Resistance mechanism and improvement strategy of CDK4/6 inhibitors in the treatment of breast cancer

Breast cancer is the most common malignancy in women, accounting for about 24.
5%
of all malignancies worldwide.
Breast cancer is divided into three subtypes based on estrogen receptor (ER), progesterone receptor (PR), and HER2 status: hormone receptor (HR) positive, HER2-positive, and triple-negative subtypes
.
Among them, HR-positive breast cancer accounts for about 60%–70%.

Currently, endocrine therapy is considered the mainstay of treatment for
HR-positive breast cancer.
Despite significant improvements in the treatment of advanced disease, a large proportion of patients eventually develop resistance to endocrine therapy
.
Studies have shown that the mechanisms leading to endocrine resistance mainly include ER loss or mutation, changes in ER pathways, dysregulation of cell cycle signaling molecules, and activation of various escape pathways
.
The emergence of resistance to endocrine therapy has created an urgent need to find new targets for breast cancer treatment, such as cyclin-dependent kinase (CDK) 4/6 inhibitors
.

Figure 1.
Summary of global cancer incidence rates in 2020

First, the biological function and pathogenic mechanism of CDK4/6

The human cyclin-dependent kinase (CDK) family contains more than 20 CDKs and up to 30 different cyclins
.
The cyclin-dependent protein kinase CDK is an important factor in cell cycle regulation and is a group of serine/threonine protein kinases that catalyze serine/threonine protein phosphorylation and synergize with cyclin cyclin to drive the cell cycle
.
CDK can be combined with cyclin to form heterodimers, of which CDK is the catalytic subunit, cyclin is the regulatory subunit, and different cyclin-CDK complexes catalyze phosphorylation of different substrates through CDK activity, and realize the promotion and transformation of different time phases of the cell cycle
.
In addition, CDK4/6 phosphorylates the retinoblastoma gene (RB), releases the transcription factor E2F, promotes the transcription of cell cycle-related genes, and promotes the cell cycle from the G1 mitosis phase to the S phase, leading to the DNA replication process (Figure 2).

Figure 2.
Mechanism of action of CDK4/6

Studies have shown that CDK4/6 is overactive in cancer, with significantly elevated
expression levels in many tumors.
Overexpressed CDK4/6 promotes G1/S transformation by directly and indirectly (by stimulating CDK2) phosphorylation of Rb and promotes tumorigenesis
.
In addition, the researchers found that CDK4 can modulate inflammatory cytokine signaling processes
through transcriptome analysis of breast cancer.
CDK6 can also induce angiogenesis, stem cell activation, immune response, etc
.
Studies have shown that many oncogenes can cause cancer by activating the CDK4/6-Rb-E2F pathway and inducing cell malignant proliferation, mainly including JAK/STAT, PI3K/Akt/mTOR, RAS/RAF/MEK/ERK, BTK/NF-κB and Wnt/β-catenin
.
In addition, mutations in tumor suppressor genes such as p53 can also activate the CDK4/6-Rb-E2F pathway
by releasing p21 CIP1 inhibitory.
Thus, CDK4/6 can act as a hub
for tumorigenic pathways.
In addition, knockout of the CDK4/6 gene was found to inhibit tumor cell growth without affecting
normal cell growth.
Therefore, based on the characteristics of CDK4/6, CDK4/6 has become a target with great clinical potential and safety
.

Figure 3.
CDK4/6-mediated pathogenesis

Second, the resistance mechanism of CDK4/6 inhibitors

Although CDK4/6 inhibitors have greatly improved disease control in patients with HR-positive breast cancer, not all patients are effective against these drugs, and most cancer patients develop acquired resistance after using CDK4/6 inhibitors
.
The resistance mechanism of CDK4/6 inhibitors can be divided into cell cycle-specific resistance and cell cycle non-specific resistance
.
Studies have shown that the resistance mechanism of CDK4/6 inhibitors mainly includes continuous G1-S phase cyclin expression and other bypass signal-mediated early and late adaptation
.
In addition, various other mechanisms exist that can also lead to intrinsic or acquired resistance to CDK4/6 inhibitors
.

