Mechanismen von Interferon-alpha-induzierten biologischen Effekten, Signaltransduktion, zellulärem Kontext, Medikamentensynergien und klinischen Biomarkern bei Myeloproliferativen Neoplasien
Zusammenfassung der Projektergebnisse
In this project, we investigated the mechanisms by which interferon alpha (IFNa) specifically reduces malignant clones in MPN patients (pts). To gain insights into these mechanisms, we used a variety of cellular systems (murine as well as human cell lines, induced pluripotent stem cells [iPSC] and primary patient material) and techniques (RNAseq and single-cell RNAseq [scRNAseq], chromatin-IP [ChIP] and ChIPseq, CRiSPR-Cas driven loss- and gain-of function experiments and other functional assays) to comprehensively analyze transcriptional and posttranscriptional pathways at the RNA and protein level. Given that the JAK2V617F mutation is the most frequent mutation in MPN, we analyzed samples from these pts in great detail. An in vitro clonal cell model was established in which primary patient cells were analyzed at the clonal level before and after their maturation. Mutantspecific reduction of the clones was quantified in this model, and all analyzed pts were grouped into molecular responders and non-responders. PV pts showed the highest rate of molecular responders (vs. ET and PMF), matching clinical observations, where PV patients show the strongest reduction in mutant VAF when treated with IFNa. However, when we analyzed the IFNa response in individual clones from the same patient, there was a high degree of functional and transcriptional heterogeneity between clones. To gain a deeper insight into this heterogeneity, scRNAseq analyses of PV patient-derived as well as healthy control clonogenic cells with and without in vitro IFNa treatment were performed. IFNa response was induced in all identified clonal cell clusters. However, the IFNa receptor expression was gradually downregulated during erythrocytic differentiation, and this was confirmed at the protein level using flow cytometry. Our detailed analysis of the scRNAseq data showed a specific reduction in PV-derived immature erythrocytic cells by IFNa treatment. We were able to trace this reduction to a significant induction of apoptosis in immature erythrocytic clusters, while differentiation was not influenced by IFNa treatment, as investigated in an in vitro differentiation model. In a last step, we were able to confirm the upregulation of key genes in in vivo treated patients. These results indicate that IFNa induces apoptosis in a specific immature fraction of MPN-derived cells, whereas other cells are not inhibited, possibly explaining a slow but longterm reduction in the mutant VAF of these patients. Together with gene expression results from CD34+ cells of IFNa-treated and -untreated MPN pts, we are currently examining off-target effects of IFNa in order to better predict the adverse events of IFNa in MPN pts. In our next approach, we assessed whether the IFNa response was dependent on the type of MPN-associated driver oncogene. Interestingly, we found that JAK2V617F mutated patients showed a significantly higher molecular response to IFNa than CALR mutated patients. Analysis of the IFNa signaling pathway revealed increased basal phosphorylation in JAK1 and STAT1 in JAK2V617F mutated cells, suggesting that JAK2V617F- but not CALR-mutated cells are primed to respond to IFNa. These results are clinically relevant, as they also suggested that higher doses of IFNa may be needed in CALR-mutated MPN pts. JAK2V617F-mutant cells also showed significantly better IFNa responses when compared to BCR-ABL-mutant cells. Here, the mechanism involved STAT2 in addition to STAT1, explaining, at least in part, the higher response of JAK2V617F-mutant MPN vs. CML pts to IFNa monotherapy. Given these maturation-, genotype- and patient-specific responses to IFNa, we generated iPSCs from MPN pts (including JAK2V617F- and CALR-mutant hetero- and homozygous clones) as well as healthy controls. In addition, JAK2V617F and CALR mutations were “repaired” back to wildtype alleles in these iPSC. We established several differentiation protocols for these iPSC, including a novel megakaryocytic differentiation protocol. We observed accelerated and TPO-independent differentiation of megakaryocytes in all mutated MPN-derived iPSC, which was abolished by the repair of the mutation. We have now generated iPSCs that no longer express the IFNa receptor (IFNAR2), and these can be used as a human cell system to analyze the IFNa response in different cell types in the future. In conclusion, the current research project on the effects of IFNa has opened new avenues of analyses with clinical implications, and we are currently expanding our experiments, both in vitro and in vivo, to the effects of IFNa on other cell populations, including the bone marrow stromal cells, which are critically involved in MPN pathogenesis.
Projektbezogene Publikationen (Auswahl)
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(2019) Differential roles of STAT1 and STAT2 in the sensitivity of JAK2V617F- vs. BCR-ABL-positive cells to interferon alpha. J Hematol Oncol 12: 36
Schubert C, Allhoff M, Tillmann S, Maie T, Costa IG, Lipka DB, Schemionek M, Feldberg K, Baumeister J, Brümmendorf TH, Chatain N, Koschmieder S
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(2019) JAK2V617F but not CALR mutations confer increased molecular responses to interferon-alpha via JAK1/STAT1 activation. Leukemia 33: 995-1010
Czech J, Cordua S, Weinbergerova B, Baumeister J, Crepcia A, Han L, Maie T, Costa IG, Denecke B, Maurer A, Schubert C, Feldberg K, Gezer D, Brümmendorf TH, Müller-Newen G, Mayer J, Racil Z, Kubesova B, Knudsen T, Sorensen AL, Holmstrom M, Kjaer L, Skov V, Larsen TS, Hasselbalch HC, Chatain N, Koschmieder S
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STAT1 Transcriptional Response Predicts Molecular Responses of PB-Derived Clonogenic Cells from MPN Patients to Interferon Alpha. Blood. 2019;134(Supplement_1):1679 (ASH Meeting Abstract)
Kalmer M, Feldberg K, Gezer D, Isfort S, Brümmendorf TH, Chatain N, Koschmieder S
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(2020) Role of inflammation in the biology of myeloproliferative neoplasms. Blood Rev 42: 100711
Koschmieder S, Chatain N
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(2021) CALR frameshift mutations in MPN patient-derived iPSCs accelerate maturation of megakaryocytes. Stem Cell Reports 16: 2768-2783
Olschok K, Han L, de Toledo MAS, Böhnke J, Grasshoff M, Costa IG, Theocharides A, Maurer A, Schuler HM, Buhl EM, Pannen K, Baumeister J, Kalmer M, Gupta S, Boor P, Gezer D, Brümmendorf TH, Zenke M, Chatain N, Koschmieder S
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(2021) Early and late stage MPN patients show distinct gene expression profiles in CD34(+) cells. Ann Hematol 100: 2943-2956
Baumeister J, Maie T, Chatain N, Gan L, Weinbergerova B, de Toledo MAS, Eschweiler J, Maurer A, Mayer J, Kubesova B, Racil Z, Schuppert A, Costa I, Koschmieder S, Brümmendorf TH, Gezer D
