ALBERT

Beschreibung: Chromosomal translocation 8;21 is found in 40% of the FAB M2 subtype of acute myeloid leukemia (AML). The resultant in-frame fusion protein AML1-ETO (AE) acts as an initiating oncogene for leukemia development. AE immortalizes human CD34+ cord blood cells in long-term culture. We assessed the transforming properties of the alternatively spliced AE isoform AE9a (or alternative splicing at exon 9), which is fully transforming in a murine retroviral model, in human cord blood cells. Full activity was realized only upon increased fusion protein expression. This effect was recapitulated in the AE9a murine AML model. Cotransduction of AE and AE9a resulted in a strong selective pressure for AE-expressing cells. In the context of AE, AE9a did not show selection for increased expression, affirming observations of human t(8;21) patient samples where full-length AE is the dominant protein detected. Mechanistically, AE9a showed defective transcriptional regulation of AE target genes that was partially corrected at high expression. Together, these results bring an additional perspective to our understanding of AE function and highlight the contribution of oncogene expression level in t(8;21) experimental models.

Beschreibung: Key Points Increased FOXO1 is oncogenic in human CD34+ cells and promotes preleukemia transition. FOXO1 is required by AE preleukemia cells for the activation of a stem cell molecular program.

Beschreibung: Abstract 580 Generating a large number of pure, functional immune cells that can be used in human patients has been a major challenge for NK cell-based immunotherapy. We have successfully established a cultivation method to generate human NK cells from CD34+ cells isolated from donor-matched cord blood and human placental derived stem cells, which were obtained from full-term human placenta. This cultivation method is feeder-free, based on progenitor expansion followed by NK differentiation supported by cytokines including thrombopoietin, stem cell factor, Flt3 ligand, IL-7, IL-15 and IL-2. A graded progression from CD34+ hematopoietic progenitor cells (HSC) to committed NK progenitor cells ultimately results in ∼90% CD3-CD56+ phenotype and is associated with an average 10,000-fold expansion achieved over 35 days. The resulting cells are CD16- and express low level of KIRs, indicating an immature NK cell phenotype, but show active in vitro cytotoxicity against a broad range of tumor cell line targets. The in vivo persistence, maturation and functional activity of HSC-derived NK cells was assessed in NSG mice engineered to express the human cytokines SCF, GM-CSF and IL-3 (NSGS mice). Human IL-2 or IL-15 was injected intraperitoneally three times per week to test the effect of cytokine supplementation on the in vivo transferred NK cells. The presence and detailed immunophenotype of NK cells was assessed in peripheral blood (PB), bone marrow (BM), spleen and liver samples at 7-day intervals up to 28 days post-transfer. Without cytokine supplementation, very few NK cells were detectable at any time-point. Administration of IL-2 resulted in a detectable but modest enhancement of human NK cell persistence. The effect of IL-15 supplementation was significantly greater, leading to the robust persistence of transferred NK cells in circulation, and likely specific homing and expansion in the liver of recipient mice. The discrete response to IL-15 versus IL-2, as well as the preferential accumulation in the liver have not been previously described following adoptive transfer of mature NK cells, and may be unique for the HSC-derived immature NK cell product. Following the in vivo transfer, a significant fraction of human CD56+ cells expressed CD16 and KIRs indicating full physiologic NK differentiation, which appears to be a unique potential of HSC-derived cells. Consistent with this, human CD56+ cells isolated ex vivo efficiently killed K562 targets in in vitro cytotoxicity assays. In contrast to PB, spleen and liver, BM contained a substantial portion of human cells that were CD56/CD16 double negative (DN) but positive for CD244 and CD117, indicating a residual progenitor function in the CD56- fraction of the CD34+ derived cell product. The BM engrafting population was higher in NK cultures at earlier stages of expansion, but was preserved in the day 35- cultured product. The frequency of these cells in the BM increased over time, and showed continued cycling based on in vivo BrdU labeling 28 days post-transfer, suggesting a significant progenitor potential in vivo. Interestingly, DN cells isolated from BM could be efficiently differentiated ex vivo to mature CD56+CD16+ NK cells with in vitro cytotoxic activity against K562. We speculate that under the optimal in vivo conditions these BM engrafting cells may provide a progenitor population to produce a mature NK cell pool in humans, and therefore could contribute to the therapeutic potential of the HSC-derived NK cell product. The in vivo activity of HSC-derived NK cells was further explored using a genetically engineered human AML xenograft model of minimal residual disease (MRD) and initial data indicates significant suppression of AML relapse in animals receiving NK cells following chemotherapy. Collectively, our data demonstrate the utility of humanized mice and in vivo xenograft models in characterizing the biodistribution, persistence, differentiation and functional assessment of human HSC-derived cell therapy products, and characterize the potential of HSC-derived NK cells to be developed as an effective off-the-shelf product for use in adoptive cell therapy approaches in AML. Disclosures: Wunderlich: Celgene Cellular Therapeutics: Research Funding. Shrestha:C: Research Funding. Kang:Celgene Cellular Therapeutics: Employment, Equity Ownership, Patents & Royalties. Law:Celgene Cellular Therapeutics: Employment, Equity Ownership, Patents & Royalties. Jankovic:Celgene Cellular Therapeutics: Employment, Equity Ownership, Patents & Royalties. Zhang:Celgene Cellular Therapeutics: Employment, Equity Ownership, Patents & Royalties. Herzberg:Celgene Cellular Therapeutics: Employment, Equity Ownership, Patents & Royalties. Abbot:Celgene Cellular Therapeutics: Employment, Equity Ownership, Patents & Royalties. Hariri:Celgene Cellular Therapeutics: Employment, Equity Ownership, Patents & Royalties. Mulloy:Celgene Cellular Therapeutics: Research Funding.

