Interferon Synergizes with Inhibition of Pro-Apoptotic Proteins to Reduce Leukemia Burden in PDX Models of ML-DS

Young children with Down syndrome have a 500-fold increased incidence of myeloid leukemia (ML-DS). Novel treatment options are needed to improve outcome in the 10-20% of patients with relapsed/refractory ML-DS who have 3-year event-free survival rate of less than 21% (Raghuram et al., 2023). The mutational landscape of ML-DS has been extensively characterized (Labuhn et al., 2019; Nikolaev et al., 2013; Sato et al., 2024; Yoshida et al., 2013). Most patients with ML-DS have mutations in the GATA1 gene encoding an essential hematopoietic transcription factor. These mutations result in the production of the N-terminally truncated short form of GATA1 (GATA1s). Additional co-operating mutations in genes such as STAG2 are required for ML-DS leukemogenesis. We modelled ML-DS by stepwise introduction of GATA1 and STAG2 mutations in induced pluripotent stem cells (iPSCs) with trisomy 21 using clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 mediated gene targeting (Barwe et al., 2022). Transcriptome analysis of megakaryoblasts generated by hematopoietic differentiation of trisomy 21 iPSCs bearing GATA1 and STAG2 mutations showed that interferon signaling via RIG-I/MDA5 pathway is suppressed. The levels of RIG-I, MDA5, and phosphorylated STAT1, the downstream activator of the interferon signaling pathway, were downregulated in two distinct iPSC models of ML-DS. The downregulation of these proteins was also seen in four different ML-DS PDX lines with diverse co-operating mutations in addition to GATA1 mutation, validating the downregulation of interferon signaling pathway in ML-DS. Treatment of all 4 ML-DS PDX lines with recombinant human interferon alpha A protein for 48 hours upregulated phospho STAT1, RIG-I and MDA5. Taken together, these data indicate that the interferon signaling is suppressed in ML-DS but can be re-activated by exogenous interferon treatment of ML-DS blasts. Furthermore, iPSC-derived ML-DS megakaryoblasts and ML-DS PDX lines treated with interferon alpha (40 ng/mL) in vitro for 48 hours showed a dose-dependent decrease in cell viability up to 39.5%. Pegylated interferon alfa-2a (Pegasys, 24 μg/Kg, s.c.) dosing weekly for 6 weeks significantly prolonged survival of ML-DS PDX models in vivo. The median survival was increased by 54, 76, 37 and 6 days in NTPL-60, NTPL-386, 20033.5, and CPCT-0010 respectively (P<0.05 for each ML-DS PDX). Transcriptome analysis of CMK cells (ML-DS cell line) treated with or without interferon revealed activation of interferon signaling pathway accompanied by an upregulation of genes encoding anti-apoptotic proteins such as BCL2L1 (encoding Bcl-xL) and MCL1. Therefore, we evaluated if inhibitors of these proteins would synergize with interferon in suppressing ML-DS cell viability. NSG-SGM3 mice were first engrafted with CPCT-0010, a PDX line generated from a patient with refractory ML-DS, and then treated with Bcl-xL inhibitor Navitoclax (100 mg/Kg p.o. daily for 14 days) or MCL1 inhibitor PRT1419 (10 mg/Kg p.o., weekly for 3 weeks) alone or in combination with Pegasys (24 μg/Kg s.c., weekly for 3 weeks). The leukemia burden was evaluated by flow cytometry of peripheral blood on day 28 post cell injection. While the untreated mice had a high percentage of human CD45+ cells (77.7±7.1%), treatment with Naviotclax, PRT1419 or Pegasys reduced this percentage to 44.9±4.8, 52.7±5.2 and 49.7±5.2 respectively (P<0.05 for each treatment in comparison with untreated). The combination of Pegasys with Navitoclax or Pegasys with PRT1419 further reduced leukemia burden to 30.9±3.5 and 31.9±3.5, with a combination index of 0.342 and 0.300 respectively (P<0.05 for each pair-wise comparison between monotherapy and combination treatment). Navitoclax, PRT1419 or Pegasys monotherapy prolonged median survival by 2 (P<0.01), 5 (P<0.01) and 4 (P<0.05) days respectively. Pegasys & Navitoclax or Pegasys & PRT1419 combination improved survival by 11 (P<0.01) and 13 (P<0.05) days respectively. These data indicate that both Navitoclax and PRT1419 synergized with Pegasys to suppress leukemia burden in ML-DS PDX model. In summary, we showed that interferon signaling is suppressed in ML-DS blasts and interferon treatment reduced ML-DS cell viability in vitro and prolonged survival in vivo. Furthermore, interferon treatment in combination with Bcl-xL or MCL1 inhibition was superior in reducing leukemia burden compared to monotherapy.