Academic literature on the topic 'Global and distal suppressors'

Abstract hnRNP K is an RNA binding protein that controls a multitude of cellular processes and is aberrantly expressed in cancers. We have previously shown that hnRNP K functions as a haploinsufficient tumor suppressor in AML patients with 9q deletions. However, overexpression of hnRNP K is the more commonly observed clinical phenomenon. We have recently discovered that hnRNP K overexpression in patients with diffuse large B-cell lymphoma correlates with dismal outcomes and directly resulted in the development of lymphomas in transgenic mice. To understand the mechanistic basis for the oncogenicity of hnRNP K overexpression and to identify therapeutic vulnerabilities, we performed RNA-sequencing, RNA immunoprecipitation following by sequencing (RIP-Seq), mass spectrometry, and polysome assays. We observed that hnRNP K regulates both global transcription and translational processes within the cell via modulation of 7SK and translation initiation proteins (such as the eIFs and PABP), respectively. Consequently, we hypothesized that aberrant hnRNP K expression primarily perturbs oncogenes with short half-lives. Mechanistically, we identified that hnRNP K binds to the RNA and regulates the expression of a plethora of critical oncogenes and tumor suppressors involved in hematologic malignancies such as c-Myc, RUNX1, and Cyclin D1. As proof of concept for clinical applications, we have demonstrated that hnRNP K-driven c-Myc overexpression renders tumors susceptible to bromodomain inhibition. Given that hnRNP K directs global transcription and translation, it is likely that hnRNP K overexpressing tumors will also be sensitive to transcriptional and translational inhibitors such as CDK9 inhibitors and omacetaxine mepesuccinate, respectively. However, since hnRNP K also regulates a plethora of additional cellular processes that extend far beyond mRNA and protein synthesis, there is a need to develop hnRNP K-specific inhibitors that will only target these activities. Thus, we have recently begun to identify small molecule compounds that can directly inhibit hnRNP K-RNA binding function on specific targets using an in vitro fluorescent binding assay. Using this assay, we are currently screening a library of 70,000 small molecule compounds to identify agents that can prevent hnRNP K-RNA interactions. In summary, we have established that hnRNP K is a bona fide oncogene that drives lymphomagenesis. Global analyses have revealed therapeutic vulnerabilities of hnRNP K overexpressing tumors. Furthermore, using our in vitro RNA binding assays, we anticipate identification of novel hnRNP K-specific inhibitors. Disclosures No relevant conflicts of interest to declare.

