Differentiation Therapy Of Acute Myeloid Leukemia Research Articles

The pro-differentiating capability of a flavonoid-rich extract of Citrus bergamia juice prompts autophagic death in THP-1 cells

Acute myeloid leukemia (AML) is a hematologic neoplasm, characterized by a blockage of differentiation and an unconstrained proliferation of immature myeloid cells. Recently, the survival of leukemia patients has increased thanks to the use of differentiating agents, though these may cause serious side effects. Hence, the search for safer differentiating compounds is necessary. Our aim was to assess the pro-differentiating effects of a flavonoid-rich extract of bergamot juice (BJe) in human monocytic leukemia THP-1 cells, an in vitro AML model. For the first time, we showed that treatment with BJe induced differentiation of THP-1 cells, changes in cell morphology and increased expression of differentiation-associated surface antigens CD68, CD11b and CD14. Moreover, BJe enhanced protein levels of autophagy-associated markers, such as Beclin-1 and LC3, as well as induced the phosphorylation of the MAPKs JNK, ERK and p38, hence suggesting a potential mechanism underlying its antiproliferative effects. Indeed, parallel experiments highlighted that BJe was able to hamper THP-1 cell growth. In conclusion, our study suggests that BJe induces the differentiation of THP-1 cells and reduces their proliferation, highlighting its potential in differentiation therapy of AML.

Parbendazole as a promising drug for inducing differentiation of acute myeloid leukemia cells with various subtypes

Acute myeloid leukemia (AML) is a malignancy characterized by differentiation arrest of hematopoietic precursor cells. Differentiation therapy is effective for patients with acute promyelocytic leukemia; however, only a few effective differentiation therapies have been established for patients with other AML subtypes. In this study, seven benzimidazole anthelmintics were examined to determine the effects of differentiation on AML cells. The expression of monocyte markers (CD11b and CD14) was elevated after treatment with most benzimidazole anthelmintics. Among these drugs, parbendazole (PBZ) induced AML cell differentiation at low concentration. PBZ induced the monocyte marker expression, KLF4/DPYSL2A gene expression, and apoptosis for 21 AML cell lines with various subtypes and a primary AML sample. Finally, an in vivo analysis using an AML patient-derived xenograft mouse model showed a significant decrease in the chimerism level and prolonged survival in PBZ-treated mice. These findings could lead to a more effective differentiation therapy for AML.

Cytarabine-induced differentiation of AML cells depends on Chk1 activation and shares the mechanism with inhibitors of DHODH and pyrimidine synthesis

Acute myeloid leukemia (AML) is characterized by arrested differentiation making differentiation therapy a promising treatment strategy. Recent success of inhibitors of mutated isocitrate dehydrogenase (IDH) invigorated interest in differentiation therapy of AML so that several new drugs have been proposed, including inhibitors of dihydroorotate dehydrogenase (DHODH), an enzyme in pyrimidine synthesis. Cytarabine, a backbone of standard AML therapy, is known to induce differentiation at low doses, but the mechanism is not completely elucidated. We have previously reported that 5-aminoimidazole-4-carboxamide ribonucleoside (AICAr) and brequinar, a DHODH inhibitor, induced differentiation of myeloid leukemia by activating the ataxia telangiectasia and Rad3-related (ATR)/checkpoint kinase 1 (Chk1) via pyrimidine depletion. In this study, using immunoblotting, flow cytometry analyses, pharmacologic inhibitors and genetic inactivation of Chk1 in myeloid leukemia cell lines, we show that low dose cytarabine induces differentiation by activating Chk1. In addition, cytarabine induces differentiation ex vivo in a subset of primary AML samples that are sensitive to AICAr and DHODH inhibitor. The results of our study suggest that leukemic cell differentiation stimulated by low doses of cytarabine depends on the activation of Chk1 and thus shares the same pathway as pyrimidine synthesis inhibitors.

Hydrophilic Realgar Nanocrystals Prolong the Survival of Refractory Acute Myeloid Leukemia Mice Through Inducing Multi-Lineage Differentiation and Apoptosis.

IntroductionAcute myeloid leukemia (AML) is a heterogeneous clonal disorder of hematopoietic progenitor cells, and the AML cells are differentiation retarded which results in the hyperproliferation of those malignant tumor cells. To stop the uncontrollable proliferation, inducing the AML cell differentiation is one highly expected therapy because it can bring relatively low systematic side effects compared to conventional chemotherapies; however, there are few options of inductive therapeutics in the clinical applications so far. This study aims to investigate the differentiation-induction effects of lab-developed hydrophilic nanocrystals of As4S4 (ee-As4S4).MethodsIn this work, ee-As4S4 was applied upon a refractory mouse model co-expressing AML1-ETO and HyC-KITD816V as well as a related human AML cell line, Kasumi-1, to investigate whether the nanocrystals can break the retardation of differentiation and drive the cells undergo apoptosis.ResultsIt was shown that ee-As4S4 induced the upregulation of surface markers CD11b, CD235a, and CD41a, which indicate granulocytic, erythroid, and megakaryocytic differentiation respectively, leading to the multiple-lineage differentiation and post-differentiation apoptosis, and the inhibition of histone deacetylase activity was largely involved with the differentiation-induction effects. In the AML mice, orally administered ee-As4S4 increased the level of Ter119, CD11b, and CD41 in bone marrow-derived leukemia cells while reducing the percentage of leukemic cells in the bone marrow. Also, ee-As4S4 improved the hemogram and relieved the hepatomegaly and splenomegaly of the AML mice. As a result, the survival of the AML mice was significantly prolonged. Importantly, ee-As4S4 did not cause acute or chronic toxicity in healthy mice.ConclusionIn conclusion, ee-As4S4 induced effective and multiple-lineage differentiation and apoptosis of AML cells in the refractory AML mouse model and cell line, suggesting that it holds promising potential as a novel inductive agent in differentiation therapy of AML.

