ALBERT

Description: Recent advances have increased the purity of hematopoietic stem cells (HSCs) isolated from young mouse bone marrow. However, little attention has been paid to the purity of HSCs from other contexts. Although Thy-1lowSca-1+Lineage-c-kit+ cells from young bone marrow are highly enriched for HSCs (1 in 5 cells gives long-term multilineage reconstitution after transplantation into irradiated mice), the same population from old, reconstituted, or cytokine-mobilized mice engrafts much less efficiently (1 in 78 to 1 in 185 cells gives long-term multilineage reconstitution). To test whether we could increase the purity of HSCs isolated from these contexts, we examined the SLAM family markers CD150 and CD48. All detectable HSCs from old, reconstituted, and cyclophosphamide/G-CSF-mobilized mice were CD150+CD48-, just as in normal young bone marrow. Thy-1lowSca-1+Lineage-c-kit+ cells from old, reconstituted, or mobilized mice included mainly CD48+ and/or CD150- cells that lacked reconstituting ability. CD150+CD48-Sca-1+Lineage-c-kit+ cells from old, reconstituted, or mobilized mice were much more highly enriched for HSCs, with 1 in 3 to 1 in 7 cells giving long-term multilineage reconstitution. SLAM family receptor expression is conserved among HSCs from diverse contexts, and HSCs from old, reconstituted, and mobilized mice engraft relatively efficiently after transplantation when contaminating cells are eliminated.

Description: Background and significance: A simple but precise method to identify hematopoietic stem cells within mobilized peripheral blood would be useful for transplantation. Our lab has recently identified a family of surface markers whose differential expression distinguishes mouse hematopoietic stem cells from other hematopoietic progenitors. The founding member of the signaling lymphocyte attractant molecule (SLAM) family, CD150, was expressed on all hematopoietic stem cells (HSCs) but not on other hematopoietic progenitors. Other SLAM-family members, including CD244 and CD48, were expressed by non-self-renewing multipotent progenitors and most colony-forming restricted progenitors, respectively. As a result, mouse stem cells can be highly purified as CD150+CD48− cells, dramatically simplifying and improving the purification of mouse HSCs. To begin to test whether SLAM family markers can facilitate the identification and purification of human hematopoietic stem cells, we have assessed the frequency of CD150+CD48− cells in mobilized peripheral blood and compared their distribution to that of CD34+CD38− cells, which are known to be highly enriched for human hematopoietic stem cells. Methods: Mobilized human peripheral blood samples were stained with anti-CD150 (conjugated to the FITC), anti-CD48 (PE), anti-CD41 (PE), anti-CD34 (APC), and anti-CD38 (PE-Cy5) antibodies. Samples were analyzed by flow-cytometry. Results: We have identified a population of CD150+CD48−CD41− cells within human mobilized peripheral blood that is present at a similar frequency as the same population in mobilized mouse peripheral blood (mean 0.039±0.11%). The CD34+CD38− population was similarly infrequent. Interestingly, 16.3±19.5% of CD150+CD48−CD41− cells were also CD34+ whereas only 1.13±3.45% of the CD34+CD38− population was CD150+CD48−CD41− raising the possibility that SLAM-family members may substantially improve the purity of human hematopoietic stem cells. Conclusion: Murine and human hematopoietic tissues have a similar frequency of CD150+CD48−CD41− cells. It is possible that the use of SLAM-family markers might enhance the identification and purification of human hematopoietic stem cells beyond what is possible using CD34 and CD38. We are currently performing reconstitution assays to test this functionally. Peripheral Blood Mononuclear Cells Peripheral Blood Mononuclear Cells

Description: Activation of the extracellular signal-regulated kinase1/2 (ERK1/2) signaling cascade mediates human multiple myeloma (MM) growth and survival triggered by cytokines and adhesion to bone marrow stromal cells (BMSCs). Here, we examined the effect of AZD6244 (ARRY-142886), a novel and specific MEK1/2 inhibitor, on human MM cell growth in the bone marrow (BM) milieu. AZD6244 blocks constitutive and cytokine-stimulated ERK1/2 phosphorylation and inhibits proliferation and survival of human MM cell lines and patient MM cells, regardless of sensitivity to conventional chemotherapy. Importantly, AZD6244 (200 nM) induces apoptosis in patient MM cells, even in the presence of exogenous interleukin-6 or BMSCs associated with triggering of caspase 3 activity. AZD6244 sensitizes MM cells to both conventional (dexamethasone) and novel (perifosine, lenalidomide, and bortezomib) therapies. AZD6244 down-regulates the expression/secretion of osteoclast (OC)–activating factors from MM cells and inhibits in vitro differentiation of MM patient PBMCs to OCs induced by ligand for receptor activator of NF-κB (RANKL) and macrophage-colony stimulating factor (M-CSF). Finally, AZD6244 inhibits tumor growth and prolongs survival in vivo in a human plasmacytoma xenograft model. Taken together, these results show that AZD6244 targets both MM cells and OCs in the BM microenvironment, providing the preclinical framework for clinical trials to improve patient outcome in MM.

