Current Advances and Trends in KRAS Targeted Therapies for Colorectal Cancer

Colorectal cancer continues to be one of the most frequent causes of cancer death in developed countries, and is on the rise in developing countries especially among Asian nations. Ranked third worldwide behind lung and stomach cancers in terms of mortality rate, colorectal cancer's overall curative rate has also not improved over the last decade with a constant 5-year survival rate of 64% (1). To date, somatic point mutations of the Kirsten Rat Sarcoma (KRAS) gene account for approximately 30% to 50% of all colorectal cancer cases (2, 3), making it the most prominently mutated proto-oncogene known to date.

Ras was the first discovered superfamily of small G proteins comprising of three proto-oncogenes (KRAS, HRAS, NRAS) and four 21 kDa subfamily proteins (KRAS4A, KRAS4B, NRAS, HRAS; refs. 4, 5). The Ras gene superfamily is mainly involved upstream of the RAF–MEK–ERK MAPK cascade and PI3K-AKT cell signaling pathway, which have an important role in cell proliferation, differentiation, and survival (6). KRAS exists as either a guanosine triphosphate (GTP)-bound active state or guanosine diphosphate (GDP)-inactive state in the GTP hydrolysis cycle. This is regulated by both switch I and switch II domains, which undergo complex conformational changes during nucleotide exchange along the inner plasma membrane. The wild-type (wt) KRAS protein is temporarily activated following RTK signal transduction, but a mutated KRAS protein will forces downstream signaling pathways to be activated all of the time, thereby promoting a tumorigenic cellular circuit that is often associated with targeted therapy resistance (7, 8). Most of these findings support the tumor suppressor function of wt KRAS (9–11), and its dysregulation can be further enhanced by gain of guanine nucleotide exchange factors (GEF) function or loss of downstream GTPase activating proteins (GAP) negative regulators (12).

The activating point mutation of the KRAS oncogene on codon 12 (exon 2) is the initiating event in the majority of colorectal cancer cases (83%; ref. 13). Point mutations can also occur at codon 13 (exon 2; 14%) and occasionally at codon 61 (exon 3; 2%). These allelic mutations are located in close proximity of the GTP binding site, thus interfering with GTPase activity. The most clinically frequent substitutions are aspartate (G12D, 36%) followed by valine (G12V, 23%), both of which are found to occur on codon 12 (14). These positions are crucial for KRAS to function as a GTPase and RAF kinase activator. Even though RAS genes share up to 90% amino acid sequence identity with near-identical structural and biochemical properties, they are nevertheless differentially expressed and mutated with different frequencies in different cancers (15, 16). This has been seen in the case of NRAS mutations, which are found mostly in cutaneous malignant melanoma on the codon 61, whereas KRAS mutations are found mostly in pancreatic ductal and colorectal adenocarcinomas on codons 12 and 13 (17, 18). These suggest that different RAS mutations exert distinct oncogenic properties in a tissue-specific context due to yet unknown reasons.

Current approaches involving anti-EGFR therapies as well as nonspecific cytotoxic chemotherapeutic regimens such as FOLFOX [folinic acid (FA), fluorouracil (5-FU), and oxaliplatin] and FOLFIRI [FA, 5-FU, and irinotecan hydrochloride] in treating patients with colorectal cancer harboring mutated (mt) KRAS genes have apparently shown little benefit. Indirect alternative approaches such as the disruption of KRAS posttranslational modification using farnesyltransferase inhibitors have also failed to demonstrate efficacy in KRAS-driven colorectal cancer trials (19, 20). To date, most of these clinically approved conventional regimens are often accompanied by dose-related toxicities, drug resistance, and undesirable physiological side effects, thus creating the need for other target-specific therapeutic alternatives. In this scoping review, the most recent targeted approaches and clinical trials tackling KRAS mutations either directly or indirectly in the clinical management of colorectal cancers are discussed and are summarised in Fig. 1.

KRAS has been long considered to be undruggable, in part because of lacking picomolar affinity targetable binding pockets for drugs on their smooth surfaces. However, recent laboratory and clinical advancements in drug discovery reveal opportunities to bring an end to KRAS-driven cancers. In 1997, Taveras and colleagues (21) developed the first ras nucleotide exchange inhibitor SCH-53239, which binds to ras-GDP protein to prevent Ras activation. However, the low toxicity and poor metabolic stability of the inorganic functional group of hydroxylamine may not be an ideal drug compound. Since then, more compounds were developed to target the RAS protein towards guanosine substrates by inhibiting the RAS–RAF interaction (22–24), Ras–SOS interaction (25, 26), or Ras–effector interaction (27). Again, the development of these compounds has been disappointing due to low potency. Perhaps the full-length Ras's hidden structure is the key to combat the untargetable KRAS.

New insights into the structure and function of KRAS proteins reveal opportunities to target the activating substitution of oncogenic Ras, and are provided in Table 1. In 2013, Ostrem and colleagues (28) were the first to identify compounds that selectively target cysteine-12 in mt KRAS G12C without inhibiting wt KRAS. These compounds were used to lock the mt KRAS G12C in its inactive GDP-bound state by binding to a pocket near the nucleotide-binding site and preventing GTP recharging. A similar direct targeted approach led to the development of adagrasib, ARS-1620, ARS-853, SML-8–73–1, and sotorasib that acts as KRAS G12C allele-specific inhibitors with promising therapeutic potential (29–33).

