BET protein proteolysis targeting chimera (PROTAC) exerts potent lethal activity against mantle cell lymphoma cells.

Bromodomain extraterminal protein (BETP) inhibitors transcriptionally repress oncoproteins and nuclear factor-κB (NF-κB) target genes that undermines the growth and survival of mantle cell lymphoma (MCL) cells. However, BET bromodomain inhibitor (BETi) treatment causes accumulation of BETPs, associated with reversible binding and incomplete inhibition of BRD4 that potentially compromises the activity of BETi in MCL cells. Unlike BETi, BET-PROTACs (proteolysis-targeting chimera) ARV-825 and ARV-771 (Arvinas, Inc.) recruit and utilize an E3-ubiquitin ligase to effectively degrade BETPs in MCL cells. BET-PROTACs induce more apoptosis than BETi of MCL cells, including those resistant to ibrutinib. BET-PROTAC treatment induced more perturbations in the mRNA and protein expressions than BETi, with depletion of c-Myc, CDK4, cyclin D1 and the NF-κB transcriptional targets Bcl-xL, XIAP and BTK, while inducing the levels of HEXIM1, NOXA and CDKN1A/p21. Treatment with ARV-771, which possesses superior pharmacological properties compared with ARV-825, inhibited the in vivo growth and induced greater survival improvement than the BETi OTX015 of immune-depleted mice engrafted with MCL cells. Cotreatment of ARV-771 with ibrutinib or the BCL2 antagonist venetoclax or CDK4/6 inhibitor palbociclib synergistically induced apoptosis of MCL cells. These studies highlight promising and superior preclinical activity of BET-PROTAC than BETi, requiring further in vivo evaluation of BET-PROTAC as a therapy for ibrutinib-sensitive or -resistant MCL.

Novel BET protein proteolysis-targeting chimera exerts superior lethal activity than bromodomain inhibitor (BETi) against post-myeloproliferative neoplasm secondary (s) AML cells.

The PROTAC (proteolysis-targeting chimera) ARV-825 recruits bromodomain and extraterminal (BET) proteins to the E3 ubiquitin ligase cereblon, leading to degradation of BET proteins, including BRD4. Although the BET-protein inhibitor (BETi) OTX015 caused accumulation of BRD4, treatment with equimolar concentrations of ARV-825 caused sustained and profound depletion (>90%) of BRD4 and induced significantly more apoptosis in cultured and patient-derived (PD) CD34+ post-MPN sAML cells, while relatively sparing the CD34+ normal hematopoietic progenitor cells. RNA-Seq, Reverse Phase Protein Array and mass cytometry 'CyTOF' analyses demonstrated that ARV-825 caused greater perturbations in messenger RNA (mRNA) and protein expressions than OTX015 in sAML cells. Specifically, compared with OTX015, ARV-825 treatment caused more robust and sustained depletion of c-Myc, CDK4/6, JAK2, p-STAT3/5, PIM1 and Bcl-xL, while increasing the levels of p21 and p27. Compared with OTX015, PROTAC ARV-771 treatment caused greater reduction in leukemia burden and further improved survival of NSG mice engrafted with luciferase-expressing HEL92.1.7 cells. Co-treatment with ARV-825 and JAK inhibitor ruxolitinib was synergistically lethal against established and PD CD34+ sAML cells. Notably, ARV-825 induced high levels of apoptosis in the in vitro generated ruxolitinib-persister or ruxolitinib-resistant sAML cells. These findings strongly support the in vivo testing of the BRD4-PROTAC based combinations against post-MPN sAML.

The genomic organization and polymorphism analysis of the human Niemann-Pick C1 gene.

