Activation Mechanism of Oncogenic Deletion Mutations in BRAF, EGFR, and HER2

Article

Activation Mechanism of Oncogenic Deletion Mutations in BRAF, EGFR, and HER2 Graphical Abstract

Authors Scott A. Foster, Daniel M. Whalen, Aysxegu¨l O¨zen, ..., Nicholas J. Skelton, Sarah G. Hymowitz, Shiva Malek

Correspondence [email protected]

In Brief Foster et al. show that oncogenic inframe deletions of BRAF, HER2, and EGFR restrain the C helix (aC) in a fixed ‘‘in’’ active conformation, resulting in resistance to commonly used aC ‘‘out’’ inhibitors, and that five amino acid deletion provides optimal kinase activation.

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BRAF, EGFR, and HER2 harbor oncogenic deletions in the b3-aC loop b3-aC deletions constrain the C helix (aC) in an active conformation Restraining aC confers resistance to aC ‘‘out’’ inhibitors vemurafenib and lapatinib b3-aC length is functionally important and conserved across specific kinase families

Foster et al., 2016, Cancer Cell 29, 1–17 April 11, 2016 ª2016 Elsevier Inc. http://dx.doi.org/10.1016/j.ccell.2016.02.010

Accession Numbers 5HI2 5HID 5HIE 5HIB 5HIC

Please cite this article in press as: Foster et al., Activation Mechanism of Oncogenic Deletion Mutations in BRAF, EGFR, and HER2, Cancer Cell (2016), http://dx.doi.org/10.1016/j.ccell.2016.02.010

Cancer Cell

Article Activation Mechanism of Oncogenic Deletion Mutations in BRAF, EGFR, and HER2 ¨ zen,3,4 Matthew J. Wongchenko,5 JianPing Yin,2 Ivana Yen,1 Scott A. Foster,1 Daniel M. Whalen,2 Aysxegu¨l O Gabriele Schaefer,6 John D. Mayfield,9 Juliann Chmielecki,9 Philip J. Stephens,9 Lee A. Albacker,9 Yibing Yan,5 Kyung Song,6 Georgia Hatzivassiliou,7 Charles Eigenbrot,2 Christine Yu,2 Andrey S. Shaw,1 Gerard Manning,8 Nicholas J. Skelton,3 Sarah G. Hymowitz,2 and Shiva Malek1,* 1Department

of Discovery Oncology of Protein Chemistry & Structural Biology 3Department of Discovery Chemistry 4Department of Early Discovery Biochemistry 5Department of Oncology Biomarker Development 6Department of Translational Oncology 7Department of Cancer Immunology 8Department of Bioinformatics & Computational Biology Genentech Inc., 1 DNA Way, South San Francisco, CA 94080, USA 9Foundation Medicine, 150 Second Street, Cambridge, MA 02141, USA *Correspondence: [email protected] http://dx.doi.org/10.1016/j.ccell.2016.02.010 2Department

SUMMARY

Activating mutations in protein kinases drive many cancers. While how recurring point mutations affect kinase activity has been described, the effect of in-frame deletions is not well understood. We show that oncogenic deletions within the b3-aC loop of HER2 and BRAF are analogous to the recurrent EGFR exon 19 deletions. We identify pancreatic carcinomas with BRAF deletions mutually exclusive with KRAS mutations. Crystal structures of BRAF deletions reveal the truncated loop restrains aC in an active ‘‘in’’ conformation, imparting resistance to inhibitors like vemurafenib that bind the aC ‘‘out’’ conformation. Characterization of loop length explains the prevalence of five amino acid deletions in BRAF, EGFR, and HER2 and highlights the importance of this region for kinase activity and inhibitor efficacy.

INTRODUCTION Kinases control diverse biological events by interpreting and propagating signals through a highly conserved structural domain (Hubbard and Till, 2000; Manning et al., 2002). A key feature of this domain is its ability to switch between active and inactive states upon a given signal, allowing the dynamics required in biological signaling systems. This switch typically involves coordinated movements of two structural elements, the activation segment (AS) and the C helix (aC). Inactive kinases commonly have a compacted AS and an outward-shifted (‘‘out’’) conformation of aC such that a salt bridge between the

catalytic lysine of the b3 strand and a glutamate of aC is disrupted (Figure S1A). Upon activation (often triggered by phosphorylation of the AS), the inactive conformation is disrupted allowing both the AS to adopt a position required for catalysis (the ‘‘DFG-in’’ state) and aC to shift ‘‘in’’ to form the catalytic salt bridge (Figure S1A) (Endicott et al., 2012). Given their significance in many biological functions, it is not surprising that kinases are frequently mutationally activated in cancer. The HER (ERBB/EGFR) family of receptor tyrosine kinases (EGFR and HER2-4) have been widely implicated in cancer with EGFR mutations seen in 10%–30% of non-small-cell lung cancers and ERBB2 mutations or amplification seen in

Significance Here we define a class of recurrent oncogenic deletions within the kinase domains of BRAF, HER2, and EGFR. Structures of BRAF deletions reveal the molecular mechanism of kinase activation and provide insight into resistance mechanisms toward aC ‘‘out’’ kinase inhibitors. Functional characterization of deletion length explains the high prevalence of five amino acid deletions in BRAF, EGFR, and HER2 in various cancers. The length of this loop is variable across the kinome yet highly conserved within individual families, likely reflecting specific regulatory mechanisms used by different kinase families. This work exploits the selective power of oncogenic mutations to highlight a conserved mechanism of kinase activation and underscores the importance of conformation-specific inhibitors to target mutationally activated kinases in cancer. Cancer Cell 29, 1–17, April 11, 2016 ª2016 Elsevier Inc. 1

