Name: Methods for Evaluating the Role of c-Fos and Dusp1 in Oncogene Dependence (Video) | JoVE Journal
Uploaded: 2019-01-07T14:00:00.000+00:00
Duration: 10 min 9 s
Description: Learn methods to validate c-Fos and Dusp1 as therapeutic targets in leukemia using genetic and chemical approaches. This protocol applies to multiple kinase-driven cancers and solid tumors.

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5 minutes

The authors are thankful to G. Q. Daley for providing the BaF3 and WEHI cells and T. Reya for the MSCV-BCR-ABL-Ires-YFP constructs. The authors are thankful to M. Carroll for providing the patient samples from the CML blast crisis. This study was supported by grants to M.A. from the NCI (1RO1CA155091), the Leukemia Research Foundation and V Foundation, and from the NHLBI (R21HL114074-01).

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The authors have nothing to disclose.

For the bulk of cancer cells, the therapeutic response to TKI is mediated by a blockade of tyrosine-kinase-oncoprotein signals to which the tumor is addicted. However, relatively little is known about how a minority of cancer cells contributing to MRD escape the oncogene dependence and therapy4. Recent studies revealed that growth factor signaling mediates drug resistance in both leukemia and solid organ tumors. This suggests that various molecular mechanisms might underlie intrinsic resistance<su...

Constitutive tyrosine kinase activity of BCR-ABL1 fusion oncogene causes CML, which provides a rationale to target the kinase activity by small molecule inhibitors. The success of TKIs in treating CML patients revolutionized the concept of targeted therapy1,2. Subsequently, anti-kinase therapy as precision medicine was developed for several other malignancies, including solid tumors. So far, more than thirty kinase inhibitors have been approved by the United State FDA for treating various malignancies. While TKI treatment is very effective in suppressing the disease, it is not curative. Besides, a small population of cancer cells persists during the treatment: the MRD3,4,5. Even patients who showed complete remission are left with MRD, which eventually results in relapse if not continuously suppressed. Therefore, the eradication of MRD cells is needed to achieve a durable or curative response. CML represents a valuable paradigm for defining the concept of precision medicine, mechanisms of oncogenesis, rational target-directed therapeutics, disease progression, and drug resistance. However, even today, the mechanism driving TKI-induced cell death in cancer cells is not fully understood, nor why MRD cells (comprised of leukemic stem cells [LSCs]) are intrinsically resistant to TKIs4,6. Nonetheless, the phenomenon of &#34;oncogene dependence&#34; to mutant kinase oncoprotein is implicated in TKI efficacy where the acute inhibition of targeted oncogene by TKIs causes an oncogenic shock that leads to a massive proapoptotic response or quiescence in cell context-dependent manner6,7,8,9. However, the mechanistic underpinning of oncogene dependence is lacking. Recent studies have implicated that growth factor signaling abrogates oncogene dependence and consequently confers resistance to TKI therapy10,11,12. Therefore, to gain insight into the mechanism of oncogene dependence, we performed whole-genome expression profiling from BCR-ABL1 addicted and nonaddicted cells (grown with growth factors), which revealed that c-Fos and Dusp1 are critical mediators of oncogene addiction13. The genetic deletion of c-Fos and Dusp1 is synthetic lethal to BCR-ABL1-expressing cells and the mice used in the experiment did not develop leukemia. Moreover, the inhibition of c-Fos and DUSP1 by small molecule inhibitors cured BCR-ABL1-induced CML in mice. The results show that the expression levels of c-Fos and Dusp1 define the apoptotic threshold in cancer cells, such that lower levels confer drug sensitivity while higher levels cause resistance to therapy13.
