Primary Acute Promyelocytic Leukemia Cells Research Articles

Targeting HDAC3 to Overcome the Resistance to ATRA or Arsenic in Acute Promyelocytic Leukemia through Ubiquitination and Degradation of PML-RARα

Acute promyelocytic leukemia (APL) is driven by the oncoprotein PML-RARα, which recruits corepressor complexes, including histone deacetylases (HDACs), to suppress cell differentiation and promote APL initiation. All-trans retinoic acid (ATRA) combined with arsenic trioxide (ATO) or chemotherapy highly improves the prognosis of APL patients. However, refractoriness to ATRA and ATO may occur, which leads to relapsed disease in a group of patients. In this study, we obtained the relationship between different HDACs with APL by analyzing transcriptional dataset of leukemia and confirmed the positive association of HDAC3 with PML-RARα in the mRNA and protein levels measured in primary human APL blasts. Experiments in vitro indicated that targeting HDAC3 but not HDAC1 or HDAC2 induced more effective differentiation and apoptosis in both NB4 cells and primary human APL cells. To examine the effects of HDAC3 inhibition on APL cells in vivo, we utilized normal FVB mice transplanted with APL cells from hMRP8-PML-RARα transgenic mice and NB4 or NB4-HDAC3Cas9 xenograft mouse model. We found that pharmacological or genetically inhibition of HDAC3 extended the survival rate of mice and alleviated spleen infiltration of leukemia cells and promoted the apoptosis and differentiation of APL cells. As to the molecular mechanism, we first examined whether HDAC3 affected PML-RARα expression level and the results demonstrated that HDAC3 inhibition reduced PML-RARα protein half-life rather than transcriptional activity via the ubiquitin proteasome system. In the subsequent experiments, we constructed a series of PML-RARα mutations. By confocal imaging system, acetylation and protein-protein interaction assays, we demonstrated that HDAC3 directly bound to the LBD and DBD domains of RARA protein and modulated the acetylation of lysine 394 of PML, which reduced PIAS1-mediated PML-RARα SUMOylation and subsequent RNF4-induced ubiquitylation. HDAC3 inhibition induced an increase of K394 acetylation, and promoted PML-RARα ubiquitylation and subsequent degradation, while the effect could be rescued by proteosome inhibition. Our results further revealed that the inhibition of HDAC3 induced degradation of both wild-type and ATRA- or ATO-resistant mutant PML-RARα (A216V, L218P, ΔF286 and R276Q). Therefore, we speculated that targeting HDAC3 could induce antileukemic effects on ATO- or ATRA-resistant APL cells. In NB4-R1, NB4-R2 and primary human APL cells, inhibition of HDAC3 increased significantly the cell differentiation and apoptosis, decreased PML-RARα expression and reduced the proliferation and colony-formation units (CFUs). For experiments in vivo, ATRA-resistant APL xenograft mouse model of NB4-R2 cells and patient-derived tumor xenograft (PDX) model were utilized to evaluate the effects of targeting HDAC3. Results showed that targeting HDAC3 extended the survival of mice, decreased the leukemia cells infiltration and increased the differentiation and apoptotic cells. In conclusion, our study identifies the role of HDAC3 as a positive regulator of the PML-RARα oncoprotein by deacetylating PML-RARα and suggests that targeting HDAC3 could be a promising strategy to treat relapsed/refractory APL. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

PRMT5-mediated RNF4 methylation promotes therapeutic resistance of APL cells to As2O3 by stabilizing oncoprotein PML-RARα.

Acute promyelocytic leukemia (APL) is a hematological malignancy driven by the oncoprotein PML-RARα, which can be treated with arsenic trioxide (As2O3) or/and all-trans retinoic acid. The protein arginine methyltransferase 5 (PRMT5) is involved in tumorigenesis. However, little is known about the biological function and therapeutic potential of PRMT5 in APL. Here, we show that PRMT5 is highly expressed in APL patients. PRMT5 promotes APL by interacting with PML-RARα and suppressing its ubiquitination and degradation. Mechanistically, PRMT5 attenuates the interaction between PML-RARα and its ubiquitin E3 ligase RNF4 by methylating RNF4 at Arg164. Notably, As2O3 treatment triggers the dissociation of PRMT5 from PML nuclear bodies, attenuating RNF4 methylation and promoting RNF4-mediated PML-RARα ubiquitination and degradation. Moreover, knockdown of PRMT5 and pharmacological inhibition of PRMT5 with the specific inhibitor EPZ015666 significantly inhibit APL cells growth. The combination of EPZ015666 with As2O3 shows synergistic effects on As2O3-induced differentiation of bone marrow cells from APL mice, as well as on apoptosis and differentiation of primary APL cells from APL patients. These findings provide mechanistic insight into the function of PRMT5 in APL pathogenesis and demonstrate that inhibition of PRMT5, alone or in combination with As2O3, might be a promising therapeutic strategy against APL.

