Leukemic Lines Research Articles

Molecular Mechanisms in Murine Syngeneic Leukemia Stem Cells.

Acute Myeloid Leukemia (AML) is a severe disease with a very high relapse rate. AML relapse may be attributable to leukemic stem cells (LSC). Notably, the "cancer stem cell" theory, which relates to LSCs, is controversial and criticized due to the technical peculiarities of the xenotransplant of human cells into mice. In this study, we searched for possible LSCs in an immunocompetent synergetic mice model. First, we found phenotypic heterogeneity in the ML23 leukemia line. We prospectively isolated a sub-population using the surface markers cKit+CD9-CD48+Mac1-/low, which have the potency to relapse the disease. Importantly, this sub-population can pass in syngeneic hosts and retrieve the heterogeneity of the parental ML23 leukemia line. The LSC sub-population resides in various organs. We present a unique gene expression signature of the LSC in the ML23 model compared to the other sub-populations. Interestingly, the ML23 LSC sub-population expresses therapeutic targeted genes such as CD47 and CD93. Taken together, we present the identification and molecular characterization of LSCs in a syngeneic murine model.

Venetoclax Resistance in B-Cell Precursor Acute Lymphoblastic Leukemia Is Characterized By Increased Mitochondrial Metabolism and Can be Overcome By Co-Targeting Oxidative Phosphorylation

Deregulated cell death pathways are a hallmark of many cancers and contribute to leukemogenesis and treatment failure in B-cell precursor acute lymphoblastic leukemia (BCP-ALL). Apoptosis is controlled by different pro- and anti-apoptotic molecules either inducing or counteracting cell death induction. Inhibition of anti-apoptotic molecules like B-cell lymphoma 2 (BCL-2) has been developed as therapeutic strategy. Venetoclax (VEN) binds selectively to anti-apoptotic BCL-2 resulting in apoptosis induction and has shown clinical activity in lymphoid malignancies, but insensitivity and resistance have been described. In BCP-ALL, we and others have shown preclinical activity of VEN and VEN is evaluated in first clinical trials. In this study, we have addressed and modeled VEN resistance in BCP-ALL and investigated underlying mechanisms in cell lines and patient-derived xenograft (PDX) samples in order to identify potential strategies to overcome VEN insensitivity. Starting from the BCP-ALL cell line RS4;11, five VEN insensitive lines were generated in parallel by exposure to increasing concentrations of VEN over time (49 passages, 8 months of continuous treatment) and five control lines were exposed to corresponding concentrations of solvent (DMSO). Measuring half maximal effective concentrations (EC50) showed increasing EC50 values from 4 nM to 26.2 µM over time in all VEN treated lines, indicating acquired resistance in our model. VEN insensitive leukemia lines displayed unchanged proliferation and same sensitivities to the prototypic apoptosis inducer staurosporine, the induction therapy drugs vincristine/dexamethasone/asparaginase or the anthracycline daunorubicin as compared to controls, indicating acquisition of VEN specific resistance. In order to further characterize the mechanism of VEN insensitivity, we performed RNA-Seq analysis comparing five RS4;11 VEN insensitive to five control lines. Interestingly, gene set enrichment analysis on significantly regulated genes showed a significant upregulation of gene sets annotated to the citric/tricarboxylic acid cycle and the respiratory electron transport chain, pointing to increased mitochondrial metabolism in VEN resistant cells. We addressed mitochondrial activity and carried out metabolic profiling (Agilent Seahorse XF Cell Mito Stress Test) of VEN resistant and control lines (N=5+5). Most interestingly, in contrast to significant reduction of oxygen consumption rates (OCR) in VEN treated control lines, we found sustained high mitochondrial metabolism indicated by persistent high OCR in VEN resistant lines. To further validate our findings, we investigated a series of BCP-ALL PDX samples (N=30) and titrated VEN EC50 values identifying heterogenous VEN activities. Metabolic profiling (Mito Stress test) was performed on sensitive as compared to highly resistant leukemias (N=3+3). Most interestingly and in line with our findings in cells with acquired resistance, PDX samples with intrinsic VEN insensitivity showed higher oxygen consumption and ATP production rates, thus further highlighting that increased mitochondrial activity is a characteristic feature of VEN resistant ALL. Along this line, significant higher mitochondrial DNA content was found in highly insensitive PDX leukemias when compared to VEN sensitive samples. Moreover, we analyzed mitochondrial morphology by electron microscopy. Although numbers of mitochondria did not differ between VEN resistant and sensitive leukemias, the overall mitochondrial area and structure was found to be significantly larger and elongated, further corroborating our finding of augmented mitochondrial metabolism in VEN resistant ALL. Finally, we addressed whether VEN resistance in ALL can be overcome by directly targeting oxidative phosphorylation (OxPhos). Using oligomycin, an inhibitor of the complex V/ATPase subunit, we found synergistic induction of apoptosis together with VEN in BCP-ALL cell lines and PDX samples, showing that acquired and intrinsic VEN insensitivity can be overcome by co-targeting BCL-2 and the OxPhos pathway. Taken together, we show that VEN resistance in BCP-ALL is characterized by high mitochondrial metabolic activity. Co-targeting of BCL-2 and oxidative phosphorylation results in synergistic anti-leukemia activity setting the basis for further preclinical and potential clinical evaluation.

Antiproliferative Activity and Apoptotic Potential of Curcumin on Human Acute Myeloid Leukemia Cell Line (HL-60)

Curcuma Longa or also known as turmeric is a prominent natural remedy in Asia that has been utilized to heal diseases for decades. It has been demonstrated to have substantial biological activities, including anticarcinogenic, antimutagenic, antioxidant, anti-inflammatory, anti-infectious, hypocholesterolemic, and chemopreventive properties. Curcumin is a therapeutic substance extracted from Curcuma Longa that is thought to have anti-cancer properties against a variety of cancer types. However, its anti-cancer potential on acute myeloid leukemia (AML) remains uncertain [1,2]. 
 
 The objective of this intervention study was to examine the anti-proliferative and apoptotic effects of curcumin on the HL-60 cell line of acute myeloid leukemia. A stock solution of pure curcumin compound was obtained by dissolving it in dimethyl sulfide (DMSO). HL-60 cells were treated with six different concentrations which were 100, 50, 25, 12.5, 6.25, 3.125 µg/ml and incubated at three distinct incubation period (24, 48 and 72 hours). The in vitro cell viability was determined using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay and quantified using a microplate reader at 570 nm. Acridine orange (AO) and propidium iodide (PI) staining were employed to evaluate morphological changes. 
 
