Lymphoblastic Leukemia Cell Line Research Articles

Simultaneous changes in expression levels of BAALC and miR-326: a novel prognostic biomarker for childhood ALL.

Multidrug resistance and consequent relapse are two major obstacles for treating children with acute lymphoblastic leukemia, the most frequent childhood malignancy. MicroRNAs have potential regulatory roles in response to chemotherapy. The goal of this study was to determine the microRNA that may have effects on the expression level of brain and acute lymphoblastic leukemia (BAALC) and to investigate the in vitro and ex vivo association between their expression levels. In silico tools were utilized to determine a putative miRNA targeting BALLC. Quantitative real-time polymerase chain reaction was used to investigate expression levels of BAALC and its predicted microRNA, miR-326, in bone marrow samples of 30 children with acute lymphoblastic leukemia and 13 controls, in addition to the resistant and parental CCRF-CEM cell lines. To assess the status of response to therapy, minimal residual disease was measured using single-strand conformation polymorphism. MiR-326 was selected due to the strong possibility of its interaction with BAALC according to the obtained in silico results. Statistical analysis showed a significant downregulation of miR-326 and overexpression of BALLC in drug-resistant acute lymphoblastic leukemia cell line and patients compared with the parental cell line and drug-sensitive patients, respectively (P=0.015, 0.005, 0.0484 and 0.0005, respectively). The expression profiles of miR-326 and BAALC were inversely correlated (P=0.028). The results introduced the inversely combined expression levels of miR-326 and BAALC as a novel, independent prognostic biomarker for pediatric acute lymphoblastic leukemia (P=0.007). Moreover, bioinformatics data showed a possible regulatory role for miR-326 on BAALC mRNA, which may possibly contribute to the development of drug resistance in patients with childhood acute lymphoblastic leukemia.

Investigating the inhibitory effect of miR-34a, miR-449a, miR-1827, and miR-106b on target genes including NOTCH1, c-Myc, and CCND1 in human T cell acute lymphoblastic leukemia clinical samples and cell line.

Objective(s):microRNAs are small non-coding molecules that regulate gene expression in various biological processes. T-cell acute lymphoblastic leukemia (T-ALL) is a malignancy accompanied with genetic aberrations and accounts for 20% of children’s and adult’s ALL. Notch signaling pathway dysregulation occurs in 60% of T-ALL cases. In the present study, we aimed to determine the relationship between miRNAs and genes involved in Notch signaling pathway. Materials and Methods:Considering the role of the pathway and its down-stream genes in proliferation, differentiation, cell cycle, and apoptosis, NOTCH1, c-Myc, and CCND1 genes were selected as target genes. Using bioinformatics studies, miR-34a, miR-449a, miR-1827, and miR-106b were selected as miRNAs targeting the above-mentioned genes. We evaluated these genes and miRNAs in T-ALL clinical samples as well as Jurkat cell line, in which NOTCH1 is overexpressed. Results:Quantitative Real-Time PCR indicated that NOTCH1, c-Myc, and CCND1 were overexpressed in samples with decreased expression of miR-34a. In addition, we observed that samples with decreased expression of miR-449a showed increased expression of NOTCH1 and CCND1. Furthermore, we analyzed the expression of miR-1827 and miR-106b, which target c-Myc and CCND1, respectively. We found out that the expression of miR-1827, miR-106b, and their respective target genes were inversely correlated in 80% and 75% of the cases (r=0.8), respectively. Furthermore, in Jurkat cell line, the expression of target genes was increased while the candidate miRNAs except miR-34a were decreased. Conclusion:These miRNAs can be proposed as biomarkers and new therapeutic targets in T-ALL patients who have NOTCH1 overexpression.

Introducing a novel chemotherapeutic drug formulated by iron nanoparticles for the clinical trial studies

AbstractIn recent years, the experiments have revealed that plants raise the cytotoxicity and anticancer potentials of iron nanoparticles against several cell lines especially tumor cell lines. In the present study, we formulated a modern drug by iron nanoparticles (FeNPs) containing Glycyrrhiza glabra L leaf for the treatment of acute lymphoblastic leukemia. Characterization of FeNPs was done by TEM, FE‐SEM, UV–Vis, and FT‐IR. FE‐SEM and TEM images revealed an average diameter of 11–18 nm for the nanoparticles. UV–Vis revealed an absorption band at 298 nm that is related to the surface plasmon resonance of FeNPs. FT‐IR results offered antioxidant compounds in the nanoparticles were the sources of reducing power, reducing iron ions to FeNPs. MTT assay was used on normal (HUVEC) and acute lymphoblastic leukemia (MOLT‐3, CEM/C2, TALL‐104, and CCRF‐CEM) cell lines for comparing the anti‐acute lymphoblastic leukemia properties of FeCl3, G. glabra, and FeNPs. Iron nanoparticles had very low cell viability dose‐dependently against MOLT‐3, CEM/C2, TALL‐104, CCRF‐CEM cell lines without any cytotoxicity on the HUVEC cell line. The best result of cytotoxicity property of FeNPs against the above cell lines was seen in the case of CCRF‐CEM cell line. These nanoparticles can be administrated in humans for the treatment of acute lymphoblastic leukemia after confirming in the in vivo and clinical trials.

Pyrvinium Pamoate Use in a B cell Acute Lymphoblastic Leukemia Model of the Bone Tumor Microenvironment.