Moving forward, Amgen and Mirati Therapeutics reported promising early clinical data evaluating two novel covalent KRAS G12C inhibitors, sotorasib (NCT03600883) and adagrasib (NCT03785249) in patients with KRAS G12C mutation solid tumors (34–36). Sotorasib appears to be safe and well-tolerated at the doses and time courses reported to date (33). An improved version of ARS-1620, JNJ-74699157 was developed by Janssen Research and Development and Wellspring Biosciences under clinical evaluation (NCT04006301). Novartis and Mirati Therapeutic had recently paired up to recruit KRAS G12C mutation advanced solid tumor patients to evaluate the combination efficacy of MRTX849 and Src homology region 2-containing protein tyrosine phosphatase 2 (SHP2) inhibitor TNO155 in a phase I/II trial (NCT04330664). Until recently, a pan-KRAS inhibitor, BI 1701963, that targets both KRAS G12C and son of sevenless 1 (SOS1), has entered clinical trials for patients with KRAS mutated solid tumors (NCT04111458; ref. 37) and colorectal cancer (NCT04627142; ref. 38). Another phase I trial with anti-PD-L1 antibody Avelumab (NCT04449874) was also recently approved for KRAS G12C mutated metastasis colorectal cancer (mCRC).

A direct targeted approach for another type of mt KRAS protein was carried out with a few promising KRAS-G12D selective targeting compounds. Welsch and colleagues (39) have developed a pan-RAS inhibitor, compound 3144 to target multiple adjacent sites on KRAS G12D protein near D38, A59, and Y32 sites. This compound was found to inhibit tumor growth in xenograft mouse cancer models. Another group identified KRpep-2d peptide that selectively inhibits protein–protein interactions (PPI) between KRAS G12D and SOS1 protein after screening a random T7 phage display peptide library (40). Computational analyses of the mt KRAS G12D protein structure make it feasible to develop KRAS allosteric ligand KAL-214040358 that selectively binds to the P110 site KRAS G12D (41). These findings suggest a new potential site for binding small molecule inhibitors of mt KRAS G12D.

Early approaches to disrupt Ras protein localization to the plasma membrane once seemed to be a promising prospects for pharmacologic interventions using farnesyl transferase inhibitors (FTI). Preclinical mouse studies using FTIs showed significant antitumor activity with no toxicity stimulated the start of clinical trials (42, 43). However, colorectal cancer clinical outcomes from phase II and III trials testing FTIs were disappointing (19, 20). This failure was predicted years earlier when Whyte and colleagues (44) found that KRAS and NRAS can alternatively be geranylgeranylated without farnesylation, a process known as alternative prenylation. Since then, more studies are focused on using combination therapy of FTIs and geranylgeranyl transferase inhibitors (GGTI) to suppress KRAS activity. In vivo study of the L-778, 123, a dual FTI and GGTI demonstrated dose-limiting toxicity; however, this compound's clinical pharmacodynamic assessment did not inhibit KRAS prenylation in human patient samples (45). Until recently, a novel FGTI-2734, dual FT, and geranylgeranyl transferase-1 (GGT-1) inhibitor was developed by Kazi and colleagues (46). This compound was found to inhibit tumor growth of mt KRAS in xenograft animal models and showed promising drug potential for KRAS cancers. Since most discoveries suggest that FTIs are not effective in managing KRAS-mutated cancers, researchers have subsequently shifted their attention towards using FTIs for HRAS-mutated cancers.

In 2013, the discovery of the farnesyl-binding pocket of phosphodiesterase 6 delta (PDEδ) provided an alternative therapeutic window against mt KRAS cancers. PDEδ inhibitors have been developed to disrupt KRAS localization of the plasma membrane by altering KRAS4B membrane trafficking and cellular activity, thereby impairs KRAS/PDEδ interaction. The first reported PDEδ inhibitor, deltarasin was identified to inhibit the prenyl-binding pocket of PDEδ with nanomolar affinity selectively and reduce tumor growth in vitro and in vivo (47). An improved PDEδ inhibitor, deltazinone 1 was subsequently reported with improved selectively and lesser cytotoxicity (48). Klein and colleagues (49) recently reported that PDEδ inhibitors specifically inhibit proliferation and survival of KRAS colorectal cancer cell lines and can be exploited for therapeutic intervention for mt KRAS cancers.

Several PI3K inhibitors have been developed and tested in preclinical studies and early clinical trials. Three main categories of PI3K inhibitors have been developed, including isoform-selective, pan-PI3K, and dual pan-PI3K inhibitors. Several clinical trials are still ongoing with isoform-selective PI3K inhibitors (p110α, β, δ, or γ) in patients with advanced solid tumors, hematologic malignancies, and lymphomas (50–53). Because of hematopoietic cell-restricted expression of p110δ and p110γ, the PI3Kδ and PI3Kγ isoform-selective inhibitors were found to achieve greater therapeutic efficacy in such hematologic malignancies rather than solid tumors (54, 55). More recent work has shown that the inhibition of either p110α or p110β leads to PI3K signaling's reactivation via p110β or p110α, respectively, limiting its efficacy in the clinic (56, 57). These data suggest that pan-PI3K inhibitors or combination therapy of PI3Kα and PI3Kβ inhibitors may require maximal PI3K pathway inhibition.

