Protective autophagy elicited by RAF→MEK→ERK inhibition suggests a treatment strategy for RAS-driven cancers.

Pancreatic ductal adenocarcinoma (PDA) was responsible for ~ 44,000 deaths in the United States in 2018 and is the epitome of a recalcitrant cancer driven by a pharmacologically intractable oncoprotein, KRAS1-4. Downstream of KRAS, the RAF→MEK→ERK signaling pathway plays a central role in pancreatic carcinogenesis5. However, paradoxically, inhibition of this pathway has provided no clinical benefit to patients with PDA6. Here we show that inhibition of KRAS→RAF→MEK→ERK signaling elicits autophagy, a process of cellular recycling that protects PDA cells from the cytotoxic effects of KRAS pathway inhibition. Mechanistically, inhibition of MEK1/2 leads to activation of the LKB1→AMPK→ULK1 signaling axis, a key regulator of autophagy. Furthermore, combined inhibition of MEK1/2 plus autophagy displays synergistic anti-proliferative effects against PDA cell lines in vitro and promotes regression of xenografted patient-derived PDA tumors in mice. The observed effect of combination trametinib plus chloroquine was not restricted to PDA as other tumors, including patient-derived xenografts (PDX) of NRAS-mutated melanoma and BRAF-mutated colorectal cancer displayed similar responses. Finally, treatment of a patient with PDA with the combination of trametinib plus hydroxychloroquine resulted in a partial, but nonetheless striking disease response. These data suggest that this combination therapy may represent a novel strategy to target RAS-driven cancers.

Publisher Correction: Protective autophagy elicited by RAF→MEK→ERK inhibition suggests a treatment strategy for RAS-driven cancers.

In the version of this article initially published, the label over the bottom schematic in Fig. 1a was "pH > 5.0"; it should have been "pH < 5.0". Further, the original article misspelt the surname of Katrin P. Guillen as "Gullien". These errors have been corrected in the print, PDF and HTML versions of the article.

Automated DNA extraction, quantification, dilution, and PCR preparation for genotyping by high-resolution melting.

Genotyping by high-resolution amplicon melting uses only two PCR primers per locus and a generic, saturating DNA dye that detects heteroduplexes as well as homoduplexes. Heterozygous genotypes have a characteristic melting curve shape and a broader width than homozygous genotypes, which are usually differentiated by their melting temperature (T(m)). The H63D mutation, associated with hemochromatosis, is a single nucleotide polymorphism, which is impossible to genotype based on T(m), as the homozygous WT and mutant amplicons melt at the same temperature. To distinguish such homozygous variants, WT DNA can be added to controls and unknown samples to create artificial heterozygotes with all genotypes distinguished by quantitative heteroduplex analysis. By automating DNA extraction, quantification, and PCR preparation, a hands-off integrated solution for genotyping is possible. A custom Biomek® NX robot with an onboard spectrophotometer and custom programming was used to extract DNA from whole blood, dilute the DNA to appropriate concentrations, and add the sample DNA to preprepared PCR plates. Agencourt® Genfind™ v.2 chemistry was used for DNA extraction. PCR was performed on a plate thermocycler, high-resolution melting data collected on a LightScanner-96, followed by analysis and automatic genotyping using custom software. In a blinded study of 42 H63D samples, 41 of the 42 sample genotypes were concordant with dual hybridization probe genotyping. The incorrectly assigned genotype was a heterozygote that appeared to be a homozygous mutant as a result of a low sample DNA concentration. Automated DNA extraction from whole blood with quantification, dilution, and PCR preparation was demonstrated using quantitative heteroduplex analysis. Accuracy is critically dependent on DNA quantification.

Multiplex amplicon genotyping by high-resolution melting.

High-resolution amplicon melting is a simple method for genotyping that uses only generic PCR primers and a saturating DNA dye. Multiplex amplicon genotyping has previously been reported in a single color, but two instruments were required: a carousel-based rapid cycler and a high-resolution melting instrument for capillaries. Manual transfer of capillaries between instruments and sequential melting of each capillary at 0.1 degrees C/s seriously limited the throughput. In this report, a single instrument that combines rapid-cycle real-time PCR with high-resolution melting [LightScanner-32 (LS-32), Idaho Technology, Salt Lake City, UT] was used for multiplex amplicon genotyping. The four most common mutations associated with thrombophilia, F5 (factor V Leiden 1691G>A), F2 (prothrombin 20210G>A), and methylenetetrahydrofolate reductase (MTHFR; 1298A>C and 677C>T) were genotyped in a single homogeneous assay with internal controls to adjust for minor chemistry and instrument variation. Forty temperature cycles required 9.2 min, and each capillary required 2.2 min by melting at 0.3 degrees C/s, 3x the prior rate. Sample volume was reduced from 20 microl to 10 microl. In a blinded study of 109 samples (436 genotypes), complete concordance with standard assays was obtained. In addition, the rare variant MTHFR 1317T>C was genotyped correctly when present. The LS-32 simplifies more complex high-resolution melting assays by reducing hands-on manipulation, total time of analysis, and reagent cost while maintaining the resolution necessary for multiplex amplicon genotyping.

Snapback primer genotyping with saturating DNA dye and melting analysis.

