The bromodomain and extra-terminal domain protein BRD4 promotes both self renewal and inflammation in TET2 mutated clonal hematopoiesis Free

Progression of clonal hematopoiesis (CH) to myeloid neoplasms (MN) is highly dependent upon inflammatory signaling. The epigenetic reader protein bromo- and extra-terminal domain 4 (BRD4) interacts with NF-kB and STAT transcription factors to promote inflammatory programs downstream of cytokines with roles in CH. Therefore, we hypothesized BRD4 inhibition would reverse abnormal hematopoiesis in CH via reduction in inflammatory signaling.

PBMCs from healthy donors or CCUS patients (n=7 TET2 mutations, 5 with other mutations,12 healthy donors) were cultured with LPS ± the BET inhibitor PLX51107. IL-1β, IL-6, IL-12p40, IL-18, IFN-γ, MCP-1, and TNFα were increased (p<0.01) following LPS in CCUS PBMCs vs healthy donors. PLX51107 reduced LPS-induced cytokines in both groups, but CCUS samples showed markedly increased sensitivity. Cytokine mRNA expression showed similar results.

We next generated a Mx1 Cre Brd4 Tet2 conditional KO mouse model to further examine Brd4's role in Tet2 CH. Because monocytes are a major cytokine source in CH, we used bone marrow derived macrophages (BMDM) to investigate Brd4's role inflammation in Tet2 KO. Brd4 Tet2 DKO BMDM showed reduced cytokine mRNA expression and produced significantly less TNF-α, IL-1β, IL-12p40, and IL-18 protein in response to LPS vs single KO or WT controls (p<0.01, n=5). Similar reductions were observed in LPS-stimulated Tet2 KO and WT BMDM treated with PLX51107. Both genetic and pharmacologic Brd4 inhibition reduced pSTAT3, NLRP3, pERK and phosphorylated p65 protein in LPS-stimulated Tet2 KO BMDM. Similar to human CCUS PBMC, LPS-induced cytokines were more strongly reduced in BETi treated murine Tet2 KO BMDM vs WT.

To assess Brd4's impact on the competitive fitness of Tet2 KO HSCs, we performed competitive repopulation studies using CD45.2+ WT, Tet2 KO, Brd4 KO, or Brd4 Tet2 DKO marrow mixed 1:1 with CD45.1+ competitor marrow and transplanted into lethally irradiated CD45.2+/CD45.1+ recipients (n=3 per group), followed by polyIC induction. As expected, Tet2 KO HSC showed increased repopulating capacity, with expansion of CD45.2+ cells over WT noted at 12 weeks (p<0.01). In contrast, DKO cells demonstrated no competitive advantage. Five months post-transplant, CD45.2+ MPP1 and CMP were significantly expanded (p<0.01) in Tet2 KO recipients, but not in DKO recipients. Furthermore, DKO marrow showed reduced colony formation and replating capability vs Tet2 KO (p<0.0001, n=5) or WT (p<0.03, n=5) marrow. Similarly, PLX51107 abrogated the increased colony formation (p<0.0001, n=5) and serial replating (p<0.01, n=5) of Tet2 KO marrow.

Tet2 KO mice exhibit exaggerated emergency granulopoiesis upon stimulation with TLR agonists like LPS. Interestingly, Brd4 Tet2 DKO mice showed significant reductions of CD11b+ Ly-6G+ neutrophils, Ly-6Chi+ monocytes and CD11b+ Gr-1+ myeloid cells vs Tet2 KO (n=6, p<0.01) 2 days after a sublethal LPS dose. At day 7 post-LPS, LSK, MPP1, and MPP3 populations were reduced (p<0.05 for all) in DKO vs Tet2 KO mice. Other progenitor populations (MPP4, CMP, MEP, GMP) were unchanged, suggesting Brd4 has developmentally specific roles in Tet2 CH. Pretreatment of Tet2 KO mice with low-dose PLX51107 (10 mg/kg daily) for one week prior to LPS yielded similar effects on myeloid populations in the blood and marrow.

To identify Brd4 dependent transcriptional programs in Tet2 KO HSC, we next performed RNA-seq on LSK isolated from LPS treated or control mice. Consistent with previous studies, GSEA of Tet2 KO LSK identified increased expression of hallmark inflammatory pathways, including TNFα signaling via NF-kB, IL6/JAK/STAT3 signaling, and IFNγ response. Interestingly, these expression signatures were among the top negatively enriched pathways in DKO vs Tet2 KO LSK. Additionally, genes with altered expression in prior human CH RNA-seq datasets were decreased in DKO LSK, including Ccl3, Ccl4, Dusp1 and Irak1. We further identified a unique gene signature of 207 Brd4-dependent genes upregulated in Tet2 KO vs DKO LSK. This signature included genes with roles in neutrophil activation, such as Cd177, Ctsg and Cxcr2, thereby linking altered HSC function to the reduced neutrophil response in DKO mice during emergency granulopoiesis.In summary, we have identified a critical role for Brd4 in self-renewal, inflammation, and myeloid expansion in Tet2 mutated CH. Collectively, these results support development of BET inhibitors to prevent progression of high risk CH to MN.