Here we describe a potential new synthetic lethal therapeutic approach for the treatment of AID-expressing B cell malignancies: attenuation of RAD51-mediated HR to inhibit repair of endogenous AID-generated DSBs, culminating in cytotoxicity. This approach uses inherent recombinase activity, rather than systemically administered genotoxic agents, to induce cell-lethal genomic damage. As such, this strategy represents a special case of synthetic lethal therapy. We establish the proof of principle, showing that AID-expressing human CLL cells are hypersensitive to DIDS, a RAD51 inhibitor. Using a combination of primary patient-derived CLLs and genetic mouse models, we establish that DIDS treatment inhibits repair of AID-mediated DSBs in AID-expressing, patient-derived CLL cells. These findings establish that HR attenuation may be a therapeutically viable approach to selectively targeted treatment of cancers (and other pathologies) involving AID-expressing cells. Because AID mRNA expression is found in a high percentage of primary CLLs, as well as other cancers, this therapeutic concept has potential to benefit a large number of patients.
Although CLL is manageable in many cases, up to half of CLL patients will require therapy at some point in their disease course (Yee and O’Brien, 2006
; Zent and Kay, 2011
). Conventional first-line therapy for CLL involves treatment with purine analogues such as fludarabine with or without other agents, especially chlorambucil and/or rituximab (Dighiero and Hamblin, 2008
). These combinations are reported to achieve complete remission in <40% of patients and usually for a duration of <3 yr before relapse with treatment refractory disease (Hallek, 2005
). Major side effects of purine analogues like fludarabine include severe hematologic events such as profound neutropenia or fatal pancytopenia; life-threatening autoimmune reactions; serious, opportunistic infections; widespread neurological effects, including peripheral neuropathy; and induction of secondary tumors, especially of the skin (Cheson et al., 1999
; Molica, 2005
; Robak and Robak, 2007
). Given that current chemotherapies for CLL and other indolent lymphoproliferative disorders are immunosuppressive and can cause devastating, long-term side effects without the promise of long-term durable remission, new treatment paradigms that target underlying mechanisms or disease-specific features are a clinical oncology imperative (Hallek, 2005
; Yee and O’Brien, 2006
; Dighiero and Hamblin, 2008
). Though our data suggest that AID-mediated cytotoxicity could result in reduction of the post-GC B cell pool, and thus a mild, transient immunosuppression, we postulate that this would be more tolerable than the widespread suppression of adaptive and innate immunity after treatment with conventional chemotherapeutics such as fludarabine. Future testing in preclinical models is needed to examine this paradigm.
Accumulating evidence has implicated AID as a functionally significant mutator enzyme in a range of human cancers, including CLL (Kou et al., 2007
; Klemm et al., 2009
; Palacios et al., 2010
; Shimizu et al., 2012
). Aberrant AID activity leads to point mutations or DSBs in cellular proto-oncogenes or tumor suppressor genes that can drive tumor formation (Okazaki et al., 2003
). The underlying cause of aberrant activation of AID in tumor cells is not yet understood, but some evidence suggests that persistent inflammatory signaling, perhaps caused by infection (Matsumoto et al., 2007
) or other stimuli (Morita et al., 2011
), can contribute to ectopic AID expression (Mechtcheriakova et al., 2012
; Shimizu et al., 2012
). We speculate that in normal cells and untreated tumor cells, efficient DNA repair systems mitigate the majority of AID-induced damage, allowing cellular survival while permitting accumulation of mutations over numerous cell divisions. Our data now indicate that inhibition of HR via DIDS treatment renders aberrant AID activity cytotoxic, owing to intolerable levels of unrepaired chromosomal breakage. Thus, AID expression may be converted from a proneoplastic role to an antineoplastic one by DNA repair inhibition.
As a putative treatment modality for AID-expressing malignancies, RAD51 inhibition is somewhat similar to, but conceptually distinct from, synthetic lethal strategies based on PARP inhibition. Synthetic lethality is defined genetically as an interaction in which two (or more) mutations that are individually tolerable result in cell lethality when combined. Clinically, this can be exploited for therapy when underlying mutations, such as BRCA1 or BRCA2 deficiency, render cancer cells susceptible to inhibition of additional pathways, such as PARP-mediated single-strand break repair, inducing conditional synthetic lethality selectively in the aberrant cells (Ashworth, 2008
). In BRCA1- or BRCA2-deficient breast and ovarian cancer, PARP inhibition results in loss of both DSB and single-strand break repair, preventing repair of replication-associated DNA lesions, culminating in cell cycle arrest or death. In contrast, the approach described here exploits the concept of synergistic toxicity, defined as an interaction in which a mutation in one pathway renders the activity of a second pathway (either of which is individually tolerable) acutely damaging when combined. Whereas PARP-based synthetic lethality aims to minimize DNA repair efficiency leading to proliferation failure, our modality aims to maximize recombinase-mediated DNA damage leading to direct cytotoxicity. As such, these represent fundamentally distinct but possibly complementary therapeutic paradigms.
Here we begin to elucidate the mechanism of a new therapeutic concept. We show that DIDS inhibits RAD51, and by extension HR, which sensitizes cells to AID-generated DSBs, leading to cell death. Whether DIDS inhibits RAD51 through preventing complex formation or nuclear localization is not understood. In this context, RAD51 lacks a known nuclear localization sequence (NLS) and is thought to be imported into the nucleus via interactions with other NLS-containing proteins (Gildemeister et al., 2009
). Thus, cytoplasmic accumulation of RAD51 caused by DIDS is likely a result of disrupted binding of RAD51 to one or more NLS-containing cofactors (Davies et al., 2001
; Tarsounas et al., 2004
; Thorslund et al., 2010
). Furthermore, AID-generated genomic deoxyuridines are processed by UNG, culminating in CSR initiation to DSBs (Schrader et al., 2005
). However, our data suggest that UNG is dispensable for the AID-dependent antileukemic effect triggered by DIDS treatment, as Ung1−/−
cells respond as WT to DIDS treatment (). This result is not surprising as UNG has been demonstrated to be dispensable for the DNA cleavage step (Begum et al., 2007
). Therefore, DSBs in collateral damage may require other glycosylases to form.
In summary, we have now established that small molecule inhibition of RAD51-mediated DSB repair sensitizes both primary and leukemic B cells to AID-initiated DSBs, culminating in growth suppression and apoptotic cell death. This suggests that pharmacologic attenuation of RAD51, and possibly other key HR factors, could be a useful therapeutic strategy to target AID-expressing tumors. Although our proof of principle experiments have focused on AID-expressing human CLL, AID expression has been noted in a wide range of other tumor types, including myeloid leukemias and a host of solid tumors. In this context, targeting HR in AID-positive acute myeloid leukemia may be especially promising, as AID has been implicated in aggressive disease associated with therapy resistance.