All-trans Retinoic Acid Resistant Cell Research Articles

All-Trans-Retinoic Acid Combined With Valproic Acid Can Promote Differentiation in Myeloid Leukemia Cells by an Autophagy Dependent Mechanism.

Acute myeloid leukemia (AML) is an aggressive blood cancer with an overall survival of 30%. One form of AML, acute promyelocytic leukemia (APL) has become more than 90% curable with differentiation therapy, consisting of all-trans-retinoic acid (ATRA) and arsenic trioxide (ATO). Application of differentiation therapy to other AML subtypes would be a major treatment advance. Recent studies have indicated that autophagy plays a key role in the differentiation of ATRA-responsive APL cells. In this study, we have investigated whether differentiation could be enhanced in ATRA resistant cells by promoting autophagy induction with valproic acid (VPA). ATRA sensitive (NB4) and resistant leukemia cells (NB4R and THP-1) were co-treated with ATRA and valproic acid, followed by assessment of autophagy and differentiation. The combination of VPA and ATRA induced autophagic flux and promoted differentiation in ATRA-sensitive and -resistant cell lines. shRNA knockdown of ATG7 and TFEB autophagy regulators impaired both autophagy and differentiation, demonstrating the importance of autophagy in the combination treatment. These data suggest that ATRA combined with valproic acid can promote differentiation in myeloid leukemia cells by mechanism involving autophagy.

Genetic Ablation of Nuclear Receptor Interacting Protein 1 (NRIP1) Sensitizes Acute Myeloid Leukemia Cells to Retinoic Acids

The success of differentiation therapy is limited to acute promyelocytic leukemia (APL), and approaches to overcome the differentiation block in non-M3 AML have been unsuccessful. Nuclear hormone receptors (NHR) belong to ligand-inducible transcription factors that govern many cellular functions like differentiation, metabolism, and development. Retinoic Acid Receptor Alpha (RXRA) is a class of NHR that, when activated by all-trans retinoic acid (ATRA), successfully alleviates differentiation block in APL.To identify the NHRs/cofactors that could mediate or prevent differentiation in AML, we examined the differentially expressed NHRs and cofactors between ATRA sensitive (ATs) (NB4 and HL60) vs. ATRA resistant (ATr) AML cell lines (KG1a, Hel, K562, MV4-11, and OCI-AML3). Nuclear Receptor Interacting Protein 1 (NRIP1), a corepressor known to prevent transactivation of ligand-activated NHRs preferentially, was one of the top upregulated targets in the ATr cell lines (3.5 fold increase in RNA expression, figure 1a ). Immunoblot analysis also showed a significant increase in NRIP1 protein expression in the ATr than ATs cell lines (Figure 1b). Further, probing for NRIP1 expression in the publicly available TCGA and MILE AML study cohorts showed decreased NRIP1 expression in the APL cohort compared to other AML subtypes. Methylation profile from CCLE database of the NRIP1 promoter in AML cell lines showed ATs cell lines to be highly methylated compared to the ATr cell lines, suggesting the involvement of NRIP1 in mediating differentiation block in non-M3 AML (Figure 1c). To further dissect the role of NRIP1 in mediating this differentiation block, we carried out experiments in the AML cell line KG1a (having primitive blast features, high expression of NRIP1, and unresponsive to ATRA). Using CRISPR-cas9, we developed an NRIP1 knock-out (KO) cell line (Figure 1d). NRIP1 KO cell line showed a significant reduction in proliferation rate (Doubling time 26.2 vs. 36.5Hrs p<0.05). Further, cell cycle analysis revealed that NRIP1 KO leads to increased accumulation of cells in the G0 phase than in the S-phase (Figure 1e & f). We next assessed the sensitivity of the NRIP1 WT/KO cells to retinoic acids ATRA and bexarotene. Cells were treated with 1µM ATRA / bexarotene or in combination for 72 hours and evaluated for differentiation using CD11b marker by flow cytometry. NRIP1 KO alone leads to a marginal increase in basal CD11b expression compared to the WT cells (Mean CD11b expression 2.03% Vs 0.91%). ATRA treatment further increased the CD11b expression to 3.8% in KO cells compared to 1.6% in the WT cells. A similar increase in CD11b expression was observed in bexarotene-treated cells (3.7% Vs 1.24%). Combination of ATRA with bexarotene showed a 3-fold increase in CD11b expression in the KO cells compared to the WT (23.9% Vs 7.2%, Figure 1g). NRIP1 KO diminishes its repressive action on ligand-activated RARA (ATRA activated) and RXRA (Bexarotene-activated), thereby allowing synergistic differentiation induction by retinoic acids in AML cells.This study suggests a potential mechanism of differentiation inhibition mediated by corepressor NRIP1 in AML cells unresponsive to retinoic acids. Further in-depth analyses of molecular pathways governed by NRIP1 during ligand activation of NHRs are warranted to design differentiation therapies for AML. [Display omitted] DisclosuresMathews: Christian Medical College: Patents & Royalties: US 2020/0345770 A1 - Pub.Date Nov.5, 2020; AML: Other: Co-Inventor.

2-Bromopalmitate targets retinoic acid receptor alpha and overcomes all-trans retinoic acid resistance of acute promyelocytic leukemia.

