CPSF1 Regulates AML1-ETO Fusion Gene Polyadenylation and Stability in t(8;21) Acute Myelogenous Leukemia

Abstract

Alternative polyadenylation (APA) can alter the three prime untranslated region (3'UTR) length of mRNA transcripts, a crucial region for regulating mRNA metabolism and gene expression. Despite the prevalence and reported importance of APA post-transcriptional regulation in cancer, changes in 3'UTR usage by APA and its contribution to leukemia development have not been thoroughly studied. In acute myelogenous leukemia (AML), t(8;21) is the most common chromosomal abnormality and encodes the AML1-ETO (AE) fusion gene, which dominantly blocks hematopoietic cell differentiation. Previous studies reported that a small amount of non-leukemic t(8;21)+ hematopoietic stem cells (HSCs) never disappeared in long-term remission bone marrows after chemotherapy. These t(8;21)+ HSCs expressed low AE mRNA compared to diagnostic AML cells and possessed normal differentiation into myeloid cells. Another study also reported that a fraction of pediatric t(8;21) AML patients had AE+ clones in their neonatal Guthrie blood spots samples. These data suggest that acquisition of AE is not sufficient for t(8;21) AML development, AE+ HSCs could be a reservoir for pre-leukemic clones, and AE up-regulation is essential for leukemic transformation. However, the mechanism of AE up-regulation has not been revealed. Uncovering this mechanism is highly significant for preventing leukemia development and relapse after treatment.The AE 3'UTR has a full length of 5.2kb (long 3'UTR) that contains 8 polyadenylation sites (PAS), such that APA can result in several mRNA isoforms with varying 3'UTR lengths. We hypothesize that changes in PAS usage, and thus AE 3'UTR length, can modulate fusion gene expression. Therefore, we first checked AE 3'UTR usage by RNA sequencing and absolute quantification using real-time qPCR. Here, we report that 3'UTR usages of total AE were restricted within 3.7kb (short 3'UTR) in t(8;21) AML (>90% in 4 of primary samples, and >98% in cell lines (Kasumi-1 and SKNO1), respectively). Actinomycin D experiments showed that the half-life of AE with the long 3'UTR was much shorter than that with the short 3'UTR (0.8 hour and 2.8 hours, respectively), supporting our hypothesis that changes in 3'UTR usage regulate AE expression. To confirm the correlation between AE expression and its 3'UTR usage, we next checked single cell gene expression patterns of various cell types in t(8;21) AML diagnostic bone marrow samples. AE mRNA was detected not only in CD34+ AML cells but also in some differentiated monocytes and granulocytes, indicating that AE+ non-leukemic cells coexist with AML cells in the leukemic bone marrow. Moreover, these differentiated AE+ cells expressed much lower levels of AE, compared with AML cells (monocytes, 0.19-fold and granulocytes, 0.11-fold). We then analyzed AE expression and long 3'UTR usage by absolute quantification of each single cell (leukemic and non-leukemic) and observed a significant negative correlation (R2=0.33, p<0.01). These data suggest that AE 3'UTR length affects mRNA stability and is an important indicator of fusion gene mRNA expression in primary patient samples.To find the key regulator of this 3'UTR change, we comprehensively knocked down all APA machinery members by shRNA in t(8;21) AML cell lines. We discovered that CPSF1 (cleavage and polyadenylation specific factor 1) knockdown increased long 3'UTR usage (37% in total AE), and down-regulated overall AE mRNA expression (0.47-fold). Importantly, CPSF1 was highly expressed in t(8;21) AML patients' leukemia cells at diagnosis compared to 5 of healthy CD34+ HSCs (2.7-fold, p = 0.01). CPSF1 knockdown also impacted apoptosis and proliferation. These data suggest that CPSF1 promotes AE 3'UTR shortening, resulting in AE stability and a growth advantage of AML cells.Collectively, we conclude that expression of polyadenylation regulator CPSF1 is significantly enhanced in t(8;21) AML, CPSF1 controls 3'UTR length of AE fusion transcripts, and extending the AE 3'UTR by knocking down CPSF1 expression reduces AE transcript stability and restricts growth of t(8;21) AML cells. These results suggest that 3'UTR shortening of oncogenic fusion gene transcripts contributes to leukemogenesis, and controlling CPSF1 and 3'UTR usage may be a useful approach to inhibit leukemia progression. DisclosuresNo relevant conflicts of interest to declare.