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subsets of lung, breast, and gastric cancers (Bose et al., 2013; Sharma et al., 2007; Stephens et al., 2004). A major signaling pathway downstream of HER kinases is the mitogen-activated protein kinase (MAPK) pathway, which transmits signals through the RAF (ARAF, BRAF, or CRAF), MEK, and ERK cascade of cytoplasmic serine/threonine kinases. MAPK pathway members are also frequently dysregulated in cancers with BRAF mutations observed in approximately 50% of malignant melanomas and subsets of lung (10%), thyroid (62%), and colorectal (20%) cancers (Cerami et al., 2012; Davies et al., 2002; Gao et al., 2013). EGFR, HER2, and BRAF require dimerization for activity and downstream signaling. HER receptor phosphorylation and activation is triggered by ligand-induced formation of an asymmetric pair of intracellular kinase domains (Schlessinger, 2000; Zhang et al., 2006). These are assembled in a head-to-tail arrangement, with one kinase functioning as the activator (also referred to as the donor) and the other functioning as the receiver (also referred to as the acceptor) (Jura et al., 2009; Zhang et al., 2006). In contrast, RAF activation occurs upon interaction with active Ras at the membrane, resulting in formation of side-to-side RAF dimers and phosphorylation through an asymmetric allosteric mechanism (Garnett et al., 2005; Hu et al., 2013; Rajakulendran et al., 2009; Weber et al., 2001). Oncogenic events circumvent the kinase activation process through a variety of mechanisms, including point mutations (e.g., BRAF V600E, EGFR L858R), in-frame deletions or insertions (e.g., EGFR exon 19 deletions or exon 20 insertions), amplifications (e.g., ERBB2), and gene fusions (e.g., BCR-Abl, BRAF fusions). Recurrent mutations in HER and RAF families (EGFR L858R and BRAF V600E) cluster in structurally homologous regions within their ASs. Charged substitutions of these residues disrupt the hydrophobically driven inactive conformation, shifting the conformational equilibrium toward the active conformation (Wan et al., 2004; Yun et al., 2007). BRAF V600E also renders the kinase independent of RAS activation and dimerization for activity (Haling et al., 2014; Yao et al., 2015). Substantial efforts have been made to find small molecule inhibitors to selectively target oncogenically activated kinases. A diverse array of inhibitors have been developed to treat BRAF-, EGFR-, and HER2-driven cancers including vemurafenib, erlotinib, gefitinib, and lapatinib. To date, their efficacy is limited to subsets of oncogenic alterations. For instance, vemurafenib has only been approved for BRAF V600E metastatic melanomas. While tumors harboring BRAF fusions have been resistant pre-clinically to this class of inhibitor, another class of RAF inhibitors such as sorafenib shows efficacy against these oncogenic alterations (Botton et al., 2013). Similarly, the EGFR inhibitor erlotinib has shown efficacy toward EGFR L858R and exon 19 deletion mutant tumors, while little clinical benefit has been reported in patients harboring tumors with exon 20 insertions (Yasuda et al., 2012). Taken together, kinase inhibitors may not be effective against all activating mutations for a given target. Indeed, mechanistic and structural studies are required to determine the differences between various oncogenic kinase alterations for the development of effective targeted therapies. The goal of this study is to understand how oncogenic short in-frame deletions within the kinase domain b3-aC loop affect kinase structure, function, and kinase inhibitor efficacy. 2 Cancer Cell 29, 1–17, April 11, 2016 ª2016 Elsevier Inc.

RESULTS b3-aC Deletions Are Activating Mutations in BRAF, EGFR, and HER2 Short in-frame deletions mapping to the homologous loop (the b3-aC loop) (see Figure S1A) within the kinase domains of BRAF, EGFR, and HER2 have been identified in patient samples of varying tumor types (Figure 1A; Tables S1–S3 and S4). Largescale sequencing of lung adenocarcinomas showed EGFR b3aC deletions (also referred to as exon 19 deletions) in
20% of tumors, similar in frequency to the L858R mutation (Ding et al., 2008; The Cancer Genome Atlas Research Network, 2014a; Imielinski et al., 2012). The most frequent b3-aC deletion is DELREA (also referred to as delE746-A750), followed by lower frequencies of DLREAT and DLRE (Figure 1A and Table S1). Analysis of lung adenocarcinomas in the Foundation Medicine dataset (n = 1,305) revealed that
20% of tumors had EGFR kinase domain alterations;
44% of which had b3-aC (exon 19) deletions (Figure 1B and Table S4). Short in-frame deletions within the HER2 kinase domain (DLRENT) have been reported at low frequency in ERBB2 non-amplified breast cancers (Figure 1A and Table S2) (Lee at al., 2006a; Shah et al., 2009; Ellis et al., 2012; Bose et al., 2013). The Foundation Medicine dataset also included ERBB2 deletions that accounted for
2% of all ERBB2 alterations (Figure 1C and Table S4). Similarly, BRAF deletions (DNVTAP, DTAPTP, or DPTPQQ) have been identified in TCGA datasets in both pancreatic and thyroid tumor samples (The Cancer Genome Atlas Research Network, 2014b; Cerami et al., 2012; Gao et al., 2013) (Figure 1A and Table S3) as well as in four cell lines (537 Mel, OV-90, NCI-H2405, and BxPC-3). Analysis of the Foundation Medicine dataset identified 29 additional patient tumor samples harboring BRAF deletions, with the highest prevalence in pancreatic carcinomas. Among all pancreatic carcinoma samples queried (n = 1772), we found
1% (13 samples) with BRAF deletions (Figure 1D and Table S4). These BRAF deletions were mutually exclusive with KRAS mutations and other BRAF alterations (Figure 1D), representing
5% of all KRAS wild-type pancreatic carcinomas. Sequence alignment of the deleted region in HER2 and BRAF with EGFR illustrates that these deletions are overlapping (Figure 1A) and occur within the conserved b3-aC loop of the kinase domain (Figure S1A). We hypothesized that BRAF and HER2 b3-aC deletions likely function mechanistically similar to the EGFR b3-aC deletions. To compare enzymatic properties of EGFR and HER2 directly, constructs containing just intracellular domains (ICDs) (Thiel and Carpenter, 2007) were expressed in 293T cells. EGFR b3-aC deletion (DELREA or DLREAT) ICDs showed high activity, comparable with the canonical activating mutation L858R, and much higher than wild-type (WT) (Figure S1B). We validated these results by expressing full-length EGFR WT or mutants in 293T cells under serum starvation. Under these overexpression conditions, EGFR WT had minimal phosphorylation with no detectable increase in pERK levels (Figure S1C). In contrast, EGFR L858R, DELREA, and DLREAT exhibited similarly high EGFR phosphorylation and pERK levels (Figure S1C). As a control, WT and mutants were similarly activated by an acute dose of EGF (Figure S1C), showing that under these starvation

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Figure 1. b3-aC Deletions Are Activating Mutations in BRAF, EGFR, and HER2 (A) Alignment of BRAF, EGFR, and HER2 highlighting the sequence from b3 (gray) to aC (blue). The region of the most frequent deletions is indicated in red letters, with specific deletions underlined. (B) Frequencies of EGFR kinase domain alterations in 1305 lung adenocarcinoma samples from Foundation Medicine. KD, kinase domain; Pt, point. (C) Frequencies of ERBB2 alterations in 134 breast carcinoma patient samples from Foundation Medicine. Amp, amplification. (D) Frequency of specific gene alterations (KRAS, BRAF, NRAS, or other) in pancreatic carcinoma samples from Foundation Medicine. The frequency and mutual exclusivity of BRAF b3-aC deletions (n = 13) and other BRAF alterations (n = 11; 6 of which are V600E) is specified. (E) Western blot of FLAG-tagged HER2 ICD (676-1255) WT or DLRENT (1:2 dilution of HER2 DNA) transiently co-expressed with a fixed concentration (equivalent to the highest HER2 DNA concentration) of MYC-tagged HER3 ICD (666–1342) in 293T cells. Lysates were analyzed for FLAG, pHER2 (Y1248), MYC, pHER3 (Y1289), and actin. (F) Western blot of FLAG-tagged BRAF full-length (FL) WT or indicated mutant transiently expressed (1:5 dilution of BRAF DNA) in HEC1A BRAF/ cells. Lysates were analyzed for BRAF, pMEK (S217/S221), and MEK. See also Figure S1 and Tables S1–S3, S4.

