Scientists Map the Hidden Genome-Wide Targets of a Key Inflammatory Switch in Leukemia

Chronic lymphocytic leukemia, the most common leukemia in adults, has long been known to thrive on signals from its microenvironment, particularly inflammatory cues that flow through Toll-like receptors and the NF-κB family of transcription factors. A new study published in iScience has now delivered the first comprehensive genome-wide map of where IκBζ, an atypical member of the IκB protein family, binds across the chromatin of primary human leukemic B cells. The work, led by Ilenia Sana, Miriam Meloni, Francesca Genova and colleagues in the laboratory of Marta Muzio at IRCCS San Raffaele Hospital, reveals that this single nuclear regulator orchestrates a surprisingly broad program spanning B cell receptor signaling, NF-κB activation, apoptosis control, and even the DNA damage response.

IκBζ, encoded by the NFKBIZ gene, is not a conventional inhibitor. While classical IκB proteins sit in the cytoplasm and restrain NF-κB until an inflammatory signal arrives, IκBζ is induced at the transcriptional level during the first wave of the inflammatory response and then accumulates in the nucleus, where it shapes the second wave of inflammation in cooperation with the p65 and p50 subunits of NF-κB. Recent structural and functional work has expanded this picture considerably: IκBζ can act as a coactivator of POU-family lineage transcription factors through two distinct interaction domains, and in melanoma cells it has been shown to enhance STAT3-dependent transcription at selected loci while repressing other targets through recruitment of chromatin-modifying enzymes such as EZH2 and HDAC3. This context-dependent versatility is precisely what made its role in B cell malignancies so intriguing and so poorly defined.

Aberrant IκBζ expression had already been implicated in lymphoid cancers. In a subset of activated B cell-like diffuse large B cell lymphomas, mutations in the 3′ untranslated region of NFKBIZ stabilize the mRNA and drive elevated protein levels, and NFKBIZ has emerged as a selective dependency gene in both B and T cell lymphomas across large-scale functional genomic screens. In chronic lymphocytic leukemia, the same 3′ UTR mutation appears only occasionally, but post-transcriptional mechanisms appear to be at work: when leukemic cells are stimulated in vitro with Toll-like receptor ligands that mimic microenvironmental signals, NFKBIZ mRNA rises sharply and IκBζ protein climbs even more dramatically. Higher IκBζ expression is found in a subset of CLL samples carrying negative prognostic markers, and pharmacologically blocking this axis with inhibitors such as dimethyl fumarate has shown significant therapeutic activity in preclinical models. What remained missing was the fundamental piece of the puzzle: the identity of the genes that IκBζ directly controls in these cells.

To close that gap, the team applied two complementary genome-binding technologies, chromatin immunoprecipitation followed by sequencing and the more recently developed CUT&Tag method, to B lymphocytes isolated from the peripheral blood of healthy donors and CLL patients. Unstimulated cells served as an internal control because they contain little to no IκBζ protein, whereas stimulation with CpG oligonucleotides, which activate the innate DNA-sensing receptor TLR9, induces high levels of the protein together with robust NF-κB activation. The two platforms behaved very differently in scale: ChIP-seq recorded roughly a fifty-five-fold increase in IκBζ binding sites after stimulation, while CUT&Tag showed about a three-fold increase, a discrepancy the authors attribute to protocol differences, with double-crosslinked ChIP capturing both direct and unstable interactions across millions of cells and native-condition CUT&Tag excelling at identifying the strongest, most direct binding events from just a few hundred nuclei.

By intersecting the consensus peak sets from both platforms at matching genomic coordinates, requiring at least 100 base pairs of reciprocal overlap, the researchers defined 421 high-confidence IκBζ binding sites that were reproducible across technologies and independent patient cohorts. Genomic annotation revealed a consistent and striking shift toward promoter-proximal binding upon stimulation: in CUT&Tag, the fraction of peaks at promoters rose from 60.8 percent to 69.0 percent, and in ChIP-seq it jumped from 9.3 percent to 33.1 percent, while intronic and intergenic peaks declined on both platforms. At the gene level, the intersection of newly gained targets across both datasets identified 654 genes recruited to IκBζ upon CpG stimulation, and only a single gene, the kinase HUNK, was lost, underscoring that the response is dominated by gain rather than loss of occupancy, exactly as expected given the near-absence of the protein in resting cells.

Pathway enrichment analysis of the 394 unique genes associated with the 421 high-confidence peaks, conducted across seven curated databases, converged on eight non-redundant biological themes. The strongest signal was B cell receptor signaling, followed closely by TNF-α/NF-κB signaling, IL-2/STAT5 signaling, and chemokine signaling. Apoptosis appeared at two levels of resolution, including the specific BCL-2-regulated program through BH3-only protein interaction, consistent with the identification of the anti-apoptotic gene BCL2 itself among the targets. Perhaps most unexpectedly, the ATM-dependent DNA damage response was significantly enriched, pointing to an unanticipated connection between IκBζ-regulated chromatin and genomic stress programs in leukemic cells. A stricter differential binding analysis, requiring genes to satisfy consensus gain, ChIP-seq differential gain, and CUT&Tag differential gain with a false discovery rate below 0.05, distilled the list to 39 top-tier candidate targets, including B cell transcriptional regulators such as FOXP1, BATF, TCF4, TCF7 and POU2F2, NF-κB pathway components such as TNIP1, TNFSF4 and CD40LG, and chromatin-modifying factors including NCOR2 and SMARCE1.

