ALBERT

Description: Key Points Biallelic inheritance of a telomerase T-motif mutation selectively impairs repeat addition processivity and results in severe disease. Computational algorithms commonly used to predict the impact of variants on protein function have limited sensitivity with regard to hTERT.

Description: The inherited bone marrow failure syndromes (IBMFS) are rare genetic disorders caused by mutations in critical components of fundamental cellular processes such as ribosome biogenesis, DNA repair, and telomere maintenance. The IBMFS Shwachman-Diamond syndrome(SDS) and Diamond-Blackfan anemia (DBA) are classified as ribosomopathies due to etiologic mutations in genes encoding factors involved in ribosome biogenesis (SBDSin the majority of patients with SDS) or ribosomal proteins (RPS19most commonly in patients with DBA). Although these disorders can be distinguished clinically and from the other IBMFS, they share with each other and with other IBMFS increased predisposition to myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). Whereas genomic instability due to defective DNA repair or telomere maintenance is thought to underlie cancer predisposition in the IBMFS Fanconi anemia and dyskeratosis congenita, respectively, the molecular mechanisms driving cancer in SDS and DBA are not fully understood. Our research has focused on DNA repair in SDS and DBA. A prior report suggested lymphoblastoid cell lines (LCLs) derived from patients with SDS arehypersensitive to ionizing radiation (IR). Consistent with this, we found SDS-LCLs had decreased survival following IR compared to control-LCLsin colony survival assays. To determine if this cellular phenotype was unique to SDS or present in the other IBMFS ribosomopathy, DBA, we examined LCLs derived from patients with DBA, including those with mutations in RPS19, RPS26, RPL5and RPL11. We found that the DBA-LCLs were similarly hypersensitive to IR as compared to control-LCLs. Further examination of γ-H2AX, a DNA damage response (DDR) factor and marker of DNA double strand breaks (DSBs), revealed that SDS- and DBA-LCLs had delayed resolution of γ-H2AX foci and increased protein levels at 24 hrs after IR as compared to control LCLs. p53, phospho-ATM, and DNA-PKcs protein levels were also higher in SDS-LCL compared to controls. The decreased survival and increased and sustained DDR following IR led us to hypothesize that SDS and DBA cells have a defect in DSB repair. There are two major pathways of DSB repair in mammals, nonhomologous end-joining (NHEJ) and homology-directed repair (HDR), and loss of either results in hypersensitivity to IR. To examine each pathway, we employed U2OS (human osteosarcoma) and HCT116 (human colon cancer) cells containing an integrated green fluorescent protein HDR or NHEJ reporter transgene. Interestingly, we found that knockdown of either SBDS or RPS19 proteins resulted in an approximately 50% reduction in HDR efficiency but no change in NHEJefficiency compared to the scrambled control in both cell lines. We next sought to determine the mechanism underlying the effect of SBDS and RPS19 deficiency on HDR. A survey of proteins required for HDR revealed a reduction in the recombinase RAD51 in SDS-LCLs and in SBDS-depleted HCT116 and U2OS cells, whereas, an initial survey in SDS-LCLs[e1] of factors involved in NHEJ did not reveal a specific NHEJ factor deficiency. Knockdown of eiF6 is known to rescue the defect in 40S and 60S ribosome subunit joining that manifests in SDS patient cells. However, we found eIF6 depletion failed to rescue the level of RAD51 protein and had no impact on HDR in SBDS-deficient cells. We conclude that decreased RAD51 levels in SBDS-deficient cells might contribute to impaired HDR, however, this decrease is independent of the ribosome subunit joining defect. Similarly, RPS19 knock down resulted in a reduction in RAD51 protein level, suggesting a potentially common pathway. We also asked whether SBDS or RPS19 might be more directly involved in the DDR or repair of DSBs. Consistent with this, we found SBDS and RPS19 recruited to chromatin surrounding an I-Sce1 site following DSB induction in chromatin immunoprecipitation assays. Collectively, these findings provide evidence that SBDS and RPS19 may be directly involved in the DDR or DSB repair and raise the possibility that loss of this function may contribute to MDS/AML predisposition in SDS and DBA patients. Disclosures No relevant conflicts of interest to declare.

Description: Shwachman-Diamond syndrome (SDS), an autosomal recessive disorder, is characterized by bone marrow dysfunction, exocrine pancreatic insufficiency, congenital abnormalities, and leukemia predisposition (Myers et al., 2012). Most patients with SDS harbor biallelic mutations in the Shwachman-Bodian-Diamond syndrome (SBDS) gene. SBDS is known to play a role in ribosome biogenesis by enabling eviction of the ribosome anti-association factor eIF6 from the 60S ribosomal subunit, to allow formation of the 80S ribosome (Wong et al., 2011). SBDS-depleted cells are, therefore, defective in ribosome assembly. In addition, absence of SBDS sensitizes cells to ultraviolet irradiation, translation inhibitors, and endoplasmic reticulum (ER) stressors, such as tunicamycin (Ball et al., 2009). A recent report indicated that lymphoblastoid cell lines (LCLs) derived from two SDS patients accumulated more DNA damage after being exposed to ionizing radiation (IR) (Morini et al., 2015). A deficiency in DNA repair was alluded to as a possible cause, however, the mechanism underlying this previously unreported phenotype was not determined. In this study, we investigated LCLs derived from five SDS patients with biallelic SBDS mutations and found all to be hypersensitive to IR in a colony survival assay. In this assay, increasing doses of IR resulted in a significantly lower survival fraction in SDS-compared to control-LCLs. We found SBDS expression to increase in control-cells when stressed with IR, suggesting that SBDS is a stress response protein and its absence in SDS-LCLs induces hypersensitivity to IR. Because knockdown of SBDS in HEK293 cells induces an ER stress response (Ball et al., 2009), we examined the expression of the ER stress response factor phospho-eIF2α in untreated and IR exposed SDS-LCLs and found phospho-eIF2α expression to be markedly increased compared to controls. This result indicated that SDS-LCLs may have an activated ER stress response, as was further confirmed by exposing these cells to additional ER stressors, tunicamycin and H2O2, and observing a similar upregulation of phospho-eIF2α. Because ER stress is known to suppress DNA double strand break (DSBR) (Yamamori et al., 2013), we examined the expression of Rad51 and Ku70, which are required for the homology-directed and nonhomologous end-joining pathways of DSBR, respectively. Surprisingly, we found Rad51 and Ku70 protein levels to be repressed in SDS-LCLs compared to controls, both with and without exposure to IR. Collectively, these data support the hypothesis that, in addition to its role in ribosome biogenesis, SBDS is a stress response protein that plays an important role in regulating the ER stress response. In SDS-cells, where SBDS is lacking, activated ER stress represses DNA repair proteins rendering cells hypersensitive to IR and other stresses. This novel pathway to ER stress induction may contribute to the bone marrow failure and cancer predisposition seen in SDS patients. Disclosures No relevant conflicts of interest to declare.

Description: Introduction: In adult populations, driver mutations often facilitate the progression of myelodysplastic syndrome (MDS) to acute myeloid leukemia (AML). Increased mutational burden is associated with decreased leukemia-free survival (Papaemmanuil et al, 2013); however, this phenomenon is less common in pediatric MDS. GATA2 mutations resulting in loss of function or haploinsufficiency are associated with MDS predisposition. Recent reports in adults with GATA2-MDS suggest that additional somatic driver mutations such as ASXL1, EZH2, and GATA1 contribute to rapidly progressive disease (Bodor et al, 2012; Fujiwara et al, 2014). Yet the spectrum of somatic mutations in pediatric GATA2-MDS is not well described. We applied a custom next-generation sequencing (NGS) panel inclusive of germline and acquired mutations commonly reported in pediatric AML/MDS to a subset of MDS patients with germline GATA2 mutations. Our results indicate that acquisition of somatic mutations in AML/MDS related genes may contribute to pediatric GATA2-MDS disease evolution and pathogenesis. Methods: Using Agilent SureDesign, we designed an NGS panel that targeted genes frequently mutated in pediatric AML/MDS, including 278 exonic, promoter, or intronic regions from 46 genes. Select known mutations in diagnostic specimens from a local pediatric AML/MDS cohort were used for panel validation. We applied this panel to DNA extracted from blood or marrow samples from 4 pediatric subjects diagnosed at ages 5-15 years with GATA2-MDS, and one subject age 8 with suspected GATA2-MDS. Sample NGS libraries were prepared, multiplexed, and sequenced on MiSeq flow cells in paired-end mode. Read alignment, variant calling, and annotation were performed using Agilent SureCall v3.0 software and NExtGENe software. Variants passing defined QC metric filters were reviewed for pathogenic significance. This research was performed under a local Institutional Review Board-approved protocol and in accord with the Declaration of Helsinki. Results: The subject with suspected GATA2-MDS was confirmed to have a deletion in GATA2 resulting in a frameshift. Presumed germline variants in telomerase reverse transcriptase (TERT) were found in one subject at allele frequencies consistent with heterozygosity. Four acquired mutations were found in two out of five GATA2-MDS subjects: a deletion in IKZF1 resulting in a frameshift, and mutations in both RUNX1 (n=2) and in SETBP1 (Table 1). Conclusions: Within a cohort of five GATA2-MDS subjects, we found four somatic mutations likely contributing to disease pathogenesis.Acquired mutations in SETBP1, including p.G870S, are an independent and poor prognostic indicator in adult AML (Makishima et al, 2013). Acquired mutations in RUNX1 are also common in adult MDS/AML, and both p.L56S and p.D198Gmutations have been described in adult cases of MDS progressing to AML (Pellagati et al, 2015). We also identified a frameshift mutation in IKZF1 presumed to be somatic in one subject. Large focal deletions, but not frameshifts, have been described as rare but recurrent events in pediatric AML in conjunction with monosomy 7, as with this case (de Rooij et al, 2015). Our results represent the first evidence of acquired driver mutations in pediatric GATA2-MDS, and demonstrate the utility of this comprehensive custom AML/MDS NGS panel to uncover key somatic mutational events that may be associated with rapidly progressive disease. Figure 1. Mutations detected in pediatric subjects with GATA2-MDS RCC=refractory cytopenia of childhood, NK=normal karyotype, VAF=variant allele frequency *This subject has a known 3MB hemizygous deletion in GATA2 that, as expected, was not detected by the Agilent NGS platform Figure 1. Mutations detected in pediatric subjects with GATA2-MDS. / RCC=refractory cytopenia of childhood, NK=normal karyotype, VAF=variant allele frequency. / *This subject has a known 3MB hemizygous deletion in GATA2 that, as expected, was not detected by the Agilent NGS platform Disclosures No relevant conflicts of interest to declare.