Notably, a PI3Kα inhibitor, alpelisib has entered phase Ib/II trial (NCT01719380) combined with a BRAFi encorafenib, and anti-EGFR mAb cetuximab in patients with mCRC has demonstrated promising results (58). Petra pharma has initiated a phase Ib/II trial (NCT04073680) to evaluate the efficacy of PI3K-α inhibitor serabelisib and sodium/glucose cotransporter 2 (SGLT2) inhibitor canagliflozin, which used to treat type 2 diabetes, for advanced solid tumors patients. Another type of PI3K-δ inhibitor parsaclisib was tested in combination with a programmed cell death protein 1 (PD-1) inhibitor pembrolizumab and compared with pembrolizumab and a JAK1 inhibitor itacitinib in patients with advanced solid tumors, which currently ongoing in a phase I trial (NCT02646748). A dual inhibitor of PI3K-δ and casein kinase (CK) 1-epsilon umbralisib was tested alone and in combination therapy with either nanoparticle albumin-bound tubulin inhibitor Nab-paclitaxel, chemotherapy drug gemcitabine, regimen FOLFOX or VEGF-A inhibitor bevacizumab for relapsed solid tumors patients. This phase I trial (NCT02574663) is completed and the results are pending.

Pan-PI3K inhibitors (Table 2) targets all the four PI3K kinase isoforms of class I PI3K (α, β, δ, and γ) in cancer. On the basis of clinical trial data, it is unlikely that pan-PI3K inhibitors have significant activity as single agents in patients with solid tumors (59–61). The key drawbacks of pan-PI3K inhibitors are the large spectrum of drug-related side effects, including anorexia, fatigue, nausea, and hyperglycemia, limiting the use of such agents at therapeutic doses. Hence, combination trials of pan-PI3K inhibitors (e.g., buparlisib, sonolisib, pictilisib) with other agents in solid tumors are required to improve the efficacy of the agents (62–66). However, a pan-PI3K inhibitor, sonolisib combined with anti-EGFR antibody cetuximab demonstrated disappointing results in mCRC patients in a phase II trial (NCT01252628; ref. 62). Ongoing phase I/II trials (NCT03735628 and NCT03711058) are currently recruiting patients with advanced colorectal cancer to explore the safety and efficacy of combination therapy of copanlisib with anti-PD-1 antibody nivolumab. To date, only one pan-PI3K inhibitor, buparlisib has progressed into phase III trial (NCT01633060) for metastatic breast cancer.

To further enhance inhibition of the PI3K–AKT–mTOR pathway, dual PI3K–mTOR inhibitors (Table 2) were developed to target all PI3K catalytic isoforms and mTORC1/2 by binding to ATP binding cleft. Thus, these drugs should effectively turn off this pathway and result in tumor regression. A preclinical study demonstrated growth-inhibitory activity in the colorectal cancer cell xenograft tumor model when treated with the dual PI3K–mTOR inhibitor dactolisib (67). Besides, dactolisib and LY3023414 were found significant treatment responses in mutated APC and PIK3CA mice with 53% and 24% reduction in the median lumen occlusion, respectively (68). The dual PI3K–mTOR inhibitor dactolisib was shown to inhibit tumor growth in the genetically engineered sporadic colorectal cancer mouse model (69). Although dactolisib demonstrated significant tumor regression in the preclinical model, the clinical efficacy in advanced solid malignancies patients has been unsatisfactory (70–73). Another two dual PI3K–mTOR inhibitors DS-7423 and voxtalisib showed disappointing results in the phase I trials (NCT01364844 and NCT01390818) for patients with advanced solid tumor (74, 75). However, it remains unclear whether dual PI3K–mTOR inhibitors are tolerable at effective doses that inhibit either PI3K and mTOR isoforms or completely inhibit one or more of the possible targets.

Most AKT inhibitors (Table 2) block AKT 1, 2, and 3, either interfering with ATP or binding partially to the ATP-binding site. Although targeting selectivity of AKT was predicted higher than PI3K inhibition, the evidence of clinical results is unsatisfactory. Adverse side effects of AKT inhibitors were included severe rash and hyperglycemia (76–78). A promising oral AKT/PI3K inhibitor, perifosine combined with a chemotherapy drug capecitabine demonstrated clinical benefits for mCRC in the phase I and II trial (NCT01048580 and NCT00398879; ref. 79). However, the phase III trial (NCT01097018) did not demonstrate better overall survival in patients with refractory colorectal cancer (80). A dual potent AKT/PKCβ inhibitor, Enzastaurin, was again shown no clinical benefits on mCRC in a phase II trial (NCT00612586; ref. 81). Two phases II clinical trials (NCT00192114 and NCT00437268) of enzastaurin for patients with colorectal cancer were completed while the results are pending.

