3219 – RNA DEADENYLATION SUBUNIT CNOT3 PROMOTES MYELOID LEUKEMIA BY DRIVING TRANSLATION OF ONCOGENIC TARGETS

Abstract

How processes that affect post-transcriptional and translational regulation impact leukemia development is not well studied. The CCR4-NOT (CNOT) complex is one of two major multi-subunit polyA deadenylation complexes which mediate the shortening of the poly(A) tails and is recruited to m6A transcripts to mediate mRNA degradation. We surveyed several genome-wide CRISPR screens and observed several subunits of the CNOT complex ranked as genes essential for survival of AML cells. The highest ranked CNOT3 adaptor subunit demonstrated elevated expression in AML patients and associated with a poor prognosis in independent AML patient cohorts. Depletion of CNOT3 in human AML cells resulted in inhibition of cell growth, increased myeloid differentiation, cell cycle arrest and delayed leukemia development in vivo. Meanwhile, CNOT3 loss in normal cord-blood CD34+ cells promoted differentiation without inducing cell death, suggesting a potential therapeutic window to target CNOT3 in leukemia. Using a CRISPR saturation mutagenesis screen, we identified NOT box in the C-term domain of CNOT3 to be most critical for cell viability, indicating a functional dependence of CNOT3 on interaction with the deadenylation complex. Transcriptomic and proteomic profiling upon CNOT3 depletion revealed activation of p53 pathways and decrease of MYC- target genes. We further observed a marked decrease in c-MYC protein without alternation in total abundancy and half-life of c-MYC mRNA upon CNOT3 ablation. Mass spectrometry analysis of CNOT3 immunoprecipitation captured proteins involved in translation machinery. Moreover, depletion of CNOT3 significantly reduced the abundancy of c-MYC mRNA in the actively translating polysome fraction. Overall, we uncovered a critical role of the CNOT deadenylation complex in myeloid leukemia and translational control of critical oncogenic targets including c-MYC. How processes that affect post-transcriptional and translational regulation impact leukemia development is not well studied. The CCR4-NOT (CNOT) complex is one of two major multi-subunit polyA deadenylation complexes which mediate the shortening of the poly(A) tails and is recruited to m6A transcripts to mediate mRNA degradation. We surveyed several genome-wide CRISPR screens and observed several subunits of the CNOT complex ranked as genes essential for survival of AML cells. The highest ranked CNOT3 adaptor subunit demonstrated elevated expression in AML patients and associated with a poor prognosis in independent AML patient cohorts. Depletion of CNOT3 in human AML cells resulted in inhibition of cell growth, increased myeloid differentiation, cell cycle arrest and delayed leukemia development in vivo. Meanwhile, CNOT3 loss in normal cord-blood CD34+ cells promoted differentiation without inducing cell death, suggesting a potential therapeutic window to target CNOT3 in leukemia. Using a CRISPR saturation mutagenesis screen, we identified NOT box in the C-term domain of CNOT3 to be most critical for cell viability, indicating a functional dependence of CNOT3 on interaction with the deadenylation complex. Transcriptomic and proteomic profiling upon CNOT3 depletion revealed activation of p53 pathways and decrease of MYC- target genes. We further observed a marked decrease in c-MYC protein without alternation in total abundancy and half-life of c-MYC mRNA upon CNOT3 ablation. Mass spectrometry analysis of CNOT3 immunoprecipitation captured proteins involved in translation machinery. Moreover, depletion of CNOT3 significantly reduced the abundancy of c-MYC mRNA in the actively translating polysome fraction. Overall, we uncovered a critical role of the CNOT deadenylation complex in myeloid leukemia and translational control of critical oncogenic targets including c-MYC.