Figure 4.
Resistance mechanisms of CDK4/6 inhibitors

1.
RB is missing

The tumor suppressor RB is a key checkpoint
in the cell cycle described above.
As the main target of CDK4/6 inhibitors, RB is considered one of the most important biomarkers of
therapeutic sensitivity.
Various preclinical studies have shown that the loss of RB is the main factor in
the development of drug resistance of CDK4/6 inhibitors.
In addition, several preclinical and clinical studies have also reported mutations in RB as the cause
of drug resistance.
A study using glioblastoma xenografted cells found that missense mutations in RB (A193T) exon 2 led to resistance to CDK4/6 inhibitors
.

2.
P16 overexpression

p16, a member of the INK4 family, is a natural inhibitor of CDK4 and an important tumor suppressor involved
in cell cycle regulation.
p16 acts as a tumor suppressor in the presence of functional RB because CDK4/6 (the target of p16) requires RB to exert its own kinase activity
.
The study found that overexpression of p16 occurs during
carcinogenic stress.
In the presence of functional RB, p16 is overexpressed, which makes CDK4/6 inhibitors resistant by inhibiting the expression of CDK4
.

3.
E2F overexpression

E2F is a downstream transcription factor
of RB.
The RB-E2F complex plays an important role
in regulating the cell cycle from the G1 phase to the S phase.
Cyclin D-CDK4/6 phosphorylates RB, releasing E2F, which leads to the expression of proteins required for cell cycle progression, including cyclin E
.
In addition, the cyclin E-CDK2 complex also phosphorylates RB, releases E2F and facilitates the entry into the S phase
.
The loss of RB is associated with an increase in the expression of E2F, leading to the constitutive activation of its downstream target protein, which in turn leads to the development
of drug resistance.

4.
Activate the FGFR pathway

The fibroblast growth factor receptor (FGFR) signaling pathway is involved in key biological processes such as proliferation, differentiation, and cell survival
.
The FGFR pathway is often activated
in a variety of cancers, including breast cancer.
Of the five FGFRs, FGFR1-4 has been reported to play an important role
in cancer progression.
In addition, FGFR1 and FGFR2 appear to be associated
with resistance to CDK4/6 inhibitors and endocrine resistance.
Mechanistic studies have shown that FGFR1 amplification activates PI3K/AKT and RAS/MEK/ERK signaling pathways
in endocrine-resistant breast cancer cells.
In addition, the FGFR pathway leads to drug resistance mainly through FGF2 amplification activation, rather than FGF3, FGF4, and FGFR1
.

5.
Activate the PI3K/AKT/mTOR pathway

The PI3K/AKT/mTOR signaling pathway is activated in approximately 30–40% of breast cancers, particularly in
the HR-positive subtype.
Abnormalities in this pathway are known to be a key factor in
resistance to endocrine therapy.
In addition, the association
of the PIK3/AKT/mTOR pathway with resistance to CDK4/6 inhibitors has recently been reported.
For example, in CDK4/6 inhibitor-resistant breast cancer cells, growth is more dependent on PI3K/AKT/mTOR signaling than ER signaling
.
In addition, it has also been noted in CDK4/6 inhibitor-resistant cell lines that phosphorylated RB and E2F reactivation may occur via pathways other than the CDK pathway, such as the mTOR pathway
.
Therefore, inhibition of mTORC1/2 can inhibit the expression of phosphorylated RB and E2F in CDK4/6 inhibitor-resistant cells, thereby restoring sensitivity
to CDK4/6 inhibitors.

