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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

MPD in Cbl/Cbl-b DKO mice is cell autonomous and only LSK cells initiate disease(A–B) Whole BM cells were transplanted into lethally-irradiated syngeneic WT recipients; PB counts performed after 4 weeks (A) and survival scored up to 16 weeks (B). Data from two experiments are pooled. Dots represent individual mice. (C–D) FACS-sorted subpopulations from WT or Cbl/Cbl-b DKO BM were transplanted with helper cells. (C) MPD (granulocyte count > 104/ml or death before the study was terminated at 16-weeks) after transplantation. (D) PB cell counts at 16-weeks. Data from two experiments are pooled (*p < 0.05).
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Figure 1: MPD in Cbl/Cbl-b DKO mice is cell autonomous and only LSK cells initiate disease(A–B) Whole BM cells were transplanted into lethally-irradiated syngeneic WT recipients; PB counts performed after 4 weeks (A) and survival scored up to 16 weeks (B). Data from two experiments are pooled. Dots represent individual mice. (C–D) FACS-sorted subpopulations from WT or Cbl/Cbl-b DKO BM were transplanted with helper cells. (C) MPD (granulocyte count > 104/ml or death before the study was terminated at 16-weeks) after transplantation. (D) PB cell counts at 16-weeks. Data from two experiments are pooled (*p < 0.05).

Mentions: While transfer of MPD by DKO BM transplant [21] supports a BM cell-intrinsic role of Cbl proteins, the precise disease-initiating cell remains unknown. BM transplants (CD45.2+ donor to CD45.1+ lethally-irradiated syngeneic recipients) confirmed that DKO BM but not the Cbl-KO, Cbl-b-KO or Cre Control BM transplant produced leukocytosis and myelomonocytosis (Figure 1A) with rapid-onset lethality (Figure 1B) in recipients assessed at 4 weeks post-transplant. We next transplanted FACS-sorted subpopulations from WT/DKO BM cells to determine disease initiating cells. Notably, transplant of DKO HSC-enriched LSKs but not that of a pool of CMP, GMP & MEP myeloid progenitors, or Lin+ c-kit− mature hematopoietic cells, led to features of MPD, identified by GRA counts > In mRNA level, p57 is more enriched 104/mm3 (at 16 weeks) or early death (Figure 1C). Leukocytosis in DKO LSK (Figure 1D) and DKO BM cell recipients (Figure 1A) was comparable. Comparable in vitro proliferation of FACS-sorted LSK cells from WT or DKO mice when cultured with DKO or control mouse sera added to growth media (Figure S2) suggests that the phenotypes are unlikely due to factors released by non-hematopoietic tissues impinging on DKO LSKs. Overall, these results demonstrate an HSC cell-intrinsic role of Cbl and Cbl-b whose abrogation allows DKO HSCs to initiate MPD.


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)

MPD in Cbl/Cbl-b DKO mice is cell autonomous and only LSK cells initiate disease(A–B) Whole BM cells were transplanted into lethally-irradiated syngeneic WT recipients; PB counts performed after 4 weeks (A) and survival scored up to 16 weeks (B). Data from two experiments are pooled. Dots represent individual mice. (C–D) FACS-sorted subpopulations from WT or Cbl/Cbl-b DKO BM were transplanted with helper cells. (C) MPD (granulocyte count > 104/ml or death before the study was terminated at 16-weeks) after transplantation. (D) PB cell counts at 16-weeks. Data from two experiments are pooled (*p < 0.05).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: MPD in Cbl/Cbl-b DKO mice is cell autonomous and only LSK cells initiate disease(A–B) Whole BM cells were transplanted into lethally-irradiated syngeneic WT recipients; PB counts performed after 4 weeks (A) and survival scored up to 16 weeks (B). Data from two experiments are pooled. Dots represent individual mice. (C–D) FACS-sorted subpopulations from WT or Cbl/Cbl-b DKO BM were transplanted with helper cells. (C) MPD (granulocyte count > 104/ml or death before the study was terminated at 16-weeks) after transplantation. (D) PB cell counts at 16-weeks. Data from two experiments are pooled (*p < 0.05).
Mentions: While transfer of MPD by DKO BM transplant [21] supports a BM cell-intrinsic role of Cbl proteins, the precise disease-initiating cell remains unknown. BM transplants (CD45.2+ donor to CD45.1+ lethally-irradiated syngeneic recipients) confirmed that DKO BM but not the Cbl-KO, Cbl-b-KO or Cre Control BM transplant produced leukocytosis and myelomonocytosis (Figure 1A) with rapid-onset lethality (Figure 1B) in recipients assessed at 4 weeks post-transplant. We next transplanted FACS-sorted subpopulations from WT/DKO BM cells to determine disease initiating cells. Notably, transplant of DKO HSC-enriched LSKs but not that of a pool of CMP, GMP & MEP myeloid progenitors, or Lin+ c-kit− mature hematopoietic cells, led to features of MPD, identified by GRA counts > In mRNA level, p57 is more enriched 104/mm3 (at 16 weeks) or early death (Figure 1C). Leukocytosis in DKO LSK (Figure 1D) and DKO BM cell recipients (Figure 1A) was comparable. Comparable in vitro proliferation of FACS-sorted LSK cells from WT or DKO mice when cultured with DKO or control mouse sera added to growth media (Figure S2) suggests that the phenotypes are unlikely due to factors released by non-hematopoietic tissues impinging on DKO LSKs. Overall, these results demonstrate an HSC cell-intrinsic role of Cbl and Cbl-b whose abrogation allows DKO HSCs to initiate MPD.

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