A novel function of STAT3β in suppressing interferon response improves outcome in acute myeloid leukemia

Signal transducer and activator of transcription 3 (STAT3) is frequently overexpressed in patients with acute myeloid leukemia (AML). STAT3 exists in two distinct alternatively spliced isoforms, the full-length isoform STAT3α and the C-terminally truncated isoform STAT3β. While STAT3α is predominantly described as an oncogenic driver, STAT3β has been suggested to act as a tumor suppressor. To elucidate the role of STAT3β in AML, we established a mouse model of STAT3β-deficient, MLL-AF9-driven AML. STAT3β deficiency significantly shortened survival of leukemic mice confirming its role as a tumor suppressor. Furthermore, RNA sequencing revealed enhanced STAT1 expression and interferon (IFN) signaling upon loss of STAT3β. Accordingly, STAT3β-deficient leukemia cells displayed enhanced sensitivity to blockade of IFN signaling through both an IFNAR1 blocking antibody and the JAK1/2 inhibitor Ruxolitinib. Analysis of human AML patient samples confirmed that elevated expression of IFN-inducible genes correlated with poor overall survival and low STAT3β expression. Together, our data corroborate the tumor suppressive role of STAT3β in a mouse model in vivo. Moreover, they provide evidence that its tumor suppressive function is linked to repression of the STAT1-mediated IFN response. These findings suggest that the STAT3β/α mRNA ratio is a significant prognostic marker in AML and holds crucial information for targeted treatment approaches. Patients displaying a low STAT3β/α mRNA ratio and unfavorable prognosis could benefit from therapeutic interventions directed at STAT1/IFN signaling.

centrifugation for 15min at 2,000g and 4°C.Supernatant was collected and stored at -80°C until further usage.Spleens were isolated and mashed through a 70µm cell strainer followed by red blood cell lysis.Bone marrow cells were harvested from hind legs.Samples were cryopreserved as single cells suspension in FBS supplemented with 10% DMSO and stored at -80°C until needed.For flow cytometry analysis the samples were thawed in PBS, stained, and analyzed simultaneously.

RNA sequencing
Leukemic blasts were isolated from bone marrow and spleen of diseased animals by sorting Venus + cells using a BD FACSymphony TM S6 Cell Sorter (BD Bioscience) and analyzed using BD FACSDiva Software.RNA was isolated using RNeasy Mini Kit (Qiagen) and sequenced with an Illumina HiSeq3000/4000 sequencer using 50bp single-end libraries.Quality checks of the raw sequence reads were done with FastQC(2) (version 0.11.9).Then we applied PRINSEQ-lite(3) (version 0.20.4) for quality based read filtering and trimming.The remaining high-quality reads were aligned against the mouse reference genome (GRCm38) using STAR(4) (version 2.5.0b) and post processed with samtools(5) (version 1.4).Read counts were obtained with featureCounts(6) (version 2.0.3) and for normalization and differential gene expression analysis we applied DESeq2(7) (version 1.40.2).GSEA (GSEA version 4.2.2) was done with genes that show an adjusted p-value < 0.05 according to the provider's protocol.(8) Gene sets were downloaded from the Molecular Signature Database (http://software.broadinstitute.org/gsea/msigdb/index.jsp) and permutations were set to 1,000.

Multiplex immunoassay
Cytokine levels were measured in plasma of diseased animals (1:2 dilution) using the ProcartaPlex TM Mouse and Rat Mix & Match panels (Invitrogen, Thermo Fisher Scientific) according to the manufacturer's protocol.All measurements were performed using a Bio-Plex 200 System (Bio-rad) following the manufacturer's instructions.

Retroviral infection
Platinum-E packaging cells were transfected with the pMSCV-MLL-AF9-IRES-Venus construct, which was a kind gift from Johannes Zuber (Research Institute of Molecular Pathology, Vienna, Austria), by calcium phosphate co-precipitation.FLCs were thawed and cultured one day prior to infection and spinoculated at 1,000g for 90min three times on two consecutive days with retroviral supernatant supplemented with 10µg/ml polybrene (Sigma-Aldrich, St. Louis, MO, USA).Two days after the first infection 2x10 5 MLL-AF9/Venus + cells were transplanted into 6 to 8 weeks old male immunocompromised NOD.Cg-Prkdc scid Il2rg tm1Wjl /SzJ mice (The Jackson Laboratory, Bar Harbor, ME, USA) via tail vein injection.The remaining cells were cultured until homogenous Venus + cells were obtained.

Growth curves
1.5x10 5 cells were seeded in triplicates in 12-well plates containing 1ml cell culture medium on day 0. For up to 4 days cell numbers were measured using a Cytoflex S (Beckman Coulter).

Cell cycle and apoptosis staining
For cell cycle analysis, cells were fixed using ice-cold 70% ethanol and stained with 5µl 7AAD (eBioscience, Thermo Fisher Scientific) followed by flow cytometry analysis.For apoptosis analysis, cells were fixed using Annexin V Binding Buffer (BioLegend, San Diego, CA, USA), followed by staining with 5µl Annexin V (BioLegend) and 5µl 7AAD in 100µl Annexin V Binding Buffer.All samples were analyzed using a Cytoflex S (Beckman Coulter).