Albumin promotes the progression of fibroblasts through late G1 into S-phase in the absence of growth factors.

G1 cell cycle progression is controlled largely by growth factors in early G1 indicating that it is appropriate to divide and by nutrients in late G1 indicating sufficient raw material for cell division. We previously mapped a late G1 cell cycle checkpoint for lipids upstream from a mammalian target of rapamycin complex 1 (mTORC1)-mediated checkpoint and downstream from a mid-G1 checkpoint known as the Restriction point. We therefore investigated a role for lipids in progression through late G1 into S-phase. Quiescent BJ-hTERT human fibroblasts were primed with 10% fetal bovine serum (FBS) for 3.5 h at which time, cells were treated with a mixture of lipids and carrier bovine serum albumin (BSA) along with [3 H]-thymidine deoxyribose ([3 H]-TdR) to monitor progression into S-phase. Surprisingly, BSA by itself was more effective than FBS in promoting progression to S-phase - the lipids had no impact on progression. While insulin strongly stimulated mTORC1 activity, it did not impact on [3 H]-TdR incorporation. Although BSA modestly elevated mTORC1 activity, rapamycin strongly inhibited BSA-induced progression to S-phase. BSA treatment promoted mitosis, but not progression through a second G1. Thus, after priming quiescent cells with FBS, albumin was sufficient to promote progression into S-phase. The BSA was not simply a source of amino acids in that amino acids were present in the culture media. We propose that the presence of albumin - the most abundant protein in serum - reflects a broader availability of essential amino acids needed for cell growth.

Rare BRAF mutations in pancreatic neuroendocrine tumors may predict response to RAF and MEK inhibition.

The clinical significance of BRAF alterations in well-differentiated (WD) metastatic pancreatic neuroendocrine tumor (panNET) is unknown, but BRAF-mutated panNET could represent a subset characterized by an identifiable and clinically actionable driver. Following the identification of two patients with WD metastatic panNET whose tumors harbored BRAF mutations, we queried the MSK-IMPACT series of 80 patients with WD metastatic panNET for additional mutations in BRAF, and in other genes involved in RAS/ RTK/ PI3K signaling pathways. BRAF mutations were identified in six samples (7.5%): two tumors harbored V600E mutations, one tumor each expressed K601E, T599K, and T310I mutations, and one tumor expressed both G596D and E451K BRAF. Few additional actionable driver alterations were identified. To determine the ERK activating capability of four BRAF mutations not previously characterized, mutant constructs were tested in model systems. Biochemical characterization of BRAF mutations revealed both high and low activity mutants. Engineered cells expressing BRAF K601E and V600E were used for in vitro drug testing of RAF and MEK inhibitors currently in clinical use. BRAF K601E demonstrated reduced sensitivity to dabrafenib compared to BRAF V600E, but the combination of RAF plus MEK inhibition was effective in cells expressing this mutation. Herein, we describe the clinical course of a patient with BRAF K601E and a patient with BRAF V600E WD metastatic panNET, and the identification of four mutations in BRAF not previously characterized. The combined clinical and biochemical data support a potential role for RAF and MEK inhibitors, or a combination of these, in a selected panNET population.

A Secondary Mutation in BRAF Confers Resistance to RAF Inhibition in a BRAFV600E-Mutant Brain Tumor.