Niemann-Pick C (NP-C) is a fatal autosomal recessive storage disorder characterized by progressive neurodegeneration and variable hepatosplenomegaly. At the cellular level, cells derived from an affected individual accumulate unesterified cholesterol in lysosomes when cultured with low-density lipoprotein. The NP-C gene was identified at 18q11. The transcript is 4.9 kb encoding a 1278-amino-acid protein. We have defined the genomic structure of NPC1 along with the 5' flanking sequence. The NPC1 gene spans greater than 47 kb and contains 25 exons. Exons range in size from 74 to 788 bp with introns ranging in size from 0.097 to 7 kb. All intron/exon boundaries follow the GT/AG rule. The 5' flanking sequence has a CpG island containing multiple Sp1 sites indicative of a promoter region. The CpG island is located in the 5' flanking sequence, exon 1 and the 5' end of intron 1. We have also identified multiple single nucleotide polymorphisms in the coding and intronic sequences.

Identification of CDK4 sequences involved in cyclin D1 and p16 binding.

Activation of CDK4 is regulated, in part, by its association with a D-type cyclin. Conversely, CDK4 activity is inhibited when it is bound to the cyclin-dependent kinase inhibitor, p16(INK4A). To investigate the molecular basis of the interactions between CDK4 and cyclin D1 or p16(INK4A) we performed site-directed mutagenesis of CDK4. The interaction was examined using in vitro translated wild type and mutant CDK4 proteins and bacterially expressed cyclin D1 and p16 fusion proteins. As mutational analysis of CDC2 suggests that its cyclin binding domain is primarily located near its amino terminus, the majority of the mutations constructed in CDK4 were located near its amino terminus. In addition, CDK4 residues homologous to CDC2 sites involved in Suc1 binding were also mutated. Our analysis indicates that cyclin D1 and p16 binding sites are overlapping and located primarily near the amino terminus. All CDK4 mutations that resulted in decreased p16 binding capability also diminished cyclin D1 binding. In contrast, amino-terminal sequences were identified, including the PSTAIRE region, that are important for cyclin D1 binding but are not involved in p16 binding.

Niemann-Pick C1 disease gene: homology to mediators of cholesterol homeostasis.

Niemann-Pick type C (NP-C) disease, a fatal neurovisceral disorder, is characterized by lysosomal accumulation of low density lipoprotein (LDL)-derived cholesterol. By positional cloning methods, a gene (NPC1) with insertion, deletion, and missense mutations has been identified in NP-C patients. Transfection of NP-C fibroblasts with wild-type NPC1 cDNA resulted in correction of their excessive lysosomal storage of LDL cholesterol, thereby defining the critical role of NPC1 in regulation of intracellular cholesterol trafficking. The 1278-amino acid NPC1 protein has sequence similarity to the morphogen receptor PATCHED and the putative sterol-sensing regions of SREBP cleavage-activating protein (SCAP) and 3-hydroxy-3-methyl-glutaryl coenzyme A (HMG-CoA) reductase.

Substantial narrowing of the Niemann-Pick C candidate interval by yeast artificial chromosome complementation.

Niemann-Pick disease type C (NP-C) is an autosomal recessive lipidosis linked to chromosome 18q11-12, characterized by lysosomal accumulation of unesterified cholesterol and delayed induction of cholesterol-mediated homeostatic responses. This cellular phenotype is identifiable cytologically by filipin staining and biochemically by measurement of low-density lipoprotein-derived cholesterol esterification. The mutant Chinese hamster ovary cell line (CT60), which displays the NP-C cellular phenotype, was used as the recipient for a complementation assay after somatic cell fusions with normal and NP-C murine cells suggested that this Chinese hamster ovary cell line carries an alteration(s) in the hamster homolog(s) of NP-C. To narrow rapidly the candidate interval for NP-C, three overlapping yeast artificial chromosomes (YACs) spanning the 1 centimorgan human NP-C interval were introduced stably into CT60 cells and analyzed for correction of the cellular phenotype. Only YAC 911D5 complemented the NP-C phenotype, as evidenced by cytological and biochemical analyses, whereas no complementation was obtained from the other two YACs within the interval or from a YAC derived from chromosome 7. Fluorescent in situ hybridization indicated that YAC 911D5 was integrated at a single site per CT60 genome. These data substantially narrow the NP-C critical interval and should greatly simplify the identification of the gene responsible in mouse and man. This is the first demonstration of YAC complementation as a valuable adjunct strategy for positional cloning of a human gene.

