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

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...

Watch this Scientific Journal Video about Methods for Evaluating the Role of c-Fos and Dusp1 in Oncogene Dependence at JoVE.com

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

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

Universidad San Sebastian

Research

JoVE Journal

Cancer Research

8.2K Views.  Cincinnati Children's Hospital Medical Center. 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.

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

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

The identification of functional driver events in cancer is central to furthering our understanding of cancer biology and indispensable for the discovery of the next generation of novel drug targets. It is becoming apparent that more complex models of cancer are required to fully appreciate the contributing factors that drive tumorigenesis in vivo and increase the efficacy of novel therapies that make the transition from pre-clinical models to clinical trials.
Here we present a methodology for generating uniform and reproducible tumor spheroids that can be subjected to siRNA functional screening. These spheroids display many characteristics that are found in solid tumors that are not present in traditional two-dimension culture. We show that several commonly used breast cancer cell lines are amenable to this protocol. Furthermore, we provide proof-of-principle data utilizing the breast cancer cell line BT474, confirming their dependency on amplification of the epidermal growth factor receptor HER2 and mutation of phosphatidylinositol-4,5-biphosphate 3-kinase (PIK3CA) when grown as tumor spheroids. Finally, we are able to further investigate and confirm the spatial impact of these dependencies using immunohistochemistry.

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.

The identification of functional driver events in cancer is central to furthering our understanding of cancer biology and indispensable for the discovery ...

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

CCCTC-binding factor (CTCF)-mediated stable topologically associating domains (TADs) play a critical role in constraining interactions of DNA elements that are located in neighboring TADs. CTCF plays an important role in regulating the spatial and temporal expression of HOX genes that control embryonic development, body patterning, hematopoiesis, and leukemogenesis. However, it remains largely unknown whether and how HOX loci associated CTCF boundaries regulate chromatin organization and HOX gene expression. In the current protocol, a specific sgRNA pooled library targeting all CTCF binding sites in the HOXA/B/C/D loci has been generated to examine the effects of disrupting CTCF-associated chromatin boundaries on TAD formation and HOX gene expression. Through CRISPR-Cas9 genetic screening, the CTCF binding site located between HOXA7/HOXA9 genes (CBS7/9) has been identified as a critical regulator of oncogenic chromatin domain, as well as being important for maintaining ectopic HOX gene expression patterns in MLL-rearranged acute myeloid leukemia (AML). Thus, this sgRNA library screening approach provides novel insights into CTCF mediated genome organization in specific gene loci and also provides a basis for the functional characterization of the annotated genetic regulatory elements, both coding and noncoding, during normal biological processes in the post-human genome project era.

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

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.

CCCTC-binding factor (CTCF)-mediated stable topologically associating domains (TADs) play a critical role in constraining interactions of DNA elements ...

Genetics

Integration of Bioinformatics Approaches and Experimental Validations to Understand the Role of Notch Signaling in Ovarian Cancer

Notch signaling is a highly conserved regulatory pathway involved in many cellular processes. Dysregulation of this signaling pathway often leads to interference with proper development and may even result in initiation or progression of cancers in certain cases. Because this pathway serves complex and versatile functions, it can be studied extensively through many different approaches. Of these, bioinformatics provides an undeniably cost-efficient, approachable, and user-friendly method of study. Bioinformatics is a useful way to extract smaller pieces of information from large-scale datasets. Through the implementation of various bioinformatics approaches, researchers can quickly, reliably, and efficiently interpret these large datasets, yielding insightful applications and scientific discoveries. Here, a protocol is presented for integration of bioinformatics approaches to investigate the role of Notch signaling in ovarian cancer. Furthermore, bioinformatics findings are validated through experimentation.

Bioinformatics is a useful way to process large-scale datasets. Through the implementation of bioinformatics approaches, researchers can quickly, reliably, and efficiently obtain insightful applications and scientific discoveries. This article demonstrates the utilization of bioinformatics in ovarian cancer research. It also successfully validates bioinformatics findings through experimentation.

Notch signaling is a highly conserved regulatory pathway involved in many cellular processes. Dysregulation of this signaling pathway often leads to ...