 MTT assays results show that curcumin has a high cytotoxicity effect on HL-60 cells. The maximal half inhibitory concentration (IC50) value of curcumin was consistently decreased when the incubation time increased. The IC50 of curcumin on HL-60 cells were reported to be 13, 8, and 3.5 µg/ml at 24, 48 and 72 hours of incubation, respectively. There was a significantly different IC50 value (p<0.05) of curcumin between the incubation periods (Figure 1). The morphological alteration was shown in AO/PI staining by the appearance of early apoptosis, late apoptosis, and necrotic cells.
 These findings illustrated that curcumin revealed an effective anti-cancer action on human acute myeloid leukemia HL-60 cell line. Further research is required to determine the mechanism of cell death in HL-60 cells treated with pure curcumin compound.
 
 In conclusion, the study revealed the role of putative targeted genes in the biological mechanism in gout pathogenesis has provides insight of the potential biomarkers in developing the personalized medicine better treatment for gout patient in ensuring better patient healthcare.

Thiopyrano[2,3-d]thiazole structures as promising scaffold with anticancer potential

Seven chromeno[4′,3':4,5]thiopyrano[2,3-d]thiazole derivatives were synthesized and screened for their cytotoxic effects on different lines of mammalian leukemia, breast adenocarcinoma, glioblastoma, and pseudo-normal and normal cells. The derivative 3 demonstrated toxicity towards tumor cells of Jurkat, K562, U251, HL-60, MCF-7, and MDA-MB-231 lines. At the same time, this compound possessed low toxicity (IC50 > 100 μM) towards cells, used as control, representing non-tumor, somatic cells: HaCaT, HEK293 cells as well as murine Balb/c 3T3 and J774.2 cells, mink Mv1Lu cells, and normal mitogen-activated human blood lymphocytes. The derivative 3 induced apoptosis in human leukemia Jurkat T-cells and glioblastoma U251 cells via mitochondria-dependent pathway and inhibition of the DNA reparation enzyme PARP-1. This compound triggered pro-apoptotic morphological changes in Jurkat and U251 cells, namely chromatin condensation, nuclei fragmentation, and membrane blebbing. However, the DNA damaging effects of compound 3 were significantly lower in normal human lymphocytes, compared with such results in tumor Jurkat and U251 cells. The DNA damaging effects of compound 3 were unrelated to its DNA-binding and/or DNA-intercalating abilities. This compound induced the accumulation of endogenous reactive oxygen species (ROS), namely superoxide radicals, in human leukemia and glioblastoma cells. Our finding indicated that compound 3 inhibited the viability of human leukemia T-cells and glioblastoma cells via induction of DNA damage and apoptosis through ROS-mediated mitochondrial pathway.

Platinum(IV) Complexes of the 1,3,5-Triamino Analogue of the Biomolecule Cis-Inositol Designed as Innovative Antineoplastic Drug Candidates.

Metal complexes occupy a special place in the field of treatment and diagnostics. Their main advantages stem from the possibility of fine-tuning their thermodynamic properties and kinetic behavior in the biological milieu by applying different approaches such as properly constructed inner coordination sphere, appropriate choice of ligands, metal oxidation state, redox potential, etc., which are specific to these compounds. Here we discuss the design and synthesis of two octahedral cationic Pt(IV) complexes of the tridentate ligand all-cis-2,4,6-triaminocyclohexane-1,3,5-triol (taci) with composition, fac-[Pt(taci)I3]+, 1 and bis-[Pt(taci)2]4+, 2 as well as the potential for their application as antineoplastic agents. The complexes have been isolated in a solid state as: fac-[Pt(taci)I3]I·3H2O (1A), fac-[Pt(taci)I3]I (1B), fac-[Pt(taci)I3]I·2DMF (1C), bis-[Pt(taci)2](CO3)2·6H2O (2A) by changing the acidity of the reaction systems, the molar ratios of the reagents and the counterions, and by re-crystallization. The ligand taci is coordinated through the NH2-groups, each molecule occupying three coordination places in the inner coordination sphere of Pt(IV). Monitoring of the hydrolysis processes of 1A and 2A at different acidity showed that while 2A remained stable over the study period, the I−-ions in 1A were successively substituted, with the main product under physiologically mimetic conditions being fac,cis-[Pt(taci)I(OH)2]+ (h2). The antiproliferative tests involved eight cancer cell models, among which chemosensitive (derived from leukemias and solid tumors) and chemoresistant human Acute myeloid leukemia lines (HL-60/Dox, HL-60/CDDP), as well as the non-malignant kidney’ cells HEK-293T showed that the complexes 1A and 2A are characterized by a fundamentally different profile of chemosensitivity and spectrum of cytotoxic activity compared to cisplatin. The new Pt(IV) complexes were shown to be more effective in selectively inhibiting the proliferation of human malignant cells compared to cisplatin. Remarkable activity was recorded for 1A, which showed an effect (IC50 = 8.9 ± 2.4) at more than 16-fold lower concentration than cisplatin (IC50 = 144.4 ± 9.8) against the resistant cell line HL-60/CDDP. In parallel, 1A exhibited virtually the same cytotoxic effect against the parental HL-60 cells (IC50 = 9.0 ± 1.2), where cisplatin displays comparable chemosensitivity (IC50 = 8.3 ± 0.8). The determined resistance indices (RI~1) show unequivocally that the resistant lines are sensitive to both compounds tested; therefore, they are capable of overcoming the mechanisms of cisplatin resistance. The structural features of these compounds and their promising pharmacological properties justify their inclusion in the group of “non-classical metal-based antitumor compounds” and are a prerequisite for the admission of alternative mechanisms of action.

The effects of acute exercise and inflammation on immune function in early-stage prostate cancer

BackgroundThe immune system plays a vital role in cancer development and progression. Strategies mobilizing cytotoxic cells of the immune system to combat immunosuppression in cancer may help to improve the treatment response of patients. To this end, we aimed to characterize the anti-cancer effect of acute exercise, including the involvement of inflammatory signals. Patients and methodsTwenty patients with early-stage prostate cancer (PCa) scheduled to undergo prostatectomy performed one bout of acute exercise consisting of a watt-max test and four high-intensity intervals. Natural Killer (NK), NKT-like and T cell phenotype, NK cell cytotoxic activity (NKCA), and NKCA per-cell against cell lines of leukemia (K562) and prostate cancer origin (LNCaP and PC-3) were assessed. Inflammatory markers (TNF-α, IL-6, and CRP) were measured in plasma. ResultsExercise increased NK, NKT-like, and CD8 T cell concentration in the circulation. Furthermore, exercise shifted immune cells towards a mature and cytotoxic phenotype e.g., NK cells exhibited higher CD57 as well as lower NKG2A expression. NKT-like and CD8 cells exhibited elevated CD57, TIGIT and Granzyme-B expression. Exercise significantly improved NKCA against K562 (+16% [5%; 27%]; p = 0.002) and LNCaP (+24% [14%; 34%]; p < 0.001) but not PC-3. NKCA per NK cell decreased during exercise and increased 1-h post exercise compared to baseline in K562, LNCap, and PC-3 cell lines. Baseline IL-6 correlated with lymphocyte, monocyte and T cell concentration pre-exercise and inversely correlated with the fold-change of mobilized lymphocytes and CD8 T cells during exercise. Furthermore, baseline IL-6 and TNF-α inversely correlated with NKCA against PC-3 cells during exercise. ConclusionsAcute exercise mobilized cytotoxic immune cells and improved NKCA in patients with PCa whereas low-grade inflammation might impair the response. Whether the observed improvements impact long-term outcomes warrant further investigation. Clinical trial numberNCT03675529.

