Transient hematopoietic expansion is followed by HSC exhaustion and BM failure in CUX1-knockdown BM transplant recipients. (A) Schematic of the BM transplant (BMT) time course. BM from Cux1^mid, Cux1^low, or Ren mice was transplanted into lethally irradiated C57BL/6 wild-type recipients. Four weeks after transplant, recipient mice were treated continuously with dox. Most mice were monitored long-term, up to 350 to 370 days of dox or time of euthanization because of disease. A cohort of mice was euthanization after 3 months of dox for analysis and to harvest BM for secondary transplantation. Secondary transplant recipients were given dox starting on the day of transplantation. (B) Kaplan-Meier plot shows decreased survival of Cux1^mid and Cux1^low BM transplant recipients (log-rank test). (C) Complete blood count analysis shows a transient WBC and platelet count expansion in Cux1 knockdown BM transplant recipients, whereas WBC, platelets, and RBCs were decreased by the time of euthanization. The mean ± SEM is shown. (D) Blood cell indices at the time of euthanization show a macrocytic anemia. The mean ± SD is shown. (E) BM from hind legs and hips, and spleen leukocyte cellularity at the indicated time points. Cell counts for Cux1^mid spleens were not determined for the 3-month time point. (F) Cux1^low BM transplant recipients have greater numbers of HSPC after 3 months of dox, as determined by flow cytometry. The mean ± SD is shown. (G) At the time that diseased mice were euthanized because of anemia and weakness, HSPC were decreased in Cux1 knockdown BM transplant recipients compared with Ren BM transplant recipients. The mean ± SD is shown. (H) Absolute LT-HSC from the BM of secondary BMT recipients is shown at the time of euthanization for disease in Cux1^low recipients and after 350 days of dox for Cux1^mid recipients. (I) Competitive BM transplantation was performed with Cux1^low, Cux1^mid, or Ren BM (CD45.2) at a 1:10 ratio with competitor BM (CD45.1). Dox was given on the day of transplant. One replicate of 3 biological replicates is shown, n = 6 mice per experimental group. Flow cytometry of the peripheral blood shows that Cux1^mid and Cux1^low HSPCs outcompete controls in the total WBC population and myeloid lineages. The mean ± SD is shown. All data were analyzed with Wilcoxon rank test, *P ≤ .05, **P ≤ .01, ***P ≤ .001.