Pyrvinium pamoate (PP) is an anthelmintic drug that has been found to have anti-cancer activity in several cancer types. In the present study, we evaluated PP for potential anti-leukemic activity in B cell acute lymphoblastic leukemia (ALL) cell lines, in an effort to evaluate the repurposing potential of this drug in leukemia. ALL cells were treated with PP at various concentrations to determine its effect on cell proliferation. Metabolic function was tested by evaluating Extracellular Acidification Rate (ECAR) and Oxygen Consumption Rate (OCR). Lastly, 3D spheroids were grown, and PP was reformulated into nanoparticles to evaluate distribution effectiveness. PP was found to inhibit ALL proliferation, with varied selectivity to different ALL cell subtypes. We also found that PP's cell death activity was specific for leukemic cells, as primary normal immune cells were resistant to PP-mediated cell death. Metabolic studies indicated that PP, in part, inhibits mitochondrial oxidative phosphorylation. To increase the targeting of PP to a hypoxic bone tumor microenvironment (BTME) niche, we successfully encapsulated PP in a nanoparticle drug delivery system and demonstrated that it retained its anti-leukemic activity in a hemosphere assay. We have demonstrated that PP is a novel therapeutic lead compound that counteracts the respiratory reprogramming found in refractory ALL cells and can be effectively formulated into a nanoparticle delivery system to target the BTME.

Ergolide, a potent sesquiterpene lactone induces cell cycle arrest along with ROS-dependent apoptosis and potentiates vincristine cytotoxicity in ALL cell lines

Ethnopharmacological relevanceInula oculus christi belongs to the family of Asteraceae and it was traditionally wide used in treatment of kidney stones and urethra infection; besides, recently the potent sesquiterpene lactones isolated from inula species has gained increasing attention in cancer treatments. This study investigates the anti-cancer properties and underlying mechanism of ergolide isolated from Inula oculus christi against leukemic cell lines. MethodsViability, metabolic activity and proliferation evaluated using different index of MTT assay such as IC50 and GI50. Human erythrocytes were used to evaluate hemolytic activity. Flow-cytometry was used to detect and measure ROS level, and the induction of apoptosis and autophagy were evaluated using Annexin V/PI, Acridine Orange staining, respectively. Moreover, qRT-PCR was performed to examine the expression of a large cohort of crucial regulatory genes. Tunel assay was also carried out to assess morphologically ergolide effects. ResultsErgolide did not exert ant cytotoxicity against non-tumorous cells and did not cause noticeable hemolysis. It also caused ROS production during early hours after treatment of cells which was then followed by cell cycle arrest in G0/G1 phase and autophagy induction. Using N-acetyl-L-cysteine (NAC), we found that ergolide could not increase ROS and induce autophagy and moreover repressed cell death, indicating that ergolide induce cell death through ROS-dependent manner by altering the expression of pro apoptotic related genes. Autophagy inhibition also potentiated ergolide-induced cell death. Furthermore, ergolide intensified vincristine cytotoxicity against acute lymphoblastic leukemia (ALL) cell lines revealed robust synergistic properties of ergolide with VCR. ConclusionHere we showed that ergolide could be considered as a potent natural compound against leukemic cells by inducing cell cycle arrest followed by dose-dependent cell death. Based on results, Autophagy response in a result of ROS accumulation acted as a survival pathway and blocking this pathway could noticeably increase ergolide cytotoxicity on ALL cell lines.

MiR-7 Functions as a Tumor Suppressor by Targeting the Oncogenes TAL1 in T-Cell Acute Lymphoblastic Leukemia.

Background:T-cell acute lymphoblastic leukemia is a hematologic malignancy characterized by T-cell proliferation, and in many cases, the ectopic expression of the oncogenic transcription factor T-cell acute lymphocytic leukemia protein 1 (TAL1). MicroRNA-7 has been shown to play a critical role in proliferation, migration, and treatment sensitivity in a diverse array of cancers. In this study, we sought to establish a novel link between microRNA-7 and T-cell acute lymphoblastic leukemia oncogenesis.Material and Method:To do so, we characterized gene expression of microRNA-7 as well as TAL1 in both T-cell acute lymphoblastic leukemia patient-derived tissue and cell lines, as well as performing functional luciferase assays to assess microRNA-7 binding to the TAL1 3′-untranslated region. We also performed growth, apoptosis, and migration experiments using 3-(4,5-dimethylthiazol-2-Yl)-2,5-diphenyltetrazolium bromide, Annexin V, and transwell assays in the context of microRNA-7 overexpression.Results:We found that microRNA-7 expression is attenuated and inversely correlated with TAL1 expression in TAL1 + T-cell acute lymphoblastic leukemia cells. Additionally, microRNA-7 directly targets and suppresses TAL1 levels. Finally, microRNA-7 overexpression reduces growth, motility, and migration while inducing apoptosis in T-cell acute lymphoblastic leukemia cells, phenotypes that can be rescued by concomitant overexpression of TAL1.Conclusions:These results indicate that microRNA-7 functions as a potent tumor suppressor by inhibiting the oncogene TAL1 and suggest microRNA-7 could function as a prognostic biomarker and possible therapeutic in the clinical management of T-cell acute lymphoblastic leukemia.

The oxidation of (-)-epigallocatechin-3-gallate inhibits T-cell acute lymphoblastic leukemia cell line HPB-ALL via the regulation of Notch1 expression.

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematological malignancy, and commonly associated with activating mutations in the Notch1 pathway. (−)-Epigallocatechin-3-gallate (EGCG) is the most abundant and active catechin and has been shown to regulate Notch signaling. Taking into account the highly oxidizable and unstable of EGCG, we proposed that EGCG oxides may have greater potential to regulate Notch signaling than EGCG. In this study, we isolated and identified EGCG oxides (compound 2–4), using a chemical oxidation strategy, and evaluated for cytotoxicity against T-cell acute lymphoblastic leukemia cell line (HPB-ALL) by using the MTS assay. We found compound 3 significantly induced cell proliferation inhibition (38.3858 ± 1.67106 μM), cell apoptosis and cell cycle arrest in a dose-dependent manner. Remarkably, compound 3 inhibited expression of Notch1 compared with EGCG in HPB-ALL cells. Meanwhile, we found that compound 3 significantly inhibited c-Myc and Hes1, which are downstream target genes of Notch1. The findings demonstrate for the first time that an oxidation product of EGCG (compound 3) inhibits T-cell acute lymphoblastic leukemia cell line (HPB-ALL) and is a promising agent for cancer therapy deserving further research.

