A Preexisting Rare <i>PIK3CA</i>E545K Subpopulation Confers Clinical Resistance to MEK plus CDK4/6 Inhibition in <i>NRAS</i> Melanoma and Is Dependent on S6K1 Signaling

Romano, Gabriele; Chen, Pei-Ling; Song, Ping; McQuade, Jennifer L.; Liang, Roger J.; Liu, Mingguang; Roh, Whijae; Duose, Dzifa Y.; Carapeto, Fernando; Li, Jun; Teh, Jessica L.F.; Aplin, Andrew E.; Chen, Merry; Zhang, Jianhua; Lazar, Alexander J.; Davies, Michael A.; Futreal, P. Andrew; Amaria, Rodabe N.; Zhang, David Y.; Wargo, Jennifer A.; Kwong, Lawrence N.

doi:10.1158/2159-8290.cd-17-0745

Cited by 60 publications

(60 citation statements)

References 33 publications

(37 reference statements)

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“…S1G and S1H) and an increase in CCND1 ( Supplementary Fig. S1H) total protein levels, both of which are consistent with previous studies (7,11). Palbociclib inhibition did not alter the MAPK signaling components RAF and MEK, however we did observe an increase in phospho-ERK levels (Fig.…”

Section: Nras-dependent Melanoma Cell Lines Are Sensitive To Pharmacosupporting

confidence: 91%

“…S2D). As there are limited clinical samples available for the evaluation of resistance to this combination (7,8), we used cBioPortal (http://www.cbioportal.org/) to determine whether proteins identified in the ORF overexpression screen were altered either through point mutation or amplification in NRAS-mutant melanoma. We observed that ARAF, KRAS, AKT1, PIK3CA, RAF1, BRAF, and AKT3 were either mutated (hotspot) or amplified in NRAS-mutant melanoma (Fig.…”

Section: Nras-dependent Melanoma Cell Lines Are Sensitive To Pharmacomentioning

confidence: 99%

“…The preclinical observation that CDK4/6 inhibition could attenuate NRAS oncogenic signaling when combined with MEK inhibition supported the use of combined MEK1/2 and CDK4/6 inhibitor combination in NRAS-mutant melanoma (6). A clinical trial (NCT01781572) designed to evaluate this combination in NRAS-mutant melanoma patients revealed multiple partial responses (7,8) and is under clinical evaluation in KRAS-mutant colon (NCT02703571), lung (NCT03170206), and pancreatic (NCT02703571) cancer. Thus, understanding the resistance landscape to MEK/CDK4/6 inhibition will be imperative for improving long-term patient responses.…”

Section: Introductionmentioning

confidence: 99%

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A Functional Landscape of Resistance to MEK1/2 and CDK4/6 Inhibition in NRAS-Mutant Melanoma