Abnormal GRHL2 Methylation Confers Malignant Progression to Acute Leukemia.

Purpose Abnormal methylation of Grainyhead-like 2 (GRHL2) is associated with a substantial role in the malignant phenotype of tumor patients. Our present research is aimed at studying the abnormal expression of GRHL2 and the association of methylation in patients with acute leukemia and its relationship with prognosis. Materials and Methods We used quantitative real-time polymerase chain reaction (qRT-PCR) for detecting the aberrant expression level of GRHL2 in 60 patients with acute leukemia and 60 normal controls. We analyzed the significant correlation between the expression level of GRHL2 with clinicopathological features and patients' prognosis in acute leukemia using the corresponding statistical methods. Secondly, we employed qRT-PCR and Western blotting to detect the mRNA and protein levels of GRHL2 in leukemia cell lines. Next, we used methylation-specific polymerase chain reaction (MSP) technology for detecting the methylation of GRHL2 in clinical samples with acute leukemia and cell lines. Then we investigated the demethylating effect of arsenic trioxide and 5-azacitidine on the mRNA and protein expression levels of GRHL2 in cell lines of acute leukemia. Finally, we studied the effects of arsenide trioxide and 5-azacitidine on the proliferation of leukemia cells and the TGF-β signaling pathway. Results We found a lower level of GRHL2 expression not only in acute leukemia patients but also in cell lines when compared with normal controls. At the same time, the expression level of GRHL2 in patients with acute leukemia was significantly correlated with leukocyte count, platelet count, and cytogenetic risk grouping. In addition, the lower GRHL2 expression group showed a significantly lower overall survival rate in acute leukemia patients than that of patients with a higher GRHL2 expression group. Univariate and multivariate analyses revealed that the expression of GRHL2 is an independent risk factor in acute leukemia patients. The methylation level of the GRHL2 promoter region in acute leukemia patients and cell lines was significantly higher than the normal control group, and we found the elevated mRNA and protein levels of GRHL2 in acute leukemia cell lines after the use of the demethylation drug arsenic trioxide and 5-azacitidine. At the same time, arsenide trioxide and 5-azacitidine are associated with the inhibition of cellular proliferation of acute leukemia cells and also promote the elevated expression of TGF-β signaling pathway-linked proteins, including TGF-β, Smad2, Smad3, and Smad4. Conclusion Increased expression and methylation level of GRHL2 are closely associated with the prognosis and malignant phenotype of acute leukemia patients and play an irreplaceable role in the occurrence and development of patients with acute leukemia.

Identification of cell type–specific correlations between ERK activity and cell viability upon treatment with ERK1/2 inhibitors

Increased MAPK signaling is a hallmark of various cancers and is a central regulator of cell survival. Direct ERK1/2 inhibition is considered a promising approach to avoid ERK1/2 reactivation caused by upstream kinases BRAF, MEK1/2, and KRAS, as well as by receptor tyrosine kinase inhibitors, but the dynamics and selectivity of ERK1/2 inhibitors are much less studied compared with BRAF or MEK inhibitors. Using ERK1/2 and downstream kinase ELK1 reporter cell lines of lung cancer (H1299; NRASQ61K), colon cancer (HCT-116; KRASG13D), neuroblastoma (SH-SY5Y), and leukemia (U937), we examined the relationship between ERK inhibition and drug-induced toxicity for five ERK inhibitors: SCH772984, ravoxertinib, LY3214996, ulixertinib, and VX-11e, as well as one MEK inhibitor, PD0325901. Comparing cell viability and ERK inhibition revealed different ERK dependencies for these cell lines. We identify several drugs, such as SCH772984 and VX-11e, which induce excessive toxicity not directly related to ERK1/2 inhibition in specific cell lines. We also show that PD0325901, LY3214996, and ulixertinib are prone to ERK1/2 reactivation over time. We distinguished two types of ERK1/2 reactivation: the first could be reversed by adding a fresh dose of inhibitors, while the second persists even after additional treatments. We also showed that cells that became resistant to the MEK1/2 inhibitor PD0325901 due to ERK1/2 reactivation remained sensitive to ERK1/2 inhibitor ulixertinib. Our data indicate that correlation of ERK inhibition with drug-induced toxicity in multiple cell lines may help to find more selective and effective ERK1/2 inhibitors.

Establishment of Drug-Resistant Cell Lines of Acute Myeloid Leukemia and Correlation of Sirt1 and PGC-1α Expression Levels with Drug Resistance

To construct cytarabine-resistant acute myeloid leukemia (AML) cell lines, and explore the correlation between Sirt1, PGC-1α expression levels and drug resistance. Human acute promyelocytic leukemia Kasumi-1 cells were induced by the method of gradually increasing the concentration of Ara-C drug. The IC50 value of Kasumi-1 cells before and after drug addition was detected by CCK-8 method, so as to construct Ara-C resistant cell lines. The expression levels of Sirt1 and PGC-1α mRNA in Kasumi-1 drug-resistant cell lines and their parental cell lines were detected by real-time fluorescence quantitative PCR, and the expression levels of Sirt1 and PGC-1α protein in kasumi-1 drug-resistant cell lines and their parental cell lines were detected by Western blot. The constructed Kasumi-1 cell line had common morphological characteristics of drug-resistant cell lines under microscope, and the drug resistance index was greater than 5, indicating that Kasumi-1 drug-resistant cells had good drug resistance after the construction. The RT-qPCR and Western blot assays showed that the expression levels of Sirt1 and PGC-1α mRNA and protein in the drug-resistant cell lines were higher than those of the parental cell lines (P<0.001). AML cell lines resistant to Ara-C can be successfully induced by the method of gradually increasing the concentration, and the co-high expression of Sirt1 and PGC-1α may mediate the drug resistance of AML cells.

