Some pharmacological properties of 4-[3-(5-bromo-2-hydroxyphenyl)-5-phenyl-3,4-dihydropyrazol-2-yl]-5H-thiazol-2-one

A series of 3,5-diaryl pyrazolyl thiazolinones were designed and synthesized as potential biologically active compounds. The study of anticancer activity of 4-[3-(5-bromo-2-hydroxyphenyl)-5-phenyl-3,4-dihydropyrazol-2-yl]-5H-thiazol-2-one (1) revealed its high antiproliferative activity against a panel of cancer cells with the lowest growth inhibition concentration (GI50) towards leukemic cell line SR (0.0351 µМ) and ovarian cancer cell line OVCAR-3 (0.248 µМ). It was also found that pyrazolyl thiazolinone 1 inhibited growth of Trypanosoma brucei brucei by 98,8% at a concentration of 10 µg/mL. The in-depth cytotoxicity study of compound 1 on human hepatocellular carcinoma HepG2 cells and non-tumorigenic murine fibroblast Balb/c 3T3 in MTT, NRU, TPC and LDH assays showed that normal cells were less sensitive to compound 1 than the cancer cells; its action had led to a disintegration of the cell membrane, inhibition of mitochondrial and lysosomal activity, and proliferation of cancer cells. The highest selectivity were detected in the LDH assay.

Inducible Knockout of HMGN1 in an In Vivo xenograft Model Reduces Down Syndrome Leukemic Burden and Increases Survival Outcomes

Introduction Down Syndrome (DS) patients are at high risk of developing hematological malignancies and ~10% are born with a pre-leukemic disorder characterised by the overproduction of immature megakaryoblasts. Children with DS have a 20-fold increased risk of developing acute lymphoblastic leukemia (ALL) of which 60% are associated with high expression of cytokine receptor like factor 2 (CRLF2) and of these, ~9% acquire the aggressive CRLF2 p.F232C mutation. DS-ALL children also experience high treatment toxicity and high relapse rates compared to non-DS leukemia patients. Genes on chromosome 21 including the high mobility group nucleosome-binding domain-containing protein 1 (HMGN1) are likely to play a role in DS leukemogenesis and may be targets for a personalized treatment approach. We aimed to determine if HMGN1 is necessary for leukemic cell proliferation using an inducible CRISPR/Cas9 guide (g)RNA murine xenograft model. Methods A DS leukemic cell line model was created using the human trisomy 21 megakaryoblastic SET-2 cell line harboring JAK2 p.V617F; the only trisomy 21 leukemic cell line currently available. SET-2 cells were transduced with CRLF2 p.F232C to model an aggressive DS-ALL mutation. NOD.Cg-Prkdcscid,Il2rgtm1Wjl/Szj (NSG) mice were each injected with 3x105 SET-2 CRLF2 p.F232C CRISPR/Cas9 cells expressing luciferase in 3 groups; Cas9 only control, HMGN1 gRNA, or JAK2 positive control gRNA. Doxycycline was administered post leukemic engraftment to induce the gRNAs and create a knockout (KO) and leukemic burden was monitored by bioluminescent imaging (BLI) twice weekly for the remainder of the experiment. Once Cas9 control mice became moribund, they were culled along with 50% of the JAK2 or HMGN1 KO mice and complete blood counts were performed. Bone marrow (BM), spleen and liver sections were stained with hematoxylin and eosin (H&E) and survival analysis was carried out for remaining JAK2 or HMGN1 KO mice. RQ-PCR was used to detect HMGN1 expression levels in KO mice organs at endpoint and DNA was extracted from cells harvested from each organ to undertake a gene editing analysis. Results Leukemic engraftment in mouse BM was observed 10 days post transplant with a radiance signal of ~1 x 104p/s/cm2/sr, therefore gRNAs were induced on day 11. On day 20, a significant reduction in tumor burden was detected in JAK2 and HMGN1 KO mice compared to Cas9 control mice (Fig. 1, Cas9: 8.4x105±1.7x105; JAK2 KO: 2.7x104±8.9x103; HMGN1 KO: 1.5x105±1.7x104 p/s/cm2/sr, prone: p<0.001, supine: p=0.005). Blood counts at day 35 indicated similar white cell counts across Cas9, JAK2 and HMGN1 KO mice, however, the Cas9 mice demonstrated thrombocytopenia and anemia (platelet count: 705±43 K/µL, HCT: 22.5±2%) which was rescued in JAK2 and HMGN1 KO mice (JAK2 KO platelet count: 3046±775 K/µL, p<0.001; HCT: 47±6.8%, p=0.002; HMGN1 KO platelet count: 1503±83 K/µL, p<0.001; HCT: 38±3.4%, p=0.004). JAK2 and HMGN1 KO mice had reduced spleen weight (JAK2 KO: 46±2 mg, p=0.019; HMGN1 KO: 51±6 mg, p=0.046; Cas9: 81±7 mg) and liver weight compared to Cas9 control mice. Megakaryoblast infiltration identified with H&E staining was evident in the BM, spleen and liver of Cas9 control mice, whereas megakaryoblasts were not observed in JAK2 or HMGN1 KO mice organs. Similarly, RQ-PCR demonstrated a 24% decrease in HMGN1 expression in the BM, 99% in the spleen and 92% in the liver; and a 38% decrease in JAK2 expression in the BM, 99% in the spleen and 70% in the liver of HMGN1 and JAK2 KO mice respectively compared to Cas9 control mice. Significantly, survival analysis of the remaining JAK2 and HMGN1 KO mice indicated a substantial survival advantage from 35 days (Cas9) to 62 and 56 days respectively for JAK2 and HMGN1 KO mice (p=0.0009). Conclusion Our CRISPR/Cas9 DS leukemic xenograft HMGN1 KO model demonstrates the important role of HMGN1 in CRLF2 p.F232C DS leukemia. Significantly, HMGN1KO decreased the leukemic burden of mice to the same extent as the JAK2 (SET-2 driver gene) KO. The HMGN1 KO mitigated ALL phenotypes including hepatosplenomegaly, anemia and thrombocytopenia, preventing leukemic progression and resulting in a significant survival advantage over Cas9 control mice. As HMGN1 has a distinct role in proliferation and survival of DS leukemic cells, it is a potential candidate for targeting with a pharmacological inhibitor as a personalized treatment for DS leukemia patients. Disclosures White: Bristol-Myers Squibb: Honoraria, Research Funding; Amgen: Honoraria.

