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Loss of Cbl and Cbl-b ubiquitin ligases abrogates hematopoietic stem cell quiescence and sensitizes leukemic disease to chemotherapy.

An W, Nadeau SA, Mohapatra BC, Feng D, Zutshi N, Storck MD, Arya P, Talmadge JE, Meza JL, Band V, Band H - Oncotarget (2015)

Bottom Line: Cell cycle analysis demonstrated that DKO HSCs exhibit reduced quiescence associated with compromised reconstitution ability and propensity to undergo exhaustion.We show that sustained c-Kit and FLT3 signaling in DKO HSCs promotes loss of colony-forming potential, and c-Kit or FLT3 inhibition in vitro protects HSCs from exhaustion.Our data reveal a novel and leukemia therapy-relevant role of Cbl and Cbl-b in the maintenance of HSC quiescence and protection against exhaustion, through negative regulation of tyrosine kinase-coupled receptor signaling.

View Article: PubMed Central - PubMed

Affiliation: Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, Omaha, NE 68198, USA.

ABSTRACT
Cbl and Cbl-b are tyrosine kinase-directed RING finger type ubiquitin ligases (E3s) that negatively regulate cellular activation pathways. E3 activity-disrupting human Cbl mutations are associated with myeloproliferative disorders (MPD) that are reproduced in mice with Cbl RING finger mutant knock-in or hematopoietic Cbl and Cbl-b double knockout. However, the role of Cbl proteins in hematopoietic stem cell (HSC) homeostasis, especially in the context of MPD is unclear. Here we demonstrate that HSC expansion and MPD development upon combined Cbl and Cbl-b deletion are dependent on HSCs. Cell cycle analysis demonstrated that DKO HSCs exhibit reduced quiescence associated with compromised reconstitution ability and propensity to undergo exhaustion. We show that sustained c-Kit and FLT3 signaling in DKO HSCs promotes loss of colony-forming potential, and c-Kit or FLT3 inhibition in vitro protects HSCs from exhaustion. In vivo, treatment with 5-fluorouracil hastens DKO HSC exhaustion and protects mice from death due to MPD. Our data reveal a novel and leukemia therapy-relevant role of Cbl and Cbl-b in the maintenance of HSC quiescence and protection against exhaustion, through negative regulation of tyrosine kinase-coupled receptor signaling.

No MeSH data available.


Related in: MedlinePlus

Impact of modulating c-Kit or FLT3 signaling on DKO HSC exhaustion(A–B) LTC-IC assays. Initial co-cultures performed without growth factors (control), or with SCF (100 ng/ml) or Imatinib (1 uM), and cell numbers counted (A), followed by colony forming assay (B) Data are from four independent experiments. (C–D) Assay was performed as in A and B, with primary co-culture without (Control), or with FLT3L (FL, 100 ng/ml), AC220 (AC, 100 nM) or FLT3L (100 ng/ml) + AC220 (100 nM). Cell numbers after initial culture (C) and colony-formation assay (D) are shown. Data are from four independent experiments. (E–G) Imatinib in vivo treatment. (E) Liver and spleen weights of mice with indicated treatment. (F) WBC counts. Dots represent individual mice. (G) BM analysis.% LSK cells in Lin- cells is shown. Data from at least three mice are pooled and shown as mean ± SD. *p < 0.05.
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Figure 4: Impact of modulating c-Kit or FLT3 signaling on DKO HSC exhaustion(A–B) LTC-IC assays. Initial co-cultures performed without growth factors (control), or with SCF (100 ng/ml) or Imatinib (1 uM), and cell numbers counted (A), followed by colony forming assay (B) Data are from four independent experiments. (C–D) Assay was performed as in A and B, with primary co-culture without (Control), or with FLT3L (FL, 100 ng/ml), AC220 (AC, 100 nM) or FLT3L (100 ng/ml) + AC220 (100 nM). Cell numbers after initial culture (C) and colony-formation assay (D) are shown. Data are from four independent experiments. (E–G) Imatinib in vivo treatment. (E) Liver and spleen weights of mice with indicated treatment. (F) WBC counts. Dots represent individual mice. (G) BM analysis.% LSK cells in Lin- cells is shown. Data from at least three mice are pooled and shown as mean ± SD. *p < 0.05.

Mentions: To link the Cbl/Cbl-b mediated negative regulation of c-Kit and FLT3 to their role in maintaining HSC self-renewal, we assessed the impact of growing BMMNC in the presence of activating ligands or kinase inhibitors of these RTKs on subsequent colony-forming ability in LTC-IC assays. Both control and DKO cells proliferated in SCF while c-Kit inhibitor imatinib abrogated (WT) or reduced (DKO) proliferation (Figure 4A). The subsequent colony-forming ability of unstimulated or SCF-expanded WT cells was comparable, while imatinib-cultured cells showed slightly higher colony-forming ability (Figure 4B). In contrast, SCF-expanded DKO cells showed significantly reduced colony-forming ability vs. the unstimulated DKO cells, and imatinib pre-treatment restored it (Figure 4B). Similar results were seen with growth in FLT3L +/− FLT3 inhibitor AC220 (Figure 4C and 4D), with the minor difference that FLT3-treated control cells showed a slightly reduced colony-forming ability.


