Journal articles on the topic 'Isogenic cellular models'
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1
Paredes-Redondo, A., and Y. Y. Lin. "Developing novel human isogenic cellular models for Duchenne muscular dystrophy." Neuromuscular Disorders 27 (March 2017): S6. http://dx.doi.org/10.1016/s0960-8966(17)30234-1.
2
Zhang, Yuting, Emily Wilt, and Xin Lu. "Human Isogenic Cell Line Models for Neutrophils and Myeloid-Derived Suppressor Cells." International Journal of Molecular Sciences 21, no. 20 (October 18, 2020): 7709. http://dx.doi.org/10.3390/ijms21207709.
Abstract:
Neutrophils with immunosuppressive activity are polymorphonuclear myeloid-derived suppressor cells (MDSCs) and may contribute to the resistance to cancer immunotherapy. A major gap for understanding and targeting these cells is the paucity of cell line models with cardinal features of human immunosuppressive neutrophils and their normal counterparts, especially in an isogenic manner. To address this issue, we employ the human promyelocytic cell line HL60 and use DMSO and cytokines (granulocyte macrophage-colony stimulating factor (GM-CSF) and interleukin 6 (IL6)) to induce the formation of either neutrophils or MDSCs. The induced MDSCs are CD11b+ CD33+ HLA-DR−/low and are heterogeneous for CD15 and CD14 expression. The induced MDSCs abrogate IL2 production and activation-induced cell death of the human T cell line Jurkat stimulated by CD3/CD28 antibodies, whereas the induced neutrophils enhance IL2 production from Jurkat cells. The induced MDSCs upregulate the expression of C/EBPβ, STAT3, VEGFR1, FATP2 and S100A8. Lastly, the immunosuppressive activity of the induced MDSCs is inhibited by all-trans retinoic acid and STAT3 inhibitor BP-1-102 through cellular differentiation and dedifferentiation mechanisms, respectively. Together, our study establishes a human isogenic cell line system for neutrophils and MDSCs and this system is expected to facilitate future studies on the biology and therapeutics of human immunosuppressive neutrophils.
3
Benarroch, Louise, Julia Madsen-Østerbye, Mohamed Abdelhalim, Kamel Mamchaoui, Jessica Ohana, Anne Bigot, Vincent Mouly, Gisèle Bonne, Anne T. Bertrand, and Philippe Collas. "Cellular and Genomic Features of Muscle Differentiation from Isogenic Fibroblasts and Myoblasts." Cells 12, no. 15 (August 3, 2023): 1995. http://dx.doi.org/10.3390/cells12151995.
Abstract:
The ability to recapitulate muscle differentiation in vitro enables the exploration of mechanisms underlying myogenesis and muscle diseases. However, obtaining myoblasts from patients with neuromuscular diseases or from healthy subjects poses ethical and procedural challenges that limit such investigations. An alternative consists in converting skin fibroblasts into myogenic cells by forcing the expression of the myogenic regulator MYOD. Here, we directly compared cellular phenotype, transcriptome, and nuclear lamina-associated domains (LADs) in myo-converted human fibroblasts and myotubes differentiated from myoblasts. We used isogenic cells from a 16-year-old donor, ruling out, for the first time to our knowledge, genetic factors as a source of variations between the two myogenic models. We show that myo-conversion of fibroblasts upregulates genes controlling myogenic pathways leading to multinucleated cells expressing muscle cell markers. However, myotubes are more advanced in myogenesis than myo-converted fibroblasts at the phenotypic and transcriptomic levels. While most LADs are shared between the two cell types, each also displays unique domains of lamin A/C interactions. Furthermore, myotube-specific LADs are more gene-rich and less heterochromatic than shared LADs or LADs unique to myo-converted fibroblasts, and they uniquely sequester developmental genes. Thus, myo-converted fibroblasts and myotubes retain cell type-specific features of radial and functional genome organization. Our results favor a view of myo-converted fibroblasts as a practical model to investigate the phenotypic and genomic properties of muscle cell differentiation in normal and pathological contexts, but also highlight current limitations in using fibroblasts as a source of myogenic cells.
4
Pavan, Eleonora, Maximiliano Ormazabal, Paolo Peruzzo, Emilio Vaena, Paula Rozenfeld, and Andrea Dardis. "CRISPR/Cas9 Editing for Gaucher Disease Modelling." International Journal of Molecular Sciences 21, no. 9 (May 5, 2020): 3268. http://dx.doi.org/10.3390/ijms21093268.
Abstract:
Gaucher disease (GD) is an autosomal recessive lysosomal storage disorder caused by mutations in the acid β-glucosidase gene (GBA1). Besides causing GD, GBA1 mutations constitute the main genetic risk factor for developing Parkinson’s disease. The molecular basis of neurological manifestations in GD remain elusive. However, neuroinflammation has been proposed as a key player in this process. We exploited CRISPR/Cas9 technology to edit GBA1 in the human monocytic THP-1 cell line to develop an isogenic GD model of monocytes and in glioblastoma U87 cell lines to generate an isogenic GD model of glial cells. Both edited (GBA1 mutant) cell lines presented low levels of mutant acid β-glucosidase expression, less than 1% of residual activity and massive accumulation of substrate. Moreover, U87 GBA1 mutant cells showed that the mutant enzyme was retained in the ER and subjected to proteasomal degradation, triggering unfolded protein response (UPR). U87 GBA1 mutant cells displayed an increased production of interleukin-1β, both with and without inflammosome activation, α-syn accumulation and a higher rate of cell death in comparison with wild-type cells. In conclusion, we developed reliable, isogenic, and easy-to-handle cellular models of GD obtained from commercially accessible cells to be employed in GD pathophysiology studies and high-throughput drug screenings.
5
Karwacka, Marianna, and Marta Olejniczak. "Advances in Modeling Polyglutamine Diseases Using Genome Editing Tools." Cells 11, no. 3 (February 2, 2022): 517. http://dx.doi.org/10.3390/cells11030517.
Abstract:
Polyglutamine (polyQ) diseases, including Huntington’s disease, are a group of late-onset progressive neurological disorders caused by CAG repeat expansions. Although recently, many studies have investigated the pathological features and development of polyQ diseases, many questions remain unanswered. The advancement of new gene-editing technologies, especially the CRISPR-Cas9 technique, has undeniable value for the generation of relevant polyQ models, which substantially support the research process. Here, we review how these tools have been used to correct disease-causing mutations or create isogenic cell lines with different numbers of CAG repeats. We characterize various cellular models such as HEK 293 cells, patient-derived fibroblasts, human embryonic stem cells (hESCs), induced pluripotent stem cells (iPSCs) and animal models generated with the use of genome-editing technology.
6
Klementieva, Natalia, Daria Goliusova, Julia Krupinova, Vladislav Yanvarev, Alexandra Panova, Natalia Mokrysheva, and Sergey L. Kiselev. "A Novel Isogenic Human Cell-Based System for MEN1 Syndrome Generated by CRISPR/Cas9 Genome Editing." International Journal of Molecular Sciences 22, no. 21 (November 8, 2021): 12054. http://dx.doi.org/10.3390/ijms222112054.
Abstract:
Multiple endocrine neoplasia type 1 (MEN1) is a rare tumor syndrome that manifests differently among various patients. Despite the mutations in the MEN1 gene that commonly predispose tumor development, there are no obvious phenotype–genotype correlations. The existing animal and in vitro models do not allow for studies of the molecular genetics of the disease in a human-specific context. We aimed to create a new human cell-based model, which would consider the variability in genetic or environmental factors that cause the complexity of MEN1 syndrome. Here, we generated patient-specific induced pluripotent stem cell lines carrying the mutation c.1252G>T, D418Y in the MEN1 gene. To reduce the genetically determined variability of the existing cellular models, we created an isogenic cell system by modifying the target allele through CRISPR/Cas9 editing with great specificity and efficiency. The high potential of these cell lines to differentiate into the endodermal lineage in defined conditions ensures the next steps in the development of more specialized cells that are commonly affected in MEN1 patients, such as parathyroid or pancreatic islet cells. We anticipate that this isogenic system will be broadly useful to comprehensively study MEN1 gene function across different contexts, including in vitro modeling of MEN1 syndrome.
7
Boussaad, Ibrahim, Emily K. Dolezal, Fabiana Perna, Stephen D. Nimer, and Eirini P. Papapetrou. "IPS Cells From Del(7q)-MDS Patients Display Impaired Proliferation and Hematopoietic Commitment." Blood 120, no. 21 (November 16, 2012): 174. http://dx.doi.org/10.1182/blood.v120.21.174.174.
Abstract:
Abstract Abstract 174 Myelodysplastic syndromes (MDS) are clonal hematologic disorders characterized by peripheral blood cytopenias and a dysplastic bone marrow (BM). Despite their relatively high incidence, these syndromes remain poorly understood and poorly studied, largely due to the unavailability of good animal models and the challenges of the ex vivo culture of primary MDS BM cells: their scarcity, poor proliferative potential and cellular heterogeneity. MDS BM cells exhibit poor growth and clonogenic capacity in culture, suggestive of a cell-intrinsic defect, but the cellular processes that are abnormal (e.g. proliferation, differentiation, cell death) remain elusive. We set to establish an in vitro system of pure clonal MDS hematopoiesis as a new platform to investigate the pathophysiology of MDS. We used reprogramming technology to derive induced pluripotent stem cells (iPSCs) from BM mononuclear cells of 3 MDS patients (RAEB by FAB) using our excisable polycistronic lentiviral vector (Papapetrou et al. Nat Biotech, 2009) or Sendai viruses. We derived 4 iPSC lines from a del(20q)-MDS patient (MDS-0), one line from a del(7q)-MDS patient (MDS-206), as well as 10 normal (wt-) iPSC lines derived in parallel in one reprogramming experiment from the same starting BM sample (MDS-206). We also derived 9 iPSC lines with chromosome 7 uniparental disomy (UPD) from a third patient (MDS-L1). Karyotyping and aCGH analyses confirmed that the MDS-iPSC lines harbored typical chromosomal deletions (20q12-q13.2 and 7q21.3-qter, respectively), identical to the starting cells. The wt- iPSCs had a normal karyotype and were confirmed to be isogenic to the del(7q) MDS-206.13 line by DNA fingerprinting. All wt- and MDS-iPSC lines display characteristic morphology and pluripotency marker expression. 6 selected lines were shown to fulfill all criteria of pluripotency, including teratoma formation. One del(7q)- and two del(20q)- iPSC lines so far studied show a 2- to 6- fold reduced proliferation rate (quantified by CFSE dilution and growth curves) compared to that of isogenic and non-isogenic wt-iPSCs, a phenotype much more pronounced in the del(7q) MDS-206.13 line, but absent from all 3 MDS-L1 UPD lines. Cell cycle analysis showed a relative accumulation in G0-G1 phase (40% in MDS-206.13 vs 23–25% in controls). Annexin V staining showed no differences in the percentage of apoptotic cells. Microarray analysis revealed 675 and 780 significantly differentially expressed genes in del(7q) MDS-206.13 and del(20q) MDS-0.12 iPSCs, respectively, compared to the wt MDS-206.12 line. In both cases, these were most enriched in the Gene Ontology categories of cellular growth and proliferation, cellular development and cell death. Ingenuity pathway analysis identified activation of p53 and FOS-JUN (AP1 transcription factor) among predominant potential regulators. Out of ∼1150 protein-coding genes residing in chromosome 7, 102 genes in 7q had reduced expression by at least 1.5-fold (23 of which by 2-fold) in the del(7q) iPSC line MDS-206.13 compared to its isogenic diploid line MDS-206.12. The hematopoietic potential of the MDS-206.13 line and its normal isogenic control MDS-206.12 was assessed in embryoid body differentiation culture with cytokine supplementation. Strikingly, after mesoderm specification for 3 days followed by 10 days of hematopoietic differentiation, less than 1% of MDS-206.13 vs 48% of MDS-206.12 cells became committed to the hematopoietic lineage (CD34+/CD45+co-expression). Consistent with this, hematopoietic colony formation in methylcellulose and further differentiation in erythroid culture of del(7q)-iPSCs was altogether absent, in contrast to the robust clonogenic and erythroid differentiation potential of the isogenic control line. Our data suggest that impaired cell proliferation may be integral to the pathophysiology of del(7q)-MDS. Since this phenotype is predominant in del(7q)-iPSCs, but absent from UPD7-iPSCs, it may be caused by reduced dosage of one or more genes on chromosome 7 (haploinsufficiency). Further studies with additional iPSC lines patient-derived and genetically engineered to harbor artificial 7/7q deletions are underway. In summary, we have developed a novel MDS model of patient-derived and isogenic normal iPSCs. This model should prove useful to study the cellular, molecular and genetic pathogenesis of MDS, identify critical genes and test therapeutic compounds. Disclosures: No relevant conflicts of interest to declare.
8
Muto, Valentina, Federica Benigni, Valentina Magliocca, Rossella Borghi, Elisabetta Flex, Valentina Pallottini, Alessandro Rosa, Claudia Compagnucci, and Marco Tartaglia. "CRISPR/Cas9 and piggyBac Transposon-Based Conversion of a Pathogenic Biallelic TBCD Variant in a Patient-Derived iPSC Line Allows Correction of PEBAT-Related Endophenotypes." International Journal of Molecular Sciences 24, no. 9 (April 28, 2023): 7988. http://dx.doi.org/10.3390/ijms24097988.
