Ropeginterferon Alfa-2b Induces Apoptosis and Differentiation of Leukemia Stem Cells and Separates GVL Effects from Gvhd after Allogeneic Hematopoietic Cell Transplantation

[Introduction] Relapse of acute myeloid leukemia (AML) is a major cause of mortality after allogeneic hematopoietic cell transplantation (allo-HCT). It has been reported that type I interferon (type I-IFN) signaling in host cells suppresses graft-versus-host disease (GVHD), while in donor T cells, it maximizes graft-versus-leukemia (GVL) effects after mouse allo-HCT (Robb JR: Blood. 2011). Ropeginterferon alfa-2b (RopegIFN), a novel mono-pegylated IFN-a with a longer half-life and an better safety profile compared to conventional pegylated IFN-a, has been recently developed as a first-line treatment for patients with polycythemia vera (NCCN Guidelines for Myeloproliferative Neoplasms v1.2024). In the current study, we tested whether RopegIFN could separate GVL effects against AML from GVHD using mouse models of allo-HCT. [Methods] Bone marrow (BM) cells from 5-fluorouracil-treated B6 or BDF1 mice were infected with a retroviral vector to express the KMT2A::MLLT3 fusion gene and eGFP. After in vivo passage, eGFP expressing cells were then used as AML cells in the following experiments. Lethally irradiated (10 Gy) BDF1 mice were transplanted with T-cell-depleted bone marrow (TCD-BM) cells, with or without purified T cells from allogeneic B6 donors. To evaluate GVL effects, recipient mice were i.v. injected with 1 × 10⁴ AML cells on the day of allo-HCT. EGFP⁺ AML cells in the peripheral blood (PB) were enumerated weekly after HCT. RopegIFN was provided by PharmaEssentia. [Results] To explore the mechanisms of the direct anti-AML effects of RopegIFN, AML cells were inoculated into syngeneic B6 mice, followed by subcutaneous (s.c.) injections of 6 µg RopegIFN on day 5,12 and 19. Flow cytometric analyses (FCM) demonstrated that RopegIFN, and to a lesser extent recombinant murine IFN-a (rmIFN-a), significantly delayed the expansion of AML cells in the PB. Importantly, RopegIFN, but not rmIFN-a, selectively reduced the proportion of leukemia stem cell (LSC)-containing granulocyte-macrophage progenitor (GMP) fraction among eGFP⁺ cells in the BM. RopegIFN significantly increased Annexin V⁺ apoptotic LSCs and reduced the absolute number of LSCs in the BM. Bulk RNA sequencing of LSC fraction demonstrated that RopegIFN induced gene sets involved in apoptosis and myeloid differentiation, while reduced LSC-related genes, suggesting that RopegIFN induces apoptosis and myeloid differentiation of LSCs. Furthermore, FCM demonstrated that RopegIFN enhanced expression of MHC class I and class II on eGFP⁺ AML cells. Although RopegIFN significantly prolonged survival of AML-inoculated mice, all mice eventually died from leukemia irrespective of RopegIFN administration. Therefore, we next tested whether RopegIFN combined with allo-HCT could enhance GVL effects and cure AML. FCM of BM cells on day 17 after allo-HCT demonstrated that RopegIFN significantly increased PD-1⁺ TOX⁺ CX3CR1⁺ CD8⁺ donor transitory exhausted T cells (transitory-Tex) expressing Granzyme B and Ki-67 (5.36 ± 0.54 × 10⁴ vs. 2.75 ± 0.52 × 10⁴, p=0.0039), which we have previously shown to maintain the allo-reactivity of donor T cells after allo-HCT (Senjo H: Blood 2023). Importantly, we found that RopegIFN significantly ameliorated pathological acute GVHD in the liver and gut, and prolonged the survival after allo-HCT. In recipients of TCD-BM alone, RopegIFN only modestly delayed AML expansion and prolonged survival. When T cells were added to the graft without RopegIFN administration, GVL effects alone also significantly delayed AML expansion and prolonged survival. Remarkably, RopegIFN eradicated AML cells in recipients of TCD-BM plus T cells, indicating that RopegIFN enhanced GVL effects against AML. As a result of synergistic interaction between RopegIFN and GVL effects, only RopegIFN-treated recipients of TCD-BM plus T cells achieved long-term survival, whereas neither GVL effects nor RopegIFN alone could cure AML. [Conclusion] We found that RopegIFN induces apoptosis and myeloid differentiation of LSCs in AML and delays leukemia-related death, but it is insufficient to eradicate leukemia cells as monotherapy. After allo-HCT, RopegIFN enhanced GVL effects in association with the expansion of donor transitory-Tex, while ameliorating GVHD. Our data indicate that RopegIFN is a promising agent for maintenance therapy following allo-HCT in AML patients, separating GVL from GVHD.

Hashimoto:Astellas Pharma Inc. Japan: Honoraria; Janssen Pharmaceutical K.K.: Honoraria; Nippon Kayaku Co.,Ltd.: Honoraria; Chugai Pharmaceutical Co., Ltd.: Honoraria; Daiichi Sankyo Co., Ltd.: Honoraria; Eisai Co., Ltd.: Honoraria; Novartis Japan: Honoraria. Teshima:Sanofi: Honoraria; LUCA Science: Research Funding; Abbvie: Honoraria; Astellas: Honoraria, Research Funding; Kyowa-Kirin: Consultancy, Honoraria, Research Funding; Novartis: Honoraria; Bristol-Myers Squibb: Honoraria; Sumitomo Pharma: Research Funding; Chugai: Honoraria, Research Funding; Otsuka: Honoraria, Research Funding; Nippon Shinyaku: Consultancy, Honoraria; Pharma Essentia Japan: Research Funding; Meiji Seika Pharma: Consultancy, Honoraria; AstraZeneca: Honoraria; MSD: Honoraria; Fuji Pharma: Honoraria, Research Funding; Asahi Kasei Pharma: Honoraria, Research Funding; Pfizer: Honoraria; Gilead: Honoraria; Takeda: Consultancy, Honoraria; Nippon Kayaku: Honoraria, Research Funding; Shionogi: Honoraria, Research Funding; Roche Diagnostics: Consultancy; Eisai: Research Funding; Genmab: Honoraria; JCR Pharma: Honoraria, Research Funding; Janssen: Honoraria; Daiichi Sankyo: Honoraria, Research Funding; Symbio: Honoraria.