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 β3-αC 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 αC in an active “in” conformation, imparting resistance to inhibitors like vemurafenib that bind the αC “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.

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 αC “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.

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 (). 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 (). 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 β3-αC loop affect kinase structure, function, and kinase inhibitor efficacy.

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 (). BRAF V600E also renders the kinase independent of RAS activation and dimerization for activity ().

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 (). 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) (). 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 ().

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 subsets of lung, breast, and gastric cancers (). 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 ().

Kinases control diverse biological events by interpreting and propagating signals through a highly conserved structural domain (). 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 (αC). Inactive kinases commonly have a compacted AS and an outward-shifted (“out”) conformation of αC such that a salt bridge between the catalytic lysine of the β3 strand and a glutamate of αC is disrupted ( Figure S1 A). 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 αC to shift “in” to form the catalytic salt bridge ( Figure S1 A) ().

Given the functional importance of the β3-αC loop length for kinase activity, we asked whether β3-αC loop length is conserved across individual families in the kinome, and whether loop length correlates with kinase activity. β3-αC loop length was inferred from the distance between the catalytic lysine and αC 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 7 E). 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 7 E). 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.

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 7 D, left) or from P490 to L485 ( Figure 7 D, 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 β3-αC loop contributes to BRAF kinase activity ( Figure 7 D). Similar to EGFR, deletions of six amino acids result in little or no activation relative to BRAF WT ( Figure 7 D). Independent of the direction of deletion, maximal activity was once again observed upon deletion of five residues ( Figure 7 D). 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.

Tumors with EGFR β3-αC 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 S7 B and Table S4 ) (). To test if the presence of a proline is advantageous for deletions shorter than five, we mutated the residue immediately following the 3′ end of the deletion to either a proline or a serine (which occurs at a much lower frequency) in the EGFR ΔLRE or ΔLREA ICD. Introduction of a proline (but not serine) in the context of both the LRE and LREA deletion significantly increased EGFR ICD activity ( Figure 7 C). Deletions longer than five frequently co-occur with mutation of proline 753, typically to serine ( Figure S7 B). Consistent with an advantage to losing this proline, P753S mutation increased EGFR ΔLREATS activity ( Figure 7 C).

Our structural observations illustrate that β3-αC deletions modulate kinase activity by restraining αC to an active “in” conformation, implying the overall length of the β3-αC deletion may modulate the degree of kinase activation. Analysis of the EGFR β3-αC deletion length from a large number of lung adenocarcinomas illustrates that five amino acid deletions are by far the most frequent ( Figures 7 A and S7 A; Table S4 ) (). To address how deletion length affects EGFR kinase activity, we deleted one to six residues stepwise from EGFR L747 to S752 ( Figure 7 B, left) or EGFR T751 to E746 ( Figure 7 B, 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 7 B). Minimal activity was observed for most other deletions with the exception of the clinically observed deletions ΔLRE, ΔLREA, and ΔLREATS ( Figure 7 B).

(E) The variability of the length between the catalytic lysine within the β3 strand and αC glutamate (K-E length) was determined for a majority of the human kinome (476 of 492 kinase domains).

(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).

(C) Western blot analysis of lysates from transient expression in 293T of FLAG-EGFR ΔLRE, ΔLREA, or ΔLREATS with additional point mutations. For ΔLRE and ΔLREA, the residue 3′ of the deletion is mutated to serine or proline. For ΔLREATS, P753 is mutated to serine.

(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).

(A) Deletion length prevalence of EGFR β3-αC deletions from the Foundation Medicine dataset. Only deletions that occur within a portion of the β3-αC loop (also referred to as LRE deletions) were included. The low frequency (14/566) of non-LRE deletions were excluded from this analysis.

