Agent All-trans Retinoic Acid Research Articles

Dual peptides-modified cationic liposomes for enhanced Lung cancer gene therapy by a gap junction regulating strategy

BackgroundGene therapy for lung cancer has emerged as a novel tumor-combating strategy for its superior tumor specificity, low systematical toxicity and huge clinical translation potential. Especially, the applications of microRNA shed led on effective tumor ablation by directly interfering with the crucial gene expression, making it one of the most promising gene therapy agents. However, for lung cancer therapy, the microRNA treatment confronted three bottlenecks, the poor tumor tissue penetration effect, the insufficient lung drug accumulation and unsatisfied gene transfection efficiency. To address these issues, an inhalable RGD-TAT dual peptides-modified cationic liposomes loaded with microRNA miR-34a and gap junction (GJ) regulation agent all-trans retinoic acid (ATRA) was proposed, which was further engineered into dry powder inhalers (DPIs).ResultsEquipped with a rough particle surface and appropriate aerodynamic size, the proposed RGD-TAT-CLPs/ARTA@miR-34a DPIs were expected to deposit into the deep lung and reach lung tumor lesions guided by targeting peptide RGD. Assisted by cellular transmembrane peptides TAT, the RGD-TAT-CLPs/ARTA@miR-34a was proven to be effectively internalized by cancer cells, enhancing gene transfection efficiency. Then, the GJ between tumor cells was upregulated by ARTA, facilitating the intercellular transport of miR-34a and boosting the gene expression in the deep tumor.ConclusionOverall, the proposed RGD-TAT-CLPs/ARTA@miR-34a DPIs could enhance tumor tissue penetration, elevate lung drug accumulation and boost gene transfection efficiency, breaking the three bottlenecks to enhancing tumor elimination in vitro and in vivo. We believe that the proposed RGD-TAT-CLPs/ARTA@miR-34a DPIs could serve as a promising pulmonary gene delivery platform for multiple lung local disease treatments.

The transcription factor HIF2α partakes in the differentiation block of acute myeloid leukemia.

One of the defining features of acute myeloid leukemia (AML) is an arrest of myeloid differentiation whose molecular determinants are still poorly defined. Pharmacological removal of the differentiation block contributes to the cure of acute promyelocytic leukemia (APL) in the absence of cytotoxic chemotherapy, but this approach has not yet been translated to non-APL AMLs. Here, by investigating the function of hypoxia-inducible transcription factors HIF1α and HIF2α, we found that both genes exert oncogenic functions in AML and that HIF2α is a novel regulator of the AML differentiation block. Mechanistically, we found that HIF2α promotes the expression of transcriptional repressors that have been implicated in suppressing AML myeloid differentiation programs. Importantly, we positioned HIF2α under direct transcriptional control by the prodifferentiation agent all-trans retinoic acid (ATRA) and demonstrated that HIF2α blockade cooperates with ATRA to trigger AML cell differentiation. In conclusion, we propose that HIF2α inhibition may open new therapeutic avenues for AML treatment by licensing blasts maturation and leukemia debulking.

Early death (ED) in elderly acute promyelocytic leukemia (APL) patients treated with lower induction doses of all-trans retinoic acid (ATRA).

e19012 Background: With the introduction of the differentiating agent ATRA, APL has progressed from being one of the most life-threatening diseases to becoming the most curable subtype of adult AML. However, despite this advancement, death within the first 30 days of treatment amongst elderly patients remains a critical issue with ED rates reportedly as high as 20-50%. There is limited data looking at the elderly population due to often being excluded from clinical trials given age associated with being prominent negative risk factor. Pharmacokinetic studies comparing 25 mg/m2/day and 45 mg/m2/day doses in adult patients have shown similar peak concentrations and mean area under the curve. Notably, low dose ATRA has been offered in pediatric patients in order to reduce incidence of differentiation syndrome. Differentiation syndrome is a key contributor to ED in elderly APL patients causing treatment failure. We propose that lower doses of ATRA in the elderly reduces the impact differentiation syndrome has on induction mortality amongst elderly APL patients and leads to a lower rate of ED. Methods: Fifteen APL patients > 60 years of age were treated. Thirteen received low doses of ATRA (equal to or less than 25mg/m2/day) and two received standard doses of ATRA (45mg/m2/day). Arsenic trioxide was also used in addition to cytoreductive agents, such as cytarabine, hydroxyurea, and idarubicin as needed during induction. Dose reductions and regimens were based on age, comorbidities, and overall clinical judgement. During the course of treatment, further dose reductions and pauses in therapy were made based on clinical course. Results: A total of 15 elderly APL patients were treated between May 2017 and November 2020 at cancer centers within Georgia and South Carolina. The age range was 65-88 years old with a median age of 71 years old. 9/15 (60%) were female and 2/15 (13%) were classified as high risk. 13/15 were Caucasian and the remaining 2 were African American. Amongst the 13 APL patients who received lower doses of ATRA, one ED was noted. This occurred in 81-year-old Caucasian male with low risk APL who had multiple significant comorbidities and elected to halt treatment and pursue hospice. Amongst the 2 low risk APL patients who received standard doses of ATRA, one ED was noted due to differentiation syndrome. This patient also had many significant comorbidities. Conclusions: The ED rate in elderly APL patients treated with low doses of ATRA during induction was noted to be 1/13 (7.7%) which is significantly lower than what has been reported in literature. [Table: see text]

