Inhibition of Dihydroorotate Dehydrogenase Overcomes Differentiation Blockade in Acute Myeloid Leukemia.

While acute myeloid leukemia (AML) comprises many disparate genetic subtypes, one shared hallmark is the arrest of leukemic myeloblasts at an immature and self-renewing stage of development. Therapies that overcome differentiation arrest represent a powerful treatment strategy. We leveraged the observation that the majority of AML, despite their genetically heterogeneity, share in the expression of HoxA9, a gene normally downregulated during myeloid differentiation. Using a conditional HoxA9 model system, we performed a high-throughput phenotypic screen and defined compounds that overcame differentiation blockade. Target identification led to the unanticipated discovery that inhibition of the enzyme dihydroorotate dehydrogenase (DHODH) enables myeloid differentiation in human and mouse AML models. In vivo, DHODH inhibitors reduced leukemic cell burden, decreased levels of leukemia-initiating cells, and improved survival. These data demonstrate the role of DHODH as a metabolic regulator of differentiation and point to its inhibition as a strategy for overcoming differentiation blockade in AML.

Transfusable neutrophil progenitors as cellular therapy for the prevention of invasive fungal infections.

The use of mature neutrophil (granulocyte) transfusions for the treatment of neutropenic patients with invasive fungal infections (IFIs) has been the focus of multiple clinical trials. Despite these efforts, the transfusion of mature neutrophils has resulted in limited clinical benefit, likely owing to problems of insufficient numbers and the very short lifespan of these donor cells. In this report, we employed a system of conditionally immortalized murine neutrophil progenitors that are capable of continuous expansion, allowing for the generation of unlimited numbers of homogenous granulocyte-macrophage progenitors (GMPs). These GMPs were assayed in vivo to demonstrate their effect on survival in 2 models of IFI: candidemia and pulmonary aspergillosis. Mature neutrophils derived from GMPs executed all cardinal functions of neutrophils. Transfused GMPs homed to the bone marrow and spleen, where they completed normal differentiation to mature neutrophils. These neutrophils were capable of homing and extravasation in response to inflammatory stimuli using a sterile peritoneal challenge model. Furthermore, conditionally immortalized GMP transfusions significantly improved survival in models of candidemia and pulmonary aspergillosis. These data confirm the therapeutic benefit of prophylactic GMP transfusions in the setting of neutropenia and encourage development of progenitor cellular therapies for the management of fungal disease in high-risk patients.

Flow Cytometry-Based Detection and Analysis of BCL-2 Family Proteins and Mitochondrial Outer Membrane Permeabilization (MOMP).

The BCL-2 family of proteins orchestrates a complex signaling network that governs the balance between cellular survival and death. A comprehensive understanding of the mechanistic interactions between these proteins continues to evolve in normal and malignant cells. The functional variation by individual BCL-2 proteins in different cell types has driven clinical therapeutic development in targeting individual BCL-2 members with the goal of fine-tuning cell death in diseased cells. Given the importance of understanding and validating the effect of activating or inhibiting BCL-2 protein interactions in individual cells, the methods used to measure apoptotic cell death have undergone increased scrutiny. Here, we describe two in vitro flow cytometry-based methods that are useful in measuring BCL-2 proteins and mitochondrial-based cell death in complex cell populations.