Precision drugging of the MAPK pathway in head and neck cancer

The mitogen-activating protein kinase (MAPK) pathway is central for cell proliferation, differentiation, and senescence. In human, germline defects of the pathway contribute to developmental and congenital head and neck disorders. Nearly 1/5 of head and neck squamous cell carcinoma (HNSCC) harbors MAPK pathway mutations, which are largely activating mutations. Yet, previous approaches targeting the MAPK pathway in HNSCC were futile. Most recent clinical evidences reveal remarkable, or even exceptional pharmacologic vulnerabilities of MAPK1-mutated, HRAS-mutated, KRAS-germline altered, as well as BRAF-mutated HNSCC patients with various targeted therapies, uncovering diverse opportunities for precision drugging this pathway at multiple “genetically condemned” nodes. Further, recent patient tumor omics unveil novel effects of MAPK aberrations on direct induction of CD8+ T cell recruitment into the HNSCC microenvironment, providing evidences for future investigation of precision immunotherapy for this large subset of patients. MAPK pathway-mutated HNSCC should warrant precision therapy assessments in vigorous manners.

In addition to external growth stimuli, somatic mutations of this pathway can cause robust constitutive MAPK activation in various solid tumors. As of today, two MAPK-driven cancers are known to be predominantly affected by somatic BRAF p.V600E activating mutations (~50% cases mutated in melanoma and thyroid cancer) 8,9 . Additional 30 cancer types also harbor noticeable subsets of MAPK pathway-mutated patients (2.99-83.4% cases) (Fig. 1). Many of the MAPK pathway mutations, though not all, have been demonstrated to be oncogenic in nature, and potentially druggable as well. These include KRAS p.G12C, MAP2K1 p.Q56P, and MAPK1 p.D321N, etc [10][11][12] . Prior to the era of genomic medicine, non-precision drugging (i.e., non-mutation based) of the MAPK pathway with many MAPK pathway inhibitors had miserably failed in clinical trials for almost all cancers. Yet, advances in precision drugging of the BRAF p.V600E mutation alone with BRAF inhibitors have now extended the survival of numerous melanoma, thyroid, and non-small cell lung cancer (NSCLC) patients worldwide 13 . However, for most other cancers, MAPK pathway has remained clinically undruggable till now despite common occurrences of MAPK mutations across all cancers.
Genetic aberrations of MAPK pathway in head and neck syndromes, and cancer Head and neck squamous cell carcinoma (HNSCC) is a highly aggressive cancer arising from the epithelial lining of the head and neck region. It has a rising global incidence of >0.83 million new cases/year (2018, International Agency for Research on Cancer, IARC 14 ). Diverse etiologies contribute to HNSCC carcinogenesis, including exposures to carcinogens, including tobacco, alcohol, betelnuts, air pollutants, oncogenic viruses (the Human Papillomaviruses, the Epstein-Barr virus), poor oral hygiene, as well as inheritance (e.g., Fanconi anemia) 15 . By and large, these carcinogens damage DNA of the head and neck epithelium and cause accumulation of genetic aberrations leading to HNSCC.
