KRAS GTPases are activated in one-third of cancers and KRAS G12C is the most common activating alteration in lung adenocarcinoma 1,2 . KRAS G12C inhibitors 3,4 are in Phase-I clinical trials and early data show partial responses in ~50% of lung cancer patients. How cancer cells bypass inhibition, to prevent maximal response to therapy, is not understood. Because KRAS G12C cycles between an active and inactive conformation [4][5][6] , and the inhibitors only bind to the latter, we tested if isogenic cell populations respond non-uniformly by studying the effect of treatment at Reprints and permissions information is available at www.nature.com/reprints.
The principles governing evolution of tumors exposed to targeted therapy are poorly understood. Here we modeled the selection and propagation of BRAF amplification (BRAFamp) in patient-derived tumor xenografts (PDX) treated with a direct ERK inhibitor, alone or in combination with other pathway inhibitors. Single cell sequencing and multiplex-fluorescence in situ hybridization mapped the emergence of extra-chromosomal amplification in parallel evolutionary tracts, arising in the same tumor shortly after treatment. The evolutionary selection of BRAFamp is determined by the fitness threshold, the barrier subclonal populations need to overcome to regain fitness in the presence of therapy. This differed for ERK signaling inhibitors, suggesting that sequential monotherapy is ineffective and selects for a progressively higher BRAF copy number. Concurrent targeting of RAF, MEK and ERK, however, imposes a sufficiently high fitness threshold to prevent the propagation of subclones with high-level amplification. Administered on an intermittent schedule, this treatment inhibited tumor growth in 11/11-lung cancer and melanoma PDX without apparent toxicity in mice. Thus, gene amplification can be acquired and expanded through parallel evolution, enabling tumors to adapt while maintaining their intratumoral heterogeneity. Treatments that impose the highest fitness threshold will likely prevent the evolution of resistance-causing alterations and merit testing in patients.
Paving the way for KRAS inhibitors KRAS is a key oncogene in multiple cancer types, but existing inhibitors target only a mutant form of KRAS containing the G12C mutation, and their function presents a mechanistic conundrum. It is known that KRAS G12C inhibitors bind to the oncoprotein in its inactive form; however, KRAS mutations such as G12C interfere with the action of proteins that normally help it hydrolyze GTP to achieve the inactive state. Li et al . have now identified a protein that enhances GTP hydrolysis by mutant KRAS, helping to explain the clinical activity of current drugs targeting this oncoprotein (see the Perspective by Cox and Der). —YN
KRAS GTPases are activated in one-third of cancers and KRAS G12C is the most common activating alteration in lung adenocarcinoma. KRAS G12C-specific inhibitors (G12Ci) are in Phase-I clinical trials and early data show only partial responses in lung cancer patients. How cancer cells bypass inhibition, to prevent maximal responses to therapy, is not understood. Because KRAS G12C cycles between an active and inactive conformation, and the covalent G12Ci only bind to the latter, we tested whether isogenic cell populations respond non-uniformly by studying the effect of treatment at a single-cell resolution. Using single-cell RNA sequencing and a fluorescent quiescence biosensor, we show that shortly after treatment, most cancer cells are sequestered in a quiescent state with low KRAS activity, while a small population reactivates KRAS to resume proliferation. This rapid divergent response is due to synthesis of new, drug-free KRAS protein, resulting from increased KRAS transcription in response to suppressed MAPK signaling. Combining cell fate-specific gene expressions and results from a CRISPR-Cas9 screen, we identified that adaptive signals such as epidermal growth-factor receptor and aurora kinase A signaling modulate the heterogeneous response to treatment with G12Ci. These upstream signals help to maintain new KRAS G12C protein in its active, drug-insensitive state, which restores KRAS signaling and transcriptional output in a subset of cells to allow escape from G12Ci-induced quiescence. Cells without these adaptive changes (or cells where they are pharmacologically inhibited) remain sensitive to G12Ci treatment, because new KRAS G12C is either not available, or it exists in its inactive, drug-sensitive state. Combined inhibition of these adaptive signals along with KRAS G12C produced more potent antitumor effects in xenograft models. The direct targeting of KRAS oncoproteins has been a longstanding objective in precision oncology. Our study uncovers a flexible non-uniform fitness mechanism that enables groups of cells within a population to rapidly bypass the effect of treatment. This adaptive process must be overcome to maximize the therapeutic potential of conformation-specific KRAS G12C inhibitors in the clinic. Citation Format: Jenny Y. Xue, Yulei Zhao, Jordan Aronowitz, Trang T. Mai, Alberto Vides, Besnik Qeriqi, Dongsung Kim, Chuanchuan Li, Elisa de Stanchina, Linas Mazutis, Davide Risso, Piro Lito. Rapid non-uniform adaptation to conformation-specific KRAS G12Cinhibition [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 622.
