The alternative NF-kB pathway activity in refractory and relapsed Hodgkin lymphoma

The refractory and relapsed disease is currently the challenge in Hodgkin Lymphoma (HL) treatment. There is no specific therapy but rescue chemotherapy schemes, which fails in 50% of the cases and associates with high risk severe toxicity. This highlights the need to deeper understand the HL molecular biology and the screening for therapeutic directed-targets.We have previously reported that HL relies on the alternative NFkB pathway, mediated by Rel-B and NIK, to survive. Its constitutive activation seems to be involved in the refractory and relapsed disease.To determine the specific Rel-B target genes we performed ChIP-Seq in UH01 and L1236 human HL cell lines for Rel-A, Rel-B, cRel, p50 and p52. We found 29,414 Rel-B peaks genome-wide distributed. To further identify genes with higher probabilities of being directly regulated by Rel-A, Rel-B, and/or cRel we concentrated on the peaks that localized within a +/- 2 kb window, relative to the gene transcription start site. The +/- 2 kb Rel-B peaks were distributed on 4,509 genes; meanwhile the +/- 2 kb cRel peaks were on 1,994 genes and the Rel-A peaks were on 830 genes. Out of the 4,509 genes with the Rel-B peaks, only 6% overlapped with the Rel-A peaks and 11% with the cRel peaks. The Rel factors distribution showed a Rel-B DNA-binding hierarchy in HL.The ChIP-Seq data were merged with gene expression arrays results. The gene expression assays were analyzed by comparing the signal from cells infected with noninduced shRNAs for the Rel factors to that from cells infected with induced shRNAs. Differentially expressed genes were identified via ANOVA analysis. Genes that were downregulated more than 2-fold with a p<0.001 were considered significant. We found that the Rel-B downstream controlled gene set was enriched for cell cycle and cell death regulation and DNA damage and repair signatures, among others.One of the exclusively Rel-B target genes was BCL2. We showed that exogenous BCL2 was able to partially rescue HL cells from dying in response to Re-B depletion. We also found that BCL2 was useful as a prognosis marker in terms of overall survival in a cohort of 96 HL patients [Log Rank Test (p=0.002)]. In this retrospective study [follow up period 47,4 (6-136) months], BCL2 was able to identify at diagnosis patients that were refractory to conventional first line treatment and patients that relapsed. The HL cell lines U-H01, L1236, SUPDHL1, KM-H2 and L540 were sensitive to the BCL2 inhibitor venetoclax. The HDM-2 cell line, which does not express BCL2, was a good control of target specificity since it did not die in response to venetoclax.In summary, we found that the alternative NFkB pathway plays an important role in the refractory and relapsed Hodgkin disease, being BCL2 a key downstream target. BCL2 performed well as a prognosis marker identifying refractory patients and those that would relapse. We propose BCL2 expression assessment in the lymph node biopsy at diagnosis and BCL2 as a directed therapy.Citation Format: Angélica María Gamboa-Cedeño, Mariángeles castillo, Victoria Otero, Natalia Schutz, Dorotea Fantl, Federico Jauk Vitali, Hernán García Rivello, Myriam Nuñez, Stella Maris Ranuncolo. The alternative NF-kB pathway activity in refractory and relapsed Hodgkin lymphoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4032.