As high as~18% of HNSCC patient tumors harbor MAPK pathway mutations 7 . Core pathway components (HRAS, BRAF, MAPK1, RPS6KA1) are mutated in~10.5% cases (54/512, TCGA-HNSCC cohort), while key scaffold proteins and negative regulators are mutated in~4% and~3% cases, respectively. Functional genomics and bioinformatics demonstrate that nearly half of the HNSCC-associated MAPK pathway mutations are activating or oncogenic in nature ( Supplementary Fig. 1 16 (Fig. 2). Interestingly, some of these defects, but in a somatic manner, have been identified in HNSCC as well. Shared MAPK pathway aberrations in HNSCC and RASopathies are shown (Fig. 2). For instance, HRAS mutations, mutated in 6.6% HNSCC (but uncommon in other solid tumors), are also germline altered in Costello syndrome. The shared MAPK-mutated pathway components found in HNSCC and RASopathies raise attention not only for head and neck pathobiology understanding but also for therapeutic development against these mutations for HNSCC and likely RASopathies.    [17][18][19] . It has been documented that HNSCC patients with high intratumor expressions of p-MAPK1/3 (p-ERK1/2) have poor survival 20 , and inhibition of p-ERK1/2 by MAPK pathway inhibitors often inhibited HNSCC cell growth in vitro [21][22][23] . However, when it came to clinical testing of MEK/MAPK inhibitors, all HNSCC clinical trials have failed with a general lack of efficacies in unstratified patients. These failed attempts with MEK/MAPK inhibitors are summarized in Table 1, which include MEK1/2 inhibitors AZD8330, selumetinib, cobimetinib, TAK-733, (or combinations) and the ERK1/2 inhibitor MK-8353. Specifically, no objective response was observed in HNSCC patients treated with AZD8330 (NCT00454090), selumetinib (NCT00085787), cobimetinib (NCT00467779), and TAK-733 (NCT00948467) [24][25][26][27] . Several combination treatments, including selumetinib with tremelimumab and/ or MEDI4736 (NCT02586987), and cobimetinib with Atezolizumab (NCT03264066), have also been tested in HNSCC patients under pan-cancer clinical trial settings without promising outcomes. Similarly, HNSCC patients did not show any objective responses towards the ERK1/2 inhibitor, MK-8353 in a Phase I trial (NCT0135833) 28 . Basically, all trials ended at Phase I settings with no further studies thereafter. In fact, other than clinical failures in HNSCC, MEK1/2 inhibitors, such as CI-1040, was also demonstrated to be ineffective against solid tumors with trials halted at Phase II settings 29,30 .
Drugging HNSCC with MAPK pathway inhibitors: potency and precision As in other cancers, the reasons behind the general lack of clinical efficacies of early MEK/MAPK inhibitor trials in HNSCC remains unclear, but likely complex. These may include drug resistance mechanisms, drug potency issues, and most importantly, potential wrong ways of drugging this pathway in HNSCC 37 .
First, various MAPK pathway inhibitors were known to cause feedback, feed-forward, cross-talk signaling that help cancer cells re-gaining MAPK activation despite initial perturbation by the inhibitors, resulting in acquired resistance to these inhibitors in cancer cells. Studies have identified NRAS or MEK activating mutations, RAF amplification, RAF heterodimerization, BRAF alternative splicing, loss of NF1, etc., as causes for acquired resistance to MAPK inhibitors 37 . Recent strategies, aiming at double-striking 2 nodes of the MAPK pathway have gained much attention as a likely and feasible way to interrupt multiple feedbacks in many cancers, though the efficacies of these strategies have not been clinically evaluated in HNSCC yet. Interestingly, a preclinical study revealed that acquired resistance against selumetinib (MEK inhibitor) was attributed by FGFR3mediated MAPK reactivation in HNSCC, suggesting combined FGFR3/MAPK to resist the development of selumetinib resistance in HNSCC 38 . Second, insufficient potencies of older generations of MAPK inhibitors may underlie their clinical failures in HNSCC in the past 39 . Indeed, in recent years, new classes of MEK inhibitors have been developed with improved potencies, and have shown some clinical promises in HNSCC, and other cancers. Of all, trametinib is one promising MEK1/2 inhibitor for HNSCC. In 2013, a Phase II window-of-opportunity trial with neoadjuvant trametinib demonstrated marked median tumor size reduction (46%) in all patients with reduction and 11 patients with partial responses among 17 evaluable patients (Stage II-IV oral cancer). Of note, 5 patients showing reduced intratumoral p-ERK1/2 levels post-treatment were all found to be clinical responders by tumor size or tumor metabolic criteria 40 . The promising trial results implicate that potent MEK/MAPK inhibitors may be required for effective treatment of HNSCC.
Third, which is the most important question to ask is: whether we have been drugging this pathway wrongly in the past among HNSCC patients? Would past failures be contributed by the unstratified patient pools (one-for-all), as we did not have ways to identify which patients are genetically-condemned with MAPK pathway dependencies? Now, with the understanding of the genomic features of HNSCC patients, shall we drug the MAPKcondemned patients differently and be able to see good clinical outcomes in those patients?
