Alisertib-Induced Polyploidy and Senescence in DH/DP-DLBCL Is Abrogated By Synthetic Lethal Interactions with Ibrutinib and Rituximab

Diffuse Large B-cell Lymphoma (DLBCL) especially the double-hit (DH)/ double-protein (DP) DLBCL is an aggressive subtype of Non-Hodgkin Lymphoma (NHL). A majority of patients will fail frontline R-CHOP-like therapy and novel agents targeting MYC/BCL2 are needed. Aurora kinases are important regulators of mitosis, frequently over expressed and strongly associated with human cancers including DLBCL. Pre-clinical research and clinical studies have shown that aurora kinase inhibitors have a ~25-30% response rate in B- and T- NHL. Here, we show that alisertib-induced polyploidy and senescence are responsible for treatment failure and can be overcome by implementing a synthetic lethal approach by targeting Bruton's Tyrosine kinase (Btk) with ibrutinib and CD20 with rituximab.

It is known that aurora kinases inhibition impede cell cycle progression through mitosis and induce polyploidy in treated cells. However, it is not yet known how these polyploid and senescent cells behave. To further investigate molecular and cellular characteristics of polyploid and senescent cells, we demonstrate these cells are metabolically 4x more active and have fragmented DNA. Though a portion of these cells undergo apoptosis using Caspase pathways, most enter a state of senescence.

To assess the effect of alisertib on DH/DP-DLBCL we investigated U2932 and VAL cell lines, where U2932 is a representative of DP-DLBCL that over-expresses MYC, BCL2, BCL6 and mutant TP53, and VAL is representative of DH-DLBCL that carries a three-way-translocation t(8;14;18) and over-expresses BCL2 and MYC. From dose vs. duration experiments, we find 50nM alisertib for 4days of treatment is most effective for investigation of polyploidy and senescent DLBCL cells. We noticed that at day-4, cells appear unusually large in size and 4x more metabolically active compared to controls. This unusually high metabolic rate may have a survival advantage in these cells. To further investigate, we evaluated the DNA integrity within these polyploidy cells. We used the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay and found that 98.8% of 8n population had fragmented DNA compared to 19.99% for the 2n population. Then we studied by flow cytometry the fate of the fragmented DNA bearing population using Caspase-3/7 and Annexin-V staining along side with PI and found that only a small fraction of these cells were undergoing apoptosis confirmed by PARP cleavage but the majority of polyploidy cells especially the 8n cells become senescent shown by C₁₂FDG a fluorescent substrate for senescent associated β-galactosidase.

We next investigated signaling pathways involved in therapy failure to alisertib treated cells and show that constitutive activation of p-Btk, p-Akt and p-Erk downstream of the BCR signaling pathway. These finding suggest that targeting Btk with ibrutinib and CD20 with rituximab would be a rational therapeutic strategy to partner with alisertib. Cell proliferation MTS assays demonstrated synergistic interactions of alisertib + ibrutinib with low combination Indices (CI) (Chou-Talalay). We next performed alisertib plus ibrutinib + rituximab with a comparison to single agent therapy for polyploidy and senescent abrogation. Triple combination was found to successfully reduce the senescence population (5x lower vs. single agent alisertib), which ultimately results in inducing apoptosis shown by a significant sub-G₀ population of cells and increased PARP cleavage. Apoptosis was further confirmed by phosphatidylserine exposure at the cell membrane.

Finally, to establish safety and efficacy of the triple combination, we tested a DH/DP-DLBCL SCID mouse U2932 xenograft model. Ibrutinib alone was not active while alisertib + ibrutinib was additive with a tumor growth inhibition (TGI) rate of ~25%. TGI for Ibrutinib + rituximab was ~50-60%, where the triple combination showed a TGI of >90% (p<0.001). All treatments were well tolerated with stable body weights (<5% body weight loss). Kaplan-Meier survival showed 67% of mice were alive at day 89 in the triple therapy group versus 20% for ibrutinib + rituximab combination, whereas no mice from other treatment group survived passed day 52. Therefore, co-targeting aurora kinase, Btk and CD20 may provide a safe and effective synthetic lethal therapeutic regimen for DH/DP-DLBCL.