2.2. Mutations in the Tyrosine Kinase Domain

Most mutations in the HER2 gene have been detected in exons 19 and 20 of the tyrosine kinase (TK) domain, at the C-α helix position of the protein [34] (Table 2). Several authors propose that mutations in this domain could be an alternative mechanism to HER2 activation and affect sensitivity to anti-HER2 therapy, as an acquired resistance mechanism to this form of therapy. The TKD mutations described to date in HER2+ breast cancer promote the activation of the functionality of the protein and increase the oncogenicity of HER2, besides inducing the phosphorylation of other cell signaling proteins [28, 34] (Table 2). This is because this domain contains the ATP binding site and its mutations are related to the enhanced phosphorylation of receptors HER2, HER3, and HER1, which causes receptor HER2 dimerization along with protein ERK (extracellular signal-regulated kinase) and AKT phosphorylation, with consequent activation of the PI3K/Akt and MAPK pathways, finally enhancing cell proliferation and angiogenesis (Figure 2). The binding site of ATP with the receptor protein forms a conformational structure with other important structures such as phosphate activation and binding loops, which could be affected by such modifications. Missense substitutions usually occur at the C-α helix, which is essential for HER2 protein activation. These alterations can promote tumorigenesis and phosphorylation of signaling proteins such as phospholipases γC1 and Cγ (PLCγ) MAPK. Many of these activating mutations have proved resistant to anti-HER2, such as those found at codons 755 or 798 [34].

Most authors have described the appearance of both intrinsic and acquired resistance to trastuzumab therapy in mutations L755S, V777L, D769Y, and K753E [32, 40, 42, 44, 45]. As these mutations are not located close to the drug's binding target, it seems that rather than blocking receptor binding of the drug, they affect resistance to its effects by increasing kinase activity and activation of the protein's oncogenic signaling pathways, independently of drug binding. All these mutations as well as D769H share the feature of sensitivity to the actions of the irreversible TK inhibitor, neratinib [28, 30, 35, 41, 42, 45]. This could be explained by the greater strength of interactions produced between this drug and the ATP-binding site. This response offers a good treatment option for patients who may have developed resistance to first-line treatments for HER2+ breast cancer. Most authors agree that resistance to lapatinib, both intrinsic and acquired, appears in L755S, D769Y, V842I, and K753E [41, 42, 44]. This indicates the importance of the electrostatic interactions that occur at the ATP binding site close to these residues. Moreover, depending on the changes produced by the amino acid substitutions, a protein conformation may arise that promotes either the active state of HER2's kinase domain impairing proper drug binding or this binding increases sensitivity toward the drug.

The L755S mutation is the most common in HER2 gene [41] and is considered a hotspot mutation [58]. The protein's codon 755 seems to be strongly involved in activating HER2 receptor kinase, which leads to the potentiated activity of the PI3K/Akt and MAPK signaling pathways, giving rise to enhanced cell proliferation and angiogenesis. In preclinical trials, this mutation has been associated with resistance to lapatinib treatment through reactivation of HER2 signaling in HER2+ breast cancer models in which the gene is overexpressed [43, 44]. In in vitro models, it was observed that cells with this mutation were resistant to treatment with lapatinib + trastuzumab, but also to trastuzumab + pertuzumab treatment [32, 40, 41]. As this mutation induces resistance to trastuzumab alone or in combination with pertuzumab, despite its location far from the drugs' binding sites on the receptor, it could be that kinase activity is so enhanced that it is able to continue signaling despite the nondimerization of the receptor after the binding of these drugs [40, 41, 44, 59]. Resistance to lapatinib can be explained by the fact that Leu 755 participates in hydrophobic interactions with the C-α helix of the TKD in the active state of HER2, while in the inactive form, L755 is found far from this helix [43]. The L755S polymorphism induces the appearance of polar interactions that stabilize the active form; this would help explain resistance to lapatinib, which only binds to the inactive conformation of HER2 [43]. This resistance could be addressed with irreversible HER1 and HER2 inhibitors such as neratinib, which has proven effective in patients with this mutation [41]. In effect, in vitro studies have shown the sensitivity of cells with the L755S mutation to afatinib plus neratinib [41, 44]. Besides intrinsic resistance, mutation L755S has been associated with resistance acquired to trastuzumab therapy in breast cancer. It appears in 7.59% of patients receiving prior trastuzumab treatment. Further, it has been reported to occur in 3 out of every 18 patients with metastasis but not those with primary tumors [41].

