AUTHORS
Megan Bywater, PhD, Ranran Zhang, PhD2, Rohit Haldar3, Yashaswini Janardhanan4, Siu Hang Natalie Chan5, Emily Cooper6, Shin-Jye Lee7, Jasmin Straube8, Steven W. Lane, MD, PhD9
AFFILIATIONS
1 QIMR Berghofer, Brisbane, Queensland, Australia, The University of Queensland, Brisbane, Queensland, Australia
2 QIMR Berghofer, Brisbane, Queensland, Australia, The University of Queensland, Brisbane, Queensland, Australia
3 QIMR Berghofer, Brisbane, Queensland, Australia
4 QIMR Berghofer, Brisbane, Queensland, Australia
5 QIMR Berghofer, Brisbane, Queensland, Australia
6 QIMR Berghofer, Brisbane, Queensland, Australia
7 PharmaEssentia Corporation, Taipei, Taiwan
8 QIMR Berghofer, Brisbane, Queensland, Australia, The University of Queensland, Brisbane, Queensland, Australia
9 QIMR Berghofer, Brisbane, Queensland, Australia, Royal Brisbane & Womens Hospital, Brisbane, Queensland, Australia, The University of Queensland, Brisbane, Queensland, Australia
ABSTRACT
Leukemic transformation from a myeloproliferative neoplasm (MPN) to acute myeloid leukemia (AML) portends a dismal prognosis. However, given the unpredictable and protracted time frame of its occurrence, clinical trials for MPN interventions are rarely powered to have AML transformation as an end point. In this study we propose to use genetically engineered mouse models that faithfully recapitulate the progression of high molecular risk MPN to determine if treatment intervention with pegylated interferon alpha (IFNα) can prevent leukemic transformation.
MPN is driven by mutations that activate JAK-STAT signalling, most commonly JAK2V617F, and progression to post-MPN AML is accompanied by the acquisition of additional mutations, including in TP53. Here, we have used genetically engineered mouse strains in combination with CRISPR/Cas9 to generate a Jak2V617F-driven model of high-molecular risk MPN with Trp53-loss.
Treatment with IFNα for 4 weeks has similar efficacy in both Jak2V617F-only and Jak2V617F/Trp53-mutant MPN, reducing splenomegaly (Jak2V617F-only vehicle: 268.8mg±35.5 vs IFNα:184.9mg±32.4, p=0.0072; Jak2V617F/Trp53-mutant vehicle: 309.2mg±86.5 vs IFNα:207.2mg±41.4, p=0.0004) and the frequency of reticulocytes (Jak2V617F-only vehicle: 11.9%±1.3 vs IFNα: 8.7%±2.6, p=0.0096, Jak2V617F/Trp53-mutant vehicle: 11.1%±2.4 vs IFNα: 8.0%±2.2, p=0.0057). This demonstrates that haematological responses of Jak2V617F MPN to cytoreductive therapy with IFNα are independent of Trp53 mutational status. Consistent with this, IFNα is able to drive cell cycle entry in MPN stem cells both with and without Trp53 mutations (%S-G2-M DNA content, Jak2V617F-only vehicle: 19.6%±8.5 vs IFNα: 37.4%±5.5, p=0.0448, Jak2V617F/Trp53-mutant vehicle: 16.6%±10.0 vs IFNα: 35.2%±12.2, p=0.0359). Despite this, Trp53-mutation provides a selective advantage to Jak2V617F cells under the pressure of IFNα treatment, evident in the peripheral blood after 6 weeks (mCherry+/GFP+, %CD45, vehicle: 34.5±6.6 vs IFNα: 53.52±8.4, p=0.0076). However, IFNα retains capacity to reduce the transplantation of Jak2V617F/Trp53-mutant MPN into secondary recipients (mCherry+/GFP+, %viable bone marrow cells at 16 weeks post-transplant, vehicle: 38.6±29.3 vs IFNα: 0.3±0.3, p<0.0001).
Untreated Jak2V617F/Trp53-mutant recipient mice, but not Jak2V617F-only controls, develop post-MPN AML characterised by an exacerbation of splenomegaly (Jak2V617F-only: 388.8mg±142.7 vs Jak2V617F/Trp53-mutant: 1200mg±444.0, p=0.0004) and leukocytosis (Jak2V617F-only: 8.0K/µl±3.4 vs Jak2V617F/Trp53-mutant: 40.2K/µl±21.4, p=0.0036). Critically, IFNα treatment of Jak2V617F/Trp53-mutant bone marrow donors is able to attenuate leukemic transformation in secondary recipients up to 16 weeks post-transplantation (spleen, vehicle: 739.2mg±468.8 vs IFNα: 130.1mg±93.5, p=0.0002). Furthermore, IFNα treatment, but not hydroxyurea, also attenuates progression of established Jak2V617F/Trp53-mutant AML (median survival, vehicle: 33day, HU: 33days, IFNα:61.5days, vehicle vs IFNα: p=0.0053).
Combined evidence from RNA sequencing and surface marker expression analysis indicates that Jak2V617F/Trp53-mutant AML undergoes enforced erythroid maturation after IFNα, in addition to activating a pro-inflammatory, pro-apoptotic transcriptional program. Consistent with this, the JAK2V617F/TP53-mutant post-MPN AML cell line HEL upregulates transcripts of the proapoptotic factors PUMA and NOXA when treated with pharmacological IFNα, Pegasys. Given that JAK2V617F/TP53-mutant AML has been shown to be highly dependent on BCL-XL for survival (Kuusanmaki et al, Blood, 2023; Wang et al, Blood, 2025), we therefore investigated the potential of combining Pegasys with a BCL-XL targeting BH3 mimetic. Encouragingly, the presence of Pegasys was able to significantly reduce the IC50 of the dual targeting BCL-2/BCL-XL BH3 mimetic Navitoclax (single agent: 273nM, +Pegasys: 132nM) and the BCL-XL specific A-1331852 (single agent: 7.4nM, +Pegasys: 0.7nM), but not the BCL-2 specific Venetoclax (single agent: 5668nM, +Pegasys: 9294nM), in HEL cells. We are now investigating the potential of this rational combination therapy in vivo.
In aggregate, these findings demonstrate the efficacy of IFNα to prevent AML in patients with high molecular risk MPN. Furthermore, combinations of IFNα and promoters of apoptosis offer a potential new avenue for therapeutic intervention in JAK2V617F/TP53-mutant post-MPN AML.
DISCLOSURES
Megan Bywater, PhD: No relevant conflicts of interest to declare.