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ELMO1 Is Upregulated in AML CD34+ Stem/Progenitor Cells, Mediates Chemotaxis and Predicts Poor Prognosis in Normal Karyotype AML

Capala, Marta E., Vellenga, Edo, Schuringa, Jan Jacob·2014·PLoS ONE
DOI 10.1371/journal.pone.0111568PMC PMC4216115
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Figure 1 ELMO1 expression is increased in AML CD34+ cells and predicts poor prognosis in normal karyotype AML patients. (A) VENN diagram displaying the overlap in AML CD34+ specific and AML CD34− specific transcriptomes compared to NBM CD34+ cells [17], [18]. (B) 1677 AML CD34+-specific genes were subjected to Gene Ontology (GO) analysis for Cellular Component (CC) of which several CCs are shown. (C) 1677 AML CD34+-specific genes were subjected to GO analysis for terms associated with adhesion and migration. (D) ELMO1 mRNA was significantly higher expressed in AML CD34+ (n = 46) as compared to AML CD34− cells (p<0.0001), as well as to NBM CD34+ cells (n = 31) (p<0.0001). (E) Increased expression of ELMO1 was further confirmed by independent Q-PCRs in a panel of 11 AML and 6 NBM samples. (F, G) The increase in ELMO1 mRNA was paralleled by increased protein levels in two representative cases. (H) High expression of ELMO1 predicts poor survival in a cohort of NK AML patients (based on [6], [29]) (p = 0.0034).
Figure 1 ELMO1 expression is increased in AML CD34+ cells and predicts poor prognosis in normal karyotype AML patients. (A) VENN diagram displaying the overlap in AML CD34+ specific and AML CD34− specific transcriptomes compared to NBM CD34+ cells [17], [18]. (B) 1677 AML CD34+-specific genes were subjected to Gene Ontology (GO) analysis for Cellular Component (CC) of which several CCs are shown. (C) 1677 AML CD34+-specific genes were subjected to GO analysis for terms associated with adhesion and migration. (D) ELMO1 mRNA was significantly higher expressed in AML CD34+ (n = 46) as compared to AML CD34− cells (p<0.0001), as well as to NBM CD34+ cells (n = 31) (p<0.0001). (E) Increased expression of ELMO1 was further confirmed by independent Q-PCRs in a panel of 11 AML and 6 NBM samples. (F, G) The increase in ELMO1 mRNA was paralleled by increased protein levels in two representative cases. (H) High expression of ELMO1 predicts poor survival in a cohort of NK AML patients (based on [6], [29]) (p = 0.0034).CC BY · PubMed Central
Figure 2 ELMO1 downmodulation in human CB CD34+ cells does not alter growth, colony formation or differentiation, but significantly decreases stem cell frequency. (A) K562 cells were transduced with control scrambled shRNA vector (shSCR) or with ELMO1-targeting shRNA vectors (shELMO1), sorted and used for RNA extraction. Quantitative PCR was performed to measure ELMO1 expression in transduced cells. ELMO1 mRNA levels were normalized against RPL27 mRNA expression. Alternatively, cells were used for Western blot analysis to determine ELMO1 protein levels. (B) FACS plots showing transduction efficiencies of cord blood (CB) CD34+ stem/progenitor cells transduced with shSCR or shELMO1. (C) 3×105 transduced and sorted cells per group were plated in liquid culture and followed for thirty days. Cumulative cell count is showed representative of 3 independent experiments. (D) 105 transduced and sorted cells per group were plated on MS5 stromal cells and kept in the co-culture for 5 weeks; cultures were demi-depopulated weekly for analysis. Weekly cumulative cell growth is shown for a representative experiment of 3 independent experiments and the average of those 3 experiments is shown in (E). (F) Suspension cells from MS5 co-cultures as described in panel D were analyzed for progenitor frequency by CFC assay. 104 cells from each co-culture were plated in a CFC assay in methylcellulose in duplicate, and colonies were evaluated 2 weeks after plating. CFC cells were then harvested and 105 cells were re-plated to form secondary CFCs. CFU-GM and BFU-E numbers are shown from a representative of 3 independent experiments; error bars indicate standard deviation. (G) LTC-IC frequencies were determined in bulk on MS5 stromal cells. After 5 weeks of co-culture methylcellulose was added to and colonies were scored two weeks later. CFU -GM, colony-forming unit-granulocyte-macrophage; BFU-E, burst forming unit-erythroid. * P<0.05, ** P<0.01, *** P<0.001.