Abstract:
Induced pluripotent stem cells (iPSCs) have been established as a reliable in vitro disease model system and represent a particularly informative tool when animal models are not available or do not recapitulate the human pathophenotype. The recognized limit in using this technology is linked to some degree of variability in the behavior of the individual patient-derived clones. The development of CRISPR/Cas9-based gene editing solves this drawback by obtaining isogenic iPSCs in which the genetic lesion is corrected, allowing a straightforward comparison with the parental patient-derived iPSC lines. Here, we report the generation of a footprint-free isogenic cell line of patient-derived TBCD-mutated iPSCs edited using the CRISPR/Cas9 and piggyBac technologies. The corrected iPSC line had no genetic footprint after the removal of the selection cassette and maintained its “stemness”. The correction of the disease-causing TBCD missense substitution restored proper protein levels of the chaperone and mitotic spindle organization, as well as reduced cellular death, which were used as read-outs of the TBCD KO-related endophenotype. The generated line represents an informative in vitro model to understand the impact of pathogenic TBCD mutations on nervous system development and physiology.
9
Li, Fenfang, Igor Cima, Jess Honganh Vo, Min-Han Tan, and Claus Dieter Ohl. "Single Cell Hydrodynamic Stretching and Microsieve Filtration Reveal Genetic, Phenotypic and Treatment-Related Links to Cellular Deformability." Micromachines 11, no. 5 (May 9, 2020): 486. http://dx.doi.org/10.3390/mi11050486.
Abstract:
Deformability is shown to correlate with the invasiveness and metastasis of cancer cells. Recent studies suggest epithelial-to-mesenchymal transition (EMT) might enable cancer metastasis. However, the correlation of EMT with cancer cell deformability has not been well elucidated. Cellular deformability could also help evaluate the drug response of cancer cells. Here, we combine hydrodynamic stretching and microsieve filtration to study cellular deformability in several cellular models. Hydrodynamic stretching uses extensional flow to rapidly quantify cellular deformability and size with high throughput at the single cell level. Microsieve filtration can rapidly estimate relative deformability in cellular populations. We show that colorectal cancer cell line RKO with the mesenchymal-like feature is more flexible than the epithelial-like HCT116. In another model, the breast epithelial cells MCF10A with deletion of the TP53 gene are also significantly more deformable compared to their isogenic wildtype counterpart, indicating a potential genetic link to cellular deformability. We also find that the drug docetaxel leads to an increase in the size of A549 lung cancer cells. The ability to associate mechanical properties of cancer cells with their phenotypes and genetics using single cell hydrodynamic stretching or the microsieve may help to deepen our understanding of the basic properties of cancer progression.
10
Patel, Ronak, Shyanne Page, and Abraham Jacob Al-Ahmad. "Isogenic blood-brain barrier models based on patient-derived stem cells display inter-individual differences in cell maturation and functionality." Journal of Neurochemistry 142, no. 1 (May 14, 2017): 74–88. http://dx.doi.org/10.1111/jnc.14040.
11
Boussaad, Ibrahim, Andriana Kotini, Emily K. Dolezal, Stephen Nimer, and Eirini P. Papapetrou. "An iPSC-Based Model Of MDS For Phenotype-Driven Gene and Drug Discovery." Blood 122, no. 21 (November 15, 2013): 859. http://dx.doi.org/10.1182/blood.v122.21.859.859.
Abstract:
Abstract Despite past and ongoing research efforts, the pathogenetic mechanisms of MDS remain far from understood sufficiently to point to single genes or molecular pathways as putative targets for generation of better mouse models or drug development. In lack of clear targets, the manipulation of disease-associated phenotypes may at present be the best opportunity for disease study and therapeutic intervention. Furthermore, because cellular phenotypes are more proximal to molecular disease mechanisms than phenotypes seen at the level of the tissue or organism (i.e. the murine or human hematopoietic system), they may provide more sensitive and relevant readouts of the disease process. With the advent of patient-specific induced pluripotent stem cells (iPSCs), disease models based on cellular phenotypes (“disease-in-a-dish”) can now be developed and their unique properties promise to revolutionize disease study and drug development. Unlimited cell numbers of biologically relevant cells can be obtained relatively easily and cost-effectively. Here we present a new MDS model based on patient-specific iPSCs and its use in two phenotype-driven screens: (a) a focused genetic screen and (b) a high-throughput chemical screen. We have derived multiple MDS-iPSC lines with deletions of chromosomes 7q or 20q (characteristic chromosomal deletions that we are using as markers of iPSCs derived from the MDS clone) from bone marrow (BM) of 3 patients with MDS and 1 patient with sAML. We also derived isogenic karyotypically normal iPSC lines in parallel from the same BM samples. We identified two cellular phenotypes specific to the MDS-iPSCs: decreased proliferation rate and decreased potential for pan-hematopoietic differentiation and reduced clonogenic capacity of their hematopoietic progeny. These phenotypes are consistent across multiple iPSC lines from different patients and absent from their isogenic normal iPSC controls. They are reminiscent of the behavior of ex vivo cultured primary MDS BM cells and are therefore likely to be relevant to the disease process. Furthermore, they are rescued by spontaneous compensation for chr7q dosage through acquisition of an extra chr7 and recapitulated by the engineering of artificial chr7q deletions in normal iPSCs. To identify critical MDS gene(s) on chr7q, we performed a screen of 62 candidate haploinsufficient genes (with reduced expression by at least 1.5-fold in our del(7q)-iPSCs compared to their isogenic normal iPSCs) for rescue of the proliferation and/or hematopoietic differentiation phenotype. We constructed a lentiviral library of all candidate ORFs (and 14 additional alternative transcripts) linked to eGFP through a P2A peptide and each tagged with a unique 4-nt barcode sequence to its 3’ UTR. The library was packaged as a pool and transduced into two different del(7q)-iPSC lines. The cells were passaged for 16 weeks and gDNA was isolated every 2 weeks. In parallel, the cells were differentiated along the hematopoietic lineage and gDNA was isolated from CD45+ cells FACS-sorted on day 15 of differentiation. High-throughput sequencing of the barcodes identified 5 ORFs that became enriched in undifferentiated iPSCs over time (rescue of proliferation) and 4 ORFs enriched in sorted CD45+hematopoietic progenitors (rescue of hematopoietic differentiation), 2 of which overlapped. All 9 hits are being further validated in more focused screens. Second, we used the same platform for a high-throughput small molecule screen. We optimized the plating conditions and densities for a 384-well format using a luminescent cell viability assay and conducted a screen of 2000 compounds comprising known drugs, natural products, and other bioactives and chemicals, in an MDS-iPSC line (2.13) and its isogenic normal control (2.8). Primary hits were defined as compounds that enhanced the growth of the MDS-iPSC line, but not of the control normal iPSC line in a compound dose-dependent manner. 38 primary hits were retested in a dose-response survival assay in one additional MDS- (2.A1-3), one additional isogenic normal iPSC line (2.12) and one sAML iPSC line over 8 concentrations and 12 compounds were prioritized for further studies. Our data highlight the potential of this new iPSC-based model of MDS for screens to identify genes or compounds that affect cellular phenotypes by acting on previously undefined targets. Disclosures: No relevant conflicts of interest to declare.
12
Haley, John A., Christian F. Ruiz, Emily D. Montal, Daifeng Wang, John D. Haley, and Geoffrey D. Girnun. "Decoupling of Nrf2 Expression Promotes Mesenchymal State Maintenance in Non-Small Cell Lung Cancer." Cancers 11, no. 10 (October 2, 2019): 1488. http://dx.doi.org/10.3390/cancers11101488.
Abstract:
Epithelial mesenchymal transition is a common mechanism leading to metastatic dissemination and cancer progression. In an effort to better understand this process we found an intersection of Nrf2/NLE2F2 (Nrf2), epithelial mesenchymal transition (EMT), and metabolic alterations using multiple in vitro and in vivo approaches. Nrf2 is a key transcription factor controlling the expression of redox regulators to establish cellular redox homeostasis. Nrf2 has been shown to exert both cancer inhibitory and stimulatory activities. Using multiple isogenic non-small cell lung cancer (NSCLC) cell lines, we observed a reduction of Nrf2 protein and activity in a prometastatic mesenchymal cell state and increased reactive oxygen species. Knockdown of Nrf2 promoted a mesenchymal phenotype and reduced glycolytic, TCA cycle and lipogenic output from both glucose and glutamine in the isogenic cell models; while overexpression of Nrf2 promoted a more epithelial phenotype and metabolic reactivation. In both Nrf2 knockout mice and in NSCLC patient samples, Nrf2low was co-correlated with markedly decreased expression of glycolytic, lipogenic, and mesenchymal RNAs. Conversely, Nrf2high was associated with partial mesenchymal epithelial transition and increased expression of metabolic RNAs. The impact of Nrf2 on epithelial and mesenchymal cancer cell states and metabolic output provide an additional context to Nrf2 function in cancer initiation and progression, with implications for therapeutic inhibition of Nrf2 in cancer treatment.
13
Ghosal, Abhisek, Stefan Jellbauer, Rubina Kapadia, Manuela Raffatellu, and Hamid M. Said. "Salmonellainfection inhibits intestinal biotin transport: cellular and molecular mechanisms." American Journal of Physiology-Gastrointestinal and Liver Physiology 309, no. 2 (July 15, 2015): G123—G131. http://dx.doi.org/10.1152/ajpgi.00112.2015.
Abstract:
Infection with the nontyphoidal Salmonella is a common cause of food-borne disease that leads to acute gastroenteritis/diarrhea. Severe/prolonged cases of Salmonella infection could also impact host nutritional status, but little is known about its effect on intestinal absorption of vitamins, including biotin. We examined the effect of Salmonella enterica serovar Typhimurium ( S. typhimurium) infection on intestinal biotin uptake using in vivo (streptomycin-pretreated mice) and in vitro [mouse (YAMC) and human (NCM460) colonic epithelial cells, and human intestinal epithelial Caco-2 cells] models. The results showed that infecting mice with wild-type S. typhimurium, but not with its nonpathogenic isogenic invA spiB mutant, leads to a significant inhibition in jejunal/colonic biotin uptake and in level of expression of the biotin transporter, sodium-dependent multivitamin transporter. In contrast, infecting YAMC, NCM460, and Caco-2 cells with S. typhimurium did not affect biotin uptake. These findings suggest that the effect of S. typhimurium infection is indirect and is likely mediated by proinflammatory cytokines, the levels of which were markedly induced in the intestine of S. typhimurium-infected mice. Consistent with this hypothesis, exposure of NCM460 cells to the proinflammatory cytokines TNF-α and IFN-γ led to a significant inhibition of biotin uptake, sodium-dependent multivitamin transporter expression, and activity of the SLC5A6 promoter. The latter effects appear to be mediated, at least in part, via the NF-κB signaling pathway. These results demonstrate that S. typhimurium infection inhibits intestinal biotin uptake, and that the inhibition is mediated via the action of proinflammatory cytokines.
14
Ceballos-Garzon, Andres, Elvira Roman, Jesús Pla, Fabrice Pagniez, Daniela Amado, Carlos J. Alméciga-Díaz, Patrice Le Pape, and Claudia M. Parra-Giraldo. "CRISPR-Cas9 approach confirms Calcineurin-responsive zinc finger 1 (Crz1) transcription factor as a promising therapeutic target in echinocandin-resistant Candida glabrata." PLOS ONE 17, no. 3 (March 18, 2022): e0265777. http://dx.doi.org/10.1371/journal.pone.0265777.
Abstract:
Invasive fungal infections, which kill more than 1.6 million patients each year worldwide, are difficult to treat due to the limited number of antifungal drugs (azoles, echinocandins, and polyenes) and the emergence of antifungal resistance. The transcription factor Crz1, a key regulator of cellular stress responses and virulence, is an attractive therapeutic target because this protein is absent in human cells. Here, we used a CRISPR-Cas9 approach to generate isogenic crz1Δ strains in two clinical isolates of caspofungin-resistant C. glabrata to analyze the role of this transcription factor in susceptibility to echinocandins, stress tolerance, biofilm formation, and pathogenicity in both non-vertebrate (Galleria mellonella) and vertebrate (mice) models of candidiasis. In these clinical isolates, CRZ1 disruption restores the susceptibility to echinocandins in both in vitro and in vivo models, and affects their oxidative stress response, biofilm formation, cell size, and pathogenicity. These results strongly suggest that Crz1 inhibitors may play an important role in the development of novel therapeutic agents against fungal infections considering the emergence of antifungal resistance and the low number of available antifungal drugs.
15
Kálmán, Sára, Edit Hathy, and János M. Réthelyi. "A Dishful of a Troubled Mind: Induced Pluripotent Stem Cells in Psychiatric Research." Stem Cells International 2016 (2016): 1–21. http://dx.doi.org/10.1155/2016/7909176.
Abstract:
Neuronal differentiation of induced pluripotent stem cells and direct reprogramming represent powerful methods for modeling the development of neuronsin vitro. Moreover, this approach is also a means for comparing various cellular phenotypes between cell lines originating from healthy and diseased individuals or isogenic cell lines engineered to differ at only one or a few genomic loci. Despite methodological constraints and initial skepticism regarding this approach, the field is expanding at a fast pace. The improvements include the development of new differentiation protocols resulting in selected neuronal populations (e.g., dopaminergic, GABAergic, hippocampal, and cortical), the widespread use of genome editing methods, and single-cell techniques. A major challenge awaitingin vitrodisease modeling is the integration of clinical data in the models, by selection of well characterized clinical populations. Ideally, these models will also demonstrate how different diagnostic categories share overlapping molecular disease mechanisms, but also have unique characteristics. In this review we evaluate studies with regard to the described developments, to demonstrate how differentiation of induced pluripotent stem cells and direct reprogramming can contribute to psychiatry.