6.
Lack of ER or PR expression

The main driver of cyclin D-CDK4/6 activity in breast cancer cells is hormone-mediated ER activation
.
Loss
of ER/PR expression was observed in a preclinical model resistant to abecilib.
In addition, in a small number of patient series, about seven out of three patients experienced loss of ER or PR expression before CDK4/6 inhibitor treatment and after disease progression
.
These data suggest that a subset of patients who develop resistance to CDK4/6 inhibitors may be associated with
changes in ER/PR levels.

7.
Immune mechanism

Immune-related pathways are associated
with the emergence of resistance to various anticancer drugs.
Overexpression
of interferon factors such as IFN-α and IFN-β has been reported in CDK4/6 inhibitor-resistant breast cancer cells.
In addition, in a preclinical study, CDK4/6 inhibitors were found to promote anti-tumor immune effects
.
Therefore, CDK4/6 inhibitors in combination with immunotherapy may play a role in overcoming resistance to CDK4/6 inhibitors

Third, the improvement strategy based on CDK4/6 inhibitor resistance

1.
Combined with other endocrine drugs

It has been reported that CDK4/6 inhibitors combined with endocrine therapy can overcome CDK4/6 inhibitor resistance
.
A Harvard Medical School study showed that switching to abemaciclib monotherapy or in combination with endocrine therapy after disease progression achieved a mPFS time
of 5.
4 months in patients who had previously received the CDK4/6 inhibitors palbociclib or ribociclib.
In addition, a phase II clinical study compared the effects of
receiving switching endocrine therapy ribociclib after CDK4/6 inhibitor progression.
The researchers found that PFS was extended by 2.
5 months
in the combination treatment group compared to the monotherapy group.
Therefore, switching to other endocrine drugs combined with CDK4/6 inhibitors is one of
the options after drug resistance.

2.
Combined with targeted anti-cancer drugs such as PI3K inhibitors and mTOR inhibitors

PIK3CA is one of
the most commonly mutated genes in breast cancer.
Approximately 40% of patients with HR+ and HER2−ABC have PIK3CA mutations
.
PIK3CA mutations promote endocrine resistance by activating the PI3K pathway, which is associated with
poor prognosis.
As a α-selective PI3K inhibitor, alpelisib was the first to demonstrate its efficacy
in the SOLAR-1 study.
In the SOLAR-1 study in patients with HR+/HER2-ABC PIK3CA mutations who progressed during or after AI (aromatase inhibitor) therapy, the alpelisib + fulvestrant group had a 5.
3 month longer PFS time and a 7.
9 months longer median overall survival (mOS
) compared with placebo + fulvestrant group 。 Therefore, endocrine therapy combined with PI3K inhibitor therapy can be an option
for patients with PIK3CA mutations who have disease progression after endocrine therapy combined with CDK4/6 inhibitor therapy.

The mTOR pathway is an important signaling pathway downstream of PI3K, so mTOR inhibitors are of the same concern
as PI3K inhibitors.
Everolimus is a representative of mTOR inhibitors, the effect of which has been confirmed
in BOLERO-2 studies.
In the BOLERO-2 study, the mPFS and mOS time was significantly longer in the everolimus + exemestane (SAI) group than in the placebo + exemestane group
in patients with disease progression after NSAI therapy.
Therefore, mTOR inhibitors appear to be a good option
for patients who are resistant to CDK4/6 inhibitors.

3.
Combined with chemotherapy drugs

Chemotherapy is also a good option
for patients with HR+/HER2− ABC who are resistant to endocrine therapy + CDK4/6 inhibitors.
In clinical practice, chemotherapy is usually the choice of follow-up regimen
.
One study showed that more than one-third of the 525 patients who developed disease progression after treatment with CDK4/6 inhibitors received follow-up chemotherapy with capecitabine and taxanes
.
Three clinical trials (NCT04251169, NCT03901339, and NCT04134884) are currently underway to evaluate the efficacy
of chemotherapy in patients with ER+/HER2- breast cancer following CDK4/6 inhibitor resistance.
Therefore, chemotherapy remains a good treatment option
in patients with HR+/HER2- ABC who are resistant to endocrine therapy + CDK4/6 inhibitors.