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Figure 2. BRAF DNVTAP Confers CRAF- and Dimer-Independent Activity

(A) Western blot analysis of lysates from transient expression of the indicated FLAG-BRAF WT or mutant (1:5 dilution) in Hec1A BRAF/ cells. The mutants include V600E, DNVTAP, or three dimer-dependent mutants (K483M, G464V, or D594V). (B) Western blot of lysates from transient expression of the indicated FLAG-BRAF WT or mutant (1:5 dilutions) in Hec1A BRAF/ cells. Dimer competent (+) refers to BRAF WT or mutant constructs with R509 while () means the indicated BRAF also carries an R509H mutation. (C) Western blot of lysates from BRAF V600E (A-375) or DNVTAP (OV-90, NCI-H2405, or 537 Mel) cell lines grown in the presence of siRNA reagents (siNTC, siBRAF, or siCRAF) for 72 hr. (D) Cellular viability, in parallel with (C), of cells grown in the presence of siRNA reagents (siNTC, siTOX, siBRAF, or siCRAF) for 8 days. Viability was determined using cell-titer glo. Error bars indicate ±SD. See also Figure S2.

conditions these EGFR mutants enhance kinase activity independent of exogenous ligand. Unlike EGFR, HER2 does not bind ligand and is dependent upon heterodimerization with other HER family members for activation in normal signaling. Previous work has suggested that HER2 DLRENT (in contrast to HER2 overexpression by ERBB2 amplification) may also require heterodimerization as it shows little activity toward itself, but exhibits increased activity toward other HER binding partners (Bose et al., 2013). We similarly observed weak phosphorylation of full-length HER2 DLRENT relative to HER2 WT but observed elevated pERK levels, possibly due to heterodimerization with endogenous EGFR in 293T cells (Figure S1D). To test heterodimerization 4 Cancer Cell 29, 1–17, April 11, 2016 ª2016 Elsevier Inc.

dependence more directly, we established an assay to measure the activity of the HER2 deletion towards HER3, the HER family member that has impaired kinase activity and is the preferred ‘‘activator’’ kinase (Jura et al., 2009). As seen in Figure 1E, the HER2 DLRENT ICD induced a significant increase in HER3 ICD phosphorylation compared with the HER2 WT ICD. Under similar conditions, we observed a slight increase in HER2 phosphorylation upon expression of the ICD deletion mutant (Figure S1E); however, the degree of activation was less dramatic compared with the analogous deletion in EGFR. Taken together, our data show that HER2 DLRENT is an activating deletion primarily through increased phosphorylation of its dimerization partner.

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We next analyzed the kinase activity of BRAF WT, V600E, and three clinically observed deletions (DNVTAP, DTAPTP, and DPTPQQ) using a cell line lacking endogenous BRAF (Hec1A BRAF/) that has nearly undetectable basal levels of phosphorylated MEK (pMEK S217/S221). Expression of BRAF V600E resulted in a significant increase of pMEK relative to WT (Figure 1F). We also observed elevated levels of pMEK for all three BRAF deletions (Figure 1F), demonstrating that these are also kinase-activating alterations in the BRAF kinase domain. BRAF DNVTAP Confers CRAF- and Dimer-Independent Activity The functional consequence of BRAF NVTAP deletion on MAPK signaling was explored by comparison with two other classes of BRAF mutations (Figure S2A): (1) the canonical high activity BRAF V600E, which confers high dimer-independent activity (Haling et al., 2014); (2) BRAF mutations in the ATP binding glycine-rich loop or essential catalytic residues, which can activate through homodimerization or heterodimerization with CRAF (Garnett et al., 2005; Heidorn et al., 2010; Yao et al., 2015). Expression of BRAF WT, V600E, DNVTAP, or dimer-dependent mutants with varying pathway activation (K483M, G464V, or D594V) in the HEC1A BRAF/ cell line shows that DNVTAP has activity comparable with V600E or G464V and much higher pathway activation than WT, K483M, or D594V (Figure 2A). To differentiate DNVTAP from the dimer-independent V600E and dimer-dependent G464V, we mutated arginine 509, a residue essential for dimerization, to histidine (R509H) in both WT and mutants (Rajakulendran et al., 2009). As expected, BRAF WT and G464V showed substantial loss in activity upon R509H mutation (Figure 2B). In contrast, the activity of DNVTAP and V600E was only mildly affected upon loss of dimerization, with the slight decrease in pMEK levels likely due to a decrease in total BRAF expression levels for the R509H constructs (Figure 2B). Similar results were observed in 293T cells (Figure S2B). Using siRNA-mediated knockdown, we tested the dependency of BRAF DNVTAP cell lines on BRAF and/or CRAF for pathway activity and cellular growth. Knockdown of BRAF, but not CRAF, blocked pathway signaling as seen by a decrease in pMEK and pERK levels at 72 hr (Figure 2C) and substantially reduced cell viability after 8 days (60%–80% loss of viability for the DNVTAP lines and 90% for the V600E line) (Figure 2D). We further evaluated CRAF dependence in a panel of cell lines with BRAF V600E, dimer-dependent, or DNVTAP mutations. As expected, all BRAF mutant cell lines showed elevated pERK levels relative to BRAF WT cell lines, and only the BRAF dimer-dependent cell lines show elevated levels of the S338 activating phosphorylation mark on CRAF (Figure S2C). Furthermore, we observed an enrichment of BRAF:CRAF complexes