Figure 2 ELMO1 downmodulation in human CB CD34+ cells does not alter growth, colony formation or differentiation, but significantly decreases stem cell frequency. (A) K562 cells were transduced with control scrambled shRNA vector (shSCR) or with ELMO1-targeting shRNA vectors (shELMO1), sorted and used for RNA extraction. Quantitative PCR was performed to measure ELMO1 expression in transduced cells. ELMO1 mRNA levels were normalized against RPL27 mRNA expression. Alternatively, cells were used for Western blot analysis to determine ELMO1 protein levels. (B) FACS plots showing transduction efficiencies of cord blood (CB) CD34+ stem/progenitor cells transduced with shSCR or shELMO1. (C) 3×105 transduced and sorted cells per group were plated in liquid culture and followed for thirty days. Cumulative cell count is showed representative of 3 independent experiments. (D) 105 transduced and sorted cells per group were plated on MS5 stromal cells and kept in the co-culture for 5 weeks; cultures were demi-depopulated weekly for analysis. Weekly cumulative cell growth is shown for a representative experiment of 3 independent experiments and the average of those 3 experiments is shown in (E). (F) Suspension cells from MS5 co-cultures as described in panel D were analyzed for progenitor frequency by CFC assay. 104 cells from each co-culture were plated in a CFC assay in methylcellulose in duplicate, and colonies were evaluated 2 weeks after plating. CFC cells were then harvested and 105 cells were re-plated to form secondary CFCs. CFU-GM and BFU-E numbers are shown from a representative of 3 independent experiments; error bars indicate standard deviation. (G) LTC-IC frequencies were determined in bulk on MS5 stromal cells. After 5 weeks of co-culture methylcellulose was added to and colonies were scored two weeks later. CFU -GM, colony-forming unit-granulocyte-macrophage; BFU-E, burst forming unit-erythroid. * P<0.05, ** P<0.01, *** P<0.001.CC BY · PubMed Central
Figure 3 BCR-ABL-transformed human CB CD34+ show proliferative disadvantage and markedly reduced replating capacity upon ELMO1 depletion. (A) CB CD34+ stem/progenitor cells were double-transduced with BCR-ABL and either control scrambled shRNA vector (shSCR) or with ELMO1-targeting shRNA vector (shELMO1). FACS plots of transduction efficiency are shown. (B) 3×105 double-transduced cells per group were plated in liquid culture and followed for 35 day. Cumulative cell count is shown representative of 3 independent experiments. (C) 5×103 double-transduced cells were sorted per group and plated on MS5 stromal cells; cultures were demi-depopulated on indicated days for analysis and replated when stroma showed signs of detaching. Cumulative cell growth is shown for a representative experiment of 3 independent experiments and the average of those 3 experiments is shown in (D). (E) Suspension cells from MS5 co-cultures as described in panel B were analyzed for progenitor frequency by CFC assay. 103 freshly transduced cells or 104 cells from each co-culture were plated in a CFC assay in methylcellulose in duplicate, and colonies were evaluated 2 weeks after plating. CFC cells were harvested and 105 cells were replated to assess secondary CFC formation. Total CFC numbers are shown from a representative of 3 independent experiments; error bars indicate standard deviation. * P<0.05, ** P<0.01, *** P<0.001.
Figure 3 BCR-ABL-transformed human CB CD34+ show proliferative disadvantage and markedly reduced replating capacity upon ELMO1 depletion. (A) CB CD34+ stem/progenitor cells were double-transduced with BCR-ABL and either control scrambled shRNA vector (shSCR) or with ELMO1-targeting shRNA vector (shELMO1). FACS plots of transduction efficiency are shown. (B) 3×105 double-transduced cells per group were plated in liquid culture and followed for 35 day. Cumulative cell count is shown representative of 3 independent experiments. (C) 5×103 double-transduced cells were sorted per group and plated on MS5 stromal cells; cultures were demi-depopulated on indicated days for analysis and replated when stroma showed signs of detaching. Cumulative cell growth is shown for a representative experiment of 3 independent experiments and the average of those 3 experiments is shown in (D). (E) Suspension cells from MS5 co-cultures as described in panel B were analyzed for progenitor frequency by CFC assay. 103 freshly transduced cells or 104 cells from each co-culture were plated in a CFC assay in methylcellulose in duplicate, and colonies were evaluated 2 weeks after plating. CFC cells were harvested and 105 cells were replated to assess secondary CFC formation. Total CFC numbers are shown from a representative of 3 independent experiments; error bars indicate standard deviation. * P<0.05, ** P<0.01, *** P<0.001.CC BY · PubMed Central