Liver fibrosis therapy based on biomimetic nanoparticles which deplete activated hepatic stellate cells.

Liver fibrosis is one of the most common liver diseases with substantial morbidity and mortality. However, effective therapy for liver fibrosis is still lacking. Considering the key fibrogenic role of activated hepatic stellate cells (aHSCs), here we reported a strategy to deplete aHSCs by inducing apoptosis as well as quiescence. Therefore, we engineered biomimetic all-trans retinoic acid (ATRA) loaded PLGA nanoparticles (NPs). HSC (LX2 cells) membranes, presenting the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), were coated on the surface of the nanoparticles, while the clinically approved agent ATRA with anti-fibrosis ability was encapsulated in the inner core. The biomimetic coating of TRAIL-expressing HSC membranes does not only provide homologous targeting to HSCs, but also effectively triggers apoptosis of aHSCs. ATRA could induce quiescence of activated fibroblasts. While TM-NPs (i.e. membrane coated NPs without ATRA) and ATRA/NPs (i.e. non-coated NPs loaded with ATRA) only showed the ability to induce apoptosis and decrease the α-SMA expression in aHSCs, respectively, TM-ATRA/NPs induced both apoptosis and quiescence in aHSCs, ultimately leading to improved fibrosis amelioration in both carbon tetrachloride-induced and methionine and choline deficient L-amino acid diet induced liver fibrosis mouse models. We conclude that biomimetic TM-ATRA/NPs may provide a novel strategy for effective antifibrosis therapy.

Abstract B027: The transcription factor HIF2alpha partakes in the differentiation block of acute myeloid leukemia

Abstract Acute myeloid leukemia (AML) is characterized by epigenetic silencing of differentiation genes and accumulation of aberrant myeloid cells arrested at different stages of myeloid development. The combination of epigenetic and differentiation therapies represents an attractive opportunity for AML patients by promoting chromatin remodeling at differentiation genes and induction of terminal maturation and leukemia debulking. The aim of our work is to define the function of the hypoxia inducible transcription factor HIF2alpha in the pathogenesis of AML. By its genetic inactivation and pharmacological inhibition in AML cell lines and patient-derived xenograft models, we found that HIF2alpha acts as a novel regulator of the AML differentiation block and promotes leukemia progression. Gene expression profiling of HIF2alpha-dependent transcriptome revealed that HIF2alpha mainly promotes the expression of genes involved in transcriptional modulation and epigenetic modifications, whilst inhibiting genes of myeloid maturation and activation. Mechanistically, inhibition of HIF2alpha results in decreased global levels of the heterochromatin marker H3K27me3, which is specifically deposited by the catalytic subunit EZH2 of the Polycomb repressive complex 2. Intriguingly, we found that HIF2alpha and EZH2 cooperate at favoring EZH2-mediated deposition of H3K27me3 at reverse hypoxia responsive elements in the regulatory regions of myeloid maturation genes. Additionally, we demonstrate that HIF2alpha is positively regulated by the pro-differentiation agent all-trans retinoic acid (ATRA), and its inhibition cooperates with ATRA in triggering AML cell differentiation. In conclusion, we provide new mechanistic insights into the role of HIF2alpha in the pathogenesis of AML, by promoting an undifferentiated state via EZH2-mediated epigenetic silencing of myeloid differentiation genes. Importantly, small molecule inhibitors of HIF2alpha have been recently generated and are tested for solid cancers. Therefore, HIF2alpha inhibition may open new therapeutic avenues for AML patients and add therapeutic value to ATRA-based therapies by interrupting HIF2alpha upregulation and promoting an epigenetic state permissive to maturation of leukemic blasts and leukemia exhaustion. Citation Format: Daniela Magliulo, Carolina Caserta, Serena Belluschi, Cristina Fracassi, Kety Giannetti, Raffaella Pini, Ettore Zapparoli, Stefano Beretta, Eleonora Draghi, Marco J. Morelli, Raffaella Di Micco, Bernhard Gentner, Luca Vago, Rosa Bernardi. The transcription factor HIF2alpha partakes in the differentiation block of acute myeloid leukemia. [abstract]. In: Proceedings of the AACR Special Conference: Cancer Epigenomics; 2022 Oct 6-8; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2022;82(23 Suppl_2):Abstract nr B027.