P53 Protein Isoform Profiles in AML: Correlation with Distinct Differentiation Stages and Response to Epigenetic Differentiation Therapy.

p53 protein isoform expression has been found to correlate with prognosis and chemotherapy response in acute myeloid leukemia (AML). We aimed to investigate how p53 protein isoforms are modulated during epigenetic differentiation therapy in AML, and if p53 isoform expression could be a potential biomarker for predicting a response to this treatment. p53 full-length (FL), p53β and p53γ protein isoforms were analyzed by 1D and 2D gel immunoblots in AML cell lines, primary AML cells from untreated patients and AML cells from patients before and after treatment with valproic acid (VPA), all-trans retinoic acid (ATRA) and theophylline. Furthermore, global gene expression profiling analysis was performed on samples from the clinical protocol. Correlation analyses were performed between p53 protein isoform expression and in vitro VPA sensitivity and FAB (French–American–British) class in primary AML cells. The results show downregulation of p53β/γ and upregulation of p53FL in AML cell lines treated with VPA, and in some of the patients treated with differentiation therapy. p53FL positively correlated with in vitro VPA sensitivity and the FAB class of AML, while p53β/γ isoforms negatively correlated with the same. Our results indicate that p53 protein isoforms are modulated by and may predict sensitivity to differentiation therapy in AML.

Dimethyl fumarate and vitamin D derivatives cooperatively enhance VDR and Nrf2 signaling in differentiating AML cells in vitro and inhibit leukemia progression in a xenograft mouse model

Acute myeloid leukemia (AML) is one of the deadliest hematological malignancies without effective treatment for most patients. Vitamin D derivatives (VDDs) - active metabolites 1α,25-dihydroxyvitamin D2 (1,25D2) and 1α,25-dihydroxyvitamin D3 (1,25D3) and their analogs - are differentiation-inducing agents which have potential for the therapy of AML. However, calcemic toxicity of VDDs limits their clinical use at doses effective against cancer cells in vivo. Here, we demonstrate that in AML cell cultures, moderate pro-differentiation effects of low concentrations of VDDs can be synergistically enhanced by structurally distinct compounds known to activate the transcription factor Nuclear Factor (Erythroid-derived 2)-Like 2 (NFE2L2 or Nrf2). Particularly, dimethyl fumarate (DMF), which is clinically approved for the treatment of multiple sclerosis and psoriasis, strongly cooperated with 1,25D3, PRI-5100 (19-nor-1,25D2; paricalcitol) and PRI-5202 (a double-point modified 19-nor analog of 1,25D2). The pro-differentiation synergy between VDDs (1,25D3 or PRI-5202) and Nrf2 activators (DMF, tert-butylhydroquinone or carnosic acid) was associated with a cooperative upregulation of the protein levels of the vitamin D receptor (VDR) and Nrf2 as well as increased mRNA expression of their respective target genes. These data support the notion that VDDs and Nrf2 activators synergize in inducing myeloid cell differentiation through the cooperative activation of the VDR and Nrf2/antioxidant response element signaling pathways. We have previously reported that PRI-5202 is more potent by approximately two orders of magnitude than 1,25D3 as a differentiation inducer in AML cell lines. In this study, we found that PRI-5202 was also at least 5-fold less calcemic in healthy mice compared to both its direct precursor PRI-1907 and 1,25D3. In addition, PRI-5202 was remarkably more resistant against degradation by the human 25-hydroxyvitamin D3-24-hydroxylase than both 1,25D2 and 1,25D3. Importantly, using a xenograft mouse model we demonstrated that co-administration of PRI-5202 and DMF resulted in a marked cooperative inhibition of human AML tumor growth without inducing treatment toxicity. Collectively, our findings provide a rationale for clinical testing of low-toxic VDD/DMF combinations as a novel approach for differentiation therapy of AML.

VEGFC Antibody Therapy Drives Differentiation of AML.

