Orally bioavailable antagonists of inhibitor of apoptosis proteins based on an azabicyclooctane scaffold.

A series of IAP antagonists based on an azabicyclooctane scaffold was designed and synthesized. The most potent of these compounds, 14b, binds to the XIAP BIR3 domain, the BIR domain of ML-IAP, and the BIR3 domain of c-IAP1 with K(i) values of 140, 38, and 33 nM, respectively. These compounds promote degradation of c-IAP1, activate caspases, and lead to decreased viability of breast cancer cells without affecting normal mammary epithelial cells. Finally, compound 14b inhibits tumor growth when dosed orally in a breast cancer xenograft model.

Impact of modeled microgravity on migration, differentiation, and cell cycle control of primitive human hematopoietic progenitor cells.

OBJECTIVE: Migration, proliferation, and differentiation of bone marrow (BM) hematopoietic stem cells (HSC) are important factors in maintaining hematopoietic homeostasis. Homeostatic control of erythrocytes and lymphocytes is perturbed in humans exposed to microgravity (micro-g), resulting in space flight-induced anemia and immunosuppression. We sought to determine whether any of these anomalies can be explained by micro-g-induced changes in migration, proliferation, and differentiation of human BM CD34+ cells, and whether such changes can begin to explain any of the shifts in hematopoietic homeostasis observed in astronauts. MATERIALS AND METHODS: BM CD34+ cells were cultured in modeled micro-g (mmicro-g) using NASA's rotating wall vessels (RWV), or in control cultures at earth gravity for 2 to 18 days. Cells were harvested at different times and CD34+ cells assessed for migration potential, cell-cycle kinetics and regulatory proteins, and maturation status. RESULTS: Culture of BM CD34+ cells in RWV for 2 to 3 days resulted in a significant reduction of stromal cell-derived factor 1 (SDF-1alpha)-directed migration, which correlated with decreased expression of F-actin. Modeled micro-g induced alterations in cell-cycle kinetics that were characterized by prolonged S phase and reduced cyclin A expression. Differentiation of primitive CD34+ cells cultured for 14 to 18 days in RWV favored myeloid cell development at the expense of erythroid development, which was significantly reduced compared to controls. CONCLUSIONS: These results illustrate that mmicro-g significantly inhibits the migration potential, cell-cycle progression, and differentiation patterns of primitive BM CD34+ cells, which may contribute to some of the hematologic abnormalities observed in humans during space flight.

Distribution of marrow repopulating cells between bone marrow and spleen early after transplantation.

Whether hematopoietic stem cells (HSCs) home selectively to bone marrow (BM) early after transplantation remains an issue of debate. Better understanding of homing mechanisms may benefit BM transplantation protocols in cases of limited graft cell number or nonmyeloablative conditioning regimens. Using flow cytometry and serial transplantation to stringently identify HSCs, trafficking patterns of long-term engrafting cells were mapped between BM and spleen early after transplantation. Low-density BM cells were tracked in irradiated or nonirradiated mice 1, 3, 6, and 20 hours after transplantation, at which time recipient BM and spleen were analyzed for recovery of primitive donor cells by phenotype and adhesion molecule expression. In addition, phenotypically defined HSC-enriched or HSC-depleted grafts were tracked 20 hours after transplantation in recipient BM and spleen and analyzed for recovery and long-term repopulating potential in mice undergoing serial transplantation. Regardless of irradiation status, recovery of donor Sca-1+ lin- cells was higher at most time points in recipient BM than in spleen, while recovery of total Sca-1+ cells was variable. A significantly higher percentage of BM-homed donor Sca-1+ cells expressed CD43, CD49e, and CD49d 20 hours after transplantation than spleen-homed cells, which contained significantly more non-HSC phenotypes. Furthermore, BM-homed cells were significantly enriched for cells capable of secondary multilineage hematopoiesis in mice undergoing serial transplantation compared with spleen-homed cells. These results support the notion of specific homing of HSCs to BM by 20 hours after transplantation and provide a basis for the enhanced engraftment potential afforded some Sca-1+ lin- cells subfractionated on the basis of adhesion molecule expression.