Protective effects of pentoxifylline on T-cell viability under inflammatory conditions

Introduction: Pentoxifylline (PTX) reduces the levels of pro-inflammatory cytokines; however, its effects on immune system is not well understood. The aim of this study was to investigate the effect of PTX on T cells under inflammatory conditions in co-culture with THP-1-derived macrophages.Methods: Toll-like receptor 4 (TLR4) and macrophage migration inhibitory factor (MIF) levels were measured after addition of PTX to lipopolysaccharide (LPS)-stimulated differentiated THP-1 cells. T cell viability and MIF levels were measured after PTX was added to prostaglandin E2(PGE2)-stimulated Jurkat T-cell leukemia line. Co-culture was conducted to determine the effect of LPS-stimulated differentiated THP-1 cells that are affected by PTX on Jurkat cells. To prevent the direct effects of LPS and PTX on Jurkat cells, LPS and PTX were washed from THP-1 cells before co-culture. T cell viability and interleukin-2 (IL-2) levels were determined in Jurkat cells. Results: Increase in the MIF concentration and TLR4expression level in differentiated THP-1 cells stimulated with LPS were reversed after PTX addition. However, PTX did not improve T cell viability in PGE2–stimulated Jurkat cells. Co-culturing Jurkat cell and LPS-stimulated differentiated THP-1 cells resulted in a decreased viability of T cells. The addition of PTX restored T cell viability to normal control levels and IL-2 expression level in Jurkat cells. Conclusion: LPS-stimulated THP-1-derived macrophages reduced the T cell viability under inflammation. However, PTX restored T cells viability and IL-2 back to normal levels. Therefore, the immunomodulatory action of PTX may be mediated by macrophage-T cell interactions.

Combinatorial Genetic Uncovers DOCK1/RAC2 As Specific Targets for the Treatment of NPM1;Cohesin Mutated AML

Acute myeloid leukemia (AML) is an aggressive malignancy of the blood and bone marrow resulting from the accumulation of multiple serially acquired mutations. The mutational complexity of the disease makes AML difficult to treat, contributing to a low 5-year survival rate. A better understanding of how different mutations interact with one another to influence disease characteristics is critical and may result in the development of targeted therapies to improve patient outcome. The most common recurrent somatic mutation in AML affects the gene NPM1 and occurs in 25-30% of patients. This mutation results in the aberrant cytoplasmic localization of the protein and is termed NPM1cA (Falini et al. 2005, Cancer Genome Atlas Research Network 2013). NPM1cA is considered to be a driver of AML, however Npm1 cA/+ mice only develop disease after a long latency (median 18 months), suggesting that other cooperating mutations are required for AML development (Vassiliou et al. 2011).Approximately 50% of patients with an NPM1cA mutation also harbor a mutation in one of four members of the cohesin complex (STAG2, SMC3, SMC1A, and RAD21). Mutations in cohesin genes are mutually exclusive and result in haploinsufficiency of the complex. As cohesin is known to regulate gene expression by facilitating promoter-enhancer interactions and three-dimensional genome organization, we wished to determine how cohesin mutation influences AML biology and gene expression in the presence of NPM1cA. We utilized the inducible Npm1 cAflox/+ and Smc3 flox/+ mouse models to examine this genetic interaction. We and others have shown that cohesin mutations result in enhanced hematopoietic stem and progenitor cell (HSPC) self-renewal (Mazumdar et al. 2015, Viny et al. 2015, Mullenders et al. 2015, Galeev et al. 2016, Fisher et al. 2017). Consistent with this, Npm1 cA/+;Smc3 Δ/+ HSPCs show enhanced self-renewal in vitro over HSPCs harboring either single mutation. Despite a shared role in self-renewal, Npm1 cA/+;Smc3 Δ/+mice developed AML with similar latency as Npm1 cA/+ mice. Interestingly, however, Npm1 cA/+;Smc3 Δ/+ HSPCs exhibited dysregulation of a unique set of genes compared to cells from either single mutant, suggesting that the Npm1 cA;cohesin mutational combination uniquely alters the transcriptional environment. Further, Npm1 cA/+;Smc3 Δ/+ leukemias had a completely unique spectrum of acquired mutations compared to Npm1 cA/+ leukemias, suggesting that the addition of Smc3 haploinsufficiency to Npm1 cA/+alters AML evolution and that different driver mutations result in the accumulation of very different somatic mutations.Among the uniquely upregulated genes in Npm1 cA/+;Smc3 Δ/+ HSPCs is Dock1, a guanine nucleotide exchange factor (GEF) for Rac1/2. As high Dock1 expression has been associated with low overall and disease-free survival in multiple cohorts of AML patients (Lee et al. 2017, Zhang et al. 2019), we hypothesized that Dock1 would be a novel target for the treatment of Npm1cA; cohesin mut leukemias. Consistent with this hypothesis, We found that knockdown of Dock1 resulted in decreased growth and adhesion and increased apoptosis in an Npm1 cA/+;Smc3 Δ/+leukemic line, but not in an Npm1 cAsingle mutant line. Higher Rac activity was also observed in Npm1 cA/+;Smc3 Δ/+ vs. Npm1 cA/+ leukemic lines. We found that DN Rac2 specifically impacted both the growth and apoptosis of an Npm1 cA/+;Smc3 Δ/+line, suggesting that Dock1 functions primarily through Rac2 to regulate survival. Importantly, the Dock1/Rac pathway is specifically targetable in Npm1 cA/+;Smc3 Δ/+ AMLs in vitro and in vivo, as knockdown of Dock1 resulted in prolonged latency in a Npm1 cA/+;Smc3 Δ/+ transplant model and slowed the growth and enhanced apoptosis of an Npm1 cA/+;Smc3 Δ/+, but not an Npm1 cA/+, leukemic line. Further, small molecule inhibitors of Dock and Rac had similar effects. Our results suggest that Dock1/Rac2 represent unique targets for the treatment of patients harboring the NPM1cA;cohesin mutational combination and provide validity to the concept that combinatorial genetics can uncover novel precision oncology targets. DisclosuresVassiliou: Kymab Ltd: Divested equity in a private or publicly-traded company in the past 24 months; STRM.BIO: Consultancy; Astrazeneca: Consultancy.