Another attempt to inhibit AKT was an oral allosteric AKT1/2 inhibitor, MK-2206. A phase II monotherapy trial (NCT01802320) for patients with recurrent mCRC was shown no significant therapeutic benefits (82). In 18 patients, 9 patients in cohort A had progressive disease whereas two cohort B patients had stable disease for 1.8 months. When combined with a MEK1/2 inhibitor selumetinib in a phase II trial (NCT01333475), patients with advanced colorectal cancer showed no objective responses (83). Two clinical trials (NCT01186705 and NCT01243762) with MK-2206 were terminated early due to insufficient patients are recruited to reach the preliminary conclusions (77). Capivasertib, developed by AstraZeneca, was reported to inhibit all AKT isoforms and AGC family kinase in vivo assays (84). Davies and colleagues (85) reported capivasertib was susceptible to wt RAS, PI3KCA, or PTEN mutated cell lines. A phase I trial of capivasertib (NCT01353781) in solid tumor patients demonstrated antitumor activity with intermittent dosing (86) and now undergoing phase II trial as combinatorial therapy (NCT02576444).

Most mTOR inhibitor (Table 2) monotherapy in clinical trials for patients with KRAS mCRC has not demonstrated therapeutic benefits (87–89). Ineffective mTOR inhibitors were found contributed to drug resistance to colorectal cancer, which correlated to the elF4E-binding protein 1 (4E-BP1) kinase (90). Because of the minimal effectiveness of a single agent, more studies are focused on the combination of mTOR inhibitor with upstream receptor inhibitors. Recent findings on combination therapy of mTOR inhibitors and other drugs have demonstrated promise in patients with colorectal cancer. For example, the oral derivative of rapamycin, everolimus that specifically inhibits mTORC1 is the most advanced in the clinic. In a phase I/II study, the combined therapy of everolimus with modified FOLFOX-6 chemotherapy regimen and VEGF-A inhibitor bevacizumab reported prolonged progression-free survival up to 6 months in 96% of patients with mCRC as first-line treatment (NCT01047293; ref. 91). This result was showed a statistically significant progression-free survival improvement than the previous study conducted by Saltz and colleagues (92). An expanded cohort phase II trial (NCT00597506) for refractory mCRC using everolimus and combination with the VEGF-A inhibitor bevacizumab demonstrated tolerable toxicity (93). In addition, a multicenter phase II trial (NCT01058655) of everolimus with another type of VEGF inhibitor tivozanib was reported well-tolerated in treating 50% of the patients with refractory mCRC (94).

When testing everolimus with the chemotherapy drug irinotecan and anti-EGFR antibody panitumumab as a second-line treatment (NCT01139138), this combination treatment fails to translate clinical benefits for patients with wt KRAS mCRC (95). However, the replacement of another EGFR inhibitor cetuximab in the same combination treatment demonstrated clinical benefits in refractory mCRC patients (NCT00478634 and NCT00522665; refs. 96, 97). In a phase I trial (NCT01154335), the maximum tolerated dose of everolimus and an insulin-like growth factor 1 receptor (IGF-1R inhibitor) linsitinib combination treatment was determined, and no clinical activity was observed in patients with refractory mCRC (98). Novartis is currently recruiting patients with CRC across seven countries for a phase I trial (NCT02890069) using everolimus in combination with a PCR001 checkpoint inhibitor, whereas Aadi is recruiting advanced or mCRC patients for phase I/II trial (NCT03439462) as first-line therapy to test the nanoparticle albumin-bound sirolimus, Nab-rapamycin in combination with chemotherapy regimen modified FOLFOX-6 and VEGF-A inhibitor bevacizumab. Besides, Aadi collaborated with Sarcoma Oncology Research Centre recruiting advanced sarcoma and solid tumors patients to evaluate the combination efficacy of Nab-rapamycin and a PD-1 inhibitor nivolumab (NCT03190714). Combinatorial therapeutic strategies using downstream PI3K pathway inhibitors effectively treat primary and mCRCs, rendering the pathway a promising therapeutic target for KRAS colorectal cancer.

Even though direct RAS inhibitors are still the best option for KRAS targeted therapies, inhibitors of the RAS/RAF/MEK/ERK (MAPK) signaling pathway may provide an alternative solution for future colorectal cancer treatments to overcome upstream drug inhibitors resistance. In this section, the latest findings and advancements related to MAPK pathway inhibitors are reviewed.

The first-generation BRAFi that target BRAF kinases have been extensively clinically investigated in patients with BRAF^V600E mCRC, such as vemurafenib, dabrafenib, encorafenib, and sorafenib (Table 2). In 2015, Kopetz and his research team (99) reported patients with BRAF^V600E colorectal cancer treated with vemurafenib in a phase I study (NCT00405587) did not benefit from monotherapy. However, when vemurafenib was tested in combination therapy, it improved the outcome of patients with colorectal cancer with promising efficacy (100–102). Shanghai Changzheng Hospital is currently recruiting patients with BRAF^V600E advanced colorectal cancer for phase II trial (NCT03727763) to investigate the effects of vemurafenib, cetuximab, and FOLFIRI combinatorial therapy. Another type of BRAFi dabrafenib demonstrated promising activity in patients with BRAF^V600E colorectal cancer. For example, an EGFRi panitumumab was added in the combinatorial therapy with dabrafenib and MEK1/2 inhibitor trametinib in a phase I/II clinical trial (NCT01750918; ref. 103). Notably, the addition of EGFRi was shown to overcome the resistance to MAPK pathway inhibition in patients with colorectal cancer. Thus, van Brummelen and colleagues (104) suggested BRAF status as a predictive biomarker for response to anti-EGFR treatment. A phase II study (NCT03668431) of dabrafenib and MEK1/2 inhibitor trametinib combined with an anti-PD-1 mAb spartalizumab is currently undergoing for patients with BRAF^V600E mCRC. Among the BRAFi, the most promising drug is encorafenib, which is currently undergoing phase III trials. The encorafenib treatment for patients with mt BRAF^V600E mCRC has been tested in combination with anti-PD-1 (nivolumab), EGFRi (cetuximab), MEK inhibitor (binimetinib), PI3Kα inhibitor (alpelisib), and other cytotoxic agents (5-FU, bevacizumab, capecitabine, FA, irinotecan, leucovorin, or oxaliplatin), which demonstrated promising clinical benefits (58, 105–107).