To identify the genes driving the oncogene dependence, we performed several whole-genome expression profiling experiments in the presence of growth factor and a TKI (imatinib) using both mouse- and CML patient-derived cells (K562). These data were analyzed in parallel with CML patient data sets obtained from CD34+ hematopoietic stem cells before and after treatment with imatinib. This analysis revealed three genes (a transcription factor [c-Fos], dual specificity phosphatase 1 [Dusp1], and an RNA-binding protein [Zfp36]) which are commonly upregulated in TKI-resistant cells. To validate the significance of these genes in conferring drug resistance, we carried out step-by-step in vitro and in vivo analysis. The expression levels of these genes were confirmed by real-time qPCR (RT-qPCR) and western blotting in drug-resistant cells. Further, cDNA overexpression and knockdown by shRNA hairpins of c-Fos, Dusp1, and Zfp36 revealed that elevated c-Fos and Dusp1 expressions are sufficient and necessary to confer TKI resistance. Therefore, we performed an in vivo validation using mouse models with c-Fos and Dusp1 only. For the genetic validation of c-Fos and Dusp1, we created ROSACreERT-inducible c-Fosfl/fl mice (conditional knockout)14 and crossed them with Dusp1-/- (straight knockout)15 to make ROSACreERT2-c-Fosfl/flDusp1-/- double-transgenic mice. Bone marrow-derived c-Kit+ cells (from c-Fosfl/fl-, Dusp1-/--, and c-Fosfl/flDusp1-/-) expressing BCR-ABL1 were analyzed in vitro in a colony-forming unit (CFU) assay, and in vivo by bone marrow transplantation in lethally irradiated mice, to test the requirement of c-Fos and Dusp1 alone or together in leukemia development. Likewise, the chemical inhibitions of c-Fos by DFC (difluorinated curcumin)16 and Dusp1 by BCI (benzylidene-3-(cyclohexylamino)-2,3-dihydro-1H-inden-1-one)17 were tested in vitro and in vivo using BCR-ABL1-expressing, bone marrow-derived c-Kit+ cells from the wild-type (WT) mouse. To confirm the requirement of c-Fos and Dusp1 in leukemic stem cells, we utilized a CML mouse model where BCR-ABL1 was specifically induced in its stem cells by doxycycline (Tet-transactivator expresses under murine stem cell leukemia (SCL) gene 3&#39; enhancer regulation)18,19. We used bone marrow Lin-Sca+c-Kit+ (LSK) cells from these mice in an in vivo transplant assay. Furthermore, we established phopsho-p38 levels and the expression of IL-6 as pharmaco-dynamic biomarkers for Dusp1 and c-Fos inhibition, respectively, in vivo. Finally, to extend the study for human relevance, patient-derived CD34+ cells (equivalent to the c-Kit+ cells from mice) were subjected to long-term in vitro culture-initiating cell assays (LTCIC) and an in vivo humanized mouse model of CML20,21. The immunodeficient mice were transplanted with CML CD34+ cells, followed by drug treatment and analysis of human leukemic cell survival.
In this project, we develop methods for target identification and validations using both genetic and chemical tools, using different preclinical models. These methods can be successfully applied to validate other targets developing chemical modalities for therapeutic development.

