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Many melanomas are resistant to standard-of-care immune checkpoint blockade and inhibitors of mutated BRAF. New effective therapies that work via immune and BRAF-independent mechanisms are urgently needed. We have previously demonstrated that an inhibitor of mitotic kinase Aurora A (AURKAi) can induce a distinct type of stable cell cycle known as senescence in melanoma tumors independent of their genetic background. Here we hypothesized that combination of AURKAi with a drug that selectively kills senescent cells, such as BCL2 inhibitor (BCLi) navitoclax, will be synthetically lethal to melanoma cells. Melanoma patient-derived organoids (PDOs) were used to test efficacy of AURKAi and BCLi combination treatment. To generate organoids, patient-derived tumor cells were cultured ex vivo in 3D matrix. Drug response was evaluated using fluorescent viability assay. Fifteen out of twenty PDOs were highly sensitive to AURKAi and BCLi combination treatment, exhibiting robust induction of cell death. No cell death was observed when either drug was added individually. NextGen DNA sequencing indicated that all sensitive PDOs retained wild-type TP53, while resistant tumors had mutations in this gene. Knockout or knockdown of TP53 abrogated AURKAi and BCLi-induced cell death in melanoma cells, suggesting that transcription factor p53 encoded by TP53 is a key mediator of drug response. Mechanistically, p53 was induced by AURKAi treatment independent of BCLi. However, single-agent AURKAi treatment resulted in activation of p53-mediated cell cycle arrest based on increased expression of CDK inhibitor p21 and decreased expression of proliferation markers. In contrast, addition of BCLi to AURKAi activated proapoptotic p53 target BAX and induced cleavage of PARP and caspases 3, 7, and 8. Treatment with pan-caspase inhibitor abrogated cell death, suggesting caspase-dependent apoptosis. Electron microscopy revealed morphologic signs of apoptosis in combination-treated cells, including cell blebbing, apoptotic bodies, and nuclear fragmentation. This suggests that BCLi shifts cell fate decision in AURKAi-treated cells towards apoptosis. In conclusion, combination treatment with AURKAi and BCl2i induced p53-dependent apoptosis. Preclinical evaluation of this drug combination in PDO model demonstrated robust efficacy against melanomas with wild-type p53. Since p53 mutations are relatively rare in melanoma, a combination of AURKAi and BCLi presents a promising therapeutic option for this deadly disease. Citation Format: Vijaya Bharti, Ashlyn Blevins, Vivian L. Weiss, Sheau C Chen, Roman Uzhachenko, Ann Richmond, Anna E. Vilgelm. Patient-derived organoids demonstrate synergistic effect of co-targeting Aurora kinase and prosurvival BCL2 family proteins [abstract]. In: Proceedings of the AACR Special Conference on the Evolving Landscape of Cancer Modeling; 2020 Mar 2-5; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2020;80(11 Suppl):Abstract nr B20.

Local tumor antigen-specific T cell-NK cell collaboration is indispensable for the elimination of tumor cells, including antigen-deficient tumor escape variants before metastasis. While mechanistic details are available for the innate instruction of the T cell responses, little is known for the adaptive control of NK cell activity. We observed in a mouse model of mastocytoma expressing a self tumor antigen P1A that effector CD8+ T cells provided a necessary “help” to dormant NK cells in eliciting their antitumor effector function. Bioluminescence imaging of mastocytoma tumors following adoptive transfer of P1A-specific T cells in RAG-/- and RAG-/-γc-/- mice showed that NK cell anti-tumor activity requires cytolytic T cells, whereas T cells can function independent of NK cells. In 2D and 3D co-culture systems, we observed that PMA/ionomycin-stimulated CD8+ T cells form multiple contacts with naïve NK lymphocytes. Data show that NK cells interacting with activated CD8+ T cells show an up-regulation of CD25 and CD69 expression mediated by intercellular contacts, and activation of NKG2D receptors and Stat2, Stat6, Jak1, Jak3, Tyk2, and PTEN signaling molecules with a decrease in the phosphorylation of Stat1, PKB/Akt, SAPK/JNK, p38. On the other hand, interacting NK cells down-regulate CD25 molecule expression on CD8+ T cells and promote differentiation of central memory CD44+CD62L+ T cells. CD8+ T cells display an elevation in the phosphorylation of Stat1 and down-regulation of Stat5 with stimulated PKB/Akt, Lck, mTOR, and p42/p44. Moreover, significant changes in the cytosolic and mitochondrial Ca2+, production of mitochondrial ROS, mitochondrial membrane potential, mitochondrial permeability transition pore, and synthesis of nitric oxide and non-protein thiols (mostly, reduced glutathion) were observed in a reciprocal T cell-NK cell interaction. These results highlight the importance of mitochondrial activity in the re-modeling of activation signaling and memory differentiation of interacting CD8 T cells and NK cells. These results will help refine cancer immunotherapeutic strategies. Citation Format: Roman V. Uzhachenko, J Shawn Goodwin, Lino Costa, Alexander Terekhov, Menaka C. Thounaojam, William H. Hofmeister, Anil Shanker. Crosstalk between CD8+ T and NK cells: fine-tuning of antitumor immune response. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4059. doi:10.1158/1538-7445.AM2015-4059