ARAF Mutations Research Articles

Abstract LB-055: Clinical acquired resistance to RAF inhibitor combinations in BRAF-mutant colorectal cancer through MAPK pathway alterations

Abstract BRAF V600E mutations occur in ∼10% of colorectal cancer (CRC), and are associated with poor prognosis. RAF inhibition alone has not been an effective treatment in BRAF-mutant (BRAFm) CRC patients, with response rates of only 5%, due to persistence of MAPK signaling. Combined RAF/EGFR, RAF/MEK, or RAF/MEK/EGFR inhibitors have produced improved efficacy in BRAFm CRC patients, yet ultimately resistance develops after an initial treatment response. Understanding the mechanisms of clinical acquired resistance that arise to RAF inhibitor combinations in BRAFm CRC patients may lead to valuable opportunities to overcome resistance and prolong clinical response. We sought to identify clinically relevant mechanisms of acquired resistance to RAF inhibitor combinations by obtaining tumor biopsies from BRAFm CRC patients upon disease progression, after initial response to RAF/EGFR or RAF/MEK inhibitor combinations. Matched pre-treatment, post-progression, and normal DNA were analyzed by whole exome sequencing (WES) and RNA-seq. In one BRAFm CRC patient with prolonged stable disease on a RAF/EGFR combination, WES identified KRAS amplification in a progressing lesion. FISH confirmed the presence of KRAS amplification in the post-progression biopsy, and RNA-seq revealed KRAS transcript overexpression. Interestingly, in resistant clones generated from BRAFm CRC cell lines selected with either RAF/EGFR or RAF/MEK inhibitors, KRAS exon 2 mutations were identified. Either KRAS amplification or KRAS mutation led to sustained MAPK pathway activity and cross-resistance to either RAF/EGFR or RAF/MEK inhibitor combinations. In a second patient with a minor response to a RAF/EGFR inhibitor combination, BRAF amplification was identified in a progressing lesion, which was confirmed by FISH and was not present in a pre-treatment biopsy of the same lesion. BRAF amplification led to cross-resistance to the BRAF/MEK inhibitor combination. In a third patient with a minor response to a RAF/MEK inhibitor combination, WES identified the presence of an ARAF Q489L mutation and a MEK1 F53L mutation in a single progressing lesion, suggesting possible intra-lesional heterogeneity of acquired resistance mechanisms. However, utilizing a cell line derived from the patient's post-progression biopsy, we found that 30 out of 30 single-cell clones harbored both the ARAF and MEK1 mutations, and that MEK1 F53L seemed to function as the primary driver of acquired resistance in these resistant cells. MEK1 F53L expression markedly abrogated the ability of RAF/MEK and RAF/EGFR inhibitor combinations to suppress MAPK signaling. Despite developing resistance to upstream MAPK pathway inhibitors, we found that each of the acquired resistance mechanisms we detected remained sensitive to ERK inhibition, which could effectively suppress MAPK signaling. Our findings demonstrate the central importance of MAPK pathway activity in BRAFm CRC, and highlight the critical need for MAPK pathway inhibition in the prevention of disease progression. Additionally, our work indicates ERK inhibitors may be valuable additions to future therapeutic combinations for BRAFm CRC patients. Further efforts to understand acquired resistance mechanisms will be vital to developing novel therapeutic strategies to overcome resistance and extend clinical benefit in this lethal CRC subtype. Citation Format: Leanne G. Ahronian, Erin M. Sennott, Eliezer M. Van Allen, Nikhil Wagle, Eunice L. Kwak, Jason E. Faris, Jason T. Godfrey, Koki Nishimura, Kerry D. Lynch, Craig H. Mermel, Elizabeth L. Lockerman, Anuj Kalsy, Joseph M. Gurski, Samira Bahl, Kristin Anderka, Lisa M. Green, Niall J. Lennon, Tiffany G. Huynh, Mari Mino-Kenudson, Gad Getz, Dora Dias-Santagata, A. John Iafrate, Jeffrey A. Engelman, Levi A. Garraway, Ryan B. Corcoran. Clinical acquired resistance to RAF inhibitor combinations in BRAF-mutant colorectal cancer through MAPK pathway alterations. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-055. doi:10.1158/1538-7445.AM2015-LB-055

Massive parallel sequencing uncovers actionable FGFR2-PPHLN1 fusion and ARAF mutations in intrahepatic cholangiocarcinoma.