Fatty acid oxidation dependency of leukemia cells has been documented in recent studies. Pharmacologic inhibition of fatty acid oxidation, thereby, displays significant effects in suppressing leukemia. 2-Bromopalmitate, a palmitate analogue, was initially identified as an inhibitor of fatty acid oxidation, and recently recognized as an inhibitor of protein palmitoylation. However, the effects of 2-Bromopalmitate on leukemia and its cellular targets remain obscure. Herein, we discover in cultured cell lines, a transplantable mouse model, and primary blasts that 2-Bromopalmitate presents synergistic differentiation induction with all-trans retinoic acid in acute promyelocytic leukemia. Moreover, 2-Bromopalmitate overcomes all-trans retinoic acid resistance in all-trans retinoic acid-resistant cells and leukemic mice. Mechanistically, 2-Bromopalmitate covalently binds at cysteine 105 and cysteine 174 of retinoic acid receptor alpha (RARα) and stabilizes RARα protein in the presence of all-trans retinoic acid which is known to induce RARα degradation, leading to enhanced transcription of RARα-target genes. Mutation of both cysteines largely abrogates the synergistic effect of 2-Bromopalmitate on all-trans retinoic acid-induced differentiation, demonstrating that 2-Bromopalmitate promotes all-trans retinoic acid-induced differentiation through binding RARα. All-trans retinoic acid-based regimens including arsenic trioxide or chemotherapy, as preferred therapy for acute promyelocytic leukemia, induce adverse events and irreversible resistance. We expect that combining all-trans retinoic acid with 2-Bromopalmitate would be a promising therapeutic strategy for acute promyelocytic leukemia, especially for overcoming all-trans retinoic acid resistance of relapsed acute promyelocytic leukemia patients.

Dasatinib synergizes with ATRA to trigger granulocytic differentiation in ATRA resistant acute promyelocytic leukemia cell lines via Lyn inhibition-mediated activation of RAF-1/MEK/ERK

All-trans retinoic acid (ATRA) resistance has been a critical problem in acute promyelocytic leukemia (APL) relapsed patients. In this study, dasatinib synergized with ATRA to trigger differentiation in ATRA-resistant APL cell lines. The combined treatment activated RAF-1, MEK and ERK as well as enhanced ATRA-promoted up-regulation of the protein level of PU.1, C/EBPβ and C/EBPε. U0126 (MEK specific inhibitor) and sorafenib tosylate (RAF-1 specific inhibitor) suppressed the combined treatment-induced differentiation, ERK phosphorylation and the up-regulation of C/EBPs and PU.1. Sorafenib tosylate also attenuated the MEK activity. However, the combined treatment did not enhance Ras activity and Ras inhibitor neither blocked MEK activation nor inhibited differentiation. Therefore, the combined treatment induced differentiation via Ras independent RAF-1/MEK/ERK. Earlier than RAF-1 activation, dasatinib suppressed Lyn activity, the predominant activated Src family kinase (SFK) and dephosphorylated RAF-1 at S259. Furthermore, SFK inhibitor, PP2 did suppress Lyn activity and mimicked the effect of dasatinib on ATRA-induced differentiation as well as decreased phosphorylation of RAF-1 at S259. Thus, it was suggested that Lyn inhibition might activate RAF-1 by the dephosphorylation of RAF at S259 and lead to differentiation. In conclusion, the combination of dasatinib and ATRA could overcome ATRA resistance through Lyn inhibition-mediated activation of RAF-1/MEK/ERK.

Abstract B37: hnRNP K in acute promyelocytic leukemia cells NB4 and NB4-R2: Involvement in cell survival and differentiation

Abstract hnRNP K has been reported to be overexpressed in myeloid leukemia and it is downregulated during all-trans retinoic acid (ATRA) induced differentiation of Acute Promyelocytic Leukemia (APL) cells. The aim of the present study was to establish role of hnRNP K in APL using responsive (NB4) and resistant (NB4-R2) cells to the all-trans retinoic acid treatment as well as the possible role of hnRNPK in ATRA-induced cellular differentiation. After demonstration, by Western blotting analysis, that hnRNP K is overexpressed (10 fold) in APL (NB4 and NB4-R2) cell lines compared to controls (bone marrow and polymorphonuclear cells), we knocked-down the protein by short hairpin RNA (shRNA) interference in these cell lines and assessed involvement of hnRNP K in ATRA-induced differentiation, by flow cytometry in cells incubated with Cd11bPE antibody, as well as the contribution of hnRNP K to proliferation, with growth curve, using Trypan Blue, and survival, by propidium iodide incorporation analysis in flow cytometer. The knockdown of hnRNP K in NB4 (72%) and NB4-R2 (60%) leads to loss of viability mainly in NB4-R2 (42,43% of viability) vs NB4 (75,33% of viability). The loss of viability was more accentuated after ATRA treatment, NB4-R2 (6,13% of viability) vs NB4 (70% of viability). hnRNP K knockdown increases APL cells differentiation and was more accentuated in association with ATRA treatment, NB4-R2 (+59,13%) vs NB4 ( +14,06%). Furthermore, knockdown of hnRNP K leads to decreased in proliferation in NB4 (-95,98%) and NB4-R2 (-97,5%). hnRNP K knockdown promotes decrease of Bcl-xl, PML-RARα, procaspase-3 and increases of SET protein levels in both cells, demonstrated by Western blotting. The extracellular signal-regulated kinase ERK is a positive hnRNP K regulator, so the treatment with the ERK inhibitor at 10 μM U0126 produces effects similar to hnRNP K knockdown on cell viability in NB4-R2 cells, and in levels of BCL-xl and SET proteins. We concluded that, hnRNP K knockdown decreases the cell proliferation and viability of NB4 and NB4-R2 cells and increases cell differentiation, via MEK-ERK-hnRNP K-SET signaling. hnRNP K represent a potential target in APL ATRA resistant cells. Citation Format: Karina Stringhetta Padovani, María Sol Brassesco Annichini, Eduardo Magalhães Rego, Carlos Curti, Lewis Joel Greene, Andréia Machado Leopoldino. hnRNP K in acute promyelocytic leukemia cells NB4 and NB4-R2: Involvement in cell survival and differentiation. [abstract]. In: Proceedings of the AACR Special Conference: Developmental Biology and Cancer; Nov 30-Dec 3, 2015; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(4_Suppl):Abstract nr B37.