Description: Abstract 3531 Poster Board III-468 The identification of hematopoietic stem cell (HSC)-specific surface markers has been a goal for functional stem cell studies and for clinical applications, including transplantation and gene therapy. In humans, the CD34 cell surface marker is largely used in clinical applications for isolation of hematopoietic stem and progenitor cells, and as a predictor of graft HSC content in hematopoietic stem cell transplant. The combination CD34+CD38- provides further enrichment, but the heterogeneity of this population precludes studies requiring levels of purity achieved in mouse models, such as comparative gene expression profiles or in situ histological analyses. Recently, a simple and broadly applicable method to isolate mouse HSCs has been developed based on expression of cell surface markers members of the Signaling Lymphocyte Activation Molecule (SLAM) family, including CD150, CD48, and CD244 (Kiel MJ et al., Cell 2005; 121:1109-1121). In transplantation assays, 1 in 4.8 CD150+CD48- bone marrow cells provide long-term, multilineage reconstitution in recipient mice. We examined the possibility of using these SLAM markers to facilitate the isolation of highly enriched populations of HSCs in humans and rhesus macaques. We tested the in vivo repopulating and colony forming potential of CD150+CD48- cells (SLAM cells) in comparison with non-SLAM cells which included all viable cells outside the SLAM gate (non-SLAM cells). Human G-CSF mobilized cells were pre-enriched for CD34+ cells to facilitate sorting of the rare population of CD150+CD48- cells. SLAM cells were absent in the CD34- fraction. Using stringent gates defined with isotype controls, we observed that 1.1% of human CD34+ cells were CD150+CD48-. In CFU assays, there was no difference in the frequency of colonies obtained between SLAM, non-SLAM and unfractionated CD34+ cell populations. Sublethally irradiated NOD/SCID/γcnull mice were transplanted by tail-vein injection with 1×103 to 1×104 human SLAM cells, 1×105 to 5×106 human non-SLAM cells, or 1×103 to 1×106 human unfractionated CD34+ cells. Mice transplanted with less than 1×105 cells also received 1×105 CD34- carrier cells. Surprisingly, in contrast to data obtained in murine models, both SLAM and non-SLAM populations had in vivo repopulating potential, and levels of human cell engraftment based on CD45 cell surface expression were similar to those obtained with unfractionated CD34+ cells. Similar results were obtained when human CB cells were used to isolate CD150+ and CD150- populations with no pre-enrichment in CD34+ cells. We next investigated whether SLAM receptors may facilitate the isolation of enriched populations of HSCs in rhesus macaques. In two independent experiments, mobilized peripheral blood CD34+ cells were obtained and further purified into SLAM and non-SLAM populations using human antibody clones cross-reactive with rhesus macaque cells. In each experiment, approximately 0.5% of CD34+ cells were CD150+CD48-. In CFU assays, the number of colonies obtained from each population was similar and comparable to the numbers of CFU derived from unfractionated CD34+ cells. Similar to findings in humans, both SLAM and non-SLAM populations had in vivo repopulating potential and levels of engraftment were similar to those obtained after transplantation of unfractionated CD34+ cells. Therefore, these combinations of antibodies against SLAM markers are not suitable for isolation of HSCs in large mammals. We conclude that human and rhesus macaque repopulating HSCs are not enriched in the CD150+CD48- SLAM population. Disclosures: No relevant conflicts of interest to declare.

