Disruption of the Retinoblastoma Lxcxe Region Induces Cell Death in AML By Activation of the Unfolded Protein Response Leading to NOXA Induction

The Retinoblastoma (Rb1) protein is expressed in high amounts in leukemia and myeloid cells. While RB1 controls cell cycle progression, it also regulates cell death through protein binding regions such as the LxCxE binding cleft. To test whether blocking the Rb1 LxCxE domain would induce specific cell death in acute myeloid leukemia (AML) cells, we developed a small molecule (AP-3-84) that can selectively bind the LxCxE domain of Rb1 blocking protein-protein interactions. Statistical analysis was performed with GraphPad Prsim.

The activity of AP-3-84 was evaluated in vitro using multiple AML cell lines (THP-1, AML-193, Kasumi-1, MOLM-14) and in vivo using luciferase-transduced THP-1 cells implanted in NGS mice. AP-3-84 induced cell death in a concentration-dependent manner in all AML cell lines tested. In vivo treatment with AP-3-84 also reduced tumor growth as measured by luciferase signal using an IVIS platform. Importantly, AP-3-84 treatment did not induce cell death in PMA-differentiated THP1 cells or non-hematopoietic cancer cell lines. PROTAC-mediated Rb1 degradation reduced AP-3-84-induced cell death in THP-1 cells, confirming Rb1 as the target for AP-3-84.

Bulk RNA sequencing showed that 30 min treatment of THP-1 cells with AP-3-84 resulted in upregulation of the NRF2-mediated oxidative stress response and unfolded protein response (UPR) and induction of ATF4 and DDIT3 (CHOP). Further evaluation of the UPR pathway via western blotting indicated increased expression of pPERK, peIF2a, ATF4, ATF3, and NOXA in a sequential time-dependent manner. Notably, the levels of BAX and BAK remained unchanged at early time points (up to 6 hours), suggesting that the UPR pathway is the predominant pathway triggered by AP-3-84. In the same experiments we observed induction of lipid peroxidation. As lipid peroxidation induce ferroptosis, treatment with ferrostatin 1 reduced the effect of AP-3-84, supporting that induction of ferroptosis is the predominant cell death pathway induced by AP-3-84. We also observed an increase in cardiolipin levels consistent with lipid peroxidation changes after AP-3-84 treatment, supporting the contribution of mitochondria to activation of cell death pathways.

We conclude that AP-3-84 triggers cell death in AML by activating the UPR via PERK-eIF2a-ATF4-NOXA pathway involving mitochondrial regulation. Our results indicate that Rb1 LxCxE domain may represent a novel therapy target in AML.

Tcyganov:AstraZenica: Current Employment. Salvino:Syndeavor Therapeutics, Inc: Consultancy; Context Therapeutics: Current equity holder in publicly-traded company; The Wistar Institute: Patents & Royalties: patents pending; Alliance Discovery, Inc.: Current equity holder in private company; Barer Institute: Current equity holder in publicly-traded company; NIH: Research Funding. Montaner:GeneOne Bioscience: Consultancy; Pennsylvania state: Research Funding; NIH: Research Funding; American foundation AIDS: Research Funding; Sauvie Inc.: Consultancy; Gilead Biosciences: Honoraria; Philadelphia Fight: Membership on an entity's Board of Directors or advisory committees.