Targeting CAMKK2 and SOC Channels as a Novel Therapeutic Approach for Sensitizing Acute Promyelocytic Leukemia Cells to All-Trans Retinoic Acid.

Calcium ions (Ca2+) play important and diverse roles in the regulation of autophagy, cell death and differentiation. Here, we investigated the impact of Ca2+ in regulating acute promyelocytic leukemia (APL) cell fate in response to the anti-cancer agent all-trans retinoic acid (ATRA). We observed that ATRA promotes calcium entry through store-operated calcium (SOC) channels into acute promyelocytic leukemia (APL) cells. This response is associated with changes in the expression profiles of ORAI1 and STIM1, two proteins involved in SOC channels activation, as well as with a significant upregulation of several key proteins associated to calcium signaling. Moreover, ATRA treatment of APL cells led to a significant activation of calcium/calmodulin-dependent protein kinase kinase 2 (CAMKK2) and its downstream effector AMP-activated protein kinase (AMPK), linking Ca2+ signaling to autophagy. Pharmacological inhibition of SOC channels and CAMKK2 enhanced ATRA-induced cell differentiation and death. Altogether, our results unravel an ATRA-elicited signaling pathway that involves SOC channels/CAMKK2 activation, induction of autophagy, inhibition of cellular differentiation and suppression of cell death. We suggest that SOC channels and CAMKK2 may constitute novel drug targets for potentiating the anti-cancer effect of ATRA in APL patients.

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

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

Abstract 4406: TRIB3 stabilizes high TWIST1 expression to promote rapid APL progression &amp; ATRA resistance

Abstract Acute promyelocytic leukemia (APL), which accounts for 10-15% of acute myeloid leukemia (AML) cases, is characterized by the t (15; 17) chromosomal translocation and is now highly curable by the combination of granulocytic differentiation induction and the PML-RARα oncoprotein-targeted agents all-trans retinoic acid (ATRA) and arsenic trioxide (ATO). Despite the striking molecular complete remission (CR) and the very few cases of relapse associated with ATRA/ATO-based regimens, mortality events typically result from early fatal bleeding, which remains the most important challenge and the largest obstacle to curing all APL patients. For instance, several studies have reported that the risk of early hemorrhagic death (HD) reaches an incidence of 10-20% during the first month of induction. Importantly, APL patients with a high white blood cell count face an increased risk of early HD. Furthermore, differentiation syndrome and resistance to ATRA/ATO treatment with PML-RARα mutations still endanger the health of a significant proportion of APL patients. Thus, we need to better understand the molecular mechanism of APL pathogenesis and design more effective therapeutic strategies to block the rapid progression of early HD and overcome resistance. Oncogenic transcription factors play an important role in the development of hematological malignancy. The dysregulation of epithelial-mesenchymal transition-inducing transcription factors (EMT-TFs), including SNAI1/SNAI2, ZEB1/ZEB2 and TWIST1/TWIST2, which are closely related to malignant progression in solid tumors, has also been explored in the context of the aggressive invasion, chemoresistance and poor prognosis of AML. In this study, we found through two database (TCGA and E-MTAB-344) gene expression searches and detailed functional verification that the EMT-TF TWIST1 is highly expressed in APL cells and is critical for leukemic cell survival. TWIST1 and TRIB3 were highly coexpressed in APL cells compared with other subtypes of AML. We subsequently demonstrated that TRIB3 could bind the bHLH and WR domains of TWIST1 and stabilize TWIST1 by inhibiting the ubiquitination of the TWIST1 protein. Based on a detailed analysis of a functional screen of synthetic peptides, we discovered a peptide analogous to the previously reported TWIST1 WR domain, which rapidly and specifically represses APL cell survival through disrupting the TRIB3/TWIST1 interaction. Our data also suggest that a peptide similar to the WR domain disturbs the TRIB3/TWIST1 interaction, impairs rapid progression during the early death of APL and reverses resistance to ATRA therapy. These results reveal the important role of a specific oncogenic transcriptional factor, TWIST1, in APL leukemogenesis and suggest a potential peptide-initiated rapid proteolysis therapeutic opportunity to protect against early death and induction therapy resistance in patients with APL. Citation Format: Wu Zhang, Jian Lin, Jie Xu. TRIB3 stabilizes high TWIST1 expression to promote rapid APL progression &amp; ATRA resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4406.

