Turkoz, G. | bioRxiv Science

bioRxiv2022

Identification of GTF2IRD1 as a novel transcription factor essential for acute myeloid leukemia

Acute myeloid leukemia (AML) is an aggressive blood malignancy characterized by clonal accumulation of immature myeloid progenitors in the bone marrow and peripheral blood. Transcription factors are the most frequently mutated and dysregulated genes in AML, and they have critical roles in AML pathogenesis and progression. In this study, we performed large-scale RNA interference screens in MLL-AF9-transformed AML cells and identified GTF2IRD1 as a novel transcription factor essential for the survival of various types of myeloid leukemic cells in vitro and in vivo, but not for primary normal hematopoietic cells. Inhibition of GTF2IRD1 reduced the frequency of primary childhood and adult AML cells, including cell populations enriched for leukemia-initiating cells. In animal models for AML, inhibition of GTF2IRD1 significantly delayed the disease progression. Loss of GTF2IRD1 promoted accumulation of AML cells in the G0 phase of the cell cycle but caused minor effects in apoptosis induction. In line with this, RNA sequencing analysis revealed a significant downregulation of E2F targets as a consequence of genetic inhibition of GTF2IRD1. Taken together, we identified GTF2IRD1 as a transcription factor with a selective importance in AML, and our findings may contribute to the development of improved therapeutic inventions for the disease.

cancer biology↗

bioRxiv2020

Lentiviral vector-based SARS-CoV-2 pseudovirus enables analysis of neutralizing activity in COVID-19 convalescent plasma

As the COVID-19 pandemic caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) continues to spread around the globe, effective vaccination protocols are under deployment. Alternatively, the use of convalescent plasma (CP) therapy relies on the transfer of the immunoglobulin repertoire of a donor that has recovered from the disease as a means of passive vaccination. While the lack of an effective antiviral treatment inadvertently increases the interest in CP products, initial clinical evaluation on COVID-19 patients revealed that critical factors determining the outcome of CP therapy need to be defined clearly if clinical efficacy is to be expected. Measurement of neutralizing activity against SARS-CoV-2 using wildtype virus presents a reliable functional assay but the availability of suitable BSL3 facilities for virus culture restricts its applicability. Instead, the use of pseudovirus particles containing elements from the SARS-CoV-2 virus is widely applied to determine the activity of CP or other neutralizing agents such as monoclonal antibodies. In this study, we present our approach to optimize GFP-encoding lentiviral particles pseudotyped with the SARS-CoV-2 Spike and Membrane proteins for use in neutralization assays. Our results show the feasibility of pseudovirus production using a C-terminal truncated Spike protein which is greatly enhanced by the incorporation of the D614G mutation. Moreover, we report that the use of Sodium Butyrate during lentiviral vector production dramatically increases pseudovirus titers. Analysis of CP neutralizing activity against particles pseudotyped with wildtype or D614G mutant Spike protein in the presence or absence the M protein revealed differential activity in CP samples that did not necessarily correlate with the amount of anti-SARS-CoV-2 antibodies. Our results indicate that the extent of neutralizing activity in CP samples depends on the quality rather than the quantity of the humoral immune responses and varies greatly between donors. Functional screening of neutralizing activity using pseudovirus-based neutralization assays must be accepted as a critical tool for choosing CP donors if clinical efficacy is to be maximized.

immunology↗