Take you to the cancer target KRAS

preface

On May 28, 2021, the US FDA announced the accelerated approval of Lumakras (sotorasib, AMG510) developed by Amgen for the treatment of non-small cell lung cancer (NSCLC) with tumors carrying the KRAS G12C mutation Patients
.
These patients have received at least one prior systemic therapy
.
This is the world's first anti-tumor drug
targeting the KRAS protein.
Lumakras is a "first-in-class" KRASG12C mutant inhibitor developed by Amgen, and the approval of Lumakras comes just 6 months
after Amgen submitted its New Drug Application (NDA).

AMG510 structure

RAS gene is found to be one of the most common mutated genes in tumors, 30% of tumors carry RAS mutations, if you count RAS regulators and signal pathway downstream mutations, covering almost all tumors, RAS mutations cause more than one million patients to die every year, worthy of being the king
of tumor genes.
This also makes people very curious, what is the magic function of RAS that makes tumor cells so dependent?

History of the discovery of KRAS

RAS was the first human oncogene to be discovered
.
The full name of the KRAS gene is Kirsten rats arcomaviral oncogene homolog, which translates to "Chinese Kirsten rat sarcoma virus oncogene homolog"
.
The protein encoded by the KAS gene is a small GTPase, which belongs to the RAS superprotein family
.

At the end of the 19th century, the research of masters such as Pasteur made modern medicine a great success
in the field of anti-infection.
In 1876, a Russian successfully transplanted tumor tissue from one dog to another
.
In 1908, two Danish scientists discovered that extracts from hen leukemia cells could infect other birds with leukemia
.
These observations and studies have led people to believe that tumors are also infectious diseases
caused by viruses.

In 1910, Peyton Rous of the Rockefeller Research Institute discovered the first tumor virus, Roussarcomavirus (RSV, Lloyd's Sarcomavirus),
from a hen from Long Island, New York.
Since then, a series of RNA and DNA viruses that cause tumors in animals have been discovered
.
The pathogenic mechanism is also plausible: these viruses (RNA viruses via reverse transcriptase) can integrate tumor genes into infected host cells, induce malignant changes and maintain the infinite division
of tumor cells.
Peyton Rous, who discovered RSV, and Howard Temin, who discovered RNA virus reverse transcriptase, won the Nobel Prize
in Physiology and Medicine in 1966 and 1975, respectively.
At this point, people are convinced that tumors are a disease
caused by a virus.

It wasn't until 1974 that J.
Michael Bishop of UCSF and his postdoc Harold Varmus accidentally discovered through DNA probes that the tumor gene src
previously found in the RSV virus was also present in uninfected normal cells.
It turns out that tumor genes have long existed in the genome of the host, and in ancient times, viruses obtained these gene fragments from host cells and modified them, which means that the tumor genes carried by viruses actually come from ourselves
.
These discoveries opened the door
to modern tumor biology based on genetic variation.
Bishop and Varmus won their third Nobel Prize
in the field in 1989.

Harvey and Kirsten et al.
discovered the mouse tumor genes HRAS and KRAS
carried by RSV-like retroviruses in the 60s, respectively.
In 1982, Weinberg and other labs also discovered HRAS in human bladder cancer cells T24/AJ, making RAS the first human tumor gene
to be discovered.

KRAS gene and its associated pathway

1.
Classification of KRAS genes

In the human genome, there are 2 KRAS genes
.
One is KRAS1, located on the short arm of chromosome 6; The other is KRAS2, located on
the short arm of chromosome 12.
Among them, KRAS1 is a "pseudogene" and cannot be transcribed into RNA, so it is not functional
.
KRAS2 is the "true gene", which can be transcribed and translated into proteins, and has biological activity
.
Usually the KRAS gene and protein studied in companies and literature refer to the "KRAS2" gene and its protein product
.

The KRAS gene belongs to the RAS gene family
.
The RAS gene family also includes NRAS (neuroblastoma-RAS) and HRAS (Harvey-RAS).