Podoplanin Expression in the Bleeding Complications of Acute Promyelocytic Leukemias

Background: Acute promyelocytic leukemia (APL), characterized by the t(15;17) translocation, represents a favorable-risk subgroup of AML patients when treated with ATRA-based regimens. The leading cause of early death and the major unmet medical need in this disease are the bleeding complications, which are mainly attributed to aberrant expression of Tissue Factor (TF) and Annexin A2 (ANXA2) on leukemic promyelocytes, leading to disseminated intravascular coagulation and to hyperfibrinolysis, respectively. APL patients are also at increased risk of venous and arterial thromboses. The mechanisms underlying APL-associated hemostatic complications are not fully elucidated.Aims and Methods: We analyzed the transcriptome of 30 APL specimens comprised in the Leucegene 430 AML cohort, as well as normal hematopoietic cell populations (n=63) aiming to better understand the hemostasis-related transcriptomic landscape of this subgroup.Results: We identified podoplanin (PDPN) as the most significantly and differentially overexpressed gene in APL (median 2.6 vs 0 RPKM for APL and AML, respectively, q-value = 7.3 x 10-29) and determined that PDPN gene expression correlates with protein surface expression as assessed by flow cytometry on primary APL cells (r=0.89). TF (6.6 vs 1.6, p=1.8x10-9) and ANXA2 (142.8 vs 69.1, p=1.9x10-5) are also more expressed in APL, but less specifically than PDPN. Podoplanin is a glycoprotein that physiologically binds to its receptor, CLEC2, on platelets to induce platelet aggregation causing the separation of blood and lymphatic vessels during embryogenesis (Uhrin et al Blood 2010). As anticipated, we found that PDPN is not expressed in all studied sorted cell subpopulations from normal blood or bone marrow specimens, including in promyelocytes (median of 0 RPKM for all), indicating that platelets are most likely not exposed to PDPN in the adult vasculature and that this protein is ectopically expressed on APL promyelocytes.Next, we used lentiviral gene transfer to engineer OCI-AML5 cells, which do not express PDPN, to ectopically express this protein (AML5PDPN). When mixed with platelet-rich plasma (PRP), we found that AML5PDPN cells have much greater platelet-binding and activating capacity than AML5CTRL cells. Using light transmission aggregometry, only AML5PDPN cells, not AML5CTRL cells, could induce platelet aggregation at tested concentrations. These results indicate that PDPN expression on leukemia cells is sufficient to induce platelet binding, activation and aggregation. Using patient-derived cells, we similarly found that PDPN-expressing primary APL cells (n=3 samples), but not PDPN-negative APL (n=2) or AML (n=5) cells, have the capacity to bind and activate platelets and to induce platelet aggregation. Our results suggest that PDPN could contribute to APL-related thromboses, as was recently reported in brain tumors (Riedl et al Blood 2017).We next tested the hypothesis that PDPN expression on human myeloid blasts leads to platelet consumption, thrombocytopenia and bleeding in vivo . NSG mice were transplanted with either AML5CTRL or AML5PDPN cells and were monitored until they showed clinical signs of leukemia. Platelet counts rapidly and significantly dropped in the AML5PDPN-cohort at day 25 and beyond when compared to control animals (median 343 vs 1005 x 109/L, p < 0.0001). Mice platelet counts were inversely correlated with circulating PDPN load (r=-0.88) and animals with PDPN+ leukemias had markedly prolonged bleeding time (median > 15:00 vs 7:45, p = 0.0003).Finally, we hypothesized that ATRA reduces bleeding complications in patients, at least partially, by decreasing PDPN expression. We found that PDPN surface protein on primary APL promyelocytes is markedly decreased within the first 24 hours of ATRA treatment in vitro, and levels reached less than 10% on day 4 when compared to untreated cells (DMSO). Arsenic trioxide or dexamethasone treatment led to a modest or no reduction in PDPN expression, respectively.Conclusion: PDPN expression is specific to APL and, by inducing platelet binding, activation and aggregation, it contributes to platelet consumption and to bleeding complications. Our findings may also expose a new promising angle for the treatment of the early hemostatic complications found in APL patients. DisclosuresNo relevant conflicts of interest to declare.

Use of Arsenic Trioxide (As2O3 ) in the Treatment of Acute Promyelocytic Leukemia (APL): I. As2O3 Exerts Dose-Dependent Dual Effects on APL Cells

AbstractRecent clinical studies in China showed that As2O3 is an effective and relatively safe drug in the treatment of acute promyelocytic leukemia (APL). We found previously that As2O3 can trigger apoptosis of APL cell line NB4 cells, which is associated with downregulation of bcl-2 gene expression and modulation of PML-RARα chimeric protein. To further understand the mechanisms of this alternative therapy for APL, we investigated in this report the effects of a wide range of concentrations of As2O3 on cultured primary APL cells, all-trans retinoic acid (ATRA)-susceptible (NB4 cells) and ATRA-resistant (MR2 subclone) APL cell lines. The results indicated that As2O3 had dose-dependent dual effects on APL cells: inducing preferentially apoptosis at relatively high concentrations (0.5 to 2 μmol/L) and inducing partial differentiation at low concentrations (0.1 to 0.5 μmol/L). The rapid modulation and degradation of PML-RARα proteins, which was induced by As2O3 at 0.1 to 2 μmol/L, could contribute to these two effects. Bone marrow and peripheral blood examination showed that myelocyte-like cells, probably as a result of partial in vivo differentiation, and degenerative cells increased after 2 to 3 weeks of continuous in vivo As2O3 treatment when leukemic promyelocytes decreased. In conclusion, combination of induction of apoptosis and partial differention could be the main cellular mechanisms of As2O3 in the treatment of APL, and PML-RARα could play an important role in determining the specific effects of As2O3 on APL cells.

