CRISPR/Cas9-Mediated Genome Editing of Y705 Residue of STAT3 for Genetic Validation of Small Molecule Inhibitors In Breast Cancer Cells​

https://openworks.mdanderson.org/sumexp23/1122/thumbnail.jp

Development and Characterization of STAT3 Y705H Mutated Breast Cancer Cell Lines for Genetic Validation of the Mechanism of Action of STAT3 Inhibitor TTI-101

https://openworks.mdanderson.org/sumexp21/1225/thumbnail.jp

Rhodium(II) Proximity-Labeling Identifies a Novel Target Site on STAT3 for Inhibitors with Potent Anti-Leukemia Activity

Nearly 40 % of children with acute myeloid leukemia (AML) suffer relapse arising from chemoresistance, often involving upregulation of the oncoprotein STAT3 (signal transducer and activator of transcription 3). Herein, rhodium(II)-catalyzed, proximity-driven modification identifies the STAT3 coiled-coil domain (CCD) as a novel ligand-binding site, and we describe a new naphthalene sulfonamide inhibitor that targets the CCD, blocks STAT3 function, and halts its disease-promoting effects in vitro, in tumor growth models, and in a leukemia mouse model, validating this new therapeutic target for resistant AML

Chemical Probes that Competitively and Selectively Inhibit Stat3 Activation

Signal transducer and activator of transcription (Stat) 3 is an oncogene constitutively activated in many cancer systems where it contributes to carcinogenesis. To develop chemical probes that selectively target Stat3, we virtually screened 920,000 small drug-like compounds by docking each into the peptide-binding pocket of the Stat3 SH2 domain, which consists of three sites—the pY-residue binding site, the +3 residue-binding site and a hydrophobic binding site, which served as a selectivity filter. Three compounds satisfied criteria of interaction analysis, competitively inhibited recombinant Stat3 binding to its immobilized pY-peptide ligand and inhibited IL-6-mediated tyrosine phosphorylation of Stat3. These compounds were used in a similarity screen of 2.47 million compounds, which identified 3 more compounds with similar activities. Examination of the 6 active compounds for the ability to inhibit IFN-γ-mediated Stat1 phosphorylation revealed that 5 of 6 were selective for Stat3. Molecular modeling of the SH2 domains of Stat3 and Stat1 bound to compound revealed that compound interaction with the hydrophobic binding site was the basis for selectivity. All 5 selective compounds inhibited nuclear-to-cytoplasmic translocation of Stat3, while 3 of 5 compounds induced apoptosis preferentially of breast cancer cell lines with constitutive Stat3 activation. Thus, virtual ligand screening of compound libraries that targeted the Stat3 pY-peptide binding pocket identified for the first time 3 lead compounds that competitively inhibited Stat3 binding to its pY-peptide ligand; these compounds were selective for Stat3 vs. Stat1 and induced apoptosis preferentially of breast cancer cells lines with constitutively activated Stat3

The ghost of memory : literary representations of slavery in post-apartheid South Africa

