Upregulated JAK/STAT signaling is considered a major factor driving the pathogenesis of myeloproliferative neoplasms (MPNs). Clinically approved JAK inhibitors alleviate constitutional symptoms of MPNs, however they do not alter underlying disease and do not target MPN-initiating malignant hematopoietic stem/ progenitor cells (HSPCs). This observation suggests that alternative signaling pathways contribute to the pathogenesis of MPNs. Detailing dysregulated pathways, besides JAK/STAT, that drive pathogenesis of MPNs specifically at the MPN stem cell level, will inform new therapeutic strategies with possible curative potential. Evidence from our laboratory indicates that loss of an adapter protein, Abelson interactor 1 (Abi-1), unexpectedly leads to development of MPN-like disease in mice, and is mechanistically linked to hyperactivated STAT3 and NF-?B signaling with only negligent upregulation of JAK2/STAT5 pathway. We also show that HSPCs and granulocytes from patients with the most severe of MPNs - primary myelofibrosis (PMF) show decreased Abi-1 transcript and protein levels. To delineate mechanistic link between Abi-1, STAT3 and NF-?B signaling we decided to use newest proximity-dependent labelling (PDL) technology in tandem with advanced proteomics to provide in depth delineation of Abi-1 interactome (Specific Aim 2 of the parent grant). Our preliminary data from this assessment, performed using cell line models stably expressing biotin ligase or biotin ligase conjugated with Abi-1 subjected to PDL followed by mass spectrometry, identified components of the TAK1 (Map3k7) signaling pathway, including TAK1, Tab1, Tab2 and NF-?B1, in addition to well-established Abi-1 interactors such as WAVE2, Sra-1 or Nap1. These preliminary data point to TAK1 being a potential new link between Abi-1 and NF-?B signaling pathways. Our central hypothesis is that Abi-1 plays an important and potentially targetable role in HSPC self-renewal and differentiation via direct negative mechanistic regulation of the STAT3/NF-?B inflammatory module of importance in MPN development. We will test this hypothesis in the Specific Aim 1 of the proposed supplementary project where we will utilize our developed PDL experimental tools, protocols and bioinformatic filtering strategy to establish STAT3 and NF-?B interactomes and to identify common interactors linking Abi-1, STAT3 and NF-?B signaling in HSPCs. Achieving this goal will not only allow us to determine mechanistic link between Abi-1/STAT3/NF-?B signaling axis newly implicated in pathogenesis of MPNs, but also to identify potential new therapeutic targets. The significance of our supplemental proposal relates to providing in depth assessment of a mechanistic cross-talk between newly identified Abi-1/STAT3/NF-?B signaling axis and identify new targetable mechanisms and disease drivers beyond JAK/STAT that contribute to malignant transformation of HSPCs, thereby leading to development of PMF and possibly other MPNs.
/RELEVANCE The JAK/STAT cascade is dysregulated in nearly all myeloproliferative neoplasms (MPNs), including most severe of MPNs ? primary myelofibrosis (PMF), however, currently available inhibitors that target the JAK/STAT pathway are not able to eradicate disease-initiating malignant hematopoietic stem cells. The objective of this research plan is to determine therapeutically targetable mechanistic link between the components of newly identified inflammatory Abelson interactor 1 (Abi-1)-STAT3-NF-?B signaling axis recently shown to be responsible for development of MPNs and systemic maintenance of MPN stem cells.
