Prospective Ropeginterferon Alfa-2b Therapy in PV & ET Influences Prothrombotic, Inflammatory and Hypoxia Inducible Factors Activities and Effect of Concomitant DNMT3A Mutation
Authors: Song Jihyun , Kim Soo Jin , Lim Yewon , Min Grace , Smith Amanda , Swomley Aaron , Tashi Tsewang , Prchal Josef T. 
Published: December 2024

Ropeg-interferon alfa 2b (Ropeg) is FDA-approved for Polycythemia Vera (PV) and used for Essential Thrombocythemia (ET) (Abu-Zeinah 2021; Kiladjian 2022). It targets JAK2V617F-positive cells and normalizes blood counts (Verger 2018). Its effects on PV and ET inflammatory, thrombotic, HIF transcriptional activities, and normal hematopoietic stem cell dormancy, remain unclear (Gangaraju 2020; Swierczek 2015; Tashi 2018).

We studied 52 PV and ET patients on Ropeg, with 64% achieving complete hematological remission (CHR) in 11.3 months (average dosage: 175.7 mcg). Ropeg starts as de novo treatment or after hydroxyurea (HU), Pegasys, and ruxolitinib. Of the 52 patients, 28 achieved CHR, 4 maintained CHR, and 10 were non-CHR. All 10 treatment-naïve patients achieved CHR. In the HU cohort, 53% (17/32) achieved CHR; in the Pegasys cohort, 71.4% (5/7); and in the ruxolitinib cohort, 60% (3/5) achieved CHR.

In 52 patients, we measured JAK2V617F variant allelic frequency (VAF) in clonal neutrophils before and after Ropeg treatment. Samples were collected at remission for CHR patients and at the longest follow-up for non-CHR patients. In the CHR group, 21.0% showed a decrease in VAF, compared to 6.7% in the non-CHR group. Females with PV and most with ET have clonal myelopoiesis (Swierczek 2015, Kralovics 2006; Nussenzweig 2007). Only interferon rescues normal dormant hematopoietic cells in PV (Liu E 2003) and ET (Tashi 2018). In our ongoing analysis of 23 females, one has converted to polyclonal myelopoiesis on Ropeg.

HIF transcriptional activity, inflammatory/thrombotic genes increased in PV & ET neutrophils and platelets (Gangaraju, 2020). The prothrombotic genes F3, THBD, SELP, SERPINE1, THBS1, anti-thrombotic gene KLF2 (Song 2023), inflammatory genes IL1B, CXCL8, IL15, TNF, IL6 and HIF-targeted genes EDN1, LDHA, SLC2A1, VEGFA were measured in neutrophils and platelets. Ropeg treatment upregulated neutrophil prothrombotic genes THBD (149%, p&lt;0.0001), SELP (149%, p&lt;0.0001), SERPINE1 (282%, p&lt;0.0001), and THBS1 (99%, p&lt;0.0001). IL1B decreased by 39% (p&lt;0.0001), while IL15 (282%, p=0.0039), TNF (47%, p=0.0016), and IL6 (85%, p=0.0131) increased. HIF target gene EDN1 decreased by 26% (p=0.0176), but SLC2A1 (47%, p=0.0001) and VEGFA (72%, p=0.0001) were upregulated. Whether the upregulation of these transcripts correlates with increased proteins and function remains to be shown. Anti-thrombotic KLF2 increased by 2.18-fold (p=0.0190). In platelets, prothrombotic genes THBD (23%, p=0.0155) and SELP (34%, p=0.0006) decreased, while KLF2 increased 1.57-fold (p=0.0016). IL1B (39%, p&lt;0.0001) and CXCL8 (23%, p=0.0097) decreased. These reductions were linked to decreases in HIF genes SLC2A1 (10%, p=0.0508) and VEGFA (38%, p=0.0008), while LDHA (28%, p=0.0022) increased.

Activated NF-kB pathway has been reported in PV and ET (Laranjeira 2024). Andean Aymara (~90%) have evolutionarily selected allele “T” of NFKB1 variant (rs230511) that is associated with increased HIF and inflammation (Song, ASH, 2018); while pro-inflammatory state decreases inflammatory gene expression (not published data). Since T allele is also found in Europeans and Asians (~30%), we genotyped the rs230511 in neutrophil DNAs. The percentage of C/T genotype was higher in the CHR compared to, while more pts with the C/C genotype were found in the nonCHR cohorts (p&lt;0.0001). Given the inflammatory milieu in PV and ET, IL1B (p=0.0447) and CXCL8 (p=0.045) transcript levels were lower in pts with T allele. This suggests that pts with T allele have lower levels of inflammation and are more likely to respond to Ropeg.

DNMT3A mutation concomitant with PV and ET-phenotype defining mutations was reported to render Pegasys treated PV pts refractory (Knudsen 2022); Usart 2024). In our cohorts, 7 pts had concomitant DNMT3A; pt#1 could not tolerate Ropeg and 6 achieved CHR on Ropeg 50-400 mcg sc (mean 183.3±134.4) compared to Ropeg dose of CHR without DNMT3A (mean 203.2±131.8).

We conclude that Ropeg improves expression of some HIF target genes, inflammatory genes, and prothrombotic genes (THBD and SELP) in neutrophils; also, HIF target genes and inflammatory gene expression in platelets compared to other PV & ET therapies. We also found that inflammation associated NFKB1 T haplotype is associated with decreased expression of inflammatory genes in neutrophils and that DNMT3A mutation does not preclude CHR by Ropeg therapy.