Combination of ERK2 and STAT3 Inhibitors Promotes Anticancer Effects on Acute Lymphoblastic Leukemia Cells.

Deregulated activation of signaling through the RAS/RAF/mitogen-activated protein kinase/extracellular signal-regulated kinase (RAS/RAF/MEK/ERK) and signal transducer and activator of transcription (STAT) pathways is involved in numerous hematological malignancies, making it an attractive therapeutic target. This study aimed to assess the effect of the combination of ERK2 inhibitor VX-11e and STAT3 inhibitor STA-21 on acute lymphoblastic leukemia cell lines REH and MOLT-4. REH and MOLT-4 cell lines were cultured with each drug alone and in combination. Cell viability, ERK activity, cell cycle distribution, apoptosis and oxidative stress induction were assessed by flow cytometry. Protein levels of STAT3, phospho-STAT3, protein tyrosine phosphatase 4A3 (PTP4A3), survivin, p53 and p21 were determined by western blotting. VX-11e in combination with STA-21 significantly inhibited cell viability, induced G0/G1 cell-cycle arrest, enhanced production of reactive oxygen species, and induced apoptosis. These effects were associated with an increased level of p21 protein in REH cells and with reduced levels of phopho-STAT3, survivin and PTP4A3 proteins in MOLT-4 cells. Our findings provide a rationale for combined inhibition of RAS/RAF/MEK/ERK and STAT3 pathways in order to enhance anticancer effects against acute lymphoblastic leukemia cells.

An artificial nucleoside that simultaneously detects and combats drug resistance to doxorubicin.

Doxorubicin is a DNA-damaging agent used to treat hematological cancers. Unfortunately, drug resistance can occur by defective DNA repair activity coupled with the ability of DNA polymerases to misreplicate unrepaired DNA lesions. This study demonstrates that the efficacy of doxorubicin can be improved by using an artificial nucleoside to efficiently and selectively inhibit this activity. In vitro studies using acute lymphoblastic leukemia cell lines define the mechanism of cell death caused by combining an artificial nucleoside with doxorubicin. Flow cytometry experiments demonstrate that combining an artificial nucleoside with doxorubicin potentiates the cell killing effects of the drug by increasing apoptosis. The potentiation effect correlates with expression of TdT, a specialized DNA polymerase overexpressed in acute lymphoblastic leukemia. Cell cycle experiments demonstrate that this combination blocks cells at S-phase prior to inducing apoptosis. Finally, the unique chemical composition of the nucleoside analog was used to visualize the replication of damaged DNA in TdT-positive cells. This represents a potential diagnostic tool to easily identify doxorubicin-resistant cancer cells. Studies demonstrate that a novel artificial nucleoside improves the therapeutic efficacy of doxorubicin, thereby reducing the risk of potential side effects caused by the DNA-damaging agent.

Preclinical Evaluation of Human Placental-Derived Allogeneic CD19 CAR-T Cells Against B Cell Malignancies

Celularity, Inc. is developing a CD19 CAR-T Cell therapy using an allogeneic platform derived from postpartum human placental cells. T cells isolated from placenta/ umbilical cord blood and genetically modified to express CD19 chimeric antigen receptor (CAR), termed Placental-derived (P-) CD19 CAR T cells, are in development for the treatment of B cell malignancies. Unlike adult peripheral blood mononuclear cell (PBMC)-derived T cells, P-T cells are mostly naïve (CD45RA+) and can be readily expanded while maintaining an earlier differentiation phenotype such as greater expression of naïve/ memory markers, lower expression of effector/ exhaustion markers, allowing for greater proliferative potential of these cells ex vivo. These cells are also known to have greater immune tolerance to HLA mismatch and display impaired allogeneic activation, contributing to lower incidences of severe graft-verse-host disease (GvHD) (Barker, et. al. Blood, 2001; Chen, et al. Biology of Blood and Marrow Transplantation, 2006), making them an attractive cell population for use as an allogeneic, adoptive cell therapy. A robust process for the isolation, transduction, and expansion of placental-derived T cells to generate "off-the-shelf" allogeneic P-CD19 CAR T cells was developed. Twenty-One day expanded, non-modified P-T cells (N=3) were compared to adult PBMCs for their allo-reactivity in a Xenogeneic GvHD model in NCG mice. P-T cells did not induce xeno-GvHD whereas PBMCs did, as evidenced by significant weight loss and death of all mice (N=5) by Day 28 post infusion. Despite expanded P-T cells demonstrating lack of in vivo GvHD, current manufacture of P-CD19 CAR T cells does include a CRISPR-mediated T-cell receptor a constant (TRAC) knockout (KO) step as an additional risk-mitigation strategy to circumvent any potential GvHD stemming from expression of endogenous T cell receptor. CD19 CAR transduction using a retrovirus provided by Sorrento Therapeutics, Inc., followed by TRAC knockout with CRISPR results in both high efficiency of CD19 CAR expression (~30% CD19 Fc+) and TCR KO (>96% CD3-/ TCR a/b-). In vitro, the functional activity of P-CD19 CAR-TRAC KO T cells against CD19+ Burkitt's Lymphoma (Daudi) and Acute lymphoblastic Leukemia (NALM6) cell lines was assessed in cytotoxicity and cytokine release assays. P-CD19 CAR T cells specifically lyse CD19+ Daudi/ Nalm6 targets in both 4-hour endpoint FACS and ACEA kinetic cytotoxicity assays, and in most cases at levels equivalent to or greater than PBMC-derived CD19 CAR T cells. When P-CD19 CAR T cells were co-cultured with CD19+ Daudi/ Nalm6 target cells for 24-hours, they secreted pro-inflammatory cytokines and effector proteins in an antigen-specific manner. In vivo, the anti-tumor activity of P-CD19 CAR T cells was assessed using a disseminated lymphoma xenograft model in NSG mice. Luciferase expressing Daudi cells (3×106) were intravenously (IV) injected on Day 0, followed by IV injection of P-CD19 CAR T cells (14×106) on Day 7. Bioluminescence Imaging (BLI) and survival were used as primary study endpoints. P- CD19 CAR T cells were well tolerated and safe. P-CD19 CAR T cells significantly reduced tumor burden, and improved survival. Four weeks after treatment, the vehicle group had a 100% mortality rate, while all animals from P-CD19 CAR T-treated group (N=5) remained alive without clinical symptoms including weight loss or changes in their fur. In summary, Celularity has defined a robust process for the generation and expansion of CD19 CAR T cells from human placenta. These cells exhibit potent anti-tumor activity both in vitro and in vivo with little evidence of acute GvHD induction, highlighting their potential as an allogeneic, adoptive cell therapeutic agent. Future in vivo GvHD studies will include assessment of both CD19 CAR and TRAC KO genetically modified P-T cells. Disclosures Karasiewicz: Celgene: Equity Ownership; Celularity, Inc.: Employment, Equity Ownership, Patents & Royalties: Patent Inventor. He:Celularity Inc: Employment. Ng:Celularity, Inc.: Employment. Tess:Celularity, Inc.: Employment. Ling:Celularity Inc: Employment. Kaufmann:Sorrento Therapeutics, Inc.: Employment, Equity Ownership, Patents & Royalties. Zeldis:Sorrento Therapeutics Inc: Employment, Equity Ownership. Ji:Celularity, Inc.: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Sorrento Therapeutics Inc: Employment, Equity Ownership, Patents & Royalties. Hariri:Celularity Inc: Employment. Zhang:Celularity Inc: Employment.

