Association of the composition of the bone marrow tumor microenvironment in BCR::ABL1-negative myeloproliferative neoplasms with IFN-γ signaling and driver mutations

Constitutive JAK/STAT pathway activation is crucial in the pathogenesis of BCR::ABL1-negative myeloproliferative neoplasms (MPN), but has not yet been linked to interferon (IFN)-γ signaling and tumor microenvironment. Human JAK2 V617F-mutated cell lines, 265 bone marrow biopsies (BMB) of two MPN cohorts, and 50 non-neoplastic BMB, revealed an intrinsic activation of IFN-γ signaling, which was confirmed by public RNA expression data. In vitro analysis of JAK2-mutated cell lines showed an activation of IFN-γ signaling pathway in the absence of IFN-γ in the cell supernatants. In addition, a heterogeneous, but increased expression of IFN-γ signaling components was found in BMB of JAK2-mutated samples with the highest expression in lymphocytes and monocytes, accompanied by increased tumor infiltrating lymphocytes (TIL). Unsupervised clustering identified a prognostic favorable cluster in both patient cohorts characterized by augmented IFN-γ signaling and TILs. This cluster was enriched with JAK2-mutated, JAK-inhibition naive MPN, mainly essential thrombocythemia and polycythemia vera with mild bone marrow fibrosis. Moreover, in silico data confirmed the link between JAK2 mutations and increased IFN-γ signaling. Multivariate Cox regression revealed TILs to be the strongest prognostic marker. In conclusion, JAK2-mutated MPN exhibit an intrinsic activation of IFN-γ signaling associated with changes in the BM TME and patients' outcome. Constitutive activation of the Janus kinases and signal transducer and activator of transcription (JAK/STAT) signaling pathway mainly mediated by mutations in the JAK2, CALR and MPL genes in pluripotent hematopoietic stem cells (HSC) is crucial for the pathogenesis of BCR::ABL1-negative myeloproliferative neoplasms (MPN). Despite the activation of JAK/STAT signaling and its influence on the proliferation of malignant cells is well studied in patient samples and JAK2- and CALR-mutated cell systems, there exists limited information about the link between interferon (IFN)-γ signaling and bone marrow (BM) environment alterations. Therefore, two human JAK2 V617F-mutated cell lines, 265 bone marrow biopsies (BMB) of MPN patients, separated in two independent cohorts, both with known clinical parameters, such as driver mutations, treatment and survival, 50 non-neoplastic BMB and five publicly available bulk and single cell RNA expression data sets of MPN samples with known JAK2 or CALR mutation status were analyzed regarding (i) the role of IFN-γ signaling, (ii) its interrelation with the composition of the local BM tumor microenvironment (TME), (iii) the expression of immune response relevant molecules and (iv) their impact on patients' survival. Created in BioRender. Bauer, M. (2025) https://BioRender.com/h133y7w .