BRCA2 Haploinsufficiency in Telomere Maintenance

Our previous studies showed an association between monoallelic BRCA2 germline mutations and dysfunctional telomeres in epithelial mammary cell lines and increased risk of breast cancer diagnosis for women with BRCA2 999del5 germline mutation and short telomeres in blood cells. In the current study, we analyzed telomere dysfunction in lymphoid cell lines from five BRCA2 999del5 mutation carriers and three Fanconi Anemia D1 patients by fluorescence in situ hybridization (FISH). Metaphase chromosomes were harvested from ten lymphoid cell lines of different BRCA2 genotype origin and analyzed for telomere loss (TL), multitelomeric signals (MTS), interstitial telomere signals (ITS) and extra chromosomal telomere signals (ECTS). TL, ITS and ECTS were separately found to be significantly increased gradually between the BRCA2+/+, BRCA2+/- and BRCA2-/- lymphoid cell lines. MTS were found to be significantly increased between the BRCA2+/+ and the BRCA2+/- heterozygous (p < 0.0001) and the BRCA2-/- lymphoid cell lines (p < 0.0001) but not between the BRCA2 mutated genotypes. Dysfunctional telomeres were found to be significantly increased in a stepwise manner between the BRCA2 genotypes indicating an effect of BRCA2 haploinsufficiency on telomere maintenance.

Functional properties of immune cells under the influence of extracts of mushrooms Ganoderma lucidum, Lentinula edodes, Boletus edulis

Тhe water extracts of Basidiomycota division mushrooms such as Boletus edulis, Ganoderma lucidum, Lentinula edodes posessed a wide spectrum of immunomodulatory activity. It was shown that Boletus edulis enhances the expression of surface markers on lymphoid cell lines Jurkat-tat and Daudi, and also increases the expression of reactive oxygen species by dendritic cells, monocytes and peripheral blood neutrophils, activates basophils. There was observed no effect of fungal extracts on NK-cells.

The Expression of Paks and Its Clinical Significance in T-Cell Lymphoblastic Lymphoma

Background: T-cell lymphoblastic lymphoma (T-LBL) is a highly aggressive non-Hodgkin's lymphoma with poor prognosis and lacks of standard treatment approaches. PAK2, a member of the p21 activated kinase (PAKs) family, is a component of the gene-expression-based classifier which made great contributions to prognostic prediction of T-LBL(Cai et al. Leukemia 2020). This study aims to analyze the expression of PAKs in human T-LBL cell lines and tissues to clarify its clinical significance and evaluate the therapeutic activity of PAKs inhibitor in T-LBL. Methods: The mRNA and protein expression levels of PAKs in human T-lymphoid cell lines (H9) and T-LBL cell lines (Jurkat, SUP-T1 and CCRF-CEM) were detected by quantitative real-time PCR and western blot, respectively. We retrospectively collected 69 Formalin-fixed, Paraffin-embedded (FFPE) samples and corresponding clinical information from T -LBL patients between September 2000 and May 2015 at Sun Yat-sen University Cancer Center (SYSUCC). Affymetrix Human Gene 2.0 ST microarray (Thermo Fisher Scientific, Waltham, MA, USA) was used to detect the expression of PAKs. Mann-Whitney U test was used for comparing the expression differences between relapsed and non-relapsed patients. Cumulative relapse-free survival (RFS) time was calculated using the Kaplan-Meier method (Expression level higher than the upper quartile (P75) was defined as PAK1 or PAK 2 high expression. High expression (median as cutoff point) of both PAK1 and PAK2 was defined as PAK1/2 high expression). Pearson's chi-square test and Fisher's exact test were used to compare the distribution of clinical variables. Correlations between PAK1, PAK2 and NOTCH1 were evaluated using Spearman's correlation coefficient. Two PAK inhibitors, PF3758309 (PF) and FRAX597, were used to block PAK kinase activity pharmacologically. Cell viability was determined using the viability assay kit CCK-8. Cell cycle and cell apoptosis were analyzed by flow cytometry. All statistical analyses were performed using SPSS 24.0 software (SPSS, Armonk, NY, USA) or GraphPad Prism 8.0 (GraphPad, La Jolla, CA, USA). A P value &lt; 0.05 was considered significant. The study protocol was approved by the Institutional Review Board of SYSUCC. Results: The mRNA and protein expression levels of PAK1 in T-LBL cell lines of Jurkat, SUP-T1 and CCRF-CEM cell lines were significantly higher than those in T-lymphoid cell lines H9 cell line (P&lt;0.05) (Figure 1). Of the 69 T-LBL patients, 44 (63.8%) were male and the median age at diagnosis was 30 years (range: 16-44). The majority (72.5%) of the patients received acute lymphoblastic leukemia (ALL)-type chemotherapy regimens. The PAK2 mRNA expression level of 32 patients with relapsed disease was significantly higher than that of 37 cases without relapse (P=0.012), and no difference was found in mRNA expression of PAK1, 3, 4, 7(Figure 2). Patients with high PAK1 and PAK2 expression had significantly shorter median RFS than those with low PAK1 and PAK2 expression (PAK1 mRFS 31.5 months vs not reached(NR), HR=3.001, P=0.028; PAK2 mRFS 25.7 months vs NR, HR=3.981, P=0.027)(Figure 3). Patients with high PAK1/2 mRNA expression experienced earlier relapse than patients with low PAK1/2 mRNA expression (mRFS 26.0 months vs NR, HR=2.721, P=0.032)(Figure 3). PAK1/2 mRNA expression was found to be associated with hemoglobin concentration, Ki-67 expression, pleural and pericardia effusion, bone marrow involvement and chemotherapy response (P&lt;0.05)(Table 1). Further, the PAK1 mRNA was correlated with the expression of NOTCH1, which is frequently mutated in T-LBL (r=0.5716, P&lt;0.0001)(Figure 4). The PAK inhibitors PF and FRAX597 demonstrated strong anti-tumor activity in vitro (Table 2). Both inhibitors suppressed cell proliferation in a time- and dose-dependent manner (Figure 5). Both inhibitors induced cell cycle arrest in the G1/0 phase, accompanied by corresponding S phase reductions in all tested cells(Figure 6). The synergistic effect between PAK inhibitor PF and doxorubicin was also observed (Figure 7). Besides, PF could inhibit the phosphorylation of PAK1/2, Cyclin D1 and NF-κB in Jurkat cell line(Figure 8). Conclusions: PAK1 and PAK2 play certain roles in the occurrence and recurrence of T-LBL, and their potential as novel biomarkers deserves further exploring. Our results underscore the potential of PAK inhibitor as effective target therapy for T-LBL. Disclosures No relevant conflicts of interest to declare.

