Leukemia-initiating cells from some acute myeloid leukemia patients with mutated nucleophosmin reside in the CD34(-) fraction

Abstract

Leukemia-initiating cells (LICs) in acute myeloid leukemia (AML) are believed to be restricted to the CD34(+) fraction. However, one of the most frequently mutated genes in AML is nucleophosmin (NPM), and this is associated with low CD34 expression. We, therefore, investigated whether NPM-mutated AMLs have LICs restricted to the CD34(+) fraction. We transplanted sorted fractions of primary NPM-mutated AML into immunodeficient mice to establish which fractions initiate leukemia. Approximately one-half of cases had LICs exclusively within the CD34(-) fraction, whereas the CD34(+) fraction contained normal multilineage hematopoietic repopulating cells. Most of the remaining cases had LICs in both CD34(+) and CD34(-) fractions. When samples were sorted based on CD34 and CD38 expression, multiple fractions initiated leukemia in primary and secondary recipients. The data indicate that the phenotype of LICs is more heterogeneous than previously realized and can vary even within a single sample. This feature of LICs may make them particularly difficult to eradicate using therapies targeted against surface antigens.

Figure 1

Classification of NPM mutant AML samples. The expression of CD34 and CD38 on 8 AML samples is shown. (A) Subtype A samples have CD34 expression less than 0.5% of cells. The phenotypes of 3 subtype A samples are shown (left, sample 1; middle, sample 3; and right, sample 4). (B) Subtype B samples have CD34 expression greater than 0.5%, but the CD34⁺CD38⁻ population is small (< 0.1%) and distinct from the CD34⁺CD38⁺ and CD34⁻ populations. The phenotypes of samples 14 (left) and 15 (right) are shown. (C) Subtype C samples are more heterogeneous than the other subtypes. The CD34⁺ fraction is greater than 0.5%, but there is no distinct and small CD34⁺CD38⁻ population. The phenotypes of samples 17 (left), 19 (middle), and 21 (right) are displayed.

Figure 2

Sorting strategy and results of transplantation from fractions of NPM mutant AML. The phenotypes and sorting strategies are displayed for subtypes A-C. Sorted fractions of the leukemias were transplanted into mice. The expression of human CD45, CD33, CD38, and CD34 on bone marrow cells from the mice is displayed in the plots on the right. The nature of the human cells was assessed by quantitative PCR, and the results are indicated by letters adjacent to the plots. M indicates mutated NPM and WT indicates wild-type NPM. (A) The phenotypes of 2 subtype A samples are displayed. The R1 region indicates the gate used for sorting CD34⁺ cells (Ai). Some subtype A samples also have a separate CD34^dim population that is marked by the R2 region (Aii). AML was initiated by the CD34⁻CD38⁺ (Aiii) and CD34⁻CD38⁻ (Av) fractions of sample 3 (subtype A), whereas CD34⁺CD38⁻ cells (Aiv) initiated normal multilineage hematopoiesis. (B) Sample 15 (subtype B) was sorted as indicated (Bi). The CD34⁻CD38⁺ (Bii) and CD34⁺CD38⁺ (Biii) fractions of sample 15 initiated leukemia, whereas CD34⁺CD38⁻ cells (Biv) initiated normal multilineage hematopoiesis. (C) Sample 17 (subtype C) was sorted as indicated (Ci). All fractions of sample 17 initiated AML (Cii-v) though both normal and leukemic cells arose from the CD34⁺CD38⁻ fraction in some mice.

Figure 3

The phenotype of LICs can change in the mice. The expression of CD14 is seen in the primary AML sample from sample 7(A). The sample was sorted, and 250 000 CD14⁺ cells were transplanted into 3 mice, but no graft was detected at 9 weeks. By contrast 250 000 CD14⁻ cells gave rise to a graft at 9 weeks in 2 of 2 mice transplanted (B), indicating that the LICs were CD14⁻. All the human CD45⁺ cells derived from the CD14⁻ fraction expressed CD14 (C). These CD14⁺ cells were capable of initiating a graft in secondary recipients (D), indicating that the LICs were now CD14. The human CD45⁺ cells in the secondary recipients were CD14⁺ (E).