Reduced SLIT2 Are Associated with Increased Cell Proliferation and Arsenic Trioxide Resistance in APL Cells

Background: Recently, the SLIT/ROBO axis became a therapeutic target for a variety of non-cerebral solid tumors because of its relevance in the regulation of angiogenesis, inflammatory cell chemotaxis, tumor cell migration and metastasis. In acute myeloid leukemia (AML), lower levels of SLIT2 expression were associated with poorer prognosis in studies that excluded patients with acute promyelocytic leukemia (APL) of the analysis (Golos et al., Arch Immunol 2019). Our group previously reported that higher SLIT2 expression was associated with improved overall survival, disease-free survival and decreased cumulative incidence of relapse. Subsequently, our in vitro functional studies indicated anti-proliferative effects exercised by exogenous SLIT2 peptide treatment in APL (Weinhauser et al., Blood 2018). Although, the expression of SLIT/ROBO was detected on leukemic cells, the fact that in healthy subjects the main source of SLIT2 derives from bone marrow stromal cells (BMSC) (Smith-Berdan et al., Cell Cycle 2012) was so far neglected in the context of hematological malignancies. Aims: Here, we expanded our data on the role of SLIT2 using lentivirally knocked down APL cells, and addressed the issue of the importance of BMSC-derived SLIT2 on APL development. Methods: NB4 and NB4R2 (RA-resistant) and primary APL cells (age, 25-47y; n=4) were transduced with shRNA-SLIT2 or shCT (control). After synchronization using double-thymidine block, transduced cells were submitted to proliferation and cell cycle assays. For the apoptosis analysis, cells were treated with ATO (1 µM) alone or in combination with RA (1 µM) for 24, 48 and 72 hours. The granulocytic differentiation in response to RA treatment alone (1 µM) or in combination with ATO (1 µM) for 48 and 72 hours, was evaluated based on the CD11b and CD11c levels. To assess the effect of SLIT2 silencing in BMSCs, HS5 and HS27A cell lines were transduced with shSLIT2 or shCT. Non-transduced NB4, NB4R2 and primary APL cells were co-cultured with engineered BMSCs and proliferation, apoptosis and granulocytic differentiation was performed in response to ATO alone or in combination with RA for 48 and 72 hours. In vivo assessment was performed using four 8-12 week-old C57/BL6PepBoy (CD45.1) lethally irradiated, transplanted with 5 x 106 hCG-PMLRARa blasts (CD45.2) transduced with shSlit2-murine and shCT. Results: Silencing SLIT2 in primary APL samples resulted in a significant increase in cell growth at the sixth day of culture compared to shCT samples (P&lt;.001). Accordingly, enhanced staining of Ki67 was detected in NB4 and NB4R2-shSLIT2 transduced compared to shCT cells. Moreover, NB4-shSLIT2 cells exhibited lower ATO-induced apoptosis either when ATO was used alone or in combination with RA. Nevertheless, SLIT2 knockdown had no effect on NB4R2 cells suggesting that RARA moiety of PML/RARA may be important to the observed effect. Concomitantly, NB4 cell differentiation rate, was greatly increased in NB4-shSLIT2 in the presence of RA after 48 and 72h (P&lt;.05) and 48h when RA was combined with ATO (P&lt;.05). In vivo transplantation of primary murine shSLIT2 hCG-PMLRARa blasts, resulted in significant increase of white blood cell count during the course of APL emergence and increased extramedullary hematopoiesis in the spleen, yet with no difference in the BM at the time of sacrifice. Next, we evaluated the number of transcripts and the function of SLIT2 in AML derived MSC cells. SLIT2 transcript levels were significantly reduced in primary AML-derived BMSC compared to BMSCs obtained from healthy donors (P&lt;.05). In vitro assays co-culturing NB4 and NB4R2 cells on engineered HS5 or HS27A BMSC cells showed no difference in drug-induced apoptosis or differentiation (P&gt;.05). In contrast to shSLIT2 APL induced cell growth, co-culture of primary APL blast cells with transduced HS27A cells had no impact on proliferation, while the expression of the neutrophilic differentiation gene ELANE2 was significantly decreased in APL cells co-cultured with HS27A-shSLIT2.ConclusionSLIT2 is expressed in APL cells and exerts an anti-proliferative effect. On the other hand, SLIT2 produced by BMSC seemed to promote APL cellular stemness. Our results reinforce that SLIT2 function is cell type dependent and may be involved in the cross-talk between microenvironment and leukemic cells. Disclosures Figueiredo-Pontes: Novartis: Honoraria.

Study of the mechanisms of crocetin-induced differentiation and apoptosis in human acute promyelocytic leukemia cells.

Crocetin, the major carotenoid in saffron, exhibits potent anticancer effects. However, the antileukemic effects of crocetin are still unclear, especially in primary acute promyelocytic leukemia (APL) cells. In the current study, the potential antipromyelocytic leukemia activity of crocetin and the underlying molecular mechanisms were investigated. Crocetin (100 µM), like standard anti-APL drugs, all-trans retinoic acid (ATRA, 10 µM) and As2 O 3 (arsenic trioxide, 50 µM), significantly inhibited proliferation and induced apoptosis in primary APL cells, as well as NB4 and HL60 cells. The effect was associated with the decreased expressions of prosurvival genes Akt and BCL2, the multidrug resistance (MDR) proteins, ABCB1 and ABCC1 and the inhibition of tyrosyl-DNA phosphodiesterase 1 (TDP1), while the expressions of proapoptotic genes CASP3, CASP9, and BAX/BCL2 ratio were significantly increased. In contrast, crocetin at relatively low concentration (10 µM), like ATRA (1 µM) and As 2 O 3 (0.5 µM), induced differentiation of leukemic cells toward granulocytic pattern, and increased the number of differentiated cells expressing CD11b and CD14, while the number of the immature cells expressing CD34 or CD33 was decreased. Furthermore, crocetin suppressed the expression of clinical marker promyelocytic leukemia/retinoic acid receptor-α ( PML/RARα) in NB4 and primary APL cells, and reduced the expression of histone deacetylase 1 ( HDAC1) in all leukemic cells. The results suggested that crocetin can be considered as a candidate for future preclinical and clinical trials of complementary APL treatment.