<table><tbody><tr><td>&lt;strong&gt;Biological Materials&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>RPMI</td><td>Cellgro (corning)</td><td>15-040-CV</td><td></td></tr><tr><td>DMEM</td><td>Cellgro (corning)</td><td>15-013-CV</td><td></td></tr><tr><td>IMDM</td><td>Cellgro (corning)</td><td>15-016-CVR</td><td></td></tr><tr><td>RetroNectin Recombinant Human Fibronectin Fragment</td><td>Takara</td><td>T100B</td><td></td></tr><tr><td>MethoCult GF M3434 (Methylcellulose for Mouse CFU)</td><td>Stem Cell</td><td>3434</td><td></td></tr><tr><td>MethoCult H4434 Classic (Methylcellulose for Human CFU)</td><td>Stem Cell</td><td>4434</td><td></td></tr><tr><td>4-Hydroxytamoxifen</td><td>Sigma</td><td>H6278</td><td></td></tr><tr><td>Recombinant Murine SCF</td><td>Prospec</td><td>CYT-275</td><td></td></tr><tr><td>Recombinant Murine Flt3-Ligand</td><td>Prospec</td><td>CYT-340</td><td></td></tr><tr><td>Recombinant Murine IL-6</td><td>Prospec</td><td>CYT-350</td><td></td></tr><tr><td>Recombinant Murine IL-7</td><td>Peprotech</td><td>217-17</td><td></td></tr><tr><td>DFC</td><td>LKT Laboratories Inc.</td><td>D3420</td><td></td></tr><tr><td>BCI</td><td>Chemzon Scientific</td><td>NZ-06-195</td><td></td></tr><tr><td>Imatinib</td><td>LC Laboratory</td><td>I-5508</td><td></td></tr><tr><td>Curcumin</td><td>Sigma</td><td>458-37-7</td><td></td></tr><tr><td>NDGA</td><td>Sigma</td><td>500-38-9</td><td></td></tr><tr><td>Penn/Strep</td><td>Cellgro (corning)</td><td>30-002-CI</td><td></td></tr><tr><td>FBS</td><td>Atlanta biological</td><td>S11150</td><td></td></tr><tr><td>Trypsin EDTA 1x</td><td>Cellgro (corning)</td><td>25-052-CI</td><td></td></tr><tr><td>1x PBS</td><td>Cellgro (corning)</td><td>21-040-CV</td><td></td></tr><tr><td>L-Glutamine</td><td>Cellgro (corning)</td><td>25-005-CL</td><td>5 mg/mL stock in water</td></tr><tr><td>Puromycin</td><td>Gibco (life technologies)</td><td>A11138-03</td><td></td></tr><tr><td>HEPES</td><td>Sigma</td><td>H7006</td><td></td></tr><tr><td>Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;.7H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Sigma</td><td>S9390</td><td></td></tr><tr><td>Protamine sulfate</td><td>Sigma</td><td>P3369</td><td>5 mg/mL stock in water</td></tr><tr><td>Trypan Blue solution (0.4%)</td><td>Sigma</td><td>T8154</td><td></td></tr><tr><td>DMSO</td><td>Cellgro (corning)</td><td>25-950-CQC</td><td></td></tr><tr><td>WST-1</td><td>Roche</td><td>11644807001</td><td></td></tr><tr><td>0.45 &amp;mu;M acro disc filter</td><td>PALL</td><td>2016-10</td><td></td></tr><tr><td>70 &amp;mu;m nylon cell stariner</td><td>Becton Dickinson</td><td>352350</td><td></td></tr><tr><td>FICOL (Histopaque 1083) (polysucrose)</td><td>Simga</td><td>1083</td><td></td></tr><tr><td>PBS</td><td>Corning</td><td>21040CV</td><td></td></tr><tr><td>LS Columns</td><td>Miltenyi</td><td>130-042-401</td><td></td></tr><tr><td>Protease Inhibitor Cocktail</td><td>Roche</td><td>CO-RO</td><td></td></tr><tr><td>Phosphatase Inhibitor Cocktail 2</td><td>Sigma</td><td>P5762</td><td></td></tr><tr><td>Nitrocullulose Membrane</td><td>Bio-Rad</td><td>1620115</td><td></td></tr><tr><td>SuperSignal West Dura Extended Duration Substrate ( chemiluminiscence substrate)</td><td>Thermo Scientific</td><td>34075</td><td></td></tr><tr><td>CD5</td><td>eBioscience</td><td>13-0051-82</td><td></td></tr><tr><td>CD11b</td><td>eBioscience</td><td>13-0112-75</td><td></td></tr><tr><td>CD45R (B220)</td><td>BD