We tested the sensitivity of EGFR β3-αC deletions toward two EGFR/HER2 kinase inhibitors with similar binding requirements as the BRAF inhibitors tested (see Figure S6 C): (1) erlotinib (similar to GDC-0879), an inhibitor that binds with DFG in, αC in; (2) lapatinib (similar to vemurafenib), an inhibitor that binds with DFG in, αC 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 6 C) (). In contrast, the β3-αC deletions ΔELREA and ΔLREAT were more resistant to lapatinib relative to EGFR WT (). We next tested the sensitivity of HER2 ΔLRENT toward lapatinib by co-expressing the HER2 ICD with the HER3 ICD (similar to Figure 1 D). The HER2 WT was strongly inhibited by lapatinib, while the deletion mutant showed moderate resistance to lapatinib treatment ( Figure 6 D) (). Taken together, these data illustrate that, as with BRAF, EGFR and HER2 β3-αC deletions have a shifted “in” αC conferring resistance to αC out inhibitors.

Like BRAF, EGFR and HER2 β3-αC deletions are of similar length and result in a similar amino acid composition (including position of a proline) in the remaining loop ( Figure 1 A), 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 (ΔELREA) using several EGFR structures as templates, which span a wide range of αC conformations ( Figure 6 A ). Our model predicts that ELREA deletion positions αC in an active-like conformation with the catalytic salt bridge formed ( Figures 6 A and 6B) consistent with the prediction byand 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 αC in the “in” conformation ( Figure S6 A). In the ΔELREA model, the N-terminal portion of αC loses helicity in MD simulations ( Figure S6 B) and is repositioned into the β3-αC 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 β3-αC deletions in HER2 as well.

(D) FLAG-HER2 ICD WT or ΔLRENT 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.

(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.

(B) Superposition of a representative active conformation EGFR structure (light gray) with the EGFR ΔELREA model (cyan) demonstrating that αC in the ΔELREA 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).

(A) Superposition of EGFR templates (all templates in light gray; PDB: 3IKA 5HIB , and 5HIC ) with the output EGFR ΔELREA model (cyan). The ELREA sequence in the β3-αC loop is indicated in magenta in template structures.

While AZ-628 and sorafenib are compatible with the BRAF ΔNVTAP αC “in” conformation, we questioned whether the β3-αC loop deletion could physically accommodate inhibitors that bind the αC “out” conformation. To assess the impact NVTAP deletion has on this class of compounds, we solved the structure of BRAF ΔNVTAP bound to dabrafenib (another DFG in, αC out inhibitor) at 3.0 Å ( Figures 5 A, S5 A, and S5B; Table S5 ). Comparison of the dabrafenib-bound BRAF ΔNVTAP and BRAF V600E structures reveals the overall kinase domain is largely unaffected (RMSD ∼0.7 Å) ( Figure 5 A ), and the dimerization interface is preserved ( Figures S5 C and S5D). In contrast, as the dabrafenib-binding mode requires the αC out conformation, dabrafenib binding to BRAF ΔNVTAP results in significant local perturbation to the β3-αC loop and αC. The shortened β3-αC loop no longer forms the type I β turn observed in the AZ-628- and sorafenib-bound BRAF ΔNVTAP structures. Instead it exhibits an extended configuration withTPQQforming the end of αC as in BRAF V600E ( Figures 5 B and 5C). In addition, the secondary structure of αC is disrupted at the site where αC 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 5 D). The relatively high B factors for αC in all four copies of dabrafenib-bound BRAF ΔNVTAP in the asymmetric unit suggest that this region is less ordered than in the AZ-628- or sorafenib-BRAF ΔNVTAP, or BRAF WT structures. Given the perturbed αC conformation induced by dabrafenib binding, and the full extension of the shortened β3-αC loop, it is difficult to envisage BRAF ΔNVTAP accommodating inhibitors that require a greater αC shift. Comparison of αC position in the dabrafenib-bound and vemurafenib-bound BRAF V600E indicates that vemurafenib induces a greater αC shift then dabrafenib ( Figure 5 B). This difference likely explains BRAF ΔNVTAP dabrafenib sensitivity versus vemurafenib resistance in both enzymatic assays and 1 hr pathway signaling in cells ( Figures 5 E and S5 E). Similar to GDC-0879, dabrafenib shows weaker affinity towards CRAF () suggesting that in the presence of dabrafenib, BRAF ΔNVTAP cells may undergo some degree of pathway reactivation. This likely explains the decreased dabrafenib sensitivity we observe in 72 hr viability assays ( Figure S5 F).

