Benjamin Legendre

Overcoming acquired resistance to AZD9291, a third generation EGFR inhibitor, through modulation of MEK/ERK-dependent Bim and Mcl-1 degradation

Purpose: The mechanisms accounting for anticancer activity of AZD9291 (osimertinib or TAGRISSO), an approved third-generation EGFR inhibitor, in EGFR-mutant non–small cell lung cancer (NSCLC) cells and particularly for the subsequent development of acquired resistance are unclear and thus are the focus of this study. Experimental Design: AZD9219-resistant cell lines were established by exposing sensitive cell lines to AZD9291. Protein alterations were detected with Western blotting. Apoptosis was measured with annexin V/flow cytometry. Growth-inhibitory effects of tested drugs were evaluated in vitro with cell number estimation and colony formation assay and in vivo with mouse xenograft models. Protein degradation was determined by comparing protein half-lives and inhibiting proteasome. Gene knockdown were achieved with siRNA or shRNA. Results: AZD9291 potently induced apoptosis in EGFR-mutant NSCLC cell lines, in which ERK phosphorylation was suppressed accompanied with Bim elevation and Mcl-1 reduction likely due to enhanced Mcl-1 degradation and increased Bim stability. Blocking Bim elevation by gene knockdown or enforcing Mcl-1 expression attenuated or abolished AZD9291-induced apoptosis. Moreover, AZD9291 lost its ability to modulate Bim and Mcl-1 levels in AZD9291-resistant cell lines. The combination of a MEK inhibitor with AZD9291 restores the sensitivity of AZD9291-resistant cells including those with C797S mutation to undergo apoptosis and growth regression in vitro and in vivo. Conclusions: Modulation of MEK/ERK-dependent Bim and Mcl-1 degradation critically mediates sensitivity and resistance of EGFR-mutant NSCLC cells to AZD9291 and hence is an effective strategy to overcome acquired resistance to AZD9291. Clin Cancer Res; 23(21); 6567–79. ©2017 AACR.

Abstract 4227: Simple identification of specific mutations using allele-specific PCR primers and micro capillary electrophoresis (WAVE MCE).

Transgenomic, Inc. has developed a size-based analysis for the detection of all EGFR Exon 19 deletions (including complex mutation) greater than 6 base pairs (ACE™ Kit EGFR Exon 19). Currently there are over 70 known EGFR Exon 19 deletions in the region important for response of non-small cell lung cancer (NSCLC) to thymidine kinase inhibitors (TKIs). The majority of these deletions are rare, but the patients may benefit from TKI treatment if the deletion is present. In most current testing paradigms, a NSCLC patient with one of these rare mutations would be considered EGFR mutation negative and not receive TKI therapy. We have designed an easy, size-based PCR assay to detect any of the deletions present in any sample after a single PCR reaction. The assay is based on the separation of PCR fragments of different sizes using microcapillary electrophyoresis (MCE). Transgenomic9s WAVE® MCE platform can discriminate the deletions from the wild-type sequence in EGFR Exon 19, however the exact nature of the mutation cannot be determined. If the exact nature of the mutation is required, the PCR fragment can be sequenced. To confirm that the platform can detect common and rare mutations, plasmids containing various EGFR Exon 19 deletions were synthesized, mixed with EGFR wild-type plasmids and analyzed on the WAVE® MCE platform. The Limit of detection for the plasmid mixing experiments is between 0.5 and 1% for all plasmid constructs tested. These plasmids include 6, 9, 15, 21, 24, and 25 bp deletions . As all samples tested have some wild-type sequence present, 6 base pair deletions cannot be resolved from the wild-type fragment. Several of the deletion constructs contain those not detected by any allele-specific kit, however patients with these deletions, as determined by Sanger sequence analysis, have benefitted from TKI therapy. In addition cell line DNA and DNA isolated from FFPE sections have been analyzed for the detection of EGFR Exon 19 deletions and similar limits of detection (0.5-1.0%) were seen. This simple, size-based analysis for EGFR Exon deletions will result in NSCLC patients with both common and rare EGFR Exon 19 deletions being able to receive the treatment option best suited for their molecular profile. Citation Format: Katherine A. Richardson, Grant Wu, Rui Lin, Yanggu Shi, Phil Eastlake, Benjamin Legendre. Simple identification of specific mutations using allele-specific PCR primers and micro capillary electrophoresis (WAVE MCE). [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4227. doi:10.1158/1538-7445.AM2013-4227