only in the two dimer-dependent cell lines (NCI-H1666 and MDA-MB-231) with both the V600E cell line (A-375) and DNVTAP cell lines (537 Mel, NCI-H2405, and OV-90) showing low basal levels of BRAF:CRAF dimers (Figure S2D). Together these results indicate BRAF b3-aC deletions (similar to V600E) can function as autonomous kinases, independent of CRAF and dimerization. BRAF DNVTAP Is Resistant to the BRAF Inhibitor Vemurafenib Biochemically, BRAF b3-aC deletions behave analogously to BRAF V600E suggesting both may show similar sensitivity to different classes of BRAF inhibitors. To address this, we tested the sensitivity of BRAF V600E or DNVTAP cell lines toward a panel of BRAF inhibitors representing three classes: GDC0879 (DFG in, aC in), AZ-628 (DFG out, aC in), and vemurafenib (DFG in, aC out). While the BRAF V600E cell line (A-375) was sensitive to all three inhibitors, the DNVTAP cell lines showed high sensitivity toward GDC-0879 and AZ-628, but complete resistance to vemurafenib (Figure 3A) after 1 hr treatment. In cell viability assays (72 hr), a panel of BRAF V600E cell lines exhibited similar sensitivity toward all three inhibitors (Figure 3B), while the DNVTAP lines were resistant to vemurafenib, partially sensitive to GDC-0879, and sensitive to AZ-628. The effects with vemurafenib and AZ-628 were largely consistent with acute pathway signaling inhibition (Figure 3A). Using the cellular thermal shift assay (CETSA) to evaluate cellular target engagement directly (Martinez Molina et al., 2013), we observe that AZ-628 strongly stabilizes BRAF in both V600E and DNVTAP cell lines, while vemurafenib stabilizes BRAF V600E but not BRAF DNVTAP (Figure S3A). This suggests that vemurafenib has differential binding properties against BRAF V600E compared with DNVTAP. We next addressed why the strong acute pathway inhibition did not translate to stronger effects on long-term viability assays in BRAF DNVTAP lines with GDC-0879. We suspected that treatment with GDC-0879 induces pathway reactivation over time through a CRAF-dependent mechanism, resulting in the dampened effects on viability. To test this, we measured MAPK signaling at an intermediate drug concentration (1 mM) at 1 or 24 hr with GDC-0879 or AZ-628. Consistent with sustained inhibitor efficacy in the V600E line, only a slight increase in pMEK levels occurred at 24 hr in the presence of GDC-0879 (Figure 3C). In contrast, a significant increase in both pMEK and pERK levels was observed at 24 hr in the DNVTAP line in the presence of GDC-0879 (Figure 3C). In both lines, AZ-628 suppressed any increase in these levels, implying that BRAF DNVTAP is more susceptible than V600E to drug-induced pathway reactivation likely through CRAF (given the increased CRAF potency of AZ-628

Figure 3. BRAF DNVTAP Is Resistant to the BRAF Inhibitor Vemurafenib (A) Western blot of lysates from BRAF V600E (A-375) or DNVTAP (537 Mel, OV-90, or NCI-H2405) cells after treatment with the indicated concentration of GDC0879, AZ-628, or vemurafenib for 1 hr. (B) Viability of BRAF V600E (501A, Colo 829, G-361, Colo 800, C32, A-375, 928 Mel, Malme-3M, or SK-Mel-28) or DNVTAP (537 Mel, OV-90, or NCI-H2405) cells grown in the presence of the indicated concentration of GDC-0879, AZ-628, or vemurafenib. (C) Western blot analysis of cell lysates from BRAF V600E (A-375) or DNVTAP (537 Mel) cells after 1 hr or 24 hr treatment with GDC-0879 or AZ-628 (both at 1 mM). (D) Phospho-MEK (Ser217/Ser221)/total MEK levels (Meso Scale Discovery) for the indicated BRAF mutants transiently expressed in 293T cells. Cells were treated with the indicated concentration of GDC-0879 or vemurafenib for 1 hr before lysis. Error bars indicate ±SD. See also Figure S3.

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over GDC-0879) (Hatzivassiliou et al., 2010). In addition, both V600E and DNVTAP lines are similarly responsive to MEK inhibitors (cobimetinib and PD-901) in viability assays, suggesting pathway suppression can be sustained if inhibited downstream of RAF (Figure S3B). Recent work suggests vemurafenib resistance of BRAF mutants other than V600 mutations is through formation of BRAF mutant homodimers (Yao et al., 2015). Similarly, the most common mechanisms of acquired resistance to vemurafenib are ones that promote BRAF dimerization: NRAS mutations (Nazarian et al., 2010), a BRAF splice variant lacking a critical negative regulatory region (Poulikakos et al., 2011), and BRAF overexpression by BRAF amplification (Shi et al., 2012). We assessed if BRAF DNVTAP confers vemurafenib resistance through dimerization by expressing dimer-competent BRAF V600E or DNVTAP or monomeric versions of these mutants (V600E R509H or DNVTAP R509H) in 293T cells. Under these conditions, dimercompetent and monomeric versions of V600E are similarly sensitive to both inhibitors (Figure 3D). In contrast, while dimercompetent and monomeric versions of DNVTAP are similarly sensitive to GDC-0879, both are resistant to vemurafenib, showing that vemurafenib resistance of BRAF DNVTAP is independent of dimerization (Figure 3D). Crystal Structure of BRAF DNVTAP Reveals Active ‘‘In’’ aC Despite extensive clinical interest in EGFR b3-aC (exon 19) deletions, crystal structures of kinase-activating deletion mutations have been elusive. To assess the effect b3-aC deletions have on kinase structure and activation state, we determined the X-ray crystal structures of the BRAF kinase domain harboring the NVTAP deletion bound to AZ-628 and the related compound sorafenib at 2.5 A˚ resolution (Figures 4A, S4A, and S4B; Table S5). Both structures superimpose well with BRAF WT bound to sorafenib with a root-mean-square deviation (RMSD) of
0.6– 0.7 A˚ (Figure 4A), indicating that deletion of NVTAP does not significantly perturb the overall kinase domain. Both AZ-628and sorafenib-bound BRAF DNVTAP exhibit a DFG out, but active-like conformation (Figures 4A and S4C) with the 483 K-501E catalytic salt bridge intact (Figure 4B) and no significant alterations to the dimer interface (Figures S4D and S4E). The AS in one of the AZ-628-bound BRAF DNVTAP protomers in the asymmetric unit is ordered, due in part to favorable crystal-packing interactions (Figure S4C). Inspection of the b3-aC region indicates the deletion is accommodated by the repositioning of residues 491TPQQ494 at the N terminus of aC (Figure 4C), which forms the initial turn of aC in BRAF WT. In BRAF

DNVTAP, these residues pivot around Q493 resulting in the unwinding of the top of aC and formation of a type I b turn between b3 and aC. This turn is stabilized by hydrogen bonds between the backbone carbonyl of T491 and the backbone amide of Q494 (Figure 4C). Comparison of this structure to the inactive conformation seen in the structure of vemurafenib bound to BRAF V600E reveals significant differences in the orientation of aC. While the aC is displaced to an ‘‘out’’ conformation bound to vemurafenib, it is sterically restrained to an active ‘‘in’’ conformation in the context of the BRAF DNVTAP (Figures 4A and 4B). To investigate whether other clinically observed BRAF b3-aC deletions result in similar structural effects, we generated homology models of these deletions. The BRAF DNVTAP homology model agrees well with our AZ-628- and sorafenib-bound crystal structures (Figures 4D and S4F–S4H), instilling confidence in our modeling algorithm. Models of three BRAF b3-aC loop deletions (DNVTAP, DTAPTP, and DPTPQQ) similarly predict an aC ‘‘in’’ conformation and formation of the catalytic salt bridge with unwinding of the first N-terminal turn of the aC (Figure S4H). Taken together, we expect that clinically observed BRAF deletions are activating through restraining aC to the ‘‘in’’ conformation. The steric restraints imposed by the shortened b3-aC loop suggests that BRAF DNVTAP would have difficulty accommodating inhibitors that induce an ‘‘out’’ conformation of aC (such as vemurafenib). This would support our previous observations that BRAF DNVTAP cell lines are resistant to vemurafenib (Figures 3A and 3B). To test if these steric restraints explain the differential sensitivities, we introduced the NVTAP deletion into BRAF V600E, using a monomeric version of V600E (V600E R509H) to eliminate the complication of dimerization. As seen in Figure 4E, monomeric BRAF V600E DNVTAP has dramatically reduced sensitivity to vemurafenib (but not to GDC-0879), implying that these steric restraints are dominant in mediating resistance to aC ‘‘out’’ inhibitors. Dabrafenib-Bound BRAF DNVTAP Reveals a Distorted aC While AZ-628 and sorafenib are compatible with the BRAF DNVTAP aC ‘‘in’’ conformation, we questioned whether the b3aC loop deletion could physically accommodate inhibitors that bind the aC ‘‘out’’ conformation. To assess the impact NVTAP deletion has on this class of compounds, we solved the structure of BRAF DNVTAP bound to dabrafenib (another DFG in, aC out inhibitor) at 3.0 A˚ (Figures 5A, S5A, and S5B; Table S5). Comparison of the dabrafenib-bound BRAF DNVTAP and BRAF V600E structures reveals the overall kinase domain is largely unaffected (RMSD
0.7 A˚) (Figure 5A), and the dimerization interface is