In fact, promising results from a recent RAS inhibitor trial with tipifarnib sheds light for effective precision drugging of HRASmutated HNSCC patients (NCT03719690) and have resulted in fasttrack drug review by the FDA (detailed discussion below). Contrasting with previous failures of MEK/MAPK inhibitors for HNSCC, this FTI is paving its way as a likely first precision medicine for a noticeable subset of HNSCC patients (~6% cases). Details on the clinical successes of tipifarnib and additional Ras inhibitors for HNSCC are discussed below. Furthermore, new clinical findings including many exceptional responder studies, and recent -omics studies do suggest new avenues for precision drugging of HNSCC patients bearing either germline or somatic MAPK aberrations, highlighting the need for future investigations in the clinic.
Tipifarnib Reveals the Need for Precision Drugging in HNSCC. RAS proteins are small GTPases activated upon GTP binding to stimulate downstream RAF/MEK/MAPK signaling for cell growth. As membrane localization of RAS proteins is required for their activation, pharmacologic inhibition of RAS post-translational modifications, such as C-terminal farnesylation (by inhibiting the enzyme farnesyl transferase that transfers farnesyl group to the C-terminal of RAS), could potentially inactivate RAS. Till now, four farnesyl transferase inhibitors (FTIs), namely L-778,123, BMS-214662, tipifarnib, and lonafarnib have been clinically evaluated in various cancers, including HNSCC (Table 2). Their effects in HNSCC patients are discussed below.
L-778,123: a Phase I trial aimed to determine L-778,123's maximal tolerated dose (MTD) in combination with radiotherapy in 9 advanced cancer patients (3 with stage IV HNSCC, 6 with NSCLC) 41 . Among the 3 HNSCC patients, despite one patient falling short of complete treatment due to toxicity, the remaining 2 patients have demonstrated complete responses with no evidence-of-disease (NOD), at follow-up examinations. Ras mutational statuses were not clearly reported. Despite complete responses observed, the MTD of the L-778,123/radiation combination could not be defined 41 . It was also noted that another Phase I clinical trial for L-778,123 was conducted by the Memorial Sloan Kettering Cancer Center and National Cancer Institute (NCT00003430) for patients with recurrent or refractory solid tumors including HNSCC, but no trial result was published.
BMS-214662: a potent FTI of the 1,4-benzodiazepine class with IC 50 value of low nM range in vitro. Two HNSCC patients involved in a mixed solid tumor Phase I trial, were treated with BMS-214662 and did not show objective responses, while another Phase I BMS-214662 trial with HNSCC patients did not report patient outcomes in detail 42,43 .
Lonafarnib (SCH66336): a tricyclic peptidomimetic inhibitor being one of the first FTIs underwent human clinical trials for cancer, including both Phase I and II trials involving HNSCC patients. An early Phase Ib trial result released in a conference proceeding reported clinical responses at all lonafarnib doses tested in patients prior surgery (100 to 300 mg twice daily), with marked tumor size reduction in 4/22 evaluable patients 44 . A later Phase II lonafarnib trial in chemo-refractory advanced HNSCC showed that among 15 patients who previously failed platinumbased therapies, 7 had stable diseases in a minimum of 3 cycles of treatment, while 1 patient was stable for 8 cycles of treatment for 220 days total. Further, lonafarnib was well-tolerated with no grade 3 or 4 hematologic toxicities 45 . Among these advanced HNSCC patients, these stable disease cases with lonafarnib suggested potential therapeutic efficacy of FTIs for HNSCC, which was later shown by tipifarnib. Interestingly, lonafarnib was recently approved by the FDA (Nov, 2020) for the treatment of a form of premature aging disease called Hutchinson-Gilford progeria syndrome, and progeroid laminopathies to reduce their risk of death by preventing the build-up of "defective progerin or progerin-like protein" that causes accelerated heart failure.
