AML-144 Uroporphyrinogen Decarboxylase (UROD) Is a Therapeutically Targetable Dependency in Acute Erythroid Leukemia

Abstract

Acute erythroid leukemia (AEL) is a disease of erythroid lineage that commonly exhibits TP53 mutations, a complex karyotype, and poor prognosis irrespective of currently available therapies. To identify AEL dependencies and novel therapeutic targets in AEL using genome-wide CRISPR/Cas9 knockout screens in mouse models of AEL. Analysis of the screens performed using the Brie library identified the Urod gene encoding uroporphyrinogen decarboxylase as a critical dependency in two Trp53/Bcor mutated mouse AEL cell lines. UROD is a cytosolic enzyme in the heme biosynthesis pathway involved in converting cytosolic uroporphyrinogen III into the key heme biosynthetic intermediate, coproporphyrinogen III. In an analysis of RNA-seq data from over 2,000 acute leukemia samples, UROD was significantly overexpressed and active by a data-driven network inference algorithm in AEL compared to other leukemia subtypes. We validated UROD as a dependency by electroporation of Cas9 and synthetic sgRNA ribonucleoprotein complex in the two screened cell lines, additional mouse leukemia cell lines, and two commercially available human AEL cell lines. Targeted knockout of UROD resulted in decreased tumor cell growth and viability compared to the non-targeting guide control cells. Sequencing analysis showed high editing efficiency (>85%) followed by recovery of the wildtype cells, indicating that UROD is a dependency in these cells. These results correlated with a significant increase of necrotic cells preceding loss of editing in the two tested human cell lines. We also observed an increase in ALAS1 expression, which is negatively regulated by cellular heme, indicating that UROD knockout leukemic cells cannot generate heme, leading to necrotic cell death. Moreover, mitochondrial function assays showed significant decreases in basal respiration, ATP production, maximal respiration, and spare capacity in a time-dependent manner, followed by the rescue of mitochondrial function after loss of editing efficiency and re-expression of UROD. We have recently developed homozygous UROD-dTAG engineered human AEL cell lines to perform in-vivo survival studies. Through whole-genome CRISPR knockout screening and functional studies, we show that UROD is a dependency in AEL and provides a basis for exploring its therapeutic inhibition.