Abstract 5170: Targeting FGFR signaling to disrupt cellular cross-talk in pancreatic cancer

Abstract Pancreatic ductal adenocarcinoma (PDAC) has a poor prognosis with a 5 year survival rate of less than 5%. PDAC tumors consist of a desmoplastic stroma, which limits the effectiveness of chemotherapy. Pancreatic stellate cells (PSCs), which form a key part of this stroma, become activated in response to tumor development. Activated PSCs enter a cross-talk with cancer cells to induce tumor cell proliferation and invasion, leading to metastatic spread. Nuclear fibroblast growth factor receptor 1 (nFGFR1) has been found in PSCs at the invasive edge of PDAC tumors. Inhibition of FGFR1 prevents its nuclear translocation in PSCs, which results in decreased invasion in 3D in vitro PDAC models. Nuclear translocation of FGFR1 in PSCs appears to be a vital mechanism that triggers the transcription of key proteins involved in PDAC invasion. We have used a powerful combination of chromatin immunoprecipitation (ChIP-seq) and sub-cellular mass spectrometry to determine the transcriptional targets of nFGFR1 and consequent sub-cellular protein flux. These techniques have allowed us to dissect the functional consequences of FGFR1 knockdown or inhibition in the PSCs. Candidate drivers of invasion are being validated in state-of-the-art 3D in vitro PDAC models. We have extended these functional studies to combination therapy with the clinical agent gemcitabine (targeting cancer cells) and all-trans retinoic acid (ATRA, modulating PSCs), providing translational relevance for our findings. We are embarking on validating this novel strategy using in vivo co-culture xenograft models with specific reference to FGFR1. Effectively disrupting the cross-talk between the tumor and stroma, either alone or in combination with other therapies, could translate to improved therapeutic responses in PDAC patients in the clinic. Citation Format: Abigail Coetzee, Edward Carter, James Heward, Faraz Mardakheh, Richard Grose, Hemant Kocher. Targeting FGFR signaling to disrupt cellular cross-talk in pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5170.

Role of Microparticles Derived from Acute Promyelocytic Leukemia Cells in Coagulopathy

To evaluate the role of microparticle (MP) derived from acute promyelocytic leukemia (APL) cells and tissue factor (TF) carried by the MP in hypercoagulable state, and the effect of treatment with cytotoxic chemotherapy/differentiating agents on procoagulant activity (PCA) of these MP. Bone marrow mononuclear cells (BMMNC) were extracted from 5 APL patients and 5 sex- and age- matched patients with iron deficiency anemia as controls. The cells were cultured in vitro for 48 h, then MP-rich culture medium and MP-free culture medium were harvested and MP was further obtained from certain volume of MP-rich culture medium. Subsequently, TF expression on MP was measured by ELISA. PCA of MP-rich culture medium or MP-free culture medium was assessed with thrombin generation assay. The role of TF on MP-related PCA was evaluated using anti-human TF antibody. In addition, APL cells were treated with all-trans retinoic acid (ATRA), arsenic trioxide (ATO) or daunorubicin (DNR) for 48 h, then MP-rich culture medium were harvested and the PCA was determined. No TF expression was found in the MP released from bone marrow MNC in control group, whereas the obvious TF expression was found in the MP originated from BMMNC of APL. MP from both APL and control BM-MNC had obvious PCA. However, compared with the MP derived from control MNC, the MP from APL BM-MNC induced significantly higher PCA. TF played a crucial role in the PCA of APL BM-MNC derived MP, while played no role in that of MP from control MNCs. DNR-treating APL BM-MNC resulted in an increase in the PCA of MP, whereas ATO or ATRA exposure lead to exactly the opposite results. MP derived from APL BM-MNC posseses obvious PCA. TF plays a crucial role in the MP-related PCA. The PCA of MP increases after treating APL BM-MNC with chemotherapy agent DNR and decreases following exposure of APL BM-MNC to differentiating agents ATRA or ATO.

Pharmacological restoration and therapeutic targeting of the B-cell phenotype in classical Hodgkin lymphoma

AbstractClassical Hodgkin lymphoma (cHL), although originating from B cells, is characterized by the virtual lack of gene products whose expression constitutes the B-cell phenotype. Epigenetic repression of B-cell–specific genes via promoter hypermethylation and histone deacetylation as well as compromised expression of B-cell–committed transcription factors were previously reported to contribute to the lost B-cell phenotype in cHL. Restoring the B-cell phenotype may not only correct a central malignant property, but it may also render cHL susceptible to clinically established antibody therapies targeting B-cell surface receptors or small compounds interfering with B-cell receptor signaling. We conducted a high-throughput pharmacological screening based on >28 000 compounds in cHL cell lines carrying a CD19 reporter to identify drugs that promote reexpression of the B-cell phenotype. Three chemicals were retrieved that robustly enhanced CD19 transcription. Subsequent chromatin immunoprecipitation-based analyses indicated that action of 2 of these compounds was associated with lowered levels of the transcriptionally repressive lysine 9-trimethylated histone H3 mark at the CD19 promoter. Moreover, the antileukemia agents all-trans retinoic acid and arsenic trioxide (ATO) were found to reconstitute the silenced B-cell transcriptional program and reduce viability of cHL cell lines. When applied in combination with a screening-identified chemical, ATO evoked reexpression of the CD20 antigen, which could be further therapeutically exploited by enabling CD20 antibody-mediated apoptosis of cHL cells. Furthermore, restoration of the B-cell phenotype also rendered cHL cells susceptible to the B-cell non-Hodgkin lymphoma-tailored small-compound inhibitors ibrutinib and idelalisib. In essence, we report here a conceptually novel, redifferentiation-based treatment strategy for cHL.

