This article reviews the research status and future of KRAS-targeted therapy in the field of RAS pathway and lung cancer

KRAS is the most common oncogenic protein in solid tumors and was once considered an "undruggable" target
.
In the past two years, several inhibitors have been shown to be effective in patients with KRAS G12C mutations and have changed current clinical practice
.
In this paper, the characteristics of RAS signaling pathways in cancer cells and tumor microenvironment are sorted out, and the treatment strategies and future research directions
for oncogenic RAS are sorted out.

RAS pathway

RAS signaling plays an important role in normal cell proliferation, and this signaling pathway is often abnormalized
during tumorigenesis.
In fact, changes in the components of this signaling pathway, especially the RAS protein itself, have a profound impact
on tumor cells.
As a result, over the past few decades, researchers have made great efforts
to target drugs for the "RAS protein" — a central member of the RAS–RAF–MEK–ERK pathway.
In 2021, decades of research finally came to fruition
.

The RAS–RAF–MEK–ERK mitogen-activated kinase (MAPK) signaling pathway is activated
by most growth factor, cytokine, and immune receptors, as well as many integrin and chemokine receptors.
The RAS family GTP kinases, including KRAS, NRAS, and HRAS, cycle
between loading the "on" state of the GTP and loading the "off" state of the GDP.
During activation-inactivation, the conformation of two regions in the RAS protein (called the Switch 1 and Switch 2 domains) changes, and Switch 2 proves critical to the eventual development of RAS inhibitors
.

Currently, a variety of inhibitors have been developed to directly target mutant RAS proteins, including inhibitors
that bind to inactive, GDP state ("KRAS-off inhibitors") and binding to active, GTP states ("KRAS-on inhibitors").
Currently, it is being evaluated
in clinical trials.
The RAS signaling pathway, which has many upstream and downstream mediators, is expected to be an attractive target in combination with RAS inhibitors to improve anti-tumor response and reduce intrinsic and acquired resistance
.

Fig.
1 RAS signaling pathway and therapies targeting this pathway in cancer

RAS mutations in cancer

RAS pathway protein abnormalities, including activation of RAS mutations, are common
in a variety of malignancies.
RAS gene mutations are common in gastrointestinal tumors and lung cancers, and most mutations are KRAS mutations
.

Factors affecting mutations in the RAS-RAF-MEK-ERK signaling pathway include RTKs, SHP2, NF1, RAS proteins, RAF family members, or MEK1/MEK2, which can lead to abnormal activation of this pathway and tumorigenesis
.
RAS mutation or amplification is one of the most common abnormalities in human cancers, and KRAS occurs most often, especially in solid tumors; NRAS mutations often occur in melanoma and hematologic malignancies; HRAS mutations are mainly seen in bladder, thyroid, cervical, and head and neck cancers
.

Fig.
2 Incidence of KRAS, NRAS and HRAS mutations in different tumor species

Overall, about 17 percent of solid tumors have KRAS mutations, including about 90 percent of pancreatic cancers, about 50 percent of colon cancers, and about 25 percent of lung adenocarcinomas
.
KRAS mutations predominate in NSCLCs, accounting for about 78%
of all RAS mutations in such tumors.
and are often mutually exclusive with other clinically targetable driver mutations such as EGFR, BRAF, and ALK
.

Different KRAS mutations have different prognostic or predictive significance
for NSCLC.
The BATTLE study showed that patients with KRAS G12C or KRAS G12V mutations associated with poorer PFS in patients with refractory NSCLC who had previously received molecularly targeted therapy compared with wild-type or other KRAS mutation types
.
Other studies have shown improved
PFS in patients with KRAS G12C mutations compared with non-G12C mutations.
Current research suggests that further research is needed to elucidate the prognostic and predictive value
of different KRAS mutations.

Coexisting mutations of KRAS and tumor suppressor genes STK11, TP53, or CDKN2A/CDKN2B are often seen in KRAS-mutated tumors
.
Patients with KRAS-STK11 co-mutations can be subdivided into KEAP1 wild or KEAP1 mutants
.
In the absence of STK11 mutations, KEAP1 can also coexist with KRAS mutations
.
Both mutation patterns are associated with treatment resistance and may lead to a worse response or poor prognosis
from immune or combination therapy.

The difficulty of KRAS target drug application

KRAS protein is a featureless, near-spherical structure with no obvious binding site, making it difficult to synthesize a compound that can target binding and inhibit its activity
.

For decades, the "struggle" with KRAS has reflected the challenges posed by three key biochemical characteristics: first, unlike other protein kinases that have a weak affinity for ATP, the RAS protein has a very strong affinity for GTP, reaching the level of picomolar concentration; Second, the high intracellular concentration of GTP (~500 nM) is 10 to ⁶ times
higher than the concentration required to bind to KRAS.
Third, the RAS protein lacks a "pocket" of binding small molecules that are "deep enough" or pharmacologically available
.
Overcoming the pimoler-level affinity of RAS for GTP will require small molecules
with unprecedented binding properties.
The GTP binding site of the KRAS protein differs in specific KRAS mutants, further complicating
the design of KRAS inhibitors.