Abstract 2090: Mutational analysis in circulating tumor cells (CTC):ScreenCell MB Filtration Unit and ICE COLD-PCR

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL The aim of this study was to validate the use of the ScreenCell® MB Filtration Unit for detection of mutations in Circulating Tumor Cells. This device allows physicians to easily isolate any CTCs using a simple blood draw which can be performed at a local doctors office. This is especially important when trying to analyze CTCs in elderly patient populations and in regions where the large medical centers are far away. Once isolated, the filter containing the CTCs can be sent to a laboratory for DNA isolation and the mutation analysis. Mutational analysis using ICE COLD-PCR can then be performed without the use of expensive or specialized equipment. The mutation was characterized using standard Sanger DNA sequencing with a detection limit of 0.5 - 0.1% mutant sequence in the wild-type background. Results will be presented from a mock experiment where PC9 cells (EGFR exon 19 deletion: p.E746\_A750del) or H1975 cells (EGFR exon 21: p.L858R) were spiked into blood and then these mock CTCs were collected on the ScreenCell Molecular Filter Device. Following DNA isolation, the mutations were easily detected at the lowest number of cells spiked in to the blood samples (10 cells). Additional data from CTCs collected from NSCLC patients detected the L858R point mutation, an exon 19 E746\_A750del mutation, and an exon 19 E746_S752delinsV. Mutations in these same samples could not be detected in DNA sequencing following standard PCR amplification. These data show that the ScreenCell MB Filtration Unit coupled to mutational analysis using ICE COLD-PCR can be an easy, cost-effective method for collection and analysis of CTCs in patient blood samples. Moreover, the device is simple enough to be used in any doctors office. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2090. doi:1538-7445.AM2012-2090

Abstract 2091: Ultrasensitive detection of mutations in the androgen receptor gene by ICE COLD-PCR

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL The Androgen Receptor plays an important role in prostate cancer, androgen-dependent (AD) and androgen-independent (AI) disease progression. Anti-androgen therapies are the hallmark of prostate cancer treatment; however spontaneous AR mutations are often detected in hormone-refractory, androgen-ablated and metastatic tumors. ICE COLD-PCR (Improved & Complete Enrichment CO-amplification at Lower Denaturation temperature) is a technology that preferentially enriches mutant DNA sequences in an excess of wild-type DNA through selective amplification of the mutant DNA population using an oligonucleotide complementary to wild-type sequence (RS-oligo). This RS-oligo prevents PCR amplification of wild-type sequences while allowing amplification of DNA containing any mutation covered in the RS-oligo region. We have developed an ICE COLD-PCR assay for the enrichment of mutations in the Androgen Receptor including the AR H875R, H875Y and T878A mutations. The mutant-enriched DNA was then analyzed by standard Sanger DNA sequencing. The analytical sensitivity for the ICE COLD-PCR assay was 1 in 10,000 (0.01%) for all the mutations, compared to 20% limit of detection by standard Sanger sequencing. The ICE COLD-PCR assay developed here provides a means to detect low level mutations in the Androgen Receptor and thus will provide a valuable methodology for the early diagnosis, treatment guidance, outcome prediction and relapse monitoring of prostate cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2091. doi:1538-7445.AM2012-2091

Abstract 4855: Use of blocker locked nucleic acid (LNA™) oligonucleotides in cycle sequencing for improved limits of detection (BLOC-Sequencing)

