A Broad Overview of Signaling in Ph-Negative Classic Myeloproliferative Neoplasms

Abstract

Ph-negative myeloproliferative neoplasms (polycythemia vera (PV), essential thrombocythemia (ET) and primary myelofibrosis (PMF)) are infrequent blood cancers characterized by signaling aberrations. Shortly after the discovery of the somatic mutations in JAK2, MPL, and CALR that cause these diseases, researchers extensively studied the aberrant functions of their mutant products. In all three cases, the main pathogenic mechanism appears to be the constitutive activation of JAK2/STAT signaling and JAK2-related pathways (MAPK/ERK, PI3K/AKT). However, some other non-canonical aberrant mechanisms derived from mutant JAK2 and CALR have also been described. Moreover, additional somatic mutations have been identified in other genes that affect epigenetic regulation, tumor suppression, transcription regulation, splicing and other signaling pathways, leading to the modification of some disease features and adding a layer of complexity to their molecular pathogenesis. All of these factors have highlighted the wide variety of cellular processes and pathways involved in the pathogenesis of MPNs. This review presents an overview of the complex signaling behind these diseases which could explain, at least in part, their phenotypic heterogeneity.

Keywords: CALR; JAK2; MPL; TPOR; myeloproliferative neoplasms; signaling pathways.

Figure 1

JAK2-related canonical signaling pathways active in Ph-negative myeloproliferative neoplasms (MPNs). Mutations in CALR (CALR^MUT), JAK2 (JAK2^V617F), and MPL (TPOR^W515) lead to the constitutive activation of JAK2/STAT, PI3K/AKT, and MAPK/ERK signaling that promotes the transport to the nucleus of several transcription factors such as STATs and FOXO. There, they regulate transcription of their target genes, causing increased proliferation and survival of mutant cells. Mutant proteins are depicted in red.

Figure 2

Main non-canonical signaling pathways activated by JAK2^V617F in Ph-negative MPNs. In PV patients, JAK2^V617F (depicted in red) activates the adhesion receptor Lu/BCAM through the RAP1-AKT signaling pathway, making their erythrocytes more adhesive. JAK2^V617F has also been described to promote aberrant signaling in the nucleus, where it prevents the binding of heterochromatin protein 1 alpha (HP1α) and inhibits the methylation of histones via protein arginine methyltransferase 5 (PRMT5) impairment. MPN patients with JAK2^V617F also seem to be insensitive to the anti-inflammatory cytokine IL-10, increasing TNF-α production through Toll-Like Receptor (TLR) signaling.

Figure 3

Major non-canonical mechanisms derived from CALR^Mut. CALR^Mut (depicted in red) shows different binding affinities for proteins implicated in the unfolding protein response (UPR) (HSPA5, HSPA9, and HSPA8), proteins of the cytoskeleton (MYL9 and APRC4), and ribosomal proteins (RP17, RSP23, and RPL11), as well as reduced binding to MSI2, a transcriptional regulator that target genes mainly involved in cell cycle regulation. Additionally, CALR^Mut seems to reduce the activation of the pro-apoptotic pathway of the UPR and increases oxidative stress and DNA damage through the downmodulation of oxidation resistance 1 (OXR1). CALR^Mut also shows decreased binding affinities for PDIA3 and has a loss-of-function effect on the peptide loading complex (PLC), which mediates the loading of cellular antigens onto major histocompatibility complex class I (MHC-I) molecules, favoring immunoevasion. Mutations in CALR increase the secretion of the protein and bind to CALR receptors in antigen presenting cells (APCs), limiting their ability to phagocytize wild-type CALR-exposing cancer cells. The main differences between the phenotypes observed in patients with type 1 (del52) and type 2 (ins5) mutations have been attributed to thrombopoietin receptor (TPOR)-independent cytosolic calcium fluxes and the binding affinity for the transcription factor FLI1.

Figure 4

Non-canonical inflammatory signaling pathways affected in Ph-negative MPNs, regardless of subtype and driver mutation. The preleukemic niche of MPNs secretes high levels of IL-1, a proinflammatory cytokine that activates multiple downstream pathways, such as p38 MAPK and NF-κB. NF-κB, in turn, generates high levels of IL-8, a proinflammatory cytokine that binds to CXCR1 or CXCR2 and activates STAT3, PI3K/AKT, p38 MAPK, FAK and PLC/PKC. NFE2 overexpression has also been reported in most MPN patients and has been associated with high IL-8 levels and increased ROS and DNA damage. On the other hand, IL-6 is a proinflammatory cytokine produced by monocytes, macrophages and T-cells that signals via JAK1/STAT3, whose levels have been found elevated in JAK2^V617F PV and PMF patients. Finally, IFN-α is a key regulator of hematopoietic stem cells (HSCs) that depletes previously dormant hematopoietic stem progenitor cells (HSPCs) and enhances the immune response. A pathogenic role of oncostatin-M, TGF-β1, platelet-derived growth factor (PDGF), basic fibroblast growth factor (BFGF), VEGF, bone morphogenic proteins, inhibitors of matrix metalloproteinases (MMPs) and lipocalin-2 has been suggested in Ph-negative MPNs.

Figure 5

Role of the bone marrow microenvironment in the pathogenesis of Ph-negative MPNs. Endothelial cells expressing JAK2^V617F increase the expression of CXCL12 and stem cell factor (SCF) and cause the expansion of HSCs and progenitor cells. On the other hand, mesenchymal stem cells (MSCs) negative for JAK2^V617F show a reduced expression of CXCL12 and SCF. MSCs also overexpress galectin-1 in all MPN subtypes and galectin-3 in PV patients, and promote the expansion of osteoblasts by cell contact and excessive TGF-1β, Notch, IL-6, IL-1β, and TNF-β signaling. Osteoblasts overproduce inflammatory cytokines and reduce CXCL12 expression. By contrast, monocytes with JAK2^V617F seem to increase osteoclast forming ability and favor the survival of clonal HSCs. Meanwhile, clonal HSCs produce high levels of IL-1β, which induces nestin-positive MSCs death. Additionally, the sympathetic nerve fibers supporting Schwann cells are reduced in the bone marrow of MPN patients. Regarding the extracellular matrix, MPN patients with JAK2^V617F show increased levels of MMP-2 and MMP-9 and patients with primary myelofibrosis (PMF) have increased levels of all LOX family members.

Figure 6

Overview of the disease-modifying genes mutated in Ph-negative MPNs and their molecular consequences. These mutations occur in genes affecting epigenetic regulation (ASXL1, EZH2, DNMT3A, TET2, IDH1, and IDH2), tumor suppression (TP53 and PPM1D), transcription regulation (RUNX1 and NFE2), splicing (SRSF2, U2AF1, SF3B1, and ZRSR2), and other signaling pathways (SH2B3, CBL, NRAS/KRAS, PTPN11). Mutant proteins are depicted in red.