2.
Structure and location of KRAS proteins

KRAS protein has 188 amino acids and its molecular weight is 21.
6KD
.
A guanine nucleoside-binding protein
with GTPase enzyme activity.

The KRAS protein is localized on the inside of the cell membrane and attached to the cell membrane by a Farnesyl modification group [1].

Farnesyl group is added to KRAS protein [3] under the action of farnesyl transferase [2]
through translational protein modification.

Source: Farnesyltransferaseinhibitorsinhematologicmalignancies: newhorizonsintherapy

3.
KRAS related pathway

From the web

Upstream signaling pathway

In normal cells, receptor monomers such as EGFR, HER2, ErbB3 and ErbB4 on the cell membrane bind to extramembrane ligands to form dimers, which phosphorylate themselves and phosphorylate downstream signaling proteins
.
One of these signaling pathways activates Grb2-Shc, which in turn activates the SOS protein, which in turn activates the KRAS protein
.

KRAS pathway

Inside the cell, the KRAS protein transitions between inactive and activated states, when KRAS binds to guanine nucleoside diphosphate (GDP), it is in an inactive state, when it binds to guanine nucleoside triphosphate (GTP), it is in an activated state, and downstream signaling pathways
can be activated.

Downstream signaling pathways

KRAS in most cells is inactivated, and when activated, multiple downstream signaling pathways can be activated, including the MAPK signaling pathway, the PI3K signaling pathway, and the Ral-GEFs signaling pathway
.
These signaling pathways play an important role
in promoting cell survival, proliferation, and cytokine release.

4.
Regulators of KRAS activity

The transition of KRAS between inactivated and activated states is regulated
by two classes of factors.

One class is guanine nucleotide exchangers (GEFs), which catalyze the binding of KRAS to GTP, thereby promoting the activation of KRAS, including SOS proteins (GEFs/Guanosine Release Factor/Guanylate Exchange Factor).

The other class is GTPase (GTPase) activating proteins (GAPs), which can promote the hydrolysis of GTP bound to KRAS to become a GDP termination active state, thereby inhibiting the activity
of KRAS.

RAS mutations in cancer

Mutations affecting components of the RAS–RAF–MEK–ERK pathway, including various RTK, SHP2, NF1, RAS proteins, Members of the RAF family, or MEK1/MEK2, cause abnormal activation of this pathway and cause cancer
.
RAS mutations or amplifications are the most common mutations in human cancers: KRAS is most commonly altered, especially in solid tumors; NRAS mutations are present in melanoma and many hematologic malignancies; HRAS mutations mainly occur in bladder, thyroid, cervical, and head and neck cancers
.

About 17% of solid tumors have KRAS mutations, including about 90% of pancreatic cancers, about 50% of colon cancers, and about 25% of lung adenocarcinomas
.
In fact, KRAS mutations dominate NSCLC, accounting for about 78%
of all RAS mutations found in such tumors.

Almost all cancer-associated RAS mutations (~95%) affect codons and lead to a significant increase in the basal RAS-GTP:RAS-GP ratio and constitutive activation
of RAS effectors.
Mutations in glycine residue 12 (G12) of KRAS are the most common, and glycine 13 (G13) is the second most commonly affected residue
of KRAS.
The most common KRAS codon mutations are G12C, G12V or G12D substitutions, accounting for 40%, 19%, and 15%
of KRAS mutations in NSCLC, respectively.
Structural studies have shown that most of these gene mutations interfere with KRAS' ability to
hydrolyze GTP.

Why is KRAS an "undruggable" target?

If KRAS is so important, and the oncogenic mutation in the KRAS gene is well understood, why has there not been a targeted drug that directly targets the KRAS gene until now? The reason is that KRAS protein is a featureless, nearly spherical structure with no obvious binding site, and it is difficult to synthesize a compound
that can target binding and inhibit its activity.
For a long time, KRAS has become synonymous with "undruggable" targets in the field of oncology drug research and development
.