Development of a Sensitive and Efficient Reporter Platform for the Detection of Chimeric Antigen Receptor T Cell Expansion, Trafficking, and Toxicity

Despite the success of chimeric antigen receptor T (CART) cell therapy, it is limited by 1) lower rates of durable responses related to inadequate CART cell expansion and trafficking to tumor sites and 2) development of life-threatening complication such as cytokine release syndrome (CRS). Development of a strategy to efficiently and robustly image and track CART cells in the clinic would allow the in vivo characterization of T cell expansion and trafficking to tumor sites as well as the development of strategies to potentially overcome these limitations. The sodium iodide symporter (NIS) is a characterized and sensitive reporter system that has been used for cell imaging in the clinic. We hypothesized that the incorporation of NIS into CART cells would be a sensitive and efficient way to assess CART cell expansion, trafficking, and toxicity. To test our hypothesis, we used two CART cell constructs that are characterized in preclinical models and studied extensively in the clinic: CART19 (41BB costimulated) and BCMA-CART cells (41BB costimulated). First, we generated NIS+CART19 and NIS+BCMA-CART cells through dual transduction of lentiviral vectors (Fig A) and revealed the exclusive 125I uptake by these NIS+CARTs and its inhibition by the NIS inhibitor KClO4in vitro (Fig B). We then analyzed T cell functions of NIS+CART cells. Here, NIS+CART19 or CART19 cells were cultured with the CD19+ acute lymphoblastic leukemia (ALL) cell line NALM6. There was no difference in CART cell cytotoxicity (Fig C), proliferation, or cytokine production (not shown) between NIS+CART19 and CART19. This indicates that the incorporation of NIS into CART cells does not impair their antitumor activity. Next, we evaluated the sensitivity of NIS+CART19 cell detection by TFB-PET in vivo; imaging was performed using an Inveon TFB-PET/CT scanner. Mice received 250 μCi 18F-TFB 45 minutes prior to image acquisition. NIS+CART cells were detectable with TFB-PET when cells were subcutaneously injected at a dose of 1.25x106 cells (Fig D). Having demonstrated that the incorporation of NIS in CART cells provides a sensitive way of their detection by TFB-PET and does not interfere with their effector functions, we tested its efficiency to assess CART cell trafficking in vivo, using multiple myeloma (MM) xenografts. Here, immunocompromised NOD-SCID-ɣ-/- (NSG) mice were engrafted with the BCMA+OPM2 MM cell line (1x106 IV). After engraftment, the tumor burden was assessed by bioluminescence imaging (BLI) and mice were randomized to receive 1) BCMA-CART or 2) NIS+BCMA-CART cells (5x106 IV). Mice were then serially imaged for 1) bioluminescence as a measure of disease burden, and 2) with TFB-PET to assess CART cell expansion and trafficking. As expected, BLI demonstrated that MM predominantly engrafts in bones (Fig E). TFB-PET confirmed trafficking of the NIS+BCMA-CART cells to the bones, corresponding to the areas involved by MM based on BLI (Fig E, right). Both BCMA-CART and NIS+BCMA-CART cells exhibited similarly potent antitumor activity in this model (not shown). Finally, we aimed to explore whether TFB-PET can detect CART massive expansion in vivo and predict the development of CRS. Here, we used an established CRS model in our laboratory. NSG mice were engrafted with patient derived relapsed ALL blasts (5x106 IV). Engraftment was confirmed by peripheral blood sampling. When the leukemic burden is >10 CD19+ cell/µl, mice were treated with either high dose NIS+CART19 cells (5x106 IV) or PBS control. One week after NIS+CART19 cell treatment, mice developed muscle weakness, hunched bodies, and weight loss (Fig F), which correlate with an extreme elevation of cytokines (Sterner et al. Blood 2018). TFB-PET revealed a significant uptake in the bone marrow, spleen, liver, and lungs (Fig G) of the diseased mice but not control mice. Mice were then euthanized, and tissues were harvested. Flow cytometric analysis confirmed an extensive infiltration of CART cells in the liver and spleen. This demonstrates the ability of TFB-PET to detect NIS+CART cell expansion in vivo, correlating with the development of CRS. In summary, our results robustly show that the incorporation of NIS into CART cells provides a sensitive, clinically applicable platform to image CART cells using TFB-PET and to assess their expansion, trafficking to tumor sites, and the development of CRS. These studies illuminate a novel way to noninvasively assess CART cell functions in vivo. Figure Disclosures Sakemura: Humanigen: Patents & Royalties. Suksanpaisan:Imanis: Employment. Cox:Humanigen: Patents & Royalties. Parikh:Janssen: Research Funding; AstraZeneca: Honoraria, Research Funding; Pharmacyclics: Honoraria, Research Funding; MorphoSys: Research Funding; AbbVie: Honoraria, Research Funding; Acerta Pharma: Research Funding; Ascentage Pharma: Research Funding; Genentech: Honoraria. Kay:Agios: Other: DSMB; Infinity Pharmaceuticals: Other: DSMB; Celgene: Other: Data Safety Monitoring Board; MorphoSys: Other: Data Safety Monitoring Board. Peng:Imanis: Equity Ownership. Russell:Imanis: Equity Ownership. Kenderian:Kite/Gilead: Research Funding; Lentigen: Research Funding; Morphosys: Research Funding; Tolero: Research Funding; Humanigen: Other: Scientific advisory board , Patents & Royalties, Research Funding; Novartis: Patents & Royalties, Research Funding.

