ALBERT

Description: Acute Myeloid Leukemia (AML) is a rapidly progressing hematologic malignancy characterized by the accumulation of clonal myeloid progenitor cells arrested in their ability to differentiate into mature blood cells. While classic chemotherapy regimens lead to remission in the majority of patients, relapse rates are very high. Relapse and also refractoriness are caused by the hierarchical organization of AML with a minor fraction of leukemic stem cells (LSC) at the apex generating leukemic progeny, which make up the majority of leukemic cells. LSC harbor self-renewal activity, relative quiescence, resistance to apoptosis, and increased drug efflux that likely render them less susceptible to conventional therapies aimed at the bulk proliferative disease. From a clinical perspective, the leukemic stem cell model implies that in order to eradicate the disease and achieve long-term remissions, treatment courses must eliminate the LSC population. To identify putative therapeutic target structures and/or pathways selectively aiming at LSC we performed high-resolution proteomic analyses of LSC and non-LSC populations of primary AML patient samples and healthy control subjects. To identify functional LSCs, we fractionated AML patient samples according to CD34 and CD38 surface expression by fluorescent-activated cell sorting and transplanted the resulting four populations per sample into immunocompromized NSG mice. Leukemic populations, which were able to initiate the AML in mice were labeled LSCs, while engraftment failures were considered non-LSCs. Six AML patients containing both LSC and non-LSC fractions were chosen for proteomic analysis. As healthy controls, CD34+CD38- hematopoietic stem and progenitor cells (HSPC) populations obtained from elderly patients without hematologic conditions undergoing hip replacement surgery were analyzed. We performed in-depth quantitative multiplex proteomic analysis employing tandem mass tag labeling and high-resolution mass spectrometry and identified more than 7,200 proteins. Our analysis revealed between 1,097 and 1,937 differentially expressed proteins when comparing LSC with non-LSC populations for each AML sample. Gene set enrichment analyses (GSEA) showed a significant enrichment for DNA replication, cell cycle, ribosome biogenesis, and protein translation in non-LSC populations, in agreement with a more quiescent state of LSC. Next to Branched Chain Amino Acid degradation, which we have recently shown to control the activity of alpha-ketoglutarate dependent dioxygenases such as TET2 and EGLN1 in AML stem cells (Raffel et al., (2017). Nature 551(7680), 384-388) other metabolic processes including lipid metabolism and oxidative phosphorylation were enriched in LSC. When compared to healthy HSPCs, especially oxidative phosphorylation, RNA processing and cell adhesion were over-represented in LSC. We will present proteins differentially expressed and pathways enriched in LSCs versus non-LSCs. These may help to understand the mechanism of LSC self-renewal and function and simultaneously represent putative novel targets to eliminate leukemic stem cells in clinical settings. Disclosures No relevant conflicts of interest to declare.

Description: Abstract 1585 Recent studies utilising surrogate leukaemic stem cell (LSC) assays have suggested that LSCs in acute lymphoblastic leukaemias (ALLs) might be neither rare, nor phenotypically or functionally distinct. However, studies of candidate LSCs in surrogate assays might not recapitulate the full leukaemic potential of candidate LSCs in patients, and in particular their responsiveness and resistance to therapeutic targeting. Therefore, we have investigated the identity, molecular and functional properties, and persistence of different subsets of candidate LSCs in childhood ALL, at diagnosis and during the course of clinical and molecular remissions in response to chemotherapy, and their relationship to subsequent relapses. First, we investigated 6 patients diagnosed with “good prognosis” TEL-AML1+ ALL, and at diagnosis we found TEL-AML1+ leukaemic cells within the immature B cell progenitor compartment (proB: 34+38+19+), mature B-cells (34-19+), as well as in a population expressing an aberrant combination of stem cell (34+38-/lo) and B-cell (19+) cell surface markers. These stem/B (34+38-/lo19+) cells were all TEL-AML1+ and not present in age-matched normal bone marrow controls. In contrast, haematopoietic stem cells (HSC: 34+38-19-) were not part of the TEL-AML1+ leukaemic clone in any of the patients. 15 days into chemotherapy, all TEL-AML1+ mature B-cells were eliminated in all patients, and this was followed by a clearance of leukaemic proB cells by day 28 of treatment. In striking contrast, leukaemic stem/B cells were still detectable at day 28, but in all TEL-AML1 patients, at later stages all leukaemic cells including the stem/B cells were undetectable, and at the same time these patients went into complete remission with less than 1 leukaemic cell in 10e4 cells detectable. A similar pattern was observed in a case of “high risk” BCR-ABL+ ALL: BCR-ABL+ proB and B-cells were efficiently eliminated by day 90 of the course of chemotherapy, and up to 180 days into the treatment only 34+38-/lo19+ stem/B cells remained part of the BCR-ABL+ clone. In agreement with the persistence of BCR-ABL+ 34+38-/lo19+ stem/B cells, this patient relapsed 17 months after the initiation of chemotherapy. In order to understand the underlying mechanisms of the observed functional and therapeutic heterogeneity seen in leukaemic subpopulations, we performed comparative gene-expression analysis of diagnostic leukaemic stem/B and proB cells of TEL-AML1+ patients. This analysis revealed a differential gene expression pattern between leukaemic stem/B and proB cells, with positive regulators of cell cycle being the most distinctly up regulated genes in leukaemic proB cells. In agreement with this, cell cycle analysis of 3 diagnostic TEL-AML1+ cases also showed proB cells to be more actively cycling compared to the more quiescent state of the leukaemic stem/B compartment (proB: G0 42%; G1 40%; S,G2,M 18% vs. stem/B: G0 81%; G1 18%; S,G2,M 1%), providing a potential mechanistic basis for the relative therapy resistance of ALL stem/B cells. Taken together the present studies suggest that quiescent 34+38-/lo19+ stem/B cells are selectively resistant to chemotherapy, and most likely the origin of relapses when these occur in childhood ALL. Disclosures: No relevant conflicts of interest to declare.

