When my supervisor suggested me to work on the deubiquitinase USP28 as a 'simple project with quick results', I had no idea that USP28 would accompany me throughout my entire PhD journey. The project began already earlier in our lab with Viola Close's groundbreaking finding about FBXW7 mutations in chronic lymphocytic leukemia (CLL) which cause pathogenic NOTCH1 signaling activity [1]. Her findings sparked our curiosity about other mechanisms that might regulate NOTCH1 signaling in this common blood cancer.

What are activating and inhibiting pieces in the NOTCH1 puzzle in CLL?

NOTCH1 signaling plays a crucial role in CLL pathogenesis. Approximately 10% of CLL patients harbor activating NOTCH1 mutations, associated with poor outcomes and disease progression. However, NOTCH1 activation is found in about 50% of CLL cases, meaning many patients show active NOTCH1 signaling without having NOTCH1 mutations [2-4]. This gap in our understanding led us to investigate alternative mechanisms.

Previous research had established that the E3-ubiquitin ligase FBXW7 targets cleaved NOTCH1 (NICD) for degradation [5]. Mutations in FBXW7 can impair this degradation, leading to NOTCH1 accumulation [1]. But what about the opposite process? This is where USP28 enters the stage. As a deubiquitinase, USP28 counteracts FBXW7-mediated protein degradation by removing ubiquitin from target proteins like NICD, leading to protein stabilization [6-7].

Intriguingly, the USP28 gene is located on chromosome 11, in a region frequently deleted in CLL [8]. This deletion, del(11q), is found in approximately 10% of early-stage CLL patients and is associated with poorer outcomes [9]. Our investigation revealed that USP28 is deleted in 90% of del(11q) CLL cases. This led to our hypothesis: Could USP28 loss with del(11q) affect NOTCH1 regulation in CLL?

USP28 deletion downregulates NOTCH1 signaling in CLL

Our research journey from using molecular biology techniques like western blotting and quantitative real-time PCR to RNA sequencing and co-immunoprecipitations led us to several key discoveries. First, we confirmed that USP28 stabilizes NICD protein levels and increases NOTCH1 activity. In experiments with cell lines and patient samples, USP28 overexpression protected NICD from degradation, while USP28 knockout decreased NICD stability.

Next, we observed that del(11q) CLL patients displayed significantly decreased USP28 protein levels. This correlated with lower NICD levels in many patient samples and reduced NOTCH1 signaling activity in del(11q) CLL cell lines.

Surprisingly, this meant that del(11q), which promotes CLL progression, actually downregulates NOTCH1 signaling. This contradicted our initial expectations and revealed a more complex picture of CLL biology. Del(11q) must drive CLL progression through other mechanisms that compensate for reduced NOTCH1 signaling, possibly through NF-κB activation via BIRC3 deletion or defective DNA damage response via ATM deletion [10].

USP28 is the conductor of a molecular orchestra

Our view of the bigger picture of USP28’s role in CLL broadened when it became clear that USP28 doesn't operate in isolation but acts as a conductor in a molecular orchestra. It interacts with multiple partners beyond NICD, including c-MYC, FBXW7, HIF-1α, and ATM, influencing various cellular pathways relevant to CLL and other cancer entities [11].

In addition, the relationship between USP28 and its targets has been proven context-dependent in earlier studies which showed that different USP28 dosages have different effects on target protein stability [12]. On top, USP28 is regulated by ATM-mediated phosphorylation, in connection with DNA damage response pathways [13]. Importantly, ATM is also affected by del(11q) in CLL and we could show that in del(11q) cell lines with additional ATM knockout NOTCH1 activity was further decreased. Which can be explained by the fact that without ATM, USP28 cannot be phosphorylated and activated, further impairing its ability to stabilize NICD.

Translating this complex interplay of USP28 with its interaction partners to the metaphor of USP28 as the conductor of a molecular orchestra, its different interaction partners like NOTCH1, c-MYC, FBXW7 and HIF-1α are the instruments. The cellular context represents the musical score, and ATM is the composer giving instructions. In the case of del(11q) the conductor is suddenly removed, and the instruments are not playing anymore. 

Therefore, we were wondering whether we could help CLL patients with pathogenically active NOTCH1 signaling by muting its conductor USP28 with pharmacological USP28 inhibition.

USP28 inhibition can be a novel therapeutic strategy in CLL

To test the novel therapeutic approach of targeting USP28 to inhibit NOTCH1 signaling in CLL we used the USP28 inhibitor AZ1. Our results demonstrated decreased NICD protein levels, NOTCH1 target gene expression, and cell viability in primary patient cells after USP28 inhibition.

Excitingly, combination of AZ1 with the clinically used BCL-2 inhibitor venetoclax showed additive effects on reduced CLL cell viability, particularly in cells with high NICD levels or NOTCH1 mutations. This combinatory effect could be especially beneficial for the treatment of NOTCH1-mutated patients, who typically respond poorly to standard therapies.