Combination Treatment with All-Trans Retinoic Acid Prevents Cisplatin-Induced Enrichment of CD133+ Tumor-Initiating Cells and Reveals Heterogeneity of Cancer Stem Cell Compartment in Lung Cancer

The existence of specific cellular subpopulations within primary tumors with increased tumorigenic potential and chemotherapy resistance (tumor-initiating cells, TICs) holds great therapeutic implications. Resistant cells can remain quiescent for long periods and be responsible for local relapses and metastasis. We and others have previously described in non–small-cell lung cancer the presence of cisplatin-resistant CD133+ cells with tumor-initiating potential and co-expression of CXCR4 as possible indicator of TICs with disseminating potential. In this study, we report, by in vitro cell fate tracing systems, heterogeneity within the TIC compartment with a highly quiescent pool and a slowly dividing subpopulation, both containing CD133+ cells but respectively enriched for CD133+/CXCR4− and CD133+/CXCR4+ cells. Pretreatment with differentiating agent all-trans retinoic acid counteracts cisplatin resistance specifically of the slowly dividing compartment indicating effect on CD133+/CXCR4+ cells. The same effects are appreciable also in vivo in patient-derived xenografts, where several cycles of all-trans retinoic acid and cisplatin treatment are able to stably reduce this fraction of TICs and tumor dissemination. Thus, partially affecting the heterogeneous TICs compartment, differentiating therapy has promising effects in counteracting cisplatin resistance of CD133+ cells, reducing both local tumor growth and dissemination. In addition, our approach discloses a further level of complexity of chemotherapy-resistant CD133+ TICs, revealing phenotypical and functional heterogeneity of the cancer stem cell compartment in lung cancer.

Biological Functions of Plasma Membrane-Derived Extracellular Vesicles and Their Role in Diseases

Plasma Membrane-derived Extracellular Vesicles (PMEVs) are membrane-coated vesicles of diameter 0.1 to 1.5µm, carrying various proteins inherent in their parental cells. PMEVs are released when cells undergo activation/apoptosis via blebbing and shedding and have a function in intercellular communication. Exposition of phosphatidylserine (PS) on the outer membrane leaflet that mark them as a biologically distinct entity could also explain a role for PMEVs in phagocytosis and thrombosis. The purpose of this review is to outline and discuss some of the functions of PMEVs in detail to throw more light on its biological effects as more research delve into emerging therapies targeting the microvesicle communication system. The role of PMEVs as a differentiation agent and therefore its possible use in differentiation therapy is discussed. In some experiments, the myeloid differentiating agents all trans retinoic acid (ATRA), phorbol 12-myristate 13-acetate (PMA) and histamine, which inhibit promonocyte proliferation, induced an intracellular Ca2+-mediated PMEV release from HL-60 promonocytes. These PMEVs caused HL-60 cells to enter G0/G1 cell cycle arrest and induce terminal monocyte-to-macrophage differentiation through TGF-β1 mediation. The review also discusses the relationship between PMEVs and diseases where it is known that patients with certain inflammatory diseases show increased PMEV levels in the plasma. The review conclude on the fact that PMEVs have a lot of biological functions that are beneficial to the physiological functions in humans and therefore more work is required to elucidate their composition and the mechanisms involved in exertion of their effects.

The IRF2BP2-RARα Fusion Generated By a Cryptic Rearrangement Transforms Hematopoietic Stem/Progenitor Cells and Induces Retinoid Sensitive Acute Promyelocytic Leukemia (APL)