A promising oral dual Raf/platelet-derived growth factor (PDGF)/VEGF inhibitor, sorafenib demonstrated clinical benefits in combination trials (NCT00989469, NCT01471353, NCT01715441, and NCT00826540) with either irinotecan, capecitabine, or bevacizumab in pretreated patients with KRAS mCRC (89, 108, 109). Besides, von Moos and colleagues (110) reported that patients with advanced KRAS-mutated colorectal cancer benefited from the combination of sorafenib and neoadjuvant radiotherapy (NCT00869570). The maximum tolerated dose of the combination between sorafenib and chemotherapy drugs pemetrexed and cisplatin was determined in a phase I trial (NCT00703638) for advanced solid tumors (111), which recommended further studies. Sorafenib combined with another EGFRi cetuximab in a phase II trial (NCT00326495) also showed no clinical benefit in 30 patients with pretreated KRAS mutated mCRC (112). Similar clinical outcome was observed for patients with mCRC treated with a combination of sorafenib and chemotherapy regimens (NCT00865709 and NCT00779311; ref. 113). To date, a total of forty clinical trials are currently in the recruiting phase for patients with leukemia and hepatocellular cancer to test the combination of sorafenib with various chemotherapy regimens or immune checkpoint inhibitors (ICI). These efforts are progressively pointing towards underexplored pathways and targets for potential encorafenib combination designs.

Despite first-generation BRAFi providing improved clinical benefit in some patients with BRAF^V600E mCRC, drug resistance to BRAF inhibition persists. This effect can be explained as BRAFi drives the binding of BRAF-CRAF heterodimers, resulting in the recovery of MEK-ERK signaling which drives tumor progression (114). To overcome the BRAF-selective inhibition, second-generation pan-RAF inhibitors were developed to inhibit both BRAF and CRAF isoforms, preventing paradoxical MAPK pathway activation when a RAS mutation is present. The pan-RAF inhibitors, LY3009120 and PLX8394 showed significant activity in BRAF, NRAS, or KRAS colorectal cancer xenografts models (115, 116). Recently, PLX8394 demonstrated promising results in a phase I/II trial (NCT02428712) with a cytochrome P450 3A4 (CYP3A4) inhibitor cobicistat and is currently recruiting patients for the phase II trial (117). However, a phase I study (NCT02014116) of the LY3009210 capsule was terminated early due to no clinical efficacy being observed in patients with advanced colorectal cancer (118).

In comparison to RAF inhibitors targeted strategy, MEK inhibitors were developed to target MEK-dependent cell signaling pathways because MEK kinase harbors fewer mutations. The most promising MEK inhibitors are binimetinib and trametinib, which are now ongoing in several phase II/III trials, as shown in Table 2.

Binimetinib is an oral inhibitor of MEK1/2 developed by Array Biopharma. The binimetinib monotherapy study (NCT00959127) reported no clinical benefits for patients with KRAS or BRAF mutated mCRC (119). When combining binimetinib with chemotherapy regimen FOLFOX in phase I trial (NCT02041481), Cho and colleagues (120) reported manageable toxicity profiles and promising antitumor activity in patients with advanced mCRC. Hence, multiple clinical trials are currently recruiting patients with mCRC to evaluate the combination efficacy of binimetinib with BRAF, EGFR, or other chemotherapy regimens (NCT03693170, NCT02928224, and NCT02613650). The results for a nonrandomized phase Ib/II study (NCT01927341) of binimetinib and anti-EGFR antibody panitumumab in patients with mCRC are still pending. Because of the clinical benefits of immunotherapy, more researchers are currently focusing on combining binimetinib with anti-PD-1 antibody nivolumab or anti-cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4) antibody ipilimumab (NCT03271047, NCT04044430).

Another type of PD-1 inhibitor, pembrolizumab was reported safe and tolerable in mCRC when treated together with mFOLFOX7 or FOLFIRI (121). Merck and Array Biopharma are currently recruiting mCRC patients for phase I trial (NCT03374254) of binimetinib and PD-1 inhibitor pembrolizumab or chemotherapy. VEGF-A inhibitor bevacizumab to binimetinib and pembrolizumab combination treatment demonstrated clinical benefits in patients with refractory colorectal cancer (122). This phase II trial (NCT03475004) is currently recruiting patients for the next stage of the study. Pfizer initiated a phase I trial (NCT01337765) to evaluate the combination efficacy of binimetinib and a dual PI3K/mTOR inhibitor dactolisib, but no results have been reported. Another MET combination treatment (MErCuRIC1) is under investigation using a dual tyrosine-protein kinase Met (c-MET)/anaplastic lymphoma kinase (ALK) inhibitor crizotinib and another MEK inhibitor mirdametinib in a phase I trial (NCT02510001) for patients with colorectal cancer with wt and mt RAS harboring aberrant c-MET expression. A phase II trial (NCT03981614) employing a combination of binimetinib and cyclin-dependent kinases CDK4/6 inhibitor palbocicib or chemotherapy trifluridine and tipiracil hydrochloride (TAS-102) is also recruiting KRAS and NRAS mt mCRC.