Tipifarnib: a potent FTI ( Supplementary Fig. 2), with IC 50 values of 0.86 nM and 7.9 nM against lamin B and K-RasB peptide substrates in vitro 46 . With the realization that HRAS mutations can cause oncogene addiction and activated MAPK signaling in cancer, tipifarnib trials have sought to investigate its effectiveness in pan-cancer patients with and without HRAS mutations. In a recent phase II trial (NCT02383927), tipifarnib demonstrated a 53% overall response rate (13/23 patients) among HNSCC patients 47 . Furthermore, significant clinical activity was noted in patients with recurrent and metastatic HNSCC harboring HRAS mutation with high variant allele frequency (VAF) of >20%. Eight out of 15 HNSCC patients meeting such a criterion had partial responses, while additional 5 demonstrated stable diseases (53% response rate) with tipifarnib 47 . Tipifarnib also demonstrated modest clinical activity in patients with recurrent, metastatic HRAS-mutant salivary gland cancer, and urothelial carcinoma. Among 12 evaluable HRAS-mutant recurrent metastatic salivary gland cancer (SGC) patients, one demonstrated partial response and 7 had stable disease with median duration of response of 9 months 48 . In this small SGC cohort, the kind of HRAS variants and allele frequency did not correspond to clinical outcome with tipifarnib. Nevertheless, these promising clinical findings provide strong evidences for the use of HRAS mutations in HNSCC as predictive biomarkers for tipifarnib sensitivity. In general, tipifarnib was well-tolerated, with fatigue, myelosuppression, nausea, and vomiting being the most common adverse events (all grades) observed 47 . Currently, the FDA has granted fast-track designation to tipifarnib for HRASmutant HNSCC whose disease progressed on platinum therapy 49 . An ongoing international Phase II trial is specifically evaluating tipifarnib efficacy in HNSCC patients with high HRAS-mutant VAF (NCT03719690). It is very likely that HRAS-mutant HNSCC may represent the first precision medicine specific for HNSCC.
Alongside, studies are ongoing to start tackling potential tipifarnib-resistance in HNSCC. Using HRAS-mutant HNSCC patient-derived xenografts (PDXs), tipifarnib-resistance was identified to involve aberrant apoptosis and angiogenesis 50 . Nevertheless, their involvement and clinical relevance in HNSCC remains unknown. A recent CRISPR-screen has already identified potential combination strategy of tipifarnib with autophagy inhibitors to prepare for even more effective tipifarnib treatment for HRASmutant HNSCC 51 .
Drugging HNSCC with KRAS Germline Variants by Cetuximab Addition. As tipifarnib demonstrates good clinical efficacies in HRAS-mutated HNSCC, salivary gland carcinoma, and urothelial   54 . In fact, AMG-510 has recently been FDA-approved for the treatment of KRAS p.G12C-mutated locally advanced or metastatic NSCLC (May, 2021). Unlike HRAS, the mutation rate of KRAS is relatively low in HNSCC (1/512; 0.2%). Yet, we recently reported that KRAS mutation rate can found iñ 3.5% of advanced HNSCC 55 . In metastatic larynx cancer, KRAS mutation may account for~6% cases 56 . Furthermore, KRAS mutations appear to be a poor prognostic biomarker for advanced HNSCC, with shorter disease-free survival in KRAS-mutant vs. WT patients 55 .
Among all new KRAS inhibitors under clinical evaluation, only AMG-510, MRTX849, and LY3499446 have entered phase II settings 57 . Yet, the low rate of KRAS somatic mutations in HNSCC presents a challenge for clinical assessment of KRAS inhibitors with KRAS-status stratification.
Interestingly, Weidhaas et al. recently performed a secondary analysis of a randomized phase III HNSCC trial and found that patients with an oncogenic germline KRAS variant (a let-7 microRNA-binding site polymorphism in the 3' untranslated region of KRAS) have significantly better clinical outcomes with cetuximab (EGFR monoclonal antibody) addition to radiotherapy plus cisplatin regimen 58 . Furthermore, these germline KRASvariant HNSCC patients were found to have increased plasma TGF-β1, potentially contributing to immunosuppression in these patients. Thus, targeting with cetuximab may help these patients overcome TGF-β1-induced immunosuppression. In fact, this very same KRAS germline variant also predicts good clinical response to cetuximab monotherapy in otherwise somatic KRAS-WT metastatic colon cancer patients 59 . Thus, KRAS germline variant guiding combination therapy with cetuximab, and potentially radiotherapy/cisplatin regimen should be further evaluated in larger prospective trial in HNSCC. The main side effects reported in the trial were skin reaction, some with grade 3 to 4 mucositis 58 .
