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Reversible Epigenetic Mechanism at Root of Treatment Resistance in T-ALL

NEW YORK (GenomeWeb News) – Treatment resistance in T-cell acute lymphoblastic leukemia can arise through a reversible epigenetic mechanism, researchers led by Bradley Bernstein from Massachusetts General Hospital and Harvard University reported in Nature Genetics yesterday.

Scientists have identified activating NOTCH1 mutations in T-ALL and tested γ-secretase inhibitors (GSIs) that block its activation to treat the disease, but response to such inhibitors has been short-lived, Bernstein and his colleagues wrote in their paper. By modeling T-ALL resistance, they found that a certain subset of T-ALL cells expanded even with the suppression of NOTCH1 signaling. Additionally, they noticed that when the GSI was removed, NOTCH signaling returned to those persister cells.

"Rare persisters are already present in naive T-ALL populations, and the reversibility of their phenotype suggests an epigenetic mechanism," Bernstein and his colleagues said.

Notably, they found that the chromatin regulator BRD4, which binds near the critical T-ALL genes MYC and BCL2, appears to be essential for persister survival.

"Our findings establish a role for epigenetic heterogeneity in leukemia resistance that may be addressed by incorporating epigenetic modulators in combination therapy," they added.

Through modeling T-ALL resistance by chronically exposing T-ALL cells to a GSI, the researchers found that GSI resistance was reversible. In persister cells, the active intracellular form of NOTCH1 was not expressed, and NOTCH1 target genes like DTX1 and HES4 were also downregulated. When the researchers removed GSI from the cells, they began to express NOTCH1 and its activated form again. Re-application of the inhibitor then led to the downregulation of NOTCH.

Persister cells, they also noted, could tolerate inhibitor levels more than 50-fold higher than naive cells.

Naive T-ALL cells treated with a GSI also experience a dip in MYC protein levels. MYC induction, the researchers noted, is thought to be one mechanism through which constitutively active NOTCH1 signaling leads to leukemic T cell transformation.

Persister cells, though, regained a moderate level of MYC protein expression, even in the face of continued inhibitor application, the researchers reported. The cells also exhibited other hints of disordered signaling as they had higher levels of phosphorylated PTEN and higher levels of phosphorylated mTOR. Rewired signaling, the researchers said, may be how the persister cells are able to continue to express MYC and proliferation without NOTCH1 activity.

This resistance, they found, seems to stem from a distinct subpopulation of T-ALL cells, rather than being induced by treatment, as they found that about 4 percent of naive T-ALL cells could expand in the presence of GSI.

The persister cells also look different from other T-ALL cells as both their cell and nuclear size was smaller, something Bernstein and colleagues attributed to global chromatin change resulting from NOTCH1 inhibition.

This change in chromatin state could offer additional targets for epigenetic therapy, they noted. To search for such possible targets, the researchers designed a lentiviral short hairpin RNA knockdown screen that targeted some 350 chromatin regulators with an average five hairpins per gene. From this, they found about 15 genes that, when knocked down, affected the survival of both naive and persister cells.

A top hit from this screen was BRD4, which is part of the BET family of bromodomain proteins that bind acetylated histones and has been linked to other cancers. The researchers also noted that BRD4 expression, at both the mRNA and protein levels, increased when NOTCH1 was inhibited and was higher in persister cells.

Using ChIP-seq, they mapped BRD4 binding in naive and persister T-ALL cells, and found that it binds promotors and putative enhancers enriched for H3K3me1 and HeK27ac marks. Further, many of the nearby genes encode known T-ALL regulations like the transcription factor ETV6, the cell cycle regulator CDK6, and the prosurvival protein BCL2.

"BRD4 binds and may sustain the activity of the regulatory elements and target genes required for the proliferation of T-ALL cells," they said.

As they noted, though, persister cells had a higher degree of chromatin compaction, leading them to suspect that enhancers in those cells are especially dependent upon BRD4 for epigenetic maintenance. Indeed, they noted that the subset of naive, GSI-tolerant T-ALL cells were highly BRD4-dependent.

Based on this, Bernstein and his colleagues suspected that combining NOTCH1 and BRD4 inhibition could be an effective T-ALL treatment. In three pediatric tumors, they tested whether those samples were sensitive to GSI, the BET inhibitor JQ1, or both in vitro and in a mouse model of the disease. In mice, combination therapy significantly improved survival compared to single-agent treatment.

"The effect of combination therapy in vivo is particularly striking given the short duration of treatment, the fact that treatment was initiated upon reaching a substantial leukemic burden, and the refractory nature of the relapsed samples examined," Bernstein and his colleagues said. "Our study provides a framework for understanding epigenetic alterations and heterogeneity in tumor pathogenesis and suggests that drug resistance may be addressed by combination therapies that incorporate epigenetic modulators."