Transcriptomic landscape of acute promyelocytic leukemia reveals aberrant surface expression of the platelet aggregation agonist Podoplanin.

Acute promyelocytic leukemia (APL) is a medical emergency because of associated lethal early bleeding, a condition preventable by prompt diagnosis and therapeutic intervention. The mechanisms underlying the hemostatic anomalies of APL are not completely elucidated. RNA-sequencing-based characterization of APL (n = 30) was performed and compared to that of other acute myeloid leukemia (n = 400) samples and normal promyelocytes. Perturbations in the transcriptome of coagulation and fibrinolysis-related genes in APL extend beyond known culprits and now include Thrombin, Factor X and Urokinase Receptor. Most intriguingly, the Podoplanin (PDPN) gene, involved in platelet aggregation, is aberrantly expressed in APL promyelocytes and is the most distinctive transcript for this disease. Using an antibody panel optimized for AML diagnosis by flow cytometry, we also found that PDPN was the most specific surface marker for APL, and that all-trans retinoic acid therapy rapidly decreases its expression. Functional studies showed that engineered overexpression of this gene in human leukemic cells causes aberrant platelet binding, activation and aggregation. PDPN-expressing primary APL cells, but not PDPN-negative primary leukemias, specifically induce platelet binding, activation and aggregation. Finally, PDPN expression on leukemia cells in a xenograft model was associated with thrombocytopenia and prolonged bleeding time in vivo. Together our results suggest that PDPN may contribute to the hemostatic perturbations found in APL.

Histone Acetyltransferase p300/CREB-binding Protein-associated Factor (PCAF) Is Required for All-trans-retinoic Acid-induced Granulocytic Differentiation in Leukemia Cells

Differentiation therapy with all-trans-retinoic acid (ATRA) improves the treatment outcome of acute promyelocytic leukemia (APL); however, the molecular mechanism by which ATRA induces granulocytic differentiation remains unclear. We previously reported that the inhibition of the NAD-dependent histone deacetylase (HDAC) SIRT2 induces granulocytic differentiation in leukemia cells, suggesting the involvement of protein acetylation in ATRA-induced leukemia cell differentiation. Herein, we show that p300/CREB-binding protein-associated factor (PCAF), a histone acetyltransferase (HAT), is a prerequisite for ATRA-induced granulocytic differentiation in leukemia cells. We found that PCAF expression was markedly increased in leukemia cell lines (NB4 and HL-60) and primary APL cells during ATRA-induced granulocytic differentiation. Consistent with these results, the expression of PCAF was markedly up-regulated in the bone marrow cells of APL patients who received ATRA-containing chemotherapy. The knockdown of PCAF inhibited ATRA-induced granulocytic differentiation in leukemia cell lines and primary APL cells. Conversely, the overexpression of PCAF induced the expression of the granulocytic differentiation marker CD11b at the mRNA level. Acetylome analysis identified the acetylated proteins after ATRA treatment, and we found that histone H3, a known PCAF acetylation substrate, was preferentially acetylated by the ATRA treatment. Furthermore, we have demonstrated that PCAF is required for the acetylation of histone H3 on the promoter of ATRA target genes, such as CCL2 and FGR, and for the expression of these genes in ATRA-treated leukemia cells. These results strongly support our hypothesis that PCAF is induced and activated by ATRA, and the subsequent acetylation of PCAF substrates promotes granulocytic differentiation in leukemia cells. Targeting PCAF and its downstream acetylation targets could serve as a novel therapeutic strategy to overcome all subtypes of AML.

Activation of G0S2 is coordinated by recruitment of PML/RARα and C/EBPɛ to its promoter during ATRA-induced APL differentiation

All-trans retinoic acid (ATRA) binds the promyelocytic leukemia/retinoic acid receptor α (PML/RARα) fusion protein and is an effective oncogene-targeted therapy for acute promyelocytic leukemia (APL). However, the molecular basis of PML/RARα-mediated transcriptional control during ATRA-induced differentiation is unclear. Previous studies have shown that the PML/RARα fusion protein behaves as a type II nuclear receptor, binding to DNA regardless of ligand status. Here, we performed a series of chromatin immunoprecipitation (ChIP)-quantitative PCR (qPCR) experiments, demonstrating that there is an additional mode of action of PML/RARα, wherein PML/RARα does not bind DNA in the absence of ATRA but binds DNA and activates adjacent genes in the presence of ATRA. This mode of action is similar to that of a type I nuclear receptor and is highlighted by activation of G0/G1 switch gene 2 (G0S2) during ATRA-induced neutrophil differentiation of leukemia cell lines (NB4 and PR9) and primary human APL cells. C/EBPε occupancy of the G0S2 promoter was elevated in parallel with recruitment of PML/RARα in ATRA-treated NB4, PR9, and primary APL cells. Furthermore, we verified that the p30 isoform of C/EBPε is crucial for activation of G0S2 and that PML/RARα interacts physically and cooperates functionally with C/EBPε to up-regulate G0S2 Our data not only demonstrate a new mode of action of PML/RARα but also suggest a novel model in which PML/RARα synergizes with C/EBPε to reactivate the C/EBPε target G0S2, thereby contributing to ATRA-mediated APL differentiation and potentially, clinical remission.

PML-RAR alpha induces the downmodulation of HHEX: a key event responsible for the induction of an angiogenetic response.

