Targeting DNA Repair to Overcome Drug Resistance in Hodgkin Lymphoma

Classical Hodgkin lymphoma (cHL) is characterized by rare tumor-initiating Hodgkin and Reed-Sternberg cells (HRS) surrounded by a microenvironment with a reactional immune cells infiltrate. cHL accounts for 15% to 25% of all lymphomas. This neoplasm is curable in the majority of cases with chemotherapy including ABVD (Doxorubicin, Bleomycin, Vinblastine, Dacarbazine) or BEACOPP (Bleomycin, etoposide, Doxorubicin, cyclophosphamide, Vincristine, procarbazine and Prednisone) and/or radiation. However, 15-20% of high-risk patients ultimately relapse and high dose chemotherapy in combination with autologous stem-cell transplantation is successful in only half of the patients with relapsed/refractory cHL (Eichenaueret al., Eur. Soc. Med. Oncol,2018). HRS cells are characterized by genetic instability, abnormal DNA damage response and repair that may play a role in drug resistance in high-risk cHL (Reichelet al., Blood,2015, Monroyet al. Mol. Carcinog,2011). In this work, we hypothesized that inhibiting DNA repair mechanisms using small molecules might represent a promising strategy to overcome drug resistance to genotoxic agents in cHL, such as cyclophosphamide and topoisomerase II inhibitors (O'Connor,Mol. Cell, 2015; Shaheenet al., Blood 2011). We characterized the drug-response of 4 cHL cell lines to DNA repair inhibitors including PJ34 (PARP inhibitor), NU7441 (DNAPK inhibitor), KU55933 (ATM inhibitor), PF477736 (CHK1 inhibitor), AZD6738 (ATR inhibitor), AZD1775 (Wee1 inhibitor), MP-470 (Rad51 inhibitor) and genotoxic agents used in standard chemotherapy (cyclophosphamide, doxorubicin and etoposide). We showed that DNA repair inhibitors targeting ATR, CHK1 or Wee1 significantly induced apoptosis of cHL cell lines (AnnexinV staining, p<0.05), significant inhibition of proliferation (BrdU incorporation). ATR inhibitor induces a significant accumulation of cHL cells in G0/G1 phase (p<0.05) whereas CHK1 and Wee1 inhibitors induced a significant accumulation in G2/M phase (p<0.01). Moreover, ATR, CHK1 and Wee1 inhibitors induced double strand breaks monitored by 53BP1 foci formation, γH2A.X staining (p<0.01) and chromosomic instability characterized by a higher frequency of micronucleation (p<0.05) and nucleoplasmic bridges formation (p<0.01). Since DNA repair pathways play a role in drug resistance, we sought to identify new synthetic lethal and synergistic combinations associating IC20 DNA repair targeted treatments with conventional genotoxic agents in cHL. Applying a standard threshold of 2 SDs below the IC50 of the genotoxic agent alone (Srivaset al., Mol. Cell, 2016), a total of two synthetic lethal combinations in cHL cells have been identified: CHK1 inhibitor (PF477736) combined with a Wee1 inhibitor (AZD1775) (IC50 reduced from 5.00 µM to 1.75 µM for L-428 and from 4.81 µM to 1.86 µM for KMH2) and cyclophosphamide combined with an ATR inhibitor (AZD6738) (IC50 reduced from 3.26 µM to 1.12 µM for L-428 and from 1.27µM to 0.10µM for KMH2). In addition, we identified 3 synergistic/additive combinations (combination index < 1): doxorubicin combined with a DNAPK inhibitor (NU7441) (IC50 reduced from 44 nM to 22 nM for L-428 and from 27 nM to 8 nM for KMH2), cyclophosphamide combined with CHK1 inhibitor (PF477736) (IC50 reduced from 2.9 µM to 2.4 µM for L-428 and from 1.2µM to 0.9µM for KMH2) and etoposide combined with an ATR inhibitor (AZD6738) (IC50 reduced from 420 nM to 195 nM for KMH2). The treatment of Hodgkin's lymphoma is successful for a majority of patients. However, patients with advanced-stage or high-risk disease are only cured in ∼70% of cases. Alternative treatment options for the subsets of patients for whom first- or second-line therapies fail are needed. These results open new perspectives to improve the treatment of relasped/refractory cHL patients and provide new strategies to overcome drug resistance. Disclosures Moreaux: Diag2Tec:Consultancy.

RDNA-06. TARGETING IDH1/2-MUTANT GLIOMAS WITH UNIQUE COMBINATIONS OF DNA REPAIR INHIBITORS

Mutations in the Isocitrate Dehydrogenase-1 and -2 (IDH1/2) genes occur in the vast majority of low-grade and secondary high-grade gliomas. These neomorphic mutations occur early on in gliomagenesis leading to the production of 2-Hydroxyglutarate (2HG). 2HG has been implicated in tumorigenesis via inhibiting α-ketoglutarate (αKG)-dependent dioxygenases. Our group recently demonstrated that the production of 2HG suppresses the high-fidelity homologous recombination (HR) DNA repair pathway, resulting in a state of “BRCAness”. We initially found that mutant IDH1/2-induced BRCAness confers exquisite sensitivity to poly(ADP-ribose) polymerase (PARP) inhibitors, a finding which now has been replicated by multiple independent laboratories. Although IDH1/2 mutations were first identified in gliomas and acute myeloid leukemia (AML) cells, multiple other tumor types have subsequently been shown to harbor these mutations. Current clinical trials are testing the efficacy of PARP inhibitors as a monotherapy, as well as in combination with other DNA repair inhibitors. Here, we demonstrate that novel combinations of DNA repair inhibitors can be utilized to synergistically target IDH1/2-mutant glioma cells. In particular, we demonstrate potent synergy with ATRi and PARPis, a finding which was validated in multiple structurally unique drugs within these classes. As this combination is active in BRCA1/2-mutant cancers, in particular after the emergence of PARPi resistance, these data suggest are consistent with an underlying HR defect in IDH1/2-mutant gliomas. These preclinical investigations will provide a blueprint for future clinical trials combining PARP and ATR inhibitors in the treatment of glioblastoma.