Development of a novel polyamide-based agent to inhibit EVI1 function

The EVI1 gene at chromosome 3q26 is associated with acute myeloid leukemogenesis, due to both chromosomal rearrangement and to overexpression in the absence of rearrangement. Some rearrangements such as t(3;3) and inv(3) result in overexpression of EVI1 protein, while translocation t(3;21) yields an AML1-MDS1-EVI1 (AME) fusion protein. EVI1 possesses two zinc finger domains, an N-terminal domain with fingers 1–7, which binds to GACAAGATA, and a C-terminal domain (fingers 8–10) which binds GAAGATGAG. Inhibition of EVI1 function with a small molecule compound may provide a targeted therapy for EVI1-expressing leukemias. As a first step towards inhibiting the leukemogenic function of EVI1, we performed structure-function studies on both EVI1 and AME protein to determine what domains are critical for malignant transformation activity. Assays were Rat1 fibroblasts in a soft agar colony forming assay for EVI1; primary bone marrow cells in a serial replating assay for AME. Both assays revealed that mutation of arginine 205 in zinc finger 6 of EVI1, which completely abrogates sequencespecific DNA binding via the N-terminal zinc finger domain, resulted in complete loss of transforming activity; mutations in other domains, such as the C-terminal zinc finger domain, CtBP binding domain, and the domains of AML1 had less of an effect or no effect on transforming activity. In an effort to inhibit EVI1 leukemogenic function, we developed a polyamide, DH-IV-298, designed to block zinc fingers 1–7 binding to the GACAAGATA motif. DNAseI footprinting revealed a specific interaction between DH-IV-298 and the GACAAGATA motif; no significant interaction was observed elsewhere; a mismatch polyamide failed to footprint at equivalent concentrations; and DH-IV-298 failed to bind to a control DNA lacking the GACAAGATA motif. Electromobility shift assay showed that, at a 1:1 polyamide:DNA ratio, DH-IV-298 lowered EVI1:DNA affinity by over 98%, while mismatch was significantly less effective (74% reduction). To assess the effect of DH-IV-298 on EVI1 binding to DNA in vivo, we performed CAT reporter assays in a NIH-3T3-derived cell line with a chromosome-embedded tet-inducible EVI1-VP16 as well as a EVI1-responsive CAT reporter. Removal of tetracycline resulted in a four-fold increase in CAT activity that was completely blocked by DH-IV-298. The mismatch polyamide was significantly less effective than DH-IV-298. Further studies are being performed to assess the effect on endogenous gene expression, and on growth of leukemic cells that express EVI1. These studies provide evidence that a cell permeable small molecule compound may effectively block the activity of a leukemogenic transcription factor

Genotype- phenotype correlation and molecular heterogeneity in pyruvate kinase deficiency

Pyruvate kinase (PK) deficiency is a rare recessive congenital hemolytic anemia caused by mutations in the PKLR gene. This study reports the molecular features of 257 patients enrolled in the PKD Natural History Study. Of the 127 different pathogenic variants detected, 84 were missense and 43 non- missense, including 20 stop- gain, 11 affecting splicing, five large deletions, four in- frame indels, and three promoter variants. Within the 177 unrelated patients, 35 were homozygous and 142 compound heterozygous (77 for two missense, 48 for one missense and one non- missense, and 17 for two non- missense variants); the two most frequent mutations were p.R510Q in 23% and p.R486W in 9% of mutated alleles. Fifty- five (21%) patients were found to have at least one previously unreported variant with 45 newly described mutations. Patients with two non- missense mutations had lower hemoglobin levels, higher numbers of lifetime transfusions, and higher rates of complications including iron overload, extramedullary hematopoiesis, and pulmonary hypertension. Rare severe complications, including lower extremity ulcerations and hepatic failure, were seen more frequently in patients with non- missense mutations or with missense mutations characterized by severe protein instability. The PKLR genotype did not correlate with the frequency of complications in utero or in the newborn period. With ICCs ranging from 0.4 to 0.61, about the same degree of clinical similarity exists within siblings as it does between siblings, in terms of hemoglobin, total bilirubin, splenectomy status, and cholecystectomy status. Pregnancy outcomes were similar across genotypes in PK deficient women. This report confirms the wide genetic heterogeneity of PK deficiency.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/154955/1/ajh25753.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/154955/2/ajh25753_am.pd

CTCF and Cohesin^SA-1 Mark Active Promoters and Boundaries of Repressive Chromatin Domains in Primary Human Erythroid Cells

