Publications by authors named "Fred Harbinski"

14 Publications

  • Page 1 of 1

SMOOT libraries and phage-induced directed evolution of Cas9 to engineer reduced off-target activity.

PLoS One 2020 16;15(4):e0231716. Epub 2020 Apr 16.

Editas Medicine, Cambridge, Massachusetts, United States of America.

RNA-guided endonucleases such as Cas9 provide efficient on-target genome editing in cells but may also cleave at off-target loci throughout the genome. Engineered variants of Streptococcus pyogenes Cas9 (SpCas9) have been developed to globally reduce off-target activity, but individual off-targets may remain, or on-target activity may be compromised. In order to evolve against activity at specific off-targets while maintaining strong on-target editing, we developed a novel M13 bacteriophage-mediated selection method. Using this method, sequential rounds of positive and negative selection are used to identify mutations to Cas9 that enhance or diminish editing activity at particular genomic sequences. We also introduce scanning mutagenesis of oligo-directed targets (SMOOT), a comprehensive mutagenesis method to create highly diverse libraries of Cas9 variants that can be challenged with phage-based selection. Our platform identifies novel SpCas9 mutants which mitigate cleavage against off-targets both in biochemical assays and in T-cells while maintaining higher on-target activity than previously described variants. We describe an evolved variant, S. pyogenes Adapted to Reduce Target Ambiguity Cas9 (SpartaCas), composed of the most enriched mutations, each of unknown function. This evolved Cas9 mutant reduces off-target cleavage while preserving efficient editing at multiple therapeutically relevant targets. Directed evolution of Cas9 using our system demonstrates an improved structure-independent methodology to effectively engineer nuclease activity.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0231716PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7161989PMC
August 2020

Recurrent ubiquitin B silencing in gynecological cancers establishes dependence on ubiquitin C.

J Clin Invest 2017 12 13;127(12):4554-4568. Epub 2017 Nov 13.

Chemical Biology and Therapeutics, Novartis Institutes for Biomedical Research, Cambridge, Massachusetts, USA.

Transcriptional repression of ubiquitin B (UBB) is a cancer-subtype-specific alteration that occurs in a substantial population of patients with cancers of the female reproductive tract. UBB is 1 of 2 genes encoding for ubiquitin as a polyprotein consisting of multiple copies of ubiquitin monomers. Silencing of UBB reduces cellular UBB levels and results in an exquisite dependence on ubiquitin C (UBC), the second polyubiquitin gene. UBB is repressed in approximately 30% of high-grade serous ovarian cancer (HGSOC) patients and is a recurrent lesion in uterine carcinosarcoma and endometrial carcinoma. We identified ovarian tumor cell lines that retain UBB in a repressed state, used these cell lines to establish orthotopic ovarian tumors, and found that inducible expression of a UBC-targeting shRNA led to tumor regression, and substantial long-term survival benefit. Thus, we describe a recurrent cancer-specific lesion at the level of ubiquitin production. Moreover, these observations reveal the prognostic value of UBB repression and establish UBC as a promising therapeutic target for ovarian cancer patients with recurrent UBB silencing.
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http://dx.doi.org/10.1172/JCI92914DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5707153PMC
December 2017

Jenkins-CI, an Open-Source Continuous Integration System, as a Scientific Data and Image-Processing Platform.

SLAS Discov 2017 03 13;22(3):238-249. Epub 2016 Dec 13.

3 Developmental and Molecular Pathways, NIBR, Postfach, Basel, Switzerland.

High-throughput screening generates large volumes of heterogeneous data that require a diverse set of computational tools for management, processing, and analysis. Building integrated, scalable, and robust computational workflows for such applications is challenging but highly valuable. Scientific data integration and pipelining facilitate standardized data processing, collaboration, and reuse of best practices. We describe how Jenkins-CI, an "off-the-shelf," open-source, continuous integration system, is used to build pipelines for processing images and associated data from high-content screening (HCS). Jenkins-CI provides numerous plugins for standard compute tasks, and its design allows the quick integration of external scientific applications. Using Jenkins-CI, we integrated CellProfiler, an open-source image-processing platform, with various HCS utilities and a high-performance Linux cluster. The platform is web-accessible, facilitates access and sharing of high-performance compute resources, and automates previously cumbersome data and image-processing tasks. Imaging pipelines developed using the desktop CellProfiler client can be managed and shared through a centralized Jenkins-CI repository. Pipelines and managed data are annotated to facilitate collaboration and reuse. Limitations with Jenkins-CI (primarily around the user interface) were addressed through the selection of helper plugins from the Jenkins-CI community.
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http://dx.doi.org/10.1177/1087057116679993DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5322829PMC
March 2017

High-Order Drug Combinations Are Required to Effectively Kill Colorectal Cancer Cells.