The targeting of USP28 offers a unique advantage - rather than directly inhibiting NOTCH1 (which has proven clinically challenging), this approach exploits the regulatory machinery controlling NOTCH1 stability. Moreover, since USP28 influences multiple oncogenic pathways, its inhibition might affect several cancer-driving mechanisms simultaneously.

Embracing complexity

What began as a supposedly straightforward project evolved into a complex investigation of molecular networks in cancer which we still haven’t elucidated completely. This experience taught me the importance of thinking beyond linear pathways and embracing the intricate webs of interaction that characterize cancer biology.

The USP28 story exemplifies how multiple regulatory layers operate in CLL, from genetic deletions and mutations to posttranslational modifications and protein-protein interactions. Understanding these networks presents challenges but also opportunities for the development of more effective targeted therapies.

USP28, which was supposed to be my 'quick win', had become the thread running through my entire doctoral research, which taught me valuable lessons about scientific discovery and persistence. 

And in a fitting conclusion to my PhD journey, our manuscript was accepted for publication on the very day of my graduation ceremony! 

Sometimes, the most illuminating scientific journeys arise from the supposedly 'simple projects' that reveal unexpected complexity. My USP28 PhD project pushed me to think more deeply and discover more broadly than I initially imagined. 

I realized that the symphony changes when the conductor is missing.

References:

[1] Close V, Close W, Kugler SJ, Reichenzeller M, Yosifov DY, Bloehdorn J, et al. FBXW7 mutations reduce binding of NOTCH1, leading to cleaved NOTCH1 accumulation and target gene activation in CLL. Blood. 2019;133(8):830-839.

[2] Fabbri G, Rasi S, Rossi D, Trifonov V, Khiabanian H, Ma J, et al. Analysis of the chronic lymphocytic leukemia coding genome: role of NOTCH1 mutational activation. J Exp Med. 2011;208(7):1389-401.

[3] Rosati E, Baldoni S, De Falco F, Del Papa B, Dorillo E, Rompietti C, et al. NOTCH1 aberrations in chronic lymphocytic leukemia. Front Oncol. 2018;27:8:229.

[4] Fabbri G, Holmes AB, Viganotti M, Scuoppo C, Belver L, Herranz D, et al. Common nonmutational NOTCH1 activation in chronic lymphocytic leukemia. Proc Natl Acad Sci U S A. 2017;114(14):E2911-E2919.

[5] Babaei-Jadidi R, Li N, Saadeddin A, et al. FBXW7 influences murine intestinal homeostasis and cancer, targeting Notch, Jun, and DEK for degradation. J Exp Med. 2011;208(2):295-312.

[6] Diefenbacher ME, Popov N, Blake SM, Schülein-Völk C, Nye E, Spencer-Dene B, et al. The deubiquitinase USP28 controls intestinal homeostasis and promotes colorectal cancer. J Clin Invest. 2014;124(8):3407-18.

[7] Diefenbacher ME, Chakraborty A, Blake SM, Mitter R, Popov N, Eilers M, et al. Usp28 counteracts Fbw7 in intestinal homeostasis and cancer. Cancer Res. 2015;75(7):1181-6.

[8] Valero R, Bayés M, Sánchez-Font MF, González-Angulo O, González-Duarte R, Marfany G. Characterization of alternatively spliced products and tissue-specific isoforms of USP28 and USP25. Genome Biol. 2001;2(10):RESEARCH0043.

[9] Hallek M, Al-Sawaf O. Chronic lymphocytic leukemia: 2022 update on diagnostic and therapeutic procedures. Am J Hematol. 2021;96:1679–1705.

[10] Quijada-Álamo M, Hernández-Sánchez M, Rodríguez-Vicente A-E, Pérez-Carretero C, Rodríguez-Sánchez A, Martín-Izquierdo M, et al. Biological significance of monoallelic and biallelic BIRC3 loss in del(11q) chronic lymphocytic leukemia progression. Blood Cancer J. 2021;11(7):127.

[11] Prieto-Garcia C, Tomašković I, Shah VJ, Dikic I, Diefenbacher M. USP28: Oncogene or tumor suppressor? A unifying paradigm for squamous cell carcinoma. Cells. 2021;10(10):2652.

[12] Schülein-Völk C, Wolf E, Zhu J, Xu W, Taranets L, Hellmann A, et al. Dual regulation of Fbw7 function and oncogenic transformation by Usp28. Cell Rep. 2014;9(3):1099-109.

[13] Zhang D, Zaugg K, Mak TW, Elledge SJ. A role for the deubiquitinating enzyme USP28 in control of the DNA-damage response. Cell. 2006;126(3):529-42.