Restoration of CCAAT Enhancer Binding Protein Alpha P42 Overcomes Myeloid Differentiation Block and All-Trans Retinoic Acid Resistance in Human Acute Promyelocytic Leukemia NB4-R1 Cells

All-trans retinoic acid (ATRA) is one of the first line agents in differentiation therapy for acute promyelocytic leukemia (APL). However, drug resistance becomes a major problem which influences the efficacy of ATRA. Mechanisms of ATRA resistance are ugly needed to be identified. Here we found that expression of C/EBPα, an important transcription factor for myeloid differentiation, was significantly suppressed in ATRA resistant APL cell line NB4-R1 compared with ATRA sensitive NB4 cells. Moreover, two forms of C/EBPα were unequally suppressed in NB4-R1 cells. Suppression of the full-length form P42 was more pronounced than the truncated form P30. Inhibition of PI3K/Akt/mTOR pathway was also observed in NB4-R1 cells. Meanwhile, C/EBPα expression was reduced by PI3K inhibitor LY294002 and mTOR inhibitor RAD001 in NB4 cell, suggesting that inactivation of the PI3K/Akt/mTOR pathway was responsible for C/EBPα suppression in APL cells. Then, we restored C/EBPα P42 and P30 by lentivirus vectors in NB4-R1 cells respectively, and found C/EBPα P42, but not P30, could increase CD11b, CD14, G-CSFR and GM-CSFR expression, which indicated the occurrence myeloid differentiation. Moreover, further upregulating of CD11b expression and differential morphological changes were found in NB4-R1 cells with restored C/EBPα P42 after ATRA treatment. However, CD11b expression and differential morphological changes could not be induced by ATRA in NB4-R1 cells infected with P30 expressing or control vector. Thus, we inferred that ATRA sensibility of NB4-R1 cells was enhanced by restoration of C/EBPα P42. In addition, we used histone deacetylase inhibitor trichostatin (TSA) to restore C/EBPα expression in NB4-R1 cells. Similar enhancement of myeloid differentiation and cell growth arrest were detected. Together, this study demonstrated that suppression of C/EBPα P42 induced by PI3K/Akt/mTOR inhibition impaired the differentiation and ATRA sensibility of APL cells. Restoring C/EBPα P42 is an attractive approach for differentiation therapy in ATRA resistant APL. DisclosuresNo relevant conflicts of interest to declare.

Restoration of CCAAT enhancer binding protein α P42 induces myeloid differentiation and overcomes all-trans retinoic acid resistance in human acute promyelocytic leukemia NB4-R1 cells.

All-trans retinoic acid (ATRA) is one of the first line agents in differentiation therapy for acute promyelocytic leukemia (APL). However, drug resistance is a major problem influencing the efficacy of ATRA. Identification of mechanisms of ATRA resistance are urgenly needed. In the present study, we found that expression of C/EBPα, an important transcription factor for myeloid differentiation, was significantly suppressed in ATRA resistant APL cell line NB4-R1 compared with ATRA sensitive NB4 cells. Moreover, two forms of C/EBPα were unequally suppressed in NB4-R1 cells. Suppression of the full-length form P42 was more pronounced than the truncated form P30. Inhibition of PI3K/Akt/mTOR pathway was also observed in NB4-R1 cells. Moreover, C/EBPα expression was reduced by PI3K inhibitor LY294002 and mTOR inhibitor RAD001 in NB4 cells, suggesting that inactivation of the PI3K/Akt/mTOR pathway was responsible for C/EBPα suppression in APL cells. We restored C/EBPα P42 and P30 by lentivirus vectors in NB4-R1 cells, respectively, and found C/EBPα P42, but not P30, could increase CD11b, CD14, G-CSFR and GM-CSFR expression, which indicated the occurrence of myeloid differentiation. Further upregulating of CD11b expression and differential morphological changes were found in NB4-R1 cells with restored C/EBPα P42 after ATRA treatment. However, CD11b expression and differential morphological changes could not be induced by ATRA in NB4-R1 cells infected with P30 expressing or control vector. Thus, we inferred that ATRA sensitivity of NB4-R1 cells was enhanced by restoration of C/EBPα P42. In addition, we used histone deacetylase inhibitor trichostatin (TSA) to restore C/EBPα expression in NB4-R1 cells. Similar enhancement of myeloid differentiation and cell growth arrest were detected. Together, the present study demonstrated that suppression of C/EBPα P42 induced by PI3K/Akt/mTOR inhibition impaired the differentiation and ATRA sensitivity of APL cells. Restoring C/EBPα P42 is an attractive approach for differentiation therapy in ATRA resistant APL.

Combined staurosporine and retinoic acid induces differentiation in retinoic acid resistant acute promyelocytic leukemia cell lines.

All-trans retinoic acid (ATRA) resistance has been a critical problem in acute promyelocytic leukemia (APL) relapsed patients. In ATRA resistant APL cell lines NB4-R1 and NB4-R2, the combination of staurosporine and ATRA synergized to trigger differentiation accompanied by significantly enhanced protein level of CCAAT/enhancer binding protein ε (C/EBPε) and C/EBPβ as well as the phosphorylation of mitogen-activated protein (MEK) and extracellular signal-regulated kinase (ERK). Furthermore, attenuation of the MEK activation blocked not only the differentiation but also the increased protein level of C/EBPε and C/EBPβ. Taken together, we concluded that the combination of ATRA and staurosporine could overcome differentiation block via MEK/ERK signaling pathway in ATRA-resistant APL cell lines.