Description: Human multiple myeloma (MM), an incurable plasma cell tumor, is highly dependent on its bone marrow (BM) microenvironment which secretes cytokines and growth factors (i.e., IL-6, IGF-1, BAFF) for myeloma cell growth, survival, and resistance to chemotherapeutic drugs. Studies of interactions of MM and BMSCs have demonstrated that activation of the extracellular signal-regulated kinase 1/2 (ERK1/2) mediates these processes. Recently, gene expression profiling studies have revealed that the c-MAF oncogene, which is overexpressed in approximately 50% of myeloma cell lines and patient MM cells, not only stimulates cell cycle progression but also promotes pathological interactions between tumor and BMSCs. In this study, we examined effects of the MEK1/2 inhibitor AZD6244 (ARRY-142886) on the c-MAF signaling and adhesion-induced c-MAF expression in MM cells. We first demonstrated that AZD6244 inhibits growth of INA6, MM1S, MM1R and MCCAR MM cells in a dose-dependent manner (see also Abstract #553572, ASH 2006). We next found that AZD6244 significantly downregulates c-MAF mRNA and c-MAF protein expression in these MM lines, as early as 2h and this was sustained until 24h after drug treatment. Hypoxia-induced factor 2α (HIF-2α), a pro-angiogenic mediator, which regulates c-MAF gene expression and is regulated by ERK1/2, was significantly inhibited in AZD6244-treated MM cell lines. Since three c-MAF target genes, integrin β7, the C-C chemokines receptor-1 (CCR1), and cyclin D2, were recently identified in MM, we next asked whether the expression of these genes was concomitantly inhibited by AZD6244 in INA6 MM cells that lacks c-MAF translocation. Integrin β7 and CCR1 were inhibited by AZD6244 in a time-dependent manner, whereas cyclin D2 was not expressed in INA6 MM cells. Importantly, downregulation of c-MAF and integrin β7 by AZD6244 is associated with decreased MM cell adhesion to BMSCs and reduced VEGF secretion. Although the role of CCR1 in MM pathophysiology remains elusive, AZD6244 significantly decreased the expression of CCR1 as well as its cognate ligand MIP-1α in INA6 MM cells, which might inhibit a potential autocrine loop in MM cells. We next determined whether MM adhesion to BMSCs could upregulate the c-MAF pathway in MM cells, in the presence or absence of AZD6244. Adhesion of INA6 MM cells to BMSCs strongly induced ERK1/2 and STAT3 activation and augmented expression of c-MAF and its downstream targets (cyclin D2 and integrin β7). The induction of c-MAF and its downstream targets was also seen, although with less extent, in IL-6-induced INA6 MM cells. Conversely, AZD6244 inhibited adhesion-induced c-MAF and its target protein expression (cyclin D2, integrin β7) in INA6 MM cells. AZD6244 further blocked autocrine and paracrine VEGF secretion in a dose-dependent manner. In conclusion, these results confirm a role for c-MAF in the pathological interactions between MM cells and BMSCs and define an important role of the MEK/ERK activation in the ~40% of c-MAF-overexpressing myeloma cells that lack c-MAF translocations. Moreover, this study strongly supports AZD6244 (ARRY-142886) as a promising targeted therapeutic intervention in MM.

Description: P2 receptors are functionally diverse cell surface receptors that bind nucleotides adenine (ADP, ATP) and uridine (UDP, UTP). P2Y receptors are metabotropic G protein-coupled receptors that mediate vascular and immune responses to injury. We previously reported the differential expression cloning of the UTP-glycoconjugate receptor, P2Y14 from quiescent primary human bone marrow (BM) hematopoietic stem cells (HSCs). Using P2Y14−/− mice, we now report that the presence of P2Y14 protects HSCs from apoptosis in the face of cytotoxic chemical injury. P2Y14 null mice develop normally and showed no significant differences in peripheral blood cell counts, BM cellularity or the absolute number/proportion of lin−cKit+Sca1+ (LKS+) and CD34−/lowLKS+ (34-LKS+) cells compared to their wildtype littermates. Similarly, cell cycle status, in vitro colony-forming cell (CFC) capacity, in vivo homing and in vivo colony-forming unit-spleen (CFU-S) function were unaffected. Since the role of nucleotide receptors in injury response have been reported, we examined BM HSC content following IP injection of 200mg/kg cyclophosphamide (CTX) and found that the relative protection of LKS+ and 34-LKS+ cells from CTX-induced apoptosis was lost in P2Y14 null animals (WT LKS+: 12.7% AnnexinV+7AAD-, KO LKS+ 36.8% AnnexinV+7AAD−, n=5 each, p=0.004; WT 34-LKS+: 13.2% AnnexinV+7AAD−, KO LKS+ 38.7% AnnexinV+7AAD−, n=5 each, p=0.007). In addition, the kinetics of long-term myeloid recovery after a single injection of 5-Fluorouracil (5FU) IP 150mg/kg was significantly more accentuated in P2Y14 null animals, with significantly greater peripheral blood Gr-1+ cell count at days 21–56 post-injection (n=10 each, p=0.009). When sorted BM LKS+ cells were exposed in vitro to UDP-glucose, a putative P2Y14 ligand known to be released from cytoplasm during cellular injury, BrDU incorporation was significantly reduced (n=3 each, p

Description: High levels of aldehyde dehydrogenase (ALDH) activity have been proposed to be a common feature of stem cells. Adult hematopoietic, neural, and cancer stem cells have all been reported to have high ALDH activity, detected using Aldefluor, a fluorogenic substrate for ALDH. This activity has been attributed to Aldh1a1, an enzyme that is expressed at high levels in stem cells and that has been suggested to regulate stem cell function. Nonetheless, Aldh1a1 function in stem cells has never been tested genetically. We observed that Aldh1a1 was preferentially expressed in mouse hematopoietic stem cells (HSCs) and expression increased with age. Hematopoietic cells from Aldh1a1-deficient mice exhibited increased sensitivity to cyclophosphamide in a non–cell-autonomous manner, consistent with its role in cyclophosphamide metabolism in the liver. However, Aldh1a1 deficiency did not affect hematopoiesis, HSC function, or the capacity to reconstitute irradiated recipients in young or old adult mice. Aldh1a1 deficiency also did not affect Aldefluor staining of hematopoietic cells. Finally, Aldh1a1 deficiency did not affect the function of stem cells from the adult central or peripheral nervous systems. Aldh1a1 is not a critical regulator of adult stem cell function or Aldefluor staining in mice.