Thesis (PhD)--Stellenbosch University, 2018.ENGLISH ABSTRACT: This study examines how authors of slave/slave-owner ancestry have constructed slave memory in selected contemporary literary texts on slavery at the Cape. The texts I study include Rayda Jacobs’s The Slave Book (1998), Therese Benadé’s Kites of Good Fortune (2004), Yvette Christiansë’s Unconfessed (2006) and André Brink’s Philida (2012). All four novels are published in the post-apartheid moment, over a century after the practice of Cape slavery ended. In their examination, I explore the lasting social and psychic effects of traumatic and repressed slave histories in the ghostly presence of a slave past in the post-apartheid present by framing my literary analysis with the concepts of cultural haunting, collective memory and rememory. My conceptualisation of haunting is centred on the idea of slavery as a ghost that haunts the present moment. The study argues that the publication of stories regarding slave pasts at this point in time indicates a haunting that is embedded in oppressive slave histories and that contemporary writers are bringing to the surface through their works. As such, the concept of haunting is embedded in this study’s three main areas of interest: firstly, the revisiting of slave memory in the post-apartheid moment; secondly, the authors’ need to revisit their ancestors’ pasts because they are themselves of slave or slave-owner ancestry; and thirdly, that some of the legacies of slavery resonate with subjectivities in present day South Africa. The chapters therefore examine the representation embedded in the neo-slave narratives by asking two questions: How do they engage with the idea of representing ‘self’ in the sense that, in writing about these slaves, the authors are also writing about their own history and ancestors? And how do they represent the ‘other’ when they write about dead and silenced slaves? My first chapter focusses on Unconfessed to foreground the trauma of slavery by developing on concepts of silence and silencing, narrative structure and fragmentation and narrative as an appropriated court room. My discussion depicts an intergenerational trajectroy in traumatic slave pasts as elucidated in the violence on slave mothers, which rendered motherhood impossible in the practice that children born to slave women inherited their maternal slave status. The second chapter on Philida problematises representation in its reading of the legacy of centuries-old policing of intimacy, white privilege and authorship. In the third chapter, I investigate the narrative of black-on-black violence formulated in inventions of blackness and racial purity in The Slave Book. My fourth chapter introduces the concept of “first person autobiographical narrative voice” as a way to read the neo-slave narratives using the case of Kites of Good Fortune. The chapter shows that racial cultural identities remain a complex issue for descendants of manumitted slaves. In conclusion, I draw a connection between the representation of slavery in the novels and the post-apartheid present of their publication. I do this in order to suggest that slave histories have not been sufficiently engaged with in ways which function to minimise individual and collective trauma and as such they emerge as ‘ghosts that have refused to be laid to rest’.AFRIKAANSE OPSOMMING: In hierdie tesis word die herskepping van slaweherinneringe in gekose kontemporêre letterkundige tekste ontleed. Die tekste wat ek bestudeer sluit Rayda Jacobs se The Slave Book (1998), Therese Benadé se Kites of Good Fortune (2004), Yvette Christiansë se Unconfessed (2006) en André Brink se Philida (2012) in. Al vier romans is gepubliseer in die post-apartheid tydsgewrig, meer as ‘n eeu nadat die praktyk van slawerny aan die Kaap beëindig is. Deur hierdie werke te ontleed, ondersoek ek die voortdurende sosiale en psigiese gevolge van traumatise en onderdrukte slawegeskiedenisse deur die raamwerk van kulturele byblywing, asook gemeenskaplike herinnerings sowel as herhalende herinnering. My konseptualisering van byblywing sentreer om die idee van slawerny as ‘n ‘spook’ wat kwellend inwerk op die hede. Daarvolgens word geargumenteer dat die publisering van verhale aangaande die slaweverlede in die huidige moment ‘n kwellende byblywing aandui wat ingebed is in slawegeskiedenisse van onderdrukking wat deur kontemporêre skrywers teruggebring word na die oppervlak. As sulks word die konsep van kwellende byblywing ingebed in hierdie studie se hoof-onderwerpe: eerstens, die herbesoek aan slaweherinnerings in die post-apartheid oomblik, tweedens, die skrywers se behoefte om hul eie voorouers se werklike slaweverlede te herbesoek en derdens die premis dat sommige van die slawerny-erfenisse aanklank vind by die belange van ‘n huidige Suid-Afrika. Die hoofstukke bestudeer dus die voorstellings van slawerny wat in die narratiewe ingebou is deur twee vrae te vra, Hoe skakel hierdie verhale met die verbeelding van die ‘self’ in díe sin dat, deur oor hierdie slawe te skryf, die outeurs ook oor hul eie voorouers en geskiedenisse skryf? En hoe beeld hulle “die ander” uit wanneer hulle oor gestorwe en stilgemaakte slawe skryf? My eerste hoofstuk fokus op Unconfessed en bring die sielkundige trauma van slawerny na die voorgrond deur die bespreking van die onmoontlikheid van slawe-moederskap as voortspruitend uit inter-geslagtelike slawerny, asook die feit dat kinders van slawe-moeders by wyse van hul geboorte slaafstatus geërf het. In die tweede hoofstuk problematiseer my analise van Philida die voorstelling van slawe deur aan te dui hoedat wit rasbevoordeling tot uiting kom by wyse van skrywerskap en die optekening van geskiedenis, terwyl in my derde hoofstuk ek die verhaal van swart-op-swart geweldpleging en rasse-identiteitskategorieë in The Slave Book analiseer. My vierde hoofstuk gebruik Kites of Good Fortune om aan te toon dat rasse-identiteitskategorieë ‘n komplekse saak bly insoverre dit die afstsammelinge van slawe betref. Ter afsluiting dui ek ‘n verband aan tussen die voorstelling van slawe en slawerny in die vier romans en die post-apartheid hede van hul publisering. Ek doen dit ten einde voor te stel dat daar huidiglik nog onvoldoende aandag aan slawegeskiedenisse gegee is, hoewel groter aandag hieraan tot vermindering van individuele en kollektiewe trauma kan lei; dus verskyn hierdie geskiedenisse as ‘spoke wat weier om tot rus te kom'

Genetic and molecular characterization of HIV-associated kidney disease in black South Africans

