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Multiple myeloma therapeutic target: CRL4 E3 ligase

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Source: Yaodu Written by: Sunset Graffiti Editor: Maruzi
multiple myeloma (MM) is the second most common hematologic malignancy and is prone to recurrence
in clinical symptoms.
The introduction of immunomodulatory drugs (IMiDs) can be said to be an important milestone
in the treatment of multiple myeloma that can significantly improve the prognosis of patients.
Currently, IMiDs is the basis for
newly diagnosed and relapsed/refractory myeloma treatment.

Studies have shown that IMiDs exert their anti-myeloma activity
primarily by binding to the substrate receptor protein Cereblon (CRBN) of the CRL4 E3 ubiquitin ligase (CRL4 CRBN⁾ complex.
By binding to CRBNs, IMiDs alter the properties of their substrates, leading to ubiquitination and proteasome degradation of proteins essential for myeloma cell survival
.

With the success of IMiDs, this has led many scholars to further explore
the possibility of treating myeloma using the activity of CRL4 CRBN complex.
In this paper, the current state of research on CRBN E3 ligase modulators (CELMODs), a new generation of IMIDs
with broader biological activity, is explored.
In addition, we discuss a novel targeted proteolysis strategy, targeted proteolytic chimeras (PROTACs), which utilize CRL4-CRBN to degrade undruggable proteins
that may be relevant for the treatment of myeloma and other malignancies.

Background

The ubiquitin-proteasome pathway plays a crucial role
in protein degradation.
This process is mediated by a series of enzymatic reactions involving ubiquitin-activating enzymes (E1), ubiquitin-binding enzymes (E2), and ubiquitin ligases (E3), which are cycled and activated by ATP throughout the ubiquitination pathway, where E3's role is to determine substrate specificity
for ubiquitination and subsequent degradation in the proteasome.

Among the more than 600 E3 ubiquitin ligases encoded in the human genome, cullin-ring ubiquitin ligases (CRLs) are the largest family of E3 ligases, involved in many cellular homeostatic processes, such as signal transduction, cell cycle regulation, DNA damage response, transcriptional regulation, and embryonic development
。 CRL4 E3 ubiquitin ligase is a complex consisting of zinc finger domain protein (ROC1, also known as RBX1), Cullin4 (Cul4) scaffold protein, and DDB1-Cul4-associated protein, which determines the substrate specificity
of CRL4 E3 activity.
Cerablon (CRBN) is one of the CRL4 E3 substrate receptors and is essential
for the action of small molecules such as immunomodulatory drugs.
Targeting CRBNs by IMiDs changes their substrate specificity for non-physiological proteins that can then be recognized and degraded
by the corresponding proteasomes.
(Figure 1A)
This mechanism of action has shown particular relevance in the treatment of multiple myeloma (MM), so the introduction of IMiDs has changed the traditional treatment of MM patients and significantly improved their prognosis
.

While E3 ligase-related functions acting on CRBNs appear to be the primary mechanism of IMiDs anti-myeloma activity, recent reports suggest that IMiDs also function by regulating other properties of CRBNs, such as chaperone function
.
Therefore, to emphasize a wider range of biological activity, the next generation of immunomodulators is called "celmod" (Cereblon E3 ligase modulator).

In recent years, CRL4 CRBN complexes and other E3 ligases have been widely explored as targets for the degradation of typical "undruggable" proteins by heterobifunctional small molecules, called proteolytic targeting chimeras (PROTACs).

This article mainly introduces a method
to regulate CRL4 CRBN E3 ligase activity in a ^{CRBN-dependent}manner in future treatments for multiple myeloma.

Figure 1 Research progress on ubiquitination of CRL4^CRBNE3 ligase complex

Immunomodulatory drugs (IMiDs)

1.
The mechanism of action of IMiDs Thalidomide introduced in 2006 is the first class of
immunomodulators and is one of
the milestones in MM treatment.
Together with its new generation of derivatives, such as lenalidomide and pomalidomide, as well as proteasome inhibitors and monoclonal antibodies, these drugs are used to treat MM patients
.
Before elucidating their mechanism of action, thalidomide and its analogues were mainly thought to regulate the function
of T cells, NK, and NK-T cells by inducing the production of cytokines such as IL-2 (interleukin) and interferon-γ.
Later, it was found that thalidomide and its analogues are called immunomodulatory drugs
in addition to their anti-angiogenesis, destruction of myeloma cell-bone marrow matrix interactions and reduction of osteoclast production.
Subsequent studies showed that CRBN expression is required for IMiDs anti-myeloma activity, as the study found that knockout of the CRBN gene led myeloma cells to develop resistance to lenalidomide and pomalidomide
.