(E) pMEK/MEK levels (Meso Scale Discovery) were determined for BRAF V600E (A-375) or ΔNVTAP (537 Mel) cells after treatment with the indicated concentration of AZ-628, vemurafenib, or dabrafenib (1 hr). Error bars indicate ±SD.

(D) As in (C), close-up of the β3-αC region highlighting the position of dabrafenib in both structures and critical αC side chains shown as sticks.

(C) Close-up of the β3-αC region from (A) of dabrafenib-bound BRAF ΔNVTAP and dabrafenib-bound BRAF V600E with critical β3-αC loop side chains shown as sticks. ∗ Denotes residues in the BRAF ΔNVTAP structure.

(B) Close-up of the β3-αC region from dabrafenib-bound BRAF ΔNVTAP (dark blue), AZ-628-bound BRAF ΔNVTAP (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 ΔNVTAP, but not in the other BRAF structures. The orientation looking down αC from the top is shown at right.

(A) Superposition of dabrafenib-bound BRAF ΔNVTAP (dark blue) on dabrafenib-bound BRAF V600E (orange with β3-αC loop in red; PDB: 4XV2 ). In both structures, residues flanking the deletion (L485 or T491) are shown in yellow.

The steric restraints imposed by the shortened β3-αC loop suggests that BRAF ΔNVTAP would have difficulty accommodating inhibitors that induce an “out” conformation of αC (such as vemurafenib). This would support our previous observations that BRAF ΔNVTAP cell lines are resistant to vemurafenib ( Figures 3 A 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 4 E, monomeric BRAF V600E ΔNVTAP has dramatically reduced sensitivity to vemurafenib (but not to GDC-0879), implying that these steric restraints are dominant in mediating resistance to αC “out” inhibitors.

To investigate whether other clinically observed BRAF β3-αC deletions result in similar structural effects, we generated homology models of these deletions. The BRAF ΔNVTAP homology model agrees well with our AZ-628- and sorafenib-bound crystal structures ( Figures 4 D and S4 F–S4H), instilling confidence in our modeling algorithm. Models of three BRAF β3-αC loop deletions (ΔNVTAP, ΔTAPTP, and ΔPTPQQ) similarly predict an αC “in” conformation and formation of the catalytic salt bridge with unwinding of the first N-terminal turn of the αC ( Figure S4 H). Taken together, we expect that clinically observed BRAF deletions are activating through restraining αC to the “in” conformation.

Despite extensive clinical interest in EGFR β3-αC (exon 19) deletions, crystal structures of kinase-activating deletion mutations have been elusive. To assess the effect β3-αC 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 Å resolution ( Figures 4 A, S4 A, 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 Å ( Figure 4 A ), indicating that deletion of NVTAP does not significantly perturb the overall kinase domain. Both AZ-628- and sorafenib-bound BRAF ΔNVTAP exhibit a DFG out, but active-like conformation ( Figures 4 A and S4 C) with theK-E catalytic salt bridge intact ( Figure 4 B) and no significant alterations to the dimer interface ( Figures S4 D and S4E). The AS in one of the AZ-628-bound BRAF ΔNVTAP protomers in the asymmetric unit is ordered, due in part to favorable crystal-packing interactions ( Figure S4 C). Inspection of the β3-αC region indicates the deletion is accommodated by the repositioning of residuesTPQQat the N terminus of αC ( Figure 4 C), which forms the initial turn of αC in BRAF WT. In BRAF ΔNVTAP, these residues pivot around Q493 resulting in the unwinding of the top of αC and formation of a type I β turn between β3 and αC. This turn is stabilized by hydrogen bonds between the backbone carbonyl of T491 and the backbone amide of Q494 ( Figure 4 C). 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 αC. While the αC is displaced to an “out” conformation bound to vemurafenib, it is sterically restrained to an active “in” conformation in the context of the BRAF ΔNVTAP ( Figures 4 A and 4B).

(C) Close-up of the β3-αC region of AZ-628-bound BRAF ΔNVTAP and sorafenib-bound BRAF WT with critical side chains shown as sticks. Residues 491 TPQQ 494 in BRAF ΔNVTAP are shown in red. Dashed line indicates hydrogen bond between T491 and Q494.