Chronic Lymphocytic Leukemia Cells with Mutated Nfkbie Are Positively Selected By Microenvironmental Signals and Display Reduced Sensitivity to Ibrutinib Treatment

Inactivating mutations in NF-kB pathway genes, such as the NF-kB inhibitor NFKBIE, are among the more frequent genetic lesions in chronic lymphocytic leukemia (CLL). However, the role of these genetic lesions in CLL pathogenesis and treatment resistance is still largely unknown and requires further study in in vivo models of the disease. To this end, we generated transplantable murine leukemias with inactivating NFKBIE mutations and investigated their impact on leukemia growth and response to ibrutinib (IBR) treatment. The NFKBIE mutations were introduced by CRISPR/Cas9 editing in two recently established autoreactive leukemia lines derived from the Eμ-TCL1 murine CLL model. These cell lines proliferate spontaneously in vitro in a BCR-dependent manner, but also respond with increased proliferation to certain microenvironmental signals, such as those generated by Toll-like receptor (TLR) stimulation (Chakraborty S et al, Blood 2021).To investigate whether NFKBIE mutations can affect the proliferation of these cell lines in vitro, we performed competition experiments with mixed cultures of cells with wild type and mutated NFKBIE. Analysis of the clonal composition after 2 weeks showed no change in the mutant allele frequency (MAF), suggesting that NFKBIE mutations do not affect the spontaneous in vitro growth of the immortalized leukemia cells. However, repeated TLR or BCR stimulation of these cells with CpG-DNA, LPS, anti-IgM or autoantigen resulted in a 2-3 fold increase in MAF, suggesting that NFKBIE mutations provide a growth advantage when the cells are exposed to certain microenvironmental signals (n=3 experiments/condition, P<0.05 for each condition).To investigate the impact of NFKBIE mutations on leukemia growth in vivo, the same cells were transplanted by intraperitoneal injection in wild type mouse recipients (n=8) and the clonal composition was determined 3 weeks later by MAF analysis of cells isolated from peritoneal cavity (PC), blood and spleen. A significant increase in MAF was observed only in leukemia cells isolated from the spleen (P<0.05), suggesting that microenvironmental signals that positively select NFKBIE-mutated cells are available only in certain tissue compartments.Because mutations in other NF-kB pathway genes have been associated with resistance to IBR in mantle cell lymphoma, we next investigated whether NFKBIE mutations can also affect the response to IBR treatment. In vitro BrdU-incorporation experiments showed that IBR inhibits the proliferation of cells with mutated NFKBIE to a significantly lesser extent compared to cells with wild type NFKBIE (% proliferating cells with wild type and mutated NFKBIE, respectively, cultured without IBR: 90% vs 88%, P=n.s., with 0.2 μM IBR: 57% vs 73%, P<0.001, with 1.0 μM IBR: 28% vs 53%, P<0.001). Consistent with this finding, positive selection of NFKBIE-mutated cells was observed in the presence of IBR after 14 days in mixed culture competition experiments (mean MAF without IBR 47%, with 0.2 μM IBR 61%, p=0.032, with 1.0 μM IBR 64%, p=0.034). The greater resistance of NFKBIE-mutated cells to IBR was further validated by in vivo competition experiments showing a significantly greater increase in MAF in mice treated with IBR compared to controls in all three investigated compartments (n=4 mice/group, PC: P=0.029, blood P=0.029, spleen: P=0.001).To validate these findings in the clinical setting, we investigated the presence of NFKBIE mutations in a cohort of 84 IBR-treated CLL patients. Mutations of NFKBIE were detected at pre-treatment in 10/84 patients, 7/10 with >10% VAF values. Kaplan Meier analysis showed a trend towards reduced progression-free and overall survival from the beginning of IBR treatment for NFKBIE-mutated cases (Figure 1A). Analysis of an extended cohort of over 200 cases is ongoing and will be presented at the meeting.Finally, to investigate whether leukemic cells with mutated NFKBIE remain sensitive to other BCR inhibitors, we tested their growth in the presence of the PI3K inhibitor idelalisib or SYK inhibitor fostamatinib (Figure 1B). In contrast to IBR, both drugs inhibited the proliferation of NFKBIE-mutated cells in vitro, with a greater effect observed with idelalisib. Collectively, these data demonstrate that NFKBIE mutations can reduce the response to IBR treatment and suggest that such cases may benefit more from treatment with a PI3K inhibitor. [Display omitted] DisclosuresMarasca: Janssen: Honoraria, Other: Travel grants; AstraZeneca: Honoraria; AbbVie: Honoraria, Other: Travel grants. Tafuri: Roche: Research Funding; Novartis: Research Funding; Celgene: Research Funding. Laurenti: Janssen: Consultancy, Honoraria; AstraZeneca: Consultancy, Honoraria; AbbVie: Consultancy, Honoraria, Research Funding; Roche: Honoraria, Research Funding; Gilead: Honoraria; BeiGene: Honoraria. Gattei: abbVie: Research Funding; Janssen: Research Funding; Menarini: Research Funding.

Fractal Sine Wave of a Histone Reductase Drug Candidate

Compounds submitted to the National Cancer Institute undergo in vitro screening against 60 malignant cell lines (NCI60). The screening for our metallized immune complex Toroglobulin showed uniform activity against leukemia lines, but not solid tumors. The drug mechanism of this effect was acknowledged by the screeners to be that of a histone reductase, with a 417 mv. electron transfer measured by cyclic voltammetry on a gold working electrode (1). Such differential cell activity in the screen has been called The Solid Tumor Barrier (2). We then searched to overcome this barrier in order to increase the drug potency and range of Toroglobulin. Experimentation led us to alter Toroglobulin by addition of the amino acids threonine and glycine. We assay the molecular magnetism (spin) by use of a magnetic electrode (Magnetic Sciences) on a Hantek Oscilloscope. This altered Toroglobulin produced a wave train in which one sine wave was integrated with another in fractal symmetry (Fig.1). This new integration with amino acids to produce magnetic sine waves, infers we have magnetic exchange (Exchange Globulin). The resulting periodic oscillation provides an electrochemical model to assay a mode of drug activity. References- US Patent No. 10,472,379 12,2019. Ruthenium Sphingomyelin Complexes and Methods for their Use in the Treatment of Tumors. Palmer, J, Peltier, R, The Solid Tumor Barrier, Proto Magazine, Mass. General Hospital, June 17, 2019. Figure 1

Mechanical stiffness softening and cell adhesion are coordinately regulated by ERM dephosphorylation in KG-1 cells.

Mechanical stiffness is closely related to cell adhesion and rounding in some cells. In leukocytes, dephosphorylation of ezrin/radixin/moesin (ERM) proteins is linked to cell adhesion events. To elucidate the relationship between surface stiffness, cell adhesion, and ERM dephosphorylation in leukocytes, we examined the relationship in the myelogenous leukemia line, KG-1, by treatment with modulation drugs. KG-1 cells have ring-shaped cortical actin with microvilli as the only F-actin cytoskeleton, and the actin structure constructs the mechanical stiffness of the cells. Phorbol 12-myristate 13-acetate and staurosporine, which induced cell adhesion to fibronectin surface and ERM dephosphorylation, caused a decrease in surface stiffness in KG-1 cells. Calyculin A, which inhibited ERM dephosphorylation and had no effect on cell adhesion, did not affect surface stiffness. To clarify whether decreasing cell surface stiffness and inducing cell adhesion are equivalent, we examined KG-1 cell adhesion by treatment with actin-attenuated cell softening reagents. Cytochalasin D clearly diminished cell adhesion, and high concentrations of Y27632 slightly induced cell adhesion. Only Y27632 slightly decreased ERM phosphorylation in KG-1 cells. Thus, decreasing cell surface stiffness and inducing cell adhesion are not equivalent, but these phenomena are coordinately regulated by ERM dephosphorylation in KG-1 cells.