BRAFV600E hyperactivates ERK and signals as a RAF inhibitor-sensitive monomer. Although RAF inhibitors can produce impressive clinical responses in patients with mutant BRAF tumors, the mechanisms of resistance to these drugs are incompletely characterized. Here, we report a complete response followed by clinical progression in a patient with a BRAFV600E-mutant brain tumor treated with dabrafenib. Whole-exome sequencing revealed a secondary BRAFL514V mutation at progression that was not present in the pretreatment tumor. Expressing BRAFV600E/L514V induces ERK signaling, promotes RAF dimer formation, and is sufficient to confer resistance to dabrafenib. Newer RAF dimer inhibitors and an ERK inhibitor are effective against BRAFL514V-mediated resistance. Collectively, our results validate a novel biochemical mechanism of RAF inhibitor resistance mediated by a secondary mutation, emphasizing that, like driver mutations in cancer, the spectrum of mutations that drive resistance to targeted therapy are heterogeneous and perhaps emerge with a lineage-specific prevalence.Significance: In contrast to receptor tyrosine kinases, in which secondary mutations are often responsible for acquired resistance, second-site mutations in BRAF have not been validated in clinically acquired resistance to RAF inhibitors. We demonstrate a secondary mutation in BRAF (V600E/L514V) following progression on dabrafenib and confirm functionally that this mutation is responsible for resistance. Cancer Discov; 8(9); 1130-41. ©2018 AACR.See related commentary by Romano and Kwong, p. 1064This article is highlighted in the In This Issue feature, p. 1047.

Activating ESR1 Mutations Differentially Affect the Efficacy of ER Antagonists.

Recent studies have identified somatic ESR1 mutations in patients with metastatic breast cancer and found some of them to promote estrogen-independent activation of the receptor. The degree to which all recurrent mutants can drive estrogen-independent activities and reduced sensitivity to ER antagonists like fulvestrant is not established. In this report, we characterize the spectrum of ESR1 mutations from more than 900 patients. ESR1 mutations were detected in 10%, with D538G being the most frequent (36%), followed by Y537S (14%). Several novel, activating mutations were also detected (e.g., L469V, V422del, and Y537D). Although many mutations lead to constitutive activity and reduced sensitivity to ER antagonists, only select mutants such as Y537S caused a magnitude of change associated with fulvestrant resistance in vivo Correspondingly, tumors driven by Y537S, but not D5358G, E380Q, or S463P, were less effectively inhibited by fulvestrant than more potent and bioavailable antagonists, including AZD9496. These data point to a need for antagonists with optimal pharmacokinetic properties to realize clinical efficacy against certain ESR1 mutants.Significance: A diversity of activating ESR1 mutations exist, only some of which confer resistance to existing ER antagonists that might be overcome by next-generation inhibitors such as AZD9496. Cancer Discov; 7(3); 277-87. ©2016 AACR.This article is highlighted in the In This Issue feature, p. 235.

Combined EGFR/MEK Inhibition Prevents the Emergence of Resistance in EGFR-Mutant Lung Cancer.

UNLABELLED: Irreversible pyrimidine-based EGFR inhibitors, including WZ4002, selectively inhibit both EGFR-activating and EGFR inhibitor-resistant T790M mutations more potently than wild-type EGFR. Although this class of mutant-selective EGFR inhibitors is effective clinically in lung cancer patients harboring EGFR(T790M), prior preclinical studies demonstrate that acquired resistance can occur through genomic alterations that activate ERK1/2 signaling. Here, we find that ERK1/2 reactivation occurs rapidly following WZ4002 treatment. Concomitant inhibition of ERK1/2 by the MEK inhibitor trametinib prevents ERK1/2 reactivation, enhances WZ4002-induced apoptosis, and inhibits the emergence of resistance in WZ4002-sensitive models known to acquire resistance via both T790M-dependent and T790M-independent mechanisms. Resistance to WZ4002 in combination with trametinib eventually emerges due to AKT/mTOR reactivation. These data suggest that initial cotargeting of EGFR and MEK could significantly impede the development of acquired resistance in EGFR-mutant lung cancer. SIGNIFICANCE: Patients with EGFR-mutant lung cancer develop acquired resistance to EGFR and mutant-selective EGFR tyrosine kinase inhibitors. Here, we show that cotargeting EGFR and MEK can prevent the emergence of a broad variety of drug resistance mechanisms in vitro and in vivo and may be a superior therapeutic regimen for these patients.