Abstract B9: Critical Early microRNA-Mediated Signalling Events During ATRA Induced Neuroblastoma Cell Differentiation

Abstract Introduction: Neuroblastoma (NB) is a childhood cancer arising from precursor cells of the sympathetic nervous system with highly heterogeneous clinical behavior, ranging from spontaneous regression to rapid progression and death due to disease. Amplification and over-expression of the MYCN transcription factor in neuroblastoma tumors is highly correlated with poor survival of patients. Children with high risk NB are given the synthetic retinoic acid, 13-cis-retinoic acid, to reduce minimal residual disease, and exposure of a number of NB cell lines to the related compound, all-trans-retinoic acid (ATRA), induces neural differentiation along with the down-regulation of MYCN. We previously determined that miRNA-10a/b plays a critical role in the process of ATRA induced neuroblastoma cell differentiation through direct targeting of nuclear co-repressor receptor 2 (NCOR2) (Foley et al Cell Death &amp; Differentiation 2011: 18:1089). Here, we have profiled three ATRA sensitive (SK-N-BE, LAN5 and SHSY-5Y) and two ATRA resistant (Kelly and SK-N-AS) neuroblastoma cell lines for the expression of 750 miRNA loci at early (3 hours) and late (7 days) time points following ATRA treatment. The purpose of our experiments was to identify critical early miRNA signaling events, as well as miRNAs that might mediate sensitivity versus resistance to ATRA. Methods: All cells were treated with 5 μmol ATRA (Sigma, St. Louis, MO) which was continuously administered by replacing the medium every 24 hrs. MiRNA expression profiling was carried out using TaqMan Low Density Arrays from Applied BioSystems (Carlsbad, CA). Results: miR-10a, -34a, -125b, -28-5p, and -183 were consistently up-regulated and miR-450-5p, -151-3p and -138-2 were consistently down-regulated &amp;gt;2 fold (p &amp;lt; 0.05) in all three ATRA sensitive cell lines at three hours post-ATRA. At 7 days post-ATRA, there were more differentially expressed miRNAs, with 13 miRNAs being up-regulated and 18 down-regulated &amp;gt; 2 fold (p &amp;lt; 0.05) across all three cell lines. Among the miRNAs differentially expressed at 3 hours post-ATRA, miR-10a, miR-125b and miR-34a remained differentially expressed at 7 days post-ATRA. Remarkably, none of the differentially expressed miRNAs in the ATRA sensitive cell lines were differentially expressed in the ATRA resistant cell lines at the three hour time point, and very few displayed altered expression in the ATRA resistant cell lines even after a 7 day exposure to ATRA. qPCR and Western blot analysis of the critical miR-10a target, NCOR2, revealed that this gene is down regulated at both mRNA and protein level at the 3 hour time point. In addition to the targeting of NCOR2 by miR-10a, we noted that two additional up-regulated miRNAs, miR-125b and miR-132 (significantly up-regulated within 7 days post-ATRA) are also predicted to target NCOR2, indicating substantial redundancy in miRNA mediated signaling. Direct targeting of NCOR2 by these additional miRNAs was experimentally validated by luciferase reporter assays. This redundancy in miRNA targeting explains why antagomir inhibition of single miRNAs prior to ATRA treatment only delays, but does not block the induction of differentiation. Finally, we present evidence that an initial early decrease in MYCN levels, which is well known to occur in neuroblastoma cells treated with ATRA, is miRNA mediated. Conclusions: We have identified critical and redundant early miRNA signaling events that take place during the process of ATRA induced neuroblastoma cell differentiation. The absence of these events in ATRA resistant cell lines suggests new avenues of investigation for elucidating potential miRNA mediated mechanisms involved with ATRA therapy failure. Citation Format: Niamh H. Foley, Isabella M. Bray, Raquel Fernandez, Raymond L. Stallings. Critical Early microRNA-Mediated Signalling Events During ATRA Induced Neuroblastoma Cell Differentiation [abstract]. In: Proceedings of the AACR Special Conference on Noncoding RNAs and Cancer; 2012 Jan 8-11; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Res 2012;72(2 Suppl):Abstract nr B9.

Discovery of Critical Functions of Histone Demethylase, PHF8, in Mediating ATRA Response in APL

Abstract 226 Introduction:Transcription deregulation plays a key role in acute leukemogenesis, which is mostly initiated by chimeric transcription factors such as PML–RARa that accounts for almost 100% of acute promyelocytic leukemia (APL). While APL is uniquely sensitive to all trans retinoic acid (ATRA) treatment and has been the paradigm of differentiation/epigenetic therapies, the underlying mechanisms remain largely unknown and are major interests in the field with a potential of extending the success to other hematological malignancies. Previously, others and we have shown that aberrant recruitment of histone deacetylases and polycomb repressive complexes by oligomeric PML-RARa are key for suppression of its downstream targets and promote cellular transformation. ATRA treatment disrupts their bindings and results in de-repression of target genes. However, the identification of the co-activator complex responsible for gene activation upon ATRA treatment remains a major hurdle that significantly hinders the progress of understanding mechanisms underlying the epigenetic therapy. Results and Discussion:Given the critical functions of JmjC-domains containing histone demethylases in mediating transcriptional regulation, we performed a systematic screening for differential interaction between JmjC-histone demethylases and PML–RARa upon ATRA treatment. We identified a highly specific interaction between PML–RARa and Plant Homeodomain Finger 8 (PHF8). To assess its effect on histone methylation, we detected specific reduction of H3K9me2 and enhancement of H3K4me3 in PHF8 transfected cells, consistent with its function as a transcriptional activator. This was further supported by results from chromatin immuno-precipitation (ChIP) assays in human APL cell line (NB4) showing that PHF8 differentially bound and mediated the same histone modifications in RARb promoter and activated its expression. To investigate the functional significance of this interaction, PHF8 was expressed in human NB4 cells harboring PML-RARa or murine primary bone marrow cells transformed by APL fusion proteins. Induction of PHF8 expression significantly decreased their in vitro transformation capacity in the presence of physiological concentrations of ATRA. Conversely, specific down-modulation of PHF8 expression by shRNAs reduced ATRA sensitivity of these cells, suggesting a critical function of PHF8 in mediating ATRA response. We hypothesized that PHF8 may be able to sensitize ATRA resistant cells to the treatment. Hence, we induced expression of PHF8 in the ATRA resistant variant of NB4 line, NB4-MR2. We were able to demonstrate that PHF8 sensitized NB4-MR2 cells to ATRA treatment in vitro. In contrast, NB4-MR2 cells expressing PHF8-F279S, a catalytically inactive mutant could not be sensitized to ATRA treatment, indicating that the enzymatic activity is critical for mediating the ATRA response. Most importantly, NB4-MR2 cells expressing wild type PHF8 were also sensitive to ATRA treatment in vivo and failed to induce leukemia in NOD/SCID mice, which would otherwise succumb to leukemia in a very brief latency.To gain further insights into the molecular regulation of PHF8 in ATRA response, we characterized the potential functions of CDK1-mediated phosphorylation of PHF8. It is known that in ATRA treated leukemic cells Cyclin A translocates into the nucleus where it interacts with CDK1. Activated CDK1 induces phosphorylation of PHF8 at two serine (S33)/ threonine (T84) phosphorylation sites. Our results showed that PHF8 in its phosphorylated form had a much higher binding affinity to PML-RARa. Consistently, ChIP analyses revealed that the binding of PHF8 to the RARb promoter and the resultant activation were significantly augmented when PHF8 was constitutively phosphorylated. Moreover, inhibition of PHF8 dephosphorylation by Okadaic Acid, sensitized NB4-MR2 cells to ATRA treatment. Conclusions:We discovered a novel function of the histone demethylase PHF8 in mediating therapeutic response in both ATRA sensitive and ATRA resistant cells. This function of PHF8 is critically dependent on two phosphorylation sites as well as its histone modification activity. Thus, therapeutic interventions such as phosphatase inhibitors that enhance the PHF8 activity might become useful tools for development of new epigenetic therapies sensitizing leukemic cells to ATRA treatment. Disclosures:No relevant conflicts of interest to declare.