KRAS GTPases are activated in one-third of cancers and KRAS G12C is the most common activating alteration in lung adenocarcinoma. KRAS G12C inhibitors are in Phase-I clinical trials and early data show only partial responses in lung cancer patients. How cancer cells bypass inhibition, to prevent maximal responses to therapy, is not understood. Because KRAS G12C cycles between an active and inactive conformation, and the inhibitors only bind to the latter, we hypothesized that isogenic cell populations respond non-uniformly. Here we studied the effect of treatment at the single cell level and showed that shortly after treatment, some cancer cells were sequestered in a quiescent state with low KRAS activity, while others reactivated KRAS to resume proliferation. By combining cell fate-specific gene expressions and results from a CRISPR-Cas9 screen, we identified that this rapid divergent response is due to new KRAS G12C produced in response to suppressed MAPK output. Upstream-acting adaptive signals, such as epidermal growth-factor receptor and aurora kinase signaling, maintain new KRAS G12C protein in its active/drug-insensitive state to restore KRAS output. Cells without these adaptive changes (or cells where they are pharmacologically inhibited) remain sensitive to drug treatment, because new KRAS G12C is either not available, or it exists in its inactive/drug-sensitive state. Combined inhibition of these adaptive signals along with KRAS G12C produced more potent antitumor effects in vivo. Our study uncovers a flexible non-uniform fitness mechanism that enables groups of cells within a population to rapidly bypass the effect of treatment. This adaptive process must be overcome to maximize the therapeutic potential of conformation-specific KRAS G12C inhibitors in the clinic. Citation Format: Jenny Xue, Yulei Zhao, Jordan Aronowitz, Trang T Mai, Alberto Vides, Besnik Qeriqi, Dongsung Kim, Chuanchuan Li, Elisa de Stanchina, Linas Mazutis, Davide Risso, Piro Lito. Rapid non-uniform adaptation to conformation-specific KRAS G12C inhibition [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr LB-A04. doi:10.1158/1535-7163.TARG-19-LB-A04
Tumors evolve as they adapt to environmental cues. The principles governing evolution of tumors under the selective pressure of targeted therapy are not well understood. We aimed to evaluate the evolution of resistance and to identify therapeutic modalities that prevent this process in BRAFV600E-mutant tumors. We modeled the selection and propagation of BRAFV600E amplification (BRAFamp) in patient-derived tumor xenografts (PDX) treated with a direct ERK inhibitor. Single-cell sequencing and multiplex-fluorescence in situ hybridization mapped the emergence of extra-chromosomal amplification in multiple subclones of the same tumor shortly after treatment. The evolutionary selection of BRAFamp is determined by the fitness threshold, the barrier subclonal populations need to overcome to regain fitness in the presence of therapy. This differed for ERK signaling inhibitors, and single-cell sequencing of a melanoma PDX model showed that drugs of the same pathway do not necessarily select for the same subclones. These data suggest that sequential monotherapy is not optimal, but concurrent targeting of RAF, MEK, and ERK, however, imposes a sufficiently high fitness threshold to prevent the propagation of subclones with high-level amplification. Administered on an intermittent schedule, this treatment inhibited tumor growth without apparent toxicity in 11/11-lung cancer and melanoma PDX models with various additional alterations. Thus, gene amplification can be acquired and expanded through parallel evolution, enabling tumors to adapt while maintaining their intratumoral heterogeneity. Treatments that impose a high fitness threshold, such as our intermittent triple therapy, will likely prevent the evolution of resistance-causing alterations and merit testing in patients. Citation Format: Yaohua Xue, Luciano Martelotto, Timour Baslan, Alberto Vides, Martha Solomon, Kalyani Chadalavada, Elisa DeStanchina, Gouri Nanjangud, Michael Berger, Scott Lowe, Jorge S. Reis-Filho, Neal Rosen, Piro Lito. An approach to suppress the evolution of resistance in BRAFV600E-mutant cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr B015.
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