Description: Infections remain a critical issue in the care of CLL patients (pts). The specific agents used and their resultant immunosuppression impact the spectrum of infection. We compared the infectious complications reported in previously untreated CLL pts enrolled on 3 sequential CALGB treatment trials. On CALGB 9011, 188 pts received single agent fludarabine (F) therapy. On CALGB 9712, 104 pts received concurrent or sequential fludarabine plus rituximab (FR). On CALGB 19901, pts were to receive 4 cycles of F, followed by 6 weeks of alemtuzumab consolidation (FA); of the 85 enrolled patients, 59 proceeded to alemtuzumab consolidation. Infections requiring hospitalization and/or parenteral antibiotics were tabulated. The 85 pts on study 19901 (FA) had significantly more of these infections during protocol therapy (32/85, 38%) compared to the 188 pts receiving F on study 9011 (43/188, 23%, p=0.01). Similarly, the 19901 study pts also had more infections than the 104 FR-treated pts on study 9712 (21/104, 20%, p=0.0007). Among the 85 pts on study 19901, 14 (16%) had infections only during the F phase of therapy, 14 (16%) had infections only during alemtuzumab consolidation, and 4 (5%) had infections during both phases of therapy. The occurrence of cytomegalovirus (CMV) and Pneumocystis (PCP) infections was also examined on these trials. No CMV and only 3 PCP infections were diagnosed among the 188 pts receiving F on study 9011. For study 9712, 3/104 pts had PCP (one on concurrent FR, two on sequential FR), and 1 pt had CMV pneumonia on sequential FR. Among pts on study 19901, one case of PCP and no cases of CMV occurred during the F phase of therapy. However, 12/59 pts developed CMV infection during alemtuzumab consolidation. Four had resolution of infection and completed alemtuzumab therapy, while 8 were hospitalized and did not complete this therapy. We conclude that the addition of rituximab to F therapy does not increase the risk or severity of infections in previously untreated CLL pts, but the use of alemtuzumab consolidation following initial F therapy does increase the overall risk of serious infectious complications, and especially CMV infections. These issues are important not only for prophylactic antimicrobial therapy and monitoring during and after therapy, but also for the potential use of alemtuzumab as a consolidation therapy to eradicate residual CLL after F treatment.

Description: Although adult mouse hematopoietic stem cells (HSCs) have been purified to near homogeneity, it remains impossible to achieve this with fetal HSCs. Adult HSC purity recently has been enhanced using the SLAM family receptors CD150, CD244, and CD48. These markers are expressed at different stages of the hematopoiesis hierarchy, making it possible to highly purify adult HSCs as CD150+CD48–CD244– cells. We found that SLAM family receptors exhibited a similar expression pattern in fetal liver. Fetal liver HSCs were CD150+CD48–CD244–, and the vast majority of colony-forming progenitors were CD48+CD244–CD150– or CD48+CD244+CD150–, just as in adult bone marrow. SLAM family markers enhanced the purification of fetal liver HSCs. Whereas 1 (11%) of every 8.9 ThylowSca-1+lineage–Mac-1+ fetal liver cells gave long-term multilineage reconstitution in irradiated mice, 1 (18%) of every 5.7 CD150+CD48–CD41– cells and 1 (37%) of every 2.7 CD150+CD48–Sca-1+lineage–Mac-1+ fetal liver cells gave long-term multilineage reconstitution. These data emphasize the robustness with which SLAM family markers distinguish progenitors at different stages of the hematopoiesis hierarchy and enhance the purification of definitive HSCs from diverse contexts. Nonetheless, CD150, CD244, and CD48 are not pan-stem cell markers, as they were not detectably expressed by stem cells in the fetal or adult nervous system.

Description: Abstract 536 Long-term outcomes following novel therapies for CLL have rarely been reported. Between 10/90 and 12/94, 509 eligible, untreated patients (pts) with symptomatic CLL were enrolled by 4 cooperative groups onto study C9011; 179 were randomized to F, 193 to C, and 137 to F+C. After slightly more than 5 years median follow up, with the time of last follow-up in June 1999, we reported in 2000 (NEJM 343:1750) that F provided significantly higher response rates and longer remission duration and progression-free survival (PFS) than C (p