All first-generation KRAS inhibitors bind to inactive, GDP-bound KRAS (KRAS-off inhibitors).

Recently, researchers have also developed "KRAS-on" inhibitors that target the active KRAS-GTP, such as RM-018 (Figure 1 and Table 1).

Table 1 Selected KRAS-targeted therapies

Clinically used KRAS G12C inhibitor

Phase I/II CodeBreaK 100 Study – Preliminary human trial data from 22 patients with advanced KRAS G12C-mutant solid tumors receiving sotorasib showed monotherapy antitumor activity
.
In a phase I study of 129 patients, the objective response rate (ORR) of 32.
2%, disease control rate (DCR) of 88.
1%, and median progression-free survival (PFS) of 6.
3 months
in 59 patients with advanced NSCLC were shown.
The Phase II CodeBreaK 100 study showed an ORR of 37.
1% in 124 patients with evaluable advanced KRAS G12C mutation NSCLC, a median duration of response (DOR) of 11.
1 months, a median PFS of 6.
8 months, and a median OS of 12.
5 months
.
These impressive results led the FDA to accelerate the approval of the indication for sotorasib for NSCLC
.
The Phase III CodeBreaK 200 trial is currently exploring the efficacy
of sotorasib versus docetaxel in patients with KRAS G12C mutations that progress after monotherapy or combination therapy.
Other studies of sotorasib are also being explored (table 1).

Adagrasib is the second KRAS G12C inhibitor
to enter clinical trials.
Based on the data from the Phase I/II KRYSTAL-1 study, adagrasib was approved as a breakthrough therapy by the FDA for the treatment of patients with advanced NSCLC with KRAS G12C
mutations 。 Data presented at ESMO 2021 showed a DCR of 96%, with 23 (45%) of 51 evaluable patients achieving a partial response (PR), and a median PFS of 11.
1 months and a median DOR of 16.
4 months
in 16 patients receiving the recommended dose of KRAS G12C in the phase I/Ib portion of the KRYSTAL-1 study 。 The phase II cohort of the KRYSTAL-1 study included 116 patients with previously treated NSCLC, with a renewal ORR of 42.
9%, a DCR of 79.
5%, a median DOR of 8.
5 months, a median PFS of 6.
5 months, a median OS of 12.
6 months, and an estimated 1-year OS rate of 50.
8%.

In addition, in the previously treated stable brain metastases subgroup (n = 33), the intracranial ORR was 33.
3%.

A large number of clinical studies on adagrasib are also underway (Table 1).

Several other KRAS G12C inhibitors with similar mechanisms of action have entered clinical development (Table 1).

Investigators are exploring the efficacy of GDC-6036 (also known as RG6330) monotherapy and combination with other kinase inhibitors and SHP2 inhibitor GDC-1971 in advanced KRAS G12C-mutant solid tumors (NCT04449874).

JDQ443 is a covalent irreversible KRAS G12C-off inhibitor that has also entered clinical trials
.
The Phase I/II KontRASt-01 trial is evaluating JDQ443 monotherapy or in combination with TNO155 (a SHP2 inhibitor) and/or tislelizumab in patients with KRAS G12C mutation NSCLC, colorectal cancer, or other advanced solid tumors
.
LY3537982, a selective KRAS G12C inhibitor, is also in phase I clinical trials
.
Other KRAS G12C inhibitors in phase I/II trials include D-1553, JNJ-74699157, BI 1823911, JAB-21822, and MK-1084
.

The great progress brought about by the success of the KRAS G12C inhibitor is worth studying and learning
from.
However, the long-term survival benefit of KRAS G12C inhibitor monotherapy is still limited, and relevant primary and acquired resistance mechanisms have been reported, and TME changes that can induce immunosuppressive states may also be associated
with drug resistance.

Overcoming resistance to KRAS G12C inhibitors

"Vertical" combination strategy

In NSCLC and colorectal cancer, KRAS G12C inhibitors can lead to the accumulation of activated upstream EGFR and/or other ERBB family members, which may lead to resistance to KRAS G12C inhibitor monotherapy
.
Therefore, the combined inhibition of KRAS G12C and EGFR is currently undergoing relevant clinical trials (Figure 1).

The CodeBreaK 101 study is exploring the value of a combination of
sotorasib + EGFR/HER2 TKI afatinib or EGFR monoclonal antibody (± FOLFIRI).
KRYSTAL-1 AND KRYSTAL-10 ARE ALSO EXPLORING THE EFFICACY
OF ADAGRASIB+ CETUXIMAB, ANOTHER EGFR MONOCLONAL ANTIBODY.
NCT04449874 is also exploring the efficacy and safety
of GDC-6036+ cetuximab or erlotinib.