High expression of VEGFC predicts adverse prognosis in acute myeloid leukemia (AML). We therefore explored VEGFC-targeting efficacy as an AML therapy using a VEGFC mAb. VEGFC antibody therapy enforced myelocytic differentiation of clonal CD34+ AML blasts. Treatment of CD34+ AML blasts with the antibody reduced expansion potential by 30% to 50% and enhanced differentiation via FOXO3A suppression and inhibition of MAPK/ERK proliferative signals. VEGFC antibody therapy also accelerated leukemia cell differentiation in a systemic humanized AML mouse model. Collectively, these results define a regulatory function of VEGFC in CD34+ AML cell fate decisions via FOXO3A and serve as a new potential differentiation therapy for patients with AML.Significance: These findings reveal VEGFC targeting as a promising new differentiation therapy in AML. Cancer Res; 78(20); 5940-8. ©2018 AACR.

Prodifferentiation Activity of Novel Vitamin D₂ Analogs PRI-1916 and PRI-1917 and Their Combinations with a Plant Polyphenol in Acute Myeloid Leukemia Cells.

1α,25-dihydroxyvitamin D3 (1,25D3) is a powerful differentiation inducer for acute myeloid leukemia (AML) cells. However, 1,25D3 doses required for differentiation of AML cells may cause lethal hypercalcemia in vivo. There is evidence that vitamin D2 is less toxic than vitamin D3 in animals. Here, we determined the differentiation effects of novel analogs of 1α,25-dihydroxyvitamin D2 (1,25D2), PRI-1916 and PRI-1917, in which the extended side chains of their previously reported precursors (PRI-1906 and PRI-1907, respectively) underwent further 24Z (24-cis) modification. Using four human AML cell lines representing different stages of myeloid maturation (KG-1a, HL60, U937, and MOLM-13), we found that the potency of PRI-1916 was slightly higher or equal to that of PRI-1906 while PRI-1917 was significantly less potent than PRI-1907. We also demonstrated that 1,25D2 was a less effective differentiation agent than 1,25D3 in these cell lines. Irrespective of their differentiation potency, all the vitamin D2 derivatives tested were less potent than 1,25D3 in transactivating the DR3-type vitamin D response elements. However, similar to 1,25D3, both 1,25D2 and its analogs could strongly cooperate with the plant polyphenol carnosic acid in inducing cell differentiation and inhibition of G1–S cell cycle transition. These results indicate that the 24Z modification has contrasting effects on the differentiation ability of PRI-1906 and PRI-1907 and that the addition of a plant polyphenol could result in a similar extent of cell differentiation induced by different vitamin D compounds. The enhanced antileukemic effects of the tested combinations may constitute the basis for the development of novel approaches for differentiation therapy of AML.

Identification of a First in Class GSK3-Alpha Selective Inhibitor As a New Differentiation Therapy for AML

Isoform-selective enzyme targeting poses a potential clinical challenge when there is significant homology in the protein activity domains. Glycogen synthase kinase 3-alpha (GSK3α) has been identified previously as a therapeutic target to promote differentiation in acute myeloid leukemia (AML) (Banerji et al, JCI, 2012). The GSK3 family consists of two highly homologous, but functionally non-redundant, isoforms (α and β). These proteins are serine-threonine kinases implicated in a diversity of cellular processes. Concurrent inhibition of both isoforms is known to induce β-catenin stabilization and can therefore increase self-renewal of leukemia initiating cells. Isoform-selective GSK3 suppression does not increase β-catenin levels (Doble et al, Dev Cell, 2007) and thus may offer a new therapeutic approach. GSK3α selective inhibitors, with cell-based activity, have not been reported. Our collaborative study aimed to identify first-in-class GSK3α selective inhibitors and evaluate their activity in AML, a disease in great need for new therapeutic approaches.A series of potent and selective inhibitors for GSK3α/β were discovered through a screening campaign supported by the NIH's Molecular Libraries Probe Centers Network (MLPCN) program. While these compounds are equipotent for GSK3α/β, they demonstrate remarkable selectivity versus the larger kinome (> 100x selective vs 311 kinases). Analysis of the high resolution x-ray crystal structure obtained with BRD9421, one of the lead compounds, and GSK3β revealed a tridentate hydrogen bond binding mode within the ATP catalytic domain. One of these interactions, is to aspartic 133 in GSK3β. This same residue in GSK3α is a glutamic acid and represents one of the two amino acid differences within the ATP binding domain between these two enzymes. GSK3α and β share 98% homology in their catalytic domains, but the exploitation of this hydrogen bond interaction through rational drug design offered an excellent opportunity to produce selective inhibitors for GSK3α and GSK3β. BRD0705 was thus identified as an 8-fold GSK3α isoform-selective inhibitor over GSK3β (IC50 GSK3α: 0.045 vs. GSK3β: 0.350 μM).We first validated BRD0705 isoform-selectivity in a panel of AML cell lines and primary patients (n=3 cell lines and n= 2 patient samples). Treatment with BRD0705 resulted in a dose- and time-dependent decrease in tyr279 GSK3α phosphorylation without any effect on tyr216 GSK3β phosphorylation. No increase in β-catenin protein levels by western blotting and no nuclear localization by immunofluorescence were observed. GSK3α inhibition with BRD0705 led to morphological and surface marker changes (CD11b, CD11c, CD14, CD117) consistent with AML differentiation in a panel of AML cell lines (n=4) and primary AML samples (n=5). BRD0705 also induced a G2/M arrest and impaired methylcellulose colony formation in AML cell lines and primary patient samples. BRD0705 possesses favorable pharmacokinetic properties and was well tolerated without any observed adverse events in C57BL/6 mice with repeated dosing. In vivo efficacy studies in AML models are ongoing.In conclusion, we identified a first-in-class GSK3α isoform-selective inhibitor and conducted preclinical studies validating BRD0705 as a promising new differentiation therapeutic candidate in AML. Small-molecule inhibition of GSK3α induced myeloid differentiation, cell cycle arrest and impaired colony formation without β-catenin stabilization in AML. DisclosuresDeAngelo:Incyte: Consultancy; Novartis: Consultancy; Amgen: Consultancy; Pfizer: Consultancy; Ariad: Consultancy; Agios: Consultancy; Bristol Myers Squibb: Consultancy; Celgene: Consultancy. Stone:Sunesis: Consultancy, Other: DSMB for clinical trial; Roche/Genetech: Consultancy; Agios: Consultancy; Karyopharm: Consultancy; Merck: Consultancy; Celgene: Consultancy; Abbvie: Consultancy; Novartis: Research Funding; Celator: Consultancy; Amgen: Consultancy; Pfizer: Consultancy; AROG: Consultancy; Juno: Consultancy. Stegmaier:Novartis Pharmaceuticals: Consultancy.

