Differentiation Therapy Research Articles

Genome-wide miRNA profiling and pivotal roles of miRs 125a-5p and 17-92 cluster in human neutrophil maturation and differentiation of acute myeloid leukemia cells.

MicroRNAs (miRNAs, miRs) are short non-coding post-transcriptional regulators of gene expression in normal physiology and disease. Acute myeloid leukemia is characterized by accumulation of malignantly transformed immature myeloid precursors, and differentiation therapy, used to overcome this differentiation blockage, has become a successful therapeutic option. The human HL-60 acute leukemia cell line serves as a cell culture model for granulocytic maturation, and dimethyl sulfoxide (DMSO) incubation leads to its differentiation towards neutrophil-like cells, as assessed by biochemical, functional and morphological parameters. DMSO-induced HL-60 cell differentiation constitutes an excellent model to examine molecular processes that turn a proliferating immortal leukemic cell line into mature non-proliferating and apoptosis-prone neutrophil-like end cells. By performing genome-wide miRNA profiling and functional assays, we have identified a signature of 86 differentially expressed canonical miRNAs (51 upregulated; 35 downregulated) during DMSO-induced granulocytic differentiation of HL-60 cells. Quantitative real-time PCR was used to validate miRNA expression. Among these differentially expressed canonical miRNAs, we found miR-125a-5p upregulation and miR-17-92 cluster downregulation acted as major regulators of granulocytic differentiation in HL-60 cells. Enforced expression of miR-125a-5p promoted granulocytic differentiation in HL-60 cells, whereas miR-17-92 ectopic expression inhibited DMSO-induced HL-60 granulocytic differentiation. Ectopic expression of miR-125a-5p also promoted granulocytic differentiation in human acute promyelocytic leukemia NB4 cells, as well as in naïve human primary CD34+-hematopoietic progenitor/stem cells. These findings provide novel molecular insights into the identification of miRNAs regulating granulocytic differentiation of human leukemia cells and normal CD34+-hematopoietic progenitor/stem cells, and may assist in the development of novel miRNA-targeted therapies for leukemia.

Comparison of survivor scores for differentiation therapy of cancer to those for checkpoint inhibition: Half full or half empty.

Differentiation therapy is directed to the self-renewing cancer stem cells, as well as their progeny transit amplifying cells, to force them to mature to terminal differentiation. Differentiation therapy is effective in treatment of neuroblastomas and myeloid leukemias. Checkpoint inhibition therapy removes blocks to cancer reactive T-killer cells and allows them to react to malignant cells and limit the growth of cancer. The percentage of patients with a given cancer that responds to either therapy is less than hoped for, and the duration of response is variable. Multiplying the response rate (percentage of patients responding to therapy) by the duration of response may be used to derive a survival score for patients treated with differentiation therapy or checkpoint inhibition. By this criterion, differentiation therapy gives better survival scores than checkpoint inhibition. Yet, checkpoint inhibition is considered a great success, mostly because it may be applied to many different types of cancer, and differentiation therapy is considered relatively ineffective because it is limited to a few specific cancers. On the other hand, the cost of checkpoint inhibition treatment is 10-20 times more per patient than that of differentiation therapy. Hopefully, future combined treatments and advances in both approaches will increase the effectiveness of these cancer treatments.

The Multifaceted p21 (Cip1/Waf1/CDKN1A) in Cell Differentiation, Migration and Cancer Therapy.

Loss of cell cycle control is characteristic of tumorigenesis. The protein p21 is the founding member of cyclin-dependent kinase inhibitors and an important versatile cell cycle protein. p21 is transcriptionally controlled by p53 and p53-independent pathways. Its expression is increased in response to various intra- and extracellular stimuli to arrest the cell cycle ensuring genomic stability. Apart from its roles in cell cycle regulation including mitosis, p21 is involved in differentiation, cell migration, cytoskeletal dynamics, apoptosis, transcription, DNA repair, reprogramming of induced pluripotent stem cells, autophagy and the onset of senescence. p21 acts either as a tumor suppressor or as an oncogene depending largely on the cellular context, its subcellular localization and posttranslational modifications. In the present review, we briefly mention the general functions of p21 and summarize its roles in differentiation, migration and invasion in detail. Finally, regarding its dual role as tumor suppressor and oncogene, we highlight the potential, difficulties and risks of using p21 as a biomarker as well as a therapeutic target.