Since its discovery in 1992, SHP2 has become a key upstream regulator of the RAS-MAPK signaling pathway and an important component of signaling by a variety of oncogenic mutant kinase signaling (Figure 1).

Preclinical studies have shown that inhibition of SHP2 may play an important role
in the treatment of KRAS-mutated NSCLC.

Based on the good results of preclinical studies, KRYSTAL-2, CodeBreak 101, and KontRASt-01 studies are currently exploring the efficacy
of SHP2 inhibitors TNO155+adagrasib, in combination with sotorasib, and in combination with JDQ443, respectively.
The CodeBreaK 101 study is also exploring the use
of sotorasib in combination with RMC-4360, a SHP2 inhibitor similar to RMC-4550.

Indirect targeting of RAS via SOS1 constitutes another form of "vertical" suppression of this signaling
pathway.
SOS1 and its paralogy, SOS2, are vital RAS-GEF members
.
SOS1/2 inhibitors represent a different strategy for inhibiting RAS mutant activity (Figure 1).

BI-3406 is an oral small molecule inhibitor that selectively binds within the active site of SOS1 and disrupts its interaction
with RAS-GDP.
BI1701963 is a small molecule
similar to BI-3406.
The KRYSTAL-14 study is exploring the efficacy
of monotherapy and combination with adagrasib.

The CodeBreaK 101 trial also evaluated trametinib in combination with sotorasib±panitumab in patients with various KRAS G12C-mutant solid tumors
.
The principle of this combined strategy is that KRAS-mutant tumor cells are primally resistant to MEK inhibitors due to RAF-mediated MEK activation (Figure 1), which can eliminate this resistance by simultaneously inhibiting mutant KRAS mutations
.
Preliminary results from the CodeBreak 101 study of sotorasib + trametinib suggest that the combination regimen is safe, tolerable, and importantly, even patients who have previously received a large number of KRAS G12C inhibitors can benefit
from combination therapy.

"Horizontal" combination strategy

Figure 1 shows mTOR inhibitors as an alternative strategy to overcome acquired resistance to KRAS (or MEK) inhibitors (Figure 1).

The CodeBreak 101 study is exploring a combination strategy
for the mTOR inhibitor everolimus + sotorasib.

Synergies between cell cycle inhibitors and KRAS inhibitors may provide another potential combination strategy (Figure 1).

CDK4/6 inhibitors are effective in treating certain cancers, particularly estrogen receptor-positive breast cancer
.
CDK4/6 inhibitors block the cell cycle processes driven by D-type cyclin, the nodes
where the RAS-MAPK and PI3K-AKT pathways converge.
CodeBreak 101 is also exploring therapeutic strategies
that combine sotorasib with palbociclib.

Immune-mediated tumor escape and combined immune strategies

KRAS mutations, like other driving oncogenes, have an impact
on the tumor microenvironment (TME) in addition to altering the behavior of the tumor cells themselves (Figure 3).

Activation of KRAS mutations has many effects on the tumor immune microenvironment, including macrophage activation and recruitment, polarization of M1 to M2 macrophages, etc
.
Changes in the tumor microenvironment may provide opportunities
for the treatment of KRAS-mutated malignancies.

Fig.
3 Effect of mutated KRAS on tumor immune microenvironment

KRAS has a variety of immunomodulatory effects and can be mediated by a variety of mechanisms (Figure 3).

The study found that KRAS oncogenic mutations can promote immune evasion by promoting immunosuppressive TME, which can be mitigated by inhibition of SHP2 – possibly a potential combined strategy
.

This combination of KRAS G12C inhibitors with immunotherapy is currently being explored in multiple clinical trials, including CodeBreaK 100, CodeBreaK 101, KRYSTAL-1 and KRYSTAL-7, GO421447, and KontRASt-01
.

In addition, other potential therapeutic strategies are being explored, including the development of KRAS-on inhibitors, RAS degraders and toxins, immunotherapies targeting KRAS, and siRNA-based approaches
.

epilogue

Since the discovery of KRAS mutations in lung cancer more than three decades ago, the discovery of KRAS-targeted drugs has made great progress, and a large number of inhibitors, combination strategies and alternative targeted therapies are currently being studied in clinical trials
.
However, data on KRAS G12C inhibitors suggest that these drugs are far from "curative.
"
This is partly because monotherapy is prone to drug
resistance.

At present, researchers are overcoming drug
resistance by "vertically" inhibiting multiple nodes of the RAS pathway.
In addition, treatment strategies include simultaneous inhibition of parallel pathways and combination immunotherapy strategies
.
At the same time, researchers are constantly developing new strategies to target RAS, which bring new hope to KRAS-targeted therapy, and so far, KRAS has shed the title
of "undruggable" target.
It is expected that the above combined strategies and new strategies can achieve long-term "cure" goals
in patients with KRAS-mutated malignancies.