16
Wang, Wei, Tiansu Wang, Andriana G. Kotini, Camelia Iancu-Rubin, Ronald Hoffman, and Eirini P. Papapetrou. "Modeling Calreticulin-Mutant Myeloproliferative Neoplasms with Isogenic Induced Pluripotent Stem Cells." Blood 132, Supplement 1 (November 29, 2018): 4319. http://dx.doi.org/10.1182/blood-2018-99-111512.
Abstract:
Abstract Myeloproliferative neoplasms (MPN) are characterized by the excessive production of one or more myeloid lineages and a propensity to progress to acute leukemia. In 2013, mutations in the CALR gene, encoding calreticulin, were identified in patients with MPN, mutually exclusive to the previously identified JAK2 and MPL (TPO-R) mutations. CALR mutations are frameshift mutations - typically a 52-bp deletion (type 1) or a 5-bp insertion (type 2) - that result in a novel C-terminus. The discovery of mutations in a ubiquitously expressed multifunctional protein like calreticulin was unanticipated. Subsequent studies found that CALR mutations lead to activation of JAK/STAT, mediated through aberrant interactions between mutant CALR and MPL, thus presenting an excellent opportunity for targeted therapy. However, the mechanism of MPL activation remains largely unexplained with prior studies using cell lines with exogenous expression of CALR and MPL following transfection. To create a more physiological cellular model to study the effects of CALR mutations, we established multiple iPSC lines from two patients with CALR-mutant MPN - one type 1-like (del34) and one type 2 (ins5) -, as well as from one patient with JAK2V617F MPN. All iPSC lines were confirmed to harbour the CALR or JAK2V617F mutation found in the corresponding patient, to express mutant calreticulin, as detected by flow cytometry using an antibody which specifically recognizes the novel calreticulin C-terminus, and to be karyotypically normal. Genetically matched iPSC lines with WT JAK2 could also be generated from the JAK2V617F (but not the CALR-mutant) patient cells in the same reprogramming round. CRISPR gene editing was used to generate isogenic CALR-corrected lines from both CALR-mutant patients. Furthermore, in order to facilitate biochemical studies, we used CRISPR to introduce a V5 epitope tag in one allele of the endogenous mutant or WT CALR gene, in mutant and isogenic corrected iPSC lines, respectively. We optimized an in vitro differentiation protocol for efficient derivation of megakaryocyte (MK) progenitors from iPSCs and found disease-relevant phenotypes, mainly TPO-independent MK colony formation in semi-solid media, which is the phenotypic hallmark of ex vivo primary MPN cells. In the absence of TPO, JAK2 V617F, CALR-mutant type 1-like and CALR-mutant type 2 iPSCs generated 52.1%, 58.7±22.2% and 59.8±3.6%, respectively, of the number of MK colonies generated in the presence of TPO, as opposed to 10%, 8.8±1.8% and 0.5±0.9%, respectively, for the matched WT JAK2, the corrected CALR-mutant type 1-like and the corrected CALR-mutant type 2 iPSCs. Isolated CALR mutant iPSC-derived CD41a+ MK progenitors had increased phosphorylation of STAT5 following cytokine starvation as compared to isogenic corrected and non-isogenic normal cells. CALR-mutant cells expressed equal transcript levels of the WT and mutant CALR alleles. However, mutant CALR protein levels were severely reduced, at levels 1~12% of those of the WT protein. This is consistent with previous studies documenting instability of mutant calreticulin. Transcriptomics (RNA-seq) and proteomics analyses of CD41a+-sorted MK progenitors derived from CALR mutant and isogenic corrected iPSCs are ongoing. These iPSC models offer the opportunity to study the effects of CALR mutations in a cellular context with both MPL and CALR (WT or mutant) expressed from their endogenous loci. They thus provide a powerful platform to investigate the disease mechanisms underlying CALR-mutant MPNs and to perform small molecule and genetic (CRISPR) screens to identify new therapeutic targets. Disclosures Iancu-Rubin: Merck: Research Funding; Incyte: Research Funding; Summer Road, LLC: Research Funding; Formation Biologics: Research Funding. Hoffman:Incyte: Research Funding; Merus: Research Funding; Formation Biologics: Research Funding; Janssen: Research Funding; Summer Road: Research Funding.
17
Хамидуллина, А. И., Э. Р. Гандалипов, Я. Е. Абраменко, К. В. Чернов, Т. А. Кирюхина, А. В. Брутер, and В. В. Татарский. "Creation of A549 and MCF7 tumor sublines with knockout of TP53 using CRISPR/Cas9." Nauchno-prakticheskii zhurnal «Medicinskaia genetika 22, no. 11 (December 19, 2023): 27–34. http://dx.doi.org/10.25557/2073-7998.2023.11.27-34.
Abstract:
Опухолевой супрессор p53 является центральным звеном защиты клетки от злокачественной трансформации. Мутации кодирующего гена TP53 наблюдаются приблизительно в половине опухолей человека и способствуют не только опухолевой прогрессии, но также устойчивости или чувствительности к противоопухолевым препаратам. Получение изогенных линий, имеющих разный статус TP53, необходимо не только для исследования его роли, но и для скрининга новых противоопухолевых препаратов и их комбинаций. Получение изогенных моделей с помощью системы CRISPR/Cas9 часто связано с одноклеточным клонированием, что приводит к клональным эффектам из-за гетерогенности опухолевых культур. В данной работе описано получение новых нокаутов TP53 в линиях MCF7 и A549 с помощью системы CRISPR/Cas9 и отбора по устойчивости к нутлину-3, позволяющей отбирать нокаутные клетки без этапа клонального отбора. Фенотипически нокаут был подтвержден по полному отсутствию белка p53, снижению экспрессии p53-зависимого гена CDKN1A и изменению чувствительности к ДНК-повреждающим противоопухолевым препаратам. The tumor suppressor p53 is the central point of cellular defense against oncogenic transformation. Mutations of TP53 gene are present in approximately half of human tumors and promote not only tumor progression, but also resistance to anticancer drugs. Creation of isogenic models, differing in their TP53 status, is valuable not only for studying its role in carcinogenesis, but also for screening of anticancer drugs and their combinations. Establishment of isogenic models using the CRISPR/Cas9 system is usually done through single cell cloning, which can lead to clonal effects, because of heterogeneity of the cell cultures. In this article we present the process of creation of new knockout cell sublines of MCF7 and A549 using CRISPR/Cas9 and selection using nutlin-3, which allows selecting cell sublines without clonal selection. Phenotypically the knockout of TP53 was confirmed by total absence of p53, absence of induction of p53-dependent gene CDKN1A, and shift in sensitivity to DNA-damaging drugs.
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Charlebois, Daniel A., Kevin Hauser, Sylvia Marshall, and Gábor Balázsi. "Multiscale effects of heating and cooling on genes and gene networks." Proceedings of the National Academy of Sciences 115, no. 45 (October 19, 2018): E10797—E10806. http://dx.doi.org/10.1073/pnas.1810858115.
Abstract:
Most organisms must cope with temperature changes. This involves genes and gene networks both as subjects and agents of cellular protection, creating difficulties in understanding. Here, we study how heating and cooling affect expression of single genes and synthetic gene circuits inSaccharomyces cerevisiae. We discovered that nonoptimal temperatures induce a cell fate choice between stress resistance and growth arrest. This creates dramatic gene expression bimodality in isogenic cell populations, as arrest abolishes gene expression. Multiscale models incorporating population dynamics, temperature-dependent growth rates, and Arrhenius scaling of reaction rates captured the effects of cooling, but not those of heating in resistant cells. Molecular-dynamics simulations revealed how heating alters the conformational dynamics of the TetR repressor, fully explaining the experimental observations. Overall, nonoptimal temperatures induce a cell fate decision and corrupt gene and gene network function in computationally predictable ways, which may aid future applications of engineered microbes in nonstandard temperatures.
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Lee, Hans C., Hua Wang, Bing-Zong Li, Zhiqiang Wang, Richard Julian Jones, Dongmin Gu, Sean O'Brien, Richard E. Davis, and Robert Z. Orlowski. "CX-5461, a Novel RNA Polymerase I Inhibitor, Is Active Against Wild-Type and Mutant p53 Myeloma Models." Blood 122, no. 21 (November 15, 2013): 4438. http://dx.doi.org/10.1182/blood.v122.21.4438.4438.
Abstract:
Background Ribosomal RNA (rRNA) forms a major component of ribosomes, which play a critical role in cellular growth and proliferation through regulation of protein synthesis. Increased rRNA transcription and ribosomal biogenesis have been associated with tumorigenesis, and recent reports have suggested that inhibition of rRNA transcription by CX-5461 (Senhwa Biosciences), a novel selective small-molecule inhibitor of RNA polymerase (pol) I, induces cell death in several human tumor types by both p53-dependent and independent mechanisms. These findings led to our hypothesis that selective RNA pol I inhibition with CX-5461 could be a rational new approach to therapy for both wild-type (wt) and mutant p53 multiple myeloma models. Methods Studies with CX-5461 were performed in wt p53 and mutant p53 cell lines, zinc-finger nuclease (ZFN) p53 knock-out (KO) isogenic myeloma cell lines, and bortezomib and carfilzomib-resistant myeloma cell lines. Results Treatment of wt p53 (MM1.S, MOLP8) and mutant p53 (U266, RPMI-8226) myeloma cell lines demonstrated a time and dose dependent decrease in cell proliferation after exposure to CX-5461 with a median inhibitory concentration (IC50) range of 50-100 nM after a 72-hour incubation. A corresponding increase in cleaved PARP, cleaved caspase-9, and cleaved caspase-3 expression was seen on Western blot, as well as increased Annexin V staining on flow cytometry analysis. Notably, the degree of Annexin V staining was less in the p53 mutant cell lines compared to the wt p53 cells at any given drug concentration, but strong apoptotic signaling could be induced in mutant p53 cell lines when using higher concentrations of CX-5461. In addition, co-culturing myeloma cells with GFP+ HS5 stromal cells to mimic the bone marrow microenvironment did decrease the therapeutic effect of CX-5461, but again could be overcome with higher drug concentrations [250-500 nM]. Similar results were seen when isogenic MM1.S ZFN p53 KO cells were used, whose sensitivity to CX-5461 was comparable to that of wt p53 cells. Finally, CX-5461 was also tested on drug-resistant myeloma cell lines that were generated by exposing cells to low concentrations of bortezomib (RMPI-8266, KAS-6/1, ANBL-6) or carfilzomib (KAS-6/1) over time. These drug-resistant cell lines showed sensitivity to CX-5461 with an IC50 in the 100-250 nM concentration range. Gene expression profiling (GEP) of isogenic MM1.S ZFN p53 KO and wt cells revealed that gene expression perturbations by CX-5461 were primarily p53-independent. Additional GEP and pathway analysis in other isogenic ZFN p53 wt and KO cell lines is currently ongoing, with a particular interest in p53-independent mechanisms that may explain the efficacy of CX-5461 in both wt and mutant p53 myeloma models. Conclusion RNA pol I inhibition by CX-5461 is a promising approach to myeloma therapy, with low nanomolar drug activity seen in wt p53, mutant p53, and drug-resistant myeloma cell line models, providing a rationale for translation of CX-5461 into the clinic for the treatment of multiple myeloma. Disclosures: O'Brien: Senhwa Biosciences, Inc: Employment. Orlowski:Bristol-Myers Squibb: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Research Funding; Celgene: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Research Funding; Millennium: The Takeda Oncology Company: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Research Funding; Onyx: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Research Funding; Resverlogix: Research Funding; Array Biopharma: Honoraria, Membership on an entity’s Board of Directors or advisory committees; Genentech: Honoraria, Membership on an entity’s Board of Directors or advisory committees; Merck: Membership on an entity’s Board of Directors or advisory committees.
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van Waardenburg, Robert C. A. M., Laurina A. de Jong, Foke van Delft, Maria A. J. van Eijndhoven, Melanie Bohlander, Mary-Ann Bjornsti, Jaap Brouwer, and Jan H. M. Schellens. "Homologous recombination is a highly conserved determinant of the synergistic cytotoxicity between cisplatin and DNA topoisomerase I poisons." Molecular Cancer Therapeutics 3, no. 4 (April 1, 2004): 393–402. http://dx.doi.org/10.1158/1535-7163.393.3.4.
Abstract:
Abstract Phase I and II clinical trails are currently investigating the antitumor activity of cisplatin and camptothecins (CPTs; DNA topoisomerase I poisons), based on the dramatic synergistic cytotoxicity of these agents in some preclinical models. However, the mechanistic basis for this synergism is poorly understood. By exploiting the evolutionary conservation of DNA repair pathways from genetically tractable organisms such as budding and fission yeasts to mammalian cells, we demonstrate that the synergism of CPT and cisplatin requires homologous recombination. In yeast and mammalian cell lines defective for RAD52 and XRCC2/3, respectively, the combination of these agents proved antagonistic, while greater than additive activity was evident in isogenic wild-type cells. Homologous recombination appears to mediate a similar interaction of X-rays and CPT, but antagonizes the synergism of cytarabine (Ara-C) with CPT. These findings suggest that homologous recombination comprises an evolutionarily conserved determinant of cellular sensitivity when CPTs are used in combination with other therapeutics.