Intrahepatic cholangiocarcinoma (iCCA) is a fatal bile duct cancer with dismal prognosis and limited therapeutic options. By performing RNA- and exome-sequencing analyses, we report a novel fusion event, FGFR2-PPHLN1 (16%), and damaging mutations in the ARAF oncogene (11%). Here we demonstrate that the chromosomal translocation t(10;12)(q26;q12) leading to FGFR2-PPHLN1 fusion possesses transforming and oncogenic activity, which is successfully inhibited by a selective FGFR2 inhibitor in vitro. Among the ARAF mutations, N217I and G322S lead to activation of the pathway and N217I shows oncogenic potential in vitro. Screening of a cohort of 107 iCCA patients reveals that FGFR2 fusions represent the most recurrent targetable alteration (45%, 17/107), while they are rarely present in other primary liver tumours (0/100 of hepatocellular carcinoma (HCC); 1/21 of mixed iCCA-HCC). Taken together, around 70% of iCCA patients harbour at least one actionable molecular alteration (FGFR2 fusions, IDH1/2, ARAF, KRAS, BRAF and FGF19) that is amenable for therapeutic targeting.

428 Clinical acquired resistance to combined RAF/EGFR or RAF/MEK inhibition in BRAF mutant colorectal cancer (CRC) patients through MAPK pathway alterations

Background: BRAF mutations occur in ~10% of CRC and confer poor prognosis. While RAF inhibitor monotherapy leads to response rates of 60−80% in BRAF mutant (BRAFm) melanoma, response rates in BRAFm CRC are poor (~5%). Promising recent studies with RAF inhibitor-based combinations, including combined RAF/EGFR or RAF/MEK inhibition, have produced higher response rates in BRAFm CRC, but a better understanding of acquired resistance mechanisms will be critical to improving therapy. Methods: Mechanisms of acquired resistance to RAF/EGFR or RAF/MEK inhibition were evaluated in BRAFm CRC cell lines and in clinical biopsies obtained from BRAFm CRC patients following disease progression after initial response or prolonged stable disease to either therapeutic combination. BRAFm CRC cell lines were cultured in the presence of combined RAF/EGFR or RAF/MEK inhibitors until resistant clones emerged. Candidate resistance mutations were identified through exome sequencing. Matched pre-treament, post-progression, and normal DNA from BRAFm CRC patients treated with either RAF/EGFR or RAF/MEK combinations were analyzed by whole exome sequencing to identify clinical acquired resistance mechanisms. Results: In resistant clones generated from BRAFm CRC cell lines selected with either RAF/EGFR or RAF/MEK inhibitor combinations, KRAS exon 2 mutations were identified. KRAS mutation led to sustained MAPK pathway activity and cross-resistance to either RAF/EGFR or RAF/MEK inhibitor combinations. Interestingly, the triple combination of RAF/EGFR/MEK inhibition was able to suppress MAPK activity and overcome resistance. In a BRAFm CRC patient with prolonged stable disease on a RAF/EGFR inhibitor combination, whole exome sequencing identified the presence of KRAS amplification in a progressing lesion. RNA sequencing of the same lesion confirmed KRAS transcript overexpression, and ~25-fold amplification of KRAS in the progressing lesion and absence of amplification in the pre-treatment biopsy was confirmed by FISH. In one patient with a minor response to a RAF/MEK inhibitor combination, whole exome sequencing identified amplification of the c-mer oncogene (MERTK) receptor tyrosine kinase as the likely acquired resistance mechanism. In another patient with a minor response to a RAF/MEK inhibitor combination, whole exome sequencing identified subclonal ARAF and MEK1 mutations in the post-progression biopsy, suggesting the presence of heterogeneous acquired resistance mechanisms in this progressing lesion. Conclusions: The identification of alterations affecting the MAPK pathway in BRAFm CRC patients who have developed clinical acquired resistance to RAF/EGFR or RAF/MEK inhibitor regimens underscores the importance of the MAPK pathway in this cancer. Understanding the mechanisms of acquired resistance can lead to novel combination strategies to overcome or delay resistance. Friday 21 November 2014