The protein kinase C agonist prostratin induces differentiation of human myeloid leukemia cells and enhances cellular differentiation by chemotherapeutic agents

As acute myeloid leukemia (AML) cells are characterized by uncontrolled self-renewal and impaired cellular differentiation, induction of terminal differentiation of leukemia cells by differentiating agents has been proposed as an attractive therapeutic strategy to treat AML. Here, we demonstrated that prostratin, a potent protein kinase C (PKC) activator, inhibited the growth of myeloid leukemia cells by a predominant G1 arrest with variable induction of apoptosis. Conversely, prostratin induced significant differentiation of AML cell lines and primary AML blasts as evidenced by morphology and immunophenotyping. The effects of prostratin were PKC dependent, and activation of mitogen-activated protein (MAP)/extracellular signal-regulated kinase (ERK) kinase (MEK) 1/2 by PKC was required for prostratin-induced cell differentiation. Consequently, prostratin reprogrammed transcriptional factor expression, and ectopic expression of c-Myc in HL-60 cells significantly eliminated prostratin-mediated cellular differentiation and cell cycle arrest, indicating an essential role for c-Myc suppression in the differentiation-inducing effects of prostratin. Finally, prostratin was able to potentiate cellular differentiation induced by chemotherapeutic agents such as Ara-C. Together, we proposed that prostratin alone or administered with other anticancer agents may be effective in differentiation therapy of AML.

Abstract 5392: Angiocidin induces differentiation of acute myeloid leukemia (AML) cells

Abstract Acute myeloid leukemia (AML) is a malignant proliferative disorder where blast stage cells fail to terminally differentiate and accumulate in the blood and bone marrow. AML represents a very significant health risk in the general population with rising incidence in aging adults. From 2004-2008 the median age at diagnosis was 67.8 years and it is estimated 12,950 adults will be diagnosed with 9,050 deaths from AML leukemia in 2011 (http://seer.cancer.gov/statfacts/html/amyl.html, accessed November 2011). Standard AML therapy requires intensive combination chemotherapy with a low rate of durable remissions often requiring stem cell transplantation and associated with significant treatment-related toxicity, especially in elderly patients. Due to this toxicity, the majority of elderly patients are not eligible for therapy. Therefore, new therapeutic options for the treatment of AML are urgently needed. In this study, we present evidence that the human protein angiocidin, discovered in our laboratory, has the potential to be used as a novel, non-toxic therapy for AML. We found that angiocidin can induce both AML cell lines and primary patient cells to increase expression of surface markers characteristic of a more normal differentiated phenotype. We evaluated expression of CD14, a major receptor for the innate immune system in macrophages, CD54 or ICAM-1, the major adhesion receptor used by macrophages for endothelial transmigration, and CD15, a neutrophil phagocytosis receptor. Expression of these receptors was measured by flow cytometry after cells were cultured for 24 hours in media alone or media containing 10 μg/ml angiocidin. Angiocidin up-regulated these receptors by more than 10 fold in 50% of the AML cells tested. The AML cells included the cell line THP-1 and 11 primary AML patient cells. Angiocidin up-regulated CD14 and CD54 in THP-1 cells and in 3 out of 5 M5 phenotype AML patient cells tested and 1 M4 AML phenotype out of 6 M4 samples tested. Angiocidin also up-regulated CD15 in 1 M4 AML patient phenotype out of 6 M4 patient cells tested. These results suggest that angiocidin can differentiate AML cells and therefore has the potential to be developed as differentiation therapy for AML. 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 5392. doi:1538-7445.AM2012-5392