Abstract Abstract 408 Background: The Ikaros (IKZF1) tumor suppressor is deleted in >80% of the cases of Ph+ ALL. While Ikaros cooperates with pre-B cell receptor signaling to induce cell cycle exit in Ph+ ALL (Trageser et al., J Exp Med, 2009), the mechanism of Ikaros-mediated tumor suppression is poorly understood. Here we report on a series of genetic experiments that show that Ikaros (i) interferes with key survival pathways downstream of the BCR-ABL1 kinase, (ii) inhibits leukemia cell proliferation through interaction with the pre-B cell receptor signaling pathway and (iii) activates the tumor suppressors p53, p21 and p27. Results: To elucidate the mechanism of Ikaros-dependent tumor suppression in BCR-ABL1-driven B cell lineage leukemia, we studied regulation of critical phosphorylation events downstream of the BCR-ABL1 kinase as a central mediators of survival and proliferation. Reconstitution of Ikaros expression in BCR-ABL1-transformed pre-B ALL cells resulted in rapid and global dephosphorylation comparable to the effect of Imatinib. A detailed analysis showed that Ikaros-induced dephosphorylation events affect activation of Stat5 (Y694), AKT (S473), ERK1/2 (T202 and Y204) and SRC (Y416). Interestingly, both Imatinib-treatment and reconstitution of pre-B cell receptor signaling using retroviral vectors for expression of the m heavy chain or the BLNK adapter molecule have the same effects as reconstitution of Ikaros. In fact, a comprehensive gene expression analysis demonstrated that Ikaros reconstitution resulted in similar gene expression changes as reconstitution of pre-B cell receptor signaling (m heavy chain or BLNK), reconstitution of PAX5, Cre-mediated deletion of Stat5 or Myc, or treatment with Imatinib. The signature of common gene expression changes shared between reconstitution of Ikaros, Pax5, m heavy chain, BLNK and inducible deletion of Stat5 or Myc and Imatinib-treatment involves known tumor suppressors including SPIB, BTG1, and BTG2. These findings suggest that reconstitution of tumor suppressive transcription factor (Ikaros, Pax5) converges with pre-B cell receptor-mediated tumor suppression. To better understand how pre-B cell receptor signaling and Ikaros intersect, we combined reconstitution of Ikaros with genetic deletion of either the (more proximal) SYK kinase or the (more distal) BLNK adapter molecule. While inducible Cre-mediated deletion of Syk had no effect on Ikaros-mediated tumor suppression, deletion of the BLNK adapter compromised the ability of Ikaros to function as tumor suppressor. These findings were confirmed in an in vivo transplantation experiment. While mice transplanted with Ikaros+ BLNK+ leukemia cells survived indefinitely, mice transplanted with Ikaros- BLNK+, Ikaros+ BLNK- or Ikaros- BLNK- leukemia cells died after 24 to 31 days post transplantation. While these findings provide genetic evidence for collaboration between the Ikaros and pre-B cell receptor tumor suppressor pathways, Ikaros and pre-B cell receptor signaling differ with respect to activation of classical tumor suppressor pathways. While reconstitution of pre-B cell receptor signaling failed to activate Arf, p53 or p27, protein levels of all these molecules were strongly upregulated by Ikaros. In agreement with these findings, reconstitution of pre-B cell receptor signaling had the same tumor suppressive effect in wildtype leukemia cells as in Arf−/−, p53−/− as well as p27−/− leukemia cells. Conversely, deletion of Arf and p53 significantly diminished the ability of Ikaros to function as tumor suppressor. Conclusion: Ikaros deletion represents a near-obligatory lesion in the pathogenesis of Ph+ ALL. Here we provide genetic evidence for three novel pathways of Ikaros-mediated tumor suppression. Like PAX5, Ikaros reconstitution results in multiple dephosphorylation events (Stat5, AKT, ERK1/2 and SRC are affected). In collaboration with the pre-B cell receptor and its downstream adapter molecule BLNK, Ikaros suppressed MYC and inhibits cell cycle progression. Induction of the Arf/p53 pathway represents a distinct function of Ikaros, which is not shared with the pre-B cell receptor signaling pathway. Disclosures: No relevant conflicts of interest to declare.

Scribble is a critical cell polarity regulator that has been shown to work as either an oncogene or tumor suppressor in a context dependent manner, and also impacts cell migration, tissue architecture and immunity. Mutations in Scribble lead to neural tube defects in mice and humans, which has been attributed to a loss of interaction with the planar cell polarity regulator Vangl2. We show that the Scribble PDZ domains 1, 2 and 3 are able to interact with the C-terminal PDZ binding motif of Vangl2 and have now determined crystal structures of these Scribble PDZ domains bound to the Vangl2 peptide. Mapping of mammalian neural tube defect mutations reveal that mutations located distal to the canonical PDZ domain ligand binding groove can not only ablate binding to Vangl2 but also disrupt binding to multiple other signaling regulators. Our findings suggest that PDZ-associated neural tube defect mutations in Scribble may not simply act in a Vangl2 dependent manner but as broad-spectrum loss of function mutants by disrupting the global Scribble-mediated interaction network.

Abstract Diffuse midline gliomas (DMGs) are very aggressive pediatric brain tumors with dismal prognosis due to therapy-resistant tumor growth and invasion. We performed the first integrated histologic/genomic/proteomic analysis of 21 tumor foci from three pontine DMG cases with supratentorial dissemination. Histone H3.3 K27M was the driver mutation, usually at high variant allele fraction due to recurrent chromosome 1q copy number gain, in combination with germline variants in ATM, FANCM and MYCN genes. Both previously reported and novel recurrent copy number variations and somatic pathogenic mutations in chromatin remodeling, DNA damage response and PI3K/MAPK growth pathways were variably detected, either in multiple or isolated foci. Proteomic analysis showed global upregulation of histone H3, lack of K27 tri-methylation, and further impairment of polycomb repressive complex 2 by ASXL1 downregulation. Activation of oncogenic pathways resulted from combined upregulation of N-Myc, SOX2, p65/p50 NF-kB and STAT3 transcription factors, EGFR, FGFR2, PDGFRa/b and MET receptor tyrosine kinases, and downregulation of PHLPP1/2, PTEN and p16/INK4A tumor suppressors. Upregulation of SMAD4, PAI-1, CD44, and c-Src in multiple foci most likely contributed to invasiveness. This integrated comprehensive analysis allowed spatiotemporal modeling of tumor progression and identified two general pathways of supratentorial invasion, and a multitude of migratory subpopulations within the infratentorial compartment. It also delineated common signaling pathways and potential therapeutic targets, revealing an unsuspected activation of a multitude of oncogenic pathways that may explain the resistance of DMG to current therapies.

