ALBERT

Description: Acute myelogenous leukemia (AML) is a heterogenous group of myeloid malignancies that are characterized by the clonal outgrowth of immature myeloid progenitor cells. For most subtypes of AML, mutations that give rise to the leukemic phenotype occur in the hematopoietic stem/progenitor cell (HSC) subset as demonstrated by studies showing that only primitive CD34+CD38− bone marrow cells could function as leukemia-initiating cells (LSC) when transferred into immunodeficient NOD-SCID mice. One rather significant challenge has been that LSC share many of the same cell-surface markers as their normal counterparts in bone marrow, thus making it difficult to functionally characterize and purify this important subset of leukemic cells from bulk bone marrow samples. To rapidly identify novel antigens that are mutation-specific and induced specifically on LSC and not on normal HSC, we have transduced highly purified HSC isolated from mouse bone marrow with retroviral vectors that co-expressed AML1-ETO along with a green fluorescent protein (GFP) reporter gene. HSC of the cell-surface phenotype c-Kit+Lin−Sca-1+Flt3− (KLSF) were transduced for 18 hours in vitro and then were resorted for GFP+ cells by flow cytometry (FACS). Purified cells were then lysed for mRNA isolation and cDNA synthesis for the generation of a probe that was then hybridized to Affymetrix oligonucleotide arrays (430 2.0 GeneChip arrays). HSC transduced with a retroviral vector that only expressed GFP were used as controls to identify genes that would normally be expressed in the HSC subset. Importantly, since retroviral vectors only integrate into cycling cells, all sorted GFP+ cells from the independent transductions would represent cycling cells, which minimizes any gene expression differences due to differential frequencies of resting versus actively cycling HSC. Changes in expression of cell-surface proteins observed at the mRNA level were then validated at the protein level using FACS. Bone marrow cells were isolated from an animal that was transplanted with cells expressing AML1-ETO and GFP from a retroviral vector. Cells were stained for the HSC/progenitor cell phenotype (KLS) as well as for the cell-surface marker of interest. For one marker, CD55, we noted a 100-fold increase in cell-surface expression specifically on HSC that express AML1-ETO and not on normal HSC. These results indicate that short-term retroviral expression of specific AML-associated mutations in HSC followed by microarray analysis of transduced cells may provide a rapid means of prospectively identifying leukemia-initiating cells in bulk patient bone marrow samples and that CD55 may be a useful therapeutic and diagnostic marker for patient samples that express the AML1-ETO chromosomal translocation. Figure 1. CD55 expression distinguishes normal HSC from HSC with the AML1-ETO translocation. Bone marrow cells were isolated from an animal that was transplanted with cells expressing AML1-ETO and the green fluorescent protein (GFP) from a retroviral vector. Cells were stained for the HSC/progenitor cell phenotype (KLS) as well as for CD55. Cells that expressed AML1-ETO are shown below as GFP+ gated cells. Note that cells that express AML1-ETO express CD55 at approximately 100-fold greater levels on the cell-surface than GFP-negative (AML1-ETO-negative) bone marrow cells. MPC=myeloid progenitor cells of the phenotype c-Kit+ Lin− Sea-1− cells. HSC are defined c-Kit+ Lin− Sca-1+ cells. Figure 1. CD55 expression distinguishes normal HSC from HSC with the AML1-ETO translocation. Bone marrow cells were isolated from an animal that was transplanted with cells expressing AML1-ETO and the green fluorescent protein (GFP) from a retroviral vector. Cells were stained for the HSC/progenitor cell phenotype (KLS) as well as for CD55. Cells that expressed AML1-ETO are shown below as GFP+ gated cells. Note that cells that express AML1-ETO express CD55 at approximately 100-fold greater levels on the cell-surface than GFP-negative (AML1-ETO-negative) bone marrow cells. MPC=myeloid progenitor cells of the phenotype c-Kit+ Lin− Sea-1− cells. HSC are defined c-Kit+ Lin− Sca-1+ cells.

Description: PU.1 is a member of the ETS family of transcription factors and is required for the development of multiple hematopoietic lineages. PU.1-/- mice die from hematopoietic failure at about embryonic day 18.5 (e18.5) and show a complete absence of B cells, mature T cells, and macrophages. This phenotype suggests that PU.1 may function at the level of the hematopoietic stem cell (HSC) or a multilineage progenitor. To investigate the role of PU.1 in the regulation of HSCs, PU.1-/- embryos were analyzed at various stages of embryonic development. The absolute number and frequency of HSCs were determined by flow cytometric analysis of c-Kit+Thy-1.1loLin-Sca-1+ (KTLS) cells. We found that KTLS cells were absent or severely reduced in PU.1-/- fetal liver from e12.5 to e15.5. Progenitor cells with a c-Kit+Lin-AA4.1+ and c-Kit+Lin-CD34+ phenotype were also severely reduced. In addition, PU.1-/- fetal liver at e14.5 lacked common myeloid progenitors (CMPs) and granulocyte-macrophage progenitors (GMPs) but retained megakaryocyteerythroid progenitors (MEPs). Consistent with the loss of HSC activity, a 10-fold reduction in erythroid progenitors (mature erythroid burst-forming units [BFUEs]) was observed between e14.5 and e16.5. These data suggest that PU.1 plays an important role in the maintenance or expansion of HSC number in murine fetal liver. (Blood. 2004;104:3894-3900)