The SUMO Pathway in Hematomalignancies and Their Response to Therapies

SUMO (Small Ubiquitin-related MOdifier) is a post-translational modifier of the ubiquitin family controlling the function and fate of thousands of proteins. SUMOylation is deregulated in various hematological malignancies, where it participates in both tumorigenesis and cancer cell response to therapies. This is the case for Acute Promyelocytic Leukemias (APL) where SUMOylation, and subsequent destruction, of the PML-RARα fusion oncoprotein are triggered by arsenic trioxide, which is used as front-line therapy in combination with retinoic acid to cure APL patients. A similar arsenic-induced SUMO-dependent degradation was also documented for Tax, a human T-cell lymphotropic virus type I (HTLV1) viral protein implicated in Adult T-cell Leukemogenesis. SUMOylation also participates in Acute Myeloid Leukemia (AML) response to both chemo- and differentiation therapies, in particular through its ability to regulate gene expression. In Multiple Myeloma, many enzymes of the SUMO pathway are overexpressed and their high expression correlates with lower response to melphalan-based chemotherapies. B-cell lymphomas overexpressing the c-Myc oncogene also overexpress most components of the SUMO pathway and are highly sensitive to SUMOylation inhibition. Targeting the SUMO pathway with recently discovered pharmacological inhibitors, alone or in combination with current therapies, might therefore constitute a powerful strategy to improve the treatment of these cancers.

Relevancia del margen de resección positivo en adenocarcinoma ductal de páncreas y otros factores pronósticos

IntroductionCurrently, R1 resection is defined by the presence of tumor cells within <1mm of the resection margin. The main aim of this study was to analyze the impact of positive margins (R1) on survival outcomes in pancreatic cancer. MethodsWe performed a retrospective analysis with multivariate regression analysis of a prospective database from 2008-2017, which included resection margin status, expanded resection margin (R1<1mm), vascular resection, lymphatic involvement, surgical complications, tumor differentiation grade and adjuvant treatment. ResultsA total of 80 patients were analyzed: 42 (52%) R1; 38 (48%) R0. No differences were found in the composition of the two groups except for the vascular resection, which was more frequent in the R1 group: 12 (21%) vs 2 (3%). Overall survival in the R0 group was 19 months vs 24 months in the R1 group (p=0.13). Wide R1 (R1<1mm) had an overall survival of 21 months versus 31 months in wide R0 (p=0.55). In the multivariate analysis, only lymph node involvement (p=0.02, HR=2.88), tumor differentiation (p=0.02, HR=3.2) and adjuvant therapy (p<0.01; HR=0.21) were found to be factors related to survival. ConclusionsR1 resection is not an independent risk factor. Lymph node involvement, differentiation grade and adjuvant treatment are prognostic factors. The benefit of expanding the resection margins should be demonstrated. More studies are needed to assess the impact of the resection margin

2000 - MONOLINEAGE ORIGIN OF RELAPSE FOLLOWING MULTILINEAGE DIFFERENTIATION THERAPY OF ACUTE MYELOID LEUKEMIA

Acute myeloid leukaemia (AML) is characterized by the accumulation of transformed immature myeloid blasts. While most AML patients treated with standard therapy have poor outcomes, in the APL disease subtype retinoic acid induces leukaemia maturation and can be curative in combination with arsenic trioxide. Recently approved mutant IDH1/2 inhibitors also induce AML maturation, renewing interest in AML differentiation therapy. To examine differentiation therapy dynamics in vivo we have generated a novel mouse AML model driven by reversible RNAi-mediated knockdown of the myeloid transcription factor PU.1. Restoration of endogenous PU.1 in established AML in vivo triggers synchronous differentiation of leukemic blasts and disease clearance. However, despite near-complete remission, mice reproducibly relapse with immature AML. Notably, in vivo time course studies reveal that one week after PU.1 restoration leukemic blasts differentiate into two mature myeloid lineages with distinct immunophenotype and morphology. AML-derived SSClowLy6G+ cells resembling neutrophils initially predominate but are rapidly eradicated in vivo. In contrast, high resolution flow and imaging indicates that mature AML-derived SSChighF4/80+SigF+ eosinophil-like cells persist at low numbers in specific organs during disease remission and appear to seed relapse. In mice transplanted with AML blasts lacking the essential eosinophil lineage transcription factor GATA1, in vivo PU.1 restoration triggers neutrophil but not eosinophil lineage differentiation and thereby eliminates residual disease. These results demonstrate that AML differentiation therapy can produce long-lived sublineages of mature AML-derived cells from which relapse can originate. Understanding the multilineage potential of AML blasts in individual patients may inform new strategies to improve differentiation therapy outcomes.

