EphA2 Is a Therapy Target in EphA2-Positive Leukemias but Is Not Essential for Normal Hematopoiesis or Leukemia

<div><p>Members of the Eph family of receptor tyrosine kinases and their membrane bound ephrin ligands have been shown to play critical roles in many developmental processes and more recently have been implicated in both normal and pathological processes in post-embryonic tissues. In particular, expression studies of Eph receptors and limited functional studies have demonstrated a role for the Eph/ephrin system in hematopoiesis and leukemogenesis. In particular, EphA2 was reported on hematopoietic stem cells and stromal cells. There are also reports of EphA2 expression in many different types of malignancies including leukemia, however there is a lack of knowledge in understanding the role of EphA2 in hematopoiesis and leukemogenesis. We explored the role of EphA2 in hematopoiesis by analyzing wild type and EphA2 knockout mice. Mature, differentiated cells, progenitors and hematopoietic stem cells derived from knockout and control mice were analyzed and no significant abnormality was detected. These studies showed that EphA2 does not have an obligatory role in normal hematopoiesis. Comparative studies using EphA2-negative MLL-AF9 leukemias derived from EphA2-knockout animals showed that there was no detectable functional role for EphA2 in the initiation or progression of the leukemic process. However, expression of EphA2 in leukemias initiated by MLL-AF9 suggested that this protein might be a possible therapy target in this type of leukemia. We showed that treatment with EphA2 monoclonal antibody IF7 alone had no effect on tumorigenicity and latency of the MLL-AF9 leukemias, while targeting of EphA2 using EphA2 monoclonal antibody with a radioactive payload significantly impaired the leukemic process. Altogether, these results identify EphA2 as a potential radio-therapeutic target in leukemias with MLL translocation.</p></div

Immunophenotyping of EphA2 knockout mice compared to wild type littermates.

<p>(<b>A</b>) Full blood counts showed no significant differences in EphA2 knockout mice compared to wild type mice (n = 15, 3 independent experiments). (<b>B</b>) Number of nucleated bone marrow and spleen cells and spleen and liver weight of EphA2 knockout mice compared to the wild type littermates did not show any significant differences (n = 5). (<b>C</b>) Erythroid maturation analysis, erythroid-1 corresponding to pro-erythroblasts (CD71^high, Ter119^mid), erythroid-2 corresponding to basophilic erythroblasts (CD71^high, Ter119^high), erythroid-3 corresponding to late basophilic and polychromatophilic erythroblasts (CD71^mid, Ter119^high) and erythroid-4 corresponding to late basophilic and polychromatophilic erythroblasts (CD71^low, Ter119^high) in the bone marrow presented as percentage of viable bone marrow cells showed no differences in erythroid maturation in EphA2 knockout compared to wild type control cells (n = 15, 3 independent experiments). (<b>D</b>) B-lymphocytes (B220), T-lymphocytes (CD3) and Granulocytes (Gr-1) analysis showed no differences in B-lymphocytes, T-lymphocytes or granulocytes population in bone marrow, spleen and blood of the EphA2 knockout mice compared to the wild type control mice (n = 15, 3 independent experiments). The data represent the mean ± SEM (standard error of the mean). Unpaired <i>t</i> test was performed for statistical analyses (n) represent the number mice used in each experiment. An unpaired <i>t</i> test was performed for statistical analyses.</p

Analysis of EphA2 protein and RNA expression on GFP-control, MLL-AF9 and BCR-ABL bone marrow and spleen.