Functional validation followed. The team first showed that dimethyl fumarate, a drug known to blunt the TLR/IκBζ axis, reduced CpG-induced expression of candidate targets in primary CLL cells. They then deployed CRISPR-Cas9 gene editing to knock out NFKBIZ in seven fresh primary leukemic samples. Real-time PCR demonstrated that two targets, NFKB2, which encodes the p100/p52 precursor of the non-canonical NF-κB pathway, and CXCR5, a chemokine receptor overexpressed in CLL compared with normal circulating B cells, were no longer induced in the absence of IκBζ. BATF showed a trend toward reduced induction, while FOXP1, whose locus carried multiple IκBζ binding regions, was not clearly dependent on the factor, suggesting that its regulation involves multiple converging transcriptional activators, in line with previous reports of complex multi-input control at this gene in B cell malignancies.

The chromatin-level analysis added a crucial layer of mechanistic insight. By profiling three histone marks in the same samples, H3K27ac for active enhancers and promoters, H3K4me3 for active promoters, and H3K27me3 for polycomb-mediated repression, the researchers found that IκBζ binding sites in unstimulated CLL cells occupied a heterogeneous, partly bivalent chromatin environment. After CpG stimulation, these sites underwent a coordinated epigenetic transition: H3K27ac rose sharply with focused enrichment at peak centers, H3K27me3 fell substantially, and H3K4me3 increased, indicating resolution of bivalency and activation of promoter-proximal regulatory regions. Notably, at the 86 statistically significant differentially gained peaks, H3K27ac was already detectable before IκBζ arrived, implying that the factor is preferentially recruited to loci carrying pre-existing partial activity rather than to fully silent regions, and that its recruitment coincides with, and may contribute to, a further gain of active marks.

Motif analysis supplied an internal validation of the entire dataset and a genuine surprise. Across all five peak sets examined, NF-κB family motifs, including p65/RelA, p52/NFKB2, REL and RELB, were consistently among the most enriched and centrally positioned, confirming IκBζ’s role as an NF-κB co-regulator. But POU-domain motifs, particularly OCT2/POU2F2, emerged as a CpG-specific signature: in the highest-confidence peak set, POU2F2 ranked as the single most significantly centrally enriched motif overall, with an adjusted p value of 1.8 × 10⁻¹⁸, edging out NFKB2 itself. Given OCT2’s established role in driving immunoglobulin transcription and B cell differentiation, this suggests that inflammatory stimulation redirects IκBζ toward regulatory elements co-occupied by lineage-specific transcription factors, a relationship that recent work in B cell lymphoma cell lines, where IκBζ was shown to be a dual-use coactivator of NF-κB and POU factors, now appears to extend into primary human leukemia.

Comparison with healthy donor B cells showed that much of this program is not leukemia-specific. CpG stimulation drove a conserved redistribution of IκBζ toward promoters in normal B cells as well, though the final promoter fraction remained far lower than in leukemic cells, 30.8 percent versus 69.0 percent, indicating that CLL mounts a quantitatively amplified version of a normal TLR9 response. A core set of targets, including FOXP1, BATF, NFKB2, TCF7 and TNFSF4, was gained in both systems, providing strong cross-system validation. Finally, experiments in mice lacking Nfkbiz specifically in the B cell lineage confirmed BATF as the single most robustly validated direct target: it was induced by CpG in wild-type B cells and significantly reduced when IκBζ was absent, while genes such as ECE1 and PIK3C2B were derepressed, revealing that IκBζ can also function as a repressor at selected loci. Taken together, the study positions IκBζ as a central transcriptional hub that couples TLR9-driven inflammation, B cell receptor signaling, lineage-specific transcription and migratory behavior in chronic lymphocytic leukemia, and strengthens the rationale for targeting this axis therapeutically in patients whose leukemic cells show the highest IκBζ levels.

Subject of Research: Genome-wide identification of IκBζ transcriptional target genes in chronic lymphocytic leukemia and normal B cells

Article Title: IκBζ target genes in chronic lymphocytic leukemia

Article References: Sana, I., Meloni, M., Genova, F., Mantione, M. E., Tascini, A. S., Zapparoli, E., Carbone Faccin, G., Paschaloudi, D., Del Nero, M., Lenzi, C., Ranghetti, P., Perotta, E., Scarfò, L., Ghia, P., & Muzio, M. (2026). IκBζ target genes in chronic lymphocytic leukemia. iScience, 29(11), Article 117828. https://doi.org/10.1016/j.isci.2026.117828

Image Credits: AI Generated

DOI: 10.1016/j.isci.2026.117828

Keywords: chronic lymphocytic leukemia, IκBζ, NFKBIZ, NF-κB, TLR9, CpG stimulation, ChIP-seq, CUT&Tag, BATF, FOXP1, NFKB2, B cell signaling