Trametinib is the first FDA-approved MEK inhibitor for BRAF^V600E metastatic melanoma with the trade name Mekinist. In a phase II trial (NCT01750918), Trametinib was shown to overcome the MAPK resistance in BRAF^V600E mutated metastatic or patients with refractory colorectal cancer in combination with BRAFi dabrafenib and EGFRi panitumumab (103). Without adding the RAF inhibitor in the combination treatment of trametinib and panitumumab, Alshammari and colleagues (123) reported no clinical benefits were observed in wt advanced colorectal cancer in an ongoing phase II trial (NCT02399943). NCI is currently recruiting patients with stage IV colorectal cancer for a phase II trial (NCT03087071) to evaluate the efficacy of trametinib and EGFRi panitumumab compared with panitumumab monotherapy. Notably, a promising FDA-approved trifluorothymidine/thymidine phosphorylase inhibitor, TAS-102 was added into the combination treatments with trametinib for patients with chemotherapy-resistant RAS-mutated (PI3KCA/PTEN-wt)-specific mCRC (NCT03317119).

Novartis is focused on developing trametinib immunotherapy combinations for patients with mCRC. A group of advanced CRC patients was recruited from nine different countries to test trametinib's efficacy combined with PD-1 inhibitor spartalizumab in a phase I trial (NCT02900664). With the addition of BRAFi dabrafenib into immunotherapy combinations with the PD-1 inhibitor spartalizumab, Novartis is currently recruiting BRAF^V600E mCRC patients for a phase II trial (NCT03668431). A collaborative study between Novartis and Bristol-Myers Squibb have entered into phase I/II clinical trials (NCT03377361) to evaluate the safety, tolerability, and efficacy of trametinib in combination with PD-1 ICI nivolumab and CTLA-4 inhibitor Ipilimumab as a potential treatment option for patients with advanced mCRC. Another type of PD-L1 inhibitor durvalumab was found to be ineffective in mCRC patients with MSS (124). Unfortunately, this phase II trial (NCT03428126) was discontinued after completing the first stage of the study. Furthermore, other phase I trials exploring combinations of trametinib plus JAK1/2 inhibitor ruxolitinib (NCT04303403), p53-MDM2 inhibitor siremadlin (NCT03714958), or dual EGFR/HER2 inhibitor lapatinib (NCT02230553) are currently recruiting patients with CRC.

In 2020, the oral MEK1/2 inhibitor selumetinib was approved by FDA for pediatric patients and currently under phase I/II study for patients with colorectal cancer with different drug combinations, such as AKT inhibitor, EGFRi, immunotherapies, and chemotherapies. The first selumetinib clinical trial (NCT01160926) was terminated early because the combination with chemotherapy drug capecitabine does not respond well in patients with locally advanced rectal cancer, even on the lowest possible dose cohort. Another early termination of a clinical trial (NCT01116271) combined with a different chemotherapy drug irinotecan was reported (125). Hence, AstraZeneca evaluated another selumetinib chemotherapy combination with docetaxel, dacarbazine, or EGFRi erlotinib or mTOR inhibitor temsirolimus in patients with advanced solid tumor. This clinical trial (NCT00600496) is currently ongoing and clinical benefits were reported in each targeted drug (126, 127). A phase II pilot study (NCT01333475) combining an AKT inhibitor MK-2206 showed no objective responses in advanced colorectal cancer (83).

Conversely, combination therapy with an anti-EGFR cetuximab antibody reported manageable safety profile and are well-tolerated in patients with KRAS mutated refractory mCRC (NCT01287130; ref. 128). Patients with colorectal cancer were included as one of the advanced refractory solid tumors patients groups to test the efficacy of the combination of selumetinib and FDA-approved immunotherapy PD-L1 checkpoint inhibitor durvalumab or CTLA-4 inhibitor tremelimumab. This multicenter trial (NCT02586987) was completed by AstraZeneca and the results are pending. Another first-in-human clinical trial (NCT02188264) is currently ongoing to evaluate the efficacy of both selumetinib and an immunosuppressant drug cyclosporin A in patients with advanced mCRC. Recently, Krishnamurthy and colleagues (129) reported that these combination treatments were well tolerated and suggested for patients with advanced solid tumor.

Unfortunately, intolerable toxicity remains an issue for the three MEK1/2 inhibitors (cobimetinib, mirdametinib, and pimasertib), when administered as monotherapy (NCT00147550) or combination therapy (NCT03340558, NCT02788279, NCT02876224, NCT02457793, NCT02039336, NCT02510001, NCT01390818; refs. 75, 130–133). However, one phase Ib trial (NCT02876224) reported an acceptable safety profile and manageable adverse effects with the combination treatment of cobimetinib, PD-L1 inhibitor atezolizumab and VEGF-A inhibitor bevacizumab in patients with mCRC, indicating that mutated RAS responded better than wt RAS colorectal cancer patients (134).

