Targeting KDM4 for treating PAX3-FOXO1-driven alveolar rhabdomyosarcoma.

Chimeric transcription factors drive lineage-specific oncogenesis but are notoriously difficult to target. Alveolar rhabdomyosarcoma (RMS) is an aggressive childhood soft tissue sarcoma transformed by the pathognomonic Paired Box 3-Forkhead Box O1 (PAX3-FOXO1) fusion protein, which governs a core regulatory circuitry transcription factor network. Here, we show that the histone lysine demethylase 4B (KDM4B) is a therapeutic vulnerability for PAX3-FOXO1+ RMS. Genetic and pharmacologic inhibition of KDM4B substantially delayed tumor growth. Suppression of KDM4 proteins inhibited the expression of core oncogenic transcription factors and caused epigenetic alterations of PAX3-FOXO1-governed superenhancers. Combining KDM4 inhibition with cytotoxic chemotherapy led to tumor regression in preclinical PAX3-FOXO1+ RMS subcutaneous xenograft models. In summary, we identified a targetable mechanism required for maintenance of the PAX3-FOXO1-related transcription factor network, which may translate to a therapeutic approach for fusion-positive RMS.

EP300 Selectively Controls the Enhancer Landscape of MYCN-Amplified Neuroblastoma.

Gene expression is regulated by promoters and enhancers marked by histone H3 lysine 27 acetylation (H3K27ac), which is established by the paralogous histone acetyltransferases (HAT) EP300 and CBP. These enzymes display overlapping regulatory roles in untransformed cells, but less characterized roles in cancer cells. We demonstrate that the majority of high-risk pediatric neuroblastoma (NB) depends on EP300, whereas CBP has a limited role. EP300 controls enhancer acetylation by interacting with TFAP2ß, a transcription factor member of the lineage-defining transcriptional core regulatory circuitry (CRC) in NB. To disrupt EP300, we developed a proteolysis-targeting chimera (PROTAC) compound termed "JQAD1" that selectively targets EP300 for degradation. JQAD1 treatment causes loss of H3K27ac at CRC enhancers and rapid NB apoptosis, with limited toxicity to untransformed cells where CBP may compensate. Furthermore, JQAD1 activity is critically determined by cereblon (CRBN) expression across NB cells. SIGNIFICANCE: EP300, but not CBP, controls oncogenic CRC-driven transcription in high-risk NB by binding TFAP2ß. We developed JQAD1, a CRBN-dependent PROTAC degrader with preferential activity against EP300 and demonstrated its activity in NB. JQAD1 has limited toxicity to untransformed cells and is effective in vivo in a CRBN-dependent manner. This article is highlighted in the In This Issue feature, p. 587.

Histone H3 Mutations: An Updated View of Their Role in Chromatin Deregulation and Cancer.

In this review, we describe the attributes of histone H3 mutants identified in cancer. H3 mutants were first identified in genes encoding H3.3, in pediatric high-grade glioma, and subsequently in chondrosarcomas and giant cell tumors of bone (GCTB) in adolescents. The most heavily studied are the lysine to methionine mutants K27M and K36M, which perturb the target site for specific lysine methyltransferases and dominantly perturb methylation of corresponding lysines in other histone H3 proteins. We discuss recent progress in defining the consequences of these mutations on chromatin, including a newly emerging view of the central importance of the disruption of H3K36 modification in many distinct K to M histone mutant cancers. We also review new work exploring the role of H3.3 G34 mutants identified in pediatric glioma and GCTB. G34 is not itself post-translationally modified, but G34 mutation impinges on the modification of H3K36. Here, we ask if G34R mutation generates a new site for methylation on the histone tail. Finally, we consider evidence indicating that histone mutations might be more widespread in cancer than previously thought, and if the perceived bias towards mutation of H3.3 is real or reflects the biology of tumors in which the histone mutants were first identified.

Hematologic reference intervals for Xenopus tropicalis with partial use of automatic counting methods and reliability of long-term stored samples.

BACKGROUND: The African frog, Xenopus tropicalis, is widely used in biomedical and toxicologic research. Reference intervals (RI) for hematologic variables, valuable to research and health status assessment, have not been established. OBJECTIVES: The purpose of the study was to determine hematologic RI of X tropicalis, and establish whether automated cell counting can facilitate routine hematologic assessment in frogs. METHODS: Blood from 41 adult healthy X tropicalis was collected via cardiac puncture, and diluted in Natt-Herrick solution. Complete WBC, RBC, and thrombocyte counts (hemocytometry), differential WBC counts (Wright-Giemsa-stained smears), PCV (centrifugation), total protein (refractometry), and automated total cell counts (WBC + RBC + thrombocytes, Sysmex particle counting) were determined. Concordance correlation coefficients calculated the agreement between total cell counts obtained by hemocytometry and automated particle counting, and between total cell counts at collection and after 2 years of storage. RESULTS: Leukocyte morphology was similar to other amphibians and mammals. PCV was similar to other frogs; RBC counts were higher, and MCV was lower than in other frog species. Neutrophils were the most numerous WBC. Agreement was good between hemocytometry and automated cell counts. Subtracting the hemocytometer WBC and thrombocyte counts from the automated total cell count reliably yielded the RBC count. Cellular integrity evaluated 2 years post collection was good, and automated counts were not clinically different from counts at collection. CONCLUSION: We provide hematologic RI for X tropicalis, suggest how automated cell counts may facilitate hematologic assessments of frogs, and establish that blood in Natt-Herrick solution is stable 2 years post collection.