FT596: Translation of First-of-Kind Multi-Antigen Targeted Off-the-Shelf CAR-NK Cell with Engineered Persistence for the Treatment of B Cell Malignancies

Induced pluripotent stem cell (iPSC)-derived effector cells offer distinct advantages for immune therapy over existing patient- or donor- derived platforms, both in terms of scalable manufacturing from a renewable starting cellular material and precision genetic engineering that is performed at the single-cell level. iPSC derived natural killer (iNK) cells offer the further advantage of innate reactivity to stress ligands and MHC downregulation and the potential to recruit downstream adaptive responses. These unique features form the basis of our multi-antigen targeted chimeric antigen receptor (CAR) CAR-iNK cell product candidate, termed FT596, which is further combined with additional functionality to enhance effector function. FT596 is consistently manufactured from a master iPSC line engineered to uniformly express an NK cell-calibrated CD19-targeting CAR (CD19-CAR), an enhanced functioning high-affinity, non-cleavable CD16 (hnCD16) and a recombinant fusion of IL-15 and IL-15 receptor alpha (IL-15RF) for cytokine-autonomous persistence. The design of the CD19-CAR involved exploiting the intrinsic polyfunctionality of NK cells, which function by engaging multiple signaling pathways activated through combinations of distinct germline encoded receptors. Using this approach, the transmembrane region of activating receptor NKG2D, combined with the intracellular signaling domains of SLAM co-receptor 2B4 and CD3ζ, proved the most effective in triggering antigen specific functional responses in NK cells. Chimerization of an anti-CD19 scFv onto this NKG2D-2B4-CD3ζ signaling platform produced specific in vitro recognition of CD19+ B cell lymphoma cells in short-term and long-term NK cytotoxicity assays (>80% and <40% clearance of tumor cells at 60H, p<0.001 respectively). The functionality of the CD19-CAR was further enhanced in combination with autonomous IL-15 signaling. Introduction of the IL-15RF enabled expansion of iNK cells without addition of soluble cytokine and greatly improved longevity and functional persistence of iNK cells both in vitro and in animal models. Moreover, iNK cells modified with IL-15RF showed enhanced functional maturation, including upregulated expression of effector molecules such as granzyme B. iNK cells with both CD19-CAR and IL-15RF resulted in enhanced CAR functionality in vitro, and mouse models for B cell malignancy demonstrated that treatment with iNK cells engineered with CD19-CAR and IL-15RF were curative against B cell lymphoma (p<0.002), when compared with iNK cells alone or iNK cells modified with CD19-CAR alone. In combination with hnCD16, co-expression of CD19-CAR and IL15-RF culminates in iNK cells capable of dual-specificity through combinatorial use with monoclonal antibodies to tackle antigen escape. In long term killing assays, FT596 alone demonstrated equivalent levels of CD19 targeted anti-tumor activity as primary CD19-targeted CAR (CAR19) T cells when tested against CD19+ CD20+ B lymphoblast target cells and demonstrated enhanced levels of activity when used in combination with anti-CD20 (rituximab). When targeting CD19- CD20+ B lymphoblast target cells and used in combination with rituximab, only FT596 was able to effectively eliminate the CD19 antigen escaped target cell (64% vs 30% clearance of tumor cells at 36H vs rituximab alone). In vivo FT596 showed equivalent levels of tumor cell clearance as primary CAR19 T cells against the CD19+ acute lymphoblastic leukemia cell line NALM6 and CD19+CD20+ Burkitts lymphoma cell line RAJI, and enhanced clearance of RAJI tumor cells in combination with rituximab (p=0.0002). Furthermore, utilizing an allogenic human CD34 engrafted NSG mouse model, FT596 demonstrated improved survival and safety over primary CAR19 T cells, either as a monotherapy or as a combination therapy with rituximab versus RAJI tumor cells. Together, these studies demonstrate FT596 provides a multi-antigen targeting, potent and persistent engineered immune cell that is derived from a master iPSC line which utilizes the intrinsic versatility of NK cells to enable a highly effective combination therapy in a single, standardized, scalable, off-the-shelf platform and supports the rational for a first-of-kind Phase I Study as a monotherapy and in combination with CD20-targeted mAbs including rituximab in subjects with relapsed/refractory B-cell lymphoma and leukemia. Figure Disclosures Goodridge: FATE THERAPEUTICS: Employment. Mahmood:Fate Therapeutics, Inc: Employment. Gaidarova:Fate Therapeutics, Inc: Employment. Bjordahl:Fate Therapeutics, Inc.: Employment. Cichocki:Fate Therapeutics, Inc: Research Funding. Chu:FATE THERAPEUTICS: Employment. Bonello:Fate Therapeutics, Inc.: Employment. Lee:Fate Therapeutics, Inc.: Employment. Groff:FATE THERAPEUTICS: Employment. Meza:FATE THERAPEUTICS: Employment. Malmberg:Vycellix: Consultancy, Membership on an entity's Board of Directors or advisory committees; Fate Therapeutics, Inc.: Consultancy, Research Funding. Miller:Moderna: Membership on an entity's Board of Directors or advisory committees; Dr. Reddys Laboratory: Membership on an entity's Board of Directors or advisory committees; CytoSen: Membership on an entity's Board of Directors or advisory committees; Fate Therapeutics, Inc: Consultancy, Research Funding; OnKImmune: Membership on an entity's Board of Directors or advisory committees; GT BioPharma: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding. Kaufman:FATE Therapeutics: Consultancy, Research Funding. Valamehr:Fate Therapeutics, Inc: Employment.