Design, Synthesis and Biological Evaluation of A Boron Containing Retinoid As a Novel Therapeutic Agent for Acute Promyelocytic Leukemia

Abstract 5008Cure rates of acute leukemia remain poor emphasizing the need to for novel therapies. All trans retinoic acid (ATRA) is an effective therapeutic agent in a subtype of acute myeloid leukemia and relieves transcriptional repression induced by the PML-RAR oncoprotein by binding to the retinoic acid receptor. Even though ATRA is effective, the treatment course is characterized by a high rate of toxicity and ATRA resistance is seen in some cases of acute promyelocytic leukemia. In an attempt to improve outcomes, we devised a methodology for creation of boronic acid and other newer retinoic acid analogues. Our lead compound, MA-21 was generated by replacing the terminal carboxyl group of ATRA with a boronic ester using Wittig reactions. Computation modeling revealed that MA-21 can fit in the RARa pocket and can form increased covalent bonding with cysteine residues within the receptor. As opposed to other synthetic retinoids, the addition of a boron atom resulted in significantly enhanced cytotoxicity in leukemic cell lines, even those that were resistant to ATRA. MA-21 at 1uM dose led to significant reduction in proliferation of ATRA sensitive NB4 APL cell line (1.8 fold decrease after 96hrs, p= 0.028) as well as in ATRA resistant cell lines NB4.007/6 (3.3 fold decrease after 72hrs and 2.1 decrease after 96hrs, p values of 0.018 and 0.046) and NB4.306 (2.6 fold decrease after 96hrs, p= 0.032). MA-21 was able to induce these effects by inducing significant G2/M cell cycle arrest and not by increased apoptosis or cellular differentiation. Cell cycle was assessed by Flow Cytometry after 96hrs of incubation and showed a significant increase in G2/M percentage in the ATRA sensitive and resistant cell lines compared to DMSO (NB4 cell line- 1.35 fold increase, p= 0.035; NB4.007/6- 1.35 fold increase, p= 0.015 and NB4.306- 2 fold increase, p= 0.023). Thus, we demonstrate novel synthetic methodology to synthesize boron containing novel retinoids and demonstrate the potential of these compounds as therapeutic agents in resistant leukemias. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Effect of Matrine on Alleviating Retinoic Acid Resisted Acute Promyelocytic Leukemia