Crispr/Cas9-Mediated ASXL1knockout in U937 Human Leukemic Cells Perturbs Myeloid Differentiation

Introduction .Mutations in the Additional Sex Combs Like1 (ASXL1) gene are frequent in myeloid malignances (myelodysplastic syndromes [MDS], myeloproliferative neoplasms, chronic myelomonocytic leukemia and acute myeloid leukemia [AML]), and they predict poor survival. As we recently published (Yoshizato T, et al., N Engl J Med 2015), mutations in ASXL1 as well as BCOR/BCORL1, PIGA, and DNMT3A are most prevalent in patients with aplastic anemia (AA). ASXL1 grouped with "unfavorable mutations" that conferred poor survival and increased risk of "clonal evolution" of AA to MDS and AML. As an epigenetic modifier, ASXL1mutations may be involved in myeloid malignant transformation (Davies C, et al., Br J Haematol 2013; Abdel-Wahab O, et al., Cancer Cell 2012) but precise mechanisms have not been delineated. Methods.The CRISPR/Cas9 system was employed to generate ASXL1-knockout clones from the U937 human leukemic cell line. Single cell clones were sorted by flow cytometry, and ASXL1 mutations were assessed by Sanger sequencing. Clones with mutations that encoded truncated proteins were used for further experiments. Characteristic features of both mutated and wild-type (WT) clones were examined: cell morphology by Wright-Giemsa staining; karyotype analysis by G-banding; and cell cycle, apoptosis, and cell differentiation by flow cytometry. RNA sequencing (RNA-Seq) was performed to screen differentially expressed genes between WT and ASXL1-knockout clones, followed by validation of gene expression using reverse transcription quantitative PCR (RT-qPCR). Results.Of 88 single cell clones, 23 clones exhibited frame-shift and nonsense mutations of ASXL1, resulting in truncated proteins. Among them, 17 clones with single nucleotide insertion (c.594insA, heterozygous or homozygous) that encoded truncated proteins Ser199Glufsx55 were used for further experiments. Karyotype analysis revealed no significant differences between WT and ASXL1-knockout clones, suggesting that neither CRISPR/Cas9 itself nor ASXL1 mutations caused chromosomal instability. No significant differences between WT and ASXL1-knockout clones were observed in cell morphology, cell proliferation, cell cycle, or 5-fluoruracil-induced cell apoptosis. When monocyte/macrophage differentiation was induced chemically by exposure to phorbol 12-myristate 13-acetate, CD11b cell surface expression was much lower in ASXL1-knockout clones than in WT. By RNA-Seq, several genes (BIRC7, CACNA2D3, CTSG, CYBB, NAIP, NTNG1, OXR1, and ACTL8)involved in pathways related to cell death and survival (including TNFR1 signaling pathway, TNFR2 signaling pathway, TWEAK signaling pathway, apoptosis signaling pathway, death receptor signaling pathway, Rac signaling pathway, and phagosome maturation pathway) were down-regulated, but were not correlated to functional deficits in cell growth or apoptosis, which was indistinguishable between ASXL1-knockout and WT clones. RT-qPCR confirmed down-regulation of genes in ASXL1-knockout compared to WT clones, including CYBB (restrictedly expressed in terminally differentiating myeloid cells) and CLEC5A (involved in granulocytic differentiation), which may contribute to disturbance in myeloid differentiation of ASXL1-knockout U937 cells. Downregulaton of NAIP, which inhibits CASP3, CASP7, and CASP9 activities, may be related to deregulation of apoptosis in ASXL1-knockout U937. Conclusion. ASXL1 mutations are frequent in both malignant and non-malignant blood diseases, and may be involved in myeloid neoplastic transformation. Using gene editing of a human leukemic cell line, we show that ASXL1-knockout perturbs myeloid differentiation and down regulates multiple genes associated with myeloid development such as CYBB and CLEC5A, providing potential mechanisms for its role in bone marrow failure and neoplastic transformation. Disclosures Young: Novartis: Research Funding.