Interconversion between Tumorigenic and Differentiated States in Acute Myeloid Leukemia.

Tumors are composed of phenotypically heterogeneous cancer cells that often resemble various differentiation states of their lineage of origin. Within thishierarchy, it is thought that an immature subpopulation of tumor-propagating cancer stem cells (CSCs) differentiates into non-tumorigenic progeny, providing a rationale for therapeutic strategies that specifically eradicate CSCs or induce their differentiation. The clinical success of these approaches depends on CSC differentiation being unidirectional rather than reversible, yet this question remains unresolved even in prototypically hierarchical malignancies, such as acute myeloid leukemia (AML). Here, we show in murine and human models of AML that, upon perturbation of endogenous expression of the lineage-determining transcription factor PU.1 or withdrawal of established differentiation therapies, some mature leukemia cells can de-differentiate and reacquire clonogenic and leukemogenic properties. Our results reveal plasticity of CSC maturation in AML, highlighting the need to therapeutically eradicate cancer cells across a range of differentiation states.

Development of functional dendrisomes based on a single molecule of polyesterbenzylether dendrimer and their application in cancer stem cell therapy

The differentiation therapy of breast cancer stem cells (CSCs) represents a significant strategy to treat cancer, but inefficient delivery to CSCs hinders the efficacy of differentiation agents. In this work, we report a type of functional dendrisomes constructed by the synthesized amphiphilic dendrimers, which enhance cellular uptake by breast CSCs, differentiate breast CSCs by carrying all-trans retinoic acid (ATRA), and increase the anticancer efficacy by carrying ATRA and docetaxel (DTX) in vitro and in breast cancer-bearing mice. The study further reveals the mechanism of cellular uptake by breast CSCs and uncovers the differentiation mechanism by analyzing relevant signal molecules, transcription factors, and cell cycle-associated signaling pathways during differentiation therapy in breast CSCs. Hence, this study offers a novel type of functional dendrisomes for differentiation therapy of breast CSCs and has significant clinical implications.

Exploring the information transmission properties of noise-induced dynamics: application to glioma differentiation

BackgroundCells operate in an uncertain environment, where critical cell decisions must be enacted in the presence of biochemical noise. Information theory can measure the extent to which such noise perturbs normal cellular function, in which cells must perceive environmental cues and relay signals accurately to make timely and informed decisions. Using multivariate response data can greatly improve estimates of the latent information content underlying important cell fates, like differentiation.ResultsWe undertake an information theoretic analysis of two stochastic models concerning glioma differentiation therapy, an alternative cancer treatment modality whose underlying intracellular mechanisms remain poorly understood. Discernible changes in response dynamics, as captured by summary measures, were observed at low noise levels. Mitigating certain feedback mechanisms present in the signaling network improved information transmission overall, as did targeted subsampling and clustering of response dynamics.ConclusionComputing the channel capacity of noisy signaling pathways present great probative value in uncovering the prevalent trends in noise-induced dynamics. Areas of high dynamical variation can provide concise snapshots of informative system behavior that may otherwise be overlooked. Through this approach, we can examine the delicate interplay between noise and information, from signal to response, through the observed behavior of relevant system components.