AbstractAbstract 4710Acute promyelocytic leukemia (APL) represents 3.3%-17.4% of acute leukemia in adults. With the application of all-trans retinoic acid (ATRA), more than 85% patient can achieve complete remission, however, high rates of relapse still exist if ATRA is used alone. Primary resistance of ATRA in PML-RARα positive APL is rare during induction therapy but the incidence markedly increases after second relapse and results in poor long time survival of patient. Matrine (MAT) is the effective constituent of Radix Sophorae Flavescentis, a commonly used traditional chinese drug, and has proven ability to inhibit carcinoma cell proliferation and can induce differentiation and apoptosis of K562 cells, however its effect on the differentiation of APL cells, especially on ATRA resistant APL cells is unclear. The aim of our research is to explore the effect of MAT on alleviating retinoic acid resistance in APL cells and address possible mechanisms involved. The characteristics of ATRA sensitive APL cell strain NB4 and resistant APL cell strains NB4-R1 and NB4-R2 were described in our former studies. Dose escalation studies using MAT and ATRA was investigated using methyl thiazolyl tetrazolium (MTT) test. The toxicity of MAT increases with increasing concentration of MAT from 0.001 mmol/L to 10 mmol/L, and the 0.1mmol/L was chosen with its inhibition ratio < 10%. IC50 of ATRA was obtained by drawing the restrain curve; the resistance coefficient (RC) of NB4-R1 and NB4-R2 to ATRA was obtained by the ratios of their IC50 to that of NB4 cell, respectively. And the reversal index (RI) was obtained by the ratios of RC of resistant cell line cells cultured with or without 0.1 mmol/L MAT for 72h. Cell differentiation was evaluated by nitro blue tetrazolium chioride (NBT) test and cell morphology. Differential effects on cell differentiation between MAT combined with 1 umol/L ATRA and controls were observed. For these studies, 1umol/L of ATRA was chosen according to the result of IC50 test, which had low toxicity to resistance cell line cells, with the inhibition ratio < 10%. Apoptosis rate of cells treated with MAT combined with 1 umol/L ATRA was tested by flow cytometry with Annexin V-PI staining. Nested PCR was used for PML/RARa PCR reaction. Our results showed, after treated with ATRA along with 0.1 mmol/L MAT, the resistance factor of NB4-R1 to ATRA decreased markedly (RI=4.96±1.15), but no difference was shown in NB4-R2 cells (RI=0.66±0.17). The positive ratio of NBT and the morphologic changes indicated that the differentiation ability of NB4 and NB4-R1was enhanced significantly with the increasing concentration of MAT combined with 1 umol/L ATRA, which reached the peak at 0.1mmol/L (Figure). The apoptosis ratio (Annexin V-FITC kit) of NB4 and NB4-R1 were also increased with increasing concentration of MAT combined with 1 umol/L ATRA, while no changes were observed using the NB4-R2 cell line. The expression of PML-RARα fusion became weak in NB4 and NB4-R1 cells after treated with 0.1 umol/L ATRA alone, and decreased notably when co-treated with 0.1 mmol/L MAT for 72h in NB4-R1, while the fusion gene expression remained negative in NB4-R2 cells exposed to both drugs. From this study, we conclude that MAT, when used together with ATRA as a low non-toxic dose, can improve the differentiation of NB4 cells and inverse the retinoic acid resistance of NB4-R1 cells, but it has no function on NB4-R2 cells. As the mechanism of ATRA resistance in NB4-R1 and NB4-R2 cells is different, with resistance to the former ascribed to abnormal cyclic adenosine monophosphate (cAMP) mediated pathways, leading to altered differentiation of NB4-R1 which can be corrected when cells are cocultured with cAMP and ATRA, and resistance of NB4-R2 is owing to the variation of PML-RARα, which can only be alleviated in the existence of RXR receptor specific agonist, MAT may play its anti-ATRA resistance role in an ATRA dependent pathway, and lead to the inhibition of the PML-RARα fusion gene. This indicates a possible application of MAT in ATRA resistance therapy. Further research will focus on the mechanism of MAT in the regulation and differentiation as well as apoptosis of APL cells. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

The novel atypical retinoid ST1926 is active in ATRA resistant neuroblastoma cells acting by a different mechanism

E-3-(4′-Hydroxy-3′-adamantylbiphenyl-4-yl)acrylic acid (ST1926) is a novel orally available compound belonging to the class of synthetic atypical retinoids. These agents are attracting growing attention because of their unique mechanism of antitumor action that appears different from that of classical retinoic acid. This study aims at investigating the antitumor activity of ST1926 in neuroblastoma (NB) preclinical models. In vitro, ST1926 was more cytotoxic than both its prototype, CD437 and all- trans-retinoic acid (ATRA) and it was active in the SK-N-AS cell line, which is refractory to ATRA. We showed that unlike ATRA, ST1926 does not induce morphological differentiation in NB cells where it produces indirect DNA damage, cell cycle arrest in late S-G2 phases and p53-independent programmed cell death. DNA damage was not mediated by oxidative stress and was repaired by 24 h after drug removal. The SK-N-DZ cell line appeared the most sensitive to the proapoptotic activity of ST1926, probably because both the extrinsic and intrinsic pathways appear involved in the process. Studies with Z-VAD-FMK, suggested that ST1926 might also mediate caspase-independent apoptosis in NB cells. In vivo, orally administered ST1926, appeared to inhibit tumor growth of NB xenografts with tolerable toxicity. Overall, our results support the view that ST1926 might represent a good drug candidate in this pediatric tumor.

급성 전골수성 백혈병 세포주간의 삼산화비소에 대한 반응

급성 전골수성 백혈병은 염색체 전위의 결과로 생긴 PML/RAR<TEX>$({\alpha})$</TEX> 융합 단백의 과발현으로 영향을 받은 전골 수세포의 분화 정지로 발생하는 골수성 백혈병의 일종이다. 삼산화 비소는 세포고사를 유발하여 급성전골수성 백혈병의 관해를 유도한다는 것이 밝혀졌으나 이 약제에 대한 감수성이 다양하여 고형암에 적용하기에는 제한점이 있다. All-trans-retinoic acid (ATRA)에 감수성인 NB4 세포주와 내성인 UF-1 세포주 모두에 삼산화 비소가 세포고사를 유도하였다. 백혈병 세포주를 삼산화 비소로 처리하여, 세포내 GSH 농도가 낮아지고 세포고사의 감수성이 높아지는 상관관계를 찾았으며 전골수성 암세포를 수지상 세포 표면 표식자를 가진 세포로 분화시켰다. ATRA에 대한 감수성인 세포주와 내성인 세포주의 삼산화 비소에 대한 반응의 차이를 이해하고, 전골수 세포가 수지상 세포로 분화하는 과정을 규명한다면, 삼산화 비소에 의한 전골수성 백혈병의 완전관해의 기전을 밝힐 수 있고 또한 임상적용을 확대할 수 있을 것이다. Acute promyelocytic leukemia (APL) is a myeloid leukemia caused by over-expression of fusion protein, PML/RAR<TEX>$({\alpha})$</TEX>, which was the result of chromosomal translocation and induces the blockage of differentiation of affected promyelocytes. Pharmacological dose of retinoic acid induces the activation of and subsequent degradation of PML/RAR<TEX>$({\alpha})$</TEX> fusion protein, and then APL cells undergo through the normal differentiation pathway. Arsenic trioxide has proved effective in causing remission of acute promyelocytic leukemia by inducing apoptosis of this tumor cells, whereas the heterogeneity of cellular susceptibility to this cytotoxic agent limited its usage on more types of tumors in clinic. This work showed that arsenic trioxide could induce apoptosis of a panel of acute promyelocytic leukemic cell lines, all-trans-retinoic acid (ATRA) sensitive NB4 cells and ATRA resistant UF-1 cell. They were investigated with regard to the correlation between the inherent or intrinsic cellular level of GSH and the apoptotic susceptibility of the cells to arsenic trioxide. We manifested, in two cell types, the inherently existed difference in intracellular GSH level reactive to the arsenic trioxide, and a positive correlation between the GSH level and their apoptotic sensitivity to arsenic trioxide. And it showed that arsenic trioxide could differentiate promyelocytic cancer cells to the cells possessed of dendritic cell surface markers. Unravelling the cause of the different susceptibility between leukemic cells and proving that promyelocyte could be differentiated to dendritic cells by arsenic trioxide will help not only to understand the mechanism underlying the complete remission of acute promyelocytic leukemia induced by arsenic trioxide, but also to expand its clinical usage.