Anti-inflammatory and prolonged protective effects of Artemisia herba-alba extracts via glutathione metabolism reinforcement

Artemisia herba-alba (AHA) is a medicinal plant known for its preventive capacities against chronic diseases. Inflammation is a naturally occurring cellular response to noxious stimuli, and both acute and chronic inflammation are involved in human disorders. Preventing and limiting the negative impact of inflammation by using phytocompounds has attracted a lot of attention. Here, we show that AHA extracts are potent anti-inflammatory products at low doses in white blood cells, treated with phorbol myristate acetate (PMA), a pro-inflammatory trigger. Regardless of the mode of extraction, i.e. maceration or microwave, all extracts reduced on dose-dependent manner reactive oxygen species (ROS) production induced by the pro-inflammatory compound PMA in white blood cells and leukemic Jurkat cell line. The induction of the anti-inflammatory effect was evaluated by the measurement of glutathione metabolism (glutathione reductase, transferase, and peroxidase), and protein-free thiols in Jurkat cells. Jurkat cells incubated with low to high concentrated AHA extracts, ranged from 10 to 100 g/mL, induced a fast cell response measured at one and six hours. Interestingly, extracts from the microwave showed a stronger induction, 5 to 10 fold changes at early time points, and maintained over 72 h. On the other hand, AHA extracts showed a moderate anti-proliferative effect in the leukemic Jurkat line. We concluded that AHA extracts enhance directly anti-inflammatory cell response, earlier via ROS induction. Later, however, vigorously maintained, independently from ROS, suggesting the intervention of the cellular metabolism in enhancing these effects. Considering the preventive and anti-inflammatory potential of AHA, regular consumption is advisable, and phytotherapeutic applications are encouraged to treat local inflammations.

Sy-1365, a Potent and Selective CDK7 Inhibitor, Exhibits Anti-Tumor Activity in Preclinical Models of Hematologic Malignancies, and Demonstrates Interactions with the BCL-XL/BCL2 Mitochondrial Apoptosis Signaling Pathway in Leukemia

Introduction: The transcriptional kinase CDK7 has been implicated in the pathogenesis of multiple malignancies, including hematologic malignancies, and may play important roles in regulation of oncogenic transcriptional dependencies and in regulation of the mitochondrial apoptosis machinery in tumors. SY-1365, a selective inhibitor of CDK7, was developed to exploit tumor dependencies driven by CDK7 and is in clinical development in patients with advanced solid tumors. We have previously reported that SY-1365 selectively induces apoptosis in leukemic cells relative to non-malignant cells in vitro, and demonstrates anti-tumor activity in AML xenografts. Here we report that SY-1365 has potent inhibitory activity against a broad panel of hematologic malignancies in vitro and ex vivo, induces regressions in AML xenografts using a clinically relevant dosing regimen, and demonstrates synergy with the BCL2 inhibitor venetoclax in AML cells in vitro.Methods: SY-1365 dose-response curves were measured using the ATP-lite assay in a panel of 85 leukemia and lymphoma cell lines in vitro, and in a panel of 226 patient-derived tumor samples from 5 leukemic indications cultured ex vivo. In vivo tumor inhibitory activity was evaluated using once weekly (QW) and twice weekly (BIW) dosing regimens in ML-2 and Kasumi-1 AML xenografts. Growth-rate (GR) adjusted dose response curves were used to classify cell lines into low and high sensitivity groups, and were evaluated relative to RNA expression data to identify markers predictive of sensitivity to SY-1365. Synergy between SY-1365 and venetoclax was evaluated in vitro using KG-1 AML cells.Results: In 42 leukemia lines, SY-1365 had a median IC50 of 11nM (range 1-79nM), with 48% (20/42) demonstrating particularly low IC50s (<10nM). In 43 lymphoma lines, the median IC50 was 6nM (range 1-39nM), with 59% (27/46) demonstrating low IC50s (<10nM). Similarly, SY-1365 demonstrated potent inhibitory activity against patient-derived leukemia samples cultured ex vivo (median IC50 of 28nM), with 82% (185/226) of the samples showing IC50 below 100nM and 8% (19/226) under 10nM. Furthermore, analysis of growth rate (GR) in hematologic tumor cell lines showed that <10nM SY-1365 induced cytotoxicity (GR<0) in 19% (8/42) of leukemia and 21% (9/43) of lymphoma lines. To evaluate SY-1365 anti-tumor activity in vivo, 2 AML cell lines that demonstrate cytotoxic responses to SY-1365 in vitro (Kasumi-1 and ML-2) were grown as subcutaneous xenografts in mice. QW and BIW dosing of 40mg/kg SY-1365 induced tumor regressions in Kasumi-1 xenografts, and BIW dosing induced tumor stasis in ML-2 xenografts. Tumor growth inhibition was detected with a 20mg/kg BIW dosing regimen in both models. Comparison of SY-1365 sensitivity with RNA expression in hematologic tumor cell lines revealed that lower expression of BCL2L1, which encodes the mitochondrial apoptosis inhibitor BCL-XL, was highly predictive of sensitivity in leukemia cell lines (Accuracy=89%, FDR<0.05); no such relationship was observed in lymphoma lines. Considering the established role of BCL2 and the mitochondrial apoptosis pathway in leukemia, we reasoned that BCL-XL low-expressing leukemias, in addition to being sensitive to SY-1365, would also be reliant on BCL2 for survival. Indeed, in vitro combination studies demonstrated synergistic inhibitory activity of SY-1365 with the BCL2 inhibitor venetoclax in KG-1 AML cells. A potential synergistic interaction with SY-1365 and venetoclax is currently being explored in vivo with AML xenografts.Conclusions: SY-1365 shows potent in vitro inhibitory activity in multiple hematological indications and can induce AML xenograft tumor regression in mice. In vitro exploratory biomarker and tumor cell inhibitory combination studies in AML suggest a role for mitochondrial apoptosis signaling in mediating sensitivity to SY-1365 and support the evaluation of a combination with venetoclax. SY-1365 is currently being assessed in a phase 1 trial in adult patients with advanced solid tumors (NCT03134638). The results described here support the potential for additional exploration in patients with hematological malignancies. DisclosuresHodgson:Syros Pharmaceuticals: Employment, Equity Ownership. Johannessen:Syros Pharmaceuticals: Employment, Equity Ownership. Rajagopal:Syros Pharmaceuticals: Employment, Equity Ownership. Hu:Syros Pharmaceuticals: Employment, Equity Ownership. Orlando:Syros Pharmaceuticals: Employment, Equity Ownership. Eaton:Syros Pharmaceuticals: Employment, Equity Ownership. McKeown:Syros Pharmaceuticals: Employment. Tyner:Takeda Pharmaceutical Company: Research Funding; Agios Pharmaceuticals: Research Funding; Incyte Corporation: Research Funding; Gilead: Research Funding; Janssen Pharmaceutica: Research Funding; Syros: Research Funding; Aptose Biosciences: Research Funding; Seattle Genetics: Research Funding; Constellation Pharmaceuticals: Research Funding; Genentech: Research Funding; Array Biopharma: Research Funding; AstraZeneca: Research Funding; Leap Oncology: Consultancy. Sprott:Syros Pharmaceuticals: Employment, Equity Ownership. Fritz:Syros Pharmaceuticals: Employment, Equity Ownership. Di Tomaso:Syros Pharmaceuticals: Employment.