<p>(<b>A</b>) EphA2 mRNA expression levels relative to 18sRNA in bone marrow of GFP-control, MLL-AF9 and BCR-ABL mice, analyzed per 1000000 18s RNA using RT-PCR. Significantly higher EphA2 expression (P = 0.0267) in the MLL-AF9 bone marrow compared to GFP-control mice. (<b>B</b>) Representative flow cytometric overlay analysis of the EphA2 expression in GFP-control, MLL-AF9 and BCR-ABL bone marrow. (<b>C</b>) Flow cytometric analysis of the EphA2 expression in bone marrow of GFP-control, MLL-AF9 and BCR-ABL mice measured as mean fluorescent intensity, showed significantly higher EphA2 expression on MLL-AF9 bone marrow compared to GFP-control bone marrow (P <0.0001) and compared to BCR-ABL leukemic bone marrow (P = 0.0019) (n = 5 GFP-control, n = 4 MLL-AF9, n = 3 BCR-ABL, 1 biological replicates, 2 technical replicates). (<b>D</b>) Flow cytometric analysis of the EphA2 expression in spleen of GFP-control, MLL-AF9 and BCR-ABL mice measured as mean fluorescent intensity, showed significantly higher EphA2 expression in spleen of the MLL-AF9 mice compared to GFP-control mice (P = 0.0143) and higher expression compared to BCR-ABL leukemic mice (n = 5 GFP-control, n = 4 MLL-AF9, n = 3 BCR-ABL, 1 biological replicates, 2 technical replicates). Each dot corresponds to one individual mouse. The data represent the mean ± SEM. Unpaired <i>t</i> test was performed for statistical analyses.</p

Expression analysis of EphA subfamily on mouse MLL-AF9 leukemic cells, on patient samples and human leukemic cell lines with MLL-rearrangement.

<p>(A) Flow cytometric analysis of EphA2, EphA7 and ephrinA5-Fc binding in EphA2 knockout and wild type bone marrow cells showed no EphA2 expression in the EphA2 knockout mice and significantly high expression of EphA2 in the wild type mice as expected (P = 0.0165). Comparable levels of EphA7 (P = 0.3099) and ephrinA5-Fc binding (P = 0.8710) expression were observed in EphA2 knockout and wild type leukemic cells. (B) RT-PCR analysis of EphA1, EphA2, EphA3, EphA4, EphA5 and EphA7 in EphA2 knockout and wild type bone marrow cells showed no EphA2 transcript in the EphA2 knockout mice compared to wild type MLL-AF9 (P = 0.0776) and comparable level of EphA1 (P = 0.3458), EphA5 (P = 0.7020) and EphA7 (P = 0.6167) transcript in EphA2 knockout and wild type mice. There was no expression of EphA3 and EphA4 observed in any of the MLL-AF9 mice (n = 4). (C) Representative overlay of flow cytometric analysis of EphA1, EphA2, EphA3, EphA7 and ephrinA5-Fc binding on patient samples and human cell lines. (D) Table summarizing the expression level of Eph receptors on patient samples (patients 1–7) and human leukemia cell lines (THP-1 and MV4-11). Each dot corresponds to one individual mouse. The data represent the mean ± SEM. Unpaired <i>t</i> test was performed for statistical analyses.</p

Survival and engraftment data from MLL-AF9 leukemic mice treated with PBS, EphA2 mAb (IF7), radiolabeled EphA2 (Lu-IF7) or radiolabeled EphA3 (Lu-IIIA4) antibody.

<p>(A) Survival of MLL-AF9 leukemic mice treated with IF7 mAb or PBS control showed no significant differences between the two groups. (B) Percentage of GFP⁺ cells in bone marrow, spleen and blood showed significantly higher GFP⁺ cells in bone marrow of IF7 mAb treated mice compared to PBS control (P = 0.0055). There were no significant differences observed in spleen and blood of the IF7 treated MLL-AF9 mice compared to wild type control mice at time of cull (n = 5 PBS treated, n = 6 IF7 treated). (C) Survival of MLL-AF9 leukemic mice treated with PBS, IF7 mAb, Lu-IIIA4 or Lu-IF7 antibody. While there were no significant differences between PBS, IF7 and Lu-IIIA4 antibody treated groups, a significant increase in the survival of Lu-IF7 mAb treated group was observed compared to PBS (P = 0.0140), IF7 mAb (P = 0.0100) and Lu-IIIA4 mAb (P = 0.0100) treated group (n = 3 PBS treated, n = 3 IF7 mAb treated, n = 3 Lu-IIIA4 antibody and n = 4 Lu-IF7 treated). (D) Survival of MLL-AF9 leukemic mice treated with PBS, unlabeled-Lu control and Lu-IF7 antibody showed no significant differences between PBS and unlabeled-Lu control antibody treated groups. In contrast there were significant increase in the survival of mice treated with a single dose of Lu-IF7 mAb (P = 0.0084) and more so with two doses of Lu-IF7 (P = 0.0039) compared to PBS control. The increase in survival after treatment with single dose of Lu-IF7 mAb (P = 0.0140) and two doses of Lu-IF7 (P = 0.0046) compared to unlabeled-Lu control (n = 4 PBS treated, n = 3 unlabeled-Lu, n = 4 Lu-IF7 antibody one dose and n = 5 Lu-IF7 antibody two doses, 3 biological replicates). Each dot corresponds to one individual mouse. The data represent mean ± SEM and unpaired <i>t</i> test was performed for statistical analyses. Kaplan-Meir curves were constructed in GraphPad and Log-rank (Mantel-Cox) tests were used to determine statistical differences.</p

