NUDT21 limits CD19 levels through alternativemRNA polyadenylation in B cell acutelymphoblastic leukemia

Witkowski MT, Lee S, Wang E, Lee AK, Talbot A, Ma C, Tsopoulidis N, Brumbaugh J, Zhao Y, Roberts KG, Hogg SJ, Nomikou S, Ghebrechristos YE, Thandapani P, Mullighan CG, Hochedlinger K, Chen W, Abdel-Wahab O, Eyquem J, Aifantis I
Source: Nature
Publication Date: (2022)
Issue: 10: 1424-1432
Research Area:
Immunotherapy / Hematology
Cells used in publication:
Endothelial, umbilical vein, human (HUVEC)
Species: human
Tissue Origin: vein
T cell, human stim.
Species: human
Tissue Origin: blood
4D-Nucleofector® 96-well Systems

T cells were activated with Dynabeads Human T-Activator CD3/CD28 (Thermo Fisher Scientific) (1:1 beads:cell) in X-VIVO 15 medium (Lonza) supplemented with 5% human serum (Gemini Bioproducts), penicillin–streptomycin (Thermo Fisher Scientific, 50 U/ml), IL-7 (Miltenyi Biotec, 5 ng/ml) and IL-15 (Miltenyi Biotec, 5 ng/ml) and cultured at 10^6 cells/ml. Medium was exchanged every 2–3 days and cells were re-suspended at 10^6 cells ml-1. After 48 hours of T cell activation, cells were detached from CD3/CD28 Dynabeads and the beads were magnetically removed. T cells were electroporated with ribonucleoprotein (RNP) using a 4D-Nucleofector 96-well unit (Lonza). RNP for each electroporation reaction was generated by co-incubating 60 pmol of recombinant Cas9 protein (QB3 MacroLab) with 120 pmol of TRAC sgRNA
(Synthego, CAGGGUUCUGGAUAUCUGU) at 37 °C for 15 min. Cells were re-suspended in P3 primary cell solution (Lonza) (2 × 10^6 live cells per electroporation) and mixed with RNP, followed by electroporation using the EH115 Nucleofector protocol. Cells were then diluted into serum-free medium (2 × 10^6 cells/ml) and incubated at 37 °C and 5% CO2.


B cell progenitor acute lymphoblastic leukemia (B-ALL) treatment has been revolutionized by T cell-based immunotherapies-including chimeric antigen receptor T cell therapy (CAR-T) and the bispecific T cell engager therapeutic, blinatumomab-targeting surface glycoprotein CD19. Unfortunately, many patients with B-ALL will fail immunotherapy due to 'antigen escape'-the loss or absence of leukemic CD19 targeted by anti-leukemic T cells. In the present study, we utilized a genome-wide CRISPR-Cas9 screening approach to identify modulators of CD19 abundance on human B-ALL blasts. These studies identified a critical role for the transcriptional activator ZNF143 in CD19 promoter activation. Conversely, the RNA-binding protein, NUDT21, limited expression of CD19 by regulating CD19 messenger RNA polyadenylation and stability. NUDT21 deletion in B-ALL cells increased the expression of CD19 and the sensitivity to CD19-specific CAR-T and blinatumomab. In human B-ALL patients treated with CAR-T and blinatumomab, upregulation of NUDT21 mRNA coincided with CD19 loss at disease relapse. Together, these studies identify new CD19 modulators in human B-ALL.