biosciences</td><td>553092</td><td></td></tr><tr><td>CD45.1-FITC</td><td>eBioscience</td><td>11-0453-85</td><td></td></tr><tr><td>CD45.2-PE</td><td>eBioscience</td><td>12-0454-83</td><td></td></tr><tr><td>hCD45-FITC</td><td>BD Biosciences</td><td>555482</td><td></td></tr><tr><td>Anti-Biotin-FITC</td><td>Miltenyi</td><td>130-090-857&amp;nbsp;</td><td></td></tr><tr><td>Anti-7-4</td><td>eBioscience</td><td>MA5-16539</td><td></td></tr><tr><td>Anti-Gr-1 (Ly-6G/c)</td><td>eBioscience</td><td>13-5931-82</td><td></td></tr><tr><td>Anti-Ter-119</td><td>eBioscience</td><td>13-5921-75</td><td></td></tr><tr><td>Ly-6 A/E (Sca1) PE Cy7</td><td>BD&amp;nbsp;</td><td>558612</td><td></td></tr><tr><td>CD117 APC&amp;nbsp;</td><td>BD&amp;nbsp;</td><td>553356</td><td></td></tr><tr><td>BD Pharm Lyse</td><td>BD&amp;nbsp;</td><td>555899</td><td></td></tr><tr><td>BD Cytofix/Cytoperm (Fixing and permeabilization solution)</td><td>BD&amp;nbsp;</td><td>554714</td><td></td></tr><tr><td>BD Perm/Wash&amp;nbsp; (permeabilization and wash solution for phospho flow)</td><td>BD&amp;nbsp;</td><td>554723</td><td></td></tr><tr><td>phospho p38</td><td>Cell Signaling Technologies</td><td>4511S</td><td></td></tr><tr><td>total p38</td><td>Cell Signaling Technologies</td><td>9212</td><td></td></tr><tr><td>Mouse IgG control</td><td>BD&amp;nbsp;</td><td>554121</td><td></td></tr><tr><td>Alexa Flour 488 conjugated&amp;nbsp;</td><td>Invitrogen</td><td>A-11034</td><td></td></tr><tr><td>Calcium Chloride</td><td>Invitrogen</td><td>K278001</td><td></td></tr><tr><td>2x HBS</td><td>Invitrogen</td><td>K278002</td><td></td></tr><tr><td>EDTA</td><td>Ambion</td><td>AM9261</td><td></td></tr><tr><td>BSA</td><td>Sigma</td><td>A7906</td><td></td></tr><tr><td>Blood Capillary Tubes</td><td>Fisher</td><td>22-260-950</td><td></td></tr><tr><td>Blood Collection Tube</td><td>Giene Bio-One</td><td>450480</td><td></td></tr><tr><td>Newborn Calf Serum</td><td>Atlanta biological</td><td>S11295</td><td></td></tr><tr><td>Erythropoiein</td><td>Amgen</td><td>5513-267-10</td><td></td></tr><tr><td>human SCF</td><td>Prospec</td><td>CYT-255</td><td></td></tr><tr><td>Human IL-3</td><td>Prospec</td><td>CYT-210</td><td></td></tr><tr><td>G-SCF</td><td>Prospec</td><td>CYT-220</td><td></td></tr><tr><td>GM-CSF</td><td>Prospec</td><td>CYT-221</td><td></td></tr><tr><td>MyeloCult (media for LTCIC assay)</td><td>Stem Cell Technologies</td><td>5100</td><td></td></tr><tr><td>Hydrocortisone Sodium Hemisuccinate</td><td>Stem Cell Technologies</td><td>7904</td><td></td></tr><tr><td>MEM alpha</td><td>Gibco</td><td>12561-056</td><td></td></tr><tr><td>1/2 cc Lo-Dose u-100 insulin syringe 28 G1/2</td><td>Becton Dickinson</td><td>329461</td><td></td></tr><tr><td>Mortor pestle</td><td>Coor tek&amp;nbsp;</td><td>60316 and 60317</td><td></td></tr><tr><td>Isoflorane (Isothesia TM)</td><td>Butler Schien</td><td>29405</td><td></td></tr><tr><td>SOC</td><td>New England Biolabs</td><td>B90920s</td><td></td></tr><tr><td>Ampicillin</td><td>Sigma</td><td>A0166</td><td>100 mg/mL stock in water</td></tr><tr><td>Bacto agar (agar)</td><td>Difco</td><td>214050</td><td></td></tr><tr><td>Terrific broth</td><td>Becton Dickinson</td><td>243820</td><td></td></tr><tr><td>Agarose</td><td>Genemate</td><td>E-3119-500</td><td></td></tr><tr><td>Doxycycline chow</td><td>TestDiet.com</td><td>52662</td><td>modified RMH1500, Autoclavable 5LK8&amp;nbsp; with 