Targeting deubiquitinases in CLL

In this issue of Blood, Agathanggelou et al demonstrate that inhibition of ubiquitin-specific protease 7 (USP7) compromises homologous recombination DNA repair in part by destabilizing the E3 ligase RAD18 leading to the accumulation of DNA damage which kills chronic lymphocytic leukemia (CLL) cells independently of ataxia telangiectasia mutated (ATM) and p53.¹ 

Deregulation of the ubiquitin-proteasome pathway is common in cancer. USP7 is a deubiquitinase that removes ubiquitin groups from target proteins to protect them from proteosomal degradation. The role of USP7 gained prominence when it was identified as a novel regulator of the tumor suppressor protein p53 based on its ability to deubiquitinate and stabilize both p53 and its negative regulators Mdm2 and MdmX. In addition, it also antagonized the stability of FOXO4, phosphatase and tensin homolog, and TIP60, a p53-activating acetyltransferase while facilitating the function of oncogenic proteins such as claspin.²  These observations made USP7 a therapeutic target in cancer and initiated efforts to develop small-molecule inhibitors of USP7.² 

CLL occurs due to the accumulation of slow-growing nonfunctional B lymphocytes that show enhanced survival. A hallmark of CLL is the presence of recurring chromosomal aberrations involving chromosomes 11q and 17p that result in the loss of 1 allele of the ATM and TP53 gene, respectively.³  Loss of ATM or p53 function results in impaired DNA-damage proapoptotic response and may in part contribute to the shortened responses to chemotherapy, clonal evolution, and inferior survival observed in CLL patients bearing these adverse prognosis markers.^3,4  In addition to sensing DNA damage, ATM and p53 also play key roles in repairing DNA damage via the homologous recombination repair (HRR) pathway.⁵ 

In this issue, Agathanggelou et al observed that the USP7 deubiquitinase was overexpressed in CLL compared with normal donors. Inhibiting USP7 induced cytotoxicity in both quiescent and proliferating CLL cells regardless of their del11q or del17p status in vitro and in vivo using CLL cell line–derived xenograft mouse models. Mechanistically, inhibition of USP7 decreased the levels of RAD18, an E3 ligase necessary for the recruitment of the DNA repair proteins FANCD2 and RAD51 to sites of damage and successful HRR. Correspondingly, the loss in RAD18 levels was associated with loss of RAD51 foci and an increase in the levels of DNA damage marker γ-H2AX. In addition to suppressing HRR, inhibition of USP7 also led to hyperactivation of poly ADP-ribose polymerase 1 (PARP1), which activates apoptotic DNA fragmentation, depletes nicotinamide adenine dinucleotide (NAD⁺); leading to a loss of ATP, and activates necrotic cell death (as modeled in Figure 6 of the Agathanggelou et al article). Finally, USP7 inhibition synergized with DNA-damaging agents such as mitomycin C (MMC) as well as cyclophosphamide in vitro and in vivo.

These results are intriguing because they identify a p53-independent role for USP7 in regulating the HRR pathway by targeting the RAD18 E3 ligase. However, HRR is likely to be active only in the smaller proliferating fraction of CLL cells, which are located in protected niches within the bone marrow or secondary lymphoid tissue and are inactive in the quiescent fraction that makes up the bulk of circulating CLL lymphocytes⁶  raising additional questions that remain to be investigated. (1) Does USP7 regulate hitherto unknown proteins in the quiescent population of CLL cells such that its inhibition is sufficient to kill these cells? (2) In this study, USP7 inhibition synergized with the chemotherapeutic agents MMC and cyclophosphamide. DNA damage caused by these agents is repaired primarily by nucleotide excision repair in addition to HRR.⁷  If that is the case, does USP7 inhibition compromise nucleotide excision repair? (3) USP7 inhibition led to the hyperactivation of PARP and PARylation in their study. PARylation is known to regulate nonhomologous end joining (NHEJ) and base excision repair and to a lesser extent in HRR.⁸  Again, does USP7 inhibition compromise NHEJ and base excision repair? (4) USP7 inhibition was effective in targeting ibrutinib-relapsed cells. Presence of mutations in Bruton tyrosine kinase or its downstream intermediary drives ibrutinib resistance,⁹  whereas presence of a complex karyotype and Del17p are predictive of progression while on ibrutinib.¹⁰  Additional work in larger cohorts of samples may be required to address whether USP7 inhibition would be sufficient to target disease in this subgroup of patients.

In conclusion, despite unresolved queries, this study identifies that USP7 is an attractive therapeutic target in CLL and warrants its investigation both mechanistically and in clinical trials.