Activity of Fluorescent Samarium Complex Containing 1,10 Phenanthroline Ligand against Human T-Cell Acute Lymphoblastic Leukaemia Cell Line

This paper is the study of the structure effect of samarium-phenanthroline complex on DNA molecule binding for cancer treatment. So the water soluble complex has been synthesized and characterized by using chemical analysis. By positive cooperative interaction, Sm complex was connected with calf thymus DNA (ct-DNA) and this interaction was investigated by circular dichroism and fluorescence spectroscopy. The result indicates that samarium complex with phenanthroline ligands can be as a fluorescence probe and intercalate in DNA additionally groove binding. Also, the cytotoxicity of this complex was investigated against human T-cell acute lymphoblastic leukemia, MOLT-4 cell line. Finally, the interaction of complex was studied by molecular docking. The results showed during DNA-Sm complex formation, the complex is surrounded by DT8, DC9, DG10, DA18, DT19 and DT20 via hydrophobic forces.

Isoquinolinamine FX-9 Exhibits Anti-Mitotic Activity in Human and Canine Prostate Carcinoma Cell Lines.

Current therapies are insufficient for metastatic prostate cancer (PCa) in men and dogs. As human castrate-resistant PCa shares several characteristics with the canine disease, comparative evaluation of novel therapeutic agents is of considerable value for both species. Novel isoquinolinamine FX-9 exhibits antiproliferative activity in acute lymphoblastic leukemia cell lines but has not been tested yet on any solid neoplasia type. In this study, FX-9′s mediated effects were characterized on two human (PC-3, LNCaP) and two canine (CT1258, 0846) PCa cell lines, as well as benign solid tissue cells. FX-9 significantly inhibited cell viability and induced apoptosis with concentrations in the low micromolar range. Mediated effects were highly comparable between the PCa cell lines of both species, but less pronounced on non-malignant chondrocytes and fibroblasts. Interestingly, FX-9 exposure also leads to the formation and survival of enlarged multinucleated cells through mitotic slippage. Based on the results, FX-9 acts as an anti-mitotic agent with reduced cytotoxic activity in benign cells. The characterization of FX-9-induced effects on PCa cells provides a basis for in vivo studies with the potential of valuable transferable findings to the benefit of men and dogs.

Identification of Novel, Potent, and Orally Available GCN2 Inhibitors with Type I Half Binding Mode.

General control nonderepressible 2 (GCN2) is a master regulator kinase of amino acid homeostasis and important for cancer survival in the tumor microenvironment under amino acid depletion. We initiated studies aiming at the discovery of novel GCN2 inhibitors as first-in-class antitumor agents and conducted modification of the substructure of sulfonamide derivatives with expected type I half binding on GCN2. Our synthetic strategy mainly corresponding to the αC-helix allosteric pocket of GCN2 led to significant enhancement in potency and a good pharmacokinetic profile in mice. In addition, compound 6d, which showed slow dissociation in binding on GCN2, demonstrated antiproliferative activity in combination with the asparagine-depleting agent asparaginase in an acute lymphoblastic leukemia (ALL) cell line, and it also displayed suppression of GCN2 pathway activation with asparaginase treatment in the ALL cell line and mouse xenograft model.

Comparison of in vitro antioxidative activities of crude methanolic extracts of three species of Passiflora from greenhouse using DPPH, ABTS and FRAP methods

Summary Introduction:. It is well documented that many species from Passifloraceae family can provide edible and nutritious fruits while the leaves of cultivated plants are renewable and waste material. This biomass may be further used in various sectors, especially as a bioactive food additive and as source of innovative pharmaceuticals, cosmetics or feed additives. The biomaterials and green chemistry are new sectors bioeconomy according to the high-level horizontal strategies and bio-based industries in Europe. In recent years, attention has been paid to the biological activity and phytochemical profiles of extracts from different species of Passiflora. However, there is little comparative studies using the same procedures and techniques in the same laboratory conditions for study of plant material obtained from the similar greenhouse conditions. Objective: This study was focused on the examination of antioxidative activities of low concentrations of crude extracts from leaves of Passiflora incarnata L., Passiflora caerulea L., and Passiflora alata Curtis. Methods: The activity was evaluated using 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2’-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) radical scavenging and ferric reducing antioxidant power (FRAP) methods. Results of study were supported by estimation of chemical composition with secondary metabolites profiling in extracts which were carried out previously for the same extracts from three Passiflora species. One-way ANOVA analysis revealed significant differences in the antioxidant activity of various concentrations of the extracts using the DPPH and ABTS radical models, and FRAP method. Results: Measurement of antioxidant capacity (expressed as trolox equivalent, TE) showed that the most active was extract of P. caerulea > P. alata > P. incarnata. Phytochemical analysis for extracts of P. caerulea and P. incarnata showed greater similarities in metabolites content than P. alata. However, comparative statistical analysis of antioxidant activity showed that despite this phytochemical similarities, extract from P. alata leaves had higher activities than extract from leaves P. incarnata. Antioxidant effect of extract from P. alata can be explain by terpenoids presented in this extract. In this work, there have been discussed activities against Acanthamoeba castellanii strain, antibacterial and antifungal activities against selected clinical microorganisms (Enterococcus faecalis, Escherichia coli, Staphylococcus aureus, and Candida albicans, Micro-sporum gypseum), and anti-leukemic activities tested in human acute lymphoblastic leukemia cell lines for this extracts, which have been described in previous authors’ publications. Conclusion: Our current and previous studies showed that the same crude extracts from leaves of P. alata, P. caerulea, P. incarnata exerted not only antioxidant potential in vitro but also few interesting properties such as antibacterial, antifungal, amoebostatic, amoebicidal activities, which indicate the possibility of using these extracts in both a healthy diet and natural cosmetics. Leaves of this species may become an interesting source of biomaterials which can exert health-promoting effects.