MAPK Expression Profile in AML cells ex Vivo changes with Differentiation Induced by 1,25-Dihydroxyvitamin D3 and Its Analogs PRI-1906 and PRI-2191

Abstract 5009 Background:The physiological form of vitamin D, 1,25-dihydroxyvitamin D3 (1,25D), has well-established anti-cancer activity, but its potential clinical use is limited by hypercalcemia. Efforts to overcome this difficulty include structural modifications of 1,25D, as well as enhancement of its activity by other compounds such as antioxidants. The PRI analogs (termed “deltanoids”) have been shown to have low calcemic activity in animals, and while the PRI-1906 (24-ene-1,25-dihydroxyvitamin D2) and PRI-2191 ([24R]-1,24-dihydroxyvitamin D3) have greater activity on established Acute Myeloid Leukemia (AML) cell lines than their congeners, their relative potency on differentiation of AML blasts is not entirely clear. We therefore studied the effects of these analogs on leukemic blasts obtained from AML patients with different FAB subtypes, and combined them in some cases with Carnosic Acid (CA), a natural benzenediol abietane diterpene found in rosemary (Rosemarinus officinalis) and common sage (Salvia offinalis). Methods:Leukemia blasts from patients with newly diagnosed, untreated AML were obtained from the peripheral blood after formal consent on an IRB approved protocol. Cells were isolated by Histopaque centrifugation, washed, and placed in tissue culture. 1,25D, PRI-1906, or PRI-2191 (each 10 nM), and CA (10 uM) were added, and cells cultured for the times shown, and then harvested for analysis. Results:Cells from 8 patients were studied (1 patient with FAB-M1, 1 patient with FAB-M2, 1 patient with FAB-M2 with mixed myeloid and lymphoid lineage; 1 patient with FAB-M3; 1 patient with FAB-M4; 3 patients with FAB-M5). Using expression of CD11b/CD14 by immunophenotyping as markers of differentiation, the 1,25D and the deltanoids each induced modest differentiation in AML FAB-M1, with greater differentiation in response to the combination of a deltanoid plus CA. Similar differentiation effects were seen in AML FAB-M2, and more dramatic effects in AML FAB-M3. Differentiation was also seen in AML FAB-M4. In AML FAB-M5, 1,25D and deltanoids induced differentiation, but CA appeared to add little to this process in 2 of the patient's samples. One patient with AML FAB-M5 did show significant differentiation in response to CA as well as 1,25D; of note, that patient had a t(11;19) involving the MLL-1 gene, with normal FLT-3 genotype). In 6 out of the 8 cases, the G1 arrest increased when AML cells were treated with CA alone, but the effect was not enhanced by CA combined with 1,25D or its analogs. MAP Kinase mRNA array analysis showed upregulation of MAP Kinase pathway molecules in response to 1,25D and the deltanoids. Conclusions: 1The analogs of 1,25D, PRI-1906 and PRI-2191 induce cell cycle arrest and differentiation of AML cells ex vivo, and carnosic acid can enhance this effect. 2. As previously shown for 1,25D, MAPK signaling network is an important regulator of this process. 3.Since the PRI analogs are less calcemic than 1,25D, and carnosic acid has no effect on calcium homeostasis in AML cells, the potential therapeutic ratio is higher for the analogs combined with carnosic acid enhancer. Thus, these compounds may be considered as candidates for chemoprevention/ differentiation therapy of AML. (Supported by NIH Grant 1RO1-CA044722-21 to GPS). Disclosures:No relevant conflicts of interest to declare.

Histone H3 Methylation Mediates All-Trans-Retinoic Acid Responsiveness in Acute Myeloid Leukemia