BackgroundRecent studies indicate that angiogenesis is important in the pathogenesis of acute myeloid leukemias (AMLs). Among the various AMLs, the bone marrow angiogenetic response is particularly pronounced in acute promyelocytic leukemia (APL). However, the molecular mechanisms responsible for this angiogenetic response are largely unknown. In the present study, we have explored the role of HHEX, a homeodomain transcription factor, as a possible mediator of the pro-angiogenetic response observed in APL. This transcription factor seems to represent an ideal candidate for this biologic function because it is targeted by PML-RARα, is capable of interaction with PML and PML-RARα, and acts as a regulator of the angiogenetic response.MethodsWe used various cellular systems of APL, including primary APL cells and leukemic cells engineered to express PML-RARα, to explore the role of the PML-RARα fusion protein on HHEX expression. Molecular and biochemical techniques have been used to investigate the mechanisms through which PML-RARα downmodulates HHEX and the functional consequences of this downmodulation at the level of the expression of various angiogenetic genes, cell proliferation and differentiation.ResultsOur results show that HHEX expression is clearly downmodulated in APL and that this effect is directly mediated by a repressive targeting of the HHEX gene promoter by PML-RARα. Studies carried out in primary APL cells and in a cell line model of APL with inducible PML-RARα expression directly support the view that this fusion protein through HHEX downmodulation stimulates the expression of various genes involved in angiogenesis and inhibits cell differentiation.ConclusionsOur data suggest that HHEX downmodulation by PML-RARα is a key event during APL pathogenesis.Electronic supplementary materialThe online version of this article (doi:10.1186/s13045-016-0262-5) contains supplementary material, which is available to authorized users.

Tetraarsenictetrasulfide and Arsenic Trioxide Exert Synergistic Effects on Induction of Apoptosis and Differentiation in Acute Promyelocytic Leukemia Cells.

Since arsenic trioxide (As3+) has been successfully used in the treatment of acute promyelocytic leukemia (APL), its adverse effects on patients have been problematic and required a solution. Considering the good therapeutic potency and low toxicity of tetraarsenictetrasulfide (As4S4) in the treatment of APL, we investigated the effects of combining As4S4 and As3+ on the apoptosis and differentiation of NB4 and primary APL cells. As4S4, acting similarly to As3+, arrested the G1/S transition, induced the accumulation of cellular reactive oxygen species, and promoted apoptosis. Additionally, low concentrations of As4S4 (0.1–0.4 μM) induced differentiation of NB4 and primary APL cells. Compared with the As4S4- or As3+-treated groups, the combination of As4S4 and As3+ obviously promoted apoptosis and differentiation of NB4 and primary APL cells. Mechanistic studies suggested that As4S4 acted synergistically with As3+ to down-regulate Bcl-2 and nuclear factor-κB expression, up-regulate Bax and p53 expression, and induce activation of caspase-12 and caspase-3. Moreover, the combination of low concentrations of As4S4 and As3+ enhanced degradation of the promyelocytic leukemia-retinoic acid receptor α oncoprotein. In summary, As4S4 and As3+ synergistically induce the apoptosis and differentiation of NB4 and primary APL cells.

Histone Acetyltransferase Pcaf Is Required for ATRA-Induced Granulocytic Differentiation in Acute Promyelocytic Leukemia Cells

Differentiation therapy with All-trans retinoic acid (ATRA) improves the treatment outcome of acute promyelocytic leukemia (APL), a subtype of acute myeloid leukemia (AML); however, its molecular mechanism remains elusive. We have previously reported that inhibition of NAD-dependent histone deacetylase SIRT2 induces granulocytic differentiation in APL cells (PLOS ONE. 2013; 8(2): e57633), suggesting a possible involvement of protein acetylation in the differentiation of APL cells by ATRA.To assess this possibility, in the present study, we examined expression of major histone-acetyltrasnferases such as GCN5, PCAF, and CBP/p300 upon ATRA-treatment, and found that PCAF-expression was dramatically increased at mRNA (1.8 to 7 fold, Figure 1A) as well as protein levels in NB4, HL-60, and APL primary cells. Consistent with this, PCAF mRNA expression was greatly induced (10 to 40 fold) in the bone marrow of APL patients who received ATRA-containing treatment (Figure 1B). A search in the public expression database revealed that PCAF-expression in bone marrow was generally reduced in not only APL but also other acute hematologic malignancies such as AML and acute lymphoblastic leukemia (ALL) (healthy donor; n = 74, APL; n=37, AML; n=505, ALL; n=750, healthy donor vs. AML, p < 0.001, healthy donor vs. APL, p < 0.001, healthy donor vs. ALL, p < 0.001, adopted from oncomine database). Interestingly, the reduction of PCAF-expression was much more evident in acute than chronic hematologic malignancies such as chronic myeloid leukemia (CML) and myelodysplastic syndrome (MDS) (CML; n = 76, MDS; n=206), suggesting that PCAF reduction is related to a blockade of proper differentiation in malignant cells. Based on these findings we hypothesized that PCAF plays an important role in ATRA-induced APL cell differentiation.To prove this, we performed a loss-of-function assay using lentivirus vectors expressing 3 independent shRNA against PCAF or non-targeting shRNA (control) (Figure 1C). When PCAF was knocked down in HL-60 cells, these cells failed to differentiate into granulocytes with ATRA-treatment (31.9±11.9% vs. 4.9±2.2%, 5.2±0.7%, 8.6±3.7%, CD11b positive cells, control vs. 3 independent PCAF shRNA) (Figure 1C). Moreover, transduction of PCAF shRNA or non-target shRNA to primary APL cells made cells resistant to ATRA-induced granulocytic differentiation (data not shown), suggesting that PCAF is required for the ATRA-induced granulocytic differentiation in APL cells. To further investigate how PCAF promotes APL cell differentiation upon ATRA-treatment, we performed an acetylome analysis to identify a downstream molecule whose activity is regulated by PCAF through acetylation. We pulled-down acetylated proteins using anti-acetylated lysine antibody from cell extracts prepared 3 or 24 hours after either mock- or ATRA-treatment, and identified 5 proteins preferentially acetylated by ATRA-treatment including histone H3, which is a known acetylation target of PCAF. These results strongly support our hypothesis that upon ATRA-treatment, PCAF induction and subsequent acetylation of PCAF substrates promotes APL cell differentiation. More detailed understanding of PCAF-dependent APL cell differentiation may lead to the development of differentiation therapy against not only APL but also other AML. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

