Unleashing Cell-Intrinsic Inflammation As a Strategy to Kill AML Blasts

Leukemic blasts are immune cells gone awry. We hypothesized that dysregulation of inflammatory pathways contributes to the maintenance of their leukemic state and can be exploited as cell-intrinsic, self-directed immunotherapy. To test the hypothesis, we analyzed three large, independent data collections from primary acute myeloid leukemia (AML) samples and identified an AML subgroup of approximately 40% of all samples enriched for immune and inflammatory pathways. Moreover, we observed a positive correlation between the enrichment of inflammatory response and a monocytic lineage signature in these primary AML samples. To discover genetic vulnerabilities in AML cells implicated in inflammatory pathways, we integrated data from the Cancer Dependency Map on 789 cancer cell lines with two independent genome-wide screens. We identified the immune modulator interferon regulatory factor 2 binding protein 2 (IRF2BP2) as a selective dependency in AML. We validated AML cell dependency on IRF2BP2 with orthogonal genetic and degradation approaches in vitro and in vivo. Genetic perturbation and degradation of IRF2BP2 translated into a reduction of AML cell line viability and decreased colony formation capacity in vitro and impaired leukemia progression in vivo in AML cell line xenografts. Moreover, inducible knock-out of IRF2BP2 significantly attenuated disease burden in multiple patient-derived xenograft models of AML and prolonged survival. Mechanistically, IRF2BP2 repression induced cell death with hallmarks of apoptosis, evidenced by an increase in annexin V/PI-positive cells and induction of cleaved caspase 3 and 8.

To decipher the role of IRF2BP2 in inflammatory signaling, we studied its localization on chromatin and its relationship to histone marks and RNA polymerase II binding in AML cells. We determined that IRF2BP2 binds at both enhancers and promotors. Global gene expression profiling, performed six hours after degradation of IRF2BP2, identified significantly more genes increased than decreased in expression. Most upregulated genes were bound by IRF2BP2 at baseline and gained H3K27ac at enhancers with IRF2BP2 degradation. All told, our data support a role for IRF2BP2 as a transcriptional repressor in AML.

Gene set enrichment analysis of the genes directly regulated by IRF2BP2 identified immune response signatures as the top enriched gene sets, with genes regulated by NF-kB in response to TNFa being the most significantly enriched. We thus hypothesized that IRF2BP2 is a repressor of NF-kB mediated TNFa signaling that, when acutely perturbed, leads to leukemia cell death. Indeed, we observed an activation of NF-κB signaling in a luciferase reporter assay, an increase in nuclear NF-kB (RELA) protein levels, and a gain in NF-κB chromatin binding following degradation of IRF2BP2 in AML cells. Moreover, a mutant "super-repressor" allele of IkBa rescued the impaired cell growth upon IRF2BP2 perturbation in AML cells, supporting cell death associated with IRF2BP2 loss being mediated through activation of NF-kB signaling. In addition, we identified IL-1ß as an enhancer of the inflammatory response repressed by IRF2BP2.

In summary, we have demonstrated that IRF2BP2 represses IL-1ß/TNFα signaling via NF-κB, and IRF2BP2 perturbation results in an acute inflammatory state leading to AML cell death. These findings elucidate a hitherto unexplored AML dependency, reveal cell-intrinsic inflammatory signaling as a mechanism priming leukemic blasts for regulated cell death, and establish IRF2BP2-mediated transcriptional repression as a mechanism for blast survival.