Histone Deacetylase 3 (HDAC3), a Component of N-CoR-TBL1/R1 Chromatin Remodeling Co-Repressor Complex, Is Recruited to the Target Gene Promoters by PML-RARα To Repress the Transcription In Vivo.

PML-RARα is a chimeric transcription factor deeply associated with acute promyelocytic leukemia (APL). PML-RARα plays an important role in the aberrant transcription repression on the target genes of wild type retinoic acid receptors (RARα). Pharmacological concentration of all-trans retinoic acid (ATRA) induces transcription de-repression on several target genes, and results in terminal differentiation of APL cells. However, the detailed mechanisms of transcription repression by PML-RARα in vivo are still unclear. Here we demonstrated that histone deacetylase 3 (HDAC3), one component of the N-CoR (nuclear receptor co-repressor)-TBL1/R1 (transducin beta-like protein 1/relating protein) transcription repressor protein complex, is a key regulator of the transcription repression by PML-RARα in vivo. Using immunoprecipitation (IP) assay, we first demonstrated that PML-RARα physically interacted with N-CoR/HDAC3 in vivo in the absence of ligand. The interaction was dissociated by adding ATRA in the dose dependent manner. Next we showed, using chromatin immunoprecipitation (ChIP) assay, that N-CoR/HDAC3 co-repressor complex was recruited to the endogenous target gene promoters (RARβ and CYP26) through PML-RARα. The neighboring histone H4 was de-acetylated and the gene expression was significantly repressed. When HDAC3 protein is knocked down by RNA interference in PML-RARα-presenting cells, the endogenous target gene expression was significantly activated. Almost the same results were also obtained when performing the luciferase reporter assay using RARβ and CYP26 promoter reporter vectors. Previously, we have shown that N-CoR-TBLR1 is recruited to the target gene promoter through PML-RARα in the absence of ligand, resulting in the transcription repression. Consistent with these data, it is strongly suggested that N-CoR/HDAC3/TBLR1 co-repressor complex is closely related to the aberrant transcription regulation by PML-RARα in APL cells. Furthermore, we also confirmed that PLZF-RARα, which is expressed in ATRA resistant APL cells, interacted with N-CoR/HDAC3/TBLR1 in ligand independent manner. These insights provide not only the basic mechanism of transcription repression by leukemia-related chimeric transcription factors, but also the new molecular targets for the transcription therapy for leukemia.

Novel Retinoic Acid Metabolism Blocking Agents (RAMBAs) Induce Differentiation in ATRA Resistant Cell Line: Potential New Theraputics for Acute Promyleocytic Leukemia (APL).

All trans retinoic acid (ATRA) has been used in differentiation therapy for APL and other types of cancers. However, the rapid emergence of ATRA resistance due in part to ATRA-induced acceleration of ATRA metabolism limits its use. A novel strategy to overcome the limitation associated with exogenous ATRA therapy has been developed by inhibiting the cytochrome P450-dependent ATRA-4-hydroxylase enzyme responsible for ATRA metabolism. These inhibitors are referred to as RAMBAs. Novel RAMBAs were developed which demonstrated a superior apoptosis, cell growth inhibition, in vivo anti-tumor effect in addition to the differentiation effect in breast cancer cell lines (Patel JB et al. J. Med. Chem 2004, 47:6716). We tested 3 RAMBAs, VN/14-1, 50-1, and 66-1 to investigate their activities against APL cell lines. RAMBAs did not confer cytotoxicity or apoptosis induction in vitro at the concentration between 0.5 to 5 μM as opposed to breast or prostate cancer cell lines. However, the differentiation effect was demonstrated by morphological and phenotypic changes using Wright-Giemsa stain and CD11b staining measured by flow cytometric analysis. VN/14-1 and VN/66-1 induced differentiation and apoptosis morphologically and phenotypically in HL60 cells. VN/14-1 and VN/50-1 showed superior differentiation in NB4 cell line compared to ATRA (70%, 69%, and 45%, respectively). Interestingly, HL60 ATRA resistant cell line was induced to undergo differentiation by VN/14-1 (0.5μM) at 55% whereas ATRA (0.5, 1, 5μM) showed less than 5% by flow cytometry analysis. VN/14-1 inhibited cell cycle at S phase whereas ATRA did not attenuate the cell cycle at the same concentration. We also tested the effect of RAMBAs on human CD34+ enriched cell colony formation. RAMBAs were added to the methylcellulose culture plates with CD34+ cells and colonies were determined after 14 days. There was no difference in the CFU-GM or BFU-E colony count between the control and the RAMBAs group. In summary, RAMBAs are promising differentiation agents in the treatment of APL, possibly through an inhibition of Cyp26A leading to increased endogenous ATRA levels. In addition, cell cycle inhibition may be a mechanism of differentiation induction in ATRA resistant cell lines. RAMBAs did not affect normal hematopoietic stem cells. We are currently testing whether RAMBAs can induce acetylation of histones in APL cell lines.

Genistein Promotes Apoptosis, Differentiation and Cell Cycle Arrest in All Trans Retinoic Acid (ATRA) Sensitive and Resistant Acute Promyelocytic Leukemia Cells.