Figure 4. Crystal Structure of BRAF DNVTAP Reveals Active ‘‘In’’ aC (A) Superposition of AZ-628-bound BRAF DNVTAP (light blue, b3-aC loop in red), sorafenib-bound BRAF WT (gray; PDB: 1UWH), and vemurafenib-bound BRAF V600E (green; PDB: 3OG7). Sorafenib-bound BRAF DNVTAP is nearly indistinguishable from AZ-628-bound BRAF DNVTAP and was omitted for simplicity. (B) Close-up of the b3-aC region from (A) showing formation of the salt bridge between E501 and K483 (dashed lines) in AZ-628-bound BRAF DNVTAP and sorafenib-bound BRAF WT, but not in vemurafenib-bound BRAF V600E. (C) Close-up of the b3-aC region of AZ-628-bound BRAF DNVTAP and sorafenib-bound BRAF WT with critical side chains shown as sticks. Residues 491TPQQ494 in BRAF DNVTAP are shown in red. Dashed line indicates hydrogen bond between T491 and Q494. (D) Superposition of AZ-628-bound BRAF DNVTAP and vemurafenib-bound BRAF V600E with the BRAF DNVTAP homology model (light gray). Dashed lines indicate the formation of the catalytic salt bridge. (E) pMEK/MEK levels (Meso Scale Discovery) in 293T cells transiently expressing the indicated BRAF mutant. Cells were treated with GDC-0879 or vemurafenib (1 hr). Error bars indicate ±SD. See also Figure S4 and Table S5.

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Figure 5. Dabrafenib-Bound BRAF DNVTAP Reveals a Distorted aC (A) Superposition of dabrafenib-bound BRAF DNVTAP (dark blue) on dabrafenib-bound BRAF V600E (orange with b3-aC loop in red; PDB: 4XV2). In both structures, residues flanking the deletion (L485 or T491) are shown in yellow. (legend continued on next page)

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preserved (Figures S5C and S5D). In contrast, as the dabrafenibbinding mode requires the aC out conformation, dabrafenib binding to BRAF DNVTAP results in significant local perturbation to the b3-aC loop and aC. The shortened b3-aC loop no longer forms the type I b turn observed in the AZ-628- and sorafenibbound BRAF DNVTAP structures. Instead it exhibits an extended configuration with 491TPQQ494 forming the end of aC as in BRAF V600E (Figures 5B and 5C). In addition, the secondary structure of aC is disrupted at the site where aC and dabrafenib interact (L505) and the register of the amino acids is altered relative to the BRAF V600E structure in this region. The helix structure and register return to ‘‘normal’’ at the C-terminal turn of the helix. Despite these alterations, the position of dabrafenib is unaltered from the complex with BRAF V600E (Figure 5D). The relatively high B factors for aC in all four copies of dabrafenib-bound BRAF DNVTAP in the asymmetric unit suggest that this region is less ordered than in the AZ-628- or sorafenib-BRAF DNVTAP, or BRAF WT structures. Given the perturbed aC conformation induced by dabrafenib binding, and the full extension of the shortened b3-aC loop, it is difficult to envisage BRAF DNVTAP accommodating inhibitors that require a greater aC shift. Comparison of aC position in the dabrafenib-bound and vemurafenib-bound BRAF V600E indicates that vemurafenib induces a greater aC shift then dabrafenib (Figure 5B). This difference likely explains BRAF DNVTAP dabrafenib sensitivity versus vemurafenib resistance in both enzymatic assays and 1 hr pathway signaling in cells (Figures 5E and S5E). Similar to GDC-0879, dabrafenib shows weaker affinity towards CRAF (Menzies et al., 2012) suggesting that in the presence of dabrafenib, BRAF DNVTAP cells may undergo some degree of pathway reactivation. This likely explains the decreased dabrafenib sensitivity we observe in 72 hr viability assays (Figure S5F). EGFR and HER2 b3-aC Deletions Are Similarly Resistant to the aC ‘‘Out’’ Inhibitor Lapatinib Like BRAF, EGFR and HER2 b3-aC deletions are of similar length and result in a similar amino acid composition (including position of a proline) in the remaining loop (Figure 1A), suggesting similar structural rearrangements are likely to occur in these deletions. To test this, a modeling protocol was performed for the most common EGFR deletion (DELREA) using several EGFR structures as templates, which span a wide range of aC conformations (Figure 6A). Our model predicts that ELREA deletion positions aC in an active-like conformation with the catalytic salt bridge formed (Figures 6A and 6B) consistent with the prediction by Gilmer et al. (2008) and analogous to the BRAF deletion models. To test these models, molecular dynamics (MD) simulations (spanning a 100-ns time frame) support the stability of the aC in the ‘‘in’’ conformation (Figure S6A). In the DELREA model,