0.0625% Doxycycline&amp;nbsp;</td></tr><tr><td>Tamoxifen</td><td>Sigma</td><td>T5648</td><td></td></tr><tr><td>Iodonitrotetrazolium chloride&amp;nbsp;</td><td>Sigma</td><td>I10406</td><td></td></tr><tr><td>&lt;strong&gt;Kits&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Dneasy Blood &amp;amp; tissue kit</td><td>Qiagen</td><td>69506</td><td></td></tr><tr><td>GoTaq Green (taq polymerase with Green loadign dye)</td><td>Promega</td><td>M1722</td><td></td></tr><tr><td>miRNeasy Mini Kit&amp;nbsp; (RNA isolation kit)</td><td>Qiagen</td><td>217084</td><td></td></tr><tr><td>DNA Free Dnase Kit (DNAse treatment for RT PCR)</td><td>Ambion, Life Technologies</td><td>AM1906</td><td></td></tr><tr><td>Superscript III First Strand Synthesis (reverse transcriptase for cDNA synthesis)</td><td>Invitrogen</td><td>18080051</td><td></td></tr><tr><td>SYBR Green (taq polymerase mix with green interchalating dye for qPCR)</td><td>Bio-Rad</td><td>1725270</td><td></td></tr><tr><td>CD117 MicroBead Kit</td><td>Miltenyi</td><td>130-091-224</td><td></td></tr><tr><td>Human Long-Term Culture Initiating Cell Assay</td><td>Stemp Cell Technologies</td><td></td><td></td></tr><tr><td>&lt;strong&gt;Instruments&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>NAPCO series 8000 WJ CO&lt;sub&gt;2&lt;/sub&gt; incubator</td><td>Thermo scientific</td><td></td><td></td></tr><tr><td>Swing bucket rotor cetrifuge 5810R</td><td>Eppendorf</td><td></td><td></td></tr><tr><td>TC-10 automated cell counter</td><td>Bio-RAD</td><td></td><td></td></tr><tr><td>C-1000 Thermal cycler</td><td>Bio-RAD</td><td></td><td></td></tr><tr><td>Mastercycler Real Plex 2</td><td>Eppendorf</td><td></td><td></td></tr><tr><td>ChemiDoc Imaging System (imaging system for gels and western blots)</td><td>Bio-RAD</td><td>17001401</td><td></td></tr><tr><td>Hemavet (boold counter)</td><td>Drew-Scientific</td><td></td><td></td></tr><tr><td>LSR II (FACS analyzer)</td><td>BD&amp;nbsp;</td><td></td><td></td></tr><tr><td>Fortessa I (FACS analyzer)</td><td>BD&amp;nbsp;</td><td></td><td></td></tr><tr><td>FACSAriaII (FACS Sorter)</td><td>BD&amp;nbsp;</td><td></td><td></td></tr><tr><td>Magnet Stand</td><td>Miltenyi</td><td></td><td></td></tr><tr><td>Irradiator</td><td>&amp;nbsp;J.L. Shepherd and Associates, San Fernando CA</td><td>Mark I Model 68A</td><td>source Cs 137</td></tr><tr><td>&lt;strong&gt;Mice&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>ROSACreERT2</td><td>Jackson Laboratory</td><td></td><td></td></tr><tr><td>Scl-tTA&amp;nbsp;</td><td>Dr. Claudia Huettner&amp;rsquo;s lab</td><td></td><td></td></tr><tr><td>BoyJ&amp;nbsp;</td><td>mouse core facility at CCHMC</td><td></td><td></td></tr><tr><td>C57Bl/6&amp;nbsp;</td><td>Jackson Laboratory</td><td></td><td></td></tr><tr><td>NSGS</td><td>mouse core facility at CCHMC</td><td></td><td></td></tr><tr><td>ROSACreERT2/c-Fos&lt;sup&gt;fl/fl &lt;/sup&gt;Dusp1&lt;sup&gt;-/-&lt;/sup&gt;&amp;nbsp;</td><td>Made in house</td><td></td><td></td></tr><tr><td>ROSACreERT2/c-Fosfl/fl</td><td>Made in house</td><td></td><td></td></tr><tr><td>&lt;strong&gt;Cells&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>BaF3</td><td>Gift from George Daley, Harvard Medical School, Boston</td><td></td><td></td></tr><tr><td>WEHI</td><td>Gift from George Daley, Harvard Medical School, Boston</td><td></td><td></td></tr><tr><td>CML-CD34+ and Normal CD34+ cells</td><td>University Hospital, University of Cincinnati</td><td></td><td></td></tr></tbody></table>