Exceptional Erlotinib responses for MAPK1-mutant HNSCC. About 5% of HNSCC patients harbor somatic mutations of MAPK1 (ERK2) gene 12 . In 2015, Van Allen and Lui et al. reported the first HNSCC exceptional responder (with Stage III advanced oral cancer) who exhibited a complete response (>30 months) to a 13-day erlotinib treatment 60 . This clinical finding is highly unexpected given another EGFR targeting agent, cetuximab often demonstrates moderate activities in HNSCC patients in general. Whole-exome characterization of pre-treatment biopsy showed no EGFR aberrations, but the presence of a somatic MAPK1 p.E322K mutation using an EGFR-network bioinformatics approach. This mutation was then functionally characterized to be a potent driver for constitutive ERK activation and HNSCC cell growth 12 . Subsequent investigation revealed its ability to drive robust EGFR hyperactivation by enhancing autocrine amphiregulin release from HNSCC cells, thus hyperactivating EGFR signaling 61 , and rendering hypersensitivity to an EGFR kinase inhibitor, erlotinib. The study was later extended to additional MAPK1 mutations in HNSCC, and led to the finding that MAPK1 p.D321N (also hyperactivates MAPK and p-EGFR) could also confer heightened sensitivity to erlotinib in HNSCC in vivo, while MAPK1 p.R135K mutation (moderately activating p-EGFR) conferred moderate level of erlotinib sensitivity 12 . Importantly, both MAPK1 p.D321N and p.R135K mutations exist in recurrent HNSCC in Asia 12 . These results suggested that MAPK (or ERK) activities could be associated with erlotinib responses in HNSCC. In fact, results from the randomized clinical trial (NCT00779389) involving the first HNSCC exceptional responder eventually concluded that baseline tumoral p-MAPK (p-ERK) levels were inversely correlated with tumor size posterlotinib treatment, showing that MAPK activation status is likely an indicator of EGFR-addiction in HNSCC, and thus predictive of clinical responses to erlotinib in HNSCC patients 62 . Overall, the clinical trial reported brief exposure to erlotinib was well-tolerated in HNSCC, with acneiform rash and diarrhea being major side effects that are commonly observed with EGFR inhibitors 61 .
Interestingly, based on current genomic findings, US TCGA-HNSCC patients with primary tumors universally harbor MAPK1 p. E322K mutations, while an Asian primary/recurrent HNSCC cohort showed a wide spectrum of p.E322K/D321N/R135 mutations 12 . As EGFR kinase inhibitors (e.g., erlotinib, gefitinib) are known to be clinically safe for treatment of lung cancer, these clinical and findings provide important scientific evidences supportive of precision trials for MAPK1-mutated, yet EGFR-addicted HNSCC.
