Final Report Summary - DBA-CHECKPOINTS (The Role of the RPL11/p53 Checkpoint in the Development and Treatment of DBA)
Our laboratory demonstrated that impairment of ribosome biogenesis, by depleting a Our laboratory demonstrated that impairment of ribosome biogenesis, by depleting a ribosomal protein (RP) of either the 40S or 60S subunit, leads to the induction of p53 and cell cycle arrest, which is dependent on RPL11 . The importance of this response is underscored by two human pathologies, which directly impair ribosome biogenesis, Diamond Blackfan Anemia (DBA) and 5q- syndrome . The pathologies are similar, with both exhibiting macrocytic anemia, erythroid hypoplasia and an increased risk of cancer in later life. DBA is caused by heterozygous mutations in a number of RPs, whereas 5q- syndrome is caused by a sporadic monoallelic deletion of the long arm of chromosome 5, including the gene for RPS14, . Although, both pathologies were initially assumed to be due to lesions in protein synthetic capacity, subsequent studies in mouse models showed that in both DBA and 5q- syndrome, it is the induction of p53, which leads to the severe anemia [4, 5]. In this proposal we set out address 3 objectives.(1) to Identify signaling components involved in the translational upregulation of RPL11 (2) to determine the mechanism of RPL11-induced inhibition of Hdm2 in response to impaired ribosome biogenesis and (3) to elucidate pharmacological treatments and to develop a mouse model for DBA. During the last two years we have achieved each of these objectives and obtained novel insights. In brief, the highlights of our findings are: (1) We have identified mRNA binding protein LARP1 as a major regulator of the stability of RPL11 mRNA. Moreover, we show that this effect is mediated through a 5’-terminal oligonucleotide sequence (5’TOP), which resides at the transcriptional start site of all RP genes. In the last 5 months we made the unexpected discovery, though not previously recognized, LARP1, like RPS14, maps within the chromosomal region deleted in 5q- syndrome. Consistent with this finding, in 5q- patients, the reduced expression of LARP1 is paralleled by a decrease in the amount of 5’TOP mRNAs, consistent with co-depletion of LARP1 dramatically enhancing the p53 stabilization induced by impaired 40S ribosome biogenesis ). (2) We have shown that RPL11 is part of a preribosomal complex, containing RPL5 and non-coding 5S rRNA, which is normally incorporated into the 60S ribosomes (see Figure, left panel), but upon impairment of ribosome biogenesis the nascent precursor complex is re-directed to the binding and inhibition of Hdm2 (see Figure, center panel and [7-9]). We now refer to this complex, as “impaired ribosome biogenesis checkpoint” (IRBC). Moreover, in recent studies we have shown that the IRBC is activated under conditions of hyperactivation of ribosome biogenesis, particularly in c-Myc driven tumors and that such tumors are exquisitely sensitive to the this checkpoint in a p53 wild type setting (see Figure, right panel). Finally in objective 3, we generated a model which recapitulates DBA, by mono-allelic RPS6 conditional deletion. However, we unexpectedly found that loss of the IRBC binding site, unlike loss of p53, does not rescue the anemia as scored for by red blood cell count or the amount of hemoglobin, suggesting an additional checkpoint in this response to depleting one allele of RPS6 RP. This has led us to DNA damage, which may be caused by impaired rRNA processing in erythropoietic cells, which have a high demand for ribosome biogenesis during early development. We used Lin- cell lysates from RPS6 deleted mice and analyzed a panel of DNA damaging pathway regulators, we observed strong induction of the phosphorylation level of Chk2 T68 and ɣH2AX S139, ATM phosphorylation sites, in CRE+ genotypes. These findings suggest that the use of ChK2 inhibitors for the treatment of DBA, and potentially 5q- syndrome. The finding of DNA damage may also explain why such patients have a propensity for cancer later in life, particularly AML.
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