Oncogenic and sorafenib-sensitive ARAF mutations in lung adenocarcinoma

Targeted cancer therapies often induce "outlier" responses in molecularly defined patient subsets. One patient with advanced-stage lung adenocarcinoma, who was treated with oral sorafenib, demonstrated a near-complete clinical and radiographic remission for 5 years. Whole-genome sequencing and RNA sequencing of primary tumor and normal samples from this patient identified a somatic mutation, ARAF S214C, present in the cancer genome and expressed at high levels. Additional mutations affecting this residue of ARAF and a nearby residue in the related kinase RAF1 were demonstrated across 1% of an independent cohort of lung adenocarcinoma cases. The ARAF mutations were shown to transform immortalized human airway epithelial cells in a sorafenib-sensitive manner. These results suggest that mutant ARAF is an oncogenic driver in lung adenocarcinoma and an indicator of sorafenib response.

Abstract C139: Oncogenic ARAF mutation in a metastatic lung adenocarcinoma from a patient exhibiting sustained sorafenib response.

Abstract Targeted cancer therapies often induce "outlier" responses in molecularly defined patient subsets. One patient with advanced-stage lung adenocarcinoma who was treated with sorafenib demonstrated a near-complete clinical and radiographic remission for five years. Whole-genome sequencing and RNA sequencing on primary tumor and normal samples from this patient identified a somatic mutation, encoding ARAF S214C, present in the cancer genome and expressed at high levels. ARAF S214 binds 14-3-3 proteins when phosphorylated, and, by analogy to the paralogous residue of RAF1, presumably interferes with binding of ARAF to activated Ras. Mutation of S214 impaired the ability of ARAF to bind 14-3-3ξ, likely contributing to the oncogenic activity of ARAF S214C. Additional mutations affecting this residue of ARAF and a nearby residue in the related kinase RAF1 were demonstrated across 1% of an independent cohort of lung adenocarcinoma cases. The ARAF and RAF1 mutants transformed immortalized human airway epithelial cells and were associated with in vitro sorafenib sensitivity. These results suggest that mutant ARAF may be a novel oncogenic driver in lung adenocarcinoma and an indicator of sorafenib response. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C139. Citation Format: Heidi E. Greulich, Marcin Imielinski, Bethany Kaplan, Luiz Araujo, Joseph Amann, Leora Horn, Miguel Villalona, Matthew Meyerson, David Carbone. Oncogenic ARAF mutation in a metastatic lung adenocarcinoma from a patient exhibiting sustained sorafenib response. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C139.

No Evidence of ARAF, CRAF and MET Mutations in BRAFT1799A Negative Human Papillary Thyroid Carcinoma

The MAPK signaling pathway plays a crucial role in tumorgenesis and cell proliferation in human papillary thyroid carcinoma (PTC). Ret/PTC rearrangements, RAS and BRAF mutations, the main non-overlapping genetic alterations all leading to MAPK cascade activation, are cumulatively identified in 60-80% of PTCs. In approximately one-fourth of the cases, oncogenic background potentially contributing to MAPK activation in PTC might be different. We therefore attempted to evaluate the mutational status of genes encoding other members of RAF family known to act upstream of MAPKs, ARAF and CRAF (RAF-1). In addition we also analyzed the MET gene that encodes hepatocyte growth factor/scatter factor receptor overexpressed in most of PTCs and a MAPK cascade contributor. In 129 Japanese patients with PTC, BRAF(T1799A) was detected in 65 cases (50.4%), and the remaining 64 tumor specimens were subjected to mutation analysis of kinase domains of ARAF, CRAF and MET genes, and hotspots of K- and N-RAS genes. No ARAF, CRAF, MET, K- and N-RAS mutations were revealed. Based on these observations, we concluded that despite the fact that ARAF, CRAF and MET are actively expressed, alterations of these genes are rare in PTC and unlikely to play a perceptible role in the molecular pathogenesis of this type of human malignancy.