Five exceptional responder reports have been documented with dabrafenib or vemurafenib monotherapies, and dabrafenib/ trametinib combination (Table 3). Kaye et al. first reported an African American ameloblastoma patient with recurrent metastases bearing somatic BRAF p.V600E mutation (detected by mutant-specific antibody with immunohistochemistry), treated with compassionate dabrafenib (150 mg twice daily) plus trametinib (2 mg once daily) and exhibited immediate marked tumor reduction at day 4, followed by disappearance of lung metastases and shrinkage of head and neck lesions at week 8, and persistent antitumor responses even at 20 weeks 69 . This combination was well-tolerated clinically. Another remarkable clinical responses to this combination was also observed in a 26-year old recurrent metastatic ameloblastoma patient showing complete dissolution of lung metastasis and primary tumor at 30 weeks after treatment (NCT02534649) 70 . The NCT02367859 trial also reported a stable disease for a BRAF p.V600E-mutated ameloblastoma patient. These cumulative responder cases strongly suggest the potential therapeutic benefits of BRAF/MEK inhibition in BRAF p.V600E-mutated ameloblastoma (note: such combination is FDA-approved for BRAF p. V600E-mutated thyroid cancer and melanoma). Besides, two other recurrent BRAF p.V600E-mutated ameloblastoma patients have also responded dramatically to dabrafenib monotherapy. These include an 83-year-old patient treated with low dose dabrafenib (75 mg twice daily) showing 75% tumor size reduction at 12 months 71 , and another recurrent patient responding to dabrafenib with >90% tumor shrinkage 72 . Lastly, besides dabrafenib, the use of vemurafenib has also caused persistent and marked tumor shrinkage (>60% for 11 months) in a recurrent BRAF p.V600E-mutated ameloblastoma patient 73 . Common reported side effects for these BRAFi, MEKi and their combinations include asthenia, rash, arthralgia, nausea, pyrexia, fatigue and headache, etc., and they, in general, showed good toxicity profiles in patients 70 . Thus, BRAF p.V600E-mutated ameloblastoma could be pharmacologically vulnerable for precision BRAFtargeting.
Remarkable clinical responses to MAPK inhibitors in BRAF-mutated HNSCC. Unlike ameloblastoma, BRAF mutations only occur iñ 1.8% of HNSCC, and the clinical responsiveness of BRAF-mutated HNSCC patients to BRAF inhibition remains under-explored. Yet, a recent Phase I trial (NCT01781429) did report a BRAF p.G469Amutated head and neck cancer patient with partial response to ulixertinib (an ERK1/2 inhibitor) per RECIST criteria (> 30% tumor size reduction) 74 . Though such a dramatic ulixertinib response only lasted for 4.9 months in this patient, a little fall short of the NCI's criteria for exceptional responders (6-months), this report does provide clinical evidences supportive of future ulixertinib trials in relation to BRAF-mutational status for this relatively safe drug. In the trial, the most common treatment-related adverse effects were rash, diarrhea, fatigue, and nausea, with no grade 4 or 5 treatment-related adverse effects reported.
MAPK mutations modulate ErbB3 activation in HNSCC: potential therapeutic consideration. ERBB3 is a new target for HNSCC. Its activation level (p-ErbB3) is significantly associated with poorer HNSCC patient survival 7 . Various ErbB3 inhibitors are under development. Among which, CDX-3379, an anti-ErbB3 monoclonal antibody, has demonstrated antitumor activity resulting in tumor shrinkage in 42% of HNSCC patients, with grade 1 to 2 diarrhea, fatigue, and acneiform dermatitis being the most often treatmentrelated toxicities 75  MAPK-mutant HNSCC are CD8 + T cell-inflamed: implications for T cellbased immunotherapies. HPV(+)HNSCC patients are known to respond well to T cell-based immunotherapies, including PD1 inhibitors pembrolizumab and nivolumab [76][77][78][79] . This is at least partially contributed by elevated immune infiltrates in HPV(+)HNSCC tumors [76][77][78][79][80][81][82] . Numerous PD1 inhibitor trials confirmed that HPV(+) HNSCC patients have reduced hazard ratios for death (HR = 0.82, p = 0.0316) when compared to standard treatment 83 . For recurrent and metastatic HNSCC, HPV(+)HNSCC patients demonstrated a better objective response rate to durvalumab than HPV(-)HNSCC patients (29.4% vs 10.8%, with median OS = 10.2 months 5.0 months) 84 . However, HPV status was not associated with clinical responses to nivolumab in the CheckMate 141 study 85 . Recent bioinformatic analysis of HNSCC patient tumors showed that MAPK-mutant HNSCC tumors, irrespective of HPV status, also have elevated CD8 + T cell and dendritic cell infiltrations, together with immune-active cytolytic and interferon-gamma signatures in situ, suggestive of an active cytolytic CD8 + T-inflamed status in these tumors. Interestingly, this CD8 + T-inflamed and cytolytic feature was not shared by HNSCC tumors bearing PI3K, NOTCH, JAK/STAT, WNT, NF-κB, or TGFβ/Smad pathway mutations 7 . The study further demonstrated that ectopic expression of MAPK pathway mutations (mouse HRAS p.G12V, mouse MAPK1 p.D319N and p.E320K), but not respective WT-counterparts, could directly shape the HNSCC immune tumor microenvironment by attracting CD8 + T cell infiltration in immunocompetent mouse allografts 7 . Whether such MAPK mutation-driven CD8 + T cell infiltration was caused by activated MAPK signaling or the associated neoantigens warrants further investigations. It also remains to be determined if these CD8 + T cells are clonal or not. Using TCGA-HNSCC dataset, Lyn et al. also showed immune-enhancing signatures associated with HRAS mutation, including elevated CD8 + markers and HLA gene expressions, high cytolytic activity, and immune scores 86 .