APL is characterised by chromosomal rearrangements involving the gene encoding the myeloid transcription factor Retinoic Acid Receptor Alpha (RARα). While RARA is fused to PML in the majority of cases consequent upon the t(15;17)(q22;q21), significant insights can be gained into pathogenesis through analysis of APL involving alternative fusion partners. It is now apparent that the fusion partner has a critical bearing on disease biology, particularly with respect to sensitivity to molecularly targeted therapies i.e. all transretinoic acid (ATRA) and arsenic trioxide (ATO). While ATO sensitivity is restricted to PML-RARA+ APL, the fusion partner influences sensitivity to ATRA, with involvement of STAT5b or PLZF (ZBTB16) being associated with resistance and poorer clinical outcome. In the European Working Party study (Blood 2000;96:1297-1308) we previously established that ~10% of morphological APL cases lack the classic t(15;17). While PML is involved in the majority due to insertion events or more complex rearrangements, the molecular basis of disease in some patients has been unexplained. Analysis was undertaken in a normal karyotype APL (WBC 3.8 x 109/l, plt 7 x 109/l) presenting in a 37 year old man, PML-RARA negative by RT-PCR, with intact PML nuclear bodies on immunofluorescence. Due to the suggestive morphology, the patient was commenced on ATRA as a single agent (45mg/m2) pending further molecular investigation. ATRA induced maturation of the leukemic blasts, apparent in bone marrows taken at day 7 and day 16, associated with normalization of the platelet count (100 x 109/l by day 25). Diagnostic material was subject to gene expression profiling (TaqMan® Array Card, Life Technologies) and on unsupervised analysis clustered with PML-RARA+ cases, with a common signature associated with upregulation of HGF and FGF13 and downregulation of HOXA7, HOXA9 and MEIS1. 5’ RACE-PCR confirmed involvement of RARA and identified the fusion partner as IRF2BP2 (Interferon Regulatory Factor 2 Binding Protein 2), which is located at 1q42. The cDNA breakpoint involved fusion of exon 1 of IRF2BP2 to exon 3 of RARA leading to retention of the regions mediating binding to DNA, retinoic acid (ligand binding domain, LBD) and RXR. Chromosome 1 painting was undertaken, which appeared normal and a reciprocal RARA-IRF2BP2 fusion transcript was not detected, consistent with formation of IRF2BP2-RARA by cryptic insertion. Whole exome sequencing identified 17 non-synonymous exonic single nucleotide variants, three (with predicted functional impact) were in genes known to be recurrently mutated in AML (LRP1B, GATA2 and EZH2). The patient’s treatment response was tracked by real-time quantitative PCR (sensitivity 1 in 10e4), which showed persistent high level IRF2BP2-RARA fusion transcripts following single agent ATRA. The patient was commenced on the PETHEMA treatment schedule, achieved molecular remission (CRm) following the first course of chemotherapy and remains in ongoing CRm in the marrow 32 months from diagnosis.IRF2BP2 is a downstream target of p53 and repressor of the cell cycle regulator NFAT1; it is fused to CDX1 as a result of the t(1;5)(q42;q32) in mesenchymal chondrosarcoma, has been reported to be mutated in CLL and primary CNS lymphoma, but has not previously been implicated in myeloid malignancy. To investigate the biology of IRF2BP2-RARα, co-immunoprecipitation assays were performed in 293T cells using tagged constructs, which revealed that the fusion protein has the capacity to self-associate. In reporter assays, IRF2BP2-RARα behaved similarly to PML-RARα, inducing repression at retinoid response elements, which was overcome by 1μM ATRA. Full length IRF2BP2-RARA was cloned into the MSCV vector, expressed in lin- murine stem/progenitor cells and replating assays performed in three conditions (SCF, IL3 + IL6/ SCF, IL3, IL6 + G-CSF/ SCF, IL3, IL6, G-CSF + GM-CSF). Expression of IRF2BP2-RARA consistently induced large numbers of third round colonies under all 3 conditions; whereas, colony formation was completely abrogated in the presence of 1μM ATRA in accordance with the sensitivity to retinoid observed in vivo.In conclusion, these data extend the range of malignancies involving IRF2BP2, identifying a novel retinoid sensitive subtype of APL and provide further evidence that forced dimerization of RARα mediated by the fusion partner plays a critical role in leukemogenesis. DisclosuresNo relevant conflicts of interest to declare.

Treatment of acute promyelocytic leukemia with arsenic trioxide: clinical results and open questions

Acute promyelocytic leukemia (APL) is a rare form of acute myeloid leukemia. The specific translocation t(15;17), which results in the fusion gene PML–RARA is the diagnostic and pathomechanistic hallmark of APL. By combination, treatment consisting of the differentiating agent all-trans retinoic acid (ATRA), which targets this molecular lesion, and cytotoxic chemotherapy, cure can be achieved in over 70% of patients. Recently, arsenic trioxide (ATO) has emerged to be the most active single agent in the treatment of APL. Previous studies employing ATO in relapse settings reported average complete remission rates of 85% and a mean overall survival of over 60%. In recent approaches installing ATO in first-line treatment, ATO-induced response rates comparable to previous combination regimen. The results of these newer studies indicate that the backbone of chemotherapy can be dramatically reduced or completely replaced by ATO and ATRA with similar or even better outcome.

