Haploinsufficiency for ribosomal protein genes has been implicated in the pathophysiology of Diamond-Blackfan anemia (DBA) and the 5q؊ syndrome, a subtype of myelodysplastic syndrome. The p53 pathway is activated by ribosome dysfunction, but the molecular basis for selective impairment of the erythroid lineage in disorders of ribosome function has not been determined. We found that p53 accumulates selectively in the erythroid lineage in primary human hematopoietic progenitor cells after expression of shRNAs targeting RPS14, the ribosomal protein gene deleted in the 5q؊ syndrome, or RPS19, the most commonly mutated gene in DBA. Induction of p53 led to lineagespecific accumulation of p21 and consequent cell cycle arrest in erythroid progenitor cells. Pharmacologic inhibition of p53 rescued the erythroid defect, whereas nutlin-3, a compound that activates p53 through inhibition of HDM2, selectively impaired erythropoiesis. In bone marrow biopsies from patients with DBA or del(5q) myelodysplastic syndrome, we found an accumulation of nuclear p53 staining in erythroid progenitor cells that was not present in control samples. Our findings indicate that the erythroid lineage has a low threshold for the induction of p53, providing a basis for the failure of erythropoiesis in the 5q؊ syndrome, DBA, and perhaps other bone marrow failure syndromes. (Blood. 2011;117(9):2567-2576) IntroductionHeterozygous deletions or mutations of ribosomal protein genes have been implicated in 2 human disorders, Diamond-Blackfan anemia (DBA) 1-3 and the 5qϪ syndrome, a subtype of myelodysplastic syndrome (MDS). 4,5 Both disorders are characterized by a severe macrocytic anemia. 2,6 In DBA, a congenital disorder, approximately 25% of patients have mutations in the RPS19 gene, 1 and mutations or deletions have been identified in at least 9 additional ribosomal protein genes. 3,7 In the 5qϪ syndrome, somatic deletion of one allele of chromosome 5q causes haploinsufficiency for the RPS14 gene. 5 A central outstanding question is how a defect in ribosomes, which are expressed in and are essential to all cells, causes a primarily erythroid phenotype.Multiple animal models have demonstrated the effects of ribosomal dysfunction on erythropoiesis and the role of p53, which is known to monitor ribosome function. 8 Morpholinos targeting RPS19 in zebrafish cause an accumulation of p53 and a block in erythropoiesis that is reversed in the absence of p53. 9 Mice with germline mutations in the RPS19 or RPS20 genes have hyperpigmented foot pads with p53 accumulation in the epidermis, a decreased hematocrit, and an increased erythrocyte mean cell volume. Crossing the RPS19 mutant mice with p53 null mice rescued the skin and hematopoietic phenotypes. 10 Finally, a conditional heterozygous deletion of the Cd74-Nid76 interval syntenic to human chromosome 5q, containing 6 genes, including RPS14, in mouse hematopoietic cells, causes a macrocytic anemia that is rescued by crossing to a p53 null background. 11Although p53 appears to play a critical role in these d...

Haploinsufficiency of ribosomal proteins (RPs) has been proposed to be the common basis for the anemia observed in Diamond-Blackfan anemia (DBA) and myelodysplastic syndrome with loss of chromosome 5q [del(5q) MDS]. We have modeled DBA and del(5q) MDS in zebrafish using antisense morpholinos to rps19 and rps14, respectively, and have demonstrated that, as in humans, haploinsufficient levels of these proteins lead to a profound anemia. To address the hypothesis that RP loss results in impaired mRNA translation, we treated Rps19 and Rps14-deficient embryos with the amino acid L-leucine, a known activator of mRNA translation. This resulted in a striking improvement of the anemia associated with RP loss. We confirmed our findings in primary human CD34 ؉ cells, after shRNA knockdown of RPS19 and RPS14. Furthermore, we showed that loss of Rps19 or Rps14 activates the mTOR pathway, and this is accentuated by L-leucine in both Rps19 and Rps14 morphants. This effect could be abrogated by rapamycin suggesting that mTOR signaling may be responsible for the improvement in anemia associated with L-leucine. Our studies support the rationale for ongoing clinical trials of L-leucine as a therapeutic agent for DBA, and potentially for patients with del(5q) MDS. (Blood. 2012; 120(11):2214-2224) IntroductionDiamond-Blackfan anemia (DBA; MIM# 105650) is a congenital bone marrow failure syndrome of childhood manifested as normochromic macrocytic anemia with absence or insufficient erythroid precursors in the bone marrow. 1,2 Twenty-five percent of DBA patients have mutations in the RPS19 gene, which encodes a component of the 40S ribosomal subunit. 3,4 A further 25% of DBA patients have been shown to have mutations in other ribosomal protein genes, 5 supporting the hypothesis that DBA is a disease of altered ribosome assembly or function. DBA shares a number of its clinical features with several other congenital syndromes that also carry heterozygous mutations affecting ribosome biogenesis, such as Shwachman-Diamond syndrome (SDS), cartilage-hair hypoplasia syndrome, and dyskeratosis congenita (DC) suggesting that all of these conditions share at least some common pathogenic mechanisms; they have thus been termed "ribosomopathies." 6 In addition, evidence suggests that the anemia associated with the 5q minus (5qϪ) syndrome (or myelodysplastic syndrome with loss of all or part of chromosome 5q [del(5q) MDS]), a distinct subtype of myelodysplastic syndrome results from somatic heterozygous loss of the ribosomal protein gene RPS14 in hematopoietic stem cells. 7,8 Efforts to understand why ribosomal protein haploinsufficiencies have such a specific and profound effect on erythroid development at the molecular level have focused on the activation and stabilization of p53 in response to ribosomal stress. 9 However, the precise mechanisms governing how p53 stabilization occurs in response to ribosomal protein haploinsufficiency have not been clearly defined. Furthermore, not all bone marrow samples from patients with del(5q) MDS or DBA sh...

Summary Deregulated HER2 is a target of many approved cancer drugs. We analyzed 111,176 patient tumors and identified recurrent HER2 transmembrane domain (TMD) and juxtamembrane domain (JMD) mutations, including G660D, R678Q, E693K and Q709L. Using a saturation mutagenesis screen and testing of patient-derived mutations we found several activating TMD and JMD mutations. Structural modeling and analysis showed that the TMD/JMD mutations function by improving the active dimer interface or stabilizing an activating conformation. Further, we found that HER2 G660D employed asymmetric kinase dimerization for activation and signaling. Importantly, anti-HER2 antibodies and small molecule kinase inhibitors blocked the activity of TMD/JMD mutants. Consistent with this, a G660D germline mutant lung cancer patient showed remarkable clinical response to HER2 blockade.

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