Resistance of t(17;19)-acute lymphoblastic leukemia cell lines to multiagents in induction therapy.

t(17;19)(q21‐q22;p13), responsible for TCF3‐HLF fusion, is a rare translocation in childhood B‐cell precursor acute lymphoblastic leukemia(BCP‐ALL). t(1;19)(q23;p13), producing TCF3‐PBX1 fusion, is a common translocation in childhood BCP‐ALL. Prognosis of t(17;19)‐ALL is extremely poor, while that of t(1;19)‐ALL has recently improved dramatically in intensified chemotherapy. In this study, TCF3‐HLF mRNA was detectable at a high level during induction therapy in a newly diagnosed t(17;19)‐ALL case, while TCF3‐PBX1 mRNA was undetectable at the end of induction therapy in most newly diagnosed t(1;19)‐ALL cases. Using 4 t(17;19)‐ALL and 16 t(1;19)‐ALL cell lines, drug response profiling was analyzed. t(17;19)‐ALL cell lines were found to be significantly more resistant to vincristine (VCR), daunorubicin (DNR), and prednisolone (Pred) than t(1;19)‐ALL cell lines. Sensitivities to three (Pred, VCR, and l‐asparaginase [l‐Asp]), four (Pred, VCR, l‐Asp, and DNR) and five (Pred, VCR, l‐Asp, DNR, and cyclophosphamide) agents, widely used in induction therapy, were significantly poorer for t(17;19)‐ALL cell lines than for t(1;19)‐ALL cell lines. Consistent with poor responses to VCR and DNR, gene and protein expression levels of P‐glycoprotein (P‐gp) were higher in t(17;19)‐ALL cell lines than in t(1;19)‐ALL cell lines. Inhibitors for P‐gp sensitized P‐gp‐positive t(17;19)‐ALL cell lines to VCR and DNR. Knockout of P‐gp by CRISPRCas9 overcame resistance to VCR and DNR in the P‐gp‐positive t(17;19)‐ALL cell line. A combination of cyclosporine A with DNR prolonged survival of NSG mice inoculated with P‐gp‐positive t(17;19)‐ALL cell line. These findings indicate involvement of P‐gp in resistance to VCR and DNR in Pgp positive t(17;19)‐ALL cell lines. In all four t(17;19)‐ALL cell lines, RAS pathway mutation was detected. Furthermore, among 16 t(1;19)‐ALL cell lines, multiagent resistance was usually observed in the cell lines with RAS pathway mutation in comparison to those without it, suggesting at least a partial involvement of RAS pathway mutation in multiagent resistance of t(17;19)‐ALL.

Abstract 871: Regulation of CDC25a expression by the ikaros and casein kinase II (CK2) in T-cell acute lymphoblastic leukemia (T-ALL)

Abstract CDC25A is a member of the CDC25 family of phosphatases that plays a major role in cell cycle progression. Here, we present evidence that expression of CDC25a in T-ALL is regulated at the transcriptional level by oncogenic Casein Kinase II (CK2) via direct phosphorylation of Ikaros, a transcription factor and tumor suppressor protein. Global chromatin immunoprecipitation coupled with next-generation sequencing (ChIP-seq) studies in both primary human acute lymphoblastic leukemia cells and cell lines, demonstrated that Ikaros binds to the promoter of the CDC25a gene. Ikaros functions as a tumor suppressor protein and deletion of which is associated with development of T-ALL. Ikaros binding to CDC25a promoter was confirmed by quantitative chromatin immunoprecipitation (qChIP) in primary T-ALL cells. Ikaros knock-down with shRNA results in increased transcription of CDC25a in T-ALL. In mice, T-ALL cells that were derived from Ikaros-knockout mice express high levels of CDC25a. Transduction of these cells with Ikaros-containing retrovirus results in sharp reduction of CDC25a expression. Overexpression of CK2 via retroviral transduction resulted in increased transcription of the CDC25a gene, as measured by qRT-PCR, as well as increased overall expression of CDC25a, as measured by Western blot. Increased expression of CK2 was associated with a loss of Ikaros binding to the CDC25a gene promoter. Molecular inhibition of CK2 using shRNA, as well as pharmacological inhibition with a specific CK2 inhibitor resulted in reduced expression of CDC25a in primary human T-ALL. CK2 inhibition was also associated with strong reduction in AKT phosphorylation, emphasizing that CK2 inhibition downregulates CDC25a and other cell cycle progression genes. Inhibition of CK2 was associated with increased Ikaros binding at the promoter of CDC25a. Ikaros knock-down restored high expression of CDC25a in T-ALL cells that were treated with CK2 inhibitors. These data showed that CK2 and Ikaros are major transcriptional regulators of CDC25a transcription in T-ALL and that CK2 inhibition represses CDC25a transcription via Ikaros-mediated repression. In conclusion, these results indicate that expression of the CDC25a oncogene in T-ALL is regulated by the CK2 which modulates Ikaros activity. Presented data revealed a novel mechanism of therapeutic action of CK2 inhibitors - repression of CDC25a expression via Ikaros. Results provide a rationale for the use of novel CK2 inhibitors in T-ALL. Citation Format: Soumya C. Iyer, Shriya Kane, Chandrika Gowda, Chunhua Song, Yali Ding, Jon Payne, Pavan Kumar Dhanyam Raju, Bihua Tan, Mary McGrath, Yevgeniya Bamme, Mario Solimon, Nathalia Moreno Cury, Dhimant Desai, Arati Sharma, Kimberly J. Payne, Sinisa Dovat. Regulation of CDC25a expression by the ikaros and casein kinase II (CK2) in T-cell acute lymphoblastic leukemia (T-ALL) [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 871.

