AML-356: High Dimensional Analytic Approaches Identify Stressor Specific Response Patterns in AML

Abstract

Cellular response to stress is diverse, ranging from adaptation to induction of cell death. We designed a multicolor flow cytometry panel to gain insight into multifaceted stress response and to assess multiple cell death modes. This enabled simultaneous interrogation of multiple cell death modes including necrosis, necroptosis, apoptosis and parthanatos as well as proliferation, autophagy and endoplasmic reticulum (ER) stress. Notably, we utilized high dimensional analytic approaches to better elucidate stress response and cell death modes. We aimed to delineate response patterns and cell death modes associated with targeted agents in the context of AML. Bcl-2 or Mcl-1 inhibition alone did not alter cellular landscape. However, treatment with MDM2 or exportin 1 (XPO1) inhibitors and mitochondrial proteasome (ClpP) activator (ONC201) elicited divergent stress responses and cell death modes. Two-dimensional UMAP plots showed differential induction of autophagy and ER stress following treatment with MDM2, XPO1 inhibitors/ClpP activators. Remarkably, we observed that MDM2i and XPO1i were associated with emergence of quiescent cells, based on high p21 expression, and higher levels of ER stress and autophagy while ClpPa induced DNA damage, and was associated with persistent Ki-67 expression and lower levels of p21. This approach enabled us to dissect single agent specific stress signatures. Remarkably, dual combinations differentially altered and skewed response patterns. Strikingly, combinatorial use of MDM2i and XPO1i almost completely eliminated all AML cells except the ones with the highest levels of ER stress and autophagy. On the other hand, addition of either Bcl-2i or MCL-1i to MDM2i markedly reduced p21, ER stress and autophagy, indicating that these anti-apoptotic molecules may play a role in cellular adaptation. We leveraged diffusion map algorithm to map cellular trajectories. This approach enabled us to identify the earliest stage of cell death, characterized by expression of LC3B, H2AX and cleaved PARP while dead cell dye (+) cells marked the latest stage. These findings provide proof of concept for the utility of single cell mapping of cellular stress in delineating stressor-specific response patterns and identifying potential resistance mechanisms. Single cell mapping of cell stress and cell death can inform the development of more effective combinatorial drug regimens.