High Level of CCL3 in the Bone Marrow Microenvironment Promotes Anemia By Suppressing the Erythropoiesis Differentiation of Hematopoietic Stem Cells in Myeloma

Background and aims:

Multiple myeloma (MM) is a hematologic malignancy characterized by monoclonal plasma cells infiltrating the bone marrow. Anemia is the most common symptom and notably characteristic of myeloma patient. The mechanism of anemia of chronic disease (ACD) is impaired underlies that of MM-related anemia. However, the mechanisms underlying myeloma related anemia have not been fully elucidated. Clarify the pathogenesis of myeloma related anemia and identify the key molecular foundation will be helpful to develop the target treatment and reverse the inferior outcome of those patients.

Methods:

The correlation of tumor burden and the level of HGB was analyzed in the cohort of 776 newly diagnosed myeloma patients. The number and the differentiation activity of HSPCs under the MM microenvironment were analyzed using both primary myeloma patient samples and NSG myeloma mouse model. The HSPC of bone marrow was isolated by the sorting of CD34 positive cells. Colony- Forming Cell (CFC) assay was performed to identify the differentiation activity of HSPC derived from myeloma patient and healthy donor. Furthermore, 5TGM1 myeloma mouse model was utilized to verify the effects of myeloma cells on the differentiation activity of HSPCs during MM progression in vivo.

Results:

Our clinical analysis showed that more than 80% (620/776) myeloma patients had anemia (HGB< 120g/L) and more than 60% patients had moderate or severe anemia (HGB< 90g/L) at newly diagnosis . The degree of anemia was positively correlated with the tumor burden of CD138⁺ cells and also highly positive correlated with bone lesions in myeloma patients (p<0.001). Moreover, the higher level of CD138⁺ cells was significantly negative correlated with the number of CD34⁺ HSPCs in the bone marrow (r=-0.526, p=0.0082). The flow cytometry data indicated that the percentage of HSPCs population in NDMM patients was significantly lower than that of normal controls (P<0.001). We did not find the decrease of HSC (Lin^-CD34⁺CD38^-) in MM bone marrow compared with healthy control. However, the population of HPC (Lin^-CD34⁺CD38⁺) significantly decreased (P<0.001). The proportion of megakaryocyte-erythrocyte progenitors (MEP) was notably decreased (p<0.0001) in MM patient samples. On the contrary, the proportion of granulocyte-macrophage progenitors (GMP) was increased in MM patients compared to healthy donors (p<0.01). These data suggested that the myeloma bone marrow microenvironment suppressed the CD34⁺ HPSC proliferation and differentiation to MEP which may contribute the myeloma related anemia. 5TGM1 mouse model of myeloma was used to verify these clinic findings. The number of HSCs (LKS⁺) and HPCs (LKS^-) during MM cell progression was monitored at different time point by flow cytometry after 5TGM1 MM cells injection. The results indicated clearly that the HSCs (LKS⁺) and HPCs (LKS^-) were suppressed in the myeloma bone marrow microenvironment. Similar to MM patient, the proportion of MEP was the most significantly suppressed population among all of the hematopoietic subsets in this MM mouse model. CFC assays indicated that the BFU-E colony formation of CD34⁺ cells from MM patients was significantly suppressed (p<0.01). RNA sequencing analysis indicated that the transcription factor in erythropoiesis differentiation, GATA1 and KLF1, was obviously decreased in myeloma CD34⁺ cells compared with that in healthy donor. Further investigated showed that high level of CCL3 in the bone marrow microenvironment suppressed the differentiation of erythropoiesis which played a pivotal role in myeloma related anemia. Administration of the CCL3 receptor (CCR1) antagonist partially recovered the yield of erythroid colonies in the presence of myeloma plasma.

Conclusion:

Bone marrow microenvironment of multiple myeloma suppresses the erythropoiesis differentiation of hematopoietic stem cells and promotes the myeloma associated anemia. High level of CCL3 may down regulate GATA1 and KLF1 and be one of the important reasons for myeloma-related anemia.