(B) Close-up of the β3-αC region from (A) showing formation of the salt bridge between E501 and K483 (dashed lines) in AZ-628-bound BRAF ΔNVTAP and sorafenib-bound BRAF WT, but not in vemurafenib-bound BRAF V600E.

(A) Superposition of AZ-628-bound BRAF ΔNVTAP (light blue, β3-αC loop in red), sorafenib-bound BRAF WT (gray; PDB: 1UWH ), and vemurafenib-bound BRAF V600E (green; PDB: 3OG7 ). Sorafenib-bound BRAF ΔNVTAP is nearly indistinguishable from AZ-628-bound BRAF ΔNVTAP and was omitted for simplicity.

Recent work suggests vemurafenib resistance of BRAF mutants other than V600 mutations is through formation of BRAF mutant homodimers (). Similarly, the most common mechanisms of acquired resistance to vemurafenib are ones that promote BRAF dimerization: NRAS mutations (), a BRAF splice variant lacking a critical negative regulatory region (), and BRAF overexpression by BRAF amplification (). We assessed if BRAF ΔNVTAP confers vemurafenib resistance through dimerization by expressing dimer-competent BRAF V600E or ΔNVTAP or monomeric versions of these mutants (V600E R509H or ΔNVTAP R509H) in 293T cells. Under these conditions, dimer-competent and monomeric versions of V600E are similarly sensitive to both inhibitors ( Figure 3 D). In contrast, while dimer-competent and monomeric versions of ΔNVTAP are similarly sensitive to GDC-0879, both are resistant to vemurafenib, showing that vemurafenib resistance of BRAF ΔNVTAP is independent of dimerization ( Figure 3 D).

We next addressed why the strong acute pathway inhibition did not translate to stronger effects on long-term viability assays in BRAF ΔNVTAP 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 μM) 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 3 C). In contrast, a significant increase in both pMEK and pERK levels was observed at 24 hr in the ΔNVTAP line in the presence of GDC-0879 ( Figure 3 C). In both lines, AZ-628 suppressed any increase in these levels, implying that BRAF ΔNVTAP is more susceptible than V600E to drug-induced pathway reactivation likely through CRAF (given the increased CRAF potency of AZ-628 over GDC-0879) (). In addition, both V600E and ΔNVTAP 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 S3 B).

Biochemically, BRAF β3-αC 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 ΔNVTAP cell lines toward a panel of BRAF inhibitors representing three classes: GDC-0879 (DFG in, αC in), AZ-628 (DFG out, αC in), and vemurafenib (DFG in, αC out). While the BRAF V600E cell line (A-375) was sensitive to all three inhibitors, the ΔNVTAP cell lines showed high sensitivity toward GDC-0879 and AZ-628, but complete resistance to vemurafenib ( Figure 3 A ) 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 3 B), while the ΔNVTAP 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 3 A). Using the cellular thermal shift assay (CETSA) to evaluate cellular target engagement directly (), we observe that AZ-628 strongly stabilizes BRAF in both V600E and ΔNVTAP cell lines, while vemurafenib stabilizes BRAF V600E but not BRAF ΔNVTAP ( Figure S3 A). This suggests that vemurafenib has differential binding properties against BRAF V600E compared with ΔNVTAP.

(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.

(C) Western blot analysis of cell lysates from BRAF V600E (A-375) or ΔNVTAP (537 Mel) cells after 1 hr or 24 hr treatment with GDC-0879 or AZ-628 (both at 1 μM).

(B) Viability of BRAF V600E (501A, Colo 829, G-361, Colo 800, C32, A-375, 928 Mel, Malme-3M, or SK-Mel-28) or ΔNVTAP (537 Mel, OV-90, or NCI-H2405) cells grown in the presence of the indicated concentration of GDC-0879, AZ-628, or vemurafenib.

(A) Western blot of lysates from BRAF V600E (A-375) or ΔNVTAP (537 Mel, OV-90, or NCI-H2405) cells after treatment with the indicated concentration of GDC-0879, AZ-628, or vemurafenib for 1 hr.