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Rakotomalala, Andria, Paul Lewandowski, Quentin Bailleul, Clara Savary, Mélanie Arcicasa, Christine Bal, Maud Hamadou, et al. "Abstract 1671: Engineering new cellular models to decipher H3.3K27M mutation role in DIPGs' resistance to therapies." Cancer Research 82, no. 12_Supplement (June 15, 2022): 1671. http://dx.doi.org/10.1158/1538-7445.am2022-1671.
Abstract:
Abstract Among pediatric brain tumors, Diffuse Intrinsic Pontine Gliomas (DIPGs) display a particularly dismal prognosis, highlighted by their median survival lower than one year. Indeed, DIPGs’ location and infiltrative properties preclude their surgical resection. Moreover, DIPGs poorly respond to chemotherapeutic agents. In this context, the only treatment for these tumors remains palliative radiotherapy, systematically followed by tumor progression. In addition to their resistance to therapies, DIPGs are characterized by recurrent histone H3 mutations. The H3.3K27M mutation is the most frequent and results from a heterozygous single nucleotide variant in the H3F3A gene, inducing the lysine 27 substitution by a methionine. Although H3.3K27M’s driver role in DIPGs tumorigenesis is now established, its role in their chemo- and radioresistance remains unclear. Aiming to decipher the potential role of this mutation in pediatric gliomas’ resistance to therapies, we established isogenic cellular models of H3.3K27M induction and reversion.We first induced H3.3K27M mutation in three initially non-mutated supratentorial pediatric glioma cell lines. Thus, we generated models that stably expressed the dominant-negative H3.3K27M or the wild type H3.3 as controls. Complementarily, to study H3.3K27M roles in a DIPG cell context, we also developed H3.3K27M reversion models in two initially mutated DIPG cell lines by applying a gene-editing strategy based on the combinatorial use of the CRISPR/Cas9 technology and an insert.We showed that H3.3K27M induction in Res259 and KNS42 cells conferred a radioresistant phenotype to a fractionated radiotherapy schedule. Besides, we performed a screening of 80 anti-cancer drugs, which revealed a differential impact of the mutation on the drug sensitivity profiles of our three H3.3K27M-induced cell lines. These results indicate that H3.3K27M can control pediatric glioma cells’ resistance to therapies, but in a heterogeneous way depending on the cellular context. Along this line, we are currently characterizing the chemo- and radiotherapy response of our new DIPG H3.3K27M-reversed models. Altogether, our first results support a role for H3.3K27M in pediatric gliomas resistance to treatments, and our complementary models pave the way for identifying new H3.3K27M-dependent mechanisms and promising targets to sensitize DIPGs to therapies. Citation Format: Andria Rakotomalala, Paul Lewandowski, Quentin Bailleul, Clara Savary, Mélanie Arcicasa, Christine Bal, Maud Hamadou, Paul Huchedé, Audrey Restouin, Remy Castellano, Yves Collette, Audrey Vincent, Pierre-Olivier Angrand, Eric Adriaenssens, Xuefen Le Bourhis, Pierre Leblond, Marie Castets, Eddy Pasquier, Alessandro Furlan, Samuel Meignan. Engineering new cellular models to decipher H3.3K27M mutation role in DIPGs' resistance to therapies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1671.
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De Souza, Cristabelle, Jill A. Madden, Dennis Minn, Vigneshwari Easwar Kumar, Dennis J. Montoya, Roshni Nambiar, Zheng Zhu, et al. "The P72R Polymorphism in R248Q/W p53 Mutants Modifies the Mutant Effect on Epithelial to Mesenchymal Transition Phenotype and Cell Invasion via CXCL1 Expression." International Journal of Molecular Sciences 21, no. 21 (October 28, 2020): 8025. http://dx.doi.org/10.3390/ijms21218025.
Abstract:
High-grade serous carcinoma (HGSC), the most lethal subtype of epithelial ovarian cancer (EOC), is characterized by widespread TP53 mutations (>90%), most of which are missense mutations (>70%). The objective of this study was to investigate differential transcriptional targets affected by a common germline P72R SNP (rs1042522) in two p53 hotspot mutants, R248Q and R248W, and identify the mechanism through which the P72R SNP affects the neomorphic properties of these mutants. Using isogenic cell line models, transcriptomic analysis, xenografts, and patient data, we found that the P72R SNP modifies the effect of p53 hotspot mutants on cellular morphology and invasion properties. Most importantly, RNA sequencing studies identified CXCL1 a critical factor that is differentially affected by P72R SNP in R248Q and R248W mutants and is responsible for differences in cellular morphology and functional properties observed in these p53 mutants. We show that the mutants with the P72 SNP promote a reversion of the EMT phenotype to epithelial characteristics, whereas its R72 counterpart promotes a mesenchymal transition via the chemokine CXCL1. These studies reveal a new role of the P72R SNP in modulating the neomorphic properties of p53 mutants via CXCL1, which has significant implications for tumor invasion and metastasis.
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Jungles, Kassidy M., Andrea M. Pesch, Nicole Hirsh, Anna R. Michmerhuizen, Kari Wilder-Romans, Benjamin C. Chandler, Meilan Liu, et al. "Abstract 216: Expression of DNA damage response proteins modifies the efficacy of CDK4/6 inhibitor-mediated radiosensitization in breast cancer models." Cancer Research 82, no. 12_Supplement (June 15, 2022): 216. http://dx.doi.org/10.1158/1538-7445.am2022-216.
Abstract:
Abstract Purpose: CDK4/6 inhibitors (CDK4/6i) are standard of care for the treatment of locally advanced and metastatic estrogen receptor-positive (ER+), HER2-negative metastatic breast cancer (BC). CDK4/6 inhibition + radiation therapy (RT) is synergistic in both ER+ and triple negative breast cancers (TNBC), but the underlying mechanism is not entirely understood. In this study, we evaluated how pre-existing or genetically engineered deficits in DNA damage response genes (BRCA1/2, RAD51, RB1, XRCC6, TP53) influence radiosensitization. We hypothesized that inhibition of homologous recombination (HR) would prevent CDK4/6i-mediated radiosensitization and blocking non-homologous end joining (NHEJ) would be synergistic. Methods: Cellular proliferation assays determined the half-maximal inhibitory concentrations (IC50) of the 3 approved CDK4/6i palbociclib, ribociclib, and abemaciclib. Clonogenic survival assays determined the radiation enhancement ratios (rERs) and evaluated the efficacy of CDK4/6i + RT. Immunofluorescence assays measured RAD51 foci formation and quantified micronuclei formation following RT and/or CDK4/6 inhibition. Immunoprecipitation with myc-RAD51 and GFP-RB assessed potential protein-protein interactions. Results: While ER+ and TNBC cell lines with wild type BRCA1 expression are radiosensitized by CDK4/6i, BRCA1-deficient SUM-149 cells are not radiosensitized by CDK4/6i at concentrations up to 1µM (rER: 0.92-1.01). In an MCF-7 isogenic model of BRCA2 knockout, CDK4/6i-mediated radiosensitization was abolished compared to Cas9 control or parental cell lines. In ER+ BC cell lines (MCF-7-p53 wt, T47D-p53 mutant), transient or genetic knockdown of RAD51 prevented CDK4/6i-induced radiosensitization. The total quantity of RT-induced RAD51 foci increased in vitro following overexpression of RB-a tumor suppressor and downstream target of CDK4/6. RB overexpression also rescued CDK4/6i-mediated radiosensitization in RB-deficient cell lines through changes in HR efficiency but not via NHEJ or altered micronuclei formation. Moreover, immunoprecipitation of RAD51 in ER+ (MCF-7) and TNBC (MDA-MB-231) cells exhibited an interaction with RB. Conversely, loss of the NHEJ-associated protein Ku70 (XRCC6) was synergistic with palbociclib + RT in MCF7 (rER: 1.76-2.44) and T47D (rER: 1.61-3.88) cells. Finally, CRISPR Cas9-mediated loss of the tumor suppressor p53 (TP53) did not affect radiosensitization induced by CDK4/6i in isogenic p53 wt ER+ (MCF-7, rER: 1.19-1.33) and p53 wt TNBC (CAL-51, rER: 1.23-1.52) cell lines with TP53 loss. Conclusions: Taken together, our results in multiple non-overlapping isogenic models of ER+ and TNBC suggest that CDK4/6i-mediated radiosensitization of BC cell lines occurs through impaired HR activity and RB signaling, and not through the actions of p53 or NHEJ-mediated DNA repair. Citation Format: Kassidy M. Jungles, Andrea M. Pesch, Nicole Hirsh, Anna R. Michmerhuizen, Kari Wilder-Romans, Benjamin C. Chandler, Meilan Liu, Lynn Lerner, Lori J. Pierce, James M. Rae, Corey W. Speers. Expression of DNA damage response proteins modifies the efficacy of CDK4/6 inhibitor-mediated radiosensitization in breast cancer models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 216.
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Chakrabarty, Anindita, Sreeraj Surendran, Neil E. Bhola, Vishnu S. Mishra, Tasaduq Hussain Wani, Khemraj S. Baghel, Carlos L. Arteaga, Rohini Garg, and Goutam Chowdhury. "The H1047R PIK3CA oncogene induces a senescence-like state, pleiotropy and acute HSP90 dependency in HER2+ mammary epithelial cells." Carcinogenesis 40, no. 10 (June 18, 2019): 1179–90. http://dx.doi.org/10.1093/carcin/bgz118.
Abstract:
Abstract In pre-clinical models, co-existence of Human Epidermal Growth Factor Receptor-2 (HER2)-amplification and PI3K catalytic subunit (PIK3CA) mutations results in aggressive, anti-HER2 therapy-resistant breast tumors. This is not always reflected in clinical setting. We speculated that the complex interaction between the HER2 and PIK3CA oncogenes is responsible for such inconsistency. We performed series of biochemical, molecular and cellular assays on genetically engineered isogenic mammary epithelial cell lines and breast cancer cells expressing both oncogenes. In vitro observations were validated in xenografts models. We showed that H1047R, one of the most common PIK3CA mutations, is responsible for endowing a senescence-like state in mammary epithelial cells overexpressing HER2. Instead of imposing a permanent growth arrest characteristic of oncogene-induced senescence, the proteome secreted by the mutant cells promotes stem cell enrichment, angiogenesis, epithelial-to-mesenchymal transition, altered immune surveillance and acute vulnerability toward HSP90 inhibition. We inferred that the pleiotropism, as observed here, conferred by the mutated oncogene, depending on the host microenvironment, contributes to conflicting pre-clinical and clinical characteristics of HER2+, mutated PIK3CA-bearing tumor cells. We also came up with a plausible model for evolution of breast tumors from mammary epithelial cells harboring these two molecular lesions.
25
Costamagna, Gianluca, Giacomo Pietro Comi, and Stefania Corti. "Advancing Drug Discovery for Neurological Disorders Using iPSC-Derived Neural Organoids." International Journal of Molecular Sciences 22, no. 5 (March 6, 2021): 2659. http://dx.doi.org/10.3390/ijms22052659.
Abstract:
In the last decade, different research groups in the academic setting have developed induced pluripotent stem cell-based protocols to generate three-dimensional, multicellular, neural organoids. Their use to model brain biology, early neural development, and human diseases has provided new insights into the pathophysiology of neuropsychiatric and neurological disorders, including microcephaly, autism, Parkinson’s disease, and Alzheimer’s disease. However, the adoption of organoid technology for large-scale drug screening in the industry has been hampered by challenges with reproducibility, scalability, and translatability to human disease. Potential technical solutions to expand their use in drug discovery pipelines include Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) to create isogenic models, single-cell RNA sequencing to characterize the model at a cellular level, and machine learning to analyze complex data sets. In addition, high-content imaging, automated liquid handling, and standardized assays represent other valuable tools toward this goal. Though several open issues still hamper the full implementation of the organoid technology outside academia, rapid progress in this field will help to prompt its translation toward large-scale drug screening for neurological disorders.
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Wenzl, Kerstin, Michelle Manske, Paola Martinez, Stephen Ansell, James R. Cerhan, and Anne J. Novak. "Isogenic Loss of TNFAIP3 in Waldenstrom Macroglobulinemia Enhances MYD88L265P-Driven Signaling." Blood 128, no. 22 (December 2, 2016): 4100. http://dx.doi.org/10.1182/blood.v128.22.4100.4100.