Abstract 2991: Targeting glutamine metabolism leads to terminal differentiation in acute myeloid leukemia (AML)

Abstract The ability of All Trans Retinoic Acid (ATRA) to induce terminal differentiation in Acute Promyelocytic Leukemia (APL) has revolutionized treatment for this deadly subset of AML. Unfortunately, identifying targets that induce differentiation in non-APL cases of AML has met with limited success. Our lab has been interested in the role of glutamine metabolism in cellular differentiation programs. As such, we have investigated the degree to which glutamine contributes to the maintenance of an undifferentiated state in AML. Herein, we report that in vitro incubation with the glutamine blocking agent, 6-diazo-5-oxo-L-norleucine (DON) robustly induces differentiation in the human AML cell line, U937. DON treatment induces phenotypic and molecular changes indicative of differentiation, including increased granularity and vacuolization, and upregulation of differentiation markers CD11b and CD14. The differentiation of these cells was confirmed by their highly abrogated growth in methylcellulose-based colony-forming assays. Differentiated AML cells were not exposed to DON during colony-forming assays, suggesting an irreversible change in cellular differentiation triggered by short-term pretreatment. We have demonstrated that DON-induced cellular differentiation is active in other AML cell lines as well. Importantly, the differentiating effect of DON can be abrogated by the addition of a cell permeable form of α-ketoglutarate (αKG), dimethyl-2-ketoglutarate (DMK). αKG is a key metabolite in glutamine metabolism and is also a critical cofactor for a range of demethylase enzymes involved in epigenetic remodeling. Indeed, we have noted a robust increase in histone methylation marks, such as H3K27Me3, during DON-induced differentiation. Further, we have observed a dramatic increase in the transcription of critical transcription factors (CEBPA, FLT3, MYB, SOX4) twenty-four hours after DON treatment, a time point immediately preceding observed phenotypic differentiation changes. In light of these findings, experiments are currently underway to use DON differentiation therapy as a means of treating mouse models of AML. Taken as a whole, these data support the use of glutamine blockade as a novel differentiating therapy in AML. Given the remarkable curative potential of differentiation therapy and the poor survival rates of adults with AML (particularly older patients), our findings represent a particularly novel approach to treating this deadly disease. Citation Format: Robert Leone, Im-Meng Sun, Roshan Chikarmane, Radhika Viswanathan, Sanjeda Patwari, Matthew Arwood, Gabriel Ghiaur, Jonathan Powell. Targeting glutamine metabolism leads to terminal differentiation in acute myeloid leukemia (AML) [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 2991.

Effect of traditional Chinese medicine syndrome differentiation and standard bundle therapy in patients with septic shock

To evaluate an effective and feasible quantitative evaluation table of traditional Chinese medicine (TCM) syndrome differentiation, and to observe the effect of combination of TCM syndrome differentiation and standard bundle therapy in patients with septic shock. A prospective randomized controlled trial was conducted. The septic shock patients with acute deficiency syndrome admitted to department of critical care medicine of Ruijin Hospital, Shanghai Jiao Tong University School of Medicine from January 1st, 2016 to December 31st, 2017 were enrolled. The patients were randomly divided into control group and Shenfu group. The patients in both groups received early application of standardized bundle therapy; those in Shenfu group received 60 mL Shenfu injection infusion in addition for 7 days. The TCM syndrome score was evaluated by classification and scoring method of TCM symptoms. The circulation and tissue perfusion, severity of disease, organ function, inflammation response, adjuvant treatment and 28-day mortality were compared between the two groups. A total of 50 patients with septic shock were enrolled in the analysis, 25 in control group and 25 in Shenfu group. The markedly effective rate of TCM symptoms score in Shenfu group was significantly higher than that in control group [60.0% (15/25) vs. 16.0% (4/25), P < 0.01]. There was no significant difference in all parameters before treatment between the two groups. After treatment, the observation indexes of both groups were improved. Compared with control group, the mean arterial pressure (MAP) in Shenfu group increased more significantly [mmHg (1 mmHg = 0.133 kPa): 13.0 (2.5, 28.5) vs. 6.0 (0, 13.5)], the lactate (Lac) and procalcitonin (PCT) decreased more significantly [Lac (mmol/L): 0.8 (0.1, 3.7) vs. 0.5 (-0.6, 1.7), PCT (μg/L): 2.0 (0.7, 32.3) vs. 0 (-1.8, 3.8)], activated partial thromboplastin time (APTT) was shortened more significantly [s: 8.5 (0, 12.9) vs. 0 (-7.2, 10.0)], and interleukins (IL-2 receptor and IL-6) levels decreased more significantly [IL-2 receptor (ng/L): 1 031.0 (533.0, 1 840.0) vs. 525.5 (186.0, 1 166.8), IL-6 (ng/L): 153.1 (21.4, 406.8) vs. 35.1 (16.3, 110.1)] with significant differences (all P < 0.05). There was no significant difference in the use time of vasoactive drugs, duration of mechanical ventilation, severity of the disease or 28-day mortality between the two groups. However, the use time of vasoactive drugs in Shenfu group was shorter than that in control group (days: 5.48±4.81 vs. 8.28±7.83), and the 28-day mortality was decreased [8.0% (2/25) vs. 20.0% (5/25)]. TCM syndrome score is helpful to evaluate the effect of TCM syndrome differentiation and treatment, and it is effective and feasible in clinical application. Septic shock patients treated with TCM syndrome differentiation and treatment combined with standard bundle therapy were significantly improved in circulation, tissue perfusion, coagulation function and inflammation reaction.