Cancer Res 2016 12 22;76(23):6950-6963. Epub 2016 Sep 22.

Translational Clinical Oncology, Novartis Institutes for Biomedical Research, Cambridge, Massachusetts.

Like classical chemotherapy regimens used to treat cancer, targeted therapies will also rely upon polypharmacology, but tools are still lacking to predict which combinations of molecularly targeted drugs may be most efficacious. In this study, we used image-based proliferation and apoptosis assays in colorectal cancer cell lines to systematically investigate the efficacy of combinations of two to six drugs that target critical oncogenic pathways. Drug pairs targeting key signaling pathways resulted in synergies across a broad spectrum of genetic backgrounds but often yielded only cytostatic responses. Enhanced cytotoxicity was observed when additional processes including apoptosis and cell cycle were targeted as part of the combination. In some cases, where cell lines were resistant to paired and tripled drugs, increased expression of antiapoptotic proteins was observed, requiring a fourth-order combination to induce cytotoxicity. Our results illustrate how high-order drug combinations are needed to kill drug-resistant cancer cells, and they also show how systematic drug combination screening together with a molecular understanding of drug responses may help define optimal cocktails to overcome aggressive cancers. Cancer Res; 76(23); 6950-63. ©2016 AACR.
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http://dx.doi.org/10.1158/0008-5472.CAN-15-3425DOI Listing
December 2016

Metabolic Enzyme Sulfotransferase 1A1 Is the Trigger for N-Benzyl Indole Carbinol Tumor Growth Suppression.

Chem Biol 2015 Sep 10;22(9):1228-37. Epub 2015 Sep 10.

Department of Developmental and Molecular Pathways, Novartis Institutes for Biomedical Research, 250 Massachusetts Avenue, Cambridge, MA 02139, USA. Electronic address:

In an attempt to identify novel therapeutics and mechanisms to differentially kill tumor cells using phenotypic screening, we identified N-benzyl indole carbinols (N-BICs), synthetic analogs of the natural product indole-3-carbinol (I3C). To understand the mode of action for the molecules we employed Cancer Cell Line Encyclopedia viability profiling and correlative informatics analysis to identify and ultimately confirm the phase II metabolic enzyme sulfotransferase 1A1 (SULT1A1) as the essential factor for compound selectivity. Further studies demonstrate that SULT1A1 activates the N-BICs by rendering the compounds strong electrophiles which can alkylate cellular proteins and thereby induce cell death. This study demonstrates that the selectivity profile for N-BICs is through conversion by SULT1A1 from an inactive prodrug to an active species that induces cell death and tumor suppression.
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http://dx.doi.org/10.1016/j.chembiol.2015.06.025DOI Listing
September 2015

Selective VPS34 inhibitor blocks autophagy and uncovers a role for NCOA4 in ferritin degradation and iron homeostasis in vivo.

Nat Cell Biol 2014 Nov 19;16(11):1069-79. Epub 2014 Oct 19.

Novartis Institutes for BioMedical Research, 250 Massachusetts Avenue, Cambridge, Massachusetts 02139, USA.

Cells rely on autophagy to clear misfolded proteins and damaged organelles to maintain cellular homeostasis. In this study we use the new autophagy inhibitor PIK-III to screen for autophagy substrates. PIK-III is a selective inhibitor of VPS34 that binds a unique hydrophobic pocket not present in related kinases such as PI(3)Kα. PIK-III acutely inhibits autophagy and de novo lipidation of LC3, and leads to the stabilization of autophagy substrates. By performing ubiquitin-affinity proteomics on PIK-III-treated cells we identified substrates including NCOA4, which accumulates in ATG7-deficient cells and co-localizes with autolysosomes. NCOA4 directly binds ferritin heavy chain-1 (FTH1) to target the iron-binding ferritin complex with a relative molecular mass of 450,000 to autolysosomes following starvation or iron depletion. Interestingly, Ncoa4(-/-) mice exhibit a profound accumulation of iron in splenic macrophages, which are critical for the reutilization of iron from engulfed red blood cells. Taken together, the results of this study provide a new mechanism for selective autophagy of ferritin and reveal a previously unappreciated role for autophagy and NCOA4 in the control of iron homeostasis in vivo.
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http://dx.doi.org/10.1038/ncb3053DOI Listing
November 2014

Identification of mixed lineage leukemia 1(MLL1) protein as a coactivator of heat shock factor 1(HSF1) protein in response to heat shock protein 90 (HSP90) inhibition.

J Biol Chem 2014 Jul 15;289(27):18914-27. Epub 2014 May 15.