PML, a suppresser of growth and proliferation in many cell types including hematopoetic cells, is localised to nuclear dot like structures known as PML oncogenic domains (POD). PML plays a crucial role in POD dependent and caspase 3 mediated apoptosis. PML-RAR, the causative agent of Acute Promyelocytic Leukaemia (APL), induces the disintegration of PODs, while Retinoid Acid (RA) mediated remission of APL is associated with reorganisation of PODs. These findings led to the concluation that disorganisation of POD is linked with the transforation of APL blasts. We have reported that nuclear hormon receptor co-repressor (N-CoR) is a component of PODs and is essential for PML mediated tumour suppressive function of Mad and Rb. (Khan MM et al. J Biol Chem.279:11814-24). Our most recent findings demonstrate that fusion protein PML-RAR triggers the ER stress and Unfolded Protein Response (UPR) by causing mis-folding and insoluble aggregation of N-CoR protein. (Khan MM et al. J Biol Chem.279:11814-24). These findings suggest that targeting the insoluble aggregation of N-CoR and the ER stress could represent an attractive therapeutic strategy for APL. Based on the hypothesis, we screened compounds known for their inhibitory effect on protein aggregation and ER stress. Among the compounds tested, genistein, a flavonoid, exhibited significant growth inhibitory effect on both RA sensitive and RA resistant APL cell NB4 in dose and time dependant manner (see fig.1 & 2). Genistein inhibited the growth on NB4 cells through collective effects on the cell cycle progression, apoptosis and differentiation. Genistein induced apoptosis in NB4 cells was mediated by the activation of caspase-3 and 9. Genistein also induced differentiation of NB4 cells as marked by appearance of cell surface differentiation marker CD11b. In cell cycle analysis, NB4 cells were accumulated in the G2-M phase after genistein treatment. In Western blotting assay, genistein unregulated and stabilized the level of PML protein in NB4 cells while promoting the degradation of PML-RAR. In protein solubility assay, genistein enhanced the solubility of N-CoR protein and reorganized the micro speckled like distribution of POD to normal dot like pattern in NB4 cells by immunocytochemical assay. In this study, we also identified that genistein abrogates PML-RAR mediated tyrosine phosphorylation of N-CoR protein. These findings, for the first time, have identified the therapeutic potential of genistein in both ATRA sensitive and resistant APL cells. [Display omitted]

P27/Kip1 mediates retinoic acid-induced suppression of ovarian carcinoma cell growth.

We have investigated the mechanisms by which all-trans retinoic acid (ATRA) causes growth inhibition of ovarian carcinoma cells. As a model, we have studied the CAOV3 cell line, which is sensitive to ATRA, and the SKOV3 cell line, which is resistant. We have found that treatment of CAOV3 cells with ATRA causes a 5-10 fold increase in the protein level of the cyclin dependent kinase inhibitor p27/Kip1. p27/Kip1 protein upregulation is important in ovarian carcinoma as primary tumors are frequently found lacking this protein. The increase in p27/Kip1 is detected by day 3 of ATRA treatment of CAOV3 cells, and is maximal by day 5. Messenger RNA levels of p27/Kip1 do not change in CAOV3 cells following ATRA treatment, however, we have shown that p27/Kip1 mRNA is more stable in ATRA treated CAOV3 cells. Conversely, the ATRA resistant cell line SKOV3 fails to show p27/Kip1 accumulation. Interestingly, the SCF component protein SKP2 appears to be decreased in CAOV3 cells treated with ATRA. We have also shown that the ATRA dependent increase in p27/kip1 protein in CAOV3 cells leads to a decrease in the kinase activity of cyclin dependent kinase 4 (CDK4) following ATRA treatment. Finally, we found that CAOV3 cells stably transfected with a p27/kip1antisense construct, which express lower levels of p27/kip1 following ATRA treatment, and have a higher CDK4 kinase activity are less sensitive to ATRA induced growth suppression. Taken together our data suggest ATRA-induced growth inhibition in CAOV3 ovarian carcinoma cells involves modulation of the CDK inhibitor p27/kip1.

Cytoplasm prepared from all-trans retinoic acid (ATRA)-treated human hematopoietic progenitor cells induces differentiation of both ATRA sensitive and ATRA resistant leukemia cells.

In this study, hematopoietic cells from human placental blood were treated with the differentiation-inducing drug all-trans retinoic acid (ATRA). Their cytoplasm was then used to culture the human myelogenous leukemia cell line HL60, NB(4) cells and the RA-resistant HL60-R and NB(4)-R(2) cells. All of these four kinds of leukemia cells underwent macrophage/monocyte differentiation and apoptosis, while their proliferation was inhibited. This suggests that the RA receptors were not essentially needed in this situation, and this method should be applicable in the treatment of RA-resistant promyelocytic leukemia as well as other kinds of leukemia.

CBP/p300 induction is required for retinoic acid sensitivity in human mammary cells

The coactivators CBP and p300 are recruited by retinoic acid receptors (RARs) during retinoid mediated transcriptional regulation. To assess the role of CBP/p300 in all- trans-retinoic acid (ATRA)-mediated growth arrest in mammary epithelial cells, two systems were tested: (1) ATRA resistant MCF-7 cells were transduced with a functional RAR-β2; (2) normal human mammary epithelial cells (HMECs) were transduced with a pan-RAR dominant negative, RAR-α403. Expression of RAR-β2 in MCF-7 cells resulted in increased sensitivity to ATRA-induced growth arrest and correlated with induction of CBP/p300 mRNA and protein. Inhibition of RAR function in HMECs resulted in resistance to ATRA-induced growth arrest and loss of CBP/p300 induction. Antisense suppression of CBP/p300 in HMECs resulted in decreased retinoic acid response element reporter trans-activation and decreased ATRA-mediated growth arrest. Thus, in human mammary epithelial cells, CBP/p300 were both modulated by an ATRA signaling pathway and were required for a normal response to ATRA.