When the Cellular Orchestra Loses Its Conductor
Exploring how dysregulated NOTCH1 signaling drives Chronic Lymphocytic Leukemia progression through FBXW7 mutations and USP28 regulation
Imagine your body's cells as a sophisticated orchestra, playing the precise rhythms of life through carefully coordinated signals. Now picture what happens when a crucial conductor goes missing—the music descends into chaos. This is precisely what occurs in chronic lymphocytic leukemia (CLL), where the harmonious regulation of cellular signals gets disrupted, particularly in the case of the NOTCH1 signaling pathway.
CLL represents the most common leukemia in adults in Western countries, characterized by the gradual accumulation of abnormal B-lymphocytes 9 .
NOTCH1 protein is synthesized in the endoplasmic reticulum and undergoes S1 cleavage by furin convertase in the Golgi apparatus 1 8 .
The mature NOTCH1 receptor travels to the cell surface, awaiting activation signals from neighboring cells.
When ligands (JAG1 or DLL proteins) bind, sequential cleavages occur: S2 by ADAM10/17 metalloproteinases and S3 by the γ-secretase complex 8 .
In CLL, NOTCH1 signaling provides significant growth and survival advantages to cancerous B-cells 1 . The activated NICD translocates to the nucleus where it forms a complex with transcription factors and turns on genes that promote cancer progression, including MYC, a powerful driver of cell proliferation 5 .
What makes NOTCH1 particularly problematic in CLL is its dual role as both a prognostic and predictive marker. Patients with NOTCH1 activation typically experience more aggressive disease, shorter overall survival, and poorer response to anti-CD20 immunotherapy 1 9 .
FBXW7 (F-box and WD40 repeat domain-containing 7) acts as a cellular quality control manager, specifically targeting NICD and other regulatory proteins for degradation 4 6 .
FBXW7 functions as part of the SCF ubiquitin ligase complex that tags proteins with ubiquitin molecules, marking them for destruction by the proteasome 6 . The recognition of NICD by FBXW7 depends on a specific region called the CDC4-phosphodegron (CPD) motif within the PEST domain of NOTCH1 2 .
When FBXW7 is mutated—as occurs in approximately 4.5% of CLL patients—this quality control system breaks down 4 6 . The mutated FBXW7 cannot properly bind to NICD, leading to accumulated active NOTCH1 that drives cancer progression.
Mutation Frequency: ~4.5%USP28 (ubiquitin-specific protease 28) acts as FBXW7's counterbalance. This deubiquitinase enzyme removes ubiquitin tags from proteins, effectively rescuing them from proteasomal degradation 2 7 .
USP28's genetic location—the chromosome 11q23 region—is frequently deleted in CLL (approximately 10% of early-stage patients) 2 . Intriguingly, USP28 is deleted in a remarkable 90% of CLL cases with del(11q), leading to significantly reduced USP28 expression 2 .
This creates a complex interplay in CLL progression, with concurrent ATM deletion (impairing DNA damage response) and BIRC3 deletion (activating NF-κB signaling) also contributing to disease aggressiveness 7 .
Deletion Frequency: ~10%Tags NICD for degradation
Normal cellular signaling
Rescues NICD from degradation
Targeted next-generation sequencing of the FBXW7 gene in 905 CLL patients, identifying mutations in 4.5% of cases 4 .
Using in silico tools like PolyPhen-2 to predict how specific FBXW7 mutations would affect NOTCH1 binding 4 .
Co-immunoprecipitation experiments to directly examine protein-protein interactions 4 6 .
Using CRISPR/Cas9 gene editing to create CLL cell lines (HG-3) with specific genetic alterations 2 6 .
Western blotting, luciferase reporters, and viability tests to measure protein levels, NOTCH1 activity, and cell survival 2 .
Analysis of 285 CLL patients demonstrated that low USP28 expression correlated with increased NOTCH1 target gene expression, independent of 11q deletion status 4 .
This suggested additional regulatory mechanisms beyond chromosomal deletions control USP28 activity in CLL.
| FBXW7 Mutation | Effect on NOTCH1 Binding | Frequency in CLL Cohort |
|---|---|---|
| G423V | Significantly reduced | 3/905 cases |
| W425C | Significantly reduced | Recurrent |
| R465C/H | Reduced (known hotspot) | Known hotspot |
| R479Q/L | Reduced (known hotspot) | Known hotspot |
| R505C | Reduced (known hotspot) | Known hotspot |
| A503V | Minimal impact | Single case |
| T15V | Maintained binding | Single case |
Direct targeting of NOTCH1 signaling has proven clinically challenging. Early attempts using γ-secretase inhibitors showed limited success, with significant gastrointestinal toxicity and modest clinical efficacy in trials 9 .
The discovery that USP28 inhibition reduces NOTCH1 signaling opens promising therapeutic avenues 2 . Rather than directly targeting NOTCH1 itself—which might disrupt its physiological functions in healthy cells—modulating its regulators offers a more nuanced strategy.
The most exciting development comes from combination approaches. Studies revealed that pairing USP28 inhibition with the BCL-2 inhibitor venetoclax creates a synergistic effect against CLL cells, particularly those with NOTCH1 dysregulation 2 .
This combination strategy could be especially beneficial for NOTCH1-mutated patients, who typically respond poorly to standard therapies.
Additionally, researchers have observed that NOTCH1 mutations frequently co-occur with trisomy 12 (an extra copy of chromosome 12), found in approximately 30% of these cases 9 . This cooperation between genetic alterations suggests that NOTCH1 activation might work in concert with genes on chromosome 12 to drive leukemia progression.
USP28 inhibitor + Venetoclax shows synergistic effects against NOTCH1-dysregulated CLL cells.
| Therapeutic Approach | Mechanism of Action | Development Stage |
|---|---|---|
| γ-secretase inhibitors | Block S3 cleavage of NOTCH1 | Clinical trials (limited by toxicity) |
| Anti-NOTCH1 antibodies | Target extracellular domain | Preclinical studies |
| USP28 inhibitors (AZ1) | Promote NICD degradation | Preclinical studies |
| Combination therapy (AZ1 + venetoclax) | Dual targeting of NOTCH and BCL-2 | Preclinical studies |
Advancements in our understanding of NOTCH1 regulation in CLL depend on sophisticated research tools and experimental models. Here are essential components of the scientific toolkit that enabled these discoveries:
Freshly isolated lymphocytes from CLL patients, providing clinically relevant experimental systems 2 .
Technique to study protein-protein interactions, essential for demonstrating FBXW7-NOTCH1 binding 4 .
Small molecule that specifically inhibits USP28 deubiquitinase activity for therapeutic testing 2 .
The journey to unravel NOTCH1 signaling in CLL exemplifies how modern cancer research has evolved from simply identifying mutated genes to understanding complex regulatory networks. The discovery of the FBXW7-USP28-NOTCH1 axis reveals how CLL cells exploit multiple strategies to activate the same pathway—either through direct NOTCH1 mutations, impaired degradation via FBXW7 mutations, or potentially through altered regulation via USP28 4 5 .
This knowledge does more than satisfy scientific curiosity—it provides clinically actionable insights that could transform patient care. The potential to target NOTCH1 signaling through USP28 inhibition, particularly in combination with existing therapies like venetoclax, offers hope for more effective treatments for high-risk CLL patients 2 7 .
"Sometimes, the most illuminating scientific journeys arise from the supposedly 'simple projects' that reveal unexpected complexity." 7
Indeed, the symphony of NOTCH1 signaling continues to reveal its secrets, promising better therapeutic strategies for patients with chronic lymphocytic leukemia.