Mutation V777L is also considered hotspot [58]. Residue V777L, located in exon 20 (at the C-terminal tail of the C-α helix), is involved in TK activity. This activating mutation promotes the TK activity of HER2, increasing the phosphorylation of signaling proteins such as HER2, HER3, EGFR, and ERK, and the transformation of breast epithelial cells [29, 33, 40, 45, 60]. This mutation causes transcriptional activation in most tumors affected by this mutation, which usually occurs independently of HER2 gene activation [60]. In effect, cases have been described in breast cancer cell lines in which increased endogenous expression levels of HER2 V777L activated signal transduction pathways, but this did not significantly increase tumor growth [61]. The effects of V777L seem enhanced by mutations in the PIK3CA gene given that, in the presence of mutation PIK3CA E545K, V777L gives rise to enhanced interaction between p58 and HER3. This suggests that reverse mutations of the HER2 gene could require other genetic alterations to promote cellular transformation and enhance interactions between signaling partners [31]. This mutation has been associated with the intrinsic development of trastuzumab resistance [45]. Although the mutation has been associated in some preclinical studies with a diminished response to lapatinib, afatinib, and neratinib, several studies have shown reduced tumor growth and signaling activity in tumors with the V777L mutation treated with lapatinib [44, 45]. A response has been observed to combined treatment with neratinib and other drugs in patients with ER + V777L breast carcinoma [35]. No cases relating this mutation to the response to pertuzumab have been described. Considering that this last drug, as does trastuzumab, binds to the extracellular domain of the protein and that resistance to trastuzumab has been described, we would expect pertuzumab to neither elicit a good response in patients with this mutation. As occurs with the L755S mutation, HER2 V777L shows strong activation of the receptor's kinase that could preserve its signaling activity even with trastuzumab and pertuzumab bound to the extracellular domain of the protein.

Interestingly, V777L and L755S mutants have been characterized using molecular dynamics simulations and in vitro studies in Ba/F3 cells expressing these mutants, showing that these mutants have a larger binding pocket volumes and therefore are more sensitive to tyrosine kinase inhibitors (TKIs) of quinazoline (afatinib and poziotinib) and indole (osimertinib and nazartinib) groups. Furthermore, in preclinical models, poziotinib upregulates HER2 cell surface expression and potentiates the activity of T-DM1, inducing a complete tumor regression with combination treatment [62]. The authors of this study suggest that poziotinib in combination with T-DMI could be a good candidate treatment for not only non-small cell lung cancer; in fact, one ongoing trial in phase II is studying the efficacy of poziotinib in metastatic breast cancer harboring HER2 mutations [21, 62]. Overall, more clinical studies are needed to test the efficacy of poziotinib in combination with T-DMI in breast cancer to rule out differences in tumor type-specific sensitivities to the same pharmacological product. In SUMMIT trial, neratinib was most effective in breast cancer patients, with patients containing L755S and V777L [33], but the same mutations were associated with resistance in other cancer types, suggesting that more research is needed to identify the mechanism involved in tumor-type-specific sensitivities.

Recently, using isogenic knock-in HER2 mutations in ER + MCF7 cells and xenografts, two activating HER2 mutations located in the kinase domain (L755S and V777L) emerged as resistance to anti-ER therapy progression [35]. These findings are corroborated by other authors, and the same mutations have been identified in metastatic biopsies of eight patients with ER + metastatic breast cancer (MBC), as mutations that were acquired under the selective pressure of ER-directed therapy such as aromatase inhibitors [36]. The same authors demonstrated that the resistance to ER-directed therapy was overcome by combining fulvestrant with the irreversible HER2 kinase inhibitor neratinib. These data suggest that the prevalence of HER2 mutations might increase in metastatic ER+ breast cancer treated with anti-ER therapy, and these mutations are a distinct mechanism of acquired resistance to ER-directed therapy in metastatic breast cancer that could be solved by the treatment with an irreversible HER2 inhibitor. Overall, these data suggest that patients with ER+/HER2 mutations would benefit from HER2-targeted therapies in combination with hormonal therapy. If ongoing clinical trials confirm these results, new approaches could be adopted in order to promote a better response in patients with ER + MBC, and one of these strategies could be to identify HER2-mutant-resistant clones to ER-directed therapy [36].