the N-terminal portion of aC loses helicity in MD simulations (Figure S6B) and is repositioned into the b3-aC loop, similar to our observations with BRAF. Given the high similarity of EGFR and HER2 kinase domains, we suspect this model is representative of the compensatory changes upon b3-aC deletions in HER2 as well. We tested the sensitivity of EGFR b3-aC deletions toward two EGFR/HER2 kinase inhibitors with similar binding requirements as the BRAF inhibitors tested (see Figure S6C): (1) erlotinib (similar to GDC-0879), an inhibitor that binds with DFG in, aC in; (2) lapatinib (similar to vemurafenib), an inhibitor that binds with DFG in, aC out. We conducted these experiments by overexpressing EGFR full-length in 293T cells in the presence of serum where we observe similar activity for EGFR WT and mutants. All EGFR mutations show a significant increase in sensitivity to erlotinib relative to EGFR WT (Figure 6C) (Sharma et al., 2007). In contrast, the b3-aC deletions DELREA and DLREAT were more resistant to lapatinib relative to EGFR WT (Gilmer et al., 2008). We next tested the sensitivity of HER2 DLRENT toward lapatinib by co-expressing the HER2 ICD with the HER3 ICD (similar to Figure 1D). The HER2 WT was strongly inhibited by lapatinib, while the deletion mutant showed moderate resistance to lapatinib treatment (Figure 6D) (Bose et al., 2013). Taken together, these data illustrate that, as with BRAF, EGFR and HER2 b3-aC deletions have a shifted ‘‘in’’ aC conferring resistance to aC out inhibitors. b3-aC Loop Length Functions as a Rheostat Controlling Kinase Activity Our structural observations illustrate that b3-aC deletions modulate kinase activity by restraining aC to an active ‘‘in’’ conformation, implying the overall length of the b3-aC deletion may modulate the degree of kinase activation. Analysis of the EGFR b3-aC deletion length from a large number of lung adenocarcinomas illustrates that five amino acid deletions are by far the most frequent (Figures 7A and S7A; Table S4) (Chung et al., 2012). To address how deletion length affects EGFR kinase activity, we deleted one to six residues stepwise from EGFR L747 to S752 (Figure 7B, left) or EGFR T751 to E746 (Figure 7B, right), centered on the LREAT deletion. Maximal activity was achieved with a deletion of five amino acids (independent of direction), with activity increasing slowly between three and four and dropping off dramatically after five (Figure 7B). Minimal activity was observed for most other deletions with the exception of the clinically observed deletions DLRE, DLREA, and DLREATS (Figure 7B). Tumors with EGFR b3-aC deletions shorter than five amino acids often also carry additional base pair substitutions, leading to amino acid changes in the truncated loop, most frequently to proline (Figure S7B and Table S4) (Chung et al., 2012). To test if

(B) Close-up of the b3-aC region from dabrafenib-bound BRAF DNVTAP (dark blue), AZ-628-bound BRAF DNVTAP (light blue), vemurafenib-bound BRAF V600E (green; PDB: 3OG7), and dabrafenib-bound BRAF V600E (orange; PDB: 4XV2) showing formation of the salt bridge between E501 and K483 (dashed line) in AZ-628-bound BRAF DNVTAP, but not in the other BRAF structures. The orientation looking down aC from the top is shown at right. (C) Close-up of the b3-aC region from (A) of dabrafenib-bound BRAF DNVTAP and dabrafenib-bound BRAF V600E with critical b3-aC loop side chains shown as sticks. *Denotes residues in the BRAF DNVTAP structure. (D) As in (C), close-up of the b3-aC region highlighting the position of dabrafenib in both structures and critical aC side chains shown as sticks. (E) pMEK/MEK levels (Meso Scale Discovery) were determined for BRAF V600E (A-375) or DNVTAP (537 Mel) cells after treatment with the indicated concentration of AZ-628, vemurafenib, or dabrafenib (1 hr). Error bars indicate ±SD. See also Figure S5 and Table S5.

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A

B

C

D

Figure 6. EGFR and HER2 b3-aC Deletions Are Similarly Resistant to the aC ‘‘Out’’ Inhibitor Lapatinib (A) Superposition of EGFR templates (all templates in light gray; PDB: 3IKA, 2JIV, 5EDQ, 5HIB, and 5HIC) with the output EGFR DELREA model (cyan). The ELREA sequence in the b3-aC loop is indicated in magenta in template structures. (B) Superposition of a representative active conformation EGFR structure (light gray) with the EGFR DELREA model (cyan) demonstrating that aC in the DELREA model is shifted further in by 10 relative to the active conformation (left). In both the template and model, the catalytic salt bridge (dashed lines) is formed (right). (C) gD-EGFR FL WT or the indicated mutant was transiently expressed into 293T cells, transfection reagents were removed after 24 hr, and cells were treated with the indicated concentration of erlotinib or lapatinib (2 hr). Lysates were analyzed by western blot. (legend continued on next page)

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the presence of a proline is advantageous for deletions shorter than five, we mutated the residue immediately following the 30 end of the deletion to either a proline or a serine (which occurs at a much lower frequency) in the EGFR DLRE or DLREA ICD. Introduction of a proline (but not serine) in the context of both the LRE and LREA deletion significantly increased EGFR ICD activity (Figure 7C). Deletions longer than five frequently co-occur with mutation of proline 753, typically to serine (Figure S7B). Consistent with an advantage to losing this proline, P753S mutation increased EGFR DLREATS activity (Figure 7C). Similar to EGFR, BRAF deletions of five amino acids are most prevalent in patient tumor samples (Tables S3 and S4). To determine if five amino acid deletions are also optimal for BRAF kinase activity, we designed an analogous deletion walking experiment with BRAF centered on the NVTAP deletion with one to six residues deleted stepwise from N486 to T491 (Figure 7D, left) or from P490 to L485 (Figure 7D, right). Partial activation was observed for deletions of two, three, or four residues, with slight differences observed depending upon the direction of the deletion, suggesting that both the amino acid context and the length of the b3-aC loop contributes to BRAF kinase activity (Figure 7D). Similar to EGFR, deletions of six amino acids result in little or no activation relative to BRAF WT (Figure 7D). Independent of the direction of deletion, maximal activity was once again observed upon deletion of five residues (Figure 7D). Taken together, the enrichment for five amino acid deletions appears to be conserved across EGFR, BRAF, and HER2, potentially due to optimal structural restraints imposed with this deletion length. b3-aC Loop Length Is Variable Across the Human Kinome, yet Highly Conserved in Individual Kinase Families Given the functional importance of the b3-aC loop length for kinase activity, we asked whether b3-aC loop length is conserved across individual families in the kinome, and whether loop length correlates with kinase activity. b3-aC loop length was inferred from the distance between the catalytic lysine and aC glutamate (K72 and E91 in PKA), across 476 human kinase domains that could be well aligned in this region (Table S7). Surprisingly, the distribution of K-E length was quite broad across the human kinome (ranging from seven to 41 amino acids), suggesting this loop likely has variable functions in different kinases (Figure 7E). Despite this broad distribution, the length was highly characteristic of individual families: 16 for all EGFR/HER family members, 17 for the three RAF family members, and 14 for all eight SrcA and B subfamily members (Figure 7E). Furthermore, these lengths are conserved throughout evolution indicating that the precise degree of flexibility accorded by this loop is key to the function of most protein kinases. DISCUSSION Oncogenic mutations within kinase genes occur in a broad spectrum of cancers and at times show tissue specificity. As