Abstract:
Abstract Recent next generation sequencing of paired normal and tumor lymphoma tissue highlights a significant role for the MYD88L265P mutations in Waldenstrom Macroglobulinemia (WM) and diffuse large B-cell lymphoma (DLBCL). In recent work we have found that MYD88L265P signaling is constitutively active in both WM and DLBCL cells leading to heightened MYD88L265P, IRAK and TRAF6 oligomerization and TAK1 and NF-kB activation. However, little else is know about the full impact of the mutation, especially in combination with other genetic events. Recent mouse models suggest that MYD88L265P alone may not be sufficient to induce tumor formation and that the pathway requires additional genetic hits. Interplay between MYD88L265P and other oncogenic events are further supported by the fact that TNFAIP3 (A20) inactivation often accompanies MYD88L265P. These data, combined with the finding that loss of TNFAIP3 promotes accumulation of MYD88L265PB-cells in a mouse model, suggest a potential relationship between these genetic events, and more importantly, a possible clinical impact. The aim of this study was to generate a human cell line model which mimics the effect of MYD88L265P andTNFAIP3 loss to further understand the cellular consequence of these genomic alterations in lymphoma. To mimic the TNFAIP3 loss in combination with the MYD88L265P mutation,we used the WM cell line MWCL1, which harbors a heterozygous mutation in MYD88. For the TNFAIP3 specific knock out, we used the transcription activator-like effector nucleases (TALENs) genome editing approach to induce a double stranded break in exon 5 of the TNFAIP3 gene. TALENs were expressed in MWCL1 cells by transient transfection, and single-cell clones were isolated. Sanger sequencing of the TNFAIP3 gene was performed to identify clones that carried the deletion and all clones were screened by Western blot to confirm knock down of A20. We successfully generated a panel of MWCL1 cell lines that harbor a heterozygous deletion in the TNFAIP3 gene. Western blot analysis confirm a 2.4 fold reduction in A20 expression in the heterozygous cell line (MWCL1A20-/A20+), Because A20 has been shown the be a central negative regulator of NF-κB activation, we first measured the impact of A20 loss on phosphorylation of NF-κB p65 and found that MWCL1A20-/A20+ cells had higher base line levels of pNF-κB compared to the wild type (WT) cell line (1.77 fold). Furthermore, time course stimulation experiments with the TLR ligand lipopolysaccharide (LPS) showed that MWCL1A20-/A20+ cells had a higher amount of pNF-κB after 30 (3 fold) and 60 (1.5 fold) minutes of stimulation. We next looked at expression of NF-κB target genes by quantitative real time PCR and found that IL-6 (2.5 fold; p≤ 0.05) and CXCL10 (IP10) (4 fold; p≤ 0.05) were upregulated in MWCL1A20-/A20+cells compared to WT. In summary, we have established a new WM cell line model which mimics the effect of the MYD88L265P mutation in combination with a heterozygous loss of the TNFAIP3 gene. We show that loss of TNFAIP3 results in a higher baseline and TLR-stimulated phosphorylation of NF-κB as well as in higher expression IL-6 and IP10. Overall, this cell line will be a useful tool to study biological consequences of TNFAIP3loss in WM patients as well as novel therapeutic strategies. Disclosures Ansell: BMS, Seattle Genetics, Merck, Celldex and Affimed: Research Funding.
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Ferri-Borgogno, Sammy, Sugata Barui, Amberly M. McGee, Tamara Griffiths, Pankaj K. Singh, Cortt G. Piett, Bidyut Ghosh, et al. "Paradoxical Role of AT-rich Interactive Domain 1A in Restraining Pancreatic Carcinogenesis." Cancers 12, no. 9 (September 21, 2020): 2695. http://dx.doi.org/10.3390/cancers12092695.
Abstract:
Background & Aims: ARID1A is postulated to be a tumor suppressor gene owing to loss-of-function mutations in human pancreatic ductal adenocarcinomas (PDAC). However, its role in pancreatic pathogenesis is not clear despite recent studies using genetically engineered mouse (GEM) models. We aimed at further understanding of its direct functional role in PDAC, using a combination of GEM model and PDAC cell lines. Methods: Pancreas-specific mutant Arid1a-driven GEM model (Ptf1a-Cre; KrasG12D; Arid1af/f or “KAC”) was generated by crossing Ptf1a-Cre; KrasG12D (“KC”) mice with Arid1af/f mice and characterized histologically with timed necropsies. Arid1a was also deleted using CRISPR-Cas9 system in established human and murine PDAC cell lines to study the immediate effects of Arid1a loss in isogenic models. Cell lines with or without Arid1a expression were developed from respective autochthonous PDAC GEM models, compared functionally using various culture assays, and subjected to RNA-sequencing for comparative gene expression analysis. DNA damage repair was analyzed in cultured cells using immunofluorescence and COMET assay. Results: Retention of Arid1a is critical for early progression of mutant Kras-driven pre-malignant lesions into PDAC, as evident by lower Ki-67 and higher apoptosis staining in “KAC” as compared to “KC” mice. Enforced deletion of Arid1a in established PDAC cell lines caused suppression of cellular growth and migration, accompanied by compromised DNA damage repair. Despite early development of relatively indolent cystic precursor lesions called intraductal papillary mucinous neoplasms (IPMNs), a subset of “KAC” mice developed aggressive PDAC in later ages. PDAC cells obtained from older autochthonous “KAC” mice revealed various compensatory (“escaper”) mechanisms to overcome the growth suppressive effects of Arid1a loss. Conclusions: Arid1a is an essential survival gene whose loss impairs cellular growth, and thus, its expression is critical during early stages of pancreatic tumorigenesis in mouse models. In tumors that arise in the setting of ARID1A loss, a multitude of “escaper” mechanisms drive progression.
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Hollywood, Jennifer A., Aneta Przepiorski, Randall F. D’Souza, Sreevalsan Sreebhavan, Ernst J. Wolvetang, Patrick T. Harrison, Alan J. Davidson, and Teresa M. Holm. "Use of Human Induced Pluripotent Stem Cells and Kidney Organoids To Develop a Cysteamine/mTOR Inhibition Combination Therapy for Cystinosis." Journal of the American Society of Nephrology 31, no. 5 (March 20, 2020): 962–82. http://dx.doi.org/10.1681/asn.2019070712.
Abstract:
BackgroundMutations in CTNS—a gene encoding the cystine transporter cystinosin—cause the rare, autosomal, recessive, lysosomal-storage disease cystinosis. Research has also implicated cystinosin in modulating the mTORC1 pathway, which serves as a core regulator of cellular metabolism, proliferation, survival, and autophagy. In its severest form, cystinosis is characterized by cystine accumulation, renal proximal tubule dysfunction, and kidney failure. Because treatment with the cystine-depleting drug cysteamine only slows disease progression, there is an urgent need for better treatments.MethodsTo address a lack of good human-based cell culture models for studying cystinosis, we generated the first human induced pluripotent stem cell (iPSC) and kidney organoid models of the disorder. We used a variety of techniques to examine hallmarks of cystinosis—including cystine accumulation, lysosome size, the autophagy pathway, and apoptosis—and performed RNA sequencing on isogenic lines to identify differentially expressed genes in the cystinosis models compared with controls.ResultsCompared with controls, these cystinosis models exhibit elevated cystine levels, increased apoptosis, and defective basal autophagy. Cysteamine treatment ameliorates this phenotype, except for abnormalities in apoptosis and basal autophagy. We found that treatment with everolimus, an inhibitor of the mTOR pathway, reduces the number of large lysosomes, decreases apoptosis, and activates autophagy, but it does not rescue the defect in cystine loading. However, dual treatment of cystinotic iPSCs or kidney organoids with cysteamine and everolimus corrects all of the observed phenotypic abnormalities.ConclusionsThese observations suggest that combination therapy with a cystine-depleting drug such as cysteamine and an mTOR pathway inhibitor such as everolimus has potential to improve treatment of cystinosis.
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Jung, Moonjung, Stefan Cordes, Jizhong Zou, Shiqin J. Yu, Xavi Guitart, So Gun Hong, Vinh Dang, et al. "GATA2 deficiency and human hematopoietic development modeled using induced pluripotent stem cells." Blood Advances 2, no. 23 (December 11, 2018): 3553–65. http://dx.doi.org/10.1182/bloodadvances.2018017137.
Abstract:
Abstract GATA2 deficiency is an inherited or sporadic genetic disorder characterized by distinct cellular deficiency, bone marrow failure, various infections, lymphedema, pulmonary alveolar proteinosis, and predisposition to myeloid malignancies resulting from heterozygous loss-of-function mutations in the GATA2 gene. How heterozygous GATA2 mutations affect human hematopoietic development or cause characteristic cellular deficiency and eventual hypoplastic myelodysplastic syndrome or leukemia is not fully understood. We used induced pluripotent stem cells (iPSCs) to study hematopoietic development in the setting of GATA2 deficiency. We performed hematopoietic differentiation using iPSC derived from patients with GATA2 deficiency and examined their ability to commit to mesoderm, hemogenic endothelial precursors (HEPs), hematopoietic stem progenitor cells, and natural killer (NK) cells. Patient-derived iPSC, either derived from fibroblasts/marrow stromal cells or peripheral blood mononuclear cells, did not show significant defects in committing to mesoderm, HEP, hematopoietic stem progenitor, or NK cells. However, HEP derived from GATA2-mutant iPSC showed impaired maturation toward hematopoietic lineages. Hematopoietic differentiation was nearly abolished from homozygous GATA2 knockout (KO) iPSC lines and markedly reduced in heterozygous KO lines compared with isogenic controls. On the other hand, correction of the mutated GATA2 allele in patient-specific iPSC did not alter hematopoietic development consistently in our model. GATA2 deficiency usually manifests within the first decade of life. Newborn and infant hematopoiesis appears to be grossly intact; therefore, our iPSC model indeed may resemble the disease phenotype, suggesting that other genetic, epigenetic, or environmental factors may contribute to bone marrow failure in these patients following birth. However, heterogeneity of PSC-based models and limitations of in vitro differentiation protocol may limit the possibility to detect subtle cellular phenotypes.
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Sidhu, Ishnoor, Sonali P. Barwe, Raju K. Pillai, and Anilkumar Gopalakrishnapillai. "Harnessing the Power of Induced Pluripotent Stem Cells and Gene Editing Technology: Therapeutic Implications in Hematological Malignancies." Cells 10, no. 10 (October 9, 2021): 2698. http://dx.doi.org/10.3390/cells10102698.
Abstract:
In vitro modeling of hematological malignancies not only provides insights into the influence of genetic aberrations on cellular and molecular mechanisms involved in disease progression but also aids development and evaluation of therapeutic agents. Owing to their self-renewal and differentiation capacity, induced pluripotent stem cells (iPSCs) have emerged as a potential source of short in supply disease-specific human cells of the hematopoietic lineage. Patient-derived iPSCs can recapitulate the disease severity and spectrum of prognosis dictated by the genetic variation among patients and can be used for drug screening and studying clonal evolution. However, this approach lacks the ability to model the early phases of the disease leading to cancer. The advent of genetic editing technology has promoted the generation of precise isogenic iPSC disease models to address questions regarding the underlying genetic mechanism of disease initiation and progression. In this review, we discuss the use of iPSC disease modeling in hematological diseases, where there is lack of patient sample availability and/or difficulty of engraftment to generate animal models. Furthermore, we describe the power of combining iPSC and precise gene editing to elucidate the underlying mechanism of initiation and progression of various hematological malignancies. Finally, we discuss the power of iPSC disease modeling in developing and testing novel therapies in a high throughput setting.
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Malladi, Srinivas. "Abstract P1-06-08: Metabolic diversity determines metastatic fitness of breast cancer brain-tropic cells." Cancer Research 82, no. 4_Supplement (February 15, 2022): P1–06–08—P1–06–08. http://dx.doi.org/10.1158/1538-7445.sabcs21-p1-06-08.
Abstract:
Abstract HER2+ breast cancer patients presenting with either synchronous (S-BM), latent residual (Lat) or metachronous (M-BM) brain metastases have poor survival outcomes. However, the basis for disparate metastatic fitness among disseminated tumor cells of similar oncotype within a distal organ remains unknown. Employing isogenic HER2+ breast cancer brain metastasis models, we show metabolic diversity and plasticity within brain-tropic cells determines metastatic fitness. Lactate secreted by aggressive metastatic cells (S-BM and M-BM) or lactate supplementation to mice bearing latent residual disease limits innate immune surveillance and triggers overt metastasis. Attenuating lactate metabolism in S-BM impedes metastasis, while M-BM adapt and survive as latent residual disease. In contrast to S-BM, Lat and M-BM survive in equilibrium with innate immune surveillance, oxidize glutamine and maintain cellular redox homeostasis through the anionic amino acid transporter xCT. Moreover, xCT expression is significantly higher in matched metachronous brain metastatic samples compared to primary tumors from HER2+ breast cancer patients. Genetic or pharmacological blockade of xCT eradicates residual disease and brain metastatic relapse in these preclinical models. Citation Format: Srinivas Malladi. Metabolic diversity determines metastatic fitness of breast cancer brain-tropic cells [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P1-06-08.
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Grantcharova, Nina, Verena Peters, Claudia Monteiro, Katherina Zakikhany, and Ute Römling. "Bistable Expression of CsgD in Biofilm Development of Salmonella enterica Serovar Typhimurium." Journal of Bacteriology 192, no. 2 (November 6, 2009): 456–66. http://dx.doi.org/10.1128/jb.01826-08.
Abstract:
ABSTRACT Bacterial persistence in the environment and in the infected host is often aided by the formation of exopolymer-enclosed communities known as biofilms. Heterogeneous gene expression takes place in microcompartments formed within the complex biofilm structure. This study describes cell differentiation within an isogenic bacterial cell population based on the example of biofilm formation by Salmonella enterica serovar Typhimurium. We analyzed the expression of the major biofilm regulator CsgD at the single-cell level with a chromosomal CsgD-green fluorescent protein (GFP) translational fusion. In individual cells, CsgD-GFP expression is mostly found in the cytoplasm. Quantitative expression analysis and results from three different models of S. Typhimurium biofilms demonstrated that CsgD is expressed in a bistable manner during biofilm development. CsgD expression is, however, monomodal when CsgD is expressed in larger amounts due to a promoter mutation or elevated levels of the secondary signaling molecule c-di-GMP. High levels of CsgD-GFP are associated with cellular aggregation in all three biofilm models. Furthermore, the subpopulation of cells expressing large amounts of CsgD is engaged in cellulose production during red, dry, and rough (rdar) morphotype development and in microcolony formation under conditions of continuous flow. Consequently, bistability at the level of CsgD expression leads to a corresponding pattern of task distribution in S. Typhimurium biofilms.