Stem/progenitor cell populations in EphA2 knockout mice compared to wild type littermates.

<p>(<b>A</b>) Gating for Progenitors (lineage^lowcKit^highSca-1^-), LKS⁺ cells (lineage^lowcKit^highSca-1⁺ enriched for hematopoietic stem cells), CMP (lineage^lowcKit^highSca-1^-CD34⁺FCGRII/III^low), MEP (lineage^lowcKit^highSca-1^-CD34^-FCGRII/III^-) and GMP (lineage^lowcKit^highSca-1^-CD34⁺FCGRII/III^high) presented as percentage of Lineage^low cells. CD34/CD135 gating for MPP (LKS⁺CD34⁺CD135⁺), ST-HSC (LKS⁺CD34⁺CD135^-) and LT-HSC (LKS+CD34-CD135-). CD48/CD150 gating for LT-HSC (LKS⁺CD150⁺CD48^-) and MPP (LKS⁺CD150^-CD48⁺). (<b>B</b>) There were no significant differences in progenitors, LKS⁺, CMP, MEP or GMP population in the EphA2 knockout mice compared to wild type control mice (n = 15, 3 independent experiments). (<b>C</b>) There were no significant differences in LT-HSC and MPP population gated by CD34/CD135 antigens in the EphA2 knockout mice compared to wild type control. There were significantly more (P = 0.0012) ST-HSCs in EphA2 knockout bone marrow compared to wild type control (n = 15, 3 independent experiments). (<b>D</b>) There were no significant differences in LT-HSC and MPP population gated using CD48/CD150 antigens in EphA2 knockout mice compared to wild type control (n = 15, 3 independent experiments). Each dot corresponds to one individual mouse. The data represent mean ± SEM. Unpaired <i>t</i> test was performed for statistical analyses.</p

EphA2 knockout bone marrow repopulating potential in primary and secondary recipients.

<p>(A) Whole-blood chimerism at 4, 8, 12 and 16 weeks after transplantation of EphA2 knockout or wild type bone marrow cells into lethally irradiated CD45.1 recipients (n = 5). (B) Analysis of bone marrow and spleen chimerism in 16 weeks after primary transplantation. (C) Whole-blood chimerism of secondary transplant at 4, 8, 12, 16, 20 and 24 weeks after transplantation of EphA2 knockout or wild type primary bone marrow cells into lethally irradiated CD45.1 recipient (n = 5). (D) Bone marrow and spleen chimerism analysis 24 weeks after secondary transplantation. Each dot on panel A and C corresponds to mean and error from all wild type or knockout blood chimerism. Each dot on panel B and D corresponds to one individual mouse. The data presented as percentage of blood, bone marrow and spleen chimerism. The data represent mean ± SEM. Unpaired <i>t</i> test was performed for statistical analyses.</p

Distal lung differentiation defects in <i>Ssb1</i>^−/− embryos.

