Targeted therapy is effective in many tumor types including lung cancer, the leading cause of cancer mortality. Paradigm defining examples are targeted therapies directed against non-small cell lung cancer (NSCLC) subtypes with oncogenic alterations in EGFR, ALK and KRAS. The success of targeted therapy is limited by drug-tolerant tumor cells which withstand and adapt to treatment and comprise the residual disease state that is typical during treatment with clinical targeted therapies. Here, we integrate studies in patient-derived and immunocompetent lung cancer models and clinical specimens obtained from patients on targeted therapy to uncover a focal adhesion kinase (FAK)-YAP signaling axis that promotes residual disease during oncogenic EGFR-, ALK-, and KRAS-targeted therapies. FAK-YAP signaling inhibition combined with the primary targeted therapy suppressed residual drug-tolerant cells and enhanced tumor responses. This study unveils a FAK-YAP signaling module that promotes residual disease in lung cancer and mechanism-based therapeutic strategies to improve tumor response.

A focal adhesion kinase-YAP signaling axis drives drug tolerant persister cells and residual disease in lung cancer

Full-length YAP was amplified by PCR using forward primer 5’- TTTGACCTCCATAGAAGATTCTAGATGGAACAAAAACTCATCTC-3’ and reverse primer 5’-AGCGATCGCAGATCCTTCGCGGCCGCTATAACCATGTAAGAAAGCTTTC-3’ from pQCXIH expression constructs encoding myc-tagged YAP-WT (Addgene #33091), YAP-5SA (Addgene #33093), and YAP-S94A (Addgene #33094), respectively. After XbaI/NotI digestion, PCR products were cloned into the lentiviral pCDH-puro plasmid backbone and correct insertion was verified by Sanger sequencing. For lentivirus production, 293T cells were co-transfected with pCDH-YAP expression plasmids and lentiviral packaging plasmids pCMV-dR8.91 and pMD2.G using the TransIT-LT1 Transfection Reagent (Mirus Bio). Viral supernatant was harvested 72 hours after transfection. Target cells were infected and selected with 1 µg/mL puromycin. YAP overexpression and YAP nuclear localization upon treatment with 0.1% DMSO or 2 µM osimertinib in stable transduced PC9 cells was monitored by Western blot analysis and confocal microscopy.

YAP-5SA overexpression

The integrity of DNA is put at risk by different lesions, among which double strand breaks (DSBs) occur at low frequency, yet remain one of the most life-threatening harms. The study of DSB repair requests tools provoking their accumulation, and include the use of chemical genotoxins, ionizing radiations or the expression of sequence-specific nucleases. While genotoxins and irradiation allow for dose-dependent studies, nucleases expression permits assessments at precise locations. In this work, we have exploited the repetitiveness of the Ty transposon elements in the genome of Saccharomyces cerevisiae and the cutting activity of the RNA-guided Cas9 nuclease to create a tool that combines sequence specificity and dose-dependency. In particular, we can achieve the controlled creation of 0, 1, 15 or 59 cuts in cells with an otherwise identical genetic background. We make the first application of this tool to better understand the behavior of the apical kinase of the DNA damage response Tel1. By comparing different strategies to create DNA breaks, we find that Tel1 is capable of forming multiple foci, in striking contrast with other DSB-related proteins. Tel1 foci are in tight contact with the nuclear periphery, therefore suggesting a role for the nuclear membrane in their congregation.

A CRISPR-Cas9-based system for the dose-dependent study of DNA double strand breaks sensing and repair

Telomere length was measured by PCR after end labeling with terminal transferase (20,21). End-labeling reactions (40 µL) contained 120 ng genomic DNA, x1 New England Biolabs™ Terminal Transferase Buffer, 1 mM dCTP, 4 units Terminal Transferase (New England Biolabs™) and were carried out at 37°C for 30 minutes followed by heat inactivation at 75°C for 10 minutes. 1/5th volume of 5 M NaCl, 1/80th volume of 1 M MgCl₂ and 1 volume of isopropanol were added to the reaction and DNA was precipitated by centrifugation at 17000×g during 15 min. Precipitated DNA was resuspended in 40 µL of ddH₂O. The end-labeled molecules were amplified by PCR using the primer 5’- GCGGATCCGGGGGGGGGGGGGGGGGG-3’ and 5’- TGTGGTGGTGGGATTAGAGTGGTAG-3’ (X) and 5’-TTAGGGCTATGTAGAAGTGCTG-3’ (Y’), respectively. PCR reactions (50 µL) contained between 40 ng and 80 ng of DNA, 1x myTaq buffer, and primers 0.4 µM each. Amplification was carried out with 5 U of MyTaq polymerase (Meridian Biosciences®). The conditions were 95°C, 5 minutes; followed by 35 cycles of 95°C, 1 minute; 56°C (Y reaction) / 60°C (X reaction), 20 seconds; 72°C, 5 minutes. Reaction was ended with 5 minutes at 72°C. Samples were visualized in a 2 % agarose gel containing 1× GelRed (Ozyme®).