From the Departments of Oncology,

Heat shock protein 90 (HSP90) inhibition inhibits cancer cell proliferation through depleting client oncoproteins and shutting down multiple oncogenic pathways. Therefore, it is an attractive strategy for targeting human cancers. Several HSP90 inhibitors, including AUY922 and STA9090, show promising effects in clinical trials. However, the efficacy of HSP90 inhibitors may be limited by heat shock factor 1 (HSF1)-mediated feedback mechanisms. Here, we identify, through an siRNA screen, that the histone H3 lysine 4 methyltransferase MLL1 functions as a coactivator of HSF1 in response to HSP90 inhibition. MLL1 is recruited to the promoters of HSF1 target genes and regulates their expression in response to HSP90 inhibition. In addition, a striking combination effect is observed when MLL1 depletion is combined with HSP90 inhibition in various human cancer cell lines and tumor models. Thus, targeting MLL1 may block a HSF1-mediated feedback mechanism induced by HSP90 inhibition and provide a new avenue to enhance HSP90 inhibitor activity in human cancers.
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http://dx.doi.org/10.1074/jbc.M114.574053DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4081932PMC
July 2014

Target identification for a Hedgehog pathway inhibitor reveals the receptor GPR39.

Nat Chem Biol 2014 May 16;10(5):343-9. Epub 2014 Mar 16.

Novartis Institutes for Biomedical Research, Cambridge, Massachusetts, USA.

Hedgehog (Hh) signaling determines cell fate during development and can drive tumorigenesis. We performed a screen for new compounds that can impinge on Hh signaling downstream of Smoothened (Smo). A series of cyclohexyl-methyl aminopyrimidine chemotype compounds ('CMAPs') were identified that could block pathway signaling in a Smo-independent manner. In addition to inhibiting Hh signaling, the compounds generated inositol phosphates through an unknown GPCR. Correlation of GPCR mRNA expression levels with compound activity across cell lines suggested the target to be the orphan receptor GPR39. RNA interference or cDNA overexpression of GPR39 demonstrated that the receptor is necessary for compound activity. We propose a model in which CMAPs activate GPR39, which signals to the Gli transcription factors and blocks signaling. In addition to the discovery of GPR39 as a new target that impinges on Hh signaling, we report on small-molecule modulators of the receptor that will enable in vitro interrogation of GPR39 signaling in different cellular contexts.
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http://dx.doi.org/10.1038/nchembio.1481DOI Listing
May 2014

Small-Molecule targeting of translation initiation for cancer therapy.

Oncotarget 2013 Oct;4(10):1606-17

Department of Medicine, Brigham and Women's Hospital, Boston.

Translation initiation plays a critical role in the regulation of cell growth and tumorigenesis. We report here that inhibiting translation initiation through induction of eIF2α phosphorylation by small-molecular-weight compounds restricts the availability of the eIF2.GTP.Met-tRNAi ternary complex and abrogates the proliferation of cancer cells in vitro and tumor growth in vivo. Restricting the availability of the ternary complex preferentially down-regulates the expression of growth-promoting proteins and up-regulates the expression of ER stress response genes in cancer cells as well as in tumors excised from either animal models of human cancer or cancer patients. These findings provide the first direct evidence for translational control of gene-specific expression by small molecules in vivo and indicate that translation initiation factors are bona fide targets for development of mechanism-specific anti-cancer agents.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3858549PMC
http://dx.doi.org/10.18632/oncotarget.1186DOI Listing
October 2013

Reporter enzyme inhibitor study to aid assembly of orthogonal reporter gene assays.

ACS Chem Biol 2013 May 26;8(5):1009-17. Epub 2013 Mar 26.

Center for Proteomic Chemistry, Novartis Institutes of Biomedical Research, 250 Massachusetts Avenue, Cambridge, MA 02139, USA.

Reporter gene assays (RGAs) are commonly used to measure biological pathway modulation by small molecules. Understanding how such compounds interact with the reporter enzyme is critical to accurately interpret RGA results. To improve our understanding of reporter enzymes and to develop optimal RGA systems, we investigated eight reporter enzymes differing in brightness, emission spectrum, stability, and substrate requirements. These included common reporter enzymes such as firefly luciferase (Photinus pyralis), Renilla reniformis luciferase, and β-lactamase, as well as mutated forms of R. reniformis luciferase emitting either blue- or green-shifted luminescence, a red-light emitting form of Luciola cruciata firefly luciferase, a mutated form of Gaussia princeps luciferase, and a proprietary luciferase termed "NanoLuc" derived from the luminescent sea shrimp Oplophorus gracilirostris. To determine hit rates and structure-activity relationships, we screened a collection of 42,460 PubChem compounds at 10 μM using purified enzyme preparations. We then compared hit rates and chemotypes of actives for each enzyme. The hit rates ranged from <0.1% for β-lactamase to as high as 10% for mutated forms of Renilla luciferase. Related luciferases such as Renilla luciferase mutants showed high degrees of inhibitor overlap (40-70%), while unrelated luciferases such as firefly luciferases, Gaussia luciferase, and NanoLuc showed <10% overlap. Examination of representative inhibitors in cell-based assays revealed that inhibitor-based enzyme stabilization can lead to increases in bioluminescent signal for firefly luciferase, Renilla luciferase, and NanoLuc, with shorter half-life reporters showing increased activation responses. From this study we suggest strategies to improve the construction and interpretation of assays employing these reporter enzymes.
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http://dx.doi.org/10.1021/cb3007264DOI Listing
May 2013

Rescue screens with secreted proteins reveal compensatory potential of receptor tyrosine kinases in driving cancer growth.

