ASH Late-Breaker Highlights for Tuesday December 8, 2020

By Lynne Lederman, PhD

Enhancer Hijacking of BCL11B Defines a Subtype of Lineage Ambiguous Acute Leukemia (LBA3)

Lindsey Montefiori, PhD, MD, St. Jude Children’s Research Hospital, Memphis, TN, presented evidence that BCL11B structural variants (SV) define a new subtype of acute leukemias of ambiguous lineage (ALAL) that includes subsets of T/myeloid mixed phenotype acute leukemia (MPAL), early T cell precursor acute lymphoblastic leukemia (ETP-ALL, acute undifferentiated leukemia (AUL) and acute myeloid leukemia (AML). ALAL are difficult to diagnose, classify, and treat. This study analyzed 2,573 pediatric and adult samples, including 1,411 B-acute lymphocytic leukemia (ALL), 262 AML 126 MPAL, and 774 T-ALL.

Among BCL11B SV, a novel, high-copy, tandem amplification of a 2.5 kb noncoding region 700 kb downstream of BCL11B on chromosome 14, termed BCL11B Enhancer Tandem Amplification (BETA) was identified and occurs in 20% of the BCL11B subtype. BCL11B breakpoints were shown to occur near CD34+ hematopoietic stem/progenitor cell (HSPC) super enhancers.

Histone H3 lysine 27 acetyl (H3K27ac) chromatin conformation capture followed by high-throughput sequencing (HiChIP) confirmed rearranged enhancers are active and loop to BCL11B. BETA therefore represents a new mechanism of de novo super enhancer formation in leukemia.

Dr. Montefiori said they are eager to develop mouse models to investigate BCL11B expression in HSPC. She said that BETA represents a unique mode of generating a transcriptional activator in any context, and that they are pursuing the possibility that it could be used as a biomarker. RNA can be detected from this region in leukemic cells, and in the absence of whole genome sequencing data they have been able to detect samples harboring the tandem amplification, reflecting its strong enhancer activity.

ETNK1 Mutations in Atypical Chronic Myeloid Leukemia Induce a Mutator Phenotype That Can be Reverted with Phosphoethanolamine (LBA5)

Recurrent somatic mutations in ETNK1 kinase, which phosphorylates ethanolamine to phosphoethanolamine (P-Et), a precursor of essential cellular phospholipids. These mutations cluster in the catalytic domain, and occur in 13% of patients with atypical chronic myeloid leukemia (aCML), as well as 3-14% of chronic myelomonocytic leukemia (CMML), and 20% of systemic mastocytosis (SM) with eosinophilia.

Diletta Fontana, PhD, University of Milano-Bicocca, Monza, Italy, discussed experiments to examine the oncogenic role of ETNK1 mutations using a cellular CRISPR/Cas9 and ETNK1 overexpression models as well as aCML patients samples.

Mitochondrial activity was significantly increased in ETNK1 mutant or knockout cells compared to wild-type. P-Et treatment restored this activity to wildtype levels. Likewise, both ETKN1 mutant or knockout cells showed increased levels of mitochondrial reactive oxygen species (ROS) production versus wild-type. Similarly, P-Et treatment restored normal ROS production.

Oxoguanine is a specific marker of ROS-induced DNA oxidative mutagenesis. ChIP-Seq data for ETNK1 mutated cells generated using an antibody raised against oxoguanine, revealed a significant increase in oxoguanine in mutated cells, compared with wild-type (P=0.018).

To see if these lesions were driving the onset of a mutator phenotype, a 6-thioguanine resistance assays in the cell models, was performed, showing that in the mutated cells there was a 5.4-fold increase in colony number compared with the wild-type line (P<.0001) that was completely reversed by P-Et treatment.

ETNK1 mutations were associated with genomic DNA double-strand breaks in the cell models as well as in cells from patients with aCML. Mitochondrial ROS production and genomic DNA damage were decreased after P-Et treatment in both the models and patient cells. Mitochondrial DNA damage was not seen.

P-Et was found to control mitochondria potential through direct inhibition of complex II, also known as of succinate dehydrogenase (SDH), by binding to the SDH catalytic domain and competitively inhibiting succinate at concentrations of at least 50µM.


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