Abstract 224During hematopoiesis, all-trans-retinoic acid (ATRA), a natural derivative of vitamin A, has been shown to induce both myelomonocytic progenitor/stem cell differentiation and self-renewal. Although these opposing effects are likely to be partly due to developmental differences, it has been shown that pro- and anti-differentiation effects of ATRA are mediated by distinct retinoic acid receptor isotypes (RARα and RARγ, respectively). With the exception of acute promyelocytic leukemia (APL), ATRA treatment as a single agent has not been successful in other types of acute myeloid leukemia (AML). We have previously hypothesized that one of the underlying reasons for poor response of non-APL AML to ATRA (pan-RAR agonist) is aberrant expression and/or activities of RAR isotypes favoring RARγ and cell growth versus differentiation. Consistently, we have reported that expression of RARα isoforms, particularly ATRA-inducible RARα2, are down-regulated in AML (Blood. 2008; 111:2374). Epigenetic analysis of patient samples revealed that relative to normal CD33+ cells, the loss of RARα2 in AML is associated with a diminution in levels of histone histone H3 lysine 4 dimethylation (H3K4me2) on the ATRA-responsive RARA2 promoter (a modification associated with transcriptional activation). Interestingly, the H3K4me1/me2 demethylase LSD1/KDM1 (AOF2) is highly expressed in AML patients (www.proteinatlas.org). A number of small molecules that target this enzyme (LSD1i) are in development and, collectively, these data predict that the use of LSD1i will facilitate induction of expression of genes that are required for differentiation of AML cells. In this study we used tranylcypromine (TCP, a monoamine oxidase used as an antidepressant and anxiolytic agent in the clinical treatment of mood and anxiety disorders, respectively), which functions a time-dependent, mechanism-based inhibitor of LSD1.Here we show that TCP unlocked the ATRA-driven therapeutic differentiation response in non-APL AML cell lines including the TEX cell line, which is derived from primitive human cord blood cells immortalized by expression of the TLS-ERG oncogene. TEX cells are >90% CD34+, respond poorly to ATRA and mimic features of primary human AML and leukemia initiating cells (Leukemia. 2005; 19:1794). Consistent with this, ATRA/TCP treatment increased differentiation in primary patient samples. ATRA alone had in general only small effects in primary AML samples and TCP showed minimal activity in most cases. Furthermore, shRNA-mediated knockdown of LSD1 confirmed a critical role for this enzyme in blocking the ATRA response in AML cells.The effects of ATRA/TCP on AML cell maturation were paralleled by enhanced induction of genes associated with myelomonocytic differentiation, including direct ATRA targets. LSD1i treatment did not lead to an increase in genome-wide H3K4me2, but did increase H3K4 dimethylation of myelomonocytic differentiation-associated genes. Importantly, treatment with ATRA/TCP dramatically diminished the clonogenic capacity of AML cells in vitro and engraftment of cells derived from AML patients in vivo, suggesting that ATRA/TCP may also target leukemic stem cells. These data strongly suggest that LSD1 may, at least in part, contribute to AML pathogenesis by inhibiting the normal function of ATRA in myelomonocytic development and pave the way for effective differentiation therapy of AML. Disclosures:No relevant conflicts of interest to declare.

Differentiation therapy of acute myeloid leukemia.

Acute Myeloid Leukemia (AML) is a predominant acute leukemia among adults, characterized by accumulation of malignantly transformed immature myeloid precursors. A very attractive way to treat myeloid leukemia, which is now called ‘differentiation therapy’, was proposed as in vitro studies have shown that a variety of agents stimulate differentiation of the cell lines isolated from leukemic patients. One of the differentiation-inducing agents, all-trans retinoic acid (ATRA), which can induce granulocytic differentiation in myeloid leukemic cell lines, has been introduced into clinics to treat patients with acute promyelocytic leukemia (APL) in which a PML-RARA fusion protein is generated by a t(15;17)(q22;q12) chromosomal translocation. Because differentiation therapy using ATRA has significantly improved prognosis for patients with APL, many efforts have been made to find alternative differentiating agents. Since 1,25-dihydroxyvitamin D3 (1,25D) is capable of inducing in vitro monocyte/macrophage differentiation of myeloid leukemic cells, clinical trials have been performed to estimate its potential to treat patients with AML or myelodysplastic syndrome (MDS). Unfortunately therapeutic concentrations of 1,25D can induce potentially fatal systemic hypercalcemia, thus limiting clinical utility of that compound. Attempts to overcome this problem have focused on the synthesis of 1,25D analogs (VDAs) which retain differentiation inducing potential, but lack its hypercalcemic effects. This review aims to discuss current problems and potential solutions in differentiation therapy of AML.

Monocytic Differentiation of Myeloid Leukemia Cell Lines Induced by ATRA and 5-Aza-2'-Deoxycitidine.