Expression of engrailed proteins in arthropods, annelids, and chordates.

engrailed is a homeobox gene that has an important role in Drosophila segmentation. Genes homologous to engrailed have been identified in several other organisms. Here we describe a monoclonal antibody that recognizes a conserved epitope in the homeodomain of engrailed proteins of a number of different arthropods, annelids, and chordates; we use this antibody to isolate the grasshopper engrailed gene. In Drosophila embryos, the antibody reveals engrailed protein in the posterior portion of each segment during segmentation, and in a segmentally reiterated subset of neuronal cells during neurogenesis. Other arthropods, including grasshopper and two crustaceans, have similar patterns of engrailed expression. However, these patterns of expression are not shared by the annelids or chordates we examined. Our results provide the most comprehensive view that has been obtained of how expression patterns of a regulatory gene vary during evolution. On the basis of these patterns, we suggest that engrailed is a gene whose ancestral function was in neurogenesis and whose function was co-opted during the evolution of segmentation in the arthropods, but not in the annelids and chordates.

The transcription unit of the Drosophila engrailed locus: an unusually small portion of a 70,000 bp gene.

Transcripts from the engrailed gene of Drosophila melanogaster have been characterized by Northern, S1 nuclease sensitivity, and primer extension analyses. The engrailed gene encodes three poly(A)+ transcripts (3.6 kb, 2.7 kb, and 1.4 kb) that derive from a 3.9-kb portion of the genome. No other transcribed regions were found up to 16 kb downstream and 48 kb upstream of the engrailed transcription unit, the portion of the genome to which engrailed mutations have been mapped. The structures of the engrailed transcripts are unaffected by lethal engrailed mutations that break the locus at points in the transcriptionally silent regions. Transcripts expressed by 145 kb of DNA that surround the engrailed locus were also identified by Northern analysis. In contrast to the large portion of the engrailed gene that is transcriptionally inactive, most of the surrounding regions are 10-fold more densely populated with transcripts. We presume that the unusually large silent region at the periphery of the engrailed transcription unit signify the presence of special mechanisms that regulate its expression.

The invected gene of Drosophila: sequence analysis and expression studies reveal a close kinship to the engrailed gene.

The invected and engrailed genes are juxtaposed in the Drosophila genome and are closely related in sequence and pattern of expression. The structure of the most abundant invected transcript was defined by obtaining the full-length cDNA sequence and by S1 nuclease sensitivity and primer extension studies; a partial sequence of the invected gene was determined; and the developmental profile of invected expression was characterized by Northern analysis and by in situ localization. The invected gene, like the engrailed gene, is expressed in the embryonic and larval cells of the posterior developmental compartments and in the embryonic hindgut, clypeolabrum, and nervous system. Like the engrailed gene, the invected gene can encode a protein of approximately 60 kD that contains a homeo box near its carboxyl terminus; indeed, a sequence of 117 amino acids in the carboxy-terminal region of both proteins is almost identical. The developmental role of the invected gene is not known.

Molecular cloning of chromosome I DNA from Saccharomyces cerevisiae: isolation and characterization of the CDC24 gene and adjacent regions of the chromosome.

