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Btg2 Regulates Myc mRNA Stability and Leukemia-Initiating Cell Dormancy in T-ALL
Kelsey Wagner1, Kevin O’Connor1,2, Leonard Murphy1, Kensei Kishimoto1,2 and Michelle Kelliher11Department of Molecular, Cell and Cancer Biology, Morningside Graduate School of Biomedical Sciences, UMass Chan Medical School, Worcester, MA 01605
2Medical Scientist Training Program, UMass Chan Medical School.
Addressing therapy failure and relapse is critical in improving outcomes for pediatric T-cell acute lymphoblastic leukemia (T-ALL) patients. Relapse is thought to represent a failure to effectively eliminate leukemia-initiating cells (L-ICs), which survive chemotherapy and self-renew, regenerating the full complement of leukemic cell populations. Limiting dilution analysis of TAL1/LMO2 (T/L) mouse T-ALLs revealed that the double negative-3 (DN3) thymic progenitor population is most enriched for L-ICs, while differentiated CD4/8 double positive cells fail to re-establish disease in mice. To hone in on the L-IC, we applied single cell RNA-sequencing (scRNA-seq) and nucleosome labeling and identified a dormant DN3 (dDN3) subpopulation that appears cell cycle restricted and shares a transcriptional signature with ALL patient samples with minimal residual disease.
Dormant DN3 leukemic cells showed enrichment of Btg2, a member of the BTG/TOB family of anti-proliferative proteins. Interestingly, in naïve T cells, Btg2 mediates quiescence by promoting formation of the CNOT7 deadenylation complex and degrading Myc and other mRNAs (Hwang et al, 2020). At the single cell level, we find Btg2 and Myc mRNA levels anti-correlated in DN3 leukemic cells, raising the possibility that Btg2 may regulate DN3 dormancy by deadenylating and destabilizing Myc mRNA. To test this, we generated isogenic T-ALL cell lines with a non-silencing control (NSC) or BTG2 shRNAs. BTG2 knockdown in human T-ALL cells promoted S-phase entry and increased leukemic cell proliferation whereas inducible expression of Btg2 in mouse T-ALL cells resulted in G1 arrest and suppression of cell growth. We tested the effect of Btg2 knockdown on Myc mRNA stability using actinomycin D and found the half-life of Myc mRNA increased by three- to six-fold in Btg2 silenced leukemic cells compared to control.
To assess the role of Btg2 in the DN3-enriched L-IC population in vivo, we transduced 3 primary T/L leukemias with NSC or with 2 independent Btg2 shRNAs and performed limiting dilution analyses. Btg2 silencing significantly increased L-IC activity 6-fold compared to NSC (NSC: 1/15,710 [95% CI: 1/7,727-1/31,943]; shBtg2: 1/2,546 [95% CI: 1/1,284-1/5,049]; P < 0.0001; n = 3 experiments) and reduced disease latency from a median survival of 50 to 39 days (p = 0.0041). Importantly, Btg2 silencing was maintained in transplanted leukemic cells and Btg2-silenced leukemic cells showed increased Myc mRNA expression compared to NSC controls. Our data suggest that knockdown of Btg2 in the DN3-enriched mouse L-IC population promotes exit from dormancy to reestablish disease in transplanted mice. Interestingly, BTG2 is enriched in quiescent and therapy resistant ALL patient cells capable of mediating disease recurrence in xenografts (Ebinger et al. 2016), thus raising the possibility that BTG2-induced quiescence may promote chemoresistance in patients.
Speakers
Kelsey Wagner