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Gerritsen, Jacqueline S., Joseph S. Faraguna, Rudy Bonavia, Frank B. Furnari, and Forest M. White. "Predictive data-driven modeling of C-terminal tyrosine function in the EGFR signaling network." Life Science Alliance 6, no. 8 (May 11, 2023): e202201466. http://dx.doi.org/10.26508/lsa.202201466.
Abstract:
The epidermal growth factor receptor (EGFR) has been studied extensively because of its critical role in cellular signaling and association with disease. Previous models have elucidated interactions between EGFR and downstream adaptor proteins or showed phenotypes affected by EGFR. However, the link between specific EGFR phosphorylation sites and phenotypic outcomes is still poorly understood. Here, we employed a suite of isogenic cell lines expressing site-specific mutations at each of the EGFR C-terminal phosphorylation sites to interrogate their role in the signaling network and cell biological response to stimulation. Our results demonstrate the resilience of the EGFR network, which was largely similar even in the context of multiple Y-to-F mutations in the EGFR C-terminal tail, while also revealing nodes in the network that have not previously been linked to EGFR signaling. Our data-driven model highlights the signaling network nodes associated with distinct EGF-driven cell responses, including migration, proliferation, and receptor trafficking. Application of this same approach to less-studied RTKs should provide a plethora of novel associations that should lead to an improved understanding of these signaling networks.
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Kotini, Andriana, Jeffrey J. Delrow, Timothy A. Graubert, Stephen Nimer, and Eirini P. Papapetrou. "Functional Dissection of Chromosome 7q Loss and Haploinsufficient Gene Discovery Using iPSC Models of MDS." Blood 124, no. 21 (December 6, 2014): 524. http://dx.doi.org/10.1182/blood.v124.21.524.524.
Abstract:
Abstract Somatic loss of one copy of the long arm of chromosome 7 [del(7q)] is a characteristic cytogenetic abnormality in MDS and other myeloid malignancies, well-recognized for decades and associated with unfavorable prognosis. Despite compelling clinical evidence that the del(7q) holds a key to the pathogenesis of MDS, the mechanism remains elusive. Gene haploinsufficiency has been proposed as a plausible mechanism, but definitive evidence is lacking. Narrowing down the responsible region and identifying the critical genes has proved challenging with existing approaches. Chr7q deletions are typically very large and modeling in the mouse is problematic, as the genomic regions syntenic to the human chr7q are dispersed into 4 different mouse chromosomes. More than one commonly deleted regions (CDRs) have been proposed by physical mapping studies in patient cells. A handful of genes on chr7q have been implicated through candidate gene approaches and knockout studies in the mouse. However, despite the intense efforts, the contribution of the del(7q) to the disease phenotype and the critical gene or genes on chr7q that mediate it remain unclear. To overcome the limitations of existing tools (primary patient cells, mouse models) to study del(7q)-MDS, we developed a new model harnessing reprogramming and genome editing technologies. First we derived del(7q)-, in parallel with isogenic karyotypically normal induced pluripotent stem cells (iPSCs) from bone marrow hematopoietic cells of two MDS patients. By whole exome sequencing, we were able to identify somatic variants of the MDS clone and show that they are present in the del(7q)-MDS-iPSCs, but not in the karyotypically normal iPSCs, which therefore unambiguously originate from residual normal cells. We used these isogenic and fully genetically characterized patient-derived iPSCs to characterize disease-relevant cellular phenotypes specific to the MDS-iPSCs, which included severely reduced hematopoietic potential and clonogenicity and increased apoptosis. We next found that iPSC clones spontaneously acquiring a second copy of chr7q had an in vitro growth advantage, which enabled us to isolate one clone that completely rescued its hematopoietic differentiation ability upon restoration of a diploid dosage of a ~30Mb chr7q telomeric region. This result provides the first definitive evidence that the del(7q) abnormality confers a profound loss of hematopoietic potential and that this defect is mediated through reduced dosage, consistent with haploinsufficiency of one or more genes. To further narrow down the critical region, we developed genome editing technologies to engineer large chromosomal deletions for the first time in human cells. Combining gene targeting with a modified Cre-loxP approach and the CRISPR/Cas9 endonuclease technology, we were able to generate a panel of 12 iPSC lines harboring hemizygous deletions of various defined segments spanning the entire long arm of chr7. By asking which of them recapitulate the MDS hematopoietic phenotype, we were able to “functionally map” the critical segment in a region spanning cytobands q32.3 - q36.1. To identify critical gene(s) on chr7q, we designed a phenotype-rescue screen. We selected 62 candidate haploinsufficient genes on the basis of significantly reduced expression in del(7q)- compared to isogenic normal iPSCs. We constructed a barcoded lentiviral library of these ORFs and performed a pooled library screen for rescue of hematopoiesis in del(7q)-MDS-iPSCs, i.e. enrichment in CD45+ hematopoietic progenitors. We selected the top 6 genes within our region that were found recurrently enriched in at least 2 independent experiments. Four of them could be individually validated: dosage complementation partially rescued hematopoiesis and knockdown studies mimicking haploinsufficiency (50% knockdown) in normal primary CD34+ hematopoietic progenitor cells had a detrimental effect in hematopoiesis. The four genes include EZH2 and LUC7L2 – two genes found to harbor recurrent heterozygous loss-of-function mutations in MDS – as well as two genes with no previously known role in MDS, located in close genomic proximity to the former two. This approach, constituting a new paradigm of functional human genetics with patient-specific iPSCs, can be more broadly applicable to the study of the phenotypic consequences of segmental chromosomal deletions and to haploinsufficient gene discovery. Disclosures No relevant conflicts of interest to declare.
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Lemaire, Sandrine, Klaudia Kosowska-Shick, Peter C. Appelbaum, Gunther Verween, Paul M. Tulkens, and Françoise Van Bambeke. "Cellular Pharmacodynamics of the Novel Biaryloxazolidinone Radezolid: Studies with Infected Phagocytic and Nonphagocytic cells, Using Staphylococcus aureus, Staphylococcus epidermidis, Listeria monocytogenes, and Legionella pneumophila." Antimicrobial Agents and Chemotherapy 54, no. 6 (April 12, 2010): 2549–59. http://dx.doi.org/10.1128/aac.01724-09.
Abstract:
ABSTRACT Radezolid is a novel biaryloxazolidinone in clinical development which shows improved activity, including against linezolid-resistant strains. In a companion paper (29), we showed that radezolid accumulates about 11-fold in phagocytic cells, with ∼60% of the drug localized in the cytosol and ∼40% in the lysosomes of the cells. The present study examines its activity against (i) bacteria infecting human THP-1 macrophages and located in different subcellular compartments (Listeria monocytogenes, cytosol; Legionella pneumophila, vacuoles; Staphylococcus aureus and Staphylococcus epidermidis, mainly phagolysosomal), (ii) strains of S. aureus with clinically relevant mechanisms of resistance, and (iii) isogenic linezolid-susceptible and -resistant S. aureus strains infecting a series of phagocytic and nonphagocytic cells. Radezolid accumulated to similar levels (∼10-fold) in all cell types (human keratinocytes, endothelial cells, bronchial epithelial cells, osteoblasts, macrophages, and rat embryo fibroblasts). At equivalent weight concentrations, radezolid proved consistently 10-fold more potent than linezolid in all these models, irrespective of the bacterial species and resistance phenotype or of the cell type infected. This results from its higher intrinsic activity and higher cellular accumulation. Time kill curves showed that radezolid's activity was more rapid than that of linezolid both in broth and in infected macrophages. These data suggest the potential interest of radezolid for recurrent or persistent infections where intracellular foci play a determinant role.
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Javier, Rodrigo, and Craig Horbinski. "TAMI-48. THE KETOGENIC DIET IS INEFFECTIVE IN PRECLINICAL MODELS OF IDH1 WILD-TYPE AND IDH1 MUTANT GLIOMA." Neuro-Oncology 23, Supplement_6 (November 2, 2021): vi208. http://dx.doi.org/10.1093/neuonc/noab196.831.
Abstract:
Abstract Despite decades of intensive research, infiltrative gliomas are still usually lethal and challenging to treat. A subset of gliomas contains mutations in isocitrate dehydrogenase 1 (IDH1mut), which disrupts cellular biochemistry; such gliomas are generally less aggressive than their IDH1 wild-type (IDH1wt) counterparts. Some preclinical studies have suggested that a ketogenic diet (KD), characterized by low-carbohydrate and high-fat content, may be beneficial against a variety of cancers, including gliomas. However, not all studies have shown promising results, and to date, no study has addressed the sensitivity of glioma cells to KD in the specific context of the endogenous IDH1mut metabolic landscape. The aim of the current study was to compare the effects of KD in preclinical models to IDH1wt versus IDH1mut gliomas. In vitro treatment of patient-derived IDH1wt and IDH1mut glioma cells with the ketone body β-hydroxybutyrate showed no significant effect on cell proliferation in a low glucose culture environment. Likewise, the in vivo flank growth rates of these patient-derived IDH1wt and IDH1mut glioma xenografts showed no significant difference when mice were fed KD versus regular diet (GBM12 p=0.98, GBM164 p=0.4, GBM196 p=0.11). Finally, KD had no effect on the survival of mice engrafted with isogenic Sleeping-Beauty transposase-engineered IDH1wt (median control survival 22 days versus treatment 23 days, p=0.23) or IDH1mut glioma cells (median control survival 26.5 days versus treatment 26 days, p=0.81). These data suggest that IDH1mut gliomas are not more responsive than IDH1wt gliomas to KD, and that clinical trials further exploring KD in this subset of glioma patients are probably not warranted.
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Frank, Karen M., Tong Zhou, Liliana Moreno-Vinasco, Brian Hollett, Joe G. N. Garcia, and Juliane Bubeck Wardenburg. "Host Response Signature to Staphylococcus aureus Alpha-Hemolysin Implicates Pulmonary Th17 Response." Infection and Immunity 80, no. 9 (June 25, 2012): 3161–69. http://dx.doi.org/10.1128/iai.00191-12.
Abstract:
ABSTRACTStaphylococcus aureuspneumonia causes significant morbidity and mortality. Alpha-hemolysin (Hla), a pore-forming cytotoxin ofS. aureus, has been identified through animal models of pneumonia as a critical virulence factor that induces lung injury. In spite of considerable molecular knowledge of how this cytotoxin injures the host, the precise host response to Hla in the context of infection remains poorly understood. We employed whole-genome expression profiling of infected lungs to define the host response to wild-typeS. aureuscompared with the response to an Hla-deficient isogenic mutant in experimental pneumonia. These data provide a complete expression profile at 4 and at 24 h postinfection, revealing a unique response to the toxin-expressing strain. Gene ontogeny analysis revealed significant differences in the extracellular matrix and cardiomyopathy pathways, both of which govern cellular interactions in the tissue microenvironment. Evaluation of individual transcript responses to Hla-secreting staphylococci was notable for upregulation of host cytokine and chemokine genes, including the p19 subunit of interleukin-23. Consistent with this observation, the cellular immune response to infection was characterized by a prominent Th17 response to the wild-type pathogen. These findings define specific host mRNA responses to Hla-producingS. aureus, coupling the pulmonary Th17 response to the secretion of this cytotoxin. Expression profiling to define the host response to a single virulence factor proved to be a valuable tool in identifying pathways for further investigation inS. aureuspneumonia. This approach may be broadly applicable to the study of bacterial toxins, defining host pathways that can be targeted to mitigate toxin-induced disease.
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Lara-Chacón, Bárbara, Sandra L. Guerrero-Rodríguez, Karla J. Ramírez-Hernández, Angélica Yamilett Robledo-Rivera, Marco Antonio Velasco Velazquez, Roberto Sánchez-Olea, and Mónica Raquel Calera. "Gpn3 Is Essential for Cell Proliferation of Breast Cancer Cells Independent of Their Malignancy Degree." Technology in Cancer Research & Treatment 18 (January 1, 2019): 153303381987082. http://dx.doi.org/10.1177/1533033819870823.
Abstract:
Successful therapies for patients with breast cancer often lose their initial effectiveness. Thus, identifying new molecular targets is a constant goal in the fight against breast cancer. Gpn3 is a protein required for RNA polymerase II nuclear targeting in both yeast and human cells. We investigated here the effect of suppressing Gpn3 expression on cell proliferation in a progression series of isogenic cell lines derived from the nontumorigenic MCF-10A breast cells that recapitulate different stages of breast carcinogenesis. Gpn3 protein levels were comparable in all malignant derivatives of the nontumorigenic MCF-10A cells. shRNA-mediated inhibition of Gpn3 expression markedly decreased cell proliferation in all MCF-10A sublines. A fraction of the largest RNA polymerase II subunit Rpb1 was retained in the cytoplasm, but most Rpb1 remained nuclear after suppressing Gpn3 in all cell lines studied. Long-term proliferation experiments in cells with suppressed Gpn3 expression resulted in the eventual loss of all isogenic cell lines but MCF-10CA1d.cl1. In MCF-10CA1d.cl1 cells, Gpn3 knockdown reduced the proliferation of breast cancer stem cells as evaluated by mammosphere assays. After the identification that Gpn3 plays a key role in cell proliferation in mammary epithelial cells independent of the degree of transformation, we also analyzed the importance of Gpn3 in other human breast cancer cell lines from different subtypes. Gpn3 was also required for cell proliferation and nuclear translocation of RNA polymerase II in such cellular models. Altogether, our results show that Gpn3 is essential for breast cancer cell proliferation regardless of the transformation level, indicating that Gpn3 could be considered a molecular target for the development of new antiproliferative therapies. Importantly, our analysis of public data revealed that Gpn3 overexpression was associated with a significant decrease in overall survival in patients with estrogen receptor-positive and Human epidermal growth factor receptor 2 (HER2+) breast cancer, supporting our proposal that targeting Gpn3 could potentially benefit patients with breast cancer.