an example, the spectrum of EGFR driver mutations differ between lung adenocarcinoma (most commonly kinase domain) and glioblastoma (most commonly extracellular) (Lee et al., 2006b; Sharma et al., 2007). While HER2 b3-aC deletions were restricted to breast tumors as may have been expected given the prevalence of ERBB2 amplifications in breast carcinomas, BRAF deletions were unexpectedly enriched in pancreatic carcinomas and less frequently observed in melanoma or thyroid tumor samples, unlike BRAF V600E. A similar trend has also been observed in lung adenocarcinomas, where non-V600E kinase domain point mutations are prevalent (The Cancer Genome Atlas Research Network, 2014a). The lower frequency of BRAF V600E and other point mutations in pancreatic tumors coupled with the low level of BRAF deletions in melanoma and thyroid samples suggest that the enrichment of a specific spectrum of mutations or deletions in each tumor type may be dependent on cellular context. The conformation of the C helix has a profound effect on kinase activity and the efficacy of different classes of kinase inhibitors. Our structural and modeling studies reveal that deletions in the b3-aC loop constrain aC to an active position by reducing a source of conformational flexibility. In essence, the shorter b3-aC loop predisposes aC to an ‘‘in’’ conformation promoting kinase activity (Figure 8A), concomitantly diminishing the efficacy of kinase inhibitors that bind an ‘‘out’’ aC conformation (Figure 8B). A surprising result was the degree to which the length of the b3-aC loop can function as a rheostat for kinase activity. Indeed, functional studies with BRAF and EGFR showed that five amino acid deletions are optimal for activity. From simple geometric considerations, reducing the length of the b3-aC loop shifts the manifold of low energy conformations accessible to aC away from the ‘‘out’’ and towards the ‘‘in’’ conformation. The extent of this shift likely depends on the sequence of the intervening loop, due to the geometric restraints imposed by its constituent amino acids on the accessible low energy conformations. In the absence of other mutations, deletion of five amino acids leads to an optimal orientation of aC for catalytic activity, while deletion of six or more amino acids leads to aC structural perturbations that degrade catalytic activity. As demonstrated with EGFR, mutations can compensate for either shorter or longer deletion lengths. For example, deletions of three or four amino acids may show optimal activity if they are accompanied by substitution of a remaining loop residue to a proline (e.g., EGFR DLRE or DLREA variants); presumably the proline ‘‘pinches off’’ part of the loop reducing its effective length. Conversely, deletions of six amino acids may also be highly activating if a proline in the remaining loop is replaced by serine to allow a more extended conformation (e.g., EGFR DLREATS). The importance of linker length in modulating the conformational dynamics of aC suggests that b3-aC loop length may modulate kinase activity more generally. Interestingly, while the length of this loop varies significantly across the kinome, loop length is highly conserved within individual families and through

(D) FLAG-HER2 ICD WT or DLRENT was transiently co-expressed with MYC-HER3 ICD (equal HER2 and HER3 DNA) in 293T cells. Transfection reagents were removed after 24 hr, cells were reseeded in six-well dishes overnight, and treated with the indicated concentration of lapatinib (2 hr). Lysates were analyzed by western blot. See also Figure S6 and Table S6.

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A

400

(n = 552)

# of Patients

300 200 100 0

3

4

5

6

7

Deletion Length

4

1

2

ΔE AT ΔR EA ΔL T R E ΔE AT LR ΔL EA R T EA TS

3

ΔE T

ΔL R

2

ΔT

ΔL R

1

Em pt y W T

E ΔL A R E ΔE AT LR ΔL EAT R EA TS

ΔL R

E ΔL

Em pt W y T

B

FLAG-EGFR pEGFR Actin 4

S -

53

51 S 51 P

P -

S

3

5

6

6

ΔLREATS Deletion

ΔLREA 50

Em pty WT ΔL RE AT

ΔLRE

-

Mutation

P7

-

T7

Controls

6

T7

6

50

C

5

A7

-

A7

-

-

Deletion Length

FLAG-EGFR pEGFR Actin 3

4

Deletion Length

6

3

4

ΔA P ΔT AP ΔV TA ΔN P VT ΔL AP N V ΔN TAP VT AP T

2

ΔP

1

Em pt y W T

ΔN

VT ΔN A VT ΔL AP N V ΔN TAP VT AP T

ΔN

VT

ΔN

V ΔN

Em

pt y W T

D

FLAG-BRAF pMEK MEK -

E

120

5

6

80

60

-

6

-

1

2

3

4

5

6

6

EGFR & HER2-4

100

# of Kinases

Deletion Length -

A/B/CRAF

SrcA & B families

40

20

0 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 32 35 36 38 41

K - E Length

(legend on next page)

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A

B

Figure 8. Models for the Activation Mechanism of b3-aC Loop Deletions and Altered Efficacy of Different Classes of Kinase Inhibitors (A) A model of a generic kinase domain highlighting how shortening of the b3-aC loop alters the equilibrium between inactive and active conformations. (B) A model of a generic kinase domain highlighting how b3-aC loop deletions cause a steric clash between aC and aC ‘‘out’’ inhibitors, reducing the efficacy of this class of inhibitor.

evolution. As an example, all eight members of the SrcA and B kinase families have b3-aC loops that are three amino acids shorter than the RAF family. Interestingly, Src kinases are constrained by intra-molecular interactions with SH2-SH3 domains, suggesting that these interactions may have co-evolved to suppress the inherent preference of aC to adopt the active conformation (Xu et al., 1999). Several families also have conserved b3-aC loop lengths similar to those of the oncogenic deletion mutants and so might be expected to be constitutively active or may require specific mechanisms to restrain activity if aC cannot be shifted ‘‘out.’’ Interestingly, this includes the known

constitutively active ‘‘casein kinase’’ families CK1 and CK2. Other conserved short-loop kinases include the non-constitutively active TGFb/activin receptors, GSK3, BUB1, and IRE families, suggesting (similar to Src kinases) that their regulatory mechanisms must have evolved to deal with, and potentially benefit from, a short loop and constrained aC. In contrast, kinases with longer linker lengths such as BRAF, EGFR, and HER2 have adopted common structural mechanisms to stabilize their active conformation, namely through dimerization upon pathway activation in normal signaling or by b3-aC loop deletions in various cancers.

Figure 7. b3-aC Loop Length Functions as a Rheostat Controlling Kinase Activity (A) Deletion length prevalence of EGFR b3-aC deletions from the Foundation Medicine dataset. Only deletions that occur within a portion of the b3-aC loop (also referred to as LRE deletions) were included. The low frequency (14/566) of non-LRE deletions were excluded from this analysis. (B) Western blot analysis of lysates from transient expression in 293T cells of FLAG-EGFR ICD WT or mutation series with sequential one amino acid deletions centered on EGFR LREAT; from left to right (E746 to T751) (left) or from right to left (S492 to L747) (right). (C) Western blot analysis of lysates from transient expression in 293T of FLAG-EGFR DLRE, DLREA, or DLREATS with additional point mutations. For DLRE and DLREA, the residue 30 of the deletion is mutated to serine or proline. For DLREATS, P753 is mutated to serine. (D) Western blot analysis of lysates from transient expression in Hec1A BRAF/ cells of FLAG-BRAF WT or mutation series with sequential one amino acid deletions centered on BRAF NVTAP; from left to right (L485 to P490) (left) or from right to left (T491 to N486) (right). (E) The variability of the length between the catalytic lysine within the b3 strand and aC glutamate (K-E length) was determined for a majority of the human kinome (476 of 492 kinase domains). See also Figure S7 and Table S7.