Preclinical Rationale for the Use of Regorafenib in Myeloid Malignancies

Background: Regorafenib is a multikinase inhibitor that targets both angiogenic, stromal and oncogenic kinases including VEGFR-1, -2, -3, TIE-2, PDGFR- β, c-KIT, rRET, RAF-1, FGFR-1, BRAF and p38 MAPK. In vitro studies have demonstrated that regorafenib inhibits these clinically relevant kinases with half maximal inhibitory concentration (IC50) values between 4-180nM. As a result of regorafenib's broad inhibition of kinases, it may have the potential to overcome the limitations of more selective kinase inhibitors, and thus to have efficacy in a broad range of myeloid malignancies since these pathways are relevant in acute myeloid leukemia (AML), myelodysplastic syndromes, and myelofibrosis. Based on this rationale, we are currently conducting a multicenter, open-label phase I trial to assess the safety and tolerability of regorafenib in patients with advanced myeloid malignancies (NCT03042689). In addition, we are characterizing the mechanism of action of regorafenib in myeloid malignancies by conducting correlative studies (molecular and cellular biomarkers) in the clinical trial and using banked specimens.Methods:Cell Line Testing: Initial testing was performed utilizing a collection of greater than 1,000 human tumor cell lines representing the spectrum of common and rare types of adult and childhood cancers in the Benes Lab at Massachusetts General Hospital. Each cell line was treated with 9 doses 2-fold apart starting with a maximum dose of 5.12 uM (Minimum dose 20 nM; Range of 256-fold). The activity of the drug was estimated by fitting viability values (treated/DMSO control). The fitted values: Area Under the dose response Curve. A total of 95 leukemic lines were tested including 24 AML lines.Genomic and Transcriptional Characterization- We performed genomic and transcriptional characterization of the leukemia cell lines tested to determine if there was a correlation between regorafenib response and mutational pattern. Genes tested included ASXL1, FLT3, Tp53, U2AF1, TET2, NRAS, MLL, BRAF, BMPR2, CDKn2A, Kd6A, Ptpn11, RB1 .Results: High-throughput testing of regorafenib across multiple tumor types showed striking sensitivity of leukemia cell lines (Figure 1). AML lines were determined to be more sensitive than other cancer subtypes, p=0.00003 (2-tail Fisher's Exact Test). Chronic myeloid leukemia lines were also seen as sensitive compared to other subtypes likely due to the broad selectivity profile of regorafenib including activity against BCR-ABL. The genomic and transcriptional characterization of the cell lines tested thus far has not identified any associations between specific mutational profiles and response to regorafenib.Of the patients treated on study so far in the clinical trial, one patient experienced a sustained and robust neutrophil count recovery after regorafenib (Figure 2A). Based on this initial finding, we examined whether differentiation of myeloid precursors may be a putative mechanism of action of regorafenib. We performed serial testing by flow cytometry to assess for differentiation. Testing in one patient suggested a change in the phenotype of the myeloid blasts on therapy from CD33+/CD13- to CD33+/CD13+ along with an increase in mature granulocytes (Figure 2B). We are currently collecting samples on study to determine if regorafenib leads to myeloid cell differentiation. In addition, we are assessing the pharmacodynamic effects of regorafenib by 1) evaluating the phosphorylation of targets such as VEGFR, FGFR, TIE-2, and cKIT, among others, and 2) measuring plasma levels of angiogenic and inflammatory biomarkers. Finally, we are measuring changes in specific markers of allele burden when applicable.Conclusion: In summary, we are conducting a clinical study of regorafenib in myeloid malignancies, which will evaluate initial activity and characterize the mechanism of action and potential biomarkers of response to this drug in myeloid malignancies. Early results of a high throughput screen on multiple tumor types suggest AML is particularly sensitive to this agent. An updated biomarker analysis will be presented at the meeting. [Display omitted] DisclosuresBrunner:Celgene: Research Funding; Takeda: Research Funding. Duda:bayer: Consultancy.

Obesity-Induced Chronic Inflammation Suppresses the Function of Non-Malignant Immune Cells While Upregulating PD-L1 on Human B-ALL Cell Lines