Description: The t(8;21) RUNX1-ETO translocation is one of the most frequent cytogenetic abnormalities in acute myeloid leukemia (AML). In RUNX1-ETO+ patient samples, differing classes of activating c-KIT receptor tyrosine kinase mutations have been observed. The most common (12%-48%) involves mutations, such as D816V, which occur in the tyrosine kinase domain, whereas another involves mutations within exon 8 in a region mediating receptor dimerization (2%-13% of cases). To test whether distinct subtypes of activating c-KIT mutations differ in their leukemogenic potential in association with RUNX1-ETO, we used a retroviral transduction/transplantation model to coexpress RUNX1-ETO with either c-KitD814V or c-KitT417IΔ418-419 in murine hematopoietic stem/progenitor cells used to reconstitute lethally irradiated mice. Analysis of reconstituted animals showed that RUNX1-ETO;c-KitD814V coexpression resulted in 3 nonoverlapping phenotypes. In 45% of animals, a transplantable AML of relatively short latency and frequent granulocytic sarcoma was noted. Other mice exhibited a rapidly fatal myeloproliferative phenotype (35%) or a lethal, short-latency pre-B-cell leukemia (20%). In contrast, RUNX1-ETO;c-KitT417IΔ418-419 coexpression promoted exclusively AML in a fraction (51%) of reconstituted mice. These observations indicate that c-KitD814V promotes a more varied and aggressive leukemic phenotype than c-KitT417IΔ418-419, which may be the result of differing potencies of the activating c-Kit alleles.

Description: Abstract 4195 The t(8;21) AML1-ETO translocation is one of the most frequent cytogenetic abnormalities observed in acute myeloid leukemia (AML), occurring in 10–12% of cases. The t(8;21) fuses the DNA binding domain of AML1 with the transcriptional repressor gene ETO. AML1-ETO dominantly blocks wild-type AML1 function, preventing activation of AML1 target genes and impairing myeloid differentiation. However, expression of AML1-ETO in mice does not promote AML in the absence of additional mutations. Another commonly mutated gene detected in AML1-ETO+ patient samples is the receptor tyrosine kinase c-KIT. Two classes of activating c-KIT mutations have been reported; one where mutations like D814V occur in the tyrosine kinase domain and the other involving a deletion within Exon 8 (E8D419) in a region that mediates receptor dimerization. These mutations are not randomly distributed with t(8;21) or with other chromosomal translocations found in AML. For instance, c-KITD814V is observed in 11–44% of t(8;21) patients, whereas c-KITE8Æ419 with t(8;21) is reported in 2–13% of cases. These observations suggest a degree of specificity with which activating c-KIT mutations can cooperate with AML1-ETO to promote AML. To test this hypothesis, we used a retroviral transduction/transplantation model to co-express AML1-ETO with c-KitD814V or c-KitE8Æ419 in murine bone marrow (BM) progenitor cells used to reconstitute lethally irradiated mice. Analysis of reconstituted mice has shown that simultaneous expression of AML1-ETO with c-KitD814V results in three non-overlapping phenotypes. The most common phenotype, occurring in 50% of animals, is a transplantable AML characterized by a relatively short disease latency that may or may not present with an associated granulocytic sarcoma. Second, a rapidly fatal non-transplantable myeloproliferative phenotype was observed in 25% of animals. Lastly, several mice (25%) developed a lethal, short latency B-cell leukemia that was transplantable to secondary recipient animals. The heterogeneity of disease phenotypes that develop in AML1-ETO;c-KITD814V mice may reflect the specific hematopoietic progenitor cell type that was initially transduced and capable of reconstituting the recipient mouse or differences in AML1-ETO and/or c-KitD814V expression levels. In contrast, co-expression of AML1-ETO with c-KitE8Æ419 promoted a transplantable AML phenotype characterized by a relatively long disease latency in a fraction (43.7%) of reconstituted mice. AML1-ETO; c-KitE8Æ419–expressing cells could persist for months without showing signs of selective expansion, which suggests that additional genetic changes are necessary for overt transformation to AML in this context. These observations indicate that when overlaid on the AML1-ETO genetic lesion, c-KitD814V promotes a more aggressive disease phenotype than c-KitE8Æ419, which displays a longer latency and reduced penetrance. These differences may account for the observed disparity in the frequencies of each c-KIT mutation concurrent with AML1-ETO in human AML cases. Disclosures: No relevant conflicts of interest to declare.

