Human Acute Myeloid Leukemia Inhibits Normal Erythroid Differentiation through the Paracrine Effects of IL-6

Acute Myeloid Leukemia (AML) is an aggressive cancer resulting in severe cytopenias related to bone marrow (BM) failure. The common assumption for AML-induced BM failure is overcrowding due to clonal expansion of immature myeloid blasts, leading to failure of normal hematopoiesis. However, in a cohort of 293 AML patients, we found that disease burden (% of blasts determined on diagnostic BM aspirate) did not predict severity of cytopenia (hemoglobin r_s=-0.053; p=0.49; WBC r_s=-0.030, p=0.70; platelet r_s=0.091, p=0.026), strongly arguing against simple crowding as the main mechanism underlying AML-induced BM failure. Thus, the goal of our study is to identify novel mechanism(s) associated with AML-induced BM failure, potentially enabling development of new therapies to improve AML management and reverse morbidity.

Conventional xenograft models of human AML do not typically exhibit cytopenias, making them unsuitable to study AML-induced BM failure. We speculated that in the mouse, increased splenic extramedullary hematopoiesis compensated for failed intramedullary hematopoiesis due to AML. To test this hypothesis, we performed surgical splenectomy on NSG mice prior to their transplantation with human AML. Strikingly, splenectomized NSG mice engrafted with primary human AML at a 30-70% disease burden (n=8 for primary AML samples, n=5-10 for each group of splenectomized NSG mice) developed leukopenia and severe anemia compared to sham-operated AML-engrafted controls. AML-engrafted splenectomized NSG mice showed early mortality compared to AML-engrafted NSG mice with intact spleens (10.1 weeks vs. 34.2 weeks p<0.0001). Thus, our splenectomized murine xenograft model uniquely recapitulates human disease and allows for novel characterization of AML-induced BM failure.

Utilizing our model, splenectomized NSG mice engrafted with human AML demonstrated depletion of normal hematopoietic progenitors (HSPCs) including HSCs (11.1 fold p<0.0001), MPPs (8.90 fold, p<0.001)), CMPs (11.4 fold p<0.0001), MEPs (1.76 fold p=0.0005) and GMPs (5.07 fold p<0.0001). Focusing on anemia, AML also depleted erythroid progenitors including proerythroblasts (1.39 fold p=0.04), normoblasts (4.96 fold p<0.0001), late normoblasts (10.0 fold p<0.0001), and reticulocytes (4.34 fold p=0.007). The relative preservation of proerythroblasts and depletion of normoblasts indicated that AML blasts impart a specific in vivo differentiation blockade in erythroid differentiation.

To explore mechanisms by which AML blasts inhibit normal HSPCs, we generated conditioned media (CM) from patient-derived AML blasts, and found that AML-CM suppressed BFU-E colony formation from normal HSPCs (3.1-5.1 fold). Furthermore, in a direct co-culture system with AML blasts and CD34⁺ HSPCs, AML blasts inhibited erythroid differentiation from the CFU-E to normoblast stage by 81%. Removing CD34⁺ cells from the AML co-culture allowed cells to resume differentiation to the proerythroblast stage. These experiments demonstrate that AML imparts a differentiation blockade along the MEP-proerythroblast axis in a cell non-autonomous, reversible fashion.

Using cytokine array analysis, we identified elevated IL-6 levels in AML sample-derived CM (n=10, 2841±766.4 pg/ml, 7.80 fold increase, p=0.03) compared to CD34⁺-derived CM (n=5, 364±36.0 pg/ml). Increased IL-6 was also found in BM aspirates from human AML engrafted splenectomized NSG mice (715±125pg/ml, p=0.001) compared to mice engrafted with normal CD34⁺ HSPCs (undetectable). Thus, we hypothesized that IL-6 produced by AML blasts acts as a paracrine factor to suppress erythropoiesis. Consistent with this hypothesis, an IL-6 neutralizing antibody reversed the inhibition of BFU-E formation imparted by AML-CM. Furthermore, the addition of recombinant IL-6 to liquid cultures of erythroid differentiation resulted in a 23% reduction in proerythroblasts.

Together, our data suggests that (1) overcrowding is not the primary mechanism resulting in BM failure in AML; (2) AML blasts play a previously unrecognized role in imparting a differentiation blockade along the MEP-proerythroblast axis, resulting in progressive anemia; and (3) this differentiation blockade is at least partially attributable to IL-6 secreted from AML blasts as a paracrine factor.