Description: Background The telomerase enzyme complex maintains telomeric DNA, the TTAGGG repeats localized to chromosome ends. Constitutional telomerase mutations are associated with short age-adjusted telomeres and a spectrum of disorders including familial pulmonary fibrosis and liver disease, aplastic anemia, myelodysplastic syndrome (MDS), and dyskeratosis congenita (DC). Notably, DC confers a 90% lifetime risk for bone marrow failure, a 200-fold risk for AML, a 2500-fold risk for MDS, and is associated with chemosensitivity in affected individuals. Exposure to intensive chemotherapy may accelerate telomere shortening and promote manifestations of a telomere biology disorder phenotype in individuals with underlying defects in telomere maintenance. Therefore, we investigated the incidence of constitutional telomerase variants in pediatric AML and their role in therapy-related adverse events (AE’s). We hypothesized that constitutional telomerase variants would be (1) more frequent in AML cases compared with controls, (2) associated with characteristics of telomere biology disorders, and (3) in addition to telomere length, would further characterize AML cases with specific AE’s. Methods We sequenced the exons and flanking intronic regions of the telomerase subunits TERT, DKC1, and TERC, as well as TINF2, a critical component in recruiting telomerase to telomeres, in a local pediatric AML/MDS cohort (n=104), a distinct Children’s Oncology Group (COG) AML AAML0531 study cohort (n=115), and a cohort of healthy controls racially and ethnically matched to our local AML/MDS cohort (n=254). We reviewed medical records in the local cohort for characteristics suggestive of DC, including first degree family history of cancer, liver, or pulmonary disease, delay in chemotherapy 〉60 days due to cytopenias(s), persistent liver or pulmonary disease, persistent cytopenias after AML therapy, history of second cancer, and specific skin, nail, and mucosal abnormalities. For the COG cohort, we compared the number of variants and remission relative telomere length (RTL), measured by qPCR, in subjects with time to absolute neutrophil count (ANC) recovery at least 1 SD above the mean for at least 2 chemotherapy courses (n=53) to those with time to ANC recovery within 1 SD above the mean for all 5 chemotherapy courses (n=62). A relationship between variants, telomere length, and specific grade 3 or 4 AE’s was also explored. Results In the local AML/MDS cohort, 13 variants resulting in missense changes or deletions were found in 21/101 subjects (20.8%). When compared with population databases, the number of novel variants in this cohort (8/13) far exceeded the expected number (p

Description: Abstract 1272 Hoyeraal Hreidarsson syndrome (HHS) is a severe form of dyskeratosis congenita (DC) characterized by bone marrow failure, intrauterine growth retardation, developmental delay, microcephaly, cerebellar hypoplasia, immunodeficiency, and extremely short telomeres. As with DC, mutations in genes that encode factors required for telomere maintenance, such as the telomerase reverse transcriptase (TERT), have been found in patients with HHS. We describe two sibling cases of HHS caused by a homozygous mutation (p.T567M) within the TERT T motif, which is a highly conserved motif specific to the telomerase-class of reverse transcriptases. This mutation resulted in a marked reduction in the capacity of telomerase to processively synthesize telomeric repeats, indicating for the first time a role for the T motif in this unique aspect of telomerase function. The consanguineous parents, heterozygous for this mutation, exhibited telomere lengths around the first percentile and no evidence of a DC phenotype. Although heterozygous processivity defects have been associated with familial adult-onset pulmonary fibrosis, these cases demonstrate the severe clinical and functional impact of biallelic processivity mutations. Thus, despite retaining the capacity to catalyze the addition of short stretches of telomeric repeats onto the shortest telomeres, the sole expression of telomerase repeat addition processivity mutants leads to a profound failure of telomere maintenance and early onset disease. In the course of our investigation of this mutant, several commonly used algorithms predicted no effect of TERT p.567M upon protein function. To examine this further, we compiled missense changes that are associated with a clinical phenotype and correlated each with its reported telomerase activity and presence of the phenotype in more than one family member. We then summarized the predicted functional impact for each of these mutations based upon four commonly used algorithms. Our results demonstrate these algorithms to be limited in their capacity for predicting the effect of missense changes upon telomerase function, emphasizing the importance of in vitro functional analyses including processivity assays for TERT variants of unknown significance. Disclosures: No relevant conflicts of interest to declare.