<p>(A) Representative image showing the comparison of lung size and morphology of E18.5 <i>Ssb1</i>^+/+, <i>Ssb1</i>^+/− and <i>Ssb1</i>^−/− embryos. <i>Scale bar = 2 mm</i>. (B) Protein level of Ssb1, Ssb2 and Actin in <i>Ssb1</i>^+/+, <i>Ssb1</i>^+/− and <i>Ssb1</i>^−/− E18.5 lungs. (C) Haematoxylin and eosin staining showing morphology of alveolar lumen formation and intra-alveolar septae thickness in <i>Ssb1</i> control (<i>Ssb1</i>^+/+, <i>Ssb1</i>^+/−) and <i>Ssb1</i>^−/− E18.5 embryos. (D) Quantitation of alveolar lumen area and (E) intra-alveolar septa area in <i>Ssb1</i> control (<i>Ssb1</i>^+/+, <i>Ssb1</i>^+/−) and <i>Ssb1</i>^−/− embryos. Data represent the mean ± SEM (<i>n</i> = 3 control and 4 <i>Ssb1</i>^−/− embryos from 2 litters. ***<i>P</i>&lt;0.001, student's <i>t</i>-test). (F) qRT-PCR of Type I and Type II alveolar epithelial cell markers in <i>Ssb1</i> control (<i>Ssb1</i>^+/+, <i>Ssb1</i>^+/−) and <i>Ssb1</i>^−/− E18.5 lungs. Data represent mean ± SEM from 4 embryos per condition (*<i>P</i>&lt;0.05, **<i>P</i>&lt;0.01,***<i>P</i>&lt;0.001; student's <i>t</i>-test).</p

Conditional <i>Ssb1</i>-deleted mice are sensitive to IR.

<p>(A) Schematic diagram of the radiation challenge assay in <i>Ssb1</i> deleted mice. <i>Rosa26</i>-CreER^T2: <i>Ssb1</i>^+/+, <i>Rosa26</i>-CreER^T2: <i>Ssb1</i>^+/− and <i>Rosa26</i>-CreER^T2: <i>Ssb1</i>^−/− mice were challenged with 8 Gy of total body irradiation (TBI). The acute lethal response of mice to TBI was evaluated over a 30-day observation period. (B) Kaplan-Meier survival analysis of irradiated mice. Kaplan-Meier survival curves compared by log-rank (Mantel-Cox) analysis showed significant difference between <i>Rosa26</i>-CreER^T2: <i>Ssb1</i>^−/− mice and the other two groups (**<i>P</i>&lt;0.01), while no difference was found between <i>Rosa26</i>-CreER^T2: <i>Ssb1^+/+</i> and <i>Rosa26</i>-CreER^T2: <i>Ssb1^+/−</i> groups. (C) Representative images of Haematoxylin and eosin, Ki67 (cell proliferation) and ApopTag (cell death) staining on small intestine sections from mice at Day 5 post 8 Gy of TBI.</p

Class switch recombination activity in B-cell specific <i>Ssb1</i>-deleted mice.

<p><i>Ssb1 ^fl/fl</i> mice were crossed with a <i>Cd19</i>-Cre transgene expressing C57BL/6J mice to specifically delete <i>Ssb1</i> in B cells. Splenic B cells were isolated and stimulated for 2, 3, and 4 days using anti-Cd40 antibodies plus IL-4 to induce CSR to IgG1. (A) Western blotting of whole cell extracts showed loss of Ssb1 protein in stimulated B cells from <i>Ssb1</i> knockout (<i>Ssb1</i> KO; <i>Cd19</i>Cre⁺: <i>Ssb1^−/−</i>) mice. Equal amounts of stimulated B cell extracts from heterozygous <i>Ssb1</i> (<i>Ssb1</i> Het; <i>Cd19</i>Cre⁺: <i>Ssb1^+/−</i>) and wild-type <i>Ssb1</i> (<i>Ssb1</i> WT; <i>Cd19</i>Cre⁺: <i>Ssb1^+/+</i>) mice were included for comparison. Equal loading was confirmed by probing for Hsp90α. (B) Western blotting of Ssb2 levels in B cells from <i>Ssb1</i> WT and <i>Ssb1</i> KO mice. (C) FACS analysis of CSR to IgG1 over time in stimulated B cells from mice of the indicated genotypes. Stimulated splenic B cells from <i>AID^−/−</i> (<i>AID</i> KO) mice served as a negative control. (D) Summary statistics of CSR activity to IgG1 and viability on day 3 of stimulation. Mean and S.E.M. from three independent experiments are shown. No statistically significant differences (two-tailed unpaired <i>t</i>-test) were found. (E) Switch region junction analysis. Sm-Sg1 junctions were amplified from IL4 plus anti-CD40 stimulated primary B cells (day 4) and sequenced. Percentage and total numbers of direct or microhomology-mediated joints are indicated. nt, nucleotides.</p