In vivo trafficking, cell cycle activity, and engraftment potential of phenotypically defined primitive hematopoietic cells after transplantation into irradiated or nonirradiated recipients.

Recent interest in bone marrow (BM) transplantation in nonconditioned or minimally conditioned recipients warrants investigation of homing patterns of transplanted hematopoietic progenitor cells (HPCs) in irradiated and nonirradiated recipients. To this end, phenotypically defined populations of BM cells were tracked in lethally irradiated or nonirradiated mice at 1, 3, 6, and 24 hours after transplantation. Recovery of transplanted cells at all time points was higher in BM of nonirradiated mice, similar to earlier suggestions. The percentage of lineage-negative Sca-1(+) cells and Sca-1(+) cells expressing CD43, CD49e, and CD49d steadily increased in BM of nonirradiated mice up to 24 hours, while fluctuating in irradiated mice. Cell cycle status and BrdU incorporation revealed that less than 20% of Sca-1(+) cells and fewer Sca-1(+)lin(-) cells had cycled by 24 hours after transplantation. To more directly examine trafficking of primitive HPCs, purified grafts of CD62L(-) or CD49e(+) subfractions of Sca-1(+)lin(-) cells, previously shown to be enriched for long-term repopulating cells, also were tracked in vivo. Recovery of purified cells was similarly increased in BM of nonirradiated mice. When 50 to 100 of these BM-homed cells were examined in serial transplantation studies, BM-homed cells from initially nonirradiated mice were enriched 5- to 30-fold for cells capable of long-term hematopoiesis in secondary recipients. Collectively, these data suggest that homing or survival of transplanted cells in irradiated recipients is less efficient than that in nonirradiated recipients, implicating an active role of radiation-sensitive microenvironmental cues in the homing process. These results may have important clinical implications in the design of BM transplantation protocols.

Treatment of circulating CD34(+) cells with SDF-1alpha or anti-CXCR4 antibody enhances migration and NOD/SCID repopulating potential.

OBJECTIVE: Stromal cell-derived factor-1alpha (SDF-1alpha) has been implicated in homing and engraftment of primitive hematopoietic progenitor cells (HPC) in studies demonstrating reduced NOD/SCID repopulating potential of HPC exposed to supra-physiologic concentrations of SDF-1alpha or anti-CXCR4. Outcome of CXCR4 signaling in some cells has been shown to be dependent on the concentration of SDF-1alpha. We aimed to determine whether similar concentration-dependent responses to CXCR4 signaling are present in CD34(+)cells. MATERIALS AND METHODS: Human peripheral blood (PB), mobilized PB (MPB), or bone marrow (BM) CD34(+) cells were incubated for 30 minutes with different concentrations of SDF-1alpha or anti-CXCR4, washed, then assessed for in vitro hematopoietic potential, migration, and NOD/SCID repopulating potential. RESULTS: Exposure of MPB or PB CD34(+) cells to 100 ng/mL SDF-1alpha increased tyrosine phosphorylation without subsequent proliferation or apoptosis. Spontaneous and SDF-1alpha-directed migration also increased in pretreated cells, despite previous exposure to SDF-1alpha. Cells exposed to 1 microg anti-CXCR4/10(6) cells displayed similar increases in activation and migration as cells exposed to SDF-1alpha, demonstrating the ability of anti-CXCR4 to activate the CXCR4 receptor. Interestingly, chimerism in NOD/SCID mice transplanted with MPB CD34(+) cells pretreated with SDF-1alpha or anti-CXCR4 was increased, while exposure of these cells to 10- to 100-fold higher concentrations of these proteins inhibited in vitro migration and NOD/SCID repopulating potential. Migration and NOD/SCID repopulating potential of BM CD34(+) cells remained unchanged after treatment with either protein. CONCLUSIONS: These results illustrate the ability of SDF-1alpha and anti-CXCR4 to augment repopulating potential of CD34(+) cells, and suggest that HPC function can be favorably modulated through specific CXCR4 signaling.