The branched-chain amino acid (BCAA) pathway and high levels of BCAA transaminase 1 (BCAT1) have recently been associated with aggressiveness in several cancer entities. However, the mechanistic role of BCAT1 in this process remains largely uncertain. Here, by performing high-resolution proteomic analysis of human acute myeloid leukaemia (AML) stem-cell and non-stem-cell populations, we find the BCAA pathway enriched and BCAT1 protein and transcripts overexpressed in leukaemia stem cells. We show that BCAT1, which transfers α-amino groups from BCAAs to α-ketoglutarate (αKG), is a critical regulator of intracellular αKG homeostasis. Further to its role in the tricarboxylic acid cycle, αKG is an essential cofactor for αKG-dependent dioxygenases such as Egl-9 family hypoxia inducible factor 1 (EGLN1) and the ten-eleven translocation (TET) family of DNA demethylases. Knockdown of BCAT1 in leukaemia cells caused accumulation of αKG, leading to EGLN1-mediated HIF1α protein degradation. This resulted in a growth and survival defect and abrogated leukaemia-initiating potential. By contrast, overexpression of BCAT1 in leukaemia cells decreased intracellular αKG levels and caused DNA hypermethylation through altered TET activity. AML with high levels of BCAT1 (BCAT1) displayed a DNA hypermethylation phenotype similar to cases carrying a mutant isocitrate dehydrogenase (IDH), in which TET2 is inhibited by the oncometabolite 2-hydroxyglutarate. High levels of BCAT1 strongly correlate with shorter overall survival in IDHTET2, but not IDH or TET2 AML. Gene sets characteristic for IDH AML were enriched in samples from patients with an IDHTET2BCAT1 status. BCAT1 AML showed robust enrichment for leukaemia stem-cell signatures, and paired sample analysis showed a significant increase in BCAT1 levels upon disease relapse. In summary, by limiting intracellular αKG, BCAT1 links BCAA catabolism to HIF1α stability and regulation of the epigenomic landscape, mimicking the effects of IDH mutations. Our results suggest the BCAA-BCAT1-αKG pathway as a therapeutic target to compromise leukaemia stem-cell function in patients with IDHTET2 AML.

Autologous haematopoietic SCT with PBSCs is regularly used to restore BM function in patients with multiple myeloma or lymphoma after myeloablative chemotherapy. Twenty-eight experts from the European Group for Blood and Marrow Transplantation developed a position statement on the best approaches to mobilising PBSCs and on possibilities of optimising graft yields in patients who mobilise poorly. Choosing the appropriate mobilisation regimen, based on patients' disease stage and condition, and optimising the apheresis protocol can improve mobilisation outcomes. Several factors may influence mobilisation outcomes, including older age, a more advanced disease stage, the type of prior chemotherapy (e.g., fludarabine or melphalan), prior irradiation or a higher number of prior treatment lines. The most robust predictive factor for poor PBSC collection is the CD34(+) cell count in PB before apheresis. Determination of the CD34(+) cell count in PB before apheresis helps to identify patients at risk of poor PBSC collection and allows pre-emptive intervention to rescue mobilisation in these patients. Such a proactive approach might help to overcome deficiencies in stem cell mobilisation and offers a rationale for the use of novel mobilisation agents.

As more efficient agents for stem cell mobilization are being developed, there is an urgent need to define which patient population might benefit from these novel drugs. For a precise and prospective definition of "poor mobilization" (PM), we have analyzed the efficiency of mobilization in patients intended to receive autologous transplantation at our center in the past 6 years. Between January 2003, and December 2008, 840 patients with the following diagnoses were scheduled to undergo leukapheresis: multiple myeloma (MM, n = 602) and non-Hodgkin lymphoma (NHL, n= 238). Most patients mobilized readily: close to 85% of the patients had a level of 20/microL to >500/microL of CD34(+) cells at the peak of stimulation. Of the 840 patients, 129 (15.3%) were considered to be PMs, defined as patients who had a peak concentration of <20/microL of CD34(+) cells upon stimulation with granulocyte-colony stimulating factor (G-CSF) subsequent to induction chemotherapy appropriate for the respective disease. Among them, 38 (4.5%) patients had CD34(+) levels between 11 and 19/microL at maximum stimulation, defined as "borderline" PM, 49 (5.8%) patients had CD34(+) levels between 6 and 10/microL, defined as "relative" PM, and 42 patients (5%) with levels of <5/microL, defined as "absolute" PM. There was no difference in the incidence of PM between patients with MM versus those with NHL. Sex, age, body weight (b.w.) and previous irradiation therapy did not make any significant difference. Only the total number of cycles of previous chemotherapy (P = .0034), and previous treatment with melphalan (Mel; P = .0078) had a significant impact on the ability to mobilize. For the good mobilizers, the median time to recovery of the white blood cells (WBCs) to 1.0/nL or more was 13 days with a range of 7 to 22 days, whereas for the PM group it was 14 days with a range of 8 to 37 days. This difference was statistically not significant. The median time to recovery of the platelets counts to an unmaintained level of >20/nL was 11 days with a range of 6 to 17 days for the good mobilizers, whereas for the PM it was 11 days with a range of 7 to 32 days. Again, this difference was not significant. The majority of the patients today intended for autologous transplantations were able to mobilize readily. As long as > or =2.0 x 10(6) of CD34(+) cells/kg b.w. have been collected, PM was not associated with inferior engraftment.