Abstract 3111Poster Board III-48 Backgrounds and IntroductionThe CCAAT/enhancer binding protein (C/EBP)αa is a transcriptional factor of hematopoietic system and plays a key role in monocytic differentiation. Recently, several studies have reported that C/EBPαa expression is down-regulated in acute myeloid leukemia (AML), leading to block of granulocytic and monocytic differentiation. Differentiation therapy of ATRA is highly effective for acute promyelocytic leukemia (APL).The mechanism of induction of differentiation in the treatment of APL is induction of a set of transcriptional factors which are responsible for myeloid differentiation. However, ATRA alone is not sufficient to treat another type of AML. Thus, it is worth to explore the agents which intensify the efficacy of ATRA. To assess the possibility of differentiation therapy in AML, except for APL, we evaluated the efficacy of demethylation agent combined with ATRA for various AML cell lines. Materials and MethodsThe five AML cell lines (K562, U-937, HL-60, THP-1, and KOCL48 expressing MLL-AF4) were treated with 50nM 5-Aza-2'-deoxycytidine (5-Aza) for two days and 1 mM ATRA for additional five days. Then, we analyzed cell growth with counting nuclei using Coulter counter. The cell cycle analysis was also performed by flow cytometry (FCM). In addition, Annexin V assay was performed to determine whether apoptosis occurred or not. To assess whether monocytic differentiation was induced or not, the expression of CD11b was evaluated by FCM. In addition, the expression of transcriptional factors, such as C/EBPαa, PU.1 and c-myc ) were analyzed by real time PCR analysis. Methylation specific PCR was also performed to evaluate the methylation status of promoter region of C/EBPαa. ResultsHL-60 was highly sensitive to ATRA (growth inhibition rate: 80%). THP-1 and KOCL-48 were moderately sensitive to ATRA (growth inhibition rate: 50-60%). Addition of 5-Aza induced suppression of the growth in these two cell lines efficiently (growth inhibition rate: 80%). K562 and U937 were resistant to ATRA (growth inhibition rate: 10-20%). Addition of 5-Aza induced suppression of cell-growth in U937 (growth inhibition rate: 70%). However, 5-Aza did not show the effect in K562. Morphological studies revealed the characteristic features, such as extended cytoplasm with vacuoles, fine granules and irregular shaped nucleus, were evident in four cell lines which was sensitive to 5-Aza. FCM analysis revealed intensification of CD11b expression. In addition, real time PCR determined the increased expression of C/EBPαa and PU.1 (Fold change: 2-3.0) in the four cell lines except for K562. On the other hand, the expression level of c-myc was decreased under treatment with ATRA and 5-Aza (Fold change: 0.2). Cell cycle analysis revealed G1 arrest was occurred. Annexin V assay also revealed that combination therapy of ATRA and 5-Aza induced apoptosis in theses cell lines except for K562. Methylation specific PCR did not identified hypermetylation of pormorter region of C/EBPαa in these cell lines except for K562. ConclusionAddition of 5-Aza to ATRA induced further expression of C/EBPαa and PU.1 efficiently, leading to monocytic differentiation in AML cell lines. Monocytic differentiation was accompanied with G1 arrest through down-regulation of c-myc, and apoptosis was induced finally. Combination of ATRA and 5-Aza might be effective therapeutic option even for AML which is resistant to differentiation therapy with ATRA. DisclosuresNo relevant conflicts of interest to declare.

X-Ray Cristallography Characterization of Human CD44 Epitopes Releasing Differentiation Blockage in Acute Myeloid Leukemia Blasts.

CD44 is a highly glycosylated type-1 transmembrane protein with multiple isoforms, including the standard one, CD44s (80kDa), which is the most abundant on hematopoietic cells, and alternatively spliced variant isoforms (CD44v). CD44 is strongly expressed on Acute Myeloid Leukemia (AML) blasts, which are immature myeloid cells which terminal differentiation is blocked at various stages, defining the distinct AML subtypes. In our laboratory, we have previously shown that several specific monoclonal antibodies (mAbs) directed to the CD44s isoform, can release the differentiation blockage of primary AML blasts (Charrad et al., Nature Medicine 1999), in all AML subtypes. These results indicated the possibility of using such mAbs to develop a CD44-targeted differentiation therapy that would be efficient in all AML subtypes. Our present aim is to characterize the molecular structure of the CD44 epitopes involved in the release of AML differentiation blockage, by using X-ray crystallography. In order to obtain high levels of soluble CD44 protein and to preserve its native conformational properties, we have constructed distinct fusion proteins comprising the glycosylated CD44s extracellular domain plus the human IgG1 Fc fragment (CD44-Fc). We have also added a polyhistidine tag, which allows to easily purify them by using Ni2+- and protein G affinity, and a TEV specific recognition sequence to cleave off the fusion partner. Eight distinct CD44-Fc constructions have been prepared, transiently expressed in COS-7 cells and the one displaying both the highest affinity for the anti-CD44 mAbs and the highest expression level, has been transfected into CHO-K1 cells. Using a stable cell clone, we have succeeded to produce large amounts of CD44-Fc (100 to 200 mg/L), in serum-free culture medium, using CELLine bioreactor flasks. This optimization of the CD44 production combined with an efficient three-step purification process (successive specific binding to G protein, to Ni2+, concluded by highly resolutive gel filtration), has allowed us to produce, for the first time, a sufficient quantity of highly pure and glycosylated CD44s extracellular domain for structural studies. SPR (Surface Plasmon Resonance) analysis have shown that the 2 differentiation-inducing anti-CD44 mAbs analyzed so far, differ in their dynamics of interaction with the CD44 protein. The molecular structure of the epitopes specifically mapped by differentiation-inducing anti-CD44 mAbs, as revealed by X-ray crystallography, will be presented and discussed, with the perspective of developing an efficient CD44-targeted differentiation therapy in AML.