Loss of Cbl and Cbl-b ubiquitin ligases abrogates hematopoietic stem cell quiescence and sensitizes leukemic disease to chemotherapy.

An W, Nadeau SA, Mohapatra BC, Feng D, Zutshi N, Storck MD, Arya P, Talmadge JE, Meza JL, Band V, Band H - Oncotarget (2015)

Impact of modulating c-Kit or FLT3 signaling on DKO HSC exhaustion(A–B) LTC-IC assays. Initial co-cultures performed without growth factors (control), or with SCF (100 ng/ml) or Imatinib (1 uM), and cell numbers counted (A), followed by colony forming assay (B) Data are from four independent experiments. (C–D) Assay was performed as in A and B, with primary co-culture without (Control), or with FLT3L (FL, 100 ng/ml), AC220 (AC, 100 nM) or FLT3L (100 ng/ml) + AC220 (100 nM). Cell numbers after initial culture (C) and colony-formation assay (D) are shown. Data are from four independent experiments. (E–G) Imatinib in vivo treatment. (E) Liver and spleen weights of mice with indicated treatment. (F) WBC counts. Dots represent individual mice. (G) BM analysis.% LSK cells in Lin- cells is shown. Data from at least three mice are pooled and shown as mean ± SD. *p < 0.05.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4496370&req=5

Figure 4: Impact of modulating c-Kit or FLT3 signaling on DKO HSC exhaustion(A–B) LTC-IC assays. Initial co-cultures performed without growth factors (control), or with SCF (100 ng/ml) or Imatinib (1 uM), and cell numbers counted (A), followed by colony forming assay (B) Data are from four independent experiments. (C–D) Assay was performed as in A and B, with primary co-culture without (Control), or with FLT3L (FL, 100 ng/ml), AC220 (AC, 100 nM) or FLT3L (100 ng/ml) + AC220 (100 nM). Cell numbers after initial culture (C) and colony-formation assay (D) are shown. Data are from four independent experiments. (E–G) Imatinib in vivo treatment. (E) Liver and spleen weights of mice with indicated treatment. (F) WBC counts. Dots represent individual mice. (G) BM analysis.% LSK cells in Lin- cells is shown. Data from at least three mice are pooled and shown as mean ± SD. *p < 0.05.
Mentions: To link the Cbl/Cbl-b mediated negative regulation of c-Kit and FLT3 to their role in maintaining HSC self-renewal, we assessed the impact of growing BMMNC in the presence of activating ligands or kinase inhibitors of these RTKs on subsequent colony-forming ability in LTC-IC assays. Both control and DKO cells proliferated in SCF while c-Kit inhibitor imatinib abrogated (WT) or reduced (DKO) proliferation (Figure 4A). The subsequent colony-forming ability of unstimulated or SCF-expanded WT cells was comparable, while imatinib-cultured cells showed slightly higher colony-forming ability (Figure 4B). In contrast, SCF-expanded DKO cells showed significantly reduced colony-forming ability vs. the unstimulated DKO cells, and imatinib pre-treatment restored it (Figure 4B). Similar results were seen with growth in FLT3L +/− FLT3 inhibitor AC220 (Figure 4C and 4D), with the minor difference that FLT3-treated control cells showed a slightly reduced colony-forming ability.

Bottom Line: Cell cycle analysis demonstrated that DKO HSCs exhibit reduced quiescence associated with compromised reconstitution ability and propensity to undergo exhaustion.We show that sustained c-Kit and FLT3 signaling in DKO HSCs promotes loss of colony-forming potential, and c-Kit or FLT3 inhibition in vitro protects HSCs from exhaustion.Our data reveal a novel and leukemia therapy-relevant role of Cbl and Cbl-b in the maintenance of HSC quiescence and protection against exhaustion, through negative regulation of tyrosine kinase-coupled receptor signaling.

View Article: PubMed Central - PubMed

Affiliation: Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, Omaha, NE 68198, USA.

ABSTRACT
Cbl and Cbl-b are tyrosine kinase-directed RING finger type ubiquitin ligases (E3s) that negatively regulate cellular activation pathways. E3 activity-disrupting human Cbl mutations are associated with myeloproliferative disorders (MPD) that are reproduced in mice with Cbl RING finger mutant knock-in or hematopoietic Cbl and Cbl-b double knockout. However, the role of Cbl proteins in hematopoietic stem cell (HSC) homeostasis, especially in the context of MPD is unclear. Here we demonstrate that HSC expansion and MPD development upon combined Cbl and Cbl-b deletion are dependent on HSCs. Cell cycle analysis demonstrated that DKO HSCs exhibit reduced quiescence associated with compromised reconstitution ability and propensity to undergo exhaustion. We show that sustained c-Kit and FLT3 signaling in DKO HSCs promotes loss of colony-forming potential, and c-Kit or FLT3 inhibition in vitro protects HSCs from exhaustion. In vivo, treatment with 5-fluorouracil hastens DKO HSC exhaustion and protects mice from death due to MPD. Our data reveal a novel and leukemia therapy-relevant role of Cbl and Cbl-b in the maintenance of HSC quiescence and protection against exhaustion, through negative regulation of tyrosine kinase-coupled receptor signaling.

No MeSH data available.


Related in: MedlinePlus