Lenti-shRNA-mediated FAM54A knockdown suppresses the proliferation and induces apoptosis of human Burkitt lymphoma cells

Background Burkitt lymphoma is a kind of non-Hodgkin B-cell-derived malignancy, derived from germinal center B cells. FAM54A has been proved to be involved in various physiological and pathological processes of cancers, but the biological function of FAM54A in Burkitt lymphoma remains unclear. Thus, the aim of our research was to elucidate the roles of FAM54A in proliferation, apoptosis and cell cycle of Burkitt lymphoma.Methods Burkitt lymphoma cell line (Namalwa) was chosen to perform the following experiments. FAM54A-shRNA and negative control-shRNA lentivirus that were synthesized, respectively by Qiagen were used to transfect targeted cells in order to knockdown FAM54A or as negative control. Then, cell proliferation, cell cycle and cell apoptosis were detected by using MTS assay, propidium iodide staining and Annexin V-APC staining, respectively.Results Our results showed that high expression of FAM54A protein was found in Namalwa cell line. Furthermore, MTS analysis exhibited that knockdown of FAM54A obviously inhibited cell proliferation in Namalwa cells. What’s more, cell cycle analysis showed that knockdown of FAM54A induced Namalwa cell apoptosis and arrested cell cycle in G2/M phase.Conclusions These findings suggest that FAM54A is essential for Namalwa cell proliferation and may be a potential therapeutic target for the treatment of Burkitt lymphoma.

a Novel Method for the Detection of Minimal Residual Disease in B-Cell Malignancies: Ion Semiconductor Sequencing for the Evaluation of Igh Gene Rearrangements

Background: Minimal residual disease (MRD) detection is of high clinical relevance in patients with B-cell malignancies and is generally a surrogate parameter to evaluate treatment response and long-term prognosis. IgH gene rearrangements can be used as molecular marker in approximately 80% of lymphoma and myeloma patients since they represent lineage-specific markers and the complementarity determining region 3 (CDR3) is unique to each clone. To date, allele specific oligonucleotide polymerase chain reaction (ASO-PCR) and real-time quantitative polymerase chain reaction (RQ-PCR) are considered the most sensitive and widely applicable methods for MRD detection. A major disadvantage of ASO-PCR and RQ-PCR assays, is the use of specific primers and probes for every individual patient. Clone-specific primers and probes are not only expensive but also time-consuming to design and to test, which limits their wide applicability in the clinical setting. The recent major improvements in next generation sequencing (NGS) technologies, provide the opportunity to identify and quantify clonotypes with consensus primers combining the benefits of high sensitivity and universal applicability. The present work was designed to overcome ASO-PCR and RQ-PCR limitations by developing a feasible method for rearranged IgH genes amplification, NGS and analysis using Ion Torrent Personal Genome Machine (IT-PGM). Methods: To define a multiplex PCR protocol, DNA from 7 CLL patients, previously shown to bare a family specific clonal VDJ rearrangement, was amplified with a pool of the seven different family-specific IgH-V primers, and a consensus JH primer (Voena et al., Leukemia 1997). After Sanger sequencing, results were compared to the ones obtained with singleplex PCR protocol. Once validated, the multiplex PCR protocol was used to amplify DNA from patients and serially diluted (up to 10-8 ) DNA from Namalwa cell line (bearing a known IgH rearrangement) and subsequently sequenced on the IT-PGM using the 316 Ion-chip. NGS data were analyzed by using the IMGT-High V-Quest web server tool and the statistical software R. RQ-PCR was used to quantify the specific VDJ rearrangement in the serially diluted Namalwa DNA solutions and in DNA from patients as previously described (Farina et al, Haematologica 2009). RQ-PCR data were analyzed through a relative quantification procedure. Results: The multiplex PCR reactions we have tested, demonstrated the same specificity as the standard singleplex PCR protocol and therefore was used to construct the DNA library required for IT-PGM-based sequencing. The IT-PGM sequencing output is represented by at least 400000 reads per sample with a minimum average coverage of the VDJ repertoire of 500x. The IMGT-High V-quest tool allows a user-friendly web based analysis and a deep molecular characterization of the IgH recombinatorial repertoire. Namalwa clonal CRD3 sequences were detected up to a dilution of 10-5 without the need for specific CDR3 primers. Comparability of NGS and ASO RQ-PCR results was assessed. The use of CDR3 specific primers, along with the specific IgH-V family fluorescent probe, enabled the identification of clonal VDJ rearrangements with a sensitivity up to 10-5 (2/3 replicates) and 10-6 (just 1/3 replicates) in Namalwa Cell Line. Similar results were obtained when we characterized the IgH recombination repertoire of two CLL patients over time. Conclusions: IgH sequencing with the IT-PGM platform showed at least the same level of sensitivity as ASO RQ-PCR, without the need for patient-specific reagents. It also allows specific and detailed molecular characterization of the clonal rearrangements and could be easily incorporated into clinical laboratories for routine testing of MRD in B-cell malignancies. Disclosures No relevant conflicts of interest to declare.