Abstract Sixty independent UGA suppressors of Saccharomyces cerevisiae have been studied. They are dominant and are divided into 16 groups (loci) by recombination. Suppressors representing these loci are divided into two classes by action spectra; four in class 1 (a broad action spectrum) and 12 in class 2 (a narrow action spectrum). Class 1 suppressors are less frequent in terms of not only total number but also number per locus than class 2 suppressors, indicating difference in either or both mutation frequency and selective pressure between suppressors of the two classes. Two of the class 1 suppressors, SUP152 and SUP161, do not recombine with SUP28 and SUP33, leucine-inserting UAA suppressors, respectively, indicating that they are mutations in genes coding for tRNA(Leu)UUA. Of the remaining two class 1 suppressors, SUP160 which causes lethality in the psi+ cytoplasm is mapped on chromosome XV very close to the centromere, and SUP165 on the right arm of chromosome XIV 44 cM distal to lys9. Of the class 2 suppressors, ten do not recombine with one or another of previously known UGA suppressors. The remaining two class 2 suppressors, SUP154 and SUP155, are mapped on the left and right arms of chromosome VII, respectively.

Most amino acid substitutions in a protein either lead to partial loss of function or are near neutral. Several studies have shown the existence of second-site mutations that can rescue defects caused by diverse loss of function mutations. Such global suppressor mutations are key drivers of protein evolution. However, the mechanisms responsible for such suppression remain poorly understood. To address this, we characterized multiple suppressor mutations both in isolation and in combination with inactive mutants. We examined six global suppressors of the bacterial toxin CcdB, the known M182T global suppressor of TEM-1 β-lactamase, the N239Y global suppressor of p53-DBD and three suppressors of the SARS-CoV-2 spike Receptor Binding Domain. When coupled to inactive mutants, they promote increased in-vivo solubilities as well as regain-of-function phenotypes. In the case of CcdB, where novel suppressors were isolated, we determined the crystal structures of three such suppressors to obtain insight into the specific molecular interactions responsible for the observed effects. While most individual suppressors result in small stability enhancements relative to wildtype, which can be combined to yield significant stability increments, thermodynamic stabilisation is neither necessary nor sufficient for suppressor action. Instead, in diverse systems, we observe that individual global suppressors greatly enhance the foldability of buried site mutants, primarily through increase in refolding rate parameters measured in vitro. In the crowded intracellular environment, mutations that slow down folding likely facilitate off-pathway aggregation. We suggest that suppressor mutations that accelerate refolding can counteract this, enhancing the yield of properly folded, functional protein in vivo.

