Alterations of Human Bone Marrow Niche in Clonal Hematopoiesis and AML

Acute myeloid leukemia (AML) is a heterogeneous disease, predominantly caused by acquisition of mutations in the hematopoietic stem and progenitor cells. Some of these mutations are acquired in a preleukemic state termed clonal hematopoiesis of indeterminate potential (CHIP). Recent studies show that alterations in the bone marrow niche can support progression and therapy resistance of myeloid neoplasia. In our study, we aim to elucidate alterations in the bone marrow niche, in particular in mesenchymal stem cells (MSC), during the evolution from healthy to CHIP and further to AML.

We collected primary human bone marrow (BM) and peripheral blood (pB) samples from hematologically healthy individuals who underwent hip replacement surgery at the LMU University Hospital as well from AML patients treated at our institution (LMU University Hospital Munich). Individuals were assigned to non-CHIP or CHIP cohorts based on targeted mutation analyses on pB samples. So far, we have included 105 healthy individuals and 92 have undergone targeted sequencing, revealing 27 individuals (29%) with CHIP. While there was no difference between the gender of the non-CHIP and CHIP groups, individuals with CHIP tended to be of older age (median, 64 y non-CHIP vs. 73.5 y CHIP). We detected DTA gene mutations as the most common CHIP alteration and 1.4 mutations per individual were found on average. Variant allele frequencies varied between 1% and 17%. The amount of mutations per individual increased with age. In the non-CHIP and CHIP individuals, we analyzed various blood parameters such as hemoglobin, leukocyte counts, mean corpuscular volume and others, as well as various comorbidities such as myocardial infarction, hypertesia, previously treated cancer, etc. We found a trend for more frequent myocardial infarction in the CHIP individuals. We did not find any significant differences between CHIP and non-CHIP for the blood parameters.

MSC from donor BM samples were isolated via plastic adherence over 10-14 days of in vitro culture. MSC were characterized regarding their surface marker expression (FACS), differentiation capacity (osteogenic, adipogenic, chondrogenic) and metabolic activity (Seahorse XF). Analysis of MSCs from non-CHIP (n=5) and CHIP (n=5) individuals revealed no difference in surface marker expression over 10 passages. Also, differentiation assays of MSCs (non-CHIP=4, CHIP= 2) showed no differences in adipogenic and chondrogenic differentiation after 21 days. For osteogenesis, we noticed a trend towards an increased differentiation potential in the MSCs of CHIP individuals. This experiment is ongoing with the goal to increase the sample number up to 10 per condition, as well as including the AML controls. The data will be shown at the ASH meeting. To gain deeper insights into the BM niche we sorted 4 non-hematopoietic and 6 hematopoietic cell populations from non-CHIP (n=3), CHIP (n=4) and AML (n=4) individuals and performed bulk-RNA sequencing on each population. So far, changes in the composition of the bone marrow niche were only detected in AML samples.

Currently, in a series of pilot experiments, we established primary MSC and HSPC cross co-cultures from non-CHIP, CHIP and AML samples. A preliminary experiment with one non-CHIP and one CHIP sample showed that both non-CHIP and CHIP HSC produced less hematopoietic cells and less colony forming units (CFUs) after co-culturing with CHIP MSCs, than with non-CHIP MSCs. The results of the main experiment with 3 individuals per condition and including the AML samples will be shown at the conference.

Subklewe:Novartis: Consultancy, Speakers Bureau; Miltenyi Biotech: Research Funding; Seagen: Research Funding; Morphosys: Research Funding; Takeda: Other: Travel support; Janssen: Consultancy, Speakers Bureau; Roche: Consultancy, Research Funding; Gilead: Consultancy, Research Funding, Speakers Bureau; Celgene/BMS: Consultancy, Speakers Bureau; Amgen: Consultancy, Research Funding, Speakers Bureau; Pfizer: Consultancy; Seattle Genetics: Research Funding; Bristol-Myers Squibb: Research Funding. Theurich:Amgen: Consultancy, Honoraria; BMS/Celgene: Consultancy, Honoraria; GSK: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria; Sanofi: Consultancy, Honoraria; Takeda: Consultancy, Honoraria. Metzeler:Pfizer: Consultancy; Daiichi Sankyo: Honoraria; Celgene/BMS: Consultancy, Honoraria, Research Funding; AbbVie: Honoraria; Novartis: Consultancy; Jazz Pharmaceuticals: Consultancy; Astellas: Honoraria.