in myeloid leukemia cells

Abstract All-trans retinoic acid (ATRA) based differentiation therapy with potentially curative outcome in acute promyelocytic leukemia (APL), has limited benefit in non-APL acute myeloid leukemia (AML). Thus, identifying key mediators of ATRA-induced differentiation/apoptosis in AML cells may reveal new putative therapeutic targets. Previously, we reported (Leukemia 20: 1809, 2006) that the therapeutic efficacy of ATRA is enhanced in topoisomerase 2β (TOP2β)-deficient AML cell lines, both APL (HL-60, AP-1060) and non-APL (KG-1), in part due to decreased expression of the antioxidant enzyme, peroxiredoxin (PRDX) 2 and increased accumulation of reactive oxygen species (ROS). Based on preliminary observations that (a) ATRA leads to down-regulation of a related antioxidant protein, PRDX1 (RNA array analysis), and (b) PRDX1 mRNA expression relative to PRDX2 mRNA is significantly higher in HL-60, KG-1, and leukemic blast cells from 20 AML patients (qRT-PCR analysis), we evaluated the role of PRDX1 in sensitizing the apoptotic effects of ATRA. Specifically, we tested the hypothesis that ATRA induced growth arrest/apoptosis is associated with decreased PRDX1 levels and increased ROS, which can be modulated by TOP2β. Accordingly, downregulation of TOP2β can enhance apoptosis by increasing ROS, whereas overexpression of TOP2β can inhibit PRDX1 downregulation by ATRA and attenuate ROS-induced apoptosis. Treatment of HL-60, KG-1 and patient AML blast cells with 1 µM ATRA in the absence or presence of 0.1 µM ICRF193 (TOP2β catalytic inhibitor) led to a 3 to 4-fold decrease in PRDX1 mRNA and protein. No effect of ICRF193, which leads to degradation of TOP2β, was observed on PRDX1 levels. Interestingly, while treatment with ATRA + ICRF 193 did not enhance PRDX1 down regulation compared to ATRA alone, it did lead to significantly (p&amp;lt;.05) more apoptosis and greater ROS than either treatment alone. Since, expression of TOP2β correlates with that of PRDX1 in blast cells of AML patients, both of which are expressed at relatively high levels, we tested whether overexpression of TOP2β affects ATRA-induced decrease in PRDX1, ROS accumulation and apoptosis. Transfection of TOP2β in an amsacrine-resistant HL-60 cell line (AR), which lacks TOP2β, leads to overexpression of TOP2β and abrogation of ATRA-induced downregulation of PRDX1, ROS accumulation and apoptosis. In summary, the present study demonstrates that PRDX1 is an ATRA regulated protein, which plays an important role in modulating ATRA-induced differentiation/apoptosis in myeloid leukemia cells. Furthermore, down regulation of PRDX1 levels by ATRA can be manipulated by overexpression of TOP2β. Since overexpression of TOP2β is observed in patient AML blasts, the increased PRDX1 levels may partly explain the resistance of non-APL acute myeloid leukemia cells to ATRA induced differentiation and ROS-mediated apoptosis. Citation Format: Eric J. Norris, Yogin C. Patel, Michaela B. Reinhart, Ram N. Ganapathi, Mahrukh K. Ganapathi. Differentiation and apoptosis induced by all-trans retinoic acid is associated with downregulation of peroxiredoxin 1 in myeloid leukemia cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4239. doi:10.1158/1538-7445.AM2014-4239

Abstract 799: Diminishing Mcl-1 protein leads to apoptosis of acute myeloid leukemia cells responding to all trans retinoic acid differentiation

