Ly6C+ Inflammatory Monocytes Create a Leukemia-Initiating Niche
Authors: Kazianka Lukas , Prchal-Murphy Michaela , Agreiter Christiane , Schweicker Carmen , Forte Maurizio , van der Kouwe Emiel , Kornauth Christoph , Pichler Alexander , Pemovska Tea , Rohrbeck Johannes , Schmeidl Margit , Troester Selina , List Julia , Sexl Veronika , Grebien Florian , Valent Peter , Tenen Daniel G. , Staber Philipp Bernhard 
Published: December 2024

Background
Physiological hematopoiesis relies on a tightly balanced equilibrium of mature blood cell formation and stem cell maintenance that is governed by cell-intrinsic as well as cell-extrinsic stimuli. While the role of transcription factors is well established in this model, the latter are exerted by a complex network of multiple cell types (such as fibroblasts, endothelial cells, bone lining cells, monocytes/macrophages, megakaryocytes, etc.) forming a functional unit that provides the bone marrow (BM) niche.
Leukemogenesis is classically perceived as a multi-step process with serial acquisition of mutations at the stem and progenitor cell level ultimately leading to a differentiation block and uncontrolled accumulation of immature cells. However, niche contributions to this process are increasingly accepted. Pronounced alterations of the BM niche were shown under stress and disease conditions, including recent work at the single-cell level. The majority of these studies focused on CD45- stromal niche populations, systematically leaving aside monocytic cells that are also members of the BM niche. We sought to characterize the role of monocytes/macrophages in the BM niche and explore a pathway through which the hematopoietic system potentially reshapes its own niche.
Methods
We use a novel mouse model of myelodysplastic syndrome (MDS) / chronic myelomonocytic leukemia (CMML) that harbours point mutations in the C/EBP binding sites at the -14kb enhancer element of Pu.1 (subsequently called Pu.1Ki/Ki). The mouse model was validated by ChIP, and extensively characterized using high-throughput multi-omics techniques and a series of transplantation experiments.
Results
Young Pu.1Ki/Ki mice displayed no overt signs of disease, but a markedly altered monocyte subset homeostasis in favour of the Ly6C+ inflammatory monocyte subset in the BM and peripheral blood (PB). This inflammatory monocyte signature also emerged as a hallmark when the BM plasma was characterized using an unbiased proteomics approach. Phenotypically this translated to an osteoporotic bone phenotype secondary to an increase in osteoclasts, proving BM niche changes on the molecular and microscopic level. Similar results were obtained with a monocyte-specific Pu.1 knockout mouse model.
Upon longer follow-up, aged Pu.1Ki/Ki mice developed a myelodysplastic phenotype characterized by thrombopenia, neutropenia and BM hypercellularity resembling human chronic myelomonocytic leukemia (CMML). Of these, thrombopenia typically arose first as an early finding of disease. The CMML phenotype was not associated with accumulation of mutations in typical myeloid candidate genes, making us speculate about a potential role of the niche in disease development.
In a series of transplant experiments, myelodysplasia (evidenced by thrombopenia and leukopenia) developed in healthy wildtype (WT) bone marrow, when WT LSK were transplanted into Pu.1Ki/Ki recipients. Importantly, co-transplantation of Pu.1Ki/Ki common monocyte progenitors (cMoP) was required to keep up monocyte-driven niche changes. Conversely, transplantation of Pu.1Ki/Ki LSK started the cascade of disturbed monocyte subset homeostasis, niche changes and increased bone turnover. Likewise, monocyte-induced niche changes fostered proliferation and disease aggressiveness of MLL-AF9 murine leukemia in vitro and in vivo. Importantly, the Pu.1Ki/Ki niche demonstrated an enhanced potential to initiate overt leukemia when the MLL-AF9 transplant was performed in a limiting dilution setup.
Summary
We delineate a novel disease mechanism that originates from Ly6C+ inflammatory monocytes that are part of the BM niche. In analogy to solid tumors, we refer to these cells as leukemia-associated monocytes (LAM) because of their capability to i) induce myelodysplasia in healthy BM, ii) increase aggressiveness of the oncogene-driven MLL-AF9 leukemia model, and iii) induce leukemia by lowering the leukemia initation potential of the niche.