Over-expression of Casein Kinase II (CK2) pro-oncogenic kinase in Acute Myelogenous Leukemia (AML) is associated with poor prognosis. Inhibition of CK2 by siRNAs or with the specific inhibitor, CX-4945, shows strong cytotoxic effects. CK2 is a ubiquitous, constitutively active serine/ threonine kinase which has been implicated in leukemia progression via multiple mechanisms. One of the well-established mechanism is CK2 mediated phosphorylation and impairment of tumor suppressor function of Ikaros transcription factor. Impaired function of Ikaros has been associated with the development of leukemia including AML. In B-cell lymphoblastic leukemia, CK2 inhibition has been shown to exert a therapeutic effect via restoration and/or enhancement of the tumor suppressor activity of the Ikaros protein. The mechanism of therapeutic action of CK2 inhibition in AML is largely unknown and in vivo efficacy CK2 inhibitors in AML has never been established. Here we report anti-leukemia effect of CX-4945 in preclinical models of AML and demonstrate mechanism of action of CK2 inhibitor via restoration of Ikaros driven global regulation of epigenetic landscape. In order to study the effect of CK2 inhibition on Ikaros function in AML, we used U937 - a human myelomonocytic leukemia cell line with high baseline CK2 expression. U937 cells were treated with specific CK2 inhibitor, CX4945 for 72 hours. CK2 activity and Ikaros phosphorylation was measured using CK2 kinase assay and in vivo labeling - radio immunoblot assay. Results showed significant decrease in CK2 activity and Ikaros phosphorylation with no change in overall expression of the Ikaros protein. Expression analysis using RNA sequencing showed that treatment with CX-4945 caused significant upregulation of genes controlling immunity, and inflammation; and downregulation of genes involved in nucleic acid metabolism, RNA processing, and translation. Analysis of global genome-wide Ikaros occupancy using Chromatin Immunoprecipitation followed by next generation sequencing (ChIP-seq) of CX4945 treated U937 cells demonstrated that CX-4945 treatment significantly increased the number of Ikaros binding sites as well as increased peak strength while minimally re-distributing Ikaros' global genomic occupancy. While increased binding to the Promoter, Gene Body, and Gene Desert elements in CX-4945-treated cells was similar, the increase in Ikaros' DNA binding to enhancers was particularly pronounced. Further analysis showed that enhanced Ikaros DNA binding following treatment with CX-4945, directly induces formation of de novo enhancers. In order to test whether enhanced DNA binding of Ikaros is accompanied by augmented Ikaros function in the global epigenetic regulation of gene expression. We determined and compared chromatin accessibility of U937 cells before and after treatment with CX-4945, using ATAC-seq. Results indicate that Ikaros-induced de novo open chromatin at distal regulatory regions controls genes involved in the negative regulation of biological processes and cellular metabolism. Overall, these data demonstrate that, augmented Ikaros DNA-binding following CK2 inhibition resulted in 1) Ikaros' pioneering activity, 2) Ikaros' ability to induce the de novo formation of enhancers and super-enhancers, and 3) Ikaros' ability to induce the de novo formation of active enhancers and to activate poised enhancers. Together, these data uncover novel Ikaros functions in regulating the epigenetic landscape and identifies CK2 as a critical regulator of Ikaros activity. Next, we treated AML xenograft model of luciferase labelled U937 cells with CX4945 via oral gavage at dose 200mg/kg/day for 3 weeks and demonstrated significantly lower leukemia burden in treated group as measured by decreased bio-luminescence imaging. In summary, these results demonstrate for the first time that CK2 inhibitor, CX-4945 has strong anti-leukemia effect in AML preclinical models. One of the mechanisms by which CX4945 exert a therapeutic effect in AML involves enhancing Ikaros' function as regulator of global epigenomic landscape. These results provide strong mechanistic basis to develop novel targeted combination therapies using CK2 inhibitors for treatment of AML. Further studies evaluating combination therapies using patient derived xenograft (PDX) models of AML are underway. Disclosures Payne: Elf Zone, Inc: Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

