Overcoming drug resistance to BET inhibitors by rational combinative strategies

Bromodomain and extra-terminal (BET) family members, including BRD2, BRD3, BRD4 and BRDT, function as critical readers of acetylated histones and globally activate oncogene expression. These proteins have emerged as attractive epigenetic targets for cancer therapy, with more than 50 clinical trials over the past two decades targeting lymphoma and solid tumors. Unfortunately, inevitable drug resistance to BET inhibitors (BETi) remains a significant clinical challenge: castration-resistant prostate cancer (CRPC) and non- small cell lung cancer (NSCLC) often exhibit intrinsic response to BET inhibition, showing limited therapeutic response at the outset of treatment; while triple-negative breast cancer (TNBC) and acute myeloid leukemia (AML) initially respond to BETi but rapidly develop acquired resistance within six months.

To date, significant progress has been made in elucidating the mechanisms underlying resistance to BET inhibitors (BETi). In CRPC, somatic mutations in the SPOP gene impair the ubiquitin degradation system, leading to BRD4 upregulation and conferring inherent resistance to BETi. Our previous work provided further insight into non-mutation-driven inherent BETi resistance in NSCLC, where non-selective inhibition of BRD3 by BET inhibitors unexpectedly activates the oncogene BCL6 to enhance mTOR pathway-mediated cell proliferation. In consistent with the oncogenic roles of BCL6, blocking such stress-related signaling by using BCL6 inhibitors or mTOR inhibitors achieved robust synergistic effects with BET inhibitors. These findings further support an effective combinatorial strategy with potential translatability to sensitize NSCLC to clinical BETi by concurrent inhibition of either BCL6 or mTOR. We believe our findings are of paramount importance, considering that clinical trials of BETi are now being conducted in intractable cancers with limited effectiveness in patients. Additionally, although recent studies revealed that distinguishing BD1 and BD2 of the BET proteins may guide future BET-targeted therapies our findings suggest that development of a BRD4-specific inhibitor may be a pivotal therapeutic option for patients with KRAS-mutant NSCLC, as selective inhibition of BRD4 avoids BCL6 upregulation.

However, in contrast to NSCLCs that exhibit poor response to BET inhibition at the beginning of treatment, TNBC trends to be sensitive to BET inhibition but rapidly develops acquired drug resistance in six months. The underlying mechanisms of acquired BETi resistance is not fully elucidated. In this study, we identified the RNA-binding protein IGF2BP2 as a key driver of acquired BETi resistance in TNBC, primarily through its role in enhancing the translation of c-MYC mRNA. Given that IGF2BP2 is not an ideal target for small-molecular drugs, we performed RNA immunoprecipitation sequencing (RIP-Seq) and discovered circRNA-BISC as a potent IGF2BP2 repressor. BISC effectively inhibited both c-MYC translation and BETi resistance. Notably, BISC contains a ‘CAC-linker-XGGX’ motif that specifically binds IGF2BP2 without interactions with IGF2BP1 and IGF2BP3. The efficacy and selectivity of BISC in targeting IGF2BP2 prompted further exploration of BISC-based RNA therapeutics for TNBC. In vitro transcribed and circularized BISC, when combined with the BET inhibitor OTX-015, demonstrated impressive tumor regression in BETi- resistant TNBC models without detectable toxicity. These findings establish BISC as a potent IGF2BP2 repressor and highlight the feasibility of circRNA-based therapeutic strategies to overcome BETi resistance in TNBC.