Downregulation of Morrbid in Tet2-Deficient Preleukemic Cells Overcomes Resistance to Inflammatory Stress and Mitigates Clonal Hematopoiesis

Abstract

Myeloid malignancies including AML, CMML, MPN and MDS are considered clonal blood disorders. Hematopoietic stem and progenitor cells (HSPCs) with mutation(s) in AML-related genes such as TET2 or DNMT3A represent what are commonly defined as preleukemic HSPCs. The selection and expansion of preleukemic-HSPC clones precedes the development of AML. Additionally, preleukemic-HSPCs can transform through serial acquisition of additional somatic mutations over time and contribute to the development of full-blown AML. What is unclear is the nature of environmental signals that might contribute to the “switch” from a preleukemic state to a leukemic state in these cells. In this context, inflammation has been hypothesized to play an essential role, but precisely how inflammatory signals influence the growth, survival, differentiation and the overall engraftment potential of preleukemic-HSPCs is yet to be determined. Mouse models carrying loss of function alleles in Tet2 or Dnmt3a manifest an expanded HSPC pool, including a hematopoietic stem cell (HSC)-enriched fraction defined by cell surface markers Lineage-/Sca-1+/c-Kit+ (LSK). Some of these genetically modified mice go on to develop CMML or MPN with modest penetration when aged. Majority of pre-leukemic mutations on their own are insufficient to cause AML in mice, suggesting that a single mutation among the above-described mutations define a pre-leukemic state and perhaps additional cooperating mutations are necessary to provide a more effective selection advantage for preleukemic-HSPC leading to the development of full-blown leukemia. Inflammation has been linked to tumor induction and transformation in solid tissues. Inflammation caused by environmental exposure, infection, autoimmunity, or ageing may result in mutations and genomic instability in somatic cells as well as in reprogramming of the tumor microenvironment (i.e. through regulating angiogenesis and expression of cytokines and chemokines). Considering that both innate and adaptive immune cells are generated from HSPCs and are involved in regulating local as well as whole-body inflammatory processes, the relationship between inflammation and hematopoietic malignancies is likely to be complex. While the influence of inflammatory stress on normal HSPCs has recently gained significant attention, little is known about how preleukemic HSPCs respond to inflammation. Because HSPCs reside in the bone marrow and are surrounded by mature immune cells, the inflammatory microenvironment is likely to influence the growth and self-renewal of these cells in part by producing pro-inflammatory cytokines and chemokines. In support of this hypothesis are epidemiologic findings demonstrating that infection may act as a trigger for AML development in humans. In the present study, we asked whether Tet2-deficient HSPCs maintain growth and survival advantage during pathological stress by examining how Tet2-KO preleukemic-HSPCs respond to acute and chronic inflammation. We show a rapid increase in the frequency and absolute number of Tet2-deficient mature myeloid cells and HSPCs in response to acute inflammatory stress, which results in enhanced production of inflammatory cytokines, including IL-6, and resistance to apoptosis. Functionally, Tet2-deficient preleukemic-HSPCs showed resistance to inflammation-induced damage and apoptosis relative to controls. IL-6 induces hyperactivation of the Shp2-Stat3 signaling axis, resulting in increased expression of a novel anti-apoptotic lncRNA Morrbid in Tet2-KO myeloid cells and HSPCs. Expression of activated Shp2 in HSPCs phenocopies Tet2 loss, with regard to hyperactivation of Stat3 and Morrbid. In vivo, pharmacologic inhibition of Shp2 or Stat3 or genetic loss of Morrbid in Tet2-deficient mice rescues inflammatory stress-induced abnormalities in HSPCs and mature myeloid cells including clonal hematopoiesis. Our results suggest that the anti-inflammatory drugs E3330 and SHP099, which suppress the hyper-activation of Morrbid, could be of clinical benefit for TET2 mutations-induced clonal hematopoiesis and leukemogenesis. Our results provide insight into the selection advantage that might render Tet2-deficient HSPCs susceptible to transformation and suggest that anti-inflammation therapy could be of clinical benefit for individuals carrying TET2 mutations. DisclosuresKelley:Apexian Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties. Mohseni:Novartis Institutes of Biomedical Research: Employment.