<div><p>Background</p><p>CTCF and cohesin^SA-1 are regulatory proteins involved in a number of critical cellular processes including transcription, maintenance of chromatin domain architecture, and insulator function. To assess changes in the CTCF and cohesin^SA-1 interactomes during erythropoiesis, chromatin immunoprecipitation coupled with high throughput sequencing and mRNA transcriptome analyses via RNA-seq were performed in primary human hematopoietic stem and progenitor cells (HSPC) and primary human erythroid cells from single donors.</p><p>Results</p><p>Sites of CTCF and cohesin^SA-1 co-occupancy were enriched in gene promoters in HSPC and erythroid cells compared to single CTCF or cohesin sites. Cell type-specific CTCF sites in erythroid cells were linked to highly expressed genes, with the opposite pattern observed in HSPCs. Chromatin domains were identified by ChIP-seq with antibodies against trimethylated lysine 27 histone H3, a modification associated with repressive chromatin. Repressive chromatin domains increased in both number and size during hematopoiesis, with many more repressive domains in erythroid cells than HSPCs. CTCF and cohesin^SA-1 marked the boundaries of these repressive chromatin domains in a cell-type specific manner.</p><p>Conclusion</p><p>These genome wide data, changes in sites of protein occupancy, chromatin architecture, and related gene expression, support the hypothesis that CTCF and cohesin^SA-1 have multiple roles in the regulation of gene expression during erythropoiesis including transcriptional regulation at gene promoters and maintenance of chromatin architecture. These data from primary human erythroid cells provide a resource for studies of normal and perturbed erythropoiesis.</p></div

Invariant and cell type-specific CTFC sites.

<p>Patterns of CTCF occupancy in HSPC and erythroid cell chromatin were compared to CTCF occupancy in several human ENCODE ChIP-seq data sets, including fibroblast, keratinocyte, endothelial, myocyte, monocyte, lymphocyte, embryonic stem (ES) cell, erythroid and HSPC cells. <b>A.</b> At the <i>TAL1</i> locus, a 3’ site of invariant CTCF binding marked by the rectangle is present in all cell types. Two sites of erythroid-specific CTCF binding, denoted by the arrows, are present 5’ of the gene. Corresponding RNA-seq tracks in HSPC and erythroid cells are shown at the top. Genomic coordinates: Chr1:47,600–47,700. <b>B.</b> At the <i>UBTF</i> and <i>SLC4A1</i> loci, there are 2 sets of invariant CTCF binding, marked by rectangles, 3’ of the <i>UBTF</i> locus, present in all cell types. One site of erythroid-specific CTCF binding, denoted by the arrows, are present 5’ of the <i>SLC4A1</i> locus. Corresponding RNA-seq tracks in HSPC and erythroid cells are shown at the top. Genomic coordinates: Chr17:42,280–42,340.</p

Gene expression and CTCF occupancy.

<p>Gene expression levels in primary human HSPC and erythroid cell mRNA were correlated with sites of CTCF occupancy by class within 1kb of the transcription start site.</p

Repressive chromatin domains and CTCF occupancy.

<p>Representative integrated genome viewer (IGV) views of CTCF occupancy, repressive chromatin domains marked by H3K27me3 enrichment, and gene expression determined by RNA-seq in erythroid cells. <b>A.</b> Repressive chromatin domains marked by CTCF occupancy at their boundaries flank the <i>SEC31B</i>, <i>NDUFB8</i>, and <i>HIF1AN</i> genes. These 3 genes are expressed in erythroid cells, while the <i>WNT8B</i> gene, located in a repressive chromatin domain, is not. Genomic coordinates: Chr10:102,220–102,380. <b>B.</b> Repressive chromatin domains marked by CTCF occupancy at their boundaries flank the <i>TROAP and C1QL4</i> genes. These 2 genes are expressed in erythroid cells, while the flanking <i>PRPH</i> and <i>DNACJ22</i> genes, located in flanking repressive chromatin domains, are not. Genomic coordinates: Chr12:49,680–49,760.</p

Repressive chromatin domains and CTCF-cohesin^SA-1 co-occupancy.

<p>Representative integrated genome viewer (IGV) views of CTCF and cohesin^SA-1 occupancy, repressive chromatin domains marked by H3K27me3 enrichment, and gene expression determined by RNA-seq in HSPC and erythroid cells. Multiple tissue-specific “exon 1s” are found at the 5’ end of the <i>ANK1</i> gene which all join in frame to exon 2, creating cDNA transcripts with unique 5’ ends. In erythroid cells (top), the sequence surrounding and including a neural-specific <i>ANK1</i> exon 1, located 5’ of the erythroid exon 1, is in a region of repressive chromatin, heavily modified by H3K27me3. At the boundary of this repressive chromatin domain are a pair of CTCF/cohesin^SA-1 sites, present in erythroid but not HSPC chromatin, followed by the transcribed exons of the <i>ANK1</i> gene. <i>ANK1</i> is not expressed in HSPCs and this entire region is modified by H3K27 trimethylation (bottom). Genomic coordinates: Chr8:41,760–41,580.</p