The difficulties are:

1) The role of KRAS is wide-ranging, the normal activity of KRAS is also the activity required for many normal cell functions, such as the selection of drugs that directly inhibit KRAS, the drug toxicity may be great, and the side effects may be strong
.
And KRAS has high homology with NRAS and HRAS, and drugs that can inhibit KRAS activity are likely to inhibit the activity
of NRAS and HRAS.
Then, the toxicity of this drug can be great
.

2) The currently known active functional domain of KRAS is mainly a pocket-shaped functional domain
of KRAS combined with GDP or GTP.
Unlike protein kinases, which have a weak affinity for ATP, KRAS binds very strongly to GTP, or GDP, with affinity coefficients of PicoMolar (picomolar, 10^-12).

The concentration of GDP and GTP in normal cells reached the MicroMolar (micromolar concentration, 10^-6) level
.
Therefore, the concentration of normal GDP and GTP in the cell is 10 to the power of 6 than the concentration required to bind to KRAS
.
RAS lacks pockets large enough to bind small molecules; Therefore, it is very difficult
to make a small molecule compound whose binding ability to KRAS can match GDP or GTP.

3) To design an active drug that only inhibits the mutant KRAS protein, while affecting the activity of the normal KRAS protein as little as possible, this compound needs to have good selectivity
for the mutant KRAS.
This is another conundrum
in drug design.

4) However, indirect targeting of KRAS strategies is also difficult, including blocking KRAS cell membrane localization and targeting of signaling molecules downstream of KRAS, such as members of the RAF, MEK, ERK and PI3K families
.
Specifically, the difficulties of indirect pathways include: (1) RAS is a necessary pathway related to normal cell growth and survival, and targeting the necessary pathway first faces serious toxic side effects, resulting in a very narrow or even no efficacy index; (2) compensatory escape mechanisms, and (3) signal feedback and redundancy
due to tight regulation.

Research progress in drug development targeting KRAS

In recent years, breakthroughs in covalent inhibitor research against KRAS mutants have made it possible
to target KRAS mutants through the allosteric site.
In the KRASG12C mutant, small molecules covalently bound to cystine produced by the mutation are more likely to bind
to the KRAS protein that binds to GDP.
This combination reduces the affinity of GTP with KRAS and hinders GEF-catalyzed GTP replacement of GDP, locking KRASG12C mutants in an inactivated state
.
The discovery of this binding "pocket" on the KRASG12C mutant has led to the emergence of a variety of small molecule covalent inhibitors
targeting the KRASG12C mutant.

In the Phase I/II CodeBreaK 100 study, the first human data from 22 patients with advanced KRASG12C mutant solid tumors treated with Sotorasib showed a partial response (PR) in 6 NSCLC patients
.
Subsequent data from 129 patients in the phase I part of this trial showed that the objective response rate (ORR) was 32.
2%, the disease control rate (DCR) of 59 patients with advanced non-small cell lung cancer was 88.
1%, the median PFS was 6.
3 months, and the ORR of 42 patients with CRC was 7.
1% and DCR was 73.
8%.
The median for PFS was 4.
0 months
.
Patients with melanoma, pancreatic cancer, and endometrial cancer also respond to the response
.
These impressive results led to the 2021 FDA accelerated approval of Sotorasib for NSCLC
.

Adagrasib is the second KRASG12C inhibitor to enter clinical trials, and based on data from the Phase I/II KRYSTAL-1 trial, the FDA granted breakthrough therapy designation
for advanced KRASG12 C-mutated non-small cell lung cancer.
Data from this trial, published in 2021 at the European Society of Medical Oncology (ESMO), showed that DCR reached 96%, 23 (45%) PR in 51 evaluable patients, and another 26 SD
。 In addition, in the Phase II update data for KRYSTAL-1, 116 previously treated NSCLC patients had an 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 a 1-year OS rate of 50.
8%
in 1-year patients.
Numerous trials of Sotorasib for other indications are
ongoing.