In Vivo Analysis of PML-RARA in a Humanized Mouse Model

Acute promyelocytic leukemia (APL) is a subtype of acute myeloid leukemia (AML) characterized by the formation of a PML-RARa fusion protein, which leads to the accumulation of abnormal promyelocytes. Xenograft mouse models with human leukemic cells have advantages for analyzing the human leukemias in vivo, especially for genetic analyses. However, human primary APL cells are difficult to engraft even in very severely immunodeficient mice, such as NOD/shi-SCID IL2Rg-/- (NOG) mice. In order to understand the mechanisms involved in human APL leukemogenesis, we established a humanized in vivo APL model using the transplantation of PML-RARA-transduced CD34+ cells from human cord blood into NOG mice. The expression of PML-RARa in the CD34+ cells disrupted the nuclear bodies in vitro. The clonogenic assay showed that PML-RARa inhibited the total colony formation, but favored the growth of myeloid colonies. When CD34+ cells with PML-RARA were transplanted, they proliferated in the NOG mice for more than three to four months after transplantation (in 24 out of the 34 mice). All 16 mice with more than 3,000 PML-RARA-transduced CD34+ cells were engrafted, while the engraftment was only detected in eight out of 18 mice when the cell density used for transplantation was less than 3,000 cells. These cells possessed abundant azurophilic abnormal granules in the cytoplasm, and some of them had bundles of Auer rods. They expressed CD13, CD33 and CD117, but not HLA-DR or CD34. In addition, the gene expression analysis revealed that these cells and human primary APL were clustered together among various types of AML, suggesting that these induced APL cells well recapitulated human primary APL. Similar to human primary APL, the induced APL cells possessed the ability for myeloid differentiation after treatment with all-trans retinoic acid in vitro and in vivo, and a very low potential for re-transplantation, which was similarly observed in both unsorted induced APL cells and the CD34- fraction. When human cord blood was fractionated before the PML-RARA transduction, the CD34+/CD38+ cells and common myeloid progenitors (CMP) in the CD34+/CD38+ cells led to the efficient development of APL in vivo. These findings demonstrate that CMP is a target for PML-RARA in APL, whereas the resultant CD34- APL cells may share the ability to maintain the leukemia. DisclosuresNo relevant conflicts of interest to declare.

Catechins induced acute promyelocytic leukemia cell apoptosis and triggered PML-RARα oncoprotein degradation.

BackgroundIt has recently been reported that the extracts of green tea polyphenol have cancer preventive effects. In this study, we investigated the effect of the natural composition from green tea leaves Catechins on acute promyelocytic leukemia (APL).MethodsIn vitro, APL cell lines NB4, retinoic acid-resistant NB4-R1 and NB4-R2 were treated with different concentrations of Catechins. Cell viability and cell apoptosis were analyzed using MTT assay and flow cytometric assay, respectively. Expression of proteins related to apoptosis and PML-RARα oncoprotein were assessed by Western blot. In vivo anti-tumor activity of Catechins was examined in nude mice xenografted with NB4 cells and in situ cell apoptosis was detected by terminal deoxytransferase-catalyzed DNA nick-end labeling assay.ResultsCatechins at micromolar concentration levels significantly inhibited APL cell proliferation and induced cell apoptosis, in association with mitochondria damage, ROS production and caspase activation. The anti-apoptotic Bcl-2 family member Bcl-xL was down regulated, with pro-apoptotic member Bax remaining unchanged. Moreover, Catechins induced the degradation of PML-RARα oncoprotein. Catechins-mediated apoptotic effect was also observed in primary APL cells without affecting normal hematopoietic progenitor cells. In the murine xenograft model, Catechins remarkably inhibited tumor growth and induced in situ leukemic cell apoptosis.ConclusionsCatechins might be a potential candidate for APL treatment by activating intrinsic apoptotic pathway and targeting PML-RARα oncoprotein.

Aquaporin 9, a promising predictor for the cytocidal effects of arsenic trioxide in acute promyelocytic leukemia cell lines and primary blasts

A close correlation between the cytocidal effects of arsenic trioxide (ATO) and aquaporin-9 (AQP9) expression levels has been proposed, yet detailed studies are still needed to confirm this association. Thus, in the present study, the correlation between the expression levels of AQP9 and sensitivity to ATO was investigated using two acute promyelocytic leukemia (APL) cell lines, NB4 and HT93A, as well as primary APL cells from newly diagnosed and relapsed APL patients. A substantially higher sensitivity to ATO-mediated induction of apoptosis was observed in the NB4 cells when compared to that in the HT93A cells. In addition, markedly higher expression levels of AQP9, as assessed using flow cytometry, along with more intracellular arsenic accumulation, were observed in the NB4 cells. More importantly, similar to APL cell lines, the trend of expression levels of AQP9 correlated closely with the differential sensitivity to ATO-mediated induction of apoptosis in primary APL cells. In contrast, no correlation was observed between ATO sensitivity associated with AQP9 expression levels and the expression profiles of cell surface markers as well as chromosomal alterations. These results provide direct evidence that the expression levels of AQP9, rather than other biomarkers such as cell surface markers and chromosomal alterations, correlate closely with the sensitivity to ATO in both APL cell lines and primary blasts. These findings suggest that the AQP9 expression status of APL patients is a predictive marker for the successful outcome of ATO treatment, since AQP9 plays a pivotal role in various arsenite-mediated biological effects on normal and cancer cells. Moreover, flow cytometry may be a new convenient and valuable tool for analyzing the AQP9 status of APL patients compared to current methods such as western blotting.