et al. 2019

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Combinatorial inhibition of MEK1/2 and CDK4/6 is currently undergoing clinical investigation in NRAS-mutant melanoma. To prospectively map the landscape of resistance to this investigational regimen, we utilized a series of gain-and loss-of-function forward genetic screens to identify modulators of resistance to clinical inhibitors of MEK1/2 and CDK4/6 alone and in combination. First, we identified NRAS-mutant melanoma cell lines that were dependent on NRAS for proliferation and sensitive to MEK1/2 and CDK4/6 combination treatment. We then used a genome-scale ORF overexpression screen and a CRISPR knockout screen to identify modulators of resistance to each inhibitor alone or in combination. These orthogonal screening approaches revealed concordant means of achieving resistance to this therapeutic modality, including tyrosine kinases, RAF, RAS, AKT, and PI3K signaling. Activated KRAS was sufficient to cause resistance to combined MEK/CDK inhibition and to replace genetic depletion of oncogenic NRAS. In summary, our comprehensive functional genetic screening approach revealed modulation of resistance to the inhibition of MEK1/2, CDK4/6, or their combination in NRAS-mutant melanoma.Significance: These findings reveal that NRAS-mutant melanomas can acquire resistance to genetic ablation of NRAS or combination MEK1/2 and CDK4/6 inhibition by upregulating activity of the RTK-RAS-RAF and RTK-PI3K-AKT signaling cascade. Materials and Methods Cell lines and reagentsCells were maintained in DMEM (Hs936T, Hs944T; Gibco), RPMI1640 (MELJUSO, SKMEL30, IPC298; Gibco), or EMEM (SKMEL-2; Gibco) supplemented with 10% FBS (Sigma), and incubated at 37 C in 5% CO 2 per ATCC guidelines. Western blot reagentsCells were lysed in RIPA buffer (25 mmol/L Tris * HCl pH 7.6, 150 mmol/L NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS; phosphatase and protease inhibitors) and resolved by Tris-Gycline SDS-PAGE. To determine the levels of activated proteins, immunoblot analyses were done with phospho-specific antibodies to AKT(S473), MEK1/2(S217/S221), RB1 (S807/811), and ERK1/2 (T202/Y204), S6 (235/236) with antibodies recognizing total AKT, RB, ERK1/2, and S6 to control for total protein expression (Cell Signaling Technologies). Antibodies to EGFR, PI3K, CCNB1, CCND1, and CCNE2 (Cell Signaling Technologies), and Active KRAS can replace loss of genetic NRAS. A and B, CellTiter MTS tetrazolium assay displaying mean cell viability of MELJUSO (A) and Hs936T (B) cell lines expressing GFP, KRAS, or NRAS with or without knockdown of NRAS (six replicates) after 96 hours, each from three independent experiments. C and D, Representative images of crystal violet stained clonogenic growth assays with MELJUSO (C) and Hs936T (D) cell lines expressing GFP, KRAS, or NRAS with or without knockdown of NRAS (duplicate) after 10 days, each from three independent experiments. E and F, Representative immunoblots displaying the effect of MELJUSO (E) and Hs936T (F) cell lines expressing GFP, KRAS, or NRAS with or without knockdown of NRAS after 96 hours on phosphor...

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Section: Nras-dependent Melanoma Cell Lines Are Sensitive To Pharmacosupporting

confidence: 91%

Section: Nras-dependent Melanoma Cell Lines Are Sensitive To Pharmacomentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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A Functional Landscape of Resistance to MEK1/2 and CDK4/6 Inhibition in NRAS-Mutant Melanoma

et al. 2019

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“…Most qPCR, Sanger, and NGS-based assays and panels for oncology targeted therapy selection [40][41][42] have mutation VAF sensitivities of between 1% and 5%, and thus are unable to identify low VAF drug resistance mutations arising from trace subclones due to tumor heterogeneity. However, under the selective pressures of targeted therapy, tumor subclones with drug resistance mutations can rapidly expand and cause treatment failure or cancer recurrence [43][44][45][46]. Thus, reliable detection of low VAF drug resistance mutations can inform personalized treatment selection, including the use of combination therapies, to improve patient outcomes.…”