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

Many cancers are characterized by chromosomal translocations which result in the expression of oncogenic fusion transcription factors. Typically, these proteins contain an intrinsically disordered domain (IDD) fused with the DNA-binding domain (DBD) of another protein and orchestrate widespread transcriptional changes to promote malignancy. These fusions are often the sole recurring genomic aberration in the cancers they cause, making them attractive therapeutic targets. However, targeting oncogenic transcription factors requires a better understanding of the mechanistic role that low-complexity, IDDs play in their function. The N-terminal domain of EWSR1 is an IDD involved in a variety of oncogenic fusion transcription factors, including EWS/FLI, EWS/ATF, and EWS/WT1. Here, we use RNA-sequencing to investigate the structural features of the EWS domain important for transcriptional function of EWS/FLI in Ewing sarcoma. First shRNA-mediated depletion of the endogenous fusion from Ewing sarcoma cells paired with ectopic expression of a variety of EWS-mutant constructs is performed. Then RNA-sequencing is used to analyze the transcriptomes of cells expressing these constructs to characterize the functional deficits associated with mutations in the EWS domain. By integrating the transcriptomic analyses with previously published information about EWS/FLI DNA binding motifs, and genomic localization, as well as functional assays for transforming ability, we were able to identify structural features of EWS/FLI important for oncogenesis and define a novel set of EWS/FLI target genes critical for Ewing sarcoma. This paper demonstrates the use of RNA-sequencing as a method to map the structure-function relationship of the intrinsically disordered domain of oncogenic transcription factors.

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.

Many cancers are characterized by chromosomal translocations which result in the expression of oncogenic fusion transcription factors. Typically, these ...

A Protocol for Genetic Induction and Visualization of Benign and Invasive Tumors in Cephalic Complexes of Drosophila melanogaster

Drosophila has illuminated our understanding of the genetic basis of normal development and disease for the past several decades and today it continues to contribute immensely to our understanding of complex diseases 1-7. Progression of tumors from a benign to a metastatic state is a complex event 8 and has been modeled in Drosophila to help us better understand the genetic basis of this disease 9. Here I present a simple protocol to genetically induce, observe and then analyze the progression of tumors in Drosophila larvae. The tumor induction technique is based on the MARCM system 10 and exploits the cooperation between an activated oncogene, RasV12 and loss of cell polarity genes (scribbled, discs large and lethal giant larvae) to generate invasive tumors 9. I demonstrate how these tumors can be visualized in the intact larvae and then how these can be dissected out for further analysis. The simplified protocol presented here should make it possible for this technique to be utilized by investigators interested in understanding the role of a gene in tumor invasion.

A Protocol for Genetic Induction and Visualization of Benign and Invasive Tumors in Cephalic Complexes of Drosophila melanogaster

Cooperation between an activated oncogene like RASV12 and mutations in cell polarity genes like scribbled, result in tumor growth in Drosophila where tumor cells also display invasive behaviors. Here a simple protocol for the induction and observation of the benign and invasive tumors is presented.

Drosophila has illuminated our  understanding of the genetic basis of normal development and disease for the  past several decades and today it continues ...

Medicine

Defining Gene Functions in Tumorigenesis by Ex vivo Ablation of Floxed Alleles in Malignant Peripheral Nerve Sheath Tumor Cells

The development of new drugs that precisely target key proteins in human cancers is fundamentally altering cancer therapeutics. However, before these drugs can be used, their target proteins must be validated as therapeutic targets in specific cancer types. This validation is often performed by knocking out the gene encoding the candidate therapeutic target in a genetically engineered mouse (GEM) model of cancer and determining what effect this has on tumor growth. Unfortunately, technical issues such as embryonic lethality in conventional knockouts and mosaicism in conditional knockouts often limit this approach. To overcome these limitations, an approach to ablating a floxed embryonic lethal gene of interest in short-term cultures of malignant peripheral nerve sheath tumors (MPNSTs) generated in a GEM model was developed.
This paper describes how to establish a mouse model with the appropriate genotype, derive short-term tumor cultures from these animals, and then ablate the floxed embryonic lethal gene using an adenoviral vector that expresses Cre recombinase and enhanced green fluorescent protein (eGFP). Purification of cells transduced with adenovirus using fluorescence-activated cell sorting (FACS) and the quantification of the effects that gene ablation exerts on cellular proliferation, viability, the transcriptome, and orthotopic allograft growth is then detailed. These methodologies provide an effective and generalizable approach to identifying and validating therapeutic targets in vitro and in vivo. These approaches also provide a renewable source of low-passage tumor-derived cells with reduced in vitro growth artifacts. This allows the biological role of the targeted gene to be studied in diverse biologic processes such as migration, invasion, metastasis, and intercellular communication mediated by the secretome.

