Background: For BL patients who relapse, the prognosis is dismal due to chemo-immunotherapy resistance (Cairo et al, JCO, 2012, Goldman/Cairo et al, Leukemia, 2013). Our group has successfully modified expanded peripheral blood Natural killer cells (exPBNK) with an anti-CD20 CAR to target rituximab sensitive/resistant CD20+ BL cells in vitro and in NSG mice (Chu/Cairo, et al, Can Imm Res 2015). Romidepsin is a histone deacetylase (HDAC) inhibitor that increases the expression of NKG2D ligands in BL (Chu/Cairo, et al, Cytotherapy, 2014).

Objective: We investigated the effect of romidepsin alone and in combination with anti-CD20 CAR modified exPBNK cells against CD20+ BL cells in vitro and in humanized BL NSG mice.

Methods: Anti-CD20 CAR modified exPBNK cells were produced as we have described (Chu/Cairo, et al, Can Imm Res 2015). HDACs levels in BL cells were examined by western blot. Raji, Raji-2R and Raji-4RH cells were treated with 10ng/ml romidepsin, generously provided by Celgene. Raji-Luc engrafted mice were injected i.p. with romidepsin (2.2mg/kg) or PBS once a week for 3 consecutive weeks followed by anti-CD20 CAR exPBNK cell or mock exPBNK cell injections 24hrs later. Tumor regression and/or progression were monitored weekly by tumor volume measurement and by in vivo bioluminescent imaging (Chu/Cairo, et al, Can Imm Res 2015).

Results: HDAC 1, 3, 6 levels were significantly increased in Raji, Raji-2R, Raji-4RH, Ramos, and Daudi compared to white blood cells. Total H3K9 acetylation was enhanced in Raji, Raji-2R, and Raji-4RH following romidepsin. More importantly, romidepsin significantly inhibited BL cell proliferation (p<0.001), induced apoptosis only in rituximab sensitive BL cells with increased active caspase 3, and induced cell cycle arrest in resistant BL cells (p<0.001). Romidepsin significantly inhibited in vivo Raji and Raji-2R cells growth in xenografted NSG mice with reduced tumor size (p<0.05) and bioluminescence (p<0.05).

In vitro cytotoxicity of anti-CD20 CAR exPBNK cells was significantly enhanced against romidepsin treated BL cells compared to the untreated at E:T = 3:1 (P<0.001; n = 4), or compared to the mock exPBNK (P<0.05; n = 4).

In humanized Raji xenograft NSG mice, survival time in romidepsin+CAR exPBNK treated mice was significantly extended compared to the untreated mice (median 28 days, P<0.001), the mock exPBNK treated mice (median 29 days, P<0.001), the CAR exPBNK treated mice (median 42.5 days, P<0.05), the romidepsin treated mice (median 30 days, P<0.001), and the romidepsin+mock exPBNK treated mice (median 34.5 days, P<0.05).

Conclusion: These results suggest the therapeutic potential of the combination of anti-CD20 CAR modified exPBNK cells and romidepsin against chemo-immunotherapy resistant BL.

Citation Format: Yaya Chu, Ashlin Yahr, Bokun Cheng, Bian Hang, Janet Ayello, Matthew Bath, Mitchell S. Cairo. Enhanced in vitro and in vivo targeting of rituximab-sensitive and -resistant Burkitt lymphoma (BL) by anti-CD20 chimeric antigen receptor (CAR)-modified expanded natural killer cells in combination with a histone deacetylase inhibitor, romidepsin. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2211.