In addition, several other KRASG12C inhibitors have also entered clinical development
.
A Phase I trial of GDC-6036 (RG6330) involving patients with various advanced KRASG12C mutant solid tumors (NCT04449874) is
ongoing.
JDQ443 is another covalent irreversible KRASG12C inhibitor, and a phase I/II trial is evaluating JDQ443 as monotherapy, or in combination with TNO155 (another SHP2 inhibitor) and/or tislelizumab (anti-PD-1 antibody) for the treatment of KRASG12C-mutant NSCLC, Patients with CRC or other advanced solid tumors (KontRASt-01; NCT04699188）
。

LY3537982 is a selective KRASG12C inhibitor that is also in Phase I clinical development for KRASG12C-mutant solid tumors (NCT04956640).

Other KRASG12C targeting compounds in the Phase I/II trial include D-1553, JNJ-74699157, BI 1823911, JAB-21822, and MK-1084
.

Drug resistance of KRAS

Although KRAS is no longer undruggable, the efficacy of KRAS inhibitors alone is far from adequate
.
In fact, plasticity and genetic instability make tumors resistant to all single-agent targeted therapies, and KRAS targeted therapies are no exception
.

Primary drug resistance

Theoretically, major resistance to KRAS inhibitors may arise from mutational heterogeneity or the presence of
specific co-mutations in tumors.
Understanding these resistance mechanisms is critical
to developing treatment strategies.
KRAS mutational heterogeneity between different disease sites in CRC patients has been described and may at least partially explain the variable response
to EGFR-targeted therapy in such patients.

Zhao et al.
evaluated 8750 pre-treatment KRAS mutant tumor specimens, and only found more than one RAS mutation in 304 samples (3.
5%); Among KRASG12C mutant tumors, 3% were found to have secondary RAS mutations
.
Currently, the understanding of the mechanisms underlying primary resistance to KRASG12C inhibitors is still extremely limited, and large-scale cohort multiomics analysis is needed to identify pretreatment factors
associated with non-response to these drugs.

Acquired resistance due to mutational escape

Mutational escape refers to drug-resistant mutations that develop at the time of treatment that cannot be detected
until treatment.
The binding sites of the commonly used KRAS inhibitors sotorasib and adagrasib are formed from amino acid residues at positions 12, 68, 95 and 96 of KRASG12C; Therefore, mutations affecting these residues are particularly relevant
for drug resistance.

For example, the second locus Y96D mutation obtained in KRASG12C confers clinical resistance to adagrasib by changing the switch pocket so that the inhibitor no longer
binds.
Koga et al.
identified 12 different secondary KRAS mutations
after treating KRASG12C mutant Ba/F3 cells with sotorasib or adagrasib in vitro.

Adaptive resistance

Adaptive resistance refers to the rapid reactivation of the RAS–MAPK pathway at a certain level, usually due to the deexpression of the MYC target gene, which inhibits ERK activity
.
Some studies have shown that similar pathway reactivation
occurs when KRASG12C inhibitors are treated.
In fact, it is unclear
whether adaptive resistance is mediated by reactivation of mutant KRAS or activation of residual wild-type KRAS, HRAS, and/or NRAS.

EMT

Epithelial-mesenchymal transition (EMT) is another potential mechanism
of intrinsic and acquired resistance to KRASG12C inhibitors.
During EMT, cells downregulate epithelial gene expression and upregulate mesenchymal gene expression, thereby increasing mobility and aggressiveness
.
Adachi et al.
used gene set enrichment analysis to demonstrate that EMT is the mechanism
by which intrinsic and acquired resistance to sotorasib develops.
It was found that in the EMT-induced KRASG12C mutant cell line, the PI3K pathway was activated by bypassing the IGFR–IRS1 signaling pathway, and sotorasib binding to the PI3K inhibitor GDC-0941 blocked AKT activation and inhibited cell proliferation
.
These preclinical observation and combination strategies warrant further clinical study
.