Figure 4 ELMO1 depletion in primary AML CD34+ cells impairs long-term expansion on stroma. AML CD34+ cells were transduced with control scrambled shRNA vector (shSCR) or with ELMO1-targeting shRNA vector (shELMO1). After washing away the virus all the cells were plated on MS5 stroma; cultures were demi-depopulated on indicated days for analysis. Cumulative cell growth is shown for each AML sample studied.
Figure 4 ELMO1 depletion in primary AML CD34+ cells impairs long-term expansion on stroma. AML CD34+ cells were transduced with control scrambled shRNA vector (shSCR) or with ELMO1-targeting shRNA vector (shELMO1). After washing away the virus all the cells were plated on MS5 stroma; cultures were demi-depopulated on indicated days for analysis. Cumulative cell growth is shown for each AML sample studied.CC BY · PubMed Central
Figure 5 Global inhibition of RAC activity severely impairs proliferation and survival of leukemic cells while ELMO1 depletion strongly affects migration. (A) CB CD34+ cells were transduced with BCR-ABL-expressing vector, sorted and plated on MS5 stroma. Cells were allowed to proliferate for 5 days after which RAC inhibitor was added to the following concentrations: 20 µM, 40 µM or 100 µM. Co-cultures were demi-depopulated on indicated days for analysis. Cumulative cell count is shown representative of 3 independent experiments. (B) CD34+ cells were sorted from BC CML patient sample and plated on MS5 stroma. Cells were allowed to proliferate for 5 days after which RAC inhibitor was added to the following concentrations: 20 µM, 40 µM or 100 µM. Co-cultures were demi-depopulated on indicated days for analysis. Cumulative cell count is shown representative of 3 independent experiments. (C) BCR-ABL transduced cells as described in (A) were treated with 50 µM NSC for 3 days after which suspension cells were harvested and stained with Annexin V to assess apoptosis. (D) TF-1 and THP-1 cells were transduced with control scrambled shRNA vector (shSCR) or with ELMO1-targeting shRNA vector (shELMO1) and migration was evaluated in a transwell assay. The percentage of migrating cells relative to control is shown as an average of 3 independent experiments.
Figure 5 Global inhibition of RAC activity severely impairs proliferation and survival of leukemic cells while ELMO1 depletion strongly affects migration. (A) CB CD34+ cells were transduced with BCR-ABL-expressing vector, sorted and plated on MS5 stroma. Cells were allowed to proliferate for 5 days after which RAC inhibitor was added to the following concentrations: 20 µM, 40 µM or 100 µM. Co-cultures were demi-depopulated on indicated days for analysis. Cumulative cell count is shown representative of 3 independent experiments. (B) CD34+ cells were sorted from BC CML patient sample and plated on MS5 stroma. Cells were allowed to proliferate for 5 days after which RAC inhibitor was added to the following concentrations: 20 µM, 40 µM or 100 µM. Co-cultures were demi-depopulated on indicated days for analysis. Cumulative cell count is shown representative of 3 independent experiments. (C) BCR-ABL transduced cells as described in (A) were treated with 50 µM NSC for 3 days after which suspension cells were harvested and stained with Annexin V to assess apoptosis. (D) TF-1 and THP-1 cells were transduced with control scrambled shRNA vector (shSCR) or with ELMO1-targeting shRNA vector (shELMO1) and migration was evaluated in a transwell assay. The percentage of migrating cells relative to control is shown as an average of 3 independent experiments.CC BY · PubMed Central

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Cited by

Metadata

Authors
Capala, M. E., Vellenga, E., & Schuringa, J. J.
Venue
PLoS ONE
Publisher
Public Library of Science (PLoS)
Published
2014-10-31
Volume
9
Issue
10
Pages
e111568
ISSN
1932-6203
DOI
10.1371/journal.pone.0111568
PMC
PMC4216115
Cited by
11
References
5
Language
en
Licence
creativecommons.org/licenses/by/4.0/ (unspecified)

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