Macrophage Involvement in the Response of Acute Myeloid Leukaemia to Chemotherapy

Acute myeloid leukemia (AML) is a clonally heterogeneous neoplasm with poor survival rates. Although chemotherapy often leads to clinical remission, the relapse rate of AML is very high and usually fatal. Chemotherapy is usually effective at debulking AML blasts however a small population capable of re-initiating the leukemia (LIC) survive ultimately causing relapse. Mouse models of AML suggest that LIC are protected from chemotherapy by a protective niche similar to hematopoietic stem cell (HSC) niches. As 1) bone marrow (BM) macrophages are key regulators of HSC niche function and 2) tumor-associated macrophages promote chemotherapy resistance in several models of solid tumors (e.g. breast, lung & ovarian) we investigated a potential role of BM non-malignant macrophages in promoting chemoresistance of AML blasts.

Using a mouse model of AML driven by the MLL-AF9 oncogene in which mouse HSC are transduced with MSC-MLL/AF9-ires-GFP and transplanted into syngeneic recipients, we examined how AML progression alters the phenotype of non-malignant GFP-negative BM macrophages by flow cytometry. 18 days post-transplant, GFP+ leukemia burden in the BM was in the 40-60% range. In non-malignant GFPneg BM macrophages, cell surface expression of MHC class II, CD80, L-selectin and neuropilin-1 was significantly decreased suggesting that AML blasts alter the function of BM macrophages.

To determine whether BM macrophages could have any role in AML response to chemotherapy, we first determine whether non-malignant BM macrophages were depleted in vivo by treatment with cytarabine (5 days, 100mg/kg) and doxorubicin (3 days, 1mg/kg). Two days after cytarabine+doxoR treatment, most CD11b+ F4/80- Ly6G+ granulocytes and CD11b+ F4/80+ CD169- monocytes were ablated from the BM with 0.1% granulocytes and 1.0 % monocytes surviving after treatment. In contrast CD11b- F4/80+ CD169+ VCAM1+ macrophages were the most resilient myeloid cells in the BM with 9.5% still present 2 days after chemotherapy.

To further investigate a possible role of these chemotherapy-resilient CD169+ macrophages in promoting response of AML blasts to chemotherapy, we transplanted MLL-AF9 leukemia cells generated with wild-type HSC into CD169^DTR/WT mouse recipients in which one CD169 allele is knocked-in with human diphtheria toxin receptor (DTR). This enables specific depletion of CD169+ macrophages in vivo by injecting diphtheria toxin (DT). In this model, DT treatment targets host CD169+ macrophages but not the AML blasts as they do not contain the DTR knock-in. In the absence of chemotherapy, depletion of recipient CD169+ macrophages did not alter leukemia burden in the BM, spleen or blood 48 hours post depletion and did not alter mouse survival (median survival 29-30 days in both CD169-DTR and WT recipients treated with DT or saline).

However when cytarabine + doxoR were administered 48 hours after DT administration, median survival of CD169-DTR recipients was significantly increased to 39.5 days compared to wild-type recipients 35 days, p<0.001. Furthermore leukemia burden in both the blood and spleen was significantly reduced.

To determine whether CD169 could by itself mediate lower response of AML to chemotherapy, we transplanted MLL-AF9 cells generated from WT HSC into CD169^DTR/DTR mice which are knocked-out for both CD169 alleles. Deletion of both CD169 alleles in recipient mice did not change mouse survival in response to chemotherapy in the absence of DT. Therefore absence of CD169 is not sufficient to prolong mouse survival in response to chemotherapy.

In conclusion, non-malignant CD169+ macrophages may protect MLL-AF9 leukemia blasts from the cytotoxic effects of cytarabine and doxorubicin. The mechanisms of this chemoprotection remain to be determined but do not involve a direct effect of CD169 on AML blasts.