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My, Ilaria, and Elisa Di Pasquale. "Genetic Cardiomyopathies: The Lesson Learned from hiPSCs." Journal of Clinical Medicine 10, no. 5 (March 9, 2021): 1149. http://dx.doi.org/10.3390/jcm10051149.
Abstract:
Genetic cardiomyopathies represent a wide spectrum of inherited diseases and constitute an important cause of morbidity and mortality among young people, which can manifest with heart failure, arrhythmias, and/or sudden cardiac death. Multiple underlying genetic variants and molecular pathways have been discovered in recent years; however, assessing the pathogenicity of new variants often needs in-depth characterization in order to ascertain a causal role in the disease. The application of human induced pluripotent stem cells has greatly helped to advance our knowledge in this field and enabled to obtain numerous in vitro patient-specific cellular models useful to study the underlying molecular mechanisms and test new therapeutic strategies. A milestone in the research of genetically determined heart disease was the introduction of genomic technologies that provided unparalleled opportunities to explore the genetic architecture of cardiomyopathies, thanks to the generation of isogenic pairs. The aim of this review is to provide an overview of the main research that helped elucidate the pathophysiology of the most common genetic cardiomyopathies: hypertrophic, dilated, arrhythmogenic, and left ventricular noncompaction cardiomyopathies. A special focus is provided on the application of gene-editing techniques in understanding key disease characteristics and on the therapeutic approaches that have been tested.
40
Manceau, Line, Julien Richard Albert, Pier-Luigi Lollini, Maxim V. C. Greenberg, Pascale Gilardi-Hebenstreit, and Vanessa Ribes. "Divergent transcriptional and transforming properties of PAX3-FOXO1 and PAX7-FOXO1 paralogs." PLOS Genetics 18, no. 5 (May 23, 2022): e1009782. http://dx.doi.org/10.1371/journal.pgen.1009782.
Abstract:
The hallmarks of the alveolar subclass of rhabdomyosarcoma are chromosomal translocations that generate chimeric PAX3-FOXO1 or PAX7-FOXO1 transcription factors. Overexpression of either PAX-FOXO1s results in related cell transformation in animal models. Yet, in patients the two structural genetic aberrations they derived from are associated with distinct pathological manifestations. To assess the mechanisms underlying these differences, we generated isogenic fibroblast lines expressing either PAX-FOXO1 paralog. Mapping of their genomic recruitment using CUT&Tag revealed that the two chimeric proteins have distinct DNA binding preferences. In addition, PAX7-FOXO1 binding results in greater recruitment of the H3K27ac activation mark than PAX3-FOXO1 binding and is accompanied by greater transcriptional activation of neighbouring genes. These effects are associated with a PAX-FOXO1-specific alteration in the expression of genes regulating cell shape and the cell cycle. Consistently, PAX3-FOXO1 accentuates fibroblast cellular traits associated with contractility and surface adhesion and limits entry into S phase. In contrast, PAX7-FOXO1 drives cells to adopt an amoeboid shape, reduces entry into M phase, and causes increased DNA damage. Altogether, our results argue that the diversity of rhabdomyosarcoma manifestation arises, in part, from the divergence between the genomic occupancy and transcriptional activity of PAX3-FOXO1 and PAX7-FOXO1.
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Kargaran, Parisa K., Jared M. Evans, Sara E. Bodbin, James G. W. Smith, Timothy J. Nelson, Chris Denning, and Diogo Mosqueira. "Mitochondrial DNA: Hotspot for Potential Gene Modifiers Regulating Hypertrophic Cardiomyopathy." Journal of Clinical Medicine 9, no. 8 (July 23, 2020): 2349. http://dx.doi.org/10.3390/jcm9082349.
Abstract:
Hypertrophic cardiomyopathy (HCM) is a prevalent and untreatable cardiovascular disease with a highly complex clinical and genetic causation. HCM patients bearing similar sarcomeric mutations display variable clinical outcomes, implying the involvement of gene modifiers that regulate disease progression. As individuals exhibiting mutations in mitochondrial DNA (mtDNA) present cardiac phenotypes, the mitochondrial genome is a promising candidate to harbor gene modifiers of HCM. Herein, we sequenced the mtDNA of isogenic pluripotent stem cell-cardiomyocyte models of HCM focusing on two sarcomeric mutations. This approach was extended to unrelated patient families totaling 52 cell lines. By correlating cellular and clinical phenotypes with mtDNA sequencing, potentially HCM-protective or -aggravator mtDNA variants were identified. These novel mutations were mostly located in the non-coding control region of the mtDNA and did not overlap with those of other mitochondrial diseases. Analysis of unrelated patients highlighted family-specific mtDNA variants, while others were common in particular population haplogroups. Further validation of mtDNA variants as gene modifiers is warranted but limited by the technically challenging methods of editing the mitochondrial genome. Future molecular characterization of these mtDNA variants in the context of HCM may identify novel treatments and facilitate genetic screening in cardiomyopathy patients towards more efficient treatment options.
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Ntai, Ioanna, Luca Fornelli, Caroline J. DeHart, Josiah E. Hutton, Peter F. Doubleday, Richard D. LeDuc, Alexandra J. van Nispen, et al. "Precise characterization of KRAS4b proteoforms in human colorectal cells and tumors reveals mutation/modification cross-talk." Proceedings of the National Academy of Sciences 115, no. 16 (April 2, 2018): 4140–45. http://dx.doi.org/10.1073/pnas.1716122115.
Abstract:
Mutations of the KRAS gene are found in human cancers with high frequency and result in the constitutive activation of its protein products. This leads to aberrant regulation of downstream pathways, promoting cell survival, proliferation, and tumorigenesis that drive cancer progression and negatively affect treatment outcomes. Here, we describe a workflow that can detect and quantify mutation-specific consequences of KRAS biochemistry, namely linked changes in posttranslational modifications (PTMs). We combined immunoaffinity enrichment with detection by top-down mass spectrometry to discover and quantify proteoforms with or without the Gly13Asp mutation (G13D) specifically in the KRAS4b isoform. The workflow was applied first to isogenic KRAS colorectal cancer (CRC) cell lines and then to patient CRC tumors with matching KRAS genotypes. In two cellular models, a direct link between the knockout of the mutant G13D allele and the complete nitrosylation of cysteine 118 of the remaining WT KRAS4b was observed. Analysis of tumor samples quantified the percentage of mutant KRAS4b actually present in cancer tissue and identified major differences in the levels of C-terminal carboxymethylation, a modification critical for membrane association. These data from CRC cells and human tumors suggest mechanisms of posttranslational regulation that are highly context-dependent and which lead to preferential production of specific KRAS4b proteoforms.
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Jacobs, Keith M., Sandeep Misri, Barbara Meyer, Suyash Raj, Cheri L. Zobel, Barry P. Sleckman, Dennis E. Hallahan, and Girdhar G. Sharma. "Unique epigenetic influence of H2AX phosphorylation and H3K56 acetylation on normal stem cell radioresponses." Molecular Biology of the Cell 27, no. 8 (April 15, 2016): 1332–45. http://dx.doi.org/10.1091/mbc.e16-01-0017.
Abstract:
Normal tissue injury resulting from cancer radiotherapy is often associated with diminished regenerative capacity. We examined the relative radiosensitivity of normal stem cell populations compared with non–stem cells within several radiosensitive tissue niches and culture models. We found that these stem cells are highly radiosensitive, in contrast to their isogenic differentiated progeny. Of interest, they also exhibited a uniquely attenuated DNA damage response (DDR) and muted DNA repair. Whereas stem cells exhibit reduced ATM activation and ionizing radiation–induced foci, they display apoptotic pannuclear H2AX-S139 phosphorylation (γH2AX), indicating unique radioresponses. We also observed persistent phosphorylation of H2AX-Y142 along the DNA breaks in stem cells, which promotes apoptosis while inhibiting DDR signaling. In addition, down-regulation of constitutively elevated histone-3 lysine-56 acetylation (H3K56ac) in stem cells significantly decreased their radiosensitivity, restored DDR function, and increased survival, signifying its role as a key contributor to stem cell radiosensitivity. These results establish that unique epigenetic landscapes affect cellular heterogeneity in radiosensitivity and demonstrate the nonubiquitous nature of radiation responses. We thus elucidate novel epigenetic rheostats that promote ionizing radiation hypersensitivity in various normal stem cell populations, identifying potential molecular targets for pharmacological radioprotection of stem cells and hopefully improving the efficacy of future cancer treatment.
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Rosenthal, Dean S., Elijah Finn, Devin Teehan, Nusrat Islam, Veerupaxagouda Patil, Bonnie Carney, Scott S. Rosenthal, Lucia Dussan, Cynthia M. Simbulan-Rosenthal, and Peter Sykora. "Abstract B035: Developing the UValidate platform to measure DNA damage and repair capacity in isogenic donor-derived skin keratinocytes, fibroblasts and melanocyte cell-lines with different Fitzpatrick phototypes." Cancer Research 84, no. 1_Supplement (January 9, 2024): B035. http://dx.doi.org/10.1158/1538-7445.dnarepair24-b035.
Abstract:
Abstract DNA damage associated with ultraviolet (UV) radiation exposure causes accelerated skin aging and cancer-initiating mutations. Over-the-counter UVA and UVB filters are required in all sunscreens, but transdermal absorption of these compounds have been reported to impair development and reproduction and metabolize into carcinogenic/mutagenic chemical intermediates. To assess the effects of sunscreen active ingredients on UVR-induced DNA damage and repair, we developed a novel high throughput (HTP) screening platform, UValidate, employing mixed populations of isogenic skin cells, exposed to single or combinations of UV filters in the presence of UVB and UVA. UValidate contains a patented LED UVR DNA damage induction system (LUDIS:V3), facilitating programmable precise induction of DNA damage and repair at multiple time points and replicates, using a 96-well configuration capable of simultaneous delivering UVA and UVB. LUDIS:V3 optimization experiments surprisingly revealed that, unlike keratinocytes, skin fibroblasts only repaired UVA DNA damage in conditioned media. We then tested sunscreen active ingredient combinations for their capacity to alter DNA repair and found that: 1) Some sunscreen ingredients, including dioxybenzone, induce a significant increase in DNA damage above endogenous levels. 2) Fibroblasts and keratinocytes respond differently to the treatment protocols. 3) Some sunscreen active compounds contribute to reduction of UVA-induced DNA damage. 4) No sunscreen completely inhibits all UVA DNA damage. We are currently employing the LUDIS:V3 system with HTP comet assays to compare DNA damage and DNA repair capacities, +/- sunscreen active ingredients, of six isogenic sets of primary epidermal keratinocytes (NHEK), melanocytes (NHEM), and fibroblasts (NHDF)` with different Fitzpatrick phototypes I-VI, representative of the diverse American population. These cells were isolated from donor skin samples, and further purified to establish banks of isogenic NHEK, NHEM, and NHDF cell lines. UVA exposure with the LUDIS:V3 and comet assays revealed higher levels of DNA damage in NHEK and NHDF, compared to NHEM, likely due to the UVR-protective effects of melanin in NHEM. Consistently, NHEK with phototype V (from darkly pigmented skin with higher melanin levels) exhibit significantly less UVA-induced DNA damage compared with phototype II NHEK. To create cellular models of dermal disease for DNA repair assays, we have successfully knocked out XPA in multiple NHDF cell lines with different phototypes using a CRISPR-Cas9 KO strategy, as confirmed by immunoblot and NGS sequence analyses. These and other engineered DNA repair-deficient skin cells will be assayed as single or mixed cell-type 2D cultures, with fluorescent labeling allowing for identification of subpopulations in comet and alkaline diffusion assays. This technology addresses the need for better platforms for rapid genotoxicity testing of safer and more effective chemical UV filters to prevent cutaneous carcinogenesis in diverse populations. Citation Format: Dean S. Rosenthal, Elijah Finn, Devin Teehan, Nusrat Islam, Veerupaxagouda Patil, Bonnie Carney, Scott S. Rosenthal, Lucia Dussan, Cynthia M. Simbulan-Rosenthal, Peter Sykora. Developing the UValidate platform to measure DNA damage and repair capacity in isogenic donor-derived skin keratinocytes, fibroblasts and melanocyte cell-lines with different Fitzpatrick phototypes [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: DNA Damage Repair: From Basic Science to Future Clinical Application; 2024 Jan 9-11; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2024;84(1 Suppl):Abstract nr B035.