2153 Poster Board II-130 Hematopoietic progenitor and stem cells (HSC) reside in the bone marrow and have to be mobilized into the circulation prior to being collected by apheresis. The number of apheresis procedures needed and the success of transplantation are determined by the efficiency of stem cell mobilization. Between January 2004 to December 2008, 840 patients (pt) with the following diagnoses were scheduled to undergo leukapheresis for autologous transplantations: multiple myeloma (MM, n=602) and non-Hodgkin's lymphoma (NHL, n=238). Mobilization data and transplantation outcome were analyzed retrospectively. Most of the pt mobilized readily: close to 85% of the pt had a level of 20/μL to >500/μL of CD34+ cells at the peak of stimulation. Of the 840 pt, 129 (15.3%) were considered to be “Poor Mobilizers” (PM), defined as pt who had a peak concentration of <20/μL of CD34+ cells upon stimulation with G-CSF subsequent to induction chemotherapy appropriate for the respective disease. Among them, 38 (4.5%) pt had CD34+ levels of between 11-19/μL at maximum stimulation, defined as borderline PM, 49 (5.8%) pt had CD34+ levels of between 6-10/μL, defined as relative PM and 42 pt (5%) with levels of <5/μL, defined as absolute PM. We have analyzed the relationship between poor mobilizations with types of disease (MM versus NHL), sex, age, body weight, previous irradiation, number of cycles of previous combination chemotherapy, and pretreatment with melphalan. There was no difference in the incidence of PM between pt with MM versus those with NHL. Sex, age, body weight and previous irradiation therapy did not make any significant difference. Only the number of cycles of previous chemotherapy (p=0.0034), and previous treatment with melphalan (p=0.0078) had a significant impact on the ability to mobilize. Secondary strategies to mobilize HSC from the 33 who failed included: (1) Administration of another cycle of induction chemotherapy + G-CSF. The goal of harvesting 2.0 × 10exp6 CD34+ cells/kg body weight could be accomplished in 7 of 21 of these patients. (2) G-CSF alone for 4 days (up to 8 days of stimulation) after hematopoietic recovery from previous induction chemotherapy. The goal could be achieved in 2 of the 9 patients thus mobilized. (3) Plerixafor within the compassionate use program. The goal was accomplished in 7 of 8 patients within one cycle of mobilization. All 8 could be transplanted successfully. (4) Bone marrow harvest in lieu of collection of peripheral HSC in 5 patients. For the good mobilizers, the median time to recovery of the WBC to 1.0/nL or granulocyte of 0.5/nL (whichever is sooner) was 13 days with a range of 7 to 22 days, whereas for the PM group it was 14 days with a range of 8 to 37 days. This difference was statistically not significant. The median time to recovery of the platelets counts to an unmaintained level of >20/nL was 11 days with a range of 6 to 17 days for the good mobilizers, whereas for the PM it was 11 days with a range of 7 to 32 days. Again this difference was statistically not significant. The majority of the patients nowadays intended for autologous transplantations were able to mobilize readily. According to the criteria proposed in this study, 15.3% were considered to be “Poor Mobilizers”, 4.5% borderline PM, 5.8% relative PM and 5% absolute PM. No significant difference was found between patients with NHL versus MM. Sex, age, body weight and previous irradiation therapy did not make any difference. Only the number of cycles of previous chemotherapy (p=0.0034), and previous treatment with melphalan (p=0.0078) had a significant impact. Above all, as long as 2.0 × 10exp6 of CD34+ cells per kg of body weight have been collected, poor mobilization was not associated with inferior engraftment. Disclosures: No relevant conflicts of interest to declare.

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