Targeting cancer stem cells in glioblastoma multiforme using mTOR inhibitors and the differentiating agent all-trans retinoic acid

Glioblastoma multiforme (GBM), the most aggressive primary brain tumor, portends a poor prognosis despite current treatment modalities. Recurrence of tumor growth is attributed to the presence of treatment-resistant cancer stem cells (CSCs). The targeting of these CSCs is therefore essential in the treatment of this disease. Mechanistic target of rapamycin (mTOR) forms two multiprotein complexes, mTORC1 and mTORC2, which regulate proliferation and migration, respectively. Aberrant function of mTOR has been shown to be present in GBM CSCs. All-trans retinoic acid (ATRA), a derivative of retinol, causes differentiation of CSCs as well as normal neural progenitor cells. The purpose of this investigation was to delineate the role of mTOR in CSC maintenance, and to establish the mechanism of targeting GBM CSCs using differentiating agents along with inhibitors of the mTOR pathways. The results demonstrated that ATRA caused differentiation of CSCs, as demonstrated by the loss of the stem cell marker Nestin. These observations were confirmed by western blotting, which demonstrated a time-dependent decrease in Nestin expression following ATRA treatment. This effect occurred despite combination with mTOR (rapamycin), PI3K (LY294002) and MEK1/2 (U0126) inhibitors. Expression of activated extracellular signal-regulated kinase 1/2 (pERK1/2) was enhanced following treatment with ATRA, independent of mTOR pathway inhibitors. Proliferation of CSCs, determined by neurosphere diameter, was decreased following treatment with ATRA alone and in combination with rapamycin. The motility of GBM cells was mitigated by treatment with ATRA, rapamycin and LY29002 alone. However, combination treatment augmented the inhibitory effect on migration suggesting synergism. These findings indicate that ATRA-induced differentiation is mediated via the ERK1/2 pathway, and underscores the significance of including differentiating agents along with inhibitors of mTOR pathways in the treatment of GBM.

The cysteinyl leukotriene 2 receptor contributes to all-transretinoic acid-induced differentiation of colon cancer cells

BackgroundCysteinyl leukotrienes (CysLTs) are potent pro-inflammatory mediators that are increased in samples from patients with inflammatory bowel diseases (IBDs). Individuals with IBDs have enhanced susceptibility to colon carcinogenesis. In colorectal cancer, the balance between the pro-mitogenic cysteinyl leukotriene 1 receptor (CysLT1R) and the differentiation-promoting cysteinyl leukotriene 2 receptor (CysLT2R) is lost. Further, our previous data indicate that patients with high CysLT1R and low CysLT2R expression have a poor prognosis. In this study, we examined whether the balance between CysLT1R and CysLT2R could be restored by treatment with the cancer chemopreventive agent all-trans retinoic acid (ATRA).MethodsTo determine the effect of ATRA on CysLT2R promoter activation, mRNA level, and protein level, we performed luciferase gene reporter assays, real-time polymerase chain reactions, and Western blots in colon cancer cell lines under various conditions.ResultsATRA treatment induces CysLT2R mRNA and protein expression without affecting CysLT1R levels. Experiments using siRNA and mutant cell lines indicate that the up-regulation is retinoic acid receptor (RAR) dependent. Interestingly, ATRA also up-regulates mRNA expression of leukotriene C4 synthase, the enzyme responsible for the production of the ligand for CysLT2R. Importantly, ATRA-induced differentiation of colorectal cancer cells as shown by increased expression of MUC-2 and production of alkaline phosphatase, both of which could be reduced by a CysLT2R-specific inhibitor.ConclusionsThis study identifies a novel mechanism of action for ATRA in colorectal cancer cell differentiation and demonstrates that retinoids can have anti-tumorigenic effects through their action on the cysteinyl leukotriene pathway.

Retinoic acid represses invasion and stem cell phenotype by induction of the metastasis suppressors RARRES1 and LXN

The mouse haematopoietic stem cell (SC) regulator Latexin (LXN) is the only known homologue of the retinoic acid receptor responder 1 (RARRES1) gene. Both genes lie adjacent on chromosome 3 and differ mostly by the presence of a transmembrane domain in RARRES1. Despite their homology, it is not known whether they possess similar regulatory mechanisms, cellular localization and function. Here, we identified RARRES1 and LXN as highly significantly downregulated genes in human prostate SCs, whose expression was induced by the pro-differentiation agent all-trans retinoic acid (atRA). AtRA induced expression in the most differentiated cells compared with the SC fraction, suggesting that this subpopulation was less responsive to atRA. Small interfering RNA suppression of RARRES1 and LXN enhanced the SC properties of primary prostate cultures, as shown by a significant increase in their colony-forming ability. Expression of both RARRES1 and LXN was co-ordinately repressed by DNA methylation in prostate cancer cell lines and inhibition of RARRES1 and LXN increased the invasive capacity of primary prostate cultures, which also fully rescued an inhibitory effect induced by atRA. Moreover, we showed that RARRES1 and LXN reside within different sub-cellular compartments, providing evidence that RARRES1 is not a plasma membrane protein as previously supposed but is located primarily in the endoplasmic reticulum; whereas LXN was detected in the nucleus of prostate epithelial cells. Thus, LXN and RARRES1 are potential tumour suppressor genes, which are co-ordinately regulated, SC-silenced genes functioning to suppress invasion and colony-forming ability of prostate cancer cells; yet the proteins reside within different sub-cellular compartments.

