Simultaneous Sessions III: New biological and translational insights into CLL
Loss of the long arm of chromosome 11, del(11q), is one of the most common alterations in CLL. ATM gene is mutated in the remaining allele in one third of del(11q) CLL patients, resulting in a complete loss-of-function of ATM protein, which is related with poor clinical outcomes. Although targeted BCR inhibition has made a paradigm shift in CLL therapy, relapsed/refractory del(11q) CLLs still show inferior responses to ibrutinib and some patients have developed resistances to these inhibitors overtime. Therefore, novel therapeutic approaches need to be explored in this high-risk subgroup of patients.
To assess the antiproliferative effects of the combination of PARP inhibiton (olaparib) and BCR inhibition (ibrutinib) in CRISPR/Cas9-engineeered del(11q)/ATM-mutated CLL cell lines as well as in primary CLL cells with del(11q) in the presence of the stromal microenvironment.
The CRISPR/Cas9 system was used to introduce del(11q) and ATM mutations (ATMKO) into CLL cell lines (HG3, MEC1). In addition, 18 CLL primary cases (9 del(11q) and 9 non-del(11q) patients) were used for ex vivo experiments. Primary CLL cells were stimulated to proliferate in a co-culture with HS-5 bone marrow stromal cells, CpG and IL-2. Viability was assessed by CellTiter-Glo luminescence assay at 5 days.
Olaparib treatment in monotherapy significantly inhibited proliferation of isogenic CRISPR/Cas9-engineered HG3-del(11q) ATMKO and MEC1 ATMKO cells, compared to HG3WT and MEC1WT cells, respectively (P = 0.006; P = 0.001). In addition, the combination olaparib+ibrutinib showed a synergistic effect in HG3-del(11q) ATMKO and MEC1 ATMKO CLL cell lines (combination index = 0.7), by viability and annexin/PI studies. To test whether this combination was effective in primary del(11q) CLL cells ex vivo, 18 primary CLL cases were co-cultured with HS-5 BMSCs, CpG and IL-2 to induce proliferation. Interestingly, dual PARP and BCR inhibition was more effective than ibrutinib monotherapy in both non-del(11q) (P = 0.03) and del(11q) (P = 0.006) CLL samples. In addition, this combination significantly reduced the viability of proliferating CLL cells with ATM monoallelic (n = 6) and biallelic loss (n = 4), compared to ATMWT CLL samples (n = 8) (P = 0.01, P = 0.001, respectively). To study the mechanisms leading to the drug synergy, we used a homologous recombination (HR) reporter assay showing that ibrutinib (1 uM) reduced the HR repair efficiency of HG3 cells (P = 0.001). Moreover, immunofluorescence and western blot experiments in HG3 and MEC1 cells revealed that ibrutinib impaired RAD51 foci formation in double strand breaks (P = 0.003; P = 0.018). RAD51 downregulation by ibrutinib was validated by qPCR in stimulated primary CLL samples, responders to ibrutinib monotherapy (P = 0.04). We further confirmed the off-target effect of ibrutinib on HR repair by comet assays, since the addition of the alkylating agent bendamustine to ibrutinib or to olaparib+ibrutinib led to a synergistic increase of DNA damage accumulation (P < 0.001), and also a decrease of viability (P < 0.001). These effects were validated in stimulated primary CLLs, showing that the triple combination (bedamustine, olaparib and ibrutinib) was more effective than dual combinations, resulting in a decrease of viability (P < 0.01).
Altogether, our results highlight that dual PARP and BCR inhibition is synergistic in del(11q) CLL cells, overcoming the induction of proliferation from the stromal microenvironment, providing a rationale for the use of this combination in del(11q) relapsed/refractory CLL patients.