In Vitro and In Vivo downregulation of Leukemogenesis by FMNL1 Inhibition In Namalwa Cell Line

Abstract 1485 Formins represent a family of proteins indispensable for fundamental actin-dependent processes; it has been suggested that they represent a group of attractive drug targets for the treatment of inflammation and malignant diseases. Formins are known for their ability to act as effectors of Rho family GTPases, proteins involved in cell organization, adhesion, and proliferation. Deregulation of Rho GTPase family members has been associated with multiple human hematological diseases such as leukemia. FMNL1 is a formin-related protein that is restrictedly expressed in hematopoietic-lineage-derived cells and overexpressed in malignant lymphoid cells such as chronic and acute lymphoid leukemia. This restricted expression supports the idea that FMNL1 may be an attractive target for novel therapies in such diseases. FMNL1 has recently been shown to interact with srGAP2, a RhoGAP family member, and this complex is temporally regulated by Rac1. Herein, we evaluated the role of FMNL1, by gene silencing, in proliferation, cell cycle, clonal growth, migration and transendothelial migration (TEM), and apoptosis in Namalwa cells, an acute lymphoblastic leukemia cell line. We also accessed the role of FMNL1 in tumor growth using a NOD/SCID xenograft model. Moreover, we confirmed the endogenous association of FMNL1 with Rac1, and studied Rac1 activity in leukemic cells lacking FMNL1 expression for the first time. To inhibit FMNL1 in the Namalwa cell line, specific shRNA-expressing lentiviral vectors targeting the FMNL1 gene (shFMNL1) or no specific sequence (shControl) were used. Cell growth was measured using the MTT colorimetric reduction in five replicates. Colony formation was carried out in semisolid methylcellulose medium and was detected after 8 days of culture by adding 1mg/mL of MTT reagent and scored by Image J quantification software. Both assays were carried out in lentiviral transduced cells treated, or not, with different concentrations of rapamycin (10 or 100nM). Migration assays were performed in triplicate using 8-mm transwells, and for TEM, HUVECs were seeded onto fibronectin-coated 5-mm transwell filters. The lower compartment for both assays was filled with SDF-1. Annexin-PI was used to access apoptosis. NOD/SCID mice were implanted with lentiviral transduced Namalwa cells in the dorsal subcutaneous site of the animal. FMNL1 association with Rac1 protein was analyzed by immunoprecipitation, and Rac1 activity was measured by a PAK-pull down assay, both followed by Western blotting. P value <0.05 was considered statistically significant. Inhibition of FMNL1 in Namalwa cells resulted in a significant decrease in proliferation of 40%, when compared with control cells (P <0.01). Interestingly, the combination of FMNL1 inhibition/rapamycin treatment showed a significantly higher reduction in cell proliferation (50%) when compared with FMNL1 inhibited cells alone (P =0.008) or rapamycin treated cells (P =0.007). Inhibition of FMNL1 induced cell cycle delay in the S phase in Namalwa cells and a consequent decrease in the number of cells in G2/M. A significant decrease of 30% in the number of colonies was observed in shFMNL1, as compared to shControl cells (P <0.0001). Again, rapamycin treatment resulted in the synergic inhibition of colony formation by 50% (P =0.007). FMNL1 silencing also resulted in a significant decrease (62%, P <0.01) in transwell and a decrease (20%) in the TEM assay. Apoptosis was unaffected by FMNL1 knockdown. In vivo experiments, the ShFMNL1 cell injected group showed a significant decrease in tumor growth during the experimental period (15 days after injection), when compared to the shControl cell injected group (P =0.002). FMNL1 was found to be associated with Rac1 in Namalwa cells, by immunoprecipitation, performed also in a reverse assay. Interestingly, Rac1 presented a four-fold increase in its activity in shFMNL1 cells in three independent PAK-pull down experiments. Aberrant Rac1 activity is known in tumorigenesis; however, in this leukemia cell model, inhibition of FMNL1 bypassed Rac1 activity. Our findings indicate that FMNL1 participates in the regulation of leukemogenesis, through different cell activities, including proliferation, colony formation, migration and tranwell migration of Namalwa cells, suggesting that FMNL1 represents a possible therapeutic target together with rapamycin, based on their synergic effects. Disclosures: No relevant conflicts of interest to declare.