Targeting the third tier of MAPK cascade, ERK was first assume have no additional clinical benefits. However, the resistance mechanism of RAF and MEK drugs was reported due to ERK1/2 reactivation (135), blocking ERK1/2 directly may overcome the current limitations of upstream RAF or MEK inhibitors. Thus, researchers are gaining renewed interest in developing ERK inhibitors, which can be combined with upstream BRAF and MEK inhibitors to multi-target in the MAPK cascade. Several ERK inhibitors (Table 2) are in the trial phase for RAF/RAS-mutated or BRAF/MEK inhibitors-resistant tumors, but it remains unclear if these agents exhibit more clinical benefits compared with MEK inhibitors.

One of the inhibitors is ravoxertinib, a potent dual inhibitor of ERK1/2. Ravoxertinib has shown promising efficacy in the phase I trial (NCT01875705) for patients with advance pancreatic adenocarcinoma and mt BRAF colorectal cancer (136). Unfortunately, the combination of ravoxertinib and MEK inhibitor cobimetinib shows adverse drug reactions for patients with mCRC in a phase Ib dose-escalation study (NCT02457793; ref. 131). LY3214996 is another potent ERK1/2 inhibitor that has demonstrated antitumor activity in a preclinical study (137). The first-in-human trial in patients with advanced cancer demonstrated an acceptable safety profile and further support LY3214996 as monotherapy or in combination treatment (138). Two clinical trials (NCT02857270 and NCT04616183) are currently recruiting patients with metastatic cancer to evaluate the combinatorial treatment, including combination with gamma-aminobuturic acid (GABA) agonist midazolam, CDK4/6 inhibitor abemaciclib, nanoparticle albumin-bound tubulin inhibitor Nab-paclitaxel, chemotherapy drug gemcitabine, BRAFi encorafenib, or EGFRi cetuximab.

Several other ERK inhibitors under development are ASN007, CC-90003, and MK-8353. ASN007 is an oral ERK1/2 inhibitor that has a long target residence time. In a phase I trial (NCT03415126), the MTD level and recommended phase II dose has been determined for patients with solid tumors harboring BRAF, MEK1, and RAS mutations (139). The irreversible ERK1/2 inhibitor CC-90003 was the first inhibitor that was terminated in a clinical trial (NCT02313012). Mita and colleagues (140) reported that the CC-90003 did not show a favorable PK profile and displayed unanticipated neurotoxicity in mt BRAF or RAS refractory metastatic patients. Monotherapy of MK-8353 phase I trial (NCT01358331) showed no clinical activity in patients with colorectal cancer with RAS and/or BRAF mutations (141). Hence, a phase I trial is ongoing to explore the combination efficacy of MK-8353 and PD-1 inhibitor pembrolizumab in patients with advanced malignancy, including colorectal cancer (NCT02972034). However, none of these compounds have entered phase III clinical trials and approved by FDA to date.

The clinical benefits of ICIs in colorectal cancer have primarily been limited to a subgroup of microsatellite instable (MSI/dMMR)-high patients. This subgroup of patients represents around 15% of all colorectal cancers (142). Such MSI-H colorectal cancer tumors are characterized by high numbers of tumor-infiltrating lymphocytes (TIL) and high tumor immunogenicity within the tumor microenvironment. Notably, high levels of PD-1, PD-L1, and CTLA-4 expressions were found in MSI-H group (143–145). Thus, the blocking of PD-1/PD-L1 and CD80/CTLA-4 interactions by ICIs could enhance T-cell activation and promote tumor cell killing. This therapeutic synergistic effect was reported in preclinical testing of either PD-1 or PD-L1 antibody combined with MEK inhibitor (146, 147). This tested combination treatment significantly suppresses tumor growth and increases intratumoral CD4⁺ and CD8⁺ T cells in vivo.

In 2018, the combination of low dose anti-CTLA-4 ipilimumab and anti-PD-1 antibody nivolumab or nivolumab as a single agent was approved to treat patients with refractory MSI-high mCRC (148). NICHE and CheckMate 142 phase II clinical trials are currently ongoing to investigate the combination of ipilimumab, nivolumab with COX-2 inhibitor celecoxib, MEK inhibitor cobimetinib, anti-CD38 antibody daratumumab, or anti-LAG-3 antibody BMS-986016 in patients with colorectal cancer (NCT03026140, NCT02060188). Concurrent studies have also reported that single dose of ipilimumab and two doses of nivolumab was very effective in treating dMMR tumor (149, 150). Recently, another immunotherapy, anti-PD-1 pembrolizumab was approved for MSI mCRC, with several trials ongoing (NCT03785249, NCT03475004, NCT03374254, NCT02563002). Le and colleagues (151) reported that MMR status was found to predict clinical benefit of ICI with pembrolizumab (NCT01876511), with progression free survival rates of 78%. Andre and colleagues (152) and Le and colleagues (153) further supported the use of pembrolizumab monotherapy for patients with MSI-high colorectal cancer. Conversely, the combinational treatment of pembrolizumab reported no clinical benefits in another subgroup of proficient MMR patients with colorectal cancer (NCT02981524; ref. 154). An ongoing phase II trial with anti-PD-L1 antibody Avelumab (NCT03150706) was approved for MSI-H or DNA polymerase epsilon (POLE) mutated mCRC. A few clinical trials are also ongoing for the anti-PD-L1 antibody Atezolizumab in treating patients with dMMR/MSI-H mCRC (NCT02997228, NCT03866239, NCT02912559).