Overcoming resistance to KRASG12C inhibitors

Several strategies to limit adaptive resistance and prolong the response to KRASG12C inhibitors are being explored.

Adaptive resistance typically involves upregulation of several RTK and RTK ligands, in NSCLC and CRC, KRASG12C Inhibition leads to the accumulation of activated upstream EGFRs and/or other ERBB family members, which may lead to escape from KRASG12C inhibitor monotherapy
。 Therefore, multiple clinical trials are currently investigating the combined inhibition
of KRASG1C and EGFR.

In the CodeBreaK 101 clinical trial (NCT04185883), sotorasib was combined
with the EGFR/HER2 tyrosine kinase inhibitor afatinib or the anti-EGFR monoclonal antibody panitumumab.
Preliminary results show a manageable safety profile, with the most common treatment-associated adverse effects (TRAE) being diarrhea and nausea
.
In both dose cohorts, efficacy signals were observed with ORRs of 20.
0% and 34.
8%, and DCRs of 70.
0% and 73.
9%,
respectively.
In addition, adagrasib was tested in conjunction with another anti-EGFR monoclonal antibody, cetuximab, in KRYSTAL-1 and KRYSTAL-10 (NCT03785249 and NCT04793958).

GDC-6036 is also being tested in combination with cetuximab and the EGFR tyrosine kinase inhibitor erlotinib (NCT04449874).

Since its discovery in 1992, SHP2 has become a key "positive" upstream regulator of the RAS–MAPK pathway and is therefore an important component of
multiple oncogenic driver kinase signaling.
SHP2 is required for tumorigenesis in several models of KRAS mutant NSCLC, suggesting that SHP2 suppression may play a role
in the treatment of KRAS mutant cancers.
Based on the increased effectiveness of SHP2 and KRAS combined inhibition in preclinical studies, the combined application of SHP2 inhibitor TNO155 with adagrasib is currently being tested in the KRYSTAL-2 trial (NCT04330664).

In addition, several other clinical trials are evaluating combinations of various SHP2 inhibitors and KRASG12C
inhibitors.

In addition to the joint rejection of the RAS signaling pathway vertically, in the horizontal direction, different combined strategies
have been tested.
For example, inhibition of mTOR as an alternative strategy
to overcome adaptive resistance to KRAS inhibitors.
In the CodeBreak 101 clinical trial, the mTOR inhibitor everolimus is being evaluated in combination with sotorasib (NCT04185883).

Cell cycle inhibition offers another potential horizontal combination strategy
for synergistic effects with KRAS inhibitors.
In addition, Aurora kinase (AURKA, AURKB and AURKC) inhibitors and mitotic checkpoint kinase WEE1 inhibitors have been tested
in combination with KRASG12C inhibitors in preclinical trials.

In addition, KRAS has a variety of immunomodulatory effects, mediated
by multiple mechanisms.
Activation of KRAS increased the production of neutrophil chemokines CXCL1, CXCL2 and CXCL5; Promote the recruitment of pro-inflammatory M1 macrophages by upregulating the expression of intercellular adhesion molecule 1 (ICAM1); Induce immunosuppressive regulatory T (Treg) cell differentiation by secretion of TGF-β and IL-10
.

Based on these findings, binding KRAS inhibitors to ICIs is a reasonable strategy
.
Currently, the method of binding KRASG12C inhibitors to ICI is being evaluated in multiple clinical trials in KRASG12C-mutant solid tumors (NCT03600883, NCT04185883, NCT03785249, NCT04613596, NCT04449874, and NCT04699188).

Future development of RAS inhibitors

Target other subtypes of KRAS

Given that KRASG12D has lower intrinsic GTPase activity than KRASG12C, most KRASG12D proteins will bind
to GTP.
Therefore, targeting KRASG12D-GTP as well as other RAS subtypes has been the focus of
drug development.