An MXD1-derived repressor peptide identifies noncoding mediators of MYC-driven cell proliferation

MYC controls the transcription of large numbers of long noncoding RNAs (lncRNAs). Since MYC is a ubiquitous oncoprotein, some of these lncRNAs probably play a significant role in cancer. We applied CRISPR interference (CRISPRi) to the identification of MYC-regulated lncRNAs that are required for MYC-driven cell proliferation in the P493-6 and RAMOS human lymphoid cell lines. We identified 320 noncoding loci that play positive roles in cell growth. Transcriptional repression of any one of these lncRNAs reduces the proliferative capacity of the cells. Selected hits were validated by RT-qPCR and in CRISPRi competition assays with individual GFP-expressing sgRNA constructs. We also showed binding of MYC to the promoter of two candidate genes by chromatin immunoprecipitation. In the course of our studies, we discovered that the repressor domain SID (SIN3-interacting domain) derived from the MXD1 protein is highly effective in P493-6 and RAMOS cells in terms of the number of guides depleted in library screening and the extent of the induced transcriptional repression. In the cell lines used, SID is superior to the KRAB repressor domain, which serves routinely as a transcriptional repressor domain in CRISPRi. The SID transcriptional repressor domain is effective as a fusion to the MS2 aptamer binding protein MCP, allowing the construction of a doxycycline-regulatable CRISPRi system that allows controlled repression of targeted genes and will facilitate the functional analysis of growth-promoting lncRNAs.

The Cell-Cycle Regulatory Protein p21CIP1/WAF1 Is Required for Cytolethal Distending Toxin (Cdt)-Induced Apoptosis

The Aggregatibacter actinomycetemcomitans cytolethal distending toxin (Cdt) induces lymphocytes to undergo cell-cycle arrest and apoptosis; toxicity is dependent upon the active Cdt subunit, CdtB. We now demonstrate that p21CIP1/WAF1 is critical to Cdt-induced apoptosis. Cdt induces increases in the levels of p21CIP1/WAF1 in lymphoid cell lines, Jurkat and MyLa, and in primary human lymphocytes. These increases were dependent upon CdtB’s ability to function as a phosphatidylinositol (PI) 3,4,5-triphosphate (PIP3) phosphatase. It is noteworthy that Cdt-induced increases in the levels of p21CIP1/WAF1 were accompanied by a significant decline in the levels of phosphorylated p21CIP1/WAF1. The significance of Cdt-induced p21CIP1/WAF1 increase was assessed by preventing these changes with a two-pronged approach; pre-incubation with the novel p21CIP1/WAF1 inhibitor, UC2288, and development of a p21CIP1/WAF1-deficient cell line (Jurkatp21−) using clustered regularly interspaced short palindromic repeats (CRISPR)/cas9 gene editing. UC2288 blocked toxin-induced increases in p21CIP1/WAF1, and JurkatWT cells treated with this inhibitor exhibited reduced susceptibility to Cdt-induced apoptosis. Likewise, Jurkatp21− cells failed to undergo toxin-induced apoptosis. The linkage between Cdt, p21CIP1/WAF1, and apoptosis was further established by demonstrating that Cdt-induced increases in levels of the pro-apoptotic proteins Bid, Bax, and Bak were dependent upon p21CIP1/WAF1 as these changes were not observed in Jurkatp21− cells. Finally, we determined that the p21CIP1/WAF1 increases were dependent upon toxin-induced increases in the level and activity of the chaperone heat shock protein (HSP) 90. We propose that p21CIP1/WAF1 plays a key pro-apoptotic role in mediating Cdt-induced toxicity.