Mesenchymal Stem Cells Induce Granulocytic Differentiation of Acute Promyelocytic Leukemic Cells via IL-6 and MEK/ERK Pathways

All-trans retinoic acid (ATRA) induces clinical remission in most acute promyelocytic leukemia (APL) patients by inducing terminal differentiation of APL cells toward mature granulocytes. Here we report that human umbilical cord-derived mesenchymal stem cells (UC-MSCs) are capable of inducing granulocytic differentiation of the APL-derived NB4 cell line as well as primary APL cells and also cooperate with ATRA in an additive manner. Transwell coculture experiments revealed that UC-MSCs' differentiation-inducing effect was mediated through some soluble factors. Differentiation attenuation by IL-6Ra neutralization and induction by addition of exogenous IL-6 confirmed that IL-6 secreted by UC-MSCs was at least partially responsible for this differentiation induction process. Moreover, we found that UC-MSCs activated the MEK/ERK signaling pathway in promyelocytic cells and pharmacological inhibition of the MEK/ERK pathway reversed UC-MSC-induced differentiation, indicating that UC-MSCs exerted effect through activation of the MEK/ERK signaling pathway. These results demonstrate for the first time a stimulatory effect of MSCs on the differentiation of APL cells and bring a new insight into the interaction between MSCs and leukemic cells. Our data suggest that UC-MSCs/ATRA combination could be used as a novel therapeutic strategy for APL patients.

Notch signaling in acute promyelocytic leukemia

Acute promyelocytic leukemia (APL) is initiated by the PML-RARA fusion oncogene and has a characteristic expression profile that includes high levels of the Notch ligand JAG1. In this study, we used a series of bioinformatic, in vitro, and in vivo assays to assess the role of Notch signaling in human APL samples, and in a PML-RARA knockin mouse model of APL (Ctsg-PML-RARA). We identified a Notch expression signature in both human primary APL cells and in Kit+Lin−Sca1+ (KLS) cells from pre-leukemic Ctsg-PML-RARA mice. Both genetic and pharmacologic inhibition of Notch signaling abrogated the enhanced self-renewal seen in hematopoietic stem/progenitor cells (HSPCs) from pre-leukemic Ctsg-PML-RARA mice, but had no influence on cells from age-matched wildtype mice. In addition, 6 of 9 murine APL tumors tested displayed diminished growth in vitro when Notch signaling was inhibited pharmacologically. Finally, we found that genetic inhibition of Notch signaling with a dominant negative MAML protein reduced APL growth in vivo in a subset of tumors. These findings expand the role of Notch signaling in hematopoietic diseases, and further define the mechanistic events important for PML-RARA-mediated leukemogenesis.

Mechanism of Regulating Fas Signaling by Promyelocytic Leukemia Protein (PML) and Its Dominant-Negative Mutant PMLRARα

Abstract 1327 Objective:Chemotherapies and irradiation depend on an intact Fas signaling system to eradicate cancer cells. Defective Fas signaling is an important cause of acquired resistance to cancer therapy. If we were able to restore Fas apoptosis or sensitize cancer cells to Fas-mediated apoptosis, we could improve the efficacy of many current cancer therapies. To elucidate defects of Fas signaling in cancer cells, we sought to identify potential modulators of Fas selectively expressed in cancers cells and target them to sensitize cancer cells to Fas-mediated apoptosis as a component of chemotherapy. Methods:Liquid chromatography tandem mass spectroscopy was used to identify Fas-associated proteins. Co-immunoprecipitation (co-IP) and Western Blot (WB) were used to detect/confirm interactions of PML and PMLRARα with Fas, and components of death-inducing signaling complex (DISC) FADD, c-FLIP, and caspase-8 cleavage in tissues from PML wild-type (WT) and knock-out (KO) mice and acute promyelocytic leukemia (APL) cells. Deletional mutagenesis was used to map protein interacting domains. PML shRNA lentivirus and As2O3 were used to downregulate PML and PMLRARα. Flow cytometry analysis of propidium iodide- and Annexin-V-stained cells was used to detect apoptosis in response to Fas stimulation. Mice transfected with PMLRARα were monitored for survival after a lethal challenge with agonistic Fas antibody Jo2 and tissues were analyzed for apoptosis by staining for cleaved caspase-3 and TUNEL. Results:The promyelocytic leukemia protein (PML) was identified as a Fas-binding protein by mass spectroscopy analysis. Using co-IP/WB analysis of tissues from PML WT and KO mice, we found PML interaction with Fas and FADD in PML WT MEF cells and liver cells but absent in KO MEF and liver cells; PML-Fas complexes were exclusively present in the membrane/cytoplasmic extracts but not in the nuclear extracts. The B-box domain of PML was found to be required for Fas binding. Knockdown of PML was associated with suppressed rates of Fas-mediated apoptosis compared to non-targeted knockdown cells; PML KO cells reconstituted with cytoplasmic PML were sensitized to Fas apoptosis. Furthermore, we found that liver cells from PML KO mouse showed impaired assembly of the Fas death-inducing signaling complex (DISC) in response to Fas activation when compared to PML WT. PML functions are known to be blocked by its dominant-negative form PMLRARα. We found PMLRARα interaction with Fas in primary human and transgenic mouse APL cells blocked Fas-mediated apoptosis. Blockage of apoptosis was mediated through PMLRARα -dependent recruitment of c-FLIPL/Sto and exclusion of procaspase-8 from the DISC. PMLRARα effects were also observed in vivo, as expression of PMLRARα protected mice against a lethal effect of agonistic anti-Fas antibody (P<.001). Livers from PMLRARα -transfected mice contained fewer cleaved caspase-3 positive/apoptotic cells when compared with control vector-transfected mice. Conclusions:PML binds to Fas and promotes Fas-mediated apoptosis through enhancing Fas DISC formation while binding of PMLRARα to Fas blocks Fas-mediated apoptosis in APL by forming an apoptotic inhibitory complex enriched in c-FLIP. Our data suggest that PML plays a critical role in initiation of Fas signaling; in contrast, the dominant-negative mutant PMLRARα is a confirmed cancer specific inhibitor of Fas-mediated apoptosis. Thus, deficiency of PML or expression of PMLRARα can contribute to cancer development and resistance to chemotherapy. The newly discovered PML-Fas and PMLRARα -Fas complexes can be sites for modulation of apoptosis. Thus, by neutralizing the inhibitory effect of Fas-binding proteins such as PMLRARα and/or promoting positive Fas modulators such as PML, we can improve responses to chemotherapy that depend on activation of death receptors for effective elimination of cancer cells. Disclosures:No relevant conflicts of interest to declare.

