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

Charmsaz S, Beckett K, Smith FM, Bruedigam C, Moore AS, Al-Ejeh F, Lane SW, Boyd AW - PLoS ONE (2015)

Bottom Line: These studies showed that EphA2 does not have an obligatory role in normal hematopoiesis.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.

View Article: PubMed Central - PubMed

Affiliation: QIMR Berghofer Medical Research Institute, Brisbane, Australia.

ABSTRACT
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.

No MeSH data available.


Related in: MedlinePlus

Expression analysis of EphA subfamily on mouse MLL-AF9 leukemic cells, on patient samples and human leukemic cell lines with MLL-rearrangement.(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 t test was performed for statistical analyses.
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pone.0130692.g006: Expression analysis of EphA subfamily on mouse MLL-AF9 leukemic cells, on patient samples and human leukemic cell lines with MLL-rearrangement.(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 t test was performed for statistical analyses.

Mentions: A possible explanation for lack of effect of EphA2 deletion in MLL-AF9 leukemia is that other Eph proteins with overlapping ephrin-binding affinities, such as EphA7 that was reported previously on MLL-AF9 leukemias [16], might show a compensatory increase in expression. To explore this possibility EphA7 and total EphA expression on EphA2 knockout and wild type MLL-AF9 leukemic cells were analyzed using flow cytometry. Total EphA expression was determined by binding of ephrinA5-Fc with high binding affinity to members of EphA subfamily. In EphA2 knockout MLL-AF9 leukemic cells, EphA7 expression was not significantly up-regulated compared to wild type MLL-AF9. However, the levels of ephrinA5-Fc binding in EphA2 knockout MLL-AF9 was not significantly different to wild type MLL-AF9 cells suggesting that, despite loss of EphA2, the complement of EphA receptors on the leukemic cells of MLL-AF9 leukemic mice was similar to control MLL-AF9 leukemic cells (Fig 6A). This supported the possibility that some compensatory increase in other Eph genes was present in the EphA2 knockout leukemic cells. To this end, we performed RT-PCR analysis of the MLL-AF9 leukemic bone marrow cells for the expression of EphA receptors in EphA2 knockout and wild type mice. This again showed no significant change in EphA7 expression in support of the flow cytometric data; there was an increase in EphA5 expression in EphA2 knockout MLL-A9 when compared to wild type MLL-AF9 leukemic cells, although this was variable and did not reach statistical significance (Fig 6B).


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

Charmsaz S, Beckett K, Smith FM, Bruedigam C, Moore AS, Al-Ejeh F, Lane SW, Boyd AW - PLoS ONE (2015)

Expression analysis of EphA subfamily on mouse MLL-AF9 leukemic cells, on patient samples and human leukemic cell lines with MLL-rearrangement.(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 t test was performed for statistical analyses.
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Related In: Results  -  Collection

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pone.0130692.g006: Expression analysis of EphA subfamily on mouse MLL-AF9 leukemic cells, on patient samples and human leukemic cell lines with MLL-rearrangement.(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 t test was performed for statistical analyses.
Mentions: A possible explanation for lack of effect of EphA2 deletion in MLL-AF9 leukemia is that other Eph proteins with overlapping ephrin-binding affinities, such as EphA7 that was reported previously on MLL-AF9 leukemias [16], might show a compensatory increase in expression. To explore this possibility EphA7 and total EphA expression on EphA2 knockout and wild type MLL-AF9 leukemic cells were analyzed using flow cytometry. Total EphA expression was determined by binding of ephrinA5-Fc with high binding affinity to members of EphA subfamily. In EphA2 knockout MLL-AF9 leukemic cells, EphA7 expression was not significantly up-regulated compared to wild type MLL-AF9. However, the levels of ephrinA5-Fc binding in EphA2 knockout MLL-AF9 was not significantly different to wild type MLL-AF9 cells suggesting that, despite loss of EphA2, the complement of EphA receptors on the leukemic cells of MLL-AF9 leukemic mice was similar to control MLL-AF9 leukemic cells (Fig 6A). This supported the possibility that some compensatory increase in other Eph genes was present in the EphA2 knockout leukemic cells. To this end, we performed RT-PCR analysis of the MLL-AF9 leukemic bone marrow cells for the expression of EphA receptors in EphA2 knockout and wild type mice. This again showed no significant change in EphA7 expression in support of the flow cytometric data; there was an increase in EphA5 expression in EphA2 knockout MLL-A9 when compared to wild type MLL-AF9 leukemic cells, although this was variable and did not reach statistical significance (Fig 6B).

Bottom Line: These studies showed that EphA2 does not have an obligatory role in normal hematopoiesis.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.

View Article: PubMed Central - PubMed

Affiliation: QIMR Berghofer Medical Research Institute, Brisbane, Australia.

ABSTRACT
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.

No MeSH data available.


Related in: MedlinePlus