Using siRNA-mediated knockdown, we tested the dependency of BRAF ΔNVTAP 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 2 C) and substantially reduced cell viability after 8 days (60%–80% loss of viability for the ΔNVTAP lines and 90% for the V600E line) ( Figure 2 D). We further evaluated CRAF dependence in a panel of cell lines with BRAF V600E, dimer-dependent, or ΔNVTAP 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 S2 C). 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 ΔNVTAP cell lines (537 Mel, NCI-H2405, and OV-90) showing low basal levels of BRAF:CRAF dimers ( Figure S2 D). Together these results indicate BRAF β3-αC deletions (similar to V600E) can function as autonomous kinases, independent of CRAF and dimerization.

The functional consequence of BRAF NVTAP deletion on MAPK signaling was explored by comparison with two other classes of BRAF mutations ( Figure S2 A): (1) the canonical high activity BRAF V600E, which confers high dimer-independent activity (); (2) BRAF mutations in the ATP binding glycine-rich loop or essential catalytic residues, which can activate through homodimerization or heterodimerization with CRAF (). Expression of BRAF WT, V600E, ΔNVTAP, or dimer-dependent mutants with varying pathway activation (K483M, G464V, or D594V) in the HEC1A BRAFcell line shows that ΔNVTAP has activity comparable with V600E or G464V and much higher pathway activation than WT, K483M, or D594V ( Figure 2 A ). To differentiate ΔNVTAP 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 (). As expected, BRAF WT and G464V showed substantial loss in activity upon R509H mutation ( Figure 2 B). In contrast, the activity of ΔNVTAP 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 2 B). Similar results were observed in 293T cells ( Figure S2 B).

(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.

(C) Western blot of lysates from BRAF V600E (A-375) or ΔNVTAP (OV-90, NCI-H2405, or 537 Mel) cell lines grown in the presence of siRNA reagents (siNTC, siBRAF, or siCRAF) for 72 hr.

(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.

(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, ΔNVTAP, or three dimer-dependent mutants (K483M, G464V, or D594V).

We next analyzed the kinase activity of BRAF WT, V600E, and three clinically observed deletions (ΔNVTAP, ΔTAPTP, and ΔPTPQQ) 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 1 F). We also observed elevated levels of pMEK for all three BRAF deletions ( Figure 1 F), demonstrating that these are also kinase-activating alterations in the BRAF kinase domain.

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 ΔLRENT (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 (). We similarly observed weak phosphorylation of full-length HER2 ΔLRENT relative to HER2 WT but observed elevated pERK levels, possibly due to heterodimerization with endogenous EGFR in 293T cells ( Figure S1 D). To test heterodimerization 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 (). As seen in Figure 1 E, the HER2 ΔLRENT 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 S1 E); however, the degree of activation was less dramatic compared with the analogous deletion in EGFR. Taken together, our data show that HER2 ΔLRENT is an activating deletion primarily through increased phosphorylation of its dimerization partner.

We hypothesized that BRAF and HER2 β3-αC deletions likely function mechanistically similar to the EGFR β3-αC deletions. To compare enzymatic properties of EGFR and HER2 directly, constructs containing just intracellular domains (ICDs) () were expressed in 293T cells. EGFR β3-αC deletion (ΔELREA or ΔLREAT) ICDs showed high activity, comparable with the canonical activating mutation L858R, and much higher than wild-type (WT) ( Figure S1 B). 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 S1 C). In contrast, EGFR L858R, ΔELREA, and ΔLREAT exhibited similarly high EGFR phosphorylation and pERK levels ( Figure S1 C). As a control, WT and mutants were similarly activated by an acute dose of EGF ( Figure S1 C), showing that under these starvation conditions these EGFR mutants enhance kinase activity independent of exogenous ligand.