Mutation V842I has been detected in various types of tumor tissue. This is also an activating mutation associated with HER2 gene amplification and increased phosphorylation of different signaling proteins [28] and also represents a hotspot in HER2 [58].

The effects of V842I on the response to anti-HER2 therapy in patients with HER2+ breast cancer have not been yet explored. Some in vitro studies indicate the resistance to trastuzumab and lapatinib of cell lines with this mutation [44]. This mutation is the most common mutation in colorectal cancers, and in vitro studies have shown that this mutant was not sensitive to neratinib [62]. However, given its recurrent expression in different tumor tissues and its association with amplification of the gene, studies are warranted to clarify its impact on the receptor's kinase activity.

The nonsynonymous mutations D769Y and D769H are among the most frequent somatic mutations of the HER2 gene. They are located in exon 19, at position 769 of the TK domain, which is important for ATP-HER2 binding [29]. Both mutations have been characterized as activators in mammary epithelium cell lines, and in vivo studies have revealed neratinib as effective at blocking tumor growth in HER2+ breast carcinomas with these mutations [28, 42].

Cases have been described of xenografts acquiring the D769Y mutation following treatment with trastuzumab, along with their subsequent resistance to trastuzumab and lapatinib, suggesting its possible role in acquired resistance to anti-HER2 therapy [42]. In mutation D769Y, the change from aspartic acid to tyrosine could lead to changes in electrostatic interactions, due to the substitution of a negatively charged acid side chain at physiological pH with the capacity to form hydrogen bridges and bind phosphate groups. As this mutation occurs at an important position for ATP binding to the receptor, this change could benefit this binding and thus diminish the impacts of lapatinib and neratinib therapy, whose mechanism of action is to impair this binding of ATP to HER2 [42]. The D769Y mutation promotes the phosphorylation of HER2, EGFR, HER3, and ERK and transformation of mammary epithelial cells. Cell lines with this mutation display sensitivity to neratinib, in smaller measure to lapatinib and resistance to trastuzumab [42], although Nagano et al. recently described sensitivity to lapatinib and afatinib in in vitro studies [44]. Some authors report that loss of the acid side chain or addition of an aromatic ring to amino acid 769 could increase HER2's TK activity due to dimeric interactions between the kinase domains of HER2 and HER3. Mutations D769H/Y may enhance hydrophobic contacts and heterodimerization of HER2. Besides, the D769H alteration could lead to activation within the HER2 monomer, adding hydrogen bonds to its own activation A-loop [46, 54].

Mutation K753E leads to a shift in charge of the amino acid's side chain, which goes from being basic to acidic, thus possibly affecting the electrostatic interactions of the protein. Several authors have related this mutation with lapatinib resistance, and this could be attributed to its close proximity with the L755S mutation which confers resistance to this drug [32, 41]. Recently, the effect of this mutation has been observed in cell lines overexpressing HER2 K753E. In HER2 K753E mutant cells resistant to lapatinib, a greater affinity of the drug for the HER2 protein was observed compared to wild-type cells and other variants. This reveals that resistance to this drug is unrelated to a lack of binding to its target [63]. It has also been related to resistance to trastuzumab and appears in 2 out of every 18 patients with metastasis [32]. While cell lines that show this mutation are resistant to lapatinib, they are sensitive to neratinib, which could benefit patients developing resistance to trastuzumab therapy [41].

Following trastuzumab therapy, the appearance of K753E and L755S mutants could suggest their potential role as drivers of developing trastuzumab resistance during HER2+ tumor progression [32].

Mutation I767M is a hotspot in gene HER2 [58] identified in patients with HER2+ breast cancer [54]. Its expression has been examined in vitro in HER2-overexpressing mammary cell lines and in HER2-negative cultures. In the former cells, the presence of this mutation along with mutations in the genes PIK3CA and TP53 conferred a significant growth benefit over cells with the wild-type HER2 gene. Further, both the mutant and wild-type protein featured similar AKT and MAPK signaling levels, although the AKT pathway remained active over time for longer in the cells expressing HER2 I767M [47]. In vitro studies conducted by Nagano et al. [44] indicate the sensitivity of I767M to therapy with both TK inhibitors (lapatinib, neratinib, and afatinib) and the monoclonal antibody trastuzumab.

According to the data from COSMIC and cBioPortal, while other mutations in this kinase domain have been described (i.e., V797A, D808E, D873G, and M889I), there are still no data regarding their role in HER2+ breast cancer.