Description: The inversion of chromosome 16 in the inv(16)(p13q22) is one of the most frequent cytogenetic abnormalities observed in acute myeloid leukemia (AML). The inv(16) fuses the core binding factor (CBF) beta subunit with the coiled-coil rod domain of smooth muscle myosin heavy chain (SMMHC). Expression of CBFβ-SMMHC in mice does not promote AML in the absence of secondary mutations. Patient samples with the inv(16) also possess mutually exclusive activating mutations in either N-RAS, K-RAS, or the receptor tyrosine kinases, c-KIT and FLT3, in almost 70% of cases. To test whether an activating mutation of FLT3 (FLT3-ITD) would cooperate with CBFβ-SMMHC to promote AML, we coexpressed both mutations in hematopoietic progenitor cells used to reconstitute lethally irradiated mice. Analysis of transplanted animals showed strong selection for CBFβ-SMMHC/FLT3-ITD–expressing cells in bone marrow and peripheral blood. Compared with animals transplanted with only CBFβ-SMMHC–expressing cells, FLT3-ITD further restricted early myeloid differentiation and promoted peripheralization of primitive myeloblasts as early as 2.5 weeks after transplantation. FLT3-ITD also accelerated disease progression in all CBFβ-SMMHC/FLT3-ITD–reconstituted animals, which died of a highly aggressive and transplantable AML within 3 to 5 months. These results indicate that FLT3-activating mutations can cooperate with CBFβ-SMMHC in an animal model of inv(16)-associated AML.

Description: Abstract 839 Objectives: Programmed death-1 (PD-1)/CD279 is an immunoinhibitory receptor that can be physiologically expressed on activated antigen-specific alpha/beta T-cells and is thought to play a role in maintaining a balance between T-cell activation and tolerance. Recently, both in vitro and in vivo, it has been shown that disrupting the interaction between PD-1 and its ligands can improve antitumor effects in preclinical and clinical models, this suggesting an important role played by this pathway in escape from immune surveillance. In comparison to healthy donors, gamma/delta-T cells found in tumor-bearing hosts can be diminished in number, or can be functionally impaired in a variety of important ways. While the mechanisms accounting for these numeric or functional defects have remained unclear, here we examine the extent to which PD-1 expression on gamma/delta-T cells may play a role in this process. Findings: We first noted that peripheral blood gamma/delta-T cells are diminished in numbers in patients newly diagnosed with cancer. In addition, these gamma/delta-T cells expanded poorly when cultured ex vivo. Similar to humans, in tumor-bearing mice, we found that peripheral blood gamma/delta-T cells are diminished in number and likewise, expand poorly when cultured ex vivo. Using FACS analysis of mouse peripheral blood, we first determined that a substantial proportion of gamma/delta-T cells are actively undergoing apoptosis in tumor-bearing mice compared to healthy mice. Further analysis revealed that PD-1 is significantly upregulated on gamma/delta-T cells taken from tumor-bearing mice compared to gamma/delta-T cells taken from healthy mice. In contrast, no difference of PD-1 expression was seen when comparing alpha/beta-T cells taken from tumor-bearing and healthy mice. Using in vitro co-culture studies, we next determined that apoptosis in gamma/delta-T cells can be induced by direct contact with malignant cells, but not by contact with non-malignant cells. We then showed in these cultures that PD-1 is upregulated on gamma/delta-T cells co-cultured with tumor cell lines. Moreover, we were able to determine that the PD-1-positive gamma/delta-T cells in these cultures were undergoing apoptosis to a greater extent than PD-1-negative gamma/delta-T cells in these same cultures. Finally, in vitro using CFSE-based methods, we showed that while gamma/delta-T cells isolated from healthy mice readily proliferate upon mitogen stimulation, in contrast, gamma/delta-T cells from tumor-bearing mice proliferate poorly under the same conditions. However, in spleen cell cultures derived from tumor-bearing mice, upon addition of a monoclonal antibody directed against PD-L1 (B7-H1), a ligand for PD-1, substantial restoration of gamma/delta-T cell proliferation occurs. Conclusion: Until now, the role played by PD-1 in the exhaustion of tumor-reactive gamma/delta T-cells has not been explored. Using in vitro and in vivo models, we show that the PD-1 pathway is a potentially important mechanism by which gamma/delta T-cells are either functionally impaired or otherwise exhausted in tumor-bearing mice. These findings suggest that by disrupting the PD-1 pathway, it may be possible to “revive” or “rescue” gamma/delta T-cells in tumor-bearing hosts. Disclosures: No relevant conflicts of interest to declare.

Description: Progenitor B cells deficient in Pax5 are developmentally multipotent, suggesting that Pax5 is necessary to maintain commitment to the B-cell lineage. Commitment may be mediated, in part, by Pax5 repression of myeloid-specific genes. To determine whether Pax5 expression in multipotential cells is sufficient to restrict development to the B-cell lineage in vivo, we enforced expression of Pax5 in hematopoietic stem cells using a retroviral vector. Peripheral blood analysis of all animals reconstituted with Pax5-expressing cells indicated that more than 90% of Pax5-expressing cells were B220+ mature B cells that were not malignant. Further analysis showed that Pax5 completely blocked T-lineage development in the thymus but did not inhibit myelopoiesis or natural killer (NK) cell development in bone marrow. These results implicate Pax5 as a critical regulator of B- versus T-cell developmental fate and suggest that Pax5 may promote commitment to the B-cell lineage by mechanisms that are independent of myeloid gene repression.