Telomere length measurement

Functional analyses of the T cell receptor (TCR) landscape can reveal critical information about protection from disease and molecular responses to vaccines. However, it has proven difficult to combine advanced next-generation sequencing technologies with methods to decode the peptide-major histocompatibility complex (pMHC) specificity of individual TCRs. Here we developed a new high-throughput approach to enable repertoire-scale functional evaluations of natively paired TCRs. In particular, we leveraged the immortalized nature of physically linked TCRα:β amplicon libraries to analyze binding against multiple recombinant pMHCs on a repertoire scale. To exemplify the utility of this approach, we also performed affinity-based functional mapping in conjunction with quantitative next-generation sequencing to track antigen-specific TCRs. These data successfully validated a new immortalization and screening platform to facilitate detailed molecular analyses of human TCRs against diverse antigen targets associated with health, vaccination, or disease.

Immortalization and Functional Screening of Natively Paired Human T Cell Receptor Repertoires

A semi-nested PCR was performed using a HotStart GoTaq Polymerase System (M500,1 Promega, Madison, WI). In addition to the forward and reverse primers used to amplify the paired TCRα:β templates, a set of blocking oligonucleotides complementary to the 3’ region of the unfused TCRα and TCRβ products was designed with several nonsense nucleotides and a phosphate group at each 3’ end54. These nonsense regions were intended to suppress non-native pairing by causing a loss of homology between the elongated non-paired TCRα and TCRβ products. The first semi-nested PCR was performed under the following conditions: 2 min at 95 °C and 27 cycles comprising 35 sec at 95 °C, 40 sec at 58 °C, and 1 min at 73 °C, with a final hold for 5 min at 73 °C. The second semi-nested PCR was performed using a KAPA HiFi HotStart PCR Kit (7958897001, Roche, Basel, Switzerland) under the following conditions: 2 min at 95 °C and 15 cycles comprising 20 sec at 98 °C, 30 sec at 63 °C, and 30 sec at 72 °C, with a final hold for 7 min at 72 °C. PCR products were excised and purified from a 1.5% SYBR Safe Agarose Gel (Thermo Fisher Scientific).

Semi-Nested PCR and Suppression PCR

Gel extractions can be a low yield affair, but overnight incubation over a spin column can increase yields. This protocol details a modified QIAquick Gel Extraction protocol.

QIAquick Gel Extraction Kit 

1. Cut the desired band out of an agarose gel using a scalpel. 

2. Weigh the slice in a snap cap tube and add 3 volumes of QG buffer per volume of gel, assuming the gel density is roughly 1µl/mg. This can be whatever gel dissolving buffer (with chaotropic salts and guanidine thiocyanate) you typically use.

3. Add 1 volume of isopropanol to the tube and vortex.

4. Place a QIAquick spin column in a collection tube. Add the sample to the column and incubate over column overnight at room temp. 

5. Centrifuge spin column for 1 minute at 13,000 RPM. 

6. Discard flow through. Repeat step 5 if the sample volume was greater than 800 µl.

7. Add 750 µl of buffer PE to column and centrifuge for 1 minute at 13,000 RPM. Discard flow through.

8. Centrifuge for an additional 1 minute to remove all wash buffer.

9. Place column in collection tube and add 50 µl EB buffer or water directly to the silica portion of the column. Incubate up to 4 minutes then centrifuge the column for 1 minute to elute DNA.

Analyze DNA using a standard Nanodrop/general spectrophotometer protocol for 260/280 absorbance ratios. Store all samples in a -20 oC freezer.

Higher Yield Gel Extractions

PCR can be performed directly from a scraped cell colony, using an extended denaturing step to cause lysis of the cells. This is a nice time saving protocol for PCRing targets directly from antibiotic selective plate media. 

Colony PCR with Patch Plating
1. If subculturing via patch plate, divide a warm LB-Amp plate into a grid (commonly 3x3), labelling each square on the back of the plate.

2. Using a sterile wooden stick, aseptically pick up a transformed colony and smear some of the colony in a PCR tube. 

3. If subculturing via patch plate, use the same stick to patch it on one of the squares. 

4. Repeat steps 2 and 3 for multiple colonies.

5. Place patch plate in 37oC incubator. Move to a 4oC fridge the next day.

6. Add reagents to the PCR tubes as described in table 1. The reagent volumes can be modified to fit whatever your typical PCR setup looks like.

7. Run the PCR cycles as detailed in table 2.

8. The resultant PCR products can be run through a gel for gel extraction or cleaned with a PCR cleanup kit.

Figure 1 displays the results of the colony PCR using all 6 patched colonies. Lanes 1 - 6 contain the colony PCR products of 6 transformed colonies. A 1 kb ladder was loaded into lane 7. Lanes 8 and 9 were PCR products of untransformed control colonies. 


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