4.
PROTAC strategy

PROTAC technology offers significant advantages
in overcoming drug resistance and targeting non-druggable targets.
PROTAC is a bifunctional molecule that, instead of inhibiting the function of the target, plays a therapeutic role
by hijacking the ubiquitin protease system (UPS) to induce degradation of the entire pathogenic target protein.

Due to the redundant role of CDK4/6 in the cell cycle, simultaneous silencing of CDK4/6 is required to achieve G1/S stagnation
.
Since CDK4/6 has similar structural characteristics and common ligands, PROTAC targeting CDK4/6 can degrade both kinase proteins
simultaneously.
Kevin Burgess et al.
reported the first potent CDK4/6 degrader, Pal-pom
, in 2019.
Pal-pom is a CDK4/6 PROTAC degrader based on pomalidomide and palbocilib (Figure 5).

The study found that Pal-pom can effectively degrade the MDA-MB-231 breast cancer cell lines CDK4 and CDK6 at low doses, with DC50 values of 13 nmol/L and 34 nmol/L, respectively, and Pal-pom can reduce Rb phosphorylation levels in a dose-dependent manner
.

Figure 5.
Chemical structure based on CDK4/6 PROTAC Pal-pom

Fourth, the research progress of CDK4/6 inhibitors

To date, a total of four CDK4/6 inhibitors have been approved for marketing worldwide (Figure 6).

They are Palbociclib developed by Pfizer, Ribociclib from Novartis and Abeciclib
from Eli Lilly.
Notably, all three drugs are administered orally and are currently approved for the treatment of
patients with advanced HR-positive breast cancer.
In addition, trilaciclib, a CDK4/6 inhibitor developed by G1 Therapeutics, is administered intravenously and is approved to reduce chemotherapy-induced bone marrow suppression
in patients with small cell lung cancer (SCLC).

Figure 6.
Summary of CDK4/6 inhibitors on the market

At present, there are also a number of CDK4/6 inhibitors in the clinical trial stage, among which Jiangsu Hengrui Pharmaceutical's SHR6390 is in clinical phase III, leading the domestic CDK4/6 track
.
In addition, pharmaceutical companies such as Beta Pharmaceuticals and Amgen are deploying in the CDK4/6 field (Figure 7).

Figure 7.
CDK4/6 inhibitor in clinical trials

V.
Summary

Over the past few decades, drug development based on breast cancer has made great progress
.
Targeting the cell cycle mechanism has emerged as an attractive strategy for the treatment of cancer, showing positive results
in both the preclinical and clinical phases.
At present, three CDK4/6 inhibitors have been approved for the treatment of HR+/HER2-breast cancer patients
worldwide.
However, resistance to these drugs inevitably emerges during clinical use, limiting the therapeutic effect
of these drugs.
At present, the drug combination strategy and PROTAC strategy have largely overcome the drug resistance problem
of CDK/6 inhibitors.
It is believed that with the continuous solution of CDK4/6 inhibitor drug resistance, adverse reactions and other issues, more CDK4/6 inhibitors will be successfully developed and marketed in the future, which is expected to rewrite the treatment pattern of breast cancer and benefit
breast cancer patients.

References:

1.
Targeting CDK4 and CDK6 in cancer Nat Rev Cancer, 2022, 22, 356–372
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CDK4/6 inhibitor resistance mechanisms and treatment strategies.
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3.
Targeting CDK4/6 for Anticancer Therapy.
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3390/biomedicines10030685

4.
PROTACs suppression of CDK4/6, crucial kinases for cell cycle regulation in cancer.
Chem.
Commun.
, 2019,55, 2704-2707.
doi.
org/10.
1039/C9CC00163H

5.
CDK4/6 inhibitors in HER2-positive breast cancer.
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