Abstract 2497: FCN-338, a novel and selective Bcl-2 inhibitor, exhibits potent anti-tumor activity in B-cell lymphoma

Abstract B-cell lymphoma-2 (Bcl-2) family proteins are key regulators of apoptosis, which is vital for proper tissue development and cellular homeostasis. Apoptosis occurs via activation of two different pathways: the extrinsic pathway, triggered by a death ligand binding to a death receptor, such as TNF-α to TNFR1; and the intrinsic pathway, regulated by Bcl-2 family proteins and their complex protein-protein interactions. Bcl-2 family proteins are functionally classified as either anti-apoptotic, such as Bcl-2, Bcl-xL and Mcl-1, or pro-apoptotic, including Bid, Bim, Bad, Bak and Bax. Anti-apoptotic Bcl-2 family members are often found to be up-regulated in many types of human cancers, such as different subtypes of B-cell lymphoma and diverse solid tumors, and are frequently been correlated with decreased susceptibility to chemotherapeutics and to increased radio-resistance. Overexpression of Bcl-2 proteins is an independent indicator of poor prognosis in tumors including chronic lymphocytic leukemia (CLL), prostate cancer, and small cell lung cancer (SCLC). Therefore, targeting Bcl-2 and its pro-survival relatives that form the core anti-apoptotic machinery, has emerged as a promising therapeutic approach in cancer. To that end, we have developed a novel and selective Bcl-2 inhibitor, FCN-338, with nanomolar affinity to Bcl-2 and physicochemical properties suitable for oral administration. FCN-338 exhibits much greater affinity to Bcl-2 than Bcl-xL, suggesting its good potential to avoid thrombocytopenia caused by the inhibition of Bcl-xL. FCN-338 has shown remarkable anti-proliferation potency against a panel of Bcl-2-addicted human B-cell lymphoma cell lines, including DOHH2 follicular lymphoma (FL) cell line (IC50 2 nM), Mino diffuse large B-cell lymphoma (DLBCL) cell line (IC50 23 nM), and RS4;11 acute lymphoblastic leukemia (ALL) cell line (IC50 35 nM), but not in Bcl-xL-dependent H146 SCLC cell line (IC50 >4,500 nM). Correspondingly, in vivo efficacy of FCN-338 was demonstrated in a variety of xenograft models derived from FL, DLBCL and ALL tumors. FCN-338 dramatically caused tumor regression in a dose-dependent manner. In the non-clinical studies, FCN-338 exhibits much improved pharmacokinetic properties with higher T1/2, dose-normalized AUC and bioavailability in dogs compared with the FDA-approved Bcl-2 inhibitor ABT-199. Preferable safety profiles of FCN-338 were shown with no potential hERG inhibitory effect and less drug-drug interaction potential, as evidenced by no inhibitory effect (IC50 >50 μM) on CYP2C9 enzyme compared with ABT-199 (IC50 1.05 μM). Together, our findings highlight the therapeutic potential of FCN-338 to be used as effective therapy across a broad range of Bcl-2-addicted B-cell malignancies. Citation Format: Shu Lin, Xingdong Zhao, Hongbin Liu, Huajie Zhang, Zhifang Chen, Lihua Jiang, Yanxin Liu, Min Lin, Yue Rong, Rui Tan, Zuwen Zhou, Zongyao Zou, Yuwei Gao, Weibo Wang. FCN-338, a novel and selective Bcl-2 inhibitor, exhibits potent anti-tumor activity in B-cell lymphoma [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 2497.

A novel method to establish glucocorticoid resistant acute lymphoblastic leukemia cell lines

BackgroundDrug-resistant cell lines, established from drug-sensitive cell lines by drug exposure in vitro, are the most useful cancer models in studies on the mechanism of chemoresistance. However, the success rate of the traditional approaches to construct such cell lines is low because a long time is required for the addition of drugs.MethodsA cell culture technique was used to establish the drug-resistant cell lines from their parental cells. Molecular and cellular biological techniques including flow cytometry, MTT assay, western blotting, and DNA fingerprinting analysis were used to characterize the drug-resistant cell lines. Nude mice were used for xenograft studies.ResultsWe established novel glucocorticoid (GC)-resistant cell lines from 3 GC-sensitive acute lymphoblastic leukemia (ALL) cell lines. First, we established a novel GC-resistant T-ALL cell line, CEM-C7/HDR, by mimicking the microenvironment of the bone marrow and culturing GC-sensitive CEM-C7–14 cells under hypoxia for 5 weeks with a single dexamethasone (Dex) treatment. The CEM-C7/HDR cells had been cultured continuously in drug-free medium under normoxia for 1 year. The IC50 and resistance index (RI) to Dex were maintained at 60~70 μM and 1500~1800, respectively, which is in consistent with the IC50 and RI of GC-resistant CEM-C1–15 cells. To clarify the reliability of the method, we subcloned CEM-C7–14 cells, and obtained Dex-resistant cell lines, CEM-C7-SC2/HDR and CEM-C7-SC14/HDR, from 2 monoclonal cells of CEM-C7–14 by the same method. Moreover, we obtained two additional Dex-resistant B-ALL cell lines, NALM-6/HDR and HXEX-ALL1/HDR, from NALM-6 and HXEX-ALL1 cells with the same approach.ConclusionsCEM-C7/HDR, NALM-6/HDR and HXEX-ALL1/HDR cell lines may serve as useful GC-resistant ALL models for both in vitro and in vivo studies. Culturing under hypoxic condition with a single Dex treatment is a novel and convenient approach for generating stable GC resistant cell lines.