BRM/BRG1 is a novel therapeutic target in ZNF384-rearranged mixed phenotype acute leukemia Free

Mixed Phenotype Acute Leukemia (MPAL) is an understudied high-risk leukemia with poor outcomes. Patients have been historically excluded from clinical trials, leading to sparse genomic characterization and a lack of standardized therapeutic management. ZNF384 rearrangements (ZNF384-r) have been identified in 50% of patients with childhood B/Myeloid MPAL. ZNF384-r give rise to a chimeric fusion protein that consists of the amino terminus of over 10 functionally diverse fusion partners juxtaposed to the entire amino-acid sequence of ZNF384 at the carboxy terminus. ZNF384 fusion proteins are established oncogenic drivers but how exactly they exert their oncogenic properties remains unclear, with no targeted therapies available.

The mammalian SWI/SNF complex (mSWI/SNF) is a chromatin remodeling complex and master epigenetic regulator of transcription. While it can act as a tumor suppressor in several malignancies, in acute leumemias, mSWI/SNF has been found to be required for aberrant oncogenic gene expression. This has led to the pre-clinical development of several agents targetting BRM and BRG1, the mutually exclusive catalytic subunits of mSWI/SNF, that have been evaluated in both acute myeloid leukemia (AML) and acute lymphoid leukemia (ALL) with encouraging results.

Given that MPAL shares features of both AML and ALL, we hypothesized that BRM/BRG1 inhibition could be a potential therapeutic target in MPAL and specifically in ZNF384-r MPAL. We treated the JIH-5 cell line, the only available ZNF384-r (EP300::ZNF384) MPAL cell line, with FHD-286, a potent dual BRM/BRG1 small molecular inhibitor or AU-24118, a BRM/BRG1 PROTAC degrader. Proliferation assays after 3 and 6 days of treatment, respectively, revealed that JIH-5 cells were sensitive to both agents, with IC50s in the nanomolar range, in keeping with previously published sensitive AML and ALL cell lines.

To broadly understand how inhibition of BRM/BRG1 affects ZNF384-r MPAL, we performed RNA seq in JIH-5 cells after 3 days of treatment with FHD-286 at 100nM. Differential gene expression analysis revealed significant downregulation of genes involved in the regulation of cell cycle such as CDK1, CDK6, CCNB1 and several E2F transcription factors.

To examine the effects of BRM/BRG1 inhibition at the chromatin level, we performed automated Cleavage Under Target & Release Using Nuclease (autoCUT&RUN) after treatment with FHD-286 at 100nM. Our analysis confirmed that treatment with FHD-286 effectively resulted in loss of BRG1 binding. In addition, we found that BRM/BRG1 inhibition resulted in widespread eviction of ZNF384 from chromatin, notably at ETS-like domains.

In summary, we demonstrate that inhibition of BRM/BRG1 is a novel therapeutic strategy in ZNF384-r MPAL. Treatment with FHD-286 or AU-24118 leads to decreased survival of JIH-5 cells in vitro, through inhibition of cell cycle. At the chromatin level, FHD-286 not only results in loss of BRG1 binding but also, interestingly, in loss of ZNF384 binding, suggesting that inhibiton of BRM/BRG1 may specifically affect the ZNF384 fusion protein. We have established cell line-derived and patient-derived xenograft models of ZNF384-r MPAL and will confirm our findings in vivo in pre-clinical studies.