Cell Cycle Kinase Inhibitors Potentiate the Effect of ¹⁷⁷lu-Lilotomab Satetraxetan in Treatment of Aggressive Diffuse Large B-Cell Lymphoma Cell Lines

Background

CD37 is an internalizing transmembrane glycoprotein widely expressed on mature B-cells and B-cell malignancies. The next generation anti-CD37 radioimmunoconjugate (RIC) ¹⁷⁷Lu-lilotomab satetraxetan (Betalutin®), containing the beta-emitting radionuclide lutetium-177, is currently being tested as one-time injection therapy in a clinical phase 2b trial for follicular lymphoma (FL) and phase 1 trial for diffuse large B-cell lymphoma (DLBCL).

We recently reported the systems biology analysis of germinal center like (GCB) and activated B-cell like (ABC) DLBCL cell lines to CD37 targeting radioimmunotherapy (RIT) (Melhus, et al., PF642 at EHA23, Stockholm, 2018). ¹⁷⁷Lu-Lilotomab satetraxetan showed generally promising activity against DLBCL cell lines, but treatment resistance was evident in a subset of cell lines. Resistance was not attributable to differences in CD37 expression nor correlated to genetic hallmarks of aggressiveness such as aberrations in TP53, BCL2 and MYC. In the present study we aimed at identifying resistance reversing drug combinations with ¹⁷⁷Lu-lilotomab satetraxetan in two RIT resistant aggressive ABC-DLBCL cell lines.

Materials and methods

U-2932 and RIVA cells were treated for 18 hours with ¹⁷⁷Lu-lilotomab satetraxetan (600 MBq/mg; 1 or 0.5 µg/ml), washed, and seeded on micro-well plates pre-printed with a drug library of 384 approved anti-cancer compounds at 10, 100, and 1000 nM (screen). For validation experiments, cells were treated with three different doses of ¹⁷⁷Lu-lilotomab satetraxetan as described above and seeded on pre-printed plates with gradients of candidate hits ranging from 1-1280 nM. Cell viability was monitored at days 3 to 6 post seeding using a cell viability assay (RealTime-Glo™). Drug combinations were scored using Bliss and Chou-Talalay theorems.

Results

Compounds scoring in both cell lines included inhibitors targeting cell cycle kinases that regulate transition through mitosis, such as CDK1/2, AURKA/B, PLK1, as well as enzymes with important roles in DNA integrity surveillance and repair, such as topoisomerases. If the compounds also have or are being tested in clinical trials for treatment of non-Hodgkin lymphoma (NHL) they were considered candidates for further studies. PLK1 inhibitors BI2536 and GSK461364 potently inhibited cell proliferation at < 20 nM, with additional, but not synergistical benefit of combination with ¹⁷⁷Lu-Lilotomab satetraxetan. The AURKA/B inhibitor Alisertib synergistically inhibited cell proliferation in combination treatment, but showed adverse effect at concentrations >160 nM, possibly owing to inhibition of a secondary target. The pan-CDK inhibitor JNJ-7706621 showed little effect alone but strongly synergized with ¹⁷⁷Lu-lilotomab satetraxetan in inhibition of proliferation at doses > 80 nM. U-2932 and RIVA cells arrested in G₂-phase with elevated DNA damage (flow cytometry; P-γ-H2AX and DNA staining) 18 hrs after treatment with ¹⁷⁷Lu-lilotomab satetraxetan. Resistance reversal by anti-mitotics may thus underlie concomitant radiation during inhibitor mediated M-phase arrest of cells that eventually evaded G₂-phase arrest.

Conclusion

In summary, combinatorial drug screening identified cell cycle kinase inhibitors as promising partners for combination treatment of aggressive DLBCL with ¹⁷⁷Lu-lilotomab satetraxetan, warranting further exploration in pre-clinical models.

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