DDX6 undergoes phase separation to modulate metabolic plasticity and chemoresistance Free

Stress granules (SGs) and processing bodies (PBs), assembled via liquid-liquid phase separation (LLPS), are critical for spatial regulation of gene expression in cytoplasm. Evidence is now mounting that SGs and PBs form in response to various cellular stresses, including chemotherapy. However, their precise roles in leukemogenesis and chemosensitivity remain poorly understood. Here, we show that DEAD-box helicase 6 (DDX6) undergoes LLPS to trigger PB assembly, thereby modulating metabolic plasticity and chemotherapy response in acute myeloid leukemia (AML).

To investigate the role of cytoplasmic LLPS granules in leukemogenesis, we developed a focused CRISPR library containing 2,084 sgRNAs targeting all the high-confidence SG and PB proteins for both in vitro (using AML cell lines and patient-derived xenograft [PDX] cells) and in vivo (using AML PDX models) screening. This analysis pinpointed DDX6 as one of the most promising vulnerabilities in AML. Knockout (KO) of DDX6 significantly suppresses AML leukemogenesis and markedly reduces PB assembly, without profound impacts on normal hematopoiesis.

Fluorescence recovery after photobleaching assay demonstrated a robust LLPS capacity of DDX6 protein (average recovery half-time: 4.2s). Droplet formation assay independently confirmed its LLPS ability; moreover, poly(U)-RNA rather than poly(C)-RNA could greatly stimulate DDX6 LLPS (P = 1.82 × 10^-6). Furthermore, we engineered a series of DDX6 truncated variants to evaluate which region of DDX6 is critical for LLPS. Meanwhile, we conducted rescue assays with those truncated variants in DDX6-deficient AML cells. These experiments identified that N-terminal intrinsically disordered region of DDX6 is not only important for its LLPS capacity and PB assembly, but also crucial for its biological function in AML.

Integrative multi-omics analysis of CLIP-seq (to profiling DDX6-binding mRNAs), RNA-seq (to profiling the dysregulated mRNAs following DDX6 KO), PB RNA-seq (to profiling PB-enriched mRNAs and PB-depleted mRNAs) revealed that: (i)DDX6 KO leads to a global reduction of DDX6-binding & PB-enriched mRNAs (87 up-regulated vs 226 down-regulated mRNAs; P < 0.05); and (iii) DDX6-binding & PB-enriched mRNAs with decreased levels upon DDX6 KO had a much lower GC content than the DDX6-binding & PB-depleted transcripts with increased levels upon DDX6 KO (P = 1.0 × 10^-89). Of note, BCAT1 (Branched-Chain Amino acid [BCAA] Transaminase 1) mRNA (GC content: 37%) stands out as the most significantly down-regulated transcript following DDX6 KO and demonstrates a significant positive correlation with DDX6 in AML. Single-molecule fluorescent in situ hybridization (smFISH) detected the localization of BCAT1 mRNA within PBs. DDX6 positively regulated BCAT1 mRNA stability and expression in an LLPS dependent manner. Moreover, Metabolic profiling combined with isotope tracing (¹³C,¹⁵N-leucine) orthogonally confirmed the crucial role of DDX6 in BCAA metabolism. These findings demonstrate that DDX6 preferentially maintains the steady-state levels of its binding transcripts, such as BCAT1, that are enriched in PBs.

Given the established role of BCAA metabolism in drug resistance and our identification of BCAT1 as a functionally essential target of DDX6, we explored the potential impact of DDX6 on the response of leukemia cells to first-line chemotherapy drugs. Notably, depletion of DDX6 or BCAT1 significantly increased the sensitivity of leukemia cells to Ara-C treatment. Additional AML “human-in-mouse” xenograft and PDX models further demonstrate that combination of Ara-C and DDX6 KO resulted in a more pronounced inhibition of AML leukemogenesis.

Altogether, this study identified PBs as “reservoirs” for the mRNAs such as BCAT1, which directly interact with DDX6 and are characterized by low GC content. By reducing BCAT1 levels, DDX6 KO reprograms amino acid metabolism and sensitizes AML cells to chemotherapy. These results uncover a previously unrecognized role of LLPS-mediated membraneless organelles in regulating leukemogenesis, metabolic plasticity, and chemotherapy response.