Cancer Discov 2012 Oct 8;2(10):948-59. Epub 2012 Aug 8.

Developmental and Molecular Pathways, Novartis Institutes for BioMedical Research, Cambridge, Massachusetts, USA.

The overall power of kinase inhibitors is substantially overshadowed by the acquisition of drug resistance. To address this issue, we systematically assessed the potential of secreted proteins to induce resistance to kinase inhibitors. To this end, we developed a high-throughput platform for screening a cDNA library encoding 3,432 secreted proteins in cellular assays. Using cancer cells originally dependent on either MET, FGFR2, or FGFR3, we observed a bypass of dependence through ligand-mediated activation of alternative receptor tyrosine kinases (RTK). Our findings indicate a broad and versatile potential for RTKs from the HER and FGFR families as well as MET to compensate for loss of each other. We further provide evidence that combined inhibition of simultaneously active RTKs can lead to an added anticancer effect.
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http://dx.doi.org/10.1158/2159-8290.CD-12-0237DOI Listing
October 2012

Chemical genetics identify eIF2α kinase heme-regulated inhibitor as an anticancer target.

Nat Chem Biol 2011 Jul 17;7(9):610-6. Epub 2011 Jul 17.

Laboratory for Translational Research, Harvard Medical School, Boston, Massachusetts, USA.

Translation initiation plays a critical role in cellular homeostasis, proliferation, differentiation and malignant transformation. Consistently, increasing the abundance of the eIF2-GTP-tRNA(i)(Met) translation initiation complex transforms normal cells and contributes to cancer initiation and the severity of some anemias. The chemical modifiers of the eIF2-GTP-tRNA(i)(Met) ternary complex are therefore invaluable tools for studying its role in the pathobiology of human disorders and for determining whether this complex can be pharmacologically targeted for therapeutic purposes. Using a cell-based assay, we identified N,N'-diarylureas as unique inhibitors of ternary complex accumulation. Direct functional-genetic and biochemical evidence demonstrated that the N,N'-diarylureas activate heme-regulated inhibitor kinase, thereby phosphorylating eIF2α and reducing the abundance of the ternary complex. Using tumor cell proliferation in vitro and tumor growth in vivo as paradigms, we demonstrate that N,N'-diarylureas are potent and specific tools for studying the role of eIF2-GTP-tRNA(i)(Met) ternary complex in the pathobiology of human disorders.
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http://dx.doi.org/10.1038/nchembio.613DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3684262PMC
July 2011

Synthesis and biological evaluation of thiazolidine-2,4-dione and 2,4-thione derivatives as inhibitors of translation initiation.

Bioorg Med Chem Lett 2004 Nov;14(21):5401-5

Laboratory for Translational Research, Harvard Medical School, Cambridge, MA 02139, USA.

In an effort to generate novel translation initiation inhibitors for cancer therapy, a series of 2'-benzyloxy-5'-substituted-5-benzylidene-thiazolidine-2,4-thione and dione derivatives was synthesized and evaluated for activity in translation initiation specific assays. Several candidates of the 5-benzylidene-thiazolidine-2,4-diones (3c, 3d, and 3f) and -thiones (2b, 2e, and 2j), inhibit cell growth with low microM GI(50) mediated by inhibition of translation initiation, which involves partial depletion of intracellular Ca(2+) stores and strong phosphorylation of eIF2alpha.
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http://dx.doi.org/10.1016/j.bmcl.2004.08.017DOI Listing
November 2004

Structure-activity requirements for the antiproliferative effect of troglitazone derivatives mediated by depletion of intracellular calcium.

Bioorg Med Chem Lett 2004 May;14(10):2547-50

Laboratory for Translational Research, Harvard Medical School, Cambridge, MA 02139, USA.

Depletion of calcium from the endoplasmic reticulum has shown to affect protein synthesis and cell proliferation. The anticancer effect of troglitazone was reported to be mediated by depletion of intracellular calcium stores resulting in inhibition of translation initiation. The unsaturated form of troglitazone displays similar anticancer properties in vitro. In this letter, we report our findings on the minimum structural requirements for both compounds to retain their calcium release and antiproliferative activities.
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http://dx.doi.org/10.1016/j.bmcl.2004.02.087DOI Listing
May 2004