BACKGROUND: DNA hairpins have been used in molecular analysis of PCR products as self-probing amplicons. Either physical separation or fluorescent oligonucleotides with covalent modifications were previously necessary. METHODS: We performed asymmetric PCR for 40-45 cycles in the presence of the saturating DNA dye, LCGreen Plus, with 1 primer including a 5' tail complementary to its extension product, but without any special covalent modifications. Samples were amplified either on a carousel LightCycler for speed or on a 96/384 block cycler for throughput. In addition to full-length amplicon duplexes, single-stranded hairpins were formed by the primer tail "snapping back" and hybridizing to its extension product. High-resolution melting was performed on a HR-1 (for capillaries) or a LightScanner (for plates). RESULTS: PCR products amplified with a snapback primer showed both hairpin melting at lower temperature and full-length amplicon melting at higher temperature. The hairpin melting temperature was linearly related to the stem length (6-28 bp) and inversely related to the log of the loop size (17-135 bases). We easily genotyped heterozygous and homozygous variants within the stem, and 100 blinded clinical samples previously typed for F5 1691G>A (Leiden) were completely concordant by snapback genotyping. We distinguished 7 genotypes in 2 regions of CFTR exon 10 with symmetric PCR using 2 snapback primers followed by product dilution to favor intramolecular hybridization. CONCLUSIONS: Snapback primer genotyping with saturating dyes provides the specificity of a probe with only 2 primers that are free of special covalent labels in a closed-tube system.

Quadruplex genotyping of F5, F2, and MTHFR variants in a single closed tube by high-resolution amplicon melting.

BACKGROUND: Multiplexed amplicon melting is a closed-tube method for genotyping that does not require probes, real-time analysis, asymmetric PCR, or allele-specific PCR; however, correct differentiation of homozygous mutant and wild-type samples by melting temperature (T(m)) analysis requires high-resolution melting analysis and controlled reaction conditions. METHODS: We designed 4 amplicons bracketing the F5 [coagulation factor V (proaccelerin, labile factor)] 1691G>A, MTHFR (NADPH) 1298A>C, MTHFR 677C>T, and F2 [coagulation factor II (thrombin)] 20210G>A gene variants to melt at different temperatures by varying amplicon length and adding GC- or AT-rich 5' tails to selected primers. We used rapid-cycle PCRs with cycles of 19-23 s in the presence of a saturating DNA dye and temperature-correction controls and then conducted a high-resolution melting analysis. Heterozygotes were identified at each locus by curve shape, and homozygous genotypes were assigned by T(m). We blinded samples previously genotyped by other methods before analysis with the multiplex melting assay (n = 110). RESULTS: All samples were correctly genotyped with the exception of 7 MTHFR 1298 samples with atypical melting profiles that could not be assigned. Sequencing revealed that these 5 heterozygotes and 2 homozygotes contained the unexpected sequence variant MTHFR 1317T>C. The use of temperature-correction controls decreased the T(m) SD within homozygotes by a mean of 38%. CONCLUSION: Rapid-cycle PCR with high-resolution melting analysis allows simple and accurate multiplex genotyping to at least a factor of 4.

Unlabeled oligonucleotides as internal temperature controls for genotyping by amplicon melting.

Amplicon melting is a closed-tube method for genotyping that does not require probes, real-time analysis, or allele-specific polymerase chain reaction. However, correct differentiation of homozygous mutant and wild-type samples by melting temperature (Tm) requires high-resolution melting and closely controlled reaction conditions. When three different DNA extraction methods were used to isolate DNA from whole blood, amplicon Tm differences of 0.03 to 0.39 degrees C attributable to the extractions were observed. To correct for solution chemistry differences between samples, complementary unlabeled oligonucleotides were included as internal temperature controls to shift and scale the temperature axis of derivative melting plots. This adjustment was applied to a duplex amplicon melting assay for the methylenetetrahydrofolate reductase variants 1298A>C and 677C>T. High- and low-temperature controls bracketing the amplicon melting region decreased the Tm SD within homozygous genotypes by 47 to 82%. The amplicon melting assay was 100% concordant to an adjacent hybridization probe (HybProbe) melting assay when temperature controls were included, whereas a 3% error rate was observed without temperature correction. In conclusion, internal temperature controls increase the accuracy of genotyping by high-resolution amplicon melting and should also improve results on lower resolution instruments.

HLA-B27 typing: evaluation of an allele-specific PCR melting assay and two flow cytometric antigen assays.

BACKGROUND: Human leukocyte antigen B27 (HLA-B27) is a major histocompatibility complex class 1 molecule that is strongly associated with the disease ankylosing spondylitis. Testing for HLA-B27 is of diagnostic value because 90% of patients with ankylosing spondylitis have the B27 antigen. Two commonly used HLA-B27 flow cytometric assays are commercially available. METHODS: An allele-specific polymerase chain reaction (PCR) melting assay for HLA-B27 was compared with two available antigen assays on 371 clinical samples. The accuracy of the assays was measured by receiver operating characteristic analysis using the PCR method and sequencing as the reference standard. RESULTS: When PCR results were compared with those of the antigen assays, complete concordance was observed except for five discrepant results that were resolved by sequence analysis. Using DNA sequencing as the gold standard, the sensitivity and specificity of PCR were 99.6 and 100.0, those of the best single antigen assay were 98.2 and 97.6, and those of a reflex combination of both antigen assays were 98.8 and 97.6. CONCLUSIONS: The allele-specific PCR melting assay for HLA-B27 genotyping is easy to perform and has better sensitivity and specificity than antigen assays. The performance of the two flow cytometric antigen assays depends on the antibody used and the positive cutoff values assigned.