Abstract 2537: Targeting acute myeloid leukemia cell death via small molecule AP-3-84 binding to the LxCxE pocket of Rb1
Authors: Bhuniya Avishek , Lu Lily , Poli Adi Narayana Reddy , Tcyganov Evgenii N. , Hart Colin , Karanam Brijesh , Sharma Paridhima , Hill Taylor , Salvino Joseph , Montaner Luis J. 
Published: April 2023

Abstract
Background: Acute myeloid leukemia (AML) is the most common form of leukemia in adults. In 2017, 120,000 new AML cases of were reported worldwide, with an expectation to reach double the number by 2040. Presently, with an 8.5 months of median survival rate and approximately 60% recurrence rate following chemotherapy treatment, leads to the opportunity to improve overall AML cancer therapy. The Retinoblastoma (Rb1) protein can control canonical cell cycle progression and be a determinant to apoptosis depending on its phosphorylation state, binding partners, and localization (specifically mitochondrial Rb1). We have developed a new small molecule, AP-3-84, which binds the LxCxE domain of Rb1 and can induce cell death in multiple acute myeloid leukemia cell lines by in vitro studies.
Methods: AML cell lines (THP-1, AML-193, Kasumi-1) were cultured at different cell concentration (75000/ml, 125,000/ml and 200,000/ml) and exposed to 5, 10, 15, 20 uM concentration of AP-3-84. Cell differentiation was induced by 100 ng/ml PMA over 24 hours. Nonhemopoietic cancer cells (ID8, PDAC) were analyzed after exposure to AP-3-84. Cell death outcomes were measured by Annexin-V using flowcytometry or viability measures by Alamar blue assay. Protein levels were measured by Western blots at intervals between 30 min and 6 hours after exposure to AP-3-84 for total Rb1, phosphorylated Rb1, ATF3, BIM, NOXA, Bak, Bax, cleaved PARP, cleaved Caspase-3. Cytoplasmic and mitochondrial lysates were examined. Confocal immunocytochemistry on Rb1 expression in nucleus versus cytoplasm was performed.
Results: Rb1 protein expression was documented by immunocytochemistry in nucleus and cytoplasm of THP1 cells consistent with its detection in nuclear and mitochondrial lysates (Western). AP-3-84 induction of cell death was observed at 10uM or higher concentration, with a 5 minute exposure sufficient to cause cell death in 70-80% of cells 24 hours later. Cell death by AP-3-84 was specific to dividing AML cells as no cell death was induced in PMA differentiated THP1 cells or nonhemopoietic cancer cells. No effect in cell division rate by AP-3-84 was present in nonhemopoitic cancer cells indicating a lack of cell cycle regulation. Mechanistically, AP-3-84 induced higher levels of ER-stress signals (higher ATF4, ATF3) and increased mitochondrial protein levels of NOXA, BIM, cleaved-PARP, and cleaved Caspase-3. Bax and Bak levels were unchanged after AP-3-84 exposure.
Conclusion: We identify a new small molecule, AP-3-84, with therapeutic potential in AML by its binding to the LxCxE pocket of Rb1 triggering mitochondria-mediated cell apoptosis.
Citation Format: Avishek Bhuniya, Lily Lu, Adi Narayana Reddy Poli, Evgenii N. Tcyganov, Colin Hart, Brijesh Karanam, Paridhima Sharma, Taylor Hill, Joseph Salvino, Luis J. Montaner. Targeting acute myeloid leukemia cell death via small molecule AP-3-84 binding to the LxCxE pocket of Rb1 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2537.