Combination of Momelotinib and Citarinostat Show Strong Synergistic Effect in Lymphoid Cell Lines Co-Targeting JAK2/STAT3 and HDAC6

Introduction. Janus kinases (JAKs) are well described signaling kinases comprising four family members JAK1, JAK2, JAK3 and TYK2 that are essential in hematological malignancy, as JAK mutations have been shown to contribute to the pathogenesis of myeloproliferative disorders. Momelotinib is a potent inhibitor of JAK1/JAK2 that demonstrated efficacy in patients with primary and secondary myelofibrosis. HDAC6 has been reported to be overexpressed in lymphoid cells and its inhibition has demonstrated activity in preclinical and clinical study of lymphoproliferative disease. Citarinostat, a second generation HDAC6 selective inhibitor, is a well-tolerated compound compared with nonselective HDAC inhibitors, with reduced potency against Class I HDACs while retaining anticancer effectiveness. Both drugs are currently under investigation in clinical trials, they show a good toxicity profile and are both orally available. Methods. Momelotinib and citarinostat alone and in combination were tested in 12 lymphoid cell lines: WSU-NHL, RL, Karpas422 (follicular lymphoma), Granta519, Jeko1 (mantle cell lymphoma), Hut78 (cutaneous T cell lymphoma), Karpas299 (anaplastic large cell lymphoma), L540, L1236 (Hodgkin's lymphoma), U266, RPMI8266 (multiple myeloma) and MEC1 (chronic lymphocytic leukemia). Synergistic interaction was evaluated using the Chou-Talalay method. Annexin V staining, ROS generation, cell cycle and migration assay were determined by flow cytometry. ATP levels and Mitochondrial Membrane Potential (ΔΨm) were evaluated by fluorometric assay. Lactate levels and Cyt-C were evaluated by colorimetric assay. Activity of caspases-8,-9 and 3 was measured using colorimetric assay. ER stress and apoptosis-related proteins and JAK2/STAT3 were detected by western blotting. Clonogenic survival was studied with the methylcellulose clonogenic assay. Co-cultures with bone marrow stromal cells were also performed. Results. The combination of momelotinib (1 μM) and citarinostat (4 μM) at 24 h showed a synergistic effect in WSU-NHL, RL, Karpas422, Jeko1, Hut78, Karpas299, L540, RPMI8226 and U266 cells with CI values < 1 and antagonist effect in L1236, Granta519 and Mec1 cells with CI > 1. We studied seven lymphoid cell lines (WSU-NHL, RL, Karpas422, Jeko1, L-540) which were particularly sensitive to the drug combination and two cell lines (L-1236, Granta-519) that showed an IC > 1. Drug combination exhibited a strong cytotoxicity, evidenced by reduction of mitochondrial depolarization, depletion of ATP and lactate levels and Cyt-C release from the mitochondria but also by increase in cellular apoptotic index and reactive oxygen species levels, leading to arrest in the sub-G0/G1 phase of the cell cycle. Apoptosis induced by the drug combination was exerted via the mitochondrial apoptotic pathway as demonstrated by upregulation of caspase-9 that was especially evident in WSU-NHL and Karpas422 with a fold increase of 3.2 and 3.8. The extrinsic apoptotic pathway was active in Karpas422 and Jeko1 cells as evaluated by upregulation of caspase-8 but not in WSU-NHL, RL and L540. The apoptosis was associated with activation of caspase-3, PARP and with increased hallmarks of ER stress and was mediated by the increased expression of the pro-apoptotic proteins Bad, Bax and Bim and downregulation of Bcl2, Bcl-xL and Mcl-1. Drug combination inhibited the migration induced by CXCL12 (chemoattractant known as stromal-cell derived factor-1, SDF-1α), reduced clonogenic survival and suppressed cell viability of lymphoid cells when co-cultured with bone marrow mesenchymal stromal cells targeting JAK2/STAT3 pathway and confirming acetylation of acetyl-α tubulin. Conclusion. Due to the good toxicity profile and the oral administration, combined therapy with momelotinib and citarinostat may represent a promising novel therapeutic modality for hematological malignancies. The study is ongoing and further investigation is required. Disclosures No relevant conflicts of interest to declare.