methods for evaluating the role of c-fos and dusp1 in oncogene dependence

Methods for Evaluating the Role of c-Fos and Dusp1 in Oncogene Dependence

The demonstration of tyrosine kinase inhibitors (TKIs) in treating chronic myeloid leukemia (CML) has heralded a new era in cancer therapeutics. However, a small population of cells does not respond to TKI treatment, resulting in minimal residual disease (MRD); even the most potent TKIs fail to eradicate these cells. These MRD cells serve as a reservoir to develop resistance to therapy. Why TKI treatment is ineffective against MRD cells is not known. Growth factor signaling is implicated in supporting the survival of MRD cells during TKI treatment, but a mechanistic understanding is lacking. Recent studies demonstrated that an elevated c-Fos and Dusp1 expression as a result of convergent oncogenic and growth factor signaling in MRD cells mediate TKI resistance. The genetic and chemical inhibition of c-Fos and Dusp1 renders CML exquisitely sensitive to TKIs and cures CML in both genetic and humanized mouse models. We identified these target genes using multiple microarrays from TKI-sensitive and -resistant cells. Here, we provide methods for target validation using in vitro and in vivo mouse models. These methods can easily be applied to any target for genetic validation and therapeutic development.

Oncogene addiction has been implicated in the therapeutic efficacy of TKIs. However, the mechanisms driving the oncogene dependence are not understood. We performed multiple unbiased gene expression analyses to identify the genetic component involved in orchestrating the addiction. These analyses revealed the upregulation of three genes, c-Fos, Dusp1, and Zfp36, in cancer cells that are not dependent on oncogenic signaling for survival and, thus, are insensitive to TKI treatment. The down...

Here, we describe protocols for the genetic and chemical validation of c-Fos and Dusp1 as a drug target in leukemia using in vitro and in vivo genetic and humanized mouse models. This method can be applied to any target for genetic validation and therapeutic development.

The demonstration of tyrosine kinase inhibitors (TKIs) in treating chronic myeloid leukemia (CML) has heralded a new era in cancer therapeutics. However, ...

methods-for-evaluating-role-c-fos-dusp1-oncogene

Research

JoVE Journal

Cancer Research

This video describes the protocol for genetic validation by transferring target genes using retroviruses in chronic myeloid leukemia cell lines. The retroviruses act as a mediator to transfer genetic material into the leukemia cell through the process called transduction, which enables long term expression of target gene.This method can be applied to any target cells for genetic validation and therapeutic development.

Retroviral-Mediated Transduction: A Method to Introduce Target Gene into Cancer Cells

The Focus Formation Assay provides a straightforward method to assess the transforming potential of a candidate oncogene.

Focus Formation: A Cell-based Assay to Determine the Oncogenic Potential of a Gene

This protocol demonstrates how to generate fluorescently marked, genetically defined clonal tumors in the Drosophila eye/antennal imaginal discs (EAD). It describes how to dissect the EAD and brain from the third instar larvae and how to process them to visualize and quantify gene expression changes and tumor invasiveness.

The Drosophila Imaginal Disc Tumor Model: Visualization and Quantification of Gene Expression and Tumor Invasiveness Using Genetic Mosaics

Identifying novel drug targets that transition from pre-clinical testing to human trials is a scientific priority. To that end, here we describe a functional genomics approach for examining the impact of gene depletion on cancer cell line spheroids, which more appropriately model human cancers in vivo.

Utilizing Functional Genomics Screening to Identify Potentially Novel Drug Targets in Cancer Cell Spheroid Cultures

This manuscript describes a Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) CRISPR-Cas9-based method for simple and expeditious investigation of the role of multiple candidate genes in Acute Myeloid Leukemia (AML) cell proliferation in parallel. This technique is scalable and can be applied in other cancer cell lines as well.

Investigation of Genetic Dependencies Using CRISPR-Cas9-based Competition Assays

A CRISPR/sgRNA library has been applied to interrogating protein-coding genes.However, the feasibility of a sgRNA library to uncover the function of a CTCF boundary in gene regulation remains unexplored. Here, we describe a HOX loci specific sgRNA library to elucidate the function of CTCF boundaries in HOX loci.

HOX Loci Focused CRISPR/sgRNA Library Screening Identifying Critical CTCF Boundaries

Intrinsically disordered domains are important for oncogenic fusion transcription factor function. To therapeutically target these proteins, a more detailed understanding of the regulatory mechanisms employed by these domains is required. Here, we use transcriptomics to map important structural features of the intrinsically disordered EWS domain in Ewing sarcoma.

Mapping the Structure-Function Relationships of Disordered Oncogenic Transcription Factors Using Transcriptomic Analysis

Here, we show the procedures for FAM83A knockdown; the assays to detect its effects on proliferation, migration, and invasion of cervical cancer cells; and the sensitization of these cells to cisplatin. This study provides a promising target gene for cervical cancer and a reference for further drug research.

Knockdown of FAM83A to Verify Its Role in Cervical Cancer Cell Growth and Cisplatin Sensitivity

This study describes a protocol for exploring the effects of altered pH on oncogene expression via RNA-seq analysis of a pancreatic ductal cell line.

Oncogene Expression Analysis with Alterations in pH in a Pancreatic Ductal Cell Line

The comet assay is an efficient method to detect DNA damage including single and double-stranded DNA breaks. We describe alkaline and neutral comet assays to measure DNA damage in cancer cells to evaluate the therapeutic effect of chemotherapy.

Evaluating In Vitro DNA Damage Using Comet Assay

Learn methods to validate c-Fos and Dusp1 as therapeutic targets in leukemia using genetic and chemical approaches. This protocol applies to multiple kinase-driven cancers and solid tumors.

Methods for Evaluating the Role of c-Fos and Dusp1 in Oncogene Dependence

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