Since CD8 + T cells are effective antitumor immune cells, its markedly increased level in MAPK-mutant HNSCC tumors may tie Resistance of MAPK-mutated HNSCC?. Activation of the RAS/RAF/ MEK/ERK signaling is known to contribute to, at least in part, resistance to several therapies in HNSCC. These include resistance to cisplatin 87 , EGFR-monotherapy (cetuximab) likely through crosstalk signaling with the PI3K/AKT pathway 5,88 , EGFR/HER-3 combination therapy 89 , PI3K inhibitor (BYL-719) via mTOR activation 90 , as well as new experimental agents such as CX-4945 (a Protein Kinase CK2 inhibitor 91 ). As for resistance to radiotherapy, a German oropharyngeal cancer study (N = 124) showed that elevated p-Erk1/2 expression levels was associated with poor clinical outcomes, suggesting the potential involvement of ERK activation in HNSCC radioresistance 92 . Future clinical trials are much anticipated for MAPK pathwaymutated HNSCC as discussed above due to the reported exceptional responders and good responders, and it is likely that resistance may also arise as in other precision medicines for many other cancers. Though resistance to "specific precision medicine treatments" of MAPK-mutated HNSCC is yet-to-be unfolded, researchers just begin to report general resistance to Cetuximab, an FDA-approved EGFR targeting antibody for HNSCC, in relation to HRAS mutations. In 2014, Rampias et al. reported an association of HRAS mutational status with de novo resistance to cetuximabbased chemotherapy (P = 0.046) in a small HNSCC cohort (7 HRASmutated vs 48 wildtype patients), suggestive of HRAS-mutantrelated RAS/Erk activation for cetuximab resistance in HNSCC 5 . Though we cannot predict how such a wide array of HNSSCassociated MAPK pathway mutations may alter sensitivity and resistance to various agents, lessons from BRAF p.V600E-mutated melanomas and thyroid cancers do alert us of such possibility of resistance to long-term monotherapy treatment through reactivation of MEK/ERK signaling (and some other mechanisms including constitutive activation of EGFR and PI3K, etc.) 93 , which can be cotargeted by BRAF-MEK inhibitor combinations (e.g., encoragenibbinimetinib; dabrafenib-trametinib, etc) as approved recently by the FDA for other cancers.

CONCLUSIONS
In conclusion, based on recent clinical and omics findings, MAPK pathway mutations can be drug-sensitizing in HNSCC. Although the evolutionary or clonal nature of MAPK pathway mutated HNSCC is still unclear but definitely of interest for further investigations, this HNSCC subset should be given more attention for therapy development. Potential precision strategies may include EGFR kinase inhibitors, monoclonal EGFR antibody cetuximab, BRAF inhibitors such as vemurafenib or darafenib, newer generations of MAPK/MEK inhibitors, and CD8 + T cell-based immunotherapies (Fig. 3). Somatic and germline mutation-based prospective trial design may help identifying new effective precision therapies for this previously undruggable MAPK pathway in HNSCC. For practical reasons due to the genetic heterogeneity of HNSCC tumors, if future MAPK precision clinical trial results are promising, it may be useful to perform a panel of MAPK pathway genes to facilitate future precision medicine implementations in the clinic.

Reporting Summary
Further information on research design is available in the Nature Research Reporting Summary linked to this article.

DATA AVAILABILITY
No datasets were generated or analyzed during the current study.