The Effect of Anti-CD44 Monoclonal Antibodies on Differentiation and Proliferation of Human Acute Myeloid Leukemia Cells

Acute myeloid leukemia (AML) is a clonal malignant disease characterized by an increasing number of immature myeloid cells arrested at various stages of granulocytic and monocytic differentiation. The stage of the blockage defines distinct AML subtypes (AML1 to AML5 are the most frequent ones). There is increasing evidence that the malignant clone is maintained by rare AML stem cells endowed with self-renewal capacity, which through extensive proliferation coupled to partial differentiation, generate leukemic progenitors and blasts, of which the vast majority have limited proliferative capacity. Contrarily to chemotherapy alone, which is still unable to cure most AML patients, the differentiation therapy, which consists in releasing the differentiation blockage of leukemic blasts, has succeeded, when it is combined with chemotherapy, to greatly improve the survival of AML3 patients, using retinoic acid as differentiating agent. However, this molecule is ineffective in other AML subtypes, which are the most frequent. We have shown that specific monoclonal antibodies (mAbs, H90 and A3D8) directed to the CD44 cell surface antigen, that is strongly expressed on human AML blasts, are capable of triggering terminal differentiation of leukemic blasts in AML1 to AML5 subtypes. These results have raised the perspective of developing a CD44-targeted differentiation therapy in most AML cases. Interestingly, these anti-CD44 mAbs can also induce the differentiation of AML cell lines, inhibit their proliferation and, in some cases, induce their apoptotic death. These results suggest that H90 and/or A3D8 mAbs may be capable to inhibit the proliferation of leukemic progenitors, to promote the differentiation of the leukemic stem cells at the expense of their self-renewal, and, perhaps, to induce their apoptotic death, thereby contributing to decrease the size of the leukemic clone. The challenges of an anti-CD44 based differentiation therapy in AML, and its importance in relation to the new other therapies developed in this malignancy, are discussed in this review.

Downregulation of c-Jun expression and cell cycle regulatory molecules in acute myeloid leukemia cells upon CD44 ligation.

In the present study, we investigated the mechanism of CD44 ligation with the anti-CD44 monoclonal antibody A3D8 to inhibit the proliferation of human acute myeloid leukemia (AML) cells. The effects of A3D8 on myeloid cells were associated with specific disruption of cell cycle events and induction of G0/G1 arrest. Induction of G0/G1 arrest was accompanied by an increase in the expression of p21, attenuation of pRb phosphorylation and associated with decreased Cdk2 and Cdk4 kinase activities. Since c-Jun is an important regulator of proliferation and cell cycle progression, we analysed its role in A3D8-mediated growth arrest. We observed that A3D8 treatment of AML patient blasts and HL60/U937 cells led to the downregulation of c-Jun expression at mRNA and protein level. Transient transfection studies showed the inhibition of c-jun promoter activity by A3D8, involving both AP-1 sites. Furthermore, A3D8 treatment caused a decrease in JNK protein expression and a decrease in the level of phosphorylated c-Jun. Ectopic overexpression of c-Jun in HL60 cells was able to induce proliferation and prevent the antiproliferative effects of A3D8. In summary, these data identify an important functional role of c-Jun in the induction of cell cycle arrest and proliferation arrest of myeloid leukemia cells because of the ligation of the cell surface adhesion receptor CD44 by anti-CD44 antibody. Moreover, targeting of G1 regulatory proteins and the resulting induction of G1 arrest by A3D8 may provide new insights into antiproliferative and differentiation therapy of AML.

Differential display as an approach to study differentiation and differentiation therapy in AML.

Different subgroups of acute myeloid leukemia (AML) can be defined by the specific non-random chromosomal translocation that is present within the abnormal cell types. In one type of AML, acute promyelocytic leukemia (APL), the block in the normal process of differentiation can be circumvented by the addition of a chemical inducer, in this case retinoic acid. This is due to the defect in APL affecting the retinoic acid receptor gene. This type of therapy has become known as differentiation therapy. However, most types of leukemia do not respond to the retinoic acid, and therefore methods of differentiation therapy need to be developed by targeting other genes involved in the leukemia process. This requires the molecular characterizations of the genes that are expressed during differentiation and in particular those genes that show a differential expression in inducer sensitive cells and those resistant to induced differentiation. Therefore, therapeutic agents could be developed to specifically target these genes. This article describes how the technique of differential display, as one of several possible methods of molecular screening, may allow the identification of genes which can be targeted to induce differentiation.

Ligation of the CD44 adhesion molecule reverses blockage of differentiation in human acute myeloid leukemia.

Blockage in myeloid differentiation characterizes acute myeloid leukemia (AML); the stage of the blockage defines distinct AML subtypes (AML1/2 to AML5). Differentiation therapy in AML has recently raised interest because the survival of AML3 patients has been greatly improved using the differentiating agent retinoic acid. However, this molecule is ineffective in other AML subtypes. The CD44 surface antigen, on leukemic blasts from most AML patients, is involved in myeloid differentiation. Here, we report that ligation of CD44 with specific anti-CD44 monoclonal antibodies or with hyaluronan, its natural ligand, can reverse myeloid differentiation blockage in AML1/2 to AML5 subtypes. The differentiation of AML blasts was evidenced by the ability to produce oxidative bursts, the expression of lineage antigens and cytological modifications, all specific to normal differentiated myeloid cells. These results indicate new possibilities for the development of CD44-targeted differentiation therapy in the AML1/2 to AML5 subtypes.