In 2014, researchers first elucidated the key mechanism
of immunomodulators.
Two studies showed that the interaction of lenalidomide with CRBNs altered its substrate specificity, thereby inducing proteasome-dependent degradation
of the transcription factor IZKF1/3.
IKZF1 and IKZF3 are defined as "new substrates" for CRBNs because they are targets for CrBN only in the presence of IMIDs
.
The degradation of IKZF1/3 regulates the expression of other genes such as IRF4 and MYC, which is essential
for the proliferation and survival of MM cells.
The disruption of the IKZF1/3-IRF4-MYC transcription axis plays a key role in MM cell survival, contrary to the study of primary effusion lymphoma cell lines, in which IMiDs trigger the expression downregulation
of IRF4 independently of IKZF1 and IKZF3.

Recent studies have reported multiple potential new substrates for IMIDs that need to be validated under physiological conditions and converted to IMIDs for clinical applications
.
Under the action of IMiDs, CRL4 CRBNE3 ligase can interact
with new substrates.
Differences in the new substrate repertoire in IMiDs activity decline may reflect various adverse events
observed during MM treatment.
The most common side effect of thalidomide is chronic axonal neuropathy, while other drugs characterized by bone marrow suppression are the most common toxicity
.
The myelosuppressive effects of lenalidomide and bomalidomide refer to the degradation of IKZF1 and the subsequent downregulation of transcription factors PU.
1 and GATA1, which lead to neutropenia and thrombocytopenia
, respectively.
Since thalidomide has a much lower degradation effect on IKZF than thaladomide and bomalidomide, it may not cause this toxicity
as much as its new derivatives.

In addition, IMiDs can also regulate the activity of CRL4 CRBNE3 ligase to degrade various proteins
with different affinities for specific new substrates.
Unique patterns of substrate specificity may translate into diversity in the clinical efficacy and toxicity profiles of these drugs
.

2.
2.
Clinical efficacy
of IMiDs Although the role of thalidomide in the treatment of MM has been steadily declining since the introduction of lenalidomide, the combination of thalidomide, dexamethasone, and bortezomib (VTD) remains a key approach for patients with undiagnosed MM who are eligible for high-dose chemotherapy followed by autologous stem cell transplantation (Auto-HSCT)
in many countries where lenalidomide is in short supply 。 More recently, VTD induction prior to Auto-HSCT (thalidomide plus vincristine V and Pyrorubicin T and dexamethasone D regimens) has been shown to exert an effective response (at least partial response to partial remission) in nearly 95% of patients, confirming previously reported results
.

As shown in the recent Phase 3 Cassiopeia trial, the clinical benefits of VTD in terms of depth of response, measurable residual disease (MRD) negative rate, and progression-free survival (PFS) can be further enhanced by the addition of Daratumumab, a state-of-the-art monoclonal antibody against CD38, That is, antigens
that are normally expressed on the surface of MM cells.

The treatment of relapsed/refractory MM is a major challenge
in clinical practice.
For patients who have not been previously exposed to lenalidomide, lenalidomide alone (especially in frail patients), or in combination with carfilzomib (Aspire test), Ixazomib (tourmaline test), daratumumab (pollox test), or Elotuzumab (Eloquent-2 test) are highly relevant treatment options
.

Conversely, for lenalidomide-refractory patients, in addition to IMID-free regimens such as DKD [CANDOR trial], DVD [Castor trial], and KD [Endeavor trial], polalidomide-based regimens are also clinically important
。 Depending on previous treatment, presentation, and comorbidities, patients with relapsed/refractory MM may be used alone or in combination with anti-CD38 antibodies (i.
e.
, dalatumab (Apollo test) or isathoximab [ICARIA-MM test], ilotuzumab (Eloquent-3 test), proteasome inhibitors (i.
e.
, bortezomib [OptimisMM test] [65], carfilzomib [66], and isazomib, and cytotoxic agents (eg, cyclophosphamide), But ultimately this depends on the patient
with relapsed/refractory MM.
The results of randomized clinical trials of immunomodulators are summarized in
(Table 1).
Table 1.
Summary of results from IMIDS randomized clinical trials

CRBN E3 ligase regulator

（CELMODs）

Although the mechanism of action between immunomodulatory drugs (IMiDs) has not been fully elucidated, studies have found that the second extended region of the structure of CELMODs (corresponding to the phthalimide ring in thalidomide) varies between each CELMoD and determines the relationship with CRBNs and the new CRL4 ^{The interaction of CRBN}E3 substrates is shown
in Figure 2A, B.
Figure 2.
Differences
in chemical structure and degradation capacity of IMIDs/CELMoDs.