Molecular cloning techniques were used to isolate and characterize the DNA including and surrounding the CDC24 and PYK1 genes on the left arm of chromosome I of the yeast Saccharomyces cerevisiae. A plasmid that complemented a temperature-sensitive cdc24 mutation was isolated from a yeast genomic DNA library in a shuttle vector. Plasmids containing pyk1-complementing DNA were obtained from other investigators. Several lines of evidence (including one-step gene replacement experiments) demonstrated that the complementing plasmids contained the bona fide CDC24 and PYK1 genes. These sequences were then used to isolate additional DNA from chromosome I by probing a yeast genomic DNA library in a lambda vector. A total of 28 kilobases (kb) of contiguous DNA surrounding the CDC24 and PYK1 genes was isolated, and a restriction map was determined. Electron microscopy of R-loop-containing DNA and RNA blot hybridization analyses indicated that an 18-kb segment contained at least seven transcribed regions, only three of which corresponded to previously known genes (CDC24, PYK1, and CYC3). Southern blot hybridization experiments suggested that none of the genes in this region was duplicated elsewhere in the yeast genome. The centers of CDC24 and PYK1 were only approximately 7.5 kb apart, although the genetic map distance between them is approximately 13 centimorgans. As previous studies with S. cerevisiae have indicated that 1 centimorgan generally corresponds to approximately 3 kb, the region between CDC24 and PYK1 appears to undergo meiotic recombination at an unusually high frequency.

Expression during embryogenesis of a mouse gene with sequence homology to the Drosophila engrailed gene.

Regions of the mouse and human genomes with strong homology to the Drosophila engrailed gene have been identified by Southern blot analysis. One mouse engrailed-like region, Mo-en.1, has been cloned and partially sequenced; homology with the engrailed gene is localized to a 180 bp engrailed-like homeo box and 63 nucleotides immediately 3' to it. The protein sequence this region can encode includes 81 amino acids, of which 60 (75%) are identical with those of the putative translation product of the corresponding engrailed sequence. These data suggest that Mo-en.1 represents a mouse homolog of a gene of the Drosophila engrailed gene complex. Mo-en.1 has been mapped to chromosome 1, indicating it is not linked to other homeo box sequences that have been mapped in the mouse genome. Analysis of poly(A)+ RNA extracted from teratocarcinoma cells and whole mouse embryos demonstrates that the conserved homeo box region of Mo-en.1 is expressed differentially during mouse embryogenesis.

Temperature-sensitive lethal mutations on yeast chromosome I appear to define only a small number of genes.

A method was developed for isolating large numbers of mutations on chromosome I of the yeast Saccharomyces cerevisiae. A strain monosomic for chromosome I (i.e., haploid for chromosome I and diploid for all other chromosomes) was mutagenized with either ethyl methanesulfonate or N-methyl-N'-nitro-N-nitrosoguanidine and screened for temperature-sensitive (Ts-) mutants capable of growth on rich, glucose-containing medium at 25 degrees but not at 37 degrees. Recessive mutations induced on chromosome I are expressed whereas those on the diploid chromosomes are usually not expressed because of the presence of wild-type alleles on the homologous chromosomes. Dominant ts mutations on all chromosomes should also be expressed, but these appeared rarely.--Of the 41 ts mutations analyzed, 32 mapped on chromosome I. These 32 mutations fell into only three complementation groups, which proved to be the previously described genes CDC15, CDC24 and PYK1 (or CDC19). We recovered 16 or 17 independent mutations in CDC15, 12 independent mutations in CDC24 and three independent mutations in PYK1. A fourth gene on chromosome I, MAK16, is known to be capable of giving rise to a ts-lethal allele, but we recovered no mutations in this gene. The remaining nine mutations isolated using the monosomic strain appeared not to map on chromosome I and were apparently expressed in the original mutants because they had become homozygous or hemizygous by mitotic recombination or chromosome loss.--The available information about the size of chromosome I suggests that it should contain approximately 60-100 genes. However, our isolation in the monosomic strain of multiple, independent alleles of just three genes suggests that only a small proportion of the genes on chromosome I is easily mutable to give a Ts--lethal phenotype.--During these studies, we located CDC24 on chromosome I and determined that it is centromere distal to PYK1 on the left arm of the chromosome.