A thesis submitted in fulfillment of the requirements for the degree of Doctor of Philosophy in Internal Medicine August, 2015Chronic kidney disease (CKD) due to HIV-associated nephropathy (HIVAN) remains an important cause of morbidity and mortality in African HIV-infected individuals. Genetic susceptibility to HIVAN has been attributed to genetic variations within MYH9 and APOL1 on chromosome 22. The expression of apolipoprotein L1 isoforms B and C coupled with high risk variants, play a crucial role in the development of kidney disease. The aims of this study were to determine the prevalence and examine the association of MYH9 and/or APOL1 high risk variants in HIVAN compared to other forms of CKD in HIV-infected individuals in a black South African population; to profile the pattern of messenger RNA N-terminal apolipoprotein L1 isoform expression in HIVAN, HIV-positive FSGS, other HIV-positive CKD, HIV-negative FSGS, other HIV-negative CKD, HIV-associated immune complex kidney disease (HIVICK) and in normal kidney; and to determine the expression of the N-terminal apolipoprotein L1 isoforms in human embryonic kidney (HEK) 293 cells. Kidney biopsies from Charlotte Maxeke Johannesburg Academic Hospital were obtained from adults (aged ≥18 years) and selected according to histological diagnoses, clinical data were collected and patients were stratified according to age, gender and clinical presentation. Single nucleotide polymorphisms (SNPs) within and flanking MYH9 and APOL1 genes were selected by identifying all SNPs documented in 2011 from 1000 base pairs upstream and downstream of the two genes, and with a minor allele frequency of ≥0.05 in African populations based on the International HapMap and 1000 Genomes Projects. SNPs with previous associations to kidney disease and literature SNPs were included, as was a panel of ancestry informative markers (AIMs). Genomic DNA was extracted from formalin fixed paraffin embedded (FFPE) kidney tissues and from whole blood. SNP genotyping was performed using the Illumina BeadXpress SNP genotyping assay and the TaqMan SNP genotyping assay. N-terminal apolipoprotein L1 mRNA expression was performed using Reverse Transcription-Polymerase Chain Reaction (RT-PCR) method. Expression of N-terminal apolipoprotein L1 isoforms in HEK 293 cells was performed using PCR-based cloning and transfection techniques. Data analysis was performed using R program and Stata v11.1. Logic regression, Fisher’s exact test, Kruskal Wallis test and Student’s t test were employed in the data analysis. A total of 228 samples [38 HIVAN, 41 HIV (+) CKD, 41 HIV (-) CKD, 54 HIV (+) Controls and 54 population controls] were successfully genotyped. Of 96 SNPs genotyped, 77 passed quality control. A total of nine MYH9 and APOL1 SNPs were significantly (P< 0.05) associated with HIVAN and not with other forms of CKD including focal segmental glomerulosclerosis (FSGS). However, the risk of MYH9 SNPs with P-values < 0.05 could not be determined. Single MYH9 E1 haplotype SNPs did not show any association, however, E1 haplotype block C-G-C showed a weaker significant factor with HIVAN (OR 3.45 P=0.008). The APOL1 risk alleles [G1 (0.560), G1 (0.500) and G2 (0.342)] had the highest prevalence among HIVAN individuals. G1GM haplotype was the most frequent in HIVAN (0.530). 19/38 (50.0%) of the HIVAN cases were compound heterozygous for G1 and G2 risk alleles; 11/38 were homozygous for either G1 or G2 risk alleles, 6/38 were heterozygous for a single risk allele and 2/38 were homozygous for non-risk allele. Thus, 30/38 (79.0%) of HIVAN but only 1/54 (2%) of population controls carried two risk alleles. Population allele frequencies were 7.3% for G1 and 11.1% for G2. In a recessive model, individuals carrying two APOL1 risk alleles had 89.0-fold higher odds 95% CI 17.68, 911.72; P=1.24x10-14 for developing HIVAN. APOL1 risk variants were not significantly associated with other forms of CKD [HIV (+) FSGS (OR=2.13 95% CI 0.03, 44.30; P=0.48) and HIVICK (OR=5.60 95% CI 0.4, 86; P=0.13) in the HIV (+) CKD group compared to HIV (+) controls] and [Primary FSGS (OR=6.30 95% CI 0.04, 248.70; P=0.26) in the HIV (-) CKD group compared to population controls]. Isoform B N-terminal apolipoprotein L1 mRNA levels were elevated in HIVAN and HIVICK while isoform C N-terminal apolipoprotein L1 levels were elevated in normal kidney. We showed that the three isoforms are not only expressed in HEK 293 cells but the expression is variable where isoforms B and C N-terminal apolipoprotein L1 that has a defective secretory domain and lack the secretory domain respectively, are seen to be more expressed within the HEK 293 cells than isoform A N-terminal apolipoprotein L1. A combination of apolipoprotein L1 isoform B expression with C-terminal risk variants is a major contributor to the development of HIVAN and possibly other kidney diseases. There is a striking increase in the prevalence of APOL1 risk variants among HIVAN compared to other forms of kidney disease and we estimate that HIV-infected South African blacks with two APOL1 risk alleles not receiving anti-retroviral therapy (ART) have the greatest risk of developing HIVAN. Further studies are required to elucidate the mechanism of apolipoprotein L1-mediated kidney disease in order to develop effective therapeutic measures