Characterizing Therapy Induced Polyploidy (TIP) Populations as a Resistance Mechanism in DH/DE-DLBCL and Identifying Synthetic Lethal Targeted Therapies

Abstract

Lymphoma is a blood cancer that involves the lymphatic system and is the 7th most common cancer in USA. Diffuse large B-cell lymphoma (DLBCL) and Peripheral T-cell lymphoma (PTCL) are the most common types of aggressive B-cell and T-cell non-Hodgkin lymphomas (NHL) respectively. Double-Hit or Double-Expresser DH/DE-DLBCL are high grade B-cell lymphomas characterized by translocation or over expression of MYC and BCL-2 which are mostly incurable with standard chemo-immunotherapy. Therefore, there is an unmet need for novel targeted therapy. Aurora kinase inhibition (alisertib) induces ~30% cell death (in vitro), while a portion of the remaining ~70% cells at day-4 escape apoptosis through polyploid populations which we called therapy induced polyploid cells (TIP). These TIP cells exhibited a high metabolic rate by increased AKT/mTOR and ERK/MAPK activity via BTK signaling through the chronic active B-cell receptor (BCR) pathway. TIP also showed increased levels of phospho-Hck and phospho-Akt indicating increased BCR signaling which is a rationale for combining ibrutinib (BTK inhibitor). Combined inhibition of AK + BTK reduced phosphorylation of AKT/mTOR and ERK-1/2, up-regulated phospho-H2A-X and Chk-2 (DNA damage), reduced Bcl-6 and decreased Bcl-2 and Bcl-xL results in induced apoptosis evident by PARP cleavage. In a DE-DLBCL SCID mouse xenograft model, ibrutinib alone was inactive, while alisertib + ibrutinib was additive with a tumor growth inhibition (TGI) rate of ~25%. However, TGI for ibrutinib + rituximab was ~50-60%. In contrast, triple therapy showed a TGI rate of >90%. Kaplan-Meier survival analysis showed 67% of mice were alive at day-89 with triple therapy versus only 20% with ibrutinib + rituximab. All treatments were well tolerated with no significant changes in body weights. Anti-DLBCL chemotherapy dosing schedules are intermittent, designed to avoid damage to normal tissue such as the mucous membranes, gut and the bone marrow. TIP are common in standard anti-DLBCL therapies (e.g. vincristine, doxorubicin) and thought to be responsible for disease relapse. Some of these TIP cells die but remaining of those are capable of re-entering the cell cycle during off-therapy periods. We discovered how these TIP cells can re-enter cell cycle and molecular mechanism underlying this resistance. We have purified AK inhibitor induced polyploid DH/DE-DLBCL cells by FACS. Time-lapse microscopy of single cells revealed that following drug removal, a subset of TIP cells divide and proliferate by reductive cell division(s). This includes multipolar mitosis, meiosis like nuclear fission or budding off daughter cells. RNA-Seq, Proteomics and Kinomics proling of TIP cells demonstrated that alisertib induced polyploid cells have up-regulated DNA damage response, replication and immune evasion; amplify receptor tyrosine kinase and T-cell receptor signaling; hijacks the spindle assembly checkpoint point control via MYC dysregulation of RanGAP1, TPX2 and KPNA2. We believe these up-regulated proteins are responsible for induction of aneuploid daughter cells and disease resistance and also provide potential opportunities for novel therapy combination that warrant further exploration. Lymphomas are systemic diseases that require a comprehensive knowledge of immune mechanism in cancer as well as targeted therapeutic approach for designing an optimal therapeutic strategy and desired synergy can be achieved by rational combination of small molecule inhibitors with immune modulatory agents that could enhance host immune response. In PTCL we have shown that expression of PD-L1 relative to PD-1 is high in PTCL biopsies ( 9-fold higher) and cell lines. Combination of alisertib with pan-PI3K inhibition or VCR significantly reduced PD-L1, NF-kB expression and inhibited phosphorylation of AKT, ERK1/2 and AK with enhanced apoptosis. In a syngeneic PTCL mouse xenograft model, alisertib demonstrated tumor growth inhibition (TGI) ~30%, whilst anti-PD-L1 therapy alone was ineffective. Alisertib + anti-PD-L1 resulted in TGI >90% indicative of a synthetic lethal interaction. PF-04691502 + alisertib + anti-PD-L1 + VCR resulted in TGI 100%. Overall, mice tolerated the treatments well. Co-targeting AK, PI3K and PD-L1 is a rational and novel therapeutic strategy for PTCL. In conclusion, we have identified therapeutic targets in aggressive B- and T-cell lymphoma which can be combined with immunotherapy that warrant investigation to disrupt rapid tumor evolution of TIP cells to mitigate disease relapse.