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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

Cbl/Cbl-b DKO HSCs show impaired in vivo reconstitution ability(A) HSC limiting-dilution transplantation. Indicated numbers of LSKs were transplanted and mice with < 0.5% donor-derived chimerism at 16 weeks were considered non-responders. Data from two experiments are pooled. (B) 2000 LSKs were transplanted and donor-cell chimerism in PB analyzed at the indicated times. Data from two experiments are pooled. (C–D) Donor LSK pool was analyzed in recipient BM at around 20 weeks. Representative FACS plot (C) and quantitation (D) are shown only for recipients with > 70% donor cell reconstitution. Data from three experiments are pooled. (E–F) Secondary transplants with 2000 donor LSKs sorted from primary recipients transplanted 20 weeks earlier. Donor cell chimerism in PB (G) and WBC counts (H) over time are shown. Data from two repeats are pooled (*p < 0.05).
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Figure 5: Cbl/Cbl-b DKO HSCs show impaired in vivo reconstitution ability(A) HSC limiting-dilution transplantation. Indicated numbers of LSKs were transplanted and mice with < 0.5% donor-derived chimerism at 16 weeks were considered non-responders. Data from two experiments are pooled. (B) 2000 LSKs were transplanted and donor-cell chimerism in PB analyzed at the indicated times. Data from two experiments are pooled. (C–D) Donor LSK pool was analyzed in recipient BM at around 20 weeks. Representative FACS plot (C) and quantitation (D) are shown only for recipients with > 70% donor cell reconstitution. Data from three experiments are pooled. (E–F) Secondary transplants with 2000 donor LSKs sorted from primary recipients transplanted 20 weeks earlier. Donor cell chimerism in PB (G) and WBC counts (H) over time are shown. Data from two repeats are pooled (*p < 0.05).

Mentions: Given our results that DKO HSCs exhibit impaired colony forming ability in vitro, we assessed the self-renewal ability of DKO vs. WT HSCs in vivo. We transplanted limiting dilutions of freshly-sorted WT or DKO LSKs, assessed engraftment at 16 weeks using peripheral blood analyses, and calculated the HSC frequency. Compared to a frequency of 1:100 for WT control, the HSC frequency of DKO LSK cells was significantly reduced (1:300) (Figure 5A). Next, we transplanted 2000 WT or DKO LSKs (sufficient for engraftment) and assessed the proportion of donor cells in PB at 4, 8 and 16 weeks post-transplant (Figure 5B). Compared to expected sustained reconstitution with WT LSKs, DKO LSK recipients showed a more variable albeit substantial reconstitution at 4 weeks; notably, several DKO LSK recipients exhibited a decrease in donor-derived WBCs with time, indicating impaired ability of DKO LSK cells to maintain long-term reconstitution. We further analyzed DKO LSK recipients that showed high donor cell reconstitution (> 70%) at 16 weeks post-transplant (to exclude mice that may have received a transplant with predominantly ST-HSC and MPPs, populations with only a short-term reconstitution ability) (Figure S6A). Notably, these mice exhibited a significantly smaller BM LSK compartment compared to WT LSK recipients (Figure 5C and 5D). Thus, even in recipients with robust reconstitution, DKO HSCs were unable to maintain the HSC pool.


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)

Cbl/Cbl-b DKO HSCs show impaired in vivo reconstitution ability(A) HSC limiting-dilution transplantation. Indicated numbers of LSKs were transplanted and mice with < 0.5% donor-derived chimerism at 16 weeks were considered non-responders. Data from two experiments are pooled. (B) 2000 LSKs were transplanted and donor-cell chimerism in PB analyzed at the indicated times. Data from two experiments are pooled. (C–D) Donor LSK pool was analyzed in recipient BM at around 20 weeks. Representative FACS plot (C) and quantitation (D) are shown only for recipients with > 70% donor cell reconstitution. Data from three experiments are pooled. (E–F) Secondary transplants with 2000 donor LSKs sorted from primary recipients transplanted 20 weeks earlier. Donor cell chimerism in PB (G) and WBC counts (H) over time are shown. Data from two repeats are pooled (*p < 0.05).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
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Figure 5: Cbl/Cbl-b DKO HSCs show impaired in vivo reconstitution ability(A) HSC limiting-dilution transplantation. Indicated numbers of LSKs were transplanted and mice with < 0.5% donor-derived chimerism at 16 weeks were considered non-responders. Data from two experiments are pooled. (B) 2000 LSKs were transplanted and donor-cell chimerism in PB analyzed at the indicated times. Data from two experiments are pooled. (C–D) Donor LSK pool was analyzed in recipient BM at around 20 weeks. Representative FACS plot (C) and quantitation (D) are shown only for recipients with > 70% donor cell reconstitution. Data from three experiments are pooled. (E–F) Secondary transplants with 2000 donor LSKs sorted from primary recipients transplanted 20 weeks earlier. Donor cell chimerism in PB (G) and WBC counts (H) over time are shown. Data from two repeats are pooled (*p < 0.05).
Mentions: Given our results that DKO HSCs exhibit impaired colony forming ability in vitro, we assessed the self-renewal ability of DKO vs. WT HSCs in vivo. We transplanted limiting dilutions of freshly-sorted WT or DKO LSKs, assessed engraftment at 16 weeks using peripheral blood analyses, and calculated the HSC frequency. Compared to a frequency of 1:100 for WT control, the HSC frequency of DKO LSK cells was significantly reduced (1:300) (Figure 5A). Next, we transplanted 2000 WT or DKO LSKs (sufficient for engraftment) and assessed the proportion of donor cells in PB at 4, 8 and 16 weeks post-transplant (Figure 5B). Compared to expected sustained reconstitution with WT LSKs, DKO LSK recipients showed a more variable albeit substantial reconstitution at 4 weeks; notably, several DKO LSK recipients exhibited a decrease in donor-derived WBCs with time, indicating impaired ability of DKO LSK cells to maintain long-term reconstitution. We further analyzed DKO LSK recipients that showed high donor cell reconstitution (> 70%) at 16 weeks post-transplant (to exclude mice that may have received a transplant with predominantly ST-HSC and MPPs, populations with only a short-term reconstitution ability) (Figure S6A). Notably, these mice exhibited a significantly smaller BM LSK compartment compared to WT LSK recipients (Figure 5C and 5D). Thus, even in recipients with robust reconstitution, DKO HSCs were unable to maintain the HSC pool.

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