STAT1 Transcriptional Response Predicts Molecular Responses of PB-Derived Clonogenic Cells from MPN Patients to Interferon Alpha
Authors: Kalmer Milena , Feldberg Kristina , Gezer Deniz , Isfort Susanne , Brümmendorf Tim H , Chatain Nicolas , Koschmieder Steffen 
Published: November 2019

Introduction: Interferon alpha (IFNa) is a cytokine with anti-viral and anti-tumoral properties which, either in its unmodified or pegylated form, is successfully used in the treatment of patients with myeloproliferative neoplasms (MPN), including polycythemia (PV), essential thrombocythemia (ET) and early primary myelofibrosis (PMF). Despite its efficacy including molecular responses and thus its potential disease modifying capabilities, IFNa carries the risk of significant adverse events, including liver toxicity, autoimmunity, and depression. This has prompted the development of interferons with improved features including better tolerability. However, biomarkers for response have been lacking, mostly due to the heterogeneity of cells analyzed and the difficulty in obtaining them from the bone marrow.

Methods: Therefore, we have set up a clonogenic assay from Ficoll-isolated mononuclear cells of the peripheral blood of patients (PBMC) with MPN (n=51, including 17 PV, 14 ET, and 14 PMF, 1 MDS/MPN, 2 Post-PV-MF, 2 Post-ET-MF, 1 MPNu) to analyze the in vitro IFNa response (either using 0.5 µg/ml ropeginterferon alfa-2b [ropegIFNa] or 500 U/ml recombinant human IFN-alpha2b [hIFNa] or no treatment control) in the cells that drive malignant clonogenic growth in the patients. After 10-14 days, the number of colonies was assessed. For each patient and condition, twenty-five of the resulting colonies were then genotyped for zygosity of JAK2V617F and three colonies per condition were analyzed for STAT1 RNA expression using RT-qPCR, an important transcriptionally induced IFNa target gene. The number of mutated alleles was determined (zero for wildtype, one for heterozygous, two for homozygous colonies), and, based upon the change in mutant alleles after in vitro hIFNa treatment, samples were categorized into responders (mutant alleles decreased) and non-responders (mutant alleles unchanged or increased). All patients provided written informed consent, and the study was approved by the local ethics committee.

Results: To assess potential differences between the two interferons used, a 14-day proliferation assay was performed in Set-2 cells, showing more sustained inhibition of proliferation by ropegIFNa than hIFNa (p=0.007), confirming the longer half-life of ropegIFNa. In the clonogenic assay, different colonies were observed depending on the MPN subtype, ranging from exclusive CFU-E (PV) to exclusive CFU-G and CFU-M (PMF) types of colonies. Both hIFNa and ropegIFNa significantly inhibited colony growth as compared to the control and reduced the colony number to 72.7 % (hIFNa) and 58.5% (ropegIFNa) of the untreated control, with ropegIFNa showing significantly stronger effects than hIFNa (p=0.0137). Interestingly, there were marked differences in the amount of JAK2 alleles in the colonies between the patients (n=24 analyzed), with 16 patients showing a reduction of up to 22% of the allele burden upon in vitro treatment with hIFNa (responders), while 8 patients showed no reduction or even an increase of up to 15% (non-responders) (p=0.0001). Basal STAT1 expression in the colonies (three colonies per patient and treatment) was significantly lower in responders than non-responders (p=0.0200). After treatment with hIFNa, STAT1 expression was induced to similar levels in both responders and non-responders (p=0.6620). As a result, STAT1 fold induction was significantly higher in responders than non-responders (p=0.0013). Interestingly, there was no correlation of the responses with current clinical treatment of the patients (previous interferon exposure did not prevent responses) or with the MPN subtype, confirming clinical reports that patients with myelofibrosis can respond to interferon.

Conclusion: In conclusion, clonogenic cells from the peripheral blood of MPN patients can be used to assess their molecular response to IFNa. In vitro, ropegIFNa induced stronger effects than non-pegylated IFNa. The responses were heterogeneous at the molecular level, but, when combined with RNA expression analysis, we were able to dissect molecular responders from non-responders. The mechanisms for these differences and their clinical impact are currently being studied.

Disclosures

Gezer: AMGEM: Membership on an entity's Board of Directors or advisory committees. Isfort:Mundipharma: Other: Travel reimbursement; Amgen: Other: Travel reimbursement; Hexal: Other: Travel reimbursement; BMS: Honoraria; Ariad: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria, Other: Travel reimbursement; Novartis: Consultancy, Honoraria, Other: Travel reimbursement; Roche: Other: Travel reimbursement; Alexion: Other: Travel reimbursement. Brümmendorf:Ariad: Consultancy; Janssen: Consultancy; Pfizer: Consultancy, Research Funding; University Hospital of the RWTH Aachen: Employment; Novartis: Consultancy, Research Funding; Merck: Consultancy. Koschmieder:Ariad: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Pfizer: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis Foundation: Research Funding; Bayer: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Shire: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; AOP Pharma: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Incyte: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Roche: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Bristol Myers-Squibb: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; CTI: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees.