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1687 Combination of 5-Azacytidine and Pegifna Is Able to Overcome Resistance in JAK2-V617F Positive MPN with Loss of Dnmt3a

Program: Oral and Poster Abstracts
Session: 631. Myeloproliferative Syndromes and Chronic Myeloid Leukemia: Basic and Translational: Poster I
Hematology Disease Topics & Pathways:
Research, Translational Research, Combination therapy, Diseases, Therapies, Myeloid Malignancies
Saturday, December 10, 2022, 5:30 PM-7:30 PM

Melissa Lock1*, Damien Luque Paz1*, Nils Hansen1*, Tiago Almeida Fonseca1*, MARC Usart2*, Shivam Rai1*, Hui Hao-Shen1*, Gabriele Mild1*, Stephan Dirnhofer3*, Radek C. Skoda1 and Jan Stetka1*

1Department of Biomedicine, Experimental Hematology, University Hospital Basel and University of Basel, Basel, Switzerland
2Department of Biomedicine, Experimental Hematology, University Hospital Basel and University of Basel, BASEL, Switzerland
3Institute of Medical Genetics and Pathology, University Hospital Basel, Basel, Switzerland

Pegylated interferon alpha (IF) can induce molecular remissions in subset of JAK2-V617F-positive MPN patients by targeting long-term hematopoietic stem cells (LT-HSCs). Patients with additional somatic mutations in genes involved in LT-HSC self-renewal have been reported to have poorer responses to IF. We found that loss of Dnmt3a increases competitiveness and self-renewal of JAK2-V617F-positive LT-HSCs and confers resistance to IF treatment in MPN mouse models and in HSCs and progenitor cells from MPN patients. Here we examined whether the resistance of double mutant JAK2-V617F;Dnmt3aΔ/Δ hematopoietic cells to IF can be overcome by addition of arsenic trioxide (At), or 5-azacytidine (Az). To test the effects of the combination treatments in vivo, we generated cohorts of mice expressing JAK2-V617F (VF) alone, or in combination with a homozygous deletion of the Dnmt3a gene (Dnmt3aΔ/Δ). We used bone marrow cells from VF or VF;Dnmt3aΔ/Δ mice that express a GFP reporter gene mixed with a 10-fold excess of bone marrow cells from a wildtype (WT) mouse to perform competitive transplantations into lethally irradiated WT recipient mice. After 7 weeks, recipient mice were randomized into 6 treatment groups including a vehicle control group. These mice were treated for 12 weeks with IF (25µg/kg; s.c. once per week), At (5mg/kg; i.p. every second day), or Az (2mg/kg; i.p. daily for two weeks followed by a break of 2 weeks), or combinations of IF+At and IF+Az (Figure 1). Peripheral blood parameters were normalized by At and Az treatment arms in both single-mutant and double-mutant mice (Figure 1A). Spleen and liver weight was decreased in all treatment groups and genotypes, except in VF;Dnmt3aΔ/Δ mice treated with IF alone, which showed a trend towards increased spleen and liver weights. Expression of GFP allowed us to follow the contribution of cells derived from the VF or VF;Dnmt3aΔ/Δ donor mice, respectively. In VF recipient mice, a pronounced decrease in GFP chimerism of peripheral blood lineages was observed in the groups treated with a combination of IF+At, or IF+Az (Figure 1A). LT-HSC from bone marrow and spleen showed reduction in GFP chimerism upon treatment with Az alone, compared to vehicle and further reduction below 10% was observed in combination of Az and IF (Figure 1B). Double-mutant VF;Dnmt3aΔ/Δ recipient mice remained resistant to the single agent regiments, but showed a significant reduction of GFP chimerism in peripheral blood and in LT-HSCs when treated with a combination of IF+Az and to a lesser degree also when treated with IF+At (Figure 1B). Thus, a combination of pegIFNa with 5-azacytidine is a promising approach to target MPN cells carrying mutations in JAK2 and Dnmt3a genes that could be also considered as a treatment option for therapy-resistant forms of MPN in patients carrying JAK2-V617F and loss-of-function DNMT3A mutations.

Disclosures: Skoda: BMS/Celgene and Novartis: Honoraria; Ajax Therapeutics: Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees.

*signifies non-member of ASH