Obesity incidence continues to rise dramatically throughout the world. Currently, 20% of school-aged children (ages 6-19) and 35% of the adult population (> 20 years of age) in the United States are obese. Obesity-related complications contributes to various diseases including insulin resistance, type 2 diabetes, and cardiovascular diseases. In addition to these diseases, obesity is a risk factor for developing childhood leukemia and significantly increases mortality rates in pediatric patients. Obesity-associated chronic inflammation is postulated to contribute to the onset and propagation of leukemia; however, this hypothesis has remained largely unexplored in experimental settings. Therefore, we established an in vitro system to delineate how non-malignant immune cells and B-cell leukemia lines respond to cytokine stimulation. Specifically, dendritic cells (DC) were isolated from the spleens of C57BL/6 mice using anti-CD11c microbeads followed by MiniMACS purification and stimulated with IL-1β, IL-6, TNF-α, or LPS (as single agents or in combination) for 24 hours prior to assaying for the upregulation of CD80,CD86, and PD-L1 using flow cytometric analysis. We found that splenic DC stimulated with IL-1β or TNF-α upregulated both CD80 and CD86 in a dose-dependent manner. Surprisingly, both cytokines also induced PD-L1 expression on DC. We next tested the effects of cytokine stimulation on Nalm6, REH, and SEM human B-ALL cell lines. Only SEM cells expressed CD86, which was not modulated by cytokine stimulation. In contrast, stimulating REH and SEM cells with cytokines upregulated PD-L1 expression with TNF-α being the most potent inducer of the immune inhibitory receptor. In order to mimic a chronic inflammatory microenvironment in vitro, mouse DC cells were treated with individual pro-inflammatory cytokines for 24 hours prior to stimulation with LPS treatment for 24 hours before assaying for functional outcomes at 48 hours post-stimulation. We observed that LPS, IL-1β, and TNF-α treatments increased CD80 and CD86 expression. Surprisingly, LPS upregulation of both CD80 and CD86 was blocked on DC when cytokine treatment preceded LPS stimulation. Similarly, DC exposure to cytokines prior LPS treatment failed to induced CD40 and PD-L1 upregulation. To examine how adipocytes-produced inflammatory cytokines modulated these functional responses in non-malignant B cells, we isolated mouse B cells from spleens of C57BL/6 mice using anti-B220 microbeads followed by MiniMACS purification. Using a modified insulin Oleate (IO) media differentiation protocol, we were able to induce adipocyte differentiation from OP9 cells (a mouse bone marrow stromal cell line). Adipocytes differentiated in vitro morphologically phenocopied primary adipocytes, and they expressed a hallmark adipocyte marker Fatty Acid Binding Protein 4 (FABP4). Importantly, these cells produced copious amounts of TNF-α. When splenic B-cells were cultured with adipocyte-produced supernatants, we found that CD86 surface expression was downregulated while PD-L1 expression increased. In conclusion, we found that IL-1β and TNF-α potently induced CD80 and CD86 on splenic DC and B-cells in acute settings. Surprisingly, these cytokines also induced PD-L1 expression on these cells most likely to prevent the pathological effects of an uncontrolled immune response. In the chronic settings, we observed blunted innate immune responses to LPS stimulation. These observations suggest that chronic inflammatory microenvironments (such as those associated with obesity) will induce an immune suppressive state in splenic DC and B-cell populations. This hypothesis was supported by our observations that adipocyte-produced soluble factors also decreased CD86 expression and upregulated PD-L1 on splenic B-cells. Pro-inflammatory cytokines also potently induced PD-L1 expression on B-ALL cells. In all, our data suggest that chronic inflammation may promote leukemogenesis by both attenuating de novo immune responses and reducing the ability to effector T-cells to kill leukemia cells when engaged due to the upregulation of immune suppressive mechanisms. Disclosures No relevant conflicts of interest to declare.

Significance of nutrient media choice for the long-term cultures of leukemic T-lymphoblasts

Correct choice of nutrient media for culturing different types of cells in various applications is one of the most important aspects of modern biotechnology, since chemical composition of the culture media largely contains the necessary metabolites to support certain cells’ growth lines outside the body. Jurkat line of human leukemic T-lymphoblast-like cells (hereinafter Jurkat T-cells) is actively used for in vitro modeling of intracellular signaling and activation of normal blood T-lymphocytes mediated by the T-cell receptor/CD3/ CD4 complex in toxicological studies of immune and secretory responses, to test medicinal substances and ions. Also, Jurkat T-cells are widely used for ex vivo testing in immunology, oncology, toxicology, orthopedics, and traumatology. The existing standards and numerous studies are mainly based on short-term in vitro cultivation of Jurkat T-cells in RPMI 1640 nutrient medium. Meanwhile, the issues of long-term maintenance of the growth of Jurkat T-cells culture are poorly presented in the research literature. This study aimed for studying the activity of Jurkat T-cells over 7 to 14 days of in vitro culture and comparing the relative value of RPMI 1640 and αMEM media for the behavior of immunocompetent tumor cells. Using flow cytometry, multiplex analysis, and phase contrast Cell-IQ microscopy, the proportions of living cells and those dying by apoptosis and necrosis, secretion of cytokines and chemokines, and the dynamics of cell biomass propagation were studied. It was found that the αMEM medium in the complete nutrient medium, as compared with RPMI 1640, is more appropriate to in vitro promotion of cell viability (increased proportion of viable cells by 13.5% at the day 14), their secretory ability for 23 из 27 tested biomolecules, shortened adaptation time (на 32%) in culture before growth initiation, 5-fold increase of the Jurkat Т-cell cellularity by the day 7. Potential significance of the chemical components of nutrient media and secreted biomolecules for these results is discussed. As based on the results obtained, we concluded on superior properties of αMEM medium for long-term in vitro cultures of Jurkat T-cells. Consequently, the in vitro testing of medical devices intended for long-term contact with the body, including those for cancer patients, using Jurkat T-cell leukemia line in RPMI 1640 medium, may lead to wrong predictions on their biocompatibility and potential antitumor activity.

CRISPR-Cas9 Editing of Human Histone Deubiquitinase Gene USP16 in Human Monocytic Leukemia Cell Line THP-1

USP16 is a histone deubiquitinase which facilitates G2/M transition during the cell cycle, regulates DNA damage repair and contributes to inducible gene expression. We mutated the USP16 gene in a high differentiation clone of the acute monocytic leukemia cell line THP-1 using the CRISPR-Cas9 system and generated four homozygous knockout clones. All were able to proliferate and to differentiate in response to phorbol ester (PMA) treatment. One line was highly proliferative prior to PMA treatment and shut down proliferation upon differentiation, like wild type. Three clones showed sustained expression of the progenitor cell marker MYB, indicating that differentiation had not completely blocked proliferation in these clones. Network analysis of transcriptomic differences among wild type, heterozygotes and homozygotes showed clusters of genes that were up- or down-regulated after differentiation in all cell lines. Prior to PMA treatment, the homozygous clones had lower levels than wild type of genes relating to metabolism and mitochondria, including SRPRB, encoding an interaction partner of USP16. There was also apparent loss of interferon signaling. In contrast, a number of genes were up-regulated in the homozygous cells compared to wild type at baseline, including other deubiquitinases (USP12, BAP1, and MYSM1). However, three homozygotes failed to fully induce USP3 during differentiation. Other network clusters showed effects prior to or after differentiation in the homozygous clones. Thus the removal of USP16 affected the transcriptome of the cells, although all these lines were able to survive, which suggests that the functions attributed to USP16 may be redundant. Our analysis indicates that the leukemic line can adapt to the extreme selection pressure applied by the loss of USP16, and the harsh conditions of the gene editing and selection protocol, through different compensatory pathways. Similar selection pressures occur during the evolution of a cancer in vivo, and our results can be seen as a case study in leukemic cell adaptation. USP16 has been considered a target for cancer chemotherapy, but our results suggest that treatment would select for escape mutants that are resistant to USP16 inhibitors.