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Despite the commonality of the structural effects of b3-aC deletions on the kinase domain, EGFR/HER2 or BRAF have uniquely co-opted these structural perturbations to optimize pathway signaling. For HER2, in addition to activating the kinase directly, the b3-aC deletions appear to confer a preference as a receiver kinase, as also suggested for the EGFR L858R (and likely the EGFR b3-aC deletions) (Red Brewer et al., 2013). In the context of BRAF, similar to BRAF V600E, b3-aC deletions are likely to dimerize (Thevakumaran et al., 2015), but kinase activity is dimerization independent (Haling et al., 2014). Interestingly, dimerization plays a central role both in normal RAF biology and in clinical acquired resistance to the aC out inhibitor vemurafenib (Poulikakos et al., 2011; Yao et al., 2015). Based on our work, we rationalize that dimerization (which results in robust pathway activation) leads to the subsequent structural shift in aC, providing an optimal way to alleviate the inhibitory effects of these inhibitors. Indeed, we have shown that introduction of the helix-shifting NVTAP deletion is sufficient to confer vemurafenib resistance to BRAF V600E, independent of dimerization. This may explain why these dimerization mechanisms are preferred and BRAF point mutations are exceedingly rare in vemurafenib-acquired resistance, unlike ‘‘gatekeeper’’ mutations in acquired resistance to kinase inhibitors that target BCR-Abl or EGFR mutants (Krishnamurty and Maly, 2010). Taken together, these data indicate that design of targeted therapies for oncogenic kinases should take into account the molecular complexity of each specific mutation and pathway. Furthermore, in the development of inhibitors toward kinase targets, the inherent structural diversity and importance of the b3-aC region should be considered. Development of conformation-specific kinase inhibitors to target a diversity of oncogenic mutations as well as anticipating acquired resistance mechanisms is critical to achieve the optimal therapeutic benefit for cancer patients. EXPERIMENTAL PROCEDURES Procedures are described in more detail in the Supplemental Experimental Procedures. Cell Culture, Constructs, and Transfections All cell lines were obtained from the Genentech cell line repository and cultured based upon ATCC specifications. Various BRAF, EGFR, HER2, and HER3 expression constructs were cloned into a cytomegalovirus-driven, non-integrating vector. Cells were transfected using Lipofectamine LTX using standard protocols. All compounds were obtained from Genentech Compound Management. Specific lysis conditions and a complete list of antibodies are provided in the Supplemental Information. Protein Production, Purification, and Crystallization of BRAF DNVTAP The His-tagged human BRAF DNVTAP kinase domain in the 16 mutation background (Joseph et al., 2010) was produced in Escherichia coli. AZ-628 or sorafenib was added at cell lysis. Complexes were purified by affinity, size-exclusion, and ion-exchange chromatography. The complexes crystallized in hanging drops composed of protein (5 mg/ml) and well solution of 20% PEG 3350 and 200 mM potassium nitrate. The His-tagged human BRAF DNVTAP kinase domain (without any additional mutations) was co-expressed in T.ni cells with FLAG-tagged human CDC37 and purified by affinity and size-exclusion chromatography. Dabrafenib was added during concentration. The complex crystallized in sitting drops composed of protein

(2.8 mg/ml) and well solution of 100 mM Tris (pH 8.5), 19.3% PEG 8K v/v, and 500 mM NaCl. X-Ray diffraction data were collected at SSRL beamline 12-2. Structures were solved by molecular replacement. Cycles of building and refinement resulted in the final models. Structural figures were created with PyMOL. Modeling Molecular modeling was performed using the Prime software (version 3.8) and is further described in the Supplemental Information. ACCESSION NUMBERS The following structures have been submitted to the PDB and given the indicated accession numbers: BRAF, DNVTAP-sorafenib (PDB: 5HI2), DNVTAPAZ-628 (PDB: 5HID), and DNVTAP-dabrafenib (PDB: 5HIE); EGFR, T790M/ L858R-G-868 (PDB: 5HIB) and T790M/L858R-G-879 (PDB: 5HIC). SUPPLEMENTAL INFORMATION Supplemental Information includes Supplemental Experimental Procedures, seven figures, and seven tables and can be found with this article online at http://dx.doi.org/10.1016/j.ccell.2016.02.010. AUTHOR CONTRIBUTIONS S.A.F. designed and conducted the biochemistry and cell biology experiments. D.W., J.P.Y., and S.G.H. crystallized and determined the structures of BRAF DNVTAP. C.Y. and C.E. determined the crystal structures of EGFR. A.O. conducted the molecular modeling and molecular dynamic simulations. G.M. carried out sequence analysis. J.C., P.J.S., L.A., and J.M. contributed to genomic analyses. S.A.F., A.O., D.W., S.G.H., G.M., N.S., and S.M. wrote the manuscript. ACKNOWLEDGMENTS We thank Weiru Wang and James Kiefer in the Genentech Structure Group for help with synchrotron data collection; the gCell group for cell lines, Dr. Todd Waldman for the HEC1A BRAF/ cell line; the Genentech Sequencing, Cloning and Expression groups for their support; David Stokoe and Nick Endres for critical review of the manuscript; and Allison Bruce for assistance in generating figures. SSRL is supported by the Department of Energy, the National Institutes of Health, the National Institute of General Medical Sciences and the National Center for Research Resources. All authors are employees and shareholders of Genentech/Roche or Foundation Medicine at the time this work was completed. Received: November 20, 2015 Revised: January 11, 2016 Accepted: February 13, 2016 Published: March 17, 2016 REFERENCES Bose, R., Kavuri, S.M., Searleman, A.C., Shen, W., Shen, D., Koboldt, D.C., Monsey, J., Goel, N., Aronson, A.B., Li, S., et al. (2013). Activating HER2 mutations in HER2 gene amplification negative breast cancer. Cancer Discov. 3, 224–237. Botton, T., Yeh, I., Nelson, T., Vemula, S.S., Sparatta, A., Garrido, M.C., Allegra, M., Rocchi, S., Bahadoran, P., McCalmont, T.H., et al. (2013). Recurrent BRAF kinase fusions in melanocytic tumors offer an opportunity for targeted therapy. Pigment Cell Melanoma Res. 26, 845–851. Cerami, E., Gao, J., Dogrusoz, U., Gross, B.E., Sumer, S.O., Aksoy, B.A., Jacobsen, A., Byrne, C.J., Heuer, M.L., Larsson, E., et al. (2012). The cBio cancer genomics portal: an open platform for exploring multidimensional cancer genomics data. Cancer Discov. 2, 401–404. Chung, K.P., Wu, S.G., Wu, J.Y., Yang, J.C., Yu, C.J., Wei, P.F., Shih, J.Y., and Yang, P.C. (2012). Clinical outcomes in non-small cell lung

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