Abstract Polyadenylation is a post-transcriptional modification where the 3' end of an mRNA is cleaved and 250-300 adenines are added. It is predicted that 70-75% of human genes have more than one polyadenylation sequence (PAS) and are subject to alternative polyadenylation (APA). APA events affect the coding sequence of a gene when a proximal PAS is located within an intron, constitutive exon, or alternative exon. Gene expression is also affected if there are multiple PAS within the distal 3' untranslated region (UTR); proximal PAS usage shortens the 3'UTR, which can remove cis-regulatory regions such as miRNA and RNA-binding protein (RBP) sites. Furthermore, global changes in APA are linked to cellular state-proximal PAS usage is associated with immature developmental phases, cell proliferation, and cancerous phenotypes. Consequently, APA is a pertinent post-transcriptional modification that regulates gene expression and isoform generation across developmental stages and tissue types. Despite its significance, there are few APA studies in the hematology field, and those that exist have focused on global shifts in PAS usage. In this study, we uniquely focus on the APA mechanism of a single gene, RUNX1, and how this event can alter hematopoietic stem cell (HSC) homeostasis and hematopoiesis. There are three main isoforms of RUNX1 that differ in promoter and/or PAS usage. RUNX1b/c use different promoters, but have identical C-terminal regions. RUNX1a utilizes the same promoter as RUNX1b, but differs from both RUNX1b/c due to usage of a proximal PAS located in alternative exon 7a. RUNX1b/c are robustly expressed in most progenitor populations and differentiated blood cell lineages, whereas RUNX1a is restricted to human CD34+ HSCs. Functionally, RUNX1b/c promote HSC differentiation and lineage commitment, whereas RUNX1a expands HSCs and their engraftment potential, a property with therapeutic advantages but leukemic potential. Due to the difference in expression pattern and distinct functionality of RUNX1a compared to RUNX1b/c, it is relevant to study the APA event that dictates isoform generation. Elucidating this mechanism could provide valuable insight into the transient control of the HSC population for therapeutic benefit and illuminate new leukemogenic pathways. To study RUNX1 APA, we cloned alternative terminal exon 7a (RUNX1a) and constitutive exon 7b (RUNX1b/c) in between the two exons of a split GFP minigene reporter, along with 500 bp of their upstream and downstream flanking introns. We hypothesized that exon 7a would be skipped during processing of the minigene construct because the proximal PAS is rarely used in vivo. Conversely, exon 7b, the penultimate exon in RUNX1b/c, would be spliced in between the GFP exons, disrupting the GFP protein. These constructs were tested in KG-1a and U937 cells. Flow cytometry for GFP fluorescence supported our hypothesis as the exon 7a minigene produced a robust GFP signal and the exon 7b minigene produced no GFP signal. We confirmed that the GFP changes were due to the hypothesized mRNA processing events by performing RT-PCR using primers specific to the two GFP exons. These data show that important cis-regulatory elements that determine RUNX1 APA are located within exon 7a, 7b, and the cloned intronic regions. Next, we altered these minigenes by strategically making chimeric constructs that consist of either exon 7a or 7b with all combinations of upstream/downstream flanking introns. We discovered that replacing the intron upstream of exon 7a confers 2-5 fold greater splicing and polyadenylation of exon 7a, indicative of RUNX1a isoform generation. Therefore, a suppressor cis-element is located in this upstream intronic region. However, placing this intron upstream of exon 7b is not sufficient to reduce its inclusion between the GFP exons. Instead, both the upstream and downstream intronic regions flanking exon 7a are required. This suggests an RNA-looping mechanism that prevents splicing and usage of the exon 7a proximal PAS. Cleavage factor (CFIm) and Polypyrimidine-tract binding protein 1 (PTBP1) are RBPs involved in splicing and polyadenylation that alter mRNA processing by RNA-looping. We aim to narrow down the suppressor region upstream of exon 7a to identify a consensus sequence and the respective RBP that diminishes RUNX1 proximal PAS usage. This knowledge can be leveraged to enhance RUNX1a production and expand HSCs for therapeutic benefit. Disclosures No relevant conflicts of interest to declare.

Abstract The glp-1 gene is essential for two cell interactions that control cell fate in Caenorhabditis elegans: induction of anterior pharynx in the embryo and induction of mitotic proliferation in the germ line. To identify other genes involved in these cell interactions, we have isolated suppressors of two temperature sensitive alleles of glp-1. Each of 14 recessive suppressors rescues both embryonic and germline glp-1(ts) defects. These suppressors are extragenic and define a set of six genes designated sog, for suppressor of glp-1. Suppression of glp-1 is the only obvious phenotype associated with sog mutations. Mutations in different sog genes show allele-specific intergenic noncomplementation, suggesting that the sog gene products may interact. In addition, we have analyzed a semidominant mutation that suppresses only the glp-1 germline phenotype and has a conditional feminized phenotype of its own. None of the suppressors rescues a glp-1 null mutation and therefore they do not bypass a requirement for glp-1. Distal tip cell function remains necessary for germline proliferation in suppressed animals. These suppressor mutations identify genes that may encode other components of the glp-1 mediated cell-signaling pathway or regulate glp-1 expression.

Abstract The glp-1 gene product mediates cell-cell interactions required for cell fate specification during development in Caenorhabditis elegans. To identify genes that interact with glp-1, we screened for dominant suppressors of two temperature-sensitive glp-1 alleles and recovered 18 mutations that suppress both germline and embryonic glp-1 phenotypes. These dominant suppressors are tightly linked to glp-1 and do not bypass the requirement for a distal tip cell, which is thought to be the source of a signal that is received and transduced by the GLP-1 protein. Using single-strand conformation polymorphism (SSCP) analysis and DNA sequencing, we found that at least 17 suppressors are second-site intragenic revertants. The suppressors, like the original glp-1(ts) mutations, are all located in the cdc10/SWI6/ankyrin domain of GLP-1. cdc10/SWI6/ankyrin motifs have been shown to mediate specific protein-protein interactions in other polypeptides. We propose that the glp-1(ts) mutations disrupt contact between GLP-1 and an as yet unidentified target protein(s) and that the dominant suppressor mutations restore appropriate protein-protein interactions.