Epidermal growth factor receptor (EGFR) antagonists, such as cetuximab and panitumumab, are therapeutic agents that can be effective in colorectal cancer (CRC) treatment. It has been shown that 40% of CRC tumors have activating K-RAS exon 2 codon 12 and 13 mutations and that these mutations may be associated with a poor response to EGFR antagonists. Very high sensitivity detection of such diagnostic biomarkers is necessary to determine the presence or emergence of drug resistant tumor cell populations. The use of Locked Nucleic Acid (LNA™) containing oligonucleotides (oligos) have been used in microRNA (sample preparation), RNA (in situ hybridization), and DNA (SNP detection using allele specific PCR) applications. The advantage of using LNA-containing oligos is that the denaturation temperature of LNA-oligo:DNA duplex is increased as compared to the DNA:DNA duplex To further the limit of detection of Sanger sequencing, a LNA-based approach has been developed to selectively block the sequencing of the wild-type DNA and allow the sequencing of DNA containing any mutation (BLOC-Sequencing). During cycle sequencing, an additional annealing step is added to hybridize the LNA containing oligo to the template DNA. Then, a denaturing step is performed at a temperature at which the LNA-containing oligo remains annealed to the wild-type sequence; however, the LNA oligo denatures from the mutant sequence. The sequencing primer then anneals to the mutant sequence and subsequently extended. Since LNA-containing oligos block amplification of the wild-type sequence, the mutant-containing DNA will be sequenced in a non-biased manner. Preliminary results using two sequences in a 1:1 ratio differing in only 2 nucleotides within the range where the LNA blocking oligo anneals demonstrated complete blocking of one sequence (sequence 1) where the LNA containing oligo was 100% complementary. The unblocked sequence (sequence 2) sequenced successfully, with minimal background due to sequence 1. Straight Sanger sequencing results of the 1:1 mixture without the LNA blocking oligo gave equal proportion of the two sequences present. To show applicability for use of this methodology in CRC samples, the limit of detection for multiple codon 12 and codon 13 K-RAS exon 2 mutations with and without the addition of the K-RAS exon 2 wild type specific LNA oligo in both the forward and reverse directions will be demonstrated. In addition, we will compare the results of 30 previously genotyped FFPE samples with this technique. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4855. doi:10.1158/1538-7445.AM2011-4855

Abstract 3063: Development of a sensitive COLD-PCR method for the detection of EGFR mutations in DNA

COLD-PCR is a method to exponentially amplify mutations in DNA while linearly amplifying wild type sequences. The method can be adapted to multiple mutation detection platforms including Sanger Sequencing and is amenable to development as a diagnostic kit. EGFR EXONS 20 & 21: Conditions for non-biased, selective amplification of DNA with mutations in the tyrosine kinase activation regions of the EGFR gene (exon 19 deletions and the T790M and L858R point mutations) have been developed. Ice COLD-PCR methodology was used for both exon 19 deletions and the exon 21 L858R mutation. Fast COLD-PCR methodology was used for the T790M mutation in exon 20. Using control plasmids containing the T790M and the L858R mutations, COLD-PCR amplification with SURVEYOR® Nuclease/WAVE® HS confirmation, demonstrated a limit of detection (LOD) of 0.05% mutant DNA in wild type DNA. COLD-PCR amplification followed by confirmation of these mutations using Sanger sequencing had a limit of detection of 0.2%. Only 5-10 copies of amplifiable DNA containing the mutation are needed for amplification using COLD-PCR followed by downstream mutation determination. Similar results were obtained using DNA isolated from the cell lines, H1650 and A549, and serum spiked with DNA containing a T790M mutation. EGFR EXON 19: For detection of deletions in EGFR exon 19, another series of plasmids containing 3, 6, 9, 15, and 21 base-pair deletions were constructed. The two 15 base-pair deletions were those which result in the most commonly observed mutation in non-small cell lung cancer, p.E746_A750del15 (c.2235-2249del15 or c.2236-2259del15). For these exon 19 deletions, the SURVEYOR Nuclease/WAVE HS was able to detect the presence of a mutation at 0.05% and Sanger sequencing was able to confirm these deletions at 0.2%. CONCLUSION: These data demonstrate that COLD-PCR enriches low number and low abundance EGFR mutations including point mutations and deletions, and suggests applicability to serum/plasma. COLD-PCR coupled with mutation detection methods such as Sanger sequencing is a new model for EGFR mutation testing with potential applicability for detection of mutations in other genes. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3063. doi:10.1158/1538-7445.AM2011-3063