Section: Melanoma Mbda Ngs Panel Reveals Frequent Heterogeneity In Tumentioning

confidence: 99%

Detecting and Quantitating Low Fraction DNA Variants with Low-Depth Sequencing

Song

Chen

Yan

et al. 2020

Preprint

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DNA sequence variants with low allele frequencies below 1% are difficult to detect and quantitate by sequencing, due to the intrinsic error of sequencing-by-synthesis (NGS). Unique molecular identifier barcodes can in principle help NGS detect mutations down to 0.1% variant allele frequency (VAF), but require extremely high sequencing depths of over 25,000x, rendering high sensitivity mutation detection out of reach for most research and clinical samples. Here, we present the multiplex blocker displacement amplification (mBDA) method to selectively enrich DNA variants by an average of 300-fold in highly multiplexed NGS settings. On a 80-plex human single nucleotide polymorphism panel, mBDA achieves a 0.019% VAF limit of detection for single nucleotide variants, using only 250x sequencing depth, and detects human cell line contamination down to 0.07%. Using this technology, we constructed a 16-plex melanoma NGS panel covering 145 actionable mutations across 9 genes, and applied it to 19 fresh/frozen tumor biopsy tissue samples with high tumor fractions. We found low VAF mutations (0.2% to 5%) in 37% of the samples (7/19, 95% confidence interval 19%-58%). These results suggest that tumor heterogeneity could be significantly more pervasive than previously recognized, and can contribute significantly to acquired drug resistance to targeted therapies. We also validate mBDA panels on clinical cell-free DNA samples from lung cancer patients.Sequencing-by-synthesis, also known as next-generation sequencing (NGS), is one of the most powerful methods available today for highly multiplexed characterization of DNA at many genetic loci [1,2]. NGS is used routinely to detect DNA sequence variants with allele frequency of ≥ 5%. However, NGS struggles to report single nucleotide variants (SNVs) with variant allele frequencies (VAFs) of below 1% because all current NGS platforms have an average intrinsic error of at least 0.2% [3,4]. Error-correction methods based on unique molecular identifiers (UMIs) have been proposed and can circumvent the NGS intrinsic error limitation to achieve SNV limits of detection of about 0.1% [5-7], but these generally require sequencing to extremely high depths of 25,000x or higher. This causes the per-sample NGS cost of even moderate-size panels to be over $1,000 (e.g. the 70-gene Guardant 360 panel [8,9]), rendering sensitive detection of SNVs with VAF of below 1% unaffordable for many researchers, clinicians, and patients.Simultaneously, there is growing research and clinical demand for high sensitivity detection of low VAF DNA sequence variants. Examples of low VAF detection need include cellfree DNA profiling for non-invasive cancer therapy guidance and post-treatment monitoring [10,11], rare microbe detection for microbiome profiling [12,13], and persistent bacterial subpopulations contributing to antibiotic resistence [14,15] Digital PCR [16,17] and allele-specific PCR [18] can be used to detect and quantitate one or a few suspected DNA variants with VAF down to 0.1%, but cannot reasonably sc...

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“…However, all of these studies are still in early-phase clinical trials. Furthermore, primary and acquired resistance to some of the combinations, such as the MEK inhibitor MEK162 with a CDK4/6 inhibitor (18,19), are emerging. Therefore, there is a continued need to understand the mechanism of resistance of NRAS mutant melanomas to MEK inhibitors and to develop new treatment strategies.…”

Section: Introductionmentioning

confidence: 99%

Inhibition of the Myocardin-Related Transcription Factor pathway increases efficacy of Trametinib in NRAS-mutant melanoma cell lines

Appleton

Palsuledesai

Misek

et al. 2019

Preprint

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The Ras/MEK/ERK pathway has been the primary focus of targeted therapies in melanoma; it is aberrantly activated in almost 80% of human cutaneous melanomas (~50% BRAF V600 mutations and ~30% NRAS mutations). While targeted therapies have yielded success in BRAF V600 mutant melanoma patients, such therapies have been ineffective in NRAS mutant melanomas in part due to their cytostatic effects and primary resistance in this patient population. Here, we demonstrate that increased Rho/MRTF-pathway activation correlates with high intrinsic resistance to the MEK inhibitor, trametinib, in a panel of NRAS mutant melanoma cell lines. Combination of trametinib with the Rho/MRTF-pathway inhibitor, CCG-222740, synergistically reduced cell viability in NRAS mutant melanoma cell lines in vitro. Furthermore, the combination of CCG-222740 with trametinib induced apoptosis and reduced clonogenicity in SK-Mel-147 cells which have a high level of trametinib resistance. These findings suggest a role of the Rho/MRTF-pathway in intrinsic trametinib resistance in a subset of NRAS mutant melanoma cell lines and highlights the potential of concurrently targeting the Rho/MRTF-pathway and MEK in NRAS mutant melanomas.

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A Preexisting Rare PIK3CAE545K Subpopulation Confers Clinical Resistance to MEK plus CDK4/6 Inhibition in NRAS Melanoma and Is Dependent on S6K1 Signaling

Cited by 60 publications

References 33 publications

A Functional Landscape of Resistance to MEK1/2 and CDK4/6 Inhibition in NRAS-Mutant Melanoma

A Functional Landscape of Resistance to MEK1/2 and CDK4/6 Inhibition in NRAS-Mutant Melanoma

Detecting and Quantitating Low Fraction DNA Variants with Low-Depth Sequencing

Inhibition of the Myocardin-Related Transcription Factor pathway increases efficacy of Trametinib in NRAS-mutant melanoma cell lines

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