Abstract All trans retinoic acid (ATRA) is successfully used for the treatment of acute promyelocytic leukemia (APL) through inducing terminal differentiation and death receptor-mediated apoptosis. However, ATRA has limited clinic efficacy in non-APL acute myeloid leukemia (AML) patients. To extend ATRA therapy to AML (non-APL), cell death and differentiation induction were tested and compared in a panel of AML cell lines after treatment with ATRA for 2, 4 and 6 days based on the staining of CD11b and annexin V, respectively. ATRA treatment in APL NB4 cells induces differentiation but not apoptosis at 2 days. ATRA differentiation peaks at 4 days and apoptosis becomes measurable at 4 days and peaks at 6 days. Western blot analysis revealed that both TRAIL and DR5 were induced by ATRA at 4 days, supporting that a death-receptor-mediated apoptosis program is initiated by ATRA. The mitochondrial apoptotic program was determined by measuring the levels of Bcl-2, Mcl-1 and the cleaved caspase-9. Although Bcl-2 protein was depleted, Mcl-1 protein was significantly increased after treatment with ATRA at 2 days. As the treatment continued to 4 and 6 days, the Mcl-1 protein levels declined and cleaved caspase 9 was detected. Similar phenomena were observed in non-APL HL-60 cells responding to ATRA-induced differentiation, but not in ATRA differentiation resistant NB4 and HL-60 subclones. Silencing of Mcl-1 with siRNA sensitized ATRA-induced apoptosis with decreased differentiation in NB4 cells treated with ATRA for 2 days. HL-60 cells with overexpression of Mcl-1 increased ATRA-induced differentiation with diminished apoptosis after treatment with ATRA for 6 days. Mechanistic studies of Mcl-1 induction by ATRA revealed that both Akt/mTOR and MEK/ERK signaling were activated associated with increased phosphorylation of glycogen synthase kinase 3β(GSK3β) and ribosomal S6 protein. Since inactivation of GSK3β by phosphorylation stabilizes Mcl-1 protein and S6 phosphorylation increases Mcl-1 protein translation, ATRA increases Mcl-1 levels through increasing translation and stability. Akt inhibitor Ly294002 and MEK inhibitor U0126, but not mTOR inhibitor rapamycin, blocked Mcl-1 induction and converted ATRA into an apoptosis inducer in HL-60 cells. More intriguingly sorafenib, a multi-kinase inhibitor, antagonized ATRA induction of Mcl-1, p-GSK3β and p-S6, but not Bcl-2 reduction, and synergistically induced apoptosis with ATRA in a panel of non-APL cell lines and primary AML cells. Our data suggest that Mcl-1 protein levels contribute to balance between cell death and differentiation of AML cells treated with ATRA and that ATRA therapy could be extended to non-APL cells by augmented cell death induction by diminishing Mcl-1 protein. Note: This abstract was not presented at the meeting. Citation Format: Rui Wang, Lijuan Xia, Janice Gabrilove, Samuel Waxman, Yongkui Jing. Diminishing Mcl-1 protein leads to apoptosis of acute myeloid leukemia cells responding to all trans retinoic acid differentiation. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 799. doi:10.1158/1538-7445.AM2014-799

5-Aminoimidazole-4-carboxamide ribonucleoside induces differentiation of acute myeloid leukemia cells

Adenosine monophosphate (AMP)-activated kinase (AMPK) modulators have been shown to exert cytotoxic activity in hematological malignancies, but their role in the differentiation of acute myeloid leukemia (AML) is less explored. In this study, the effects of AMPK agonists on all-trans retinoic acid (ATRA)-mediated differentiation of acute promyelocytic leukemia (APL) and non-APL AML cell lines were investigated. The results show that AMPK agonists inhibit the growth of myeloblastic HL-60, promyelocytic NB4 and monocytic U937 cells. 5-Aminoimidazole-4-carboxamide ribonucleoside (AICAR), an AMPK activator, enhances ATRA-mediated differentiation of NB4 cells. In U937 cells, AICAR alone induces the expression of cell surface markers associated with mature monocytes and macrophages. In both cell lines, AICAR increases the activity of mitogen-activated protein kinase (MAPK), and the presence of a MAPK inhibitor reduces the expression of differentiation markers. These results reveal beneficial effects of AICAR in AML, including differentiation of non-APL AML cells.