Abstract 529: Obatoclax, a safe BH3-mimetic, is noninferior to neurotoxic oxaliplatin when combined with adenovirus to treat colorectal carcinoma

Oxaliplatin (Ox) in an important inducer of Immunogenic Cell Death (ICD) that causes endoplasmic reticulum (ER) stress and externalizes Calreticulin (ectoCALR) to attract and activate immune cells agnostically. However, since OX is quite toxic, we hypothesize that Obatoclax (OB), a clinically safe BH3 mimetic, that causes ER stress through mitochondrial stress can replace OX in our vaccine model. Our model involves a single intralesional injection of 80 µM OX combined with an oncolytic chimeric Ad5/3-NP2.ADP (ADP) adenovirus into > 6 mm CT26 colorectal carcinomas (CRC) dwelling in BALB/c hosts for 14 or more days. Treatment causes 90% regression with 20% durable complete responses - without checkpoint blockade. Treatment is accompanied by a four-fold increase in intratumoral CD8+ T cells from 99% PD-1HiEOMESInt-Highin control tumor bearers to 79% PD-1LoEOMESLo or PD-1LoEOMESInt CD8+ T cells in treated mice along with new in vitro cytotoxicity to CT26 cells and rejection of distant CT26 cells in survivors. We postulate that OB synergizes with ADP to increase ectoCALR, enhance viral infectivity and inhibit the growth of murine and human CRC in vitro that can be used in vivo as a vaccine. The approach used cytometry to quantify proliferation, CALR protein expression, viral production, autophagy and ER stress, and their response to inhibitors. In both murine CT26 and human Clone A (DLD-1) CRC cells, OB induces more ectoCALR than OX as a monotherapy within 2 hours at dosages equivalent to 50% of clinical Cmax for each agent. OB increased superoxide production with an EC50 50.2 nM in CT26 cells confirming mitochondrial stress. OB and OX both cause necroptosis in CT26 and Clone A cells which is critical for ICD. OB synergizes with ADP to inhibit growth in vitro in CT26 cells (OB alone IC50 1.1 nM, ADP IC50 for Multiplicity of Infection (MOI) 69.2 MOI which decreases to an IC50 of 0.3 MOI with OB at 1 nM). Similar synergy occurred with Clone A cells. Clone A was used In further studies for interaction with ADP because human adenovirus does not replicate in mouse cells. Both OB and OX activate PERK, increase DDIT3/CHOP expression, and enhance red/green fluorescence ratio (R/GFIR) in acridine orange staining. PERK (PERK-IN-2) and ER stress (4-phenylbutyric Acid) inhibitors decrease these responses and ectoCALR expression but increase proliferation of Clone A cells. Ecto-CALR expression with OB-ADP on Clone A cells was higher than that of Clone A cells after OX-ADP after three days in vitro with a similar growth inhibition. In summary, OB is an agent that rapidly initiates ICD and supports viral infectivity in CRC cells to achieve ICD with necroptosis and autophagy. OB-ADP treatment is noninferior to OX-AdV therapy in vitro for initiation of ICD. These results support using OB in an agnostic therapeutic vaccine in vivo, particularly due to its systemic injection capability. Citation Format: John Milburn Jessup, Sameer Tiwari, Ali Andalibi, Yungtao Wu. Obatoclax, a safe BH3-mimetic, is noninferior to neurotoxic oxaliplatin when combined with adenovirus to treat colorectal carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 529.