In 2020, Zhang et al.
identified three unique cyclic peptide ligands that preferentially bind within the switch 2 groove of KRASG12D-GTP and inhibit their interaction
with RAF proteins.
Notably, these compounds had no significant effect on wild-type KRAS, which exemplified the different characteristics of
the GTP-bound states of KRAS mutants.

Another novel strategy targeting mutant KRAS and/or other mutant RAS subtypes uses mechanisms
similar to the immunosuppressants cyclosporine, FK506, and rapamycin.
Various compounds
that bind to cyclophilin A and subsequently form inhibitory trimeric complexes with various RAS proteins have been developed.
Recent preclinical data on the new generation of "trimeric complex" KRAS-on inhibitor RMC-6291, indicating that it is superior to the KRAS-off inhibitor adagrasib, supports the feasibility
of this targeted approach.

Target RAS degradation

RAS degradation is another novel way
to target RAS-mutant malignancies.
Proteolytic targeting chimeras (PROTACs) promote proteasome degradation
of disease-associated proteins by binding to the target protein and E3 ubiquitin ligase.
LC-2 is a PROTAC designed for KRASG12C, and similar designs of pan-KRAS and other mutation-specific KRAS degraders are in preclinical development
.

The use of chimeric toxins is another alternative to RAS targeting
.
RRSP–DTB is one such toxin consisting of translocated B fragments of RAS/RAP1-specific endopeptidase (RRSP) and diphtheria toxin (DTB) derived from Vibrio vulnificus.

The toxin enters cells via heparin-bound EGF-like growth factor (HB-EGF)-mediated endocytosis followed by cleavage of RAS
in switch 1 region.

Immunotherapy targeting KRAS

In 2016, Tran et al.
first described an endogenous immune response against KRAS mutant cancer cells by identifying CD8+TIL with a T cell receptor (TCR) that recognizes new epitopes of peptides presented by MHC I (HLA-C*08:02) derived from KRASG12D
。 ACT using these specific TILs resolved all seven lung metastases in patients isolated with KRASG12D mutant CRC
.
TCR-T cells targeting KRASG12V mutant cells have entered clinical trials
.

Therapeutic cancer vaccines constitute another immune-based approach
to targeting KRAS mutant tumors.
An mRNA neoantigen vaccine mRNA-5671/V941
targeting the G12D, G12V, G13D and G12C variants of KRAS has been developed.
mRNA-5671/V941 has entered Phase I clinical trials
.
In addition, long peptide vaccines with activity against KRAS G12C, G12V, G12D, G12A, G13D and G12R variants are currently being evaluated in combination with ICIs in a Phase I trial (NCT04117087).

siRNA-based methods

Nanoparticle-based platforms have been developed to deliver KRAS-specific small interfering RNAs (siRNAs).

This technique has been shown to be adequately delivered to cancer cells and effectively reduces their KRAS levels, resulting in anti-cancer activity
.
Preclinical studies suggest that siRNA-based approaches against KRAS-driven cancers may be a viable therapeutic strategy
.

brief summary

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 approaches, and new alternative targeted approaches are currently in clinical studies
.
However, data on KRASG12C inhibitors suggest that these drugs are far from cure
.
This is at least partly due to the fact that monotherapy almost always develops resistance
.

Currently, attempts are being made to overcome the resistance mechanism
by vertically inhibiting multiple nodes of the RAS pathway.
In addition, the therapeutic strategy also includes a combination strategy of inhibiting parallel pathways, combined with immunotherapy strategies to induce a durable anti-tumor memory immune response
.
Finally, new approaches and therapeutic strategies to target RAS are being developed, and we expect these new approaches to achieve the long-sought cure in the KRAS mutant malignancy population
.
"

References:

1.
The current state of the art and future trends in RAS-targeted cancer therapies.
Nat Rev Clin Oncol.
2022 Aug 26 : 1–19.

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