One of the key features that distinguish CELMoDs from IMiDs is that CELMoDs enhances affinity for
CRBNs.
Nevertheless, the CRBN affinity of CELMoDs is approximately 10-20 times
higher compared to lenalidomide or bomalidoxamine.
A stronger potency of CELMODs in IKZF1 and IKZF3 degradation was observed compared to classical IMiDs (Figure 2C).

Figure 2.
Comparison of degradation efficacy of IMIDs and CELMODs in IKZF: 3.
2 Clinical efficacy
of CELMODS (1) CC-92480 (mezigmide).

Preliminary results
from the Phase 1/2 CC-92480-MM-002 study have recently been reported.
A total of 19 patients with relapsed/refractory MM received a combination of CC-92480 (mezidomide), bortezomib, and dexamethasone after the previous treatment median of 3 (range, 2-4) lines
.

(2) Iberdoamine (CC-220) According to the preliminary results of the phase 1/2 CC-220-MM-001 study (NCT02773030), the triple use of iberdoamine (CC-220)
Good safety and clinical activity
have been shown in patients with MM with severe pretreatment.
The Iberdomiddaratumab-dexamethasone (IBERDD) cohort included 63% and 58% of patients with DaratumumaMability and quadruple refractory (defined as refractory
to ≥ 1 imine, 1 PI, 1 anti-CD38 monoclonal antibody, and 1 steroid).
Similarly, in the iberdoamide-bortezomib-dexamethasone (IBERVD) cohort, a high proportion of refractory patients (PI refractory, 76%; Type 4 refractory, 48%)
.
However, objective response rates were 35% in the IBERDD group and 50%
in the IBERVD group, respectively.

Importantly, the reaction to Ibervd had nothing to do with
Daratumumab and Bortezomiberefrictory.
It is worth emphasizing that a large proportion of patients receive clinical benefit from iberdoamine-based therapy because minimal response or stable disease
is obtained.
Clinical benefit rates and disease control rates were 47% and 88% (IBERDD cohort) and 65% and 85% (IBERVD cohort),
respectively.
Cytopenia is the most common complication
of combination therapy.
IBERDD combination therapy for relapsed/refractory MM is planned to be compared
to DRD in the Phase 3 Excaliber-RRMM trial (NCT04975997).

In addition, IBERVD as a first-line treatment for MM patients who are not eligible for HDT-Auto-HSCT will be evaluated
in the Phase 2 Borealis trial (NCT05272826).
Ibertomide in combination with dexamethasone was evaluated
in a phase 1/2 CC-220-MM-001 study (NCT02773030).
Almost all of the 107 enrolled patients were triple-refractory (IMID, PI, and anti-CD38 monoclonal antibodies), 25% had extramedullary disease, and 30% had high-risk cytogenetics
.
Treatment with ebedomide and dexamethasone resulted in response in 26% of patients
.
The median PFS and OS were 3 months and 11 months
, respectively.

Interestingly, patients who had previously received anti-BCMA therapy had similar response rates (ORR of 25%), and no new concerns emerged
about the toxicity of combination therapy 。 The efficacy and safety of iberdomib-dexamethasone in combination with other anti-myeloma drugs such as carfilzomib (NCT05199311, NCT02773030), isazomib (NCT04998786), cyclophosphamide (NCT04392037) and idecabtagene Vicelel (Karmma-7 trial, NCT04855136) is currently being investigated
in several early-stage studies.

Target proteolytic chimeras

（PROTACs）

Selective protein degradation technology is a therapeutic strategy with great clinical potential, and this treatment method is not only suitable for MM patients
.
The PROTAC strategy is to recruit E3 ubiquitin ligases to ubiquitinate and degrade
the targeted proteins (POIs).
In recent years, the development of PROTAC degraders has been promoted with the extensive study of the mechanism of
ubiquitination.
The PROTAC molecule consists of three sites:
(1) a small molecule compound
that binds specifically to the protein of interest.
(2) A compound
that binds specifically to E3 ubiquitin ligase.
(3) A linkage chain linking a target protein ligand and an E3 ubiquitin ligand
.