Up until now, ICIs have been very successful in the subgroup of dMMR/MSI-H patients, but not in the subgroup of proficient MMR colorectal cancer, rendering these tumors insensitive to immunotherapy. Further immunotherapeutic efforts are needed to determine which subgroup of patients should receive ICI combinatorial treatment to identify those who may benefit from immune checkpoint blockade interventions. That being said, we do expect ICIs to become a standard treatment for dMMR mCRC soon.

Among the most common mutations in KRAS, mutated codon 13 colorectal cancer is found frequently in poorly differentiated tumors with microsatellite-stable phenotype and significantly correlated with worst overall survival (155, 156). However, these findings are controversial with Bai and colleagues (157) and Li and colleagues (158). They reported that the KRAS mutated codon 12 colorectal cancers, especially G12D and G12V, are more aggressive tumor phenotypes with the worst prognosis when compared with KRAS mutated codon 13 colorectal cancers. Besides, other reports conflict with the correlation between KRAS mutations and clinical outcomes (159–161). These findings suggest that KRAS mutations are not created equally and have distinct mutation profiles representing different tumor clones. Even the same patients with KRAS mutation colorectal cancer treated with the same treatment may experience different treatment outcomes. Massive evidence on tumor heterogeneity in colorectal cancer suggests that not all KRAS mutations confer equivalent resistance to anti-EGFR agents (162–164). This explains why most patients with wt KRAS mCRC do not respond to anti-EGFR treatment, whereas a minority of them responded well (165). Because of the tumor heterogeneity in colorectal cancer, KRAS mutations can only partially explain the development of tumorigenesis. This study was further examined by Laurent-Puig and colleagues (166), who reported that a minor subclone of resistant cells was detected during the anti-EGFR treatment. Their findings were in line with the observation in both tissue and blood biopsy analyses in patients with mCRC treated with anti-EGFR therapy (167) and further confirmed the existence of intratumoral heterogeneity. These findings suggested that therapeutic interventions targeted specific molecular aberrations may impose an evolutionary pressure on cancer and are likely to benefit only a small percentage of patients.

To date, patients with colorectal cancer treated with chemotherapies are still highly vulnerable towards the occurrence of secondary drug resistance. The unexpected therapeutic failure can be attributed to both inter- and intratumoral heterogeneity, as the detection of tumor heterogeneity is limited by routine diagnostic testing. Current clinical practice for colorectal cancer diagnosis and treatment is still largely dependent on the status of RAS mutation, DNA MMR, and BRAF mutational status, even though prognostic or predictive biomarkers are rapidly expanding. Over the past decade, several distinct subtypes of colorectal cancers were identified based on gene expression data. For example, the colon cancer molecular subtyping divided patients into six groups, with C4 and C6 subtypes associated with a more inferior relapse-free survival rate than C1, C2, C3, and C5 subtypes (168). The consensus molecular subtyping categorized colorectal cancers into four subtypes, in which one of the subtypes, CMS4 displayed worse overall survival and relapse-free survival (169). However, this subtype was significantly associated with a positive clinical outcome when patients were treated with chemotherapy FOLFIRI. Recently, Buechler and colleagues (170) further expanded the consensus molecular subtypes into 40 gene signatures developed a ColoType assay for clinical diagnostics using formalin-fixed, paraffin-embedded tissue samples. Intriguingly, Sadanandam and colleagues (171) had proposed the colorectal cancer classification with six subtypes based on differential response to anti-EGFR cetuximab therapy. Among all the subtypes, the stem cell subtype displayed inferior disease-free survival. Hence, this subtype of patients was selected for chemotherapy FOLFIRI treatment, and clinical benefits were shown in both the adjuvant treated and metastatic patients. These findings indicate that the comprehensive genetic profiles of colorectal cancer are essential for developing a personalized treatment for patients with colorectal cancer to prevent them from evolving therapeutic resistance mechanisms.

Colorectal cancer is a complex dynamic disease, and its treatment strategy has greatly evolved alongside advancements in molecular therapy. Early attempts to prevent mTOR and ERK activation were initially focused on developing upstream PI3K and RAF inhibitors because the protein kinase cascade were thought to be a linear pathway. Researchers later found out that RAS cancer cells can dynamically reorganize their downstream signaling networks to restore mTOR or ERK activity and induce resistance to upstream inhibitors. It has become clear that molecular subtyping has a significant influence on the prognosis and therapeutic choices for different stages of colorectal cancer. This implies that future colorectal cancer clinical trials should consider the diverse drug resistance mechanisms, molecular subtypes of interest, and patient stratification based on prognostic and predictive biomarkers. More trials combining targeted inhibitors and immunotherapies are now being investigated. Therefore, study approaches should be designed to also consider the presence of simultaneous resistance and escape mechanism accordingly. There is also a need to identify biomarker-driven therapies in the clinic to complement the dawn of a personalized medicine approach for patients with colorectal cancer in future trials.

No disclosures were reported.

This work was supported by the UCSI University Research Excellence & Innovation Grants: REIG-FAS-2020/030 (principal investigator, Dr Michelle Teo Yee Mun) and REIG-FAS-2020/004 (principal investigator, Dr Lionel In Lian Aun).