Abstract 3798: Acetate supplementation eliminates hypoxia mediated resistance to differentiation therapy in neuroblastoma cells

Abstract Neuroblastoma is the most common extracranial solid tumor in childhood. Retinoic acid (RA) leads to cell cycle arrest and induces cell differentiation in neuroblastoma cells, thus it has been used to treat neuroblastoma as differentiating agent for decades. However, therapy resistance often occurs and the overall survival remains low. Hypoxia, a common metabolic stress existing in tumor microenvironment, increases tumor resistance to RA by promoting dedifferentiation of neuroblastoma cells. The aim of this study was to elucidate the mechanisms underlying hypoxia-mediated therapy resistance and develop therapeutic approach to improve the response to differentiation therapy. By RNA-Seq analysis, we identified a group of neuron-specific differentiation markers that induced by RA treatment, while the induction was diminished under hypoxia. Associated with the silencing of differentiation markers expression, a significant loss of histone acetylation of H3K9, H3K27 and total H3 was observed under hypoxia. Metabolomics analysis indicated hypoxia inhibited mitochondrial oxidative phosphorylation to decrease intracellular acetyl-CoA and citrate, which provides acetyl-CoA for histone acetyltransferase. Importantly, acetate supplementation restored cellular acetyl-CoA level, histone acetylation, and the expression of differentiation markers under hypoxia. Differentiation morphology was also restored by acetate supplementation under hypoxia. In conclusion, our finding suggested hypoxia contributed to the resistance to RA-induced differentiation by reprogramming cellular metabolism and inducing histone hypoacetylation. Acetate supplementation is an efficacious therapeutic approach to improve the sensitivity of hypoxic tumor to RA-based differentiation therapy. Citation Format: Yang Li, Yiren Zhou, John M. Maris, Amato J. Giaccia, Jiangbin Ye. Acetate supplementation eliminates hypoxia mediated resistance to differentiation therapy in neuroblastoma cells [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 3798.

Abstract 3695: A protein tyrosine phosphatase 4A3 (PRL-3)/Wnt signaling axis as a novel therapeutic target in acute lymphoblastic leukemia (ALL) relapse