Beschreibung: Chromosome rearrangements involving the mixed-lineage leukemia gene (MLL) create MLL-fusion proteins, which could drive both acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML). The lineage decision of MLL-fusion leukemia is influenced by the fusion partner and microenvironment. To investigate the interplay of fusion proteins and microenvironment in lineage choice, we transplanted human hematopoietic stem and progenitor cells (HSPCs) expressing MLL-AF9 or MLL-Af4 into immunodeficient NSGS mice, which strongly promote myeloid development. Cells expressing MLL-AF9 efficiently developed AML in NSGS mice. In contrast, MLL-Af4 cells, which were fully oncogenic under lymphoid conditions present in NSG mice, displayed compromised transformation capacity in a myeloid microenvironment. MLL-Af4 activated a self-renewal program in a lineage-dependent manner, showing the leukemogenic activity of MLL-Af4 was interlinked with lymphoid lineage commitment. The C-terminal homology domain (CHD) of Af4 was sufficient to confer this linkage. Although the MLL-CHD fusion protein failed to immortalize HSPCs in myeloid conditions in vitro, it could successfully induce ALL in NSG mice. Our data suggest that defective self-renewal ability and leukemogenesis of MLL-Af4 myeloid cells could contribute to the strong B-cell ALL association of MLL-AF4 leukemia observed in the clinic.

Beschreibung: Abstract 3304 Although significant progress has been made in the treatment of leukemia, relapse continues to be a major problem, particularly in acute myeloid leukemia (AML). The prognosis for relapsed leukemia is poor, indicating an area for potential improvements. However, animal models to study the response of human AML to chemotherapeutics and subsequent relapse are lacking. Recently we developed an improved NOD/SCID mouse with IL2RG knockout and transgenic expression of myelo-supportive cytokines SCF, GM-CSF, and IL-3 (the NSGS mouse). This mouse is remarkable in its ability to accept human AML grafts more efficiently than all other available strains. When coupled with in vitro derived AML cells, the NSGS mouse allows for a more predictable AML model with shorter latency and smaller range of death than in other mouse strains, including NSG mice. Importantly, very low numbers of cells reliably generate fatal AML in roughly 40 days, even in non-irradiated NSGS mice, allowing for rapid experimental conclusions and reduced toxicity. With the benefits of these unique tools, we sought to develop a model system to evaluate the efficacy of chemotherapeutic agents on human AML cells in vivo. Engrafted mice received a chemotherapy regimen over a 5-day treatment period consisting of a daily dose of cytarabine with simultaneous injection of doxorubicin during the first three days. Treated mice experienced striking weight loss during the treatment period with a nadir at days 8–10 post-treatment. Mice recovered body weight within 3 weeks. Serial complete blood counts indicated a rapid transient drop in total white blood cell and neutrophil counts and a delayed transient drop in red blood cell and platelet numbers, reminiscent of the effects observed in patients undergoing chemotherapy. The drugs successfully targeted the cells of the bone marrow, as evidenced by a profound loss of cellularity in treated mice relative to controls. When mice harboring N-Ras(G12D) positive AML cells were treated at early time points post-transplant, a significant reduction of tumor burden was observed in the BM and PB, with the grafts of treated mice essentially undetectable for weeks after treatment cessation. Nevertheless, treated mice inevitably succumbed to disease, although with a significantly prolonged latency compared to mock treated mice. However, when AML cells containing the FLT3-ITD mutation were used, a shift in disease latency was not reproducibly seen. This data correlates well with patient data showing that FLT3-ITD mutant AML has a worse prognosis than AML samples with N-Ras mutations. Importantly, the reappearance of AML within weeks of treatment affords the opportunity to model drug resistance and relapse, as well as the potential synergistic effects of experimental compounds used in combination with traditional chemotherapy. Additionally, the period following treatment may allow for studies of minimal residual disease as well as the testing of potential maintenance therapies. Finally, this approach permits a detailed analysis of the critical few cancer stem cells that remain after induction therapy with the goal of identifying novel compounds capable of targeting these cells. Disclosures: No relevant conflicts of interest to declare.