Promotion of all-trans retinoic acid (ATRA)-induced differentiation of acute promyelocytic leukemia cells by Bufalin

All-trans retinoic acid (ATRA) has been shown as a promising agent for the treatment of acute promyelocytic leukemia (APL). However, the clinical application of ATRA is limited by the resistance of APL to ATRA-induced differentiation. Bufalin is a steroid isolated from Chan’su and could induce leukemia cell differentiation. This study is aimed to determine whether bufalin treatment could promote ATRA-induced differentiation of APL cell lines and primary cells. The cell surface differentiation marker CD11b, nitro blue tetrazolium (NBT) reduction and morphological changes in the cells were examined. The results showed that bufalin promoted ATRA-induced differentiation of APL primary cells and NB4 cell line, although low dose of bufalin alone had no direct effects on APL cell differentiation. In addition, bufalin and ATRA had synergistic effects to down-regulate survivin expression in NB4 cells. Taken together, these data suggest that bufalin is a potential regimen to be used in combination with ATRA to promote the differentiation of APL cells, which could achieve better therapy efficacy. Key words: Bufalin, all-trans retinoic acid, surviving, acute promyelocytic leukemia,differentiation.

Abstract 4965: Oncoprotein PMLRARα directly suppresses Fas-mediated apoptosis through forming an apoptotic inhibitory complex with c-FLIP in vivo

Abstract Objective: Many genotoxic therapies, including radiation, depend on intact Fas signaling to eradicate cancer cells. Defective Fas signaling is an important cause of cancer resistance to therapy. Restoring Fas apoptosis or sensitizing cancer cells to Fas-mediated apoptosis would improve the efficacy of many cancer therapies. To elucidate a role for specific regulators of Fas signaling in cancer cells, we sought to identify potential modulators of Fas expressed in cancers and target them to selectively sensitize cancer cells to Fas-mediated apoptosis as a component of chemotherapy. Methods: Liquid chromatography tandem mass spectrometry was used to identify Fas-associated proteins; co-immunoprecipitation and Western blot were used to detect interactions of PMLRARα, PML, c-FLIP and Fas, and to examine the components of death-inducing signaling complex (DISC) and caspase-8 cleavage. Deletional mutagenesis was used to map the interaction domains. PML shRNA lentivirus and As2O3 were used to knock down PML and PMLRARα. Flow cytometry analysis of propidium iodide- and Annexin-V-stained cells was used to detect apoptosis. Mice were transfected with PMLRARα, monitored for survival, and tissues were analyzed for apoptosis by staining for cleaved caspase-3 and TUNEL. Results: We identified promyelocytic leukemia protein (PML) as a Fas-interacting protein using mass spectrometry analysis. The function of PML is blocked by its dominant-negative form PMLRARα. We found PMLRARα interaction with Fas in acute promyelocytic leukemia (APL)-derived cells and APL primary cells, and PML-Fas complexes in normal tissues. Binding of PMLRARα to Fas was mapped to the B-box domain of PML moiety and death domain of Fas. PMLRARα blockage of Fas apoptosis was demonstrated in U937/PR9 cells, human APL cells and transgenic mouse APL cells, in which PMLRARα recruited c-FLIPL/S and excluded procaspase-8 from Fas death signaling complex. PMLRARα expression in mice protected the mice against a lethal dose of agonistic anti-Fas antibody (P&amp;lt;.001) and the protected tissues contained Fas-PMLRARα-cFLIP complexes. Livers from PMLRARα-transfected mice contained fewer cleaved caspase-3 positive/apoptotic cells when compared with control vector-transfected mice. Conclusions: PMLRARα binds to Fas and blocks Fas-mediated apoptosis in APL by forming an apoptotic inhibitory complex with c-FLIP. These data suggest that PMLRARα is a cancer specific Fas-binding inhibitor of Fas-mediated apoptosis and thus, can contribute to cancer development and resistance to therapy. The newly discovered PMLRARα-Fas and PML-Fas complexes can be sites for modulation of apoptosis. By neutralizing the effect of death receptor inhibitors, such as PMLRARα, we can improve responses to many chemotherapeutic treatments that depend on activation of death receptors for effective elimination of cancer cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4965. doi:1538-7445.AM2012-4965