Abstract Acute Lymphoblastic Leukemia (ALL) is the most common pediatric malignancy and 15-20% of patients experience disease relapse, which is frequently more aggressive and treatment resistant than the primary disease, and often has unfavorable outcome. Relapse occurs because conventional chemotherapies are unable to reliably and completely eliminate leukemia stem cells (LSCs), which are the only cells within the leukemia with the ability to self-renew and remake or replenish a leukemia from a single cell. Eliminating LSCs can greatly improve patient outcome; for example, differentiation therapy, which blocks LSC self-renewal, has improved the survival of Acute Pro-myelocytic Leukemia to &amp;gt;95%. We have previously completed a &amp;gt;8,000 animal screen in a zebrafish Myc-induced ALL model and identified a panel of zebrafish ALL, in which the leukemia stem cell frequency is ~1 in every 10 cells, and identified a unique leukemia stem cell signature. The Wnt pathway was highly expressed by LSCs, and has also emerged in mouse studies as having an important role in LSC self-renewal in T-ALL. Current Wnt inhibitors have unacceptably toxicity in the clinic. I have found that Protein Tyrosine Phosphatase 4A3 (PTP4A3 or PRL3) is highly expressed by ALL cells that also express Wnt pathways genes, and is not expressed by normal cell types. In a zebrafish Myc-induce ALL model, PRL3 expression significantly enhanced leukemia stem cell frequency, while inhibition of PRL3 reduced LSC numbers in vivo. In human cells, I found that PRL3 regulates the expression of downstream Wnt pathway target genes, and we are currently testing the effects of small molecule inhibition of PRL3 on the phosphorylation status of proteins involved in Wnt signaling, to define the mechanism of PRL3 action in this pathway. Apart from identifying novel interacting partners in the Wnt pathway, I have also developed a novel zebrafish reporter line that can be used to identify other small molecules that block Wnt signaling. In these fish, Wnt expressing leukemia stem cells are fluorescently tagged. These leukemias will be used for in vivo drug screens to identify FDA-approved compounds that block Wnt signaling and/or target leukemia stem cells without adverse effects on developing zebrafish larvae. In total, my research will define a novel role for the phosphatase PRL3 in self-renewal of leukemia stem cells via activation of Wnt signaling. These data will demonstrate that “untargetable” oncogenes like Wnt can be targeted by inhibiting easily druggable critical interacting proteins, such as phosphatases like PRL3. This work is also likely to have a positive translational impact by identifying FDA-approved drugs that block LSC self-renewal in ALL and other types of Wnt and/or PRL3 dependent cancers. Citation Format: Meghan G. Haney, Kristin O'Leary, Jessica S. Blackburn. A protein tyrosine phosphatase 4A3 (PRL-3)/Wnt signaling axis as a novel therapeutic target in acute lymphoblastic leukemia (ALL) relapse [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 3695.

The Role of AMPK/mTOR Modulators in the Therapy of Acute Myeloid Leukemia.

Differentiation therapy of acute promyelocytic leukemia with all-trans retinoic acid represents the most successful pharmacological therapy of acute myeloid leukemia (AML). Numerous studies demonstrate that drugs that inhibit mechanistic target of rapamycin (mTOR) and activate AMP-kinase (AMPK) have beneficial effects in promoting differentiation and blocking proliferation of AML. Most of these drugs are already in use for other purposes; rapalogs as immunosuppressants, biguanides as oral antidiabetics, and 5-amino-4-imidazolecarboxamide ribonucleoside (AICAr, acadesine) as an exercise mimetic. Although most of these pharmacological modulators have been widely used for decades, their mechanism of action is only partially understood. In this review, we summarize the role of AMPK and mTOR in hematological malignancies and discuss the possible role of pharmacological modulators in proliferation and differentiation of leukemia cells.

Abstract 3064: Epigenetic manipulation can sensitize AML cells to differentiate with ATRA

Abstract Human acute myeloid leukemia (AML), the most common type of acute leukemia, has approximately eight subtypes, many of which have poor prognosis. Most of these subtypes are associated with specific, recurrent chromosome translocations. These translocations result in fusion genes, which encode oncoproteins that block differentiation and promote proliferation of immature cells. The Myeloid Lymphoid Leukemia gene (MLL) is frequently involved in these translocations, and is considered a driver of the AML. Differentiation promoting drugs, such as all-trans-retinoic acid (ATRA) are an attractive alternative to cytotoxic chemotherapy, but few types of AML, other than acute promyelocytic leukemia (APL), respond to ATRA. We hypothesize that specific genes must be activated or inhibited in AML for drugs like ATRA to induce differentiation, and that gene activation or inhibition may be the result of specific epigenetic modification. We also hypothesize that AML with different genetic alterations may respond differently to specific epigenetic inhibitors. Our initial studies have focused on two MLL-driven AML cell lines, MV4;11 and THP-1, showing translocations t(4;11) and t(9;11), respectively, and one non-MLL related AML cell line, U937. MV4;11, THP-1, and U937 were treated with specific epigenetic modifiers, including tranylcypromine (TCP), an inhibitor of histone demethylase KMD1A/LSD1, N-acetyl-dinaline (CI-994), a general histone deacetylase inhibitor, and 3-deazaneplanocin A (DZNep), a S-adenosylhomocystein hydrolase inhibitor that depletes EZH2 and thus its associated H3K27me3 activity. Evidence for differentiation was noted in a variety of assays, including reduced cell proliferation as measured directly and by MTT assay, upregulation of myeloid-specific cell surface markers such as CD11b as measured by fluorescence-activated cell sorting (FACS), myeloid-related nuclear morphological changes noted with cytospin analysis of cells, and decreased AML-associated gene expression with qPCR. All three drugs seemed to sensitize U937 and THP-1 cells to differentiate when treated with ATRA. MV4;11 cells were sensitized to differentiate by CI-994 and DZNep with or without ATRA, although there was no indication of CD11b upregulation. However, MV4;11 was not sensitized by TCP to differentiate with or without ATRA. These experiments suggest epigenetic inhibitors may increase sensitivity to ATRA differentiation therapy, but since the two MLL cell lines responded differently the response may still be dependent on the specific MLL partner gene driving the AML. Citation Format: Kalsi Heimdal, Bikalpa Ghimire, Edjay Ralph Hernandez, Heidi J. Gill Super. Epigenetic manipulation can sensitize AML cells to differentiate with ATRA [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 3064.