Beschreibung: AML1-ETO (AE) is a fusion product of translocation (8;21) that accounts for 40% of M2 type acute myeloid leukemia (AML). In addition to its role in promoting preleukemic hematopoietic cell self-renewal, AE represses DNA repair genes, which leads to DNA damage and increased mutation frequency. Although this latter function may promote leukemogenesis, concurrent p53 activation also leads to an increased baseline apoptotic rate. It is unclear how AE expression is able to counterbalance this intrinsic apoptotic conditioning by p53 to promote survival and self-renewal. In this report, we show that Bcl-xL is up-regulated in AE cells and plays an essential role in their survival and self-renewal. Further investigation revealed that Bcl-xL expression is regulated by thrombopoietin (THPO)/MPL-signaling induced by AE expression. THPO/MPL-signaling also controls cell cycle reentry and mediates AE-induced self-renewal. Analysis of primary AML patient samples revealed a correlation between MPL and Bcl-xL expression specifically in t(8;21) blasts. Taken together, we propose that survival signaling through Bcl-xL is a critical and intrinsic component of a broader self-renewal signaling pathway downstream of AML1-ETO–induced MPL.

Beschreibung: The Rac family of small Rho GTPases coordinates diverse cellular functions in hematopoietic cells including adhesion, migration, cytoskeleton rearrangements, gene transcription, proliferation, and survival. The integrity of Rac signaling has also been found to critically regulate cellular functions in the initiation and maintenance of hematopoietic malignancies. Using an in vivo gene targeting approach, we demonstrate that Rac2, but not Rac1, is critical to the initiation of acute myeloid leukemia in a retroviral expression model of MLL-AF9 leukemogenesis. However, loss of either Rac1 or Rac2 is sufficient to impair survival and growth of the transformed MLL-AF9 leukemia. Rac2 is known to positively regulate expression of Bcl-2 family proteins toward a prosurvival balance. We demonstrate that disruption of downstream survival signaling through antiapoptotic Bcl-2 proteins is implicated in mediating the effects of Rac2 deficiency in MLL-AF9 leukemia. Indeed, overexpression of Bcl-xL is able to rescue the effects of Rac2 deficiency and MLL-AF9 cells are exquisitely sensitive to direct inhibition of Bcl-2 family proteins by the BH3-mimetic, ABT-737. Furthermore, concurrent exposure to NSC23766, a small-molecule inhibitor of Rac activation, increases the apoptotic effect of ABT-737, indicating the Rac/Bcl-2 survival pathway may be targeted synergistically.

Beschreibung: Key Points A relevant xenograft chemotherapy model was developed by using standard AML induction therapy drugs and primary human AML patient samples. Human AML cells show significantly increased sensitivity to in vivo chemotherapy treatment compared with murine LSK and total bone marrow cells.

Beschreibung: Abstract 1536 The Rac family of small Rho GTPases has attracted interest as a therapeutic target in hematologic malignancies due to their central role in coordinating diverse cellular processes such as adhesion, cytoskeletal organization, proliferation, and survival. Rac activity is increased in MLL-AF9 (MA9) acute myeloid leukemia, and Rac inhibition using the small-molecule NSC23766 induces apoptosis in MA9 cells. We recently found that loss of Rac2 delays development of MA9 leukemia in a murine genetic model. Furthermore, latency of disease can be rescued by ectopic expression of Bcl-xL in the Rac2 knockout cells implicating Rac2 regulation of pro-survival Bcl-2 proteins in MA9 leukemogenesis. Whether Rac survival signaling through Bcl-2 proteins can be exploited therapeutically in the developed MA9 leukemia is untested. We hypothesized that Rac2 signaling is critical for MA9 leukemia cell survival, and that inhibition of Rac or downstream Bcl-2 proteins could be an effective therapeutic strategy alone or in combination. We tested our hypothesis in MA9 cells derived from human CD34+ umbilical cord blood (UCB) cells expressing MA9 by retroviral transduction. Lentiviral knockdown was used to determine the specific contribution of Rac2 to MA9 cell survival and maintenance. Decreased levels of Bcl-xL and Bcl-2 were seen in MA9 cells expressing Rac2-targeting shRNA, compared to non-targeting control. Rac2 knockdown induced apoptosis and impaired growth of MA9 cells in culture. Furthermore, Rac2 deficiency reduced in vitro colony-forming ability indicating impairment of the clonogenic MA9 cell. MA9 cells expressing two different Rac2 shRNA vs. non-targeting control were injected into NOD/LtSz-scid-SGM3 (NSS) mice to determine whether Rac2 deficiency impairs engraftment and progression of the MA9 leukemia stem cell in a xenotransplantation assay. Flow cytometric analysis of bone marrow aspirates showed markedly reduced MA9 engraftment in the Rac2 knockdown groups. Whereas all mice in the control group eventually died of MA9 leukemia (N=5), only one death from MA9 cells expressing Rac2 shRNA was seen in the knockdown groups (N=10). We next evaluated the effects of direct inhibition of Bcl-2 proteins downstream from Rac using the BH3-mimetic ABT-737. Three different MA9 cell lines, as well as the THP-1 cell line bearing an MLL-AF9 fusion, were highly sensitive (IC50 ∼30 nM) to ABT-737, with no toxicity seen in control UCB cells in the dose range tested. To determine in vivo efficacy, ABT-737 was administered to NSS mice engrafted with human MA9 cells. End of treatment aspirates showed a marked decrease in leukemia engraftment in the ABT-737 treatment group compared to vehicle control (