Antisense Long Non-Coding RNA in the LEF1 Locus Regulates Sense LEF1 Expression in Leukemic Cell Line KG1

Aberrant regulation of the WNT signaling pathway is a signature in numerous human cancers. Lymphoid enhancer-binding factor-1 (LEF1) is an important transcription factor downstream of this pathway. LEF1 over-expression induces AML in mice and plays a critical role in hematopoietic cell differentiation (Petropoulos et al JME 2008). Reduction of LEF1 expression through the progression of myelodysplastic syndrome has been reported and further supports the relevance of this gene in the disease pathogenesis (Pellagatti et al Br J Haematol. 2009). Our previous work using microarray technology revealed a decreased expression of a long non-coding RNA antisense to LEF1 (LEF1-AS) in MDS patients (Baratti et al BMC Medical Genomics 2010). Mounting evidence suggests that long non-coding transcripts play important roles in the epigenetic regulation of coding genes. In this context it is not surprising that long non-coding RNAs are emerging as key players in disease development and progression. Non-coding expression overlapping coding genes is very common and several examples of local regulation have been described in the literature. Here we investigate for the first time the role of LEF1 antisense long non-coding in hematopoiesis and demonstrated its contribution in the regulation of the LEF1 locus in a leukemic cell line. To explore a possible role of LEF1-AS in differentiation, we evaluated the expression pattern of LEF1-AS through erythroid cell differentiation using qRT-PCR. CD34+ HSC cells from 6 healthy donors were induced to differentiate into erythrocytes by addition of erythropoietin during 12 days. We observed that LEF1-AS is modulated during erythroid differentiation. It was significantly down-regulated during the first stages of differentiation from CD34+ HSC to erythroblast (from collection day 6 to day 8 after addition of erythropoietin, 78% mean reduction, P<0.0001) and it was up-regulated at the end-point of collection, day 12 (not significant). Lef1 coding gene displayed a similar expression pattern, consistent with previous reports of Lef1 expression during erythroid maturation (Edmaier et al Leukemia 2014). To explore a possible regulatory role of LEF1-AS, we cloned and over-expressed the transcript in KG1 CD34+ leukemia cell line. Transient over-expression of Lef1-AS led to a significant up-regulation of Lef1 gene (22% increase, P<0.05). We also observed an increase in cell viability (19% increase P<0.05), measured by MTT, which is consistent with the up-regulation of LEF1, a pro-proliferative and anti-apoptotic transcription factor. Our preliminary results from over-expressing LEF1-AS in CD34+ HSCs suggest a similar regulatory effect of LEF1-AS upon its coding counterpart, LEF1. Since aberrant expression of LEF1 is known to disrupt normal differentiation of CD34+ cells, LEF1-AS could potentially affect differentiation through the modulation of LEF1 coding gene. Our results reveal LEF1-AS transcript as a novel player in hematopoiesis and hematologic malignancy. Disclosures No relevant conflicts of interest to declare.