PROTAC technology does not require high binding to the protein of interest and therefore offers great advantages
in overcoming the potential limitations of classical small molecule protein inhibitors (transient targeting of non-covalent inhibitors, resistance due to protein overexpression or point mutation).
This new strategy can effectively target "undruggable" targets, such as key oncoproteins
.

Figure 3.

Currently, most PROTACs use CRL4 CRBN and VHL, two E3 ubiquitin ligases, as recruitment ligases
.
As a result, IMIDs are often considered precursors to the "molecular glue" portion of PROTAC because they facilitate the discovery
of CRBN interactions with many new substrates associated with treatment.

In 2015, the first CRBN-based PROTAC targeting the degradation of BRD4 was developed, with thalidomide as the CRBN E3 ubiquitin ligand and the bromine domain as the target protein
.
The results showed that DBET1 induced highly selective CRBN-dependent BET protein degradation
in MM and AML cell lines.
The next generation of PROTACs based on CRL4 CRBN-polymaridomide interaction also targeted BET protein (ARV825), which showed strong inhibitory activity against MM cells and also showed good inhibitory effect
in mouse transplanted tumor models.
In addition, the researchers have developed potent PROTACs
against other promising oncoproteins for MM, such as CDK4 and CDK6 and MCL-1.

Of note, acquired resistance to MM cells is inevitable during treatment with IMiD or CELMoD, which may result in altered CRBN expression and genetic mutations encoding CRBNs to affect the efficacy of CRL4 CRBNE3 ubiquitin ligase-based PROTACs
。 Fortunately, the human genome encodes more than 600 E3 ubiquitin ligases
.
So far, only a few have been used for the generation of PROTACs: VHL, MDM2, IAP (Apoptotic Protein Inhibitor), and CRBN.

In August 2022, clinical trials of ARV-110 (Adrent, NCT0388861) and ARV-471 (Veritac, NCT04072952) are undergoing Phase II trials
.
In the field of hematology, the first oral BTK degrader with IMiD-like activity (NX-2127) is currently recruiting patients with relapsed/refractory B-cell malignancies (NCT04830137).

Similarly, another IMID-based BTK degrader (NX-5948) has entered Phase 1 trials in patients with relapsed/refractory B-cell malignancies, including primary central nervous system lymphoma (NCT05131022).

Recently, the same IMID-based STAT3 degrader (KT-333) was approved for the treatment of adult refractory B-cell non-Hodgkin lymphoma, T-cell lymphoma, and solid tumors and is in Phase I clinical trials
.

brief summary

The introduction of IMIDS has revolutionized the outlook for multiple myeloma treatment and has significantly improved MM outcomes
in combination with anti-cancer therapies.
The discovery of thalidomide from teratogenic "dark drugs" to the first class of IMID, and the extensive study of its mechanism of action, provides us with a new perspective
on the precise and re-understanding of the protein ubiquitination process.
Although IMIDs are one of the most important drugs in the treatment of MM, indications for IMIDs are also expanding to other malignancies, such as hematologic malignancies
.
For example, lenalidomide is effective
in recurrent mantle cell lymphoma, follicular lymphoma, and marginal lymphoma.
It is expected that more IMIDs will be approved for marketing and bring good news
to patients.

References

[1] Cullin-RING E3 Ubiquitin Ligases: Bridges to Destruction.
Macromol.
Protein Complexes 2017, 83, 323–347

[2] The Myeloma Drug Lenalidomide Promotes the Cereblon-Dependent Destruction of Ikaros Proteins.
Science 2014, 343, 305–309.

[3] Cereblon loss and up-regulation of c-Myc are associated with lenalidomide resistance in multiple myeloma patients.
Haematologica 2018, 103, e368–e371.

[4] Rapid Progress in the Use of Immunomodulatory Drugs and Cereblon E3 Ligase

Modulators in the Treatment of Multiple Myeloma.
Cancers 2021, 13, 4666.

[5] CRL4 E3 Ligase Complex as a Therapeutic Target in Multiple Myeloma.
[J] .
Cancers (Basel), 2022, 14: undefined.

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.
This article does not constitute any investment advice and is not a recommendation
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