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Vötsch, Désirée, Maren Willenborg, Walter M. R. Oelemann, Graham Brogden, and Peter Valentin-Weigand. "Membrane Binding, Cellular Cholesterol Content and Resealing Capacity Contribute to Epithelial Cell Damage Induced by Suilysin of Streptococcus suis." Pathogens 9, no. 1 (December 30, 2019): 33. http://dx.doi.org/10.3390/pathogens9010033.
Abstract:
Streptococcus (S.) suis is a major cause of economic losses in the pig industry worldwide and is an emerging zoonotic pathogen. One important virulence-associated factor is suilysin (SLY), a toxin that belongs to the family of cholesterol-dependent pore-forming cytolysins (CDC). However, the precise role of SLY in host–pathogen interactions is still unclear. Here, we investigated the susceptibility of different respiratory epithelial cells to SLY, including immortalized cell lines (HEp-2 and NPTr cells), which are frequently used in in vitro studies on S. suis virulence mechanisms, as well as primary porcine respiratory cells, which represent the first line of barrier during S. suis infections. SLY-induced cell damage was determined by measuring the release of lactate dehydrogenase after infection with a virulent S. suis serotype 2 strain, its isogenic SLY-deficient mutant strain, or treatment with the recombinant protein. HEp-2 cells were most susceptible, whereas primary epithelial cells were hardly affected by the toxin. This prompted us to study possible explanations for these differences. We first investigated the binding capacity of SLY using flow cytometry analysis. Since binding and pore-formation of CDC is dependent on the membrane composition, we also determined the cellular cholesterol content of the different cell types using TLC and HPLC. Finally, we examined the ability of those cells to reseal SLY-induced pores using flow cytometry analysis. Our results indicated that the amount of membrane-bound SLY, the cholesterol content of the cells, as well as their resealing capacity all affect the susceptibility of the different cells regarding the effects of SLY. These findings underline the differences of in vitro pathogenicity models and may further help to dissect the biological role of SLY during S. suis infections.
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Meng, Qingyuan, Xiao Ding, Xiaosong Liu, Hailong Wang, Ling Wang, Junwen Qiao, Hua Cao, et al. "Abstract 503: ISM3412, a novel and selective MAT2A inhibitor for the treatment of cancer." Cancer Research 83, no. 7_Supplement (April 4, 2023): 503. http://dx.doi.org/10.1158/1538-7445.am2023-503.
Abstract:
Abstract The enzyme methionine adenosyltransferase 2α (MAT2A) has an important role in metabolism and epigenetics as the major producer of the universal methyl donor S-adenosyl methionine (SAM). Despite broad cellular functions, MAT2A was recently implicated as a synthetic lethal target in cancers that have a deletion of the methylthioadenosine phosphorylase (MTAP) gene, which is found in approximately 15% of all cancers. Here, we report ISM3412 is a novel potent MAT2A inhibitor with high selectivity. In enzymatic assays, ISM3412 demonstrated a strong inhibition of MAT2A with an IC50 of 19.4 nM. In both HCT116 MTAP-/- cells and HCT116 parental cells, the compound inhibited SAM production, with IC50 values of 5.97 nM and 3.66 nM, respectively. Additionally, it inhibited SDMA production in HCT116 MTAP-/- cells (IC50 of 6.37 nM), but not in HCT116 parental cells (IC50 > 2000 nM). Consistent with the large SDMA inhibitory potency difference between the HCT116 isogenic cell pair, ISM3412 also exhibited very good selectivity in cellular proliferation assays. While it inhibited cellular growth in HCT116 MTAP-/-cells with an IC50 of 199nM, its inhibitory potency on HCT116 parental cells was very weak (IC50 of >21117nM), revealing more than a 100-fold selectivity in inhibiting growth in this isogenic pair cell line. Furthermore, while ISM3412 strongly inhibited proliferation in multiple cancer cell lines with natural MTAP deficiency (including pancreatic cancer, bladder cancer, and NSCLC), the same was not observed in multiple MTAP-intact cell lines (IC50 > 30 uM). Consistent with the in vitro data, ISM3412 demonstrated robust mono-therapeutic efficacy in an MTAP-/- HCT116 CDX model with tumor growth inhibition of 66% at 3 mg/kg QD. Similarly, ISM3412 also demonstrated robust single-agent efficacy in CDX models of pancreatic cancer, bladder cancer and NSCLC, as well as in a PDX model of esophageal cancer. Notably, ISM3412 showed improved efficacy when combined with docetaxel, compared to the individual monotherapy. ISM3412, at 10 uM, showed no obvious agonistic or antagonistic effect on 44 selected targets in a mini safety panel, demonstrating its selectivity as a MAT2A inhibitor. In addition to its strong biological potency and selectivity, ISM3412 exhibited favorable drug-likeness properties, including low molecular weight, good solubility and permeability, and low plasma protein binding. In vivo PK data revealed low clearance and high oral-bioavailability. Further, it was well tolerated with no significant hepatobiliary toxicity. In conclusion, ISM3412 is a novel and highly selective MAT2A inhibitor for MTAP-deficient cancers, and merits clinical evaluation. Citation Format: Qingyuan Meng, Xiao Ding, Xiaosong Liu, Hailong Wang, Ling Wang, Junwen Qiao, Hua Cao, Yushu Yin, Congcong Zhu, Xing Liang, Xin Cai, Yuanyuan Xu, Chenxi Xu, Shan Chen, Sujata Rao, Feng Ren, Alex Zhavoronkov. ISM3412, a novel and selective MAT2A inhibitor for the treatment of cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 503.
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Jenkins, Samir V., Shruti Shah, Azemat Jamshidi-Parsian, Amir Mortazavi, Gunnar Boysen, Kieng B. Vang, Robert J. Griffin, Narasimhan Rajaram, and Ruud P. Dings. "Abstract 1088: Acquired radiation resistance induces thiol-dependent cisplatin cross-resistance." Cancer Research 83, no. 7_Supplement (April 4, 2023): 1088. http://dx.doi.org/10.1158/1538-7445.am2023-1088.
Abstract:
Abstract Purpose. Resistance to radiation, both intrinsic and acquired, remains a significant clinical challenge. It is therefore import to identify the underlying molecular and cellular features involved in this resistance. Materials and Methods. We generated genetically-matched counterparts of various degrees of radiation sensitivity in non-small cell lung carcinoma (NSCLC) tumor models to define characteristics of acquired resistance through repeated exposures to conventional X-ray radiation. Results. As assessed by cell viability and clonogenic assays murine Lewis lung carcinoma (LLC) and human A549 cell lines acquired an approximate 1.5 - 2-fold increase in radiation resistance (RR) as compared to its parental counterpart. Morphologically, we found that A549-RR exhibited a greater nucleus-to-cytosol (N/C) ratio as compared to its respective counterpart. Since the N/C ratio has been linked to the differentiation state, we next investigated the epithelial-to-mesenchymal transition (EMT) phenotype and cellular plasticity. We found that A549 have a greater radiation-induced plasticity, as measured by E-cadherin, vimentin and double positive (DP) modulation, as compared to LLC. Additionally, migration was suppressed in A549-RR, as compared to A549. Subsequently, we confirmed that the LLC-RR and A549-RR are also more resistant to radiotherapy than their isogenic-matched counterpart in vivo. Remarkably, we found that the acquired radiation resistance also induced resistance to first-line chemotherapeutic cisplatin, but not carboplatin or oxaliplatin. This cross-resistance was attributed to induced elevated thiol levels. Decreasing the amount of thiols with buthionine sulfoximine (BSO), a gamma-glutamylcysteine synthetase inhibitor, sensitized the resistant cells to cisplatin, to levels similar as parental cells. Conclusions. By generating genetically-matched radiation resistant NSCLC tumor models we were able to identify and overcome cisplatin cross-resistance. This is an important finding arguing for multi-modality treatment regimens with the potential of improving clinical outcomes in the future. Citation Format: Samir V. Jenkins, Shruti Shah, Azemat Jamshidi-Parsian, Amir Mortazavi, Gunnar Boysen, Kieng B. Vang, Robert J. Griffin, Narasimhan Rajaram, Ruud P. Dings. Acquired radiation resistance induces thiol-dependent cisplatin cross-resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1088.
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Tawil, Nadim, Rayhaan Bassawon, Brian Meehan, Laura Montermini, Dongsic Choi, Ali Nehme, Hamed Najafabadi, et al. "OPTC-5. Molecular signatures of podoplanin expressing glioblastoma cell subsets with putative role in cancer associated thrombosis and microthrombosis." Neuro-Oncology Advances 3, Supplement_2 (July 1, 2021): ii7. http://dx.doi.org/10.1093/noajnl/vdab070.026.
Abstract:
Abstract Vascular anomalies, including thrombosis, are a hallmark of glioblastoma (GBM) and an aftermath of dysregulated cancer cell genome and epigenome. Upregulation of podoplanin (PDPN) by cancer cells has recently been linked to an increased risk of venous thromboembolism in glioblastoma patients. Thus, regulation of this platelet activating transmembrane protein by transforming events and release from cancer cells into the circulation are of considerable interest. We took advantage of single-cell and bulk GBM transcriptome dataset mining and investigated the pattern of PDPN expression across several databases. Our analysis indicated that PDPN is expressed by distinct (mesenchymal) glioblastoma cell subpopulations and is downregulated by oncogenic mutations of EGFR and IDH1 genes, via changes in chromatin modifications (EZH2) and DNA methylation, respectively. Additionally, we utilized isogenic and stem GBM cell lines, xenograft models in mice, ELISA assays for PDPN, tissue factor (TF), platelet factor 4 (PF4) and clotting activation markers (D-dimer), and multicolor nano-flow cytometry to show that GBM cells exteriorize PDPN and/or TF as cargo of exosome-like coagulant extracellular vesicles EVs. We also documented an increase of platelet activation (PF4) or coagulation markers (D-dimer) in mice harboring the corresponding PDPN- or TF-expressing glioma xenografts, respectively. While PDPN was a dominant regulator of systemic platelet activation, co-expression of PDPN and TF impacted local microthrombosis. Our work suggests that distinct cellular subsets drive multiple facets of GBM-associated thrombosis and may represent targets for diagnosis and intervention.
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Maurissen, Thomas L., Masahide Kawatou, Víctor López-Dávila, Kenji Minatoya, Jun K. Yamashita, and Knut Woltjen. "Modeling mutation-specific arrhythmogenic phenotypes in isogenic human iPSC-derived cardiac tissues." Scientific Reports 14, no. 1 (January 31, 2024). http://dx.doi.org/10.1038/s41598-024-52871-1.
Abstract:
AbstractDisease modeling using human induced pluripotent stem cells (hiPSCs) from patients with genetic disease is a powerful approach for dissecting pathophysiology and drug discovery. Nevertheless, isogenic controls are required to precisely compare phenotypic outcomes from presumed causative mutations rather than differences in genetic backgrounds. Moreover, 2D cellular models often fail to exhibit authentic disease phenotypes resulting in poor validation in vitro. Here we show that a combination of precision gene editing and bioengineered 3D tissue models can establish advanced isogenic hiPSC-derived cardiac disease models, overcoming these drawbacks. To model inherited cardiac arrhythmias we selected representative N588D and N588K missense mutations affecting the same codon in the hERG potassium channel gene KCNH2, which are reported to cause long (LQTS) and short (SQTS) QT syndromes, respectively. We generated compound heterozygous variants in normal hiPSCs, and differentiated cardiomyocytes (CMs) and mesenchymal cells (MCs) to form 3D cardiac tissue sheets (CTSs). In hiPSC-derived CM monolayers and 3D CTSs, electrophysiological analysis with multielectrode arrays showed prolonged and shortened repolarization, respectively, compared to the isogenic controls. When pharmacologically inhibiting the hERG channels, mutant 3D CTSs were differentially susceptible to arrhythmic events than the isogenic controls. Thus, this strategy offers advanced disease models that can reproduce clinically relevant phenotypes and provide solid validation of gene mutations in vitro.
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Lebedeva, Irina V., Michelle V. Wagner, Sunil Sahdeo, Yi-Fan Lu, Anuli Anyanwu-Ofili, Matthew B. Harms, Jehangir S. Wadia, Gunaretnam Rajagopal, Michael J. Boland, and David B. Goldstein. "Precision genetic cellular models identify therapies protective against ER stress." Cell Death & Disease 12, no. 8 (August 2021). http://dx.doi.org/10.1038/s41419-021-04045-4.
Abstract:
AbstractRare monogenic disorders often share molecular etiologies involved in the pathogenesis of common diseases. Congenital disorders of glycosylation (CDG) and deglycosylation (CDDG) are rare pediatric disorders with symptoms that range from mild to life threatening. A biological mechanism shared among CDG and CDDG as well as more common neurodegenerative diseases such as Alzheimer’s disease and amyotrophic lateral sclerosis, is endoplasmic reticulum (ER) stress. We developed isogenic human cellular models of two types of CDG and the only known CDDG to discover drugs that can alleviate ER stress. Systematic phenotyping confirmed ER stress and identified elevated autophagy among other phenotypes in each model. We screened 1049 compounds and scored their ability to correct aberrant morphology in each model using an agnostic cell-painting assay based on >300 cellular features. This primary screen identified multiple compounds able to correct morphological phenotypes. Independent validation shows they also correct cellular phenotypes and alleviate each of the ER stress markers identified in each model. Many of the active compounds are associated with microtubule dynamics, which points to new therapeutic opportunities for both rare and more common disorders presenting with ER stress, such as Alzheimer’s disease and amyotrophic lateral sclerosis.