Description: Acute Myeloid Leukemia (AML) is a heterogeneous group of myeloid malignancies. The classification of AML subtypes is based on recurrent genetic abnormalities and typical histopathological features, which impact on the patient’s prognosis. AML is also the model disease for the cancer stem cell model with leukemia stem cells (LSCs) residing at the top of a hierarchical organization. LSCs have self-renewal activity and generate leukemic progeny, which make up the majority of leukemic cells. Because LSCs can be quiescent and reside in specific niches in the bone marrow, rendering them resistant to conventional chemotherapy approaches, they are considered the source of relapse. Hence, further strategies to eradicate LSCs are pivotal to improve patient outcomes of this dismal disease. LSCs present within cell populations can be detected by their capacity to re-initiate the leukemia after xenotransplantation into immuno-compromised mice. However, using current methods, it is neither possible to prospectively isolate pure functional LSCs nor to distinguish them reliably from normal hematopoietic stem cells (HSCs). To define functional LSCs we FACS-sorted primary patient samples of different AML subtypes according to surface-expression of CD34 and CD38 and transplanted each of the resulting four cell populations into conditioned NSG recipients. Several AML samples showed human leukemic engraftment in at least one of the subsets, dissecting LSC-containing and LSC-free subpopulations within the same patient. AML engraftment was mainly observed within the CD34+CD38- fraction, but several cases showed LSC activity also in the CD34+CD38+ fraction or even in the CD34- subsets. As age-matched healthy controls, we collected bone marrow hematopoietic stem and progenitor cells (HSPCs, Lineage -CD34+CD38-) from individuals older than 60 years, undergoing hip replacement surgery. In-depth quantitative multiplex proteomic analysis was performed by employing tandem mass tag (TMT) labeling and high-resolution mass spectrometry. Using this approach, more than 7,000 proteins were quantified from 10 LSC-containing and 8 LSC-free fractions from six individual AML samples of different subtypes. Importantly, our data include many low abundance proteins or others that tend to be difficult to detect by mass spectrometry, such as transcription factors and membrane proteins. We identified up to 1500 differentially expressed proteins between LSC-containing and LSC-free fractions. Most interestingly, differentially expressed proteins also clustered according to AML subtype, indicating subtype-specific proteome differences at the level of AML LSCs. Along the same lines, Gene Ontology and Gene Set Enrichment Analyses showed distinct (in some instances even opposing) differences between AML subtypes. For example, interferon- and integrin-signaling were enriched in LSC-fractions of FLT3-ITD, NPM1-mutated AMLs while reduced in LSC-fractions of FLT3-wt, NPM1-wt AML samples. In summary, our data indicate that also at the level of functionally validated LSC populations, subtype-specific differences in protein expression are remarkably evident. This heterogeneity should be taken into account with respect to the development of targeting strategies for LSCs aiming to improve the clinical outcome of AML patients. Disclosures No relevant conflicts of interest to declare.