Short in-frame deletions within the HER2 kinase domain (ΔLRENT) have been reported at low frequency in ERBB2 non-amplified breast cancers ( Figure 1 A and Table S2 ) (). The Foundation Medicine dataset also included ERBB2 deletions that accounted for ∼2% of all ERBB2 alterations ( Figure 1 C and Table S4 ). Similarly, BRAF deletions (ΔNVTAP, ΔTAPTP, or ΔPTPQQ) have been identified in TCGA datasets in both pancreatic and thyroid tumor samples () ( Figure 1 A 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 1 D and Table S4 ). These BRAF deletions were mutually exclusive with KRAS mutations and other BRAF alterations ( Figure 1 D), 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 1 A) and occur within the conserved β3-αC loop of the kinase domain ( Figure S1 A).

Short in-frame deletions mapping to the homologous loop (the β3-αC loop) (see Figure S1 A) within the kinase domains of BRAF, EGFR, and HER2 have been identified in patient samples of varying tumor types ( Figure 1 A ; Tables S1–S3 and S4 ). Large-scale sequencing of lung adenocarcinomas showed EGFR β3-αC deletions (also referred to as exon 19 deletions) in ∼20% of tumors, similar in frequency to the L858R mutation (). The most frequent β3-αC deletion is ΔELREA (also referred to as delE746-A750), followed by lower frequencies of ΔLREAT and ΔLRE ( Figure 1 A 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 β3-αC (exon 19) deletions ( Figure 1 B and Table S4 ).

(E) Western blot of FLAG-tagged HER2 ICD (676-1255) WT or ΔLRENT (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.

(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 β3-αC deletions (n = 13) and other BRAF alterations (n = 11; 6 of which are V600E) is specified.

(A) Alignment of BRAF, EGFR, and HER2 highlighting the sequence from β3 (gray) to αC (blue). The region of the most frequent deletions is indicated in red letters, with specific deletions underlined.

Discussion

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Haber D.A. Epidermal growth factor receptor mutations in lung cancer. The Cancer Genome Atlas Research Network, 2014a The Cancer Genome Atlas Research Network Comprehensive molecular profiling of lung adenocarcinoma. 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) (). While HER2 β3-αC 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 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.

Figure 8 Models for the Activation Mechanism of β3-αC Loop Deletions and Altered Efficacy of Different Classes of Kinase Inhibitors Show full caption (A) A model of a generic kinase domain highlighting how shortening of the β3-αC loop alters the equilibrium between inactive and active conformations. (B) A model of a generic kinase domain highlighting how β3-αC loop deletions cause a steric clash between αC and αC “out” inhibitors, reducing the efficacy of this class of inhibitor. 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 β3-αC loop constrain αC to an active position by reducing a source of conformational flexibility. In essence, the shorter β3-αC loop predisposes αC to an “in” conformation promoting kinase activity ( Figure 8 A ), concomitantly diminishing the efficacy of kinase inhibitors that bind an “out” αC conformation ( Figure 8 B). A surprising result was the degree to which the length of the β3-αC 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 β3-αC loop shifts the manifold of low energy conformations accessible to αC 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 αC for catalytic activity, while deletion of six or more amino acids leads to αC 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 ΔLRE or ΔLREA 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 ΔLREATS).

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Harrison S.C. Crystal structures of c-Src reveal features of its autoinhibitory mechanism. The importance of linker length in modulating the conformational dynamics of αC suggests that β3-αC 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 evolution. As an example, all eight members of the SrcA and B kinase families have β3-αC 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 αC to adopt the active conformation (). Several families also have conserved β3-αC 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 αC 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 TGFβ/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 αC. 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 β3-αC loop deletions in various cancers.

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Maly D.J. Biochemical mechanisms of resistance to small-molecule protein kinase inhibitors. Despite the commonality of the structural effects of β3-αC 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 β3-αC deletions appear to confer a preference as a receiver kinase, as also suggested for the EGFR L858R (and likely the EGFR β3-αC deletions) (). In the context of BRAF, similar to BRAF V600E, β3-αC deletions are likely to dimerize (), but kinase activity is dimerization independent (). Interestingly, dimerization plays a central role both in normal RAF biology and in clinical acquired resistance to the αC out inhibitor vemurafenib (). Based on our work, we rationalize that dimerization (which results in robust pathway activation) leads to the subsequent structural shift in αC, 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 ().

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 β3-αC 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.