ALBERT

Description: PHF6 is an X-chromosome gene showing recurrent loss-of-function mutations in acute and chronic myeloid leukemias and T-lymphoblastic leukemia, indicating that it acts as a tumor suppressor in both myeloid and lymphoid hematopoietic lineages. PHF6 protein is localized to the nucleolus, the site of ribosome biogenesis, where it is reported to regulate rDNA transcription. It is also localized to the nucleoplasm, where it binds chromatin and may regulate gene transcription. However, these mechanisms are incompletely established, no animal model of PHF6 loss has been reported, and there is limited insight into the precise role of PHF6 in hematopoiesis, both mechanistically and as a leukemia suppressor. To study the in vivo role of Phf6, we generated mice with hematopoietic knockout of Phf6 using the Vav-Cre recombinase system, achieving a 〉95% deletion efficiency. In comparison with Vav-Cre mice (WT), mice with Vav-Cre;Phf6-flox genotype (Phf6 KO) showed, at 8-12 weeks of age, a 1.25-fold expansion of the LSK (Lin-Sca1+Kit+) compartment in the bone marrow (see figure), accompanied with a similar increase in the common myeloid progenitor CMP compartment (Lin-Kit+Sca1-CD34+FcRIII-). Within the LSK compartment, there was a 2-fold and 1.7-fold expansion of the myeloid-biased multipotent progenitor compartments MPP2 (LSK,Flk2-CD150+CD48+) and MPP3 (LSK,Flk2-CD150-CD48+) respectively. The lymphoid-biased MPP4 compartment was not changed, nor was the common lymphoid progenitor CLP compartment (not shown). Conversely, the number of stringently defined HSCs (LSK,Flk2-CD150+CD48-CD34-) was reduced by 40%. This suggests depletion of HSCs through loss of dormancy, accompanied by myeloid skewing. At 8-12 weeks of age, there was no change in overall bone marrow cellularity or spleen size/cellularity, though flowcytometric analysis of spleen showed identical reduction of HSC and expansion of MPP2 compartments in Phf6 KO. As of 40 weeks of age, Phf6 KO mice did not show any gross peripheral blood count abnormalities. We also used CRISPR/Cas9 to generate PHF6 knockout clones from the THP-1 human AML cell line. RNA-Seq and quantitative proteomics in knockout cells showed downregulation of mature myeloid genes and increased expression of hematopoietic progenitor gene sets, including increased expression of cell surface receptor KIT. KO cells showed increased proliferation when cultured with KIT ligand. Using IP-mass spectrometry in WT and KO clones, we identified ribosomal proteins RPL12 and RPLP0 as the most abundant and specific binding partners of PHF6. In summary, young Phf6 knockout mice show HSC depletion and expansion of myeloid-skewed progenitors without overt peripheral blood abnormalities. Further work is in progress to characterize HSC dormancy and competitiveness, progression of Phf6 KO phenotype with age, and mechanisms of gene regulation by Phf6 through binding of ribosomal proteins. Figure. Figure. Disclosures No relevant conflicts of interest to declare.

Description: Mammalian cells express thousands of lineage-restricted long noncoding RNAs (lncRNAs), although the functions of most are unknown. "Knockout" studies to investigate lncRNA function by gene deletion in cells or whole animals have been informative. However, since most lncRNAs show open chromatin and transcription factor (TF) binding at their promoters as well as elsewhere in their genomic loci, it is essential to distinguish the roles of the RNA transcript from those of potential cis-regulatory elements, both of which are eliminated in deletion studies. We identified a novel mouse lncRNA named Drop27 (Downstream of p27) that is transcribed 4 kb downstream of the Cdkn1b gene encoding the cell-cycle inhibitor p27, which is strongly upregulated during erythroid maturation. Drop27 is conserved between mice and humans, has two exons separated by a 21 kb intron, and produces a 400 bp spliced, polyadenylated RNA that is abundantly transcribed and highly enriched in mouse erythroblasts compared to other tissues. We used the CRISPR/Cas system to delete the entire Drop27 locus in the G1E erythroid cell line. Microarray analysis showed reduced Cdkn1b mRNA in Drop27-deleted cells with very few other changes in the transcriptome. Quantitative PCR verified this finding by demonstrating that heterozygous deletion of Drop27 caused reduced Cdkn1b mRNA by 35%, while homozygous deletion led to a 70% reduction. This effect also occurred at the primary transcript level, demonstrating that deletion of the Drop27 reduced Cdkn1b transcription. To distinguish the effects of loss of the lncRNA transcribed from Drop27 from the potential loss of a cis-acting regulatory module in the Drop27 genomic locus, we used homologous recombination to insert a bovine growth hormone (BGH) polyadenylation (polyA) cassette into the first exon of Drop27, 80 bp downstream of the transcription start site. This caused premature termination of the lncRNA transcript while maintaining all genomic sequences, including potential cis-regulatory elements. Analysis of multiple clones showed that homozygous polyA cassette integration reduced full-length Drop27 transcript levels by more than 99%, although Cdkn1b mRNA levels were unaffected. These findings demonstrate that Drop27 lncRNA is dispensable for Cdkn1b transcription, while its genomic sequences are required, indicating that the Drop27 gene locus contains an erythroid cis-regulatory element (enhancer) for the Cdkn1b gene. Several strong candidates for the proposed enhancer are found in the Drop27 gene. Multiple epigenetic features strongly associated with enhancers map in several distinct locations in the Drop27 locus in erythroid and other hematopoietic cells, including DNase hypersensitivity, p300 binding, and multiple transcription factor sites. A functional role for the Drop27 lncRNA is not identified by this experiment, and it is possible that it arose as a byproduct of enhancer activity. Our findings provide new insight into mechanisms of hematopoietic gene expression and are of more broad relevance to the lncRNA field in general. In particular, we demonstrate that the genomic loci of some lncRNA genes may function as cis elements, irrespective of the transcripts arising from them. Disclosures No relevant conflicts of interest to declare.