Description: Patients with acute myeloid leukaemia (AML) often achieve remission but subsequently die of relapse driven by chemotherapy resistant leukemic stem cells (LSCs). To initiate and maintain cancer, LSCs must also escape immunosurveillance. However, in vivo studies on human LSCs largely disregard lymphocyte mediated anti-tumor immunity due to the use of immunocompromised mice. Here we investigate the immunosurveillance mediated by NKG2D, a danger detector expressed by cytotoxic lymphocytes such as natural killer (NK) cells that recognizes stress-induced ligands (NKG2DL) of the MIC and ULBP protein families on AML cells. Staining of n=175 de novo AML with antibodies against MICA, MICB and ULB2/5/6 or an NKG2D-Fc chimeric protein recognizing pan-NKG2DL expression revealed NKG2DL to heterogeneously express among leukemic cells of the same patient (Fig. 1a). As expected, NKG2DLpos AML cells were efficiently cleared by natural killer (NK) cells, while NKG2DLneg leukemic cells escaped NK cell lysis. Interestingly, these NKG2DLneg AML cells also showed immature morphology, enhanced in vitro clonogenicity (39±47 colonies vs. 1±4, p

Description: Background: Signals provided by the microenvironment can modify and circumvent pathway activities that are therapeutically targeted by drugs. However, a systems-level understanding of how the microenvironment and the genetic and molecular alterations of the tumor interact with each other and contribute to drug resistance is lacking. Methods: To address this unmet need, we established an automated microscopy-based phenotyping platform that uses co-culture conditions mimicking the bone marrow environment. We cultured primary tumor cells from more than 100 leukemia patients (CLL, AML, MCL, T-PLL, HCL) with and without bone marrow stroma cell support in DMEM and 10% human serum and treated each condition with 57 drugs in 3 concentrations. After 72h of incubation, 22 000 images per patient were acquired and processed by our custom made image analysis pipeline. Our set-up allows us to increase sensitivity far beyond simple viability testing, as it reads out additional cell type specific features such as cell morphology, autophagy and cell-cell interactions. Results: Quality assessment revealed that in contrast to mono-culture conditions, assay plate edge effects can be avoided under stable stroma cell co-culture conditions. Correlation of replicated patient samples were comparable between mono- and co-cultures (R2〉0.75). In the absence of their native microenvironment, primary leukemia cells undergo spontaneous apoptosis ex-vivo. Viability at culture start was always 〉90% and dropped to a median of 51% (viability range: 17%-90%) after 72h in mono-cultures. Among CLL samples spontaneous apoptosis was not dependent on either IGHV mutation status or any major cytogenetic risk group. Bone marrow stroma cell co-culture conditions protected tumor cells from spontaneous apoptosis (p=8.2e-6, paired t-test). Patient samples with a high degree of spontaneous apoptosis benefited most from co-culture conditions (p=7.2e-10, Pearson correlation). To model interactions of stroma cell conditions and drug-induced apoptosis we established the following linear model: Viability ~ drug-effect + culture-model + drug-effect:culture-model. While activity of some drugs was significantly altered under co-culture conditions, we could also identify drugs with similar activity in mono- and co-cultures. For instance, the activity of common chemotherapeutics (fludarabine: p=0.002 at 0.6µM, cytarabine: p=0.001 at 1.5µM, ANOVA) or bromodomain inhibitors (I-BET-762: p=5.9e-5 at 4.5µM, JQ1: p=1.5e-8 at 1.5µM, ANOVA) was significantly reduced under co-culture conditions. In contrast, PI3K inhibitors idelalisib and duvelisib had a similar activity in mono-culture and stroma co-culture conditions and might represent a starting point to overcome stroma cell mediated drug resistance. In CLL, we identified IGHV mutation status and trisomy 12 as important determinants of response to kinase inhibitors. We confirmed these findings in stroma cell co-cultures, e.g. a better activity of B-cell receptor inhibitors in trisomy 12 and IGHV unmutated CLL. A systematic comparison of ex-vivo drug response pattern in mono- and co-cultures across 171 drug conditions will be presented. Conclusion: Our results suggest that high throughput co-culture drug testing can be robustly performed and provide an unprecedented understanding of how the stroma cell microenvironment and the genetic make-up of tumor cells contribute to drug resistance and sensitivity. Figure: Over 2 million microscopy images were acquired and analysed to assess drug resistance and sensitivity in a co-culture model of primary leukemia and bone marrow stroma cells. blue= Hoechst33342, green=Calcein AM, red=lysosomal dye NIR Figure 1. Figure 1. Disclosures No relevant conflicts of interest to declare.

Description: Radiotherapy and chemotherapy are known to be mutagenic and may cause cytogenetic aberrations. Secondary neoplasms, which also arise after hematopoietic stem cell transplantation, are frequently associated with cytogenetic aberrations, which might be caused by DNA-alterations due to radio- and chemotherapy. Skin samples were examined for cytogenetic aberrations in patients after allogeneic stem cell transplantation (SCT). In a retrospective study skin samples were taken and examined from 18 patients 23–216 months (range 