Long-Term Efficacy of Low-Dose All-Trans Retinoic Acid Plus Individually Adapted Chemotherapy Induction Followed by Arsenic Trioxide Based Post-Remission Therapy in Adults with Newly Diagnosed Acute Promyelocytic Leukemia

AbstractAbstract 1480Acute promyelocytic leukemia (APL) is a distinct subtype of acute myelogenous leukemia (AML), which usually presents with pancytopenia, coagulopathies and bleeding. Molecular studies have revealed that it was caused by leukemogenic PML-RARA fusion gene resulting from a specific chromosomal translocation t(15;17). The administration of target agent all-trans-retinoic acid (ATRA) combined with anthracycline-based chemotherapy for induction and consolidation followed by ATRA plus low-dose chemotherapy maintenance is the standard strategies for patients with newly diagnosed APL. However, despite the high cure rate, early death and leukemia relapse are the two main important obstacles. We evaluated the efficacy of low-dose All-trans-retinoic acid (ATRA) plus individually adapted chemotherapy for induction followed by arsenic trioxide (ATO) based post-remission therapy in newly diagnosed acute promyelocytic leukemia (APL). From January 2004 to September 2011, 109 patients with APL were enrolled the study. The complete remission (CR) rate was 96.3%. The early death rate was 0.9%. Two arms were assigned according to post-remission protocols: ATO group cases were treated with standard chemotherapy, ATO, and ATRA. Without ATO group cases were subsequently treated with chemotherapy and ATRA only. Patients were monitored by reverse transcriptase-polymerase chain reaction (RT-PCR) during and after treatment. The six-year relapse-free survival (RFS) was significantly better for patients in ATO group than in without ATO group, 94.4% versus 50.6% (P < 0.0001) and the six-year overall survival (OS) rate was 95.7% versus 64.1%, in two groups (P = 0.003). This study shows that low-dose ATRA plus tailored chemotherapy is effective in induction therapy, and the addition of ATO to post-remission therapy significantly improves the long-term outcome. Disclosures:No relevant conflicts of interest to declare.

Treatment of Acute Promyelocytic Leukemia (APL) At Tawam Hospital UAE

AbstractAbstract 4347Acute Promyelocytic Leukemia (APL) is a unique sub-type of Acute Myeloid leukemia associated with a balanced reciprocal translocation between chromosomes 15 and 17 and 80% of the cases present with bleeding diathesis caused by severe coagulopathy. The translocation generates a fusion transcript joining the PML(promyelocyte) and RAR-α (retinoic acid receptor-α) genes. The therapy of APL has been revolutionized by the introduction of differentiating agents All Trans Retinoic Acid (ATRA) and Arsenic Trioxide.All patients were treated as per Pethema protocol. Initially LPA99 (Sanz MA. Blood. 1999 Nov 1;94(9):3015-21) and since January 2012 a risk adapted therapy based on LPA2005 (Sanz MA et al. Blood June 24, 2010 vol. 115 no. 25 5137–5146). Treatment included induction followed by 3 cycles of consolidation and two years of maintenance.Nineteen patients were diagnosed with APL between January 2009 and June 2012. Three patients are excluded from the analysis as karyotyping and/or PCR did not confirm the diagnosis. The median age was 35 years (range 22–53 years). Male to female ratio was 4:1. Nine (56%) patients were stratified as high risk (WBC ≥ 10 ×109/l) while, seven (44%) as intermediate risk and low risk (WBC < 10 ×109/l). Three (19%) patients had early death despite treatment and supportive care. The cause of death was intracranial hemorrhage (1) pulmonary hemorrhage (1) and multi-organ failure (1). Thirteen patients achieved a complete morphological and molecular remission (80%). There has been only 1 case of treatment failure (high risk at presentation). This patient was successfully re-induced with arsenic trioxide and achieved second molecular complete remission. Unfortunately he relapsed a second time and is currently alive in third morphological remission but remains PCR positive for PML/RARα 33 months after diagnosis.Our limited experience shows favorable outcome (CR 80%) for the treatment of APL using the Pethema protocol compared to published data (Tallman MS. Blood 2002;99(3):759–767). Early death rate remains high despite intensive supportive care. The only variable is the availability and initiation of ATRA at the clinical suspicion of diagnosis both at the referring hospitals and treatment center. Disclosures:No relevant conflicts of interest to declare.