Description: Introduction: Hematopoietic stem cell transplantation (HSCT) is associated with severe intestinal microbiota injury as a consequence of broad spectrum antibiotics, mucosal damage from conditioning, and dietary changes. Intestinal dysbiosis has been correlated with adverse outcomes post-transplant. We aimed to characterize and compare the patterns of microbiota injury that occur post autologous and allogeneic transplant. We characterized the specific risk factors associated with decreased microbial diversity in HSCT, and defined specific bacterial taxa that are enriched or depleted in patients based on the type of transplant and various other risk factors Methods: Stool samples from 35 patients (pts) undergoing HSCT were collected immediately pre-conditioning, at day 14, 28, and 100 post-transplant. 17 pts underwent an autologous (auto-) and 18 pts underwent an allogeneic transplant (allo-HSCT). The stool microbiome was characterized by sequencing of the V3V4 16s rRNA region; a- and b-diversity were determined and differential abundance analysis of OTUs was performed using DESeq2. Results: In the auto-HSCT cohort 70% of pts had plasma cell dyscrasia, and 24% had non-Hodgkin lymphoma. 83% of pts in the allo-HSCT cohort had a myeloid neoplasm, and 17% had a T cell neoplasm. 33% of pts undergoing allo-HSCT developed grade 2-4 GVHD within 100 days and 56% developed GVHD within 6 months. The pre-transplant intestinal microbiome α-diversity was significantly higher in pts undergoing auto-HSCT compared to allo-HSCT (Fig 1). The etiology for this was likely multifactorial. More pts in the allo-HSCT cohort had received antibiotics within 30 days prior to transplant, compared to the auto-HSCT cohort (56% vs 18%). Pts in the allo-HSCT cohort had a higher proportion of VRE or MDR Gram-negative bacteria on screening cultures (44% vs 29%), which may be associated with domination of certain species. In both the allo- and auto-HSCT cohorts, the α-diversity significantly decreased at days 14 and 28, but recovered to pre-transplant levels by day 100. Pts in the auto-HSCT cohort displayed faster recovery kinetics, and had a significantly higher α-diversity at day 28 compared to the allo-HSCT cohort (Fig 1). The microbiome recovery in the auto-HSCT cohort was consistent across the whole group and was characterized by a much smaller variance compared to the allo-HSCT cohort. This may reflect lower total antibiotic exposure in the auto-HSCT cohort (Fig 2). Other factors specific to the allo-HSCT cohort were the occurrence of GVHD and the use of TBI, both of which were associated with decrease in α-diversity in our population (Fig 3). Of note, Faecalibacterium prausnitzii was found to have a 〉20 log reduction in both the auto and allo-HSCT cohorts. It is a commensal gut bacterium, and its depletion has previously been reported in inflammatory bowel disease. There is very limited data on this bacterium in the peri-transplant setting. It has an anti-inflammatory effect on the intestinal microenvironment through production of butyrate, inhibition of NF-kB, upregulation of regulatory T cell production, and release of metabolites that enhance intestinal barrier function (Lopez-Siles et al, ISME 2017). Transplantation of F. prausnitzii has been shown to decrease inflammation in mice models of colitis (Sokol 2009). We then evaluated the association between microbiota injury and GVHD. α-diversity was significantly decreased at day 14 and day 28 in pts who developed GVHD within 100 days. In differential abundance analysis of samples from pts who developed acute GVHD within 6 months, there was a 〉20 log reduction in the Bifidobacterium, Blautia, and Eubacterium Dolichum. Decrease in Blautia has previously been associated with increased GVHD related mortality. Bifidobacterium and Eubacterium Dolichum have not been reported in association with GVHD in humans before. All three bacterial taxa reported here are involved in production of short chain fatty acids, which may provide a common protective mechanism. In summary, this study showed that pts undergoing autologous transplant have a healthier pre-transplant microbial diversity, and that microbiota injury heals quicker after transplant. We have identified TBI as a risk factor for microbiome injury, which has not been previously studied in the context of HSCT. We also identify previously unreported bacterial taxa whose depletion is strongly associated with GVHD. Disclosures Assal: Incyte corporation: Consultancy, Research Funding; boston biomedical: Consultancy. Uhlemann:Merk: Research Funding; GSK: Research Funding; Allergan: Research Funding. Reshef:Kite, a Gilead Company: Consultancy, Honoraria, Research Funding; Atara: Consultancy, Research Funding; Magenta: Consultancy; Pfizer: Consultancy; Pharmacyclics: Consultancy, Research Funding; Celgene: Research Funding; Incyte: Consultancy, Research Funding; Shire: Research Funding; BMS: Consultancy.