Defining Gene Functions in Tumorigenesis by Ex vivo Ablation of Floxed Alleles in Malignant Peripheral Nerve Sheath Tumor Cells

Here, we present a protocol for performing gene knockouts that are embryonic lethal in vivo in genetically engineered mouse model-derived tumors and then assessing the effect that the knockout has on tumor growth, proliferation, survival, migration, invasion, and the transcriptome in vitro and in vivo.

The development of new drugs that precisely target key proteins in human cancers is fundamentally altering cancer therapeutics. However, before these ...

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

Malignant transformation of cells is typically associated with increased proliferation, loss of contact inhibition, acquisition of anchorage-independent growth potential, and the ability to form tumors in experimental animals1. In NIH 3T3 cells, the Ras signal transduction pathway is known to trigger many of these events, what is known as Ras transformation. The introduction of an overexpressed gene in NIH 3T3 cells may promote morphological transformation and loss of contact inhibition, which can help determine the oncogenic potential of that gene of interest. An assay that provides a straightforward method to assess one aspect of the transforming potential of an oncogene is the Focus Formation Assay (FFA)2. When NIH 3T3 cells divide normally in culture, they do so until they reach a confluent monolayer. However, in the presence of an overexpressed oncogene, these cells can begin to grow in dense, multilayered foci1 that can be visualized and quantified by crystal violet or Hema 3 staining. In this article we describe the FFA protocol with retroviral transduction of the gene of interest into NIH 3T3 cells, and how to quantify the number of foci through staining. Retroviral transduction offers a more efficient method of gene delivery than transfection, and the use of an ecotropic murine retrovirus provides a biosafety control when working with potential human oncogenes.

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

Malignant transformation of cells is typically associated with increased proliferation, loss of contact inhibition, acquisition of anchorage-independent ...

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

The fourth leading cause of cancer-related death, pancreatic ductal adenocarcinoma (PDAC), has a 12% five-year survival rate. This disease has a poor prognosis and is characterized by a rigid stromal microenvironment, which represents a tangible challenge in its treatment. Chronic pancreatitis patients have a 10-fold greater risk of developing PDAC; in these patients, the ductal pH decreases from pH 8.0 to pH 6.0 due to bicarbonate insufficiency and the inflammatory milieu. Our goal was to understand the role of the acidic environment observed in chronic pancreatitis on oncogene expression in a pancreatic ductal cell line.
Therefore, 80% confluent human pancreatic ductal epithelial cells were incubated at pH 6.0 to pH 7.2 for 6 h. Total RNA from the cells was processed to enrich the total mRNA in the samples. Gene expression was evaluated via next-generation sequencing (NGS) of biological replicates. RNA-seq analysis was carried out with the aid of an online tool, and the differentially expressed genes (FCs &#60; &#177; 2.0) were identified; there were 90, 148, and 109 upregulated genes and 20, 14, and 23 downregulated genes at pH 6.0, 6.5, and 7.0, respectively. Four oncogenes were upregulated at pH 6.0, seven were upregulated at pH 6.5, and seven were upregulated at pH 7.0. The common genes that were upregulated at pH 6.0, pH 6.5, and pH 7.0 were lymphocyte cell-specific protein-tyrosine kinase (LCK) [pH 6.0, FC: 2.93; pH 6.5, FC: 2.93; pH 7.0, FC: 3.32], FGR proto-oncogene, Src family tyrosine kinase (FGR) [pH 6.0, FC: 4.17; pH 6.5, FC: 5.25; pH 7.0, FC: 5.09], and ArfGAP With SH3 domain, ankyrin repeat, and PH domain 3 (ASAP3) [pH 6.0, FC: 2.37; pH 6.5, FC: 3.84; pH 7.0, FC: 2.51]. The acidic environment triggers the activation of proto-oncogenes, which may trigger tumor initiation in chronic pancreatitis.

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.

The fourth leading cause of cancer-related death, pancreatic ductal adenocarcinoma (PDAC), has a 12% five-year survival rate. This disease has a poor ...

