ALBERT

Description: Approximately 53% of patients with Diamond-Blackfan Anemia (DBA) have mutations in one of nine ribosomal protein components (RPS7, RPS10, RPS17, RPS19, RPS24, RPS26, RPL5, RPL11 and RPL35A), with RPS19 being the most commonly affected (25% of all patients). Mutations found in patients result in protein haploinsufficiency and subsequent ribosomal stress, which increases the expression and activity of p53, leading to a failure of HSC differentiation during erythropoiesis. While RPS19-/- mice are not viable, we have generated a model system using different shRNA constructs to generate varying degrees of RPS19 knockdown in cultured cells and animal models. Cultured cells (including CD34+ HSCs derived from fetal liver or cord blood) recapitulate features of DBA erythropoietic failure in vitro and in vivo, including p53 upregulation and defects in erythrocyte differentiation, while anemias and skeletal defects are observed in zebrafish models. Using next-generation miRNA sequencing on the Illumina HiSeq2000 platform, we identified a number of miRNAs differentially expressed between human fetal liver CD34+ HSCs expressing normal or reduced RPS19. Preprocessing, alignment, and miRNA quantification were carried out using miRDeep2 [1] software. Differential expression analysis was performed with DESeq2 [2], which tests for differential expression based on a model using negative binomial distribution for count data from high-throughput sequencing assays. Candidates underwent a second round of screening based on robustness of expression differential (fold increase/decrease, variation of fold increase/decrease across replicates, and increase/decrease relative to RPS19 expression) and possible links to hematopoiesis and erythropoiesis, as reported in the scientific record. All three miR-34 isoforms (a, b and c) are robustly upregulated upon RPS19 reduction, with the extent of miR-34 upregulation being inversely proportional to RPS19 expression. When RPS19 protein expression is suppressed by approximately 50% (as observed in DBA patients), miR-34a, b and c isoforms are upregulated by 3.2, 2.2 and 2.6 fold respectively. In other systems, increased miR-34 results in modulation of a number of factors linked to erythropoiesis, including downregulation of c-Myc, Myb and NOTCH signaling. Genome-wide transcriptional analysis using next-generation sequencing on the Illumina HiSeq platform in CD34+ HSCs indicates c-Myc (4.2 fold), c-Myb (2.8 fold) and a number of NOTCH effectors [SIRT1 (1.8 fold) andHES1 (1.9 fold)] are indeed downregulated at the transcriptional level. Results were validated with qRT-PCR and western blot analysis, however, while transcriptional modulation fully accounts for c-Myc downregulation at the mRNA and protein level, we propose that c-Myb protein levels are modulated both transcriptionally and post-translationally, as protein expression is significantly further downregulated than mRNA message (6.3 fold). We propose that miR-34 upregulation in RPS19-depleted cells occurs through p53 and initiates genomic changes incompatible with erythropoiesis through downregulation of transcription factors c-Myc, c-Myb and the NOTCH signaling pathway. Disclosures No relevant conflicts of interest to declare.

Description: Introduction Pediatric chronic myeloid leukemia (CML) accounts for 10-15% of pediatric myeloid leukemias and 2-9% of all pediatric leukemias. There are several unique characteristics of CML diagnosed in children, adolescents, and young adults, compared to adults. They present with higher white blood counts and larger spleens, suggesting that the biology of pediatric CML is different from adult CML. We hypothesize that the differences in clinical presentation of pediatric CML patients are due to unique molecular characteristics that differ from adult CML patients. To test this hypothesis, we studied the transcriptomic signature of pediatric CD34+ CML cells compared to adult CML and normal age-matched bone marrow CD34+ cells. Methods CD34+ cells were isolated by FACS from pediatric CML (n=9), adult CML (n=10), pediatric normal (n=10) and adult normal (n=10) bone marrow samples. Total RNA was isolated from cells, and cDNA libraries were generated. Prepared libraries were sequenced on the Illumina HiSeq 4000 instrument. Raw sequences were trimmed and aligned to the hg38 reference genome with STAR/2.5.1b aligner. Gene level counts were determined with STAR -quantMode option using gene annotations from GENCODE (p5). Differential gene expression and pathway analysis were conducted with R/3.5.3. Counts were normalized with trimmed mean of M-values (TMM) from the EdgeR/ 3.24.3 package and further transformed with VOOM from the Limma/ 3.38.3 package. A linear model using the empirical Bayes analysis pipeline also from Limma was then used to obtain p-values, adjusted p-values and log-fold changes (LogFC). We performed three comparisons: (1) Pediatric CML vs Normal, (2) Adult CML vs Normal, and (3) Pediatric CML vs Adult CML. A False Discovery Rate (FDR) of £ .05 and absolute log2 fold-change 〉 1 was used to define differentially expressed genes in each comparison. Over-representation analysis was used to identify potentially unique pathways based on differentially expressed genes. Clinical and demographic features at diagnosis were extracted for pediatric and adult CML patients and compared using Fisher's exact test (categorical variables) or Wilcoxon rank sum test (continuous variables). Results Pediatric patients were diagnosed with CML at a median of 11 years (interquartile range (IQR): 10-14) compared to 54 years (IQR: 33-62) for adult patients. At diagnosis, pediatric patients had higher platelet counts (p=0.001) and larger spleen sizes (p=0.010) than adult patients, whereas the white blood cell count and phase at diagnosis did not differ. We found 606 genes (210 up- and 396 down-regulated) differentially expressed in pediatric CML CD34+ cells compared to pediatric normal controls. Interestingly, transcriptional regulators involved in blood cell differentiation including GATA1, TAL1, and KLF1 were differentially enriched in pediatric CML. In comparing adult CML patients to normal adult CD34+ cells, we found 920 genes (379 up- and 541 down-regulated) differentially expressed. Among all dysregulated genes we identified (1352 genes), 174 genes (54 up- and 120-down-regulated) overlapped when comparing pediatric and adult CML patients. Significantly enriched pathways in both adult and pediatric CML cells included PI3K/AKT signaling, MAPK signaling, and Notch/Wnt signaling, which have been previously reported. We found 437 unique genes that were dysregulated only in pediatric CML (270 up- and 167 down-regulated). Notch/Wnt signaling and Rho signaling pathways were significantly enriched. DLC1, a tumor suppressor gene that encodes a RhoGTPase-activating protein, has been known to be downregulated in solid tumors and hematologic malignancies. Interestingly, our data showed that DLC1 is significantly upregulated by 3-fold (p=0.0238) in pediatric CML, but not adult CML CD34+ cells. In addition, we observed that ABR, an inducer of C/EBPa that encodes an activator of RhoGEF and GTPase, was significantly downregulated by 2-fold (p=0.0119) in pediatric but not in adult CML CD34+ cells. Conclusion These results demonstrate unique molecular characteristics of pediatric CML that may contribute to the clinical differences at presentation between adult and pediatric disease. A better understanding of the particular biology of pediatric CML might impact the treatment of those patients in the future. Disclosures Gotlib: Deciphera: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: co-chair of the Study Steering Committee and Research Funding; Blueprint Medicines Corporation: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Chair of the Response Adjudication Committee and Research Funding, Research Funding.