CDK inhibitors reduce cell proliferation and reverse hypoxia-induced metastasis of neuroblastoma tumours in a chick embryo model

Neuroblastoma is a paediatric cancer with a poor prognosis. This is in part due to widespread metastasis at time of presentation, which is refractory to current treatment modalities. New therapeutic agents that can control not only tumour growth but also metastasis are urgently needed. The differentiation therapy, retinoic acid, is currently used in clinic, leading to terminal differentiation of neuroblastoma cells thus reducing tumour growth in the primary tumour as well as at metastatic sites. However, retinoic acid only works in a subset of patients. We investigated the potential of CDK inhibitors, Palbociclib and RO-3306, on neuroblastoma cell differentiation, tumour progression and metastasis by utilising a 3R compliant cost effective preclinical chick embryo model. In both SK-N-AS and BE(2)C cell lines, when engrafted on the chorioallantoic membrane of chick embryos, we observed a reduction of tumour cell proliferation as well as a reduction in hypoxia preconditioning-driven metastasis by 60%. In addition, the expression of a panel of genes with known roles in metastasis, which increased upon hypoxia-preconditioning, was largely reduced by a CDK1 inhibitor. These results provide a promising alternative to currently existing therapies and might aid the development of new treatment protocols for retinoic acid-resistant patients.

Prediction of neuroblastoma cell response to treatment with natural or synthetic retinoids using selected protein biomarkers.

Although the administration of retinoids represents an important part of treatment for children suffering from high-risk neuroblastomas, approximately 50% of these patients do not respond to this therapy or develop resistance to retinoids during treatment. Our study focused on the comparative analysis of the expression of five genes and corresponding proteins (DDX39A, HMGA1, HMGA2, HOXC9 and PBX1) that have recently been discussed as possible predictive biomarkers of clinical response to retinoid differentiation therapy. Expression of these five candidate biomarkers was evaluated at both the mRNA and protein level in the same subset of 8 neuroblastoma cell lines after treatment with natural or synthetic retinoids. We found that the cell lines that were HMGA2-positive and/or HOXC9-negative have a reduced sensitivity to retinoids. Furthermore, the experiments revealed that the retinoid-sensitive cell lines showed a uniform pattern of change after treatment with both natural and sensitive retinoids: increased DDX39A and decreased PBX1 protein levels. Our results showed that in NBL cells, these putative protein biomarkers are associated with sensitivity or resistance to retinoids, and their endogenous or induced expression can distinguish between these two phenotypes.

Investigation of specific binding of designed oligodeoxynucleotide decoys to transcription factors in HT29 cell line undergoing epithelial-mesenchymal transition (EMT).

Expression of master transcriptional regulators of stem cells (Oct4 and Sox2) is associated with mediating tumor proliferation and tumor differentiation. The main goal of this study is the investigation of specific binding of designed Oct4-Sox2 transcription factors decoy oligodeoxynucleotides (ODNs) sequence to their nucleus-extracted proteins in HT29-ShE cells containing enriched cancer stem-like cells (SCLCs). First, gene expression of Oct4, Sox2, and E-cadherin revealed the overexpression of Oct4 and Sox2 and downregulation of E-cadherin in HT29-ShE cells compared with HT29 wild-type and HT29-ShC cells. Next, Oct4-Sox2 complex decoy ODNs were designed according to their elements in the promoter region of Sox2 gene. Then, the interactions of Oct4 and Sox2 proteins to designed ODNs were evaluated in silico. Finally, DNA-protein interactions of decoy ODNs and their corresponding proteins were examined by electrophoretic mobility shift assay (EMSA). Analysis of gel shift retardation assay admitted the specific binding of designed ODNs sequence to the nuclear extracted Oct4 and Sox2 proteins. The results will be a promising approach to target cancer stem cells for potential use in differentiation therapy before chemotherapy and radiotherapy of cancers.