Preparation of Cell-lines for Conditional Knockdown of Gene Expression and Measurement of the Knockdown Effects on E4orf4-Induced Cell Death

Functional inactivation of gene expression in mammalian cells is crucial for the study of the contribution of a protein of interest to various pathways1,2. However, conditional knockdown of gene expression is required in cases when constitutive knockdown is not tolerated by cells for a long period of time3-5. Here we describe a protocol for preparation of cell lines allowing conditional knockdown of subunits of the ACF chromatin remodeling factor. These cell lines facilitate the determination of the contribution of ACF to induction of cell death by the adenovirus E4orf4 protein6. Sequences encoding short hairpin RNAs for the Acf1 and SNF2h subunits of the ACF chromatin remodeling factor were cloned next to a doxycycline-inducible promoter in a plasmid also containing a gene for the neomycin resistance gene. Neomycin-resistant cell clones were selected in the presence of G418 and isolated. The resulting cell lines were induced by doxycycline treatment, and once Acf1 or SNF2h expression levels were reduced, the cells were transfected with a plasmid encoding E4orf4 or an empty vector. To confirm the specific effect of the shRNA constructs, Acf1 or SNF2h protein levels were restored to WT levels by cotransfection with a plasmid expressing Acf1 or SNF2h which were rendered resistant to the shRNA by introduction of silent mutations. The ability of E4orf4 to induce cell death in the various samples was determined by a DAPI assay, in which the frequency of appearance of nuclei with apoptotic morphologies in the transfected cell population was measured7-9.
The protocol described here can be utilized for determination of the functional contribution of various proteins to induction of cell death by their protein partners in cases when constitutive knockdown may be cell lethal.

Contribution of the ACF chromatin remodeling factor to E4orf4-induced cell death was measured. The protocol includes selection of cell clones in which doxycycline treatment induces conditional knockdown of the ACF subunits Acf1 and SNF2h, and use of the DAPI assay to measure E4orf4-induced cell death in the inducible cell lines.

Functional inactivation of gene expression in mammalian cells is crucial for the study of the contribution of a protein of interest to various ...

Biology

The c-FOS Protein Immunohistological Detection: A Useful Tool As a Marker of Central Pathways Involved in Specific Physiological Responses In Vivo and Ex Vivo

Many studies seek to identify and map the brain regions involved in specific physiological regulations. The proto-oncogene c-fos, an immediate early gene, is expressed in neurons in response to various stimuli. The protein product can be readily detected with immunohistochemical techniques leading to the use of c-FOS detection to map groups of neurons that display changes in their activity. In this article, we focused on the identification of brainstem neuronal populations involved in the ventilatory adaptation to hypoxia or hypercapnia. Two approaches were described to identify involved neuronal populations in vivo in animals and ex vivo in deafferented brainstem preparations. In vivo, animals were exposed to hypercapnic or hypoxic gas mixtures. Ex vivo, deafferented preparations were superfused with hypoxic or hypercapnic artificial cerebrospinal fluid. In both cases, either control in vivo animals or ex vivo preparations were maintained under normoxic and normocapnic conditions. The comparison of these two approaches allows the determination of the origin of the neuronal activation i.e., peripheral and/or central. In vivo and ex vivo, brainstems were collected, fixed, and sliced into sections. Once sections were prepared, immunohistochemical detection of the c-FOS protein was made in order to identify the brainstem groups of cells activated by hypoxic or hypercapnic stimulations. Labeled cells were counted in brainstem respiratory structures. In comparison to the control condition, hypoxia or hypercapnia increased the number of c-FOS labeled cells in several specific brainstem sites that are thus constitutive of the neuronal pathways involved in the adaptation of the central respiratory drive.

The c-FOS Protein Immunohistological Detection: A Useful Tool As a Marker of Central Pathways Involved in Specific Physiological Responses In Vivo and Ex Vivo

Here, we present a protocol based on c-FOS protein immunohistological detection, a classical technique used for the identification of neuronal populations involved in specific physiological responses in vivo and ex vivo.

Many studies seek to identify and map the brain regions involved in specific physiological regulations. The proto-oncogene c-fos, an immediate early gene, ...

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

Summary

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

Preparation of Cell-lines for Conditional Knockdown of Gene Expression and Measurement of the Knockdown Effects on E4orf4-Induced Cell Death

The c-FOS Protein Immunohistological Detection: A Useful Tool As a Marker of Central Pathways Involved in Specific Physiological Responses In Vivo and Ex Vivo