Andean Enriched NFKB1 Haplotype Reduces Inflammation and Improves Response to Ropeginterferon Alfa-2b in Polycythemia Vera (PV) and Essential Thrombocythemia (ET)

Chronic inflammation is a feature of polycythemia vera (PV), essential thrombocythemia (ET), and other myeloproliferative neoplasms (MPNs). NF-κB plays a key role in regulating pro-inflammatory cytokines. In PV and ET, activation of the NF-κB pathway is a primary driver of inflammation and augments hypoxia-inducible factors (HIFs) activity, increasing transcription of prothrombotic genes. Suppression of the NF-κB pathway has been shown to reduce disease burden in MPNs (Laranjeira, Blood, 2024).

We investigated the genetic basis underlying elevated hemoglobin in Andean natives, Aymara, by using granulocyte transcriptome data from both Aymara and Europeans from La Paz, Bolivia. We found their differentially expressed and spliced genes are associated with inflammatory and HIFs pathways. We identified three novel, alternatively spliced NFKB1 transcripts (AS-NFKB1) in Aymara, with skipped exon 4, exon 5, or both. The AS-NFKB1 transcript with exon 5 skipped was translated into protein but showed a defect in nuclear translocation, while the other two AS-NFKB1 transcripts were not translated, indicating a partial or complete loss of NFKB1’s function as an NF-κB suppressor. AS-NFKB1 also augmented activity of HIFs, explaining high Aymara hemoglobin. Using integrative analysis of whole-genome sequencing and granulocyte transcriptome, we identified splicing quantitative trait locus associated with AS-NFKB1; NFKB1 rs230511 is linked to high AS-NFKB1 expression. Its T allele (C/T or T/T genotype) is present in approximately 90% of Aymara but is also present in ~30% of Europeans, Hispanics, and Asians. Under TNF-induced inflammatory stress, unlike wild-type NFKB1, AS-NFKB1 suppressed inflammatory gene expression, suggesting a protective mechanism against inflammation (under review, Nature Communication).

Thus, we examined NFKB1 rs230511 association with inflammatory markers and response to ropeginterferon alfa-2b (Ropeg) therapy in PV and ET. We genotyped 30 PV and 15 ET patients, assessing inflammatory gene expression (IL1B, CXCL8, IL6, IL15, and TNF) in neutrophils and platelets. In platelets, individuals with the C/T genotype showed lower levels of these inflammatory transcripts compared to those with the C/C genotype and IL1B, CXCL8, and TNF levels were also reduced in the T/T genotype. In neutrophils, IL1B and CXCL8 were decreased in the T/T genotype, and IL15 and TNF levels were lower in the C/T genotype.

Given that AS-NFKB1 and NFKB1 rs230511 are linked to elevated transcriptional activity of HIFs, we analyzed the expression of HIF-target genes. LDHA, SLC2A1, and VEGFA transcripts were significantly lower in the C/T genotype in both PV and ET neutrophils and platelets. Since increased inflammation and HIF transcriptional activity are associated with thrombosis in PV and ET (Gangaraju, Blood Advances, 2020), we also measured expression levels of prothrombotic genes (F3, THBD, SELP, SERPINE1). In both platelets and granulocytes, F3 and THBD transcript levels were significantly lower in individuals with the C/T genotype compared to those with the C/C genotype.

Ropeg modulates inflammation via interferon signaling and attacks PV and ET clones (Verger, Blood Cancer J., 2018). We found that the C/T genotype (58.6%) was more prevalent among patients who achieved complete hematological response (CHR) compared to the non-CHR cohort, which included patients who did not reach CHR or had cytopenia (33.3%) (p<0.0001). This finding also correlated with lower transcript levels of inflammatory, HIF-targeted, and thrombotic genes in the CHR group compared to non-CHR group.

Our study indicates that presence of Aymara enriched NFKB1 rs230511 haplotype decreases PV and ET inflammatory, prothrombotic, and HIF’s activity compared to NFKB1 wild allele. The NFKB1 rs230511 haplotype also modulates response to Ropeg in PV and ET. The presence of NFKB1 rs230511 T allele is associated with reduced expression of inflammatory and HIF-targeted genes, potentially decreasing inflammation and thrombosis risk in PV and ET. Patients with the C/T genotype were more likely to achieve CHR, suggesting this NFKB1 haplotype may impact Ropeg therapy efficacy and serve as a potential biomarker for response assessment in PV and ET patients. These novel insights offer valuable implications for both of our understanding of inflammatory and prothrombotic processes and may also lead to novel PV and ET therapies.