Dual Delivery of HNF4α and Cisplatin by Mesoporous Silica Nanoparticles Inhibits Cancer Pluripotency and Tumorigenicity in Hepatoma-Derived CD133-Expressing Stem Cells.

Hepatocellular carcinoma (HCC) is one of the most prevalent and deadly malignancies characterized by high rate of recurrence. Tumor recurrence is often attributed to the presence of a subpopulation of cells with stem cell properties, referred to as cancer stem cells (CSCs). Traditionally, cancer therapies target the entire bulk of tumor cells; however, they are poorly effective against CSCs, characterized by higher drug resistance. Therefore, approaches targeting CSCs may be required in addition to conventional chemotherapy to prevent tumor recurrence. In this study, we investigated an approach to target HCC by combining the conventional chemotherapeutic drug, cisplatin, to target the bulk of tumor cells, and differentiation therapy by delivering the gene encoding HNF4α, an important regulator of hepatocyte differentiation, to target CSCs. We used the Huh7 cell line as an in vitro model of HCC, which is characterized by a high proportion of CD133-expressing CSCs. By using flow cytometry, we separated CD133+ and CD133- Huh7 cell subpopulations and have shown that the former has highly pronounced in vivo tumorigenic capacity in contrast to the latter, which could not generate tumors in vivo. For the dual delivery of HNF4α-encoding plasmid and cisplatin, we used polyethyleneimine-modified mesoporous silica nanoparticles (PMSNs) as the nanocarriers. Here, we show that the treatment of CD133-expressing Huh7 cells with HNF4α-loaded PMSNs can suppress their proliferation rate, decrease the proportion of CSCs, downregulate stemness-associated genes, and increase the expression of mature hepatocyte-associated genes. At the same time, the treatment of Huh7 with PMSNs loaded with both HNF4α-encoding plasmid and cisplatin could block them in the S-phase of the cell cycle and cause apoptosis. In addition, dually loaded PMSNs were the most efficient formulation in suppressing tumor growth in vivo. To summarize, in this study, we tested the nanoparticle-based delivery system as both chemotherapy and gene-based therapy agents, which has great potential for development of effective treatment of HCC.

Epigenetic drug library screening identified an LSD1 inhibitor to target UTX-deficient cells for differentiation therapy

UTX (also known as KDM6A), a histone 3 lysine 27 demethylase, is among the most frequently mutated epigenetic regulators in myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). Recent studies have suggested that UTX mutations promote MDS and AML by blocking the differentiation of hematopoietic stem and progenitor cells (HSPCs). Here, we performed an epigenetic drug library screening for small molecules able to release the differentiation block on HSPCs induced by UTX deficiency. We found that SP2509, a selective inhibitor of LSD1, specifically promoted the differentiation of Utx-null HSPCs while sparing wild-type HSPCs. Transcriptome profiling showed that Utx loss reduced the expression of differentiation-related and tumor suppressor genes, correlating with their potential roles in HSPC self-renewal and leukemogenesis. In contrast, SP2509 treatment reversed these changes in gene expression in Utx-null HSPCs. Accordingly, Utx loss decreased H3K4 methylation level probably through the COMPASS-like complex, while LSD1 inhibition by SP2509 partially reversed the reduction of H3K4 methylation in Utx-deficient HSPCs. Further, SP2509 promoted the differentiation of Utx-null AML cells in vitro and in vivo and, therefore, extended the survival of these leukemic mice. Thus, our study identified a novel strategy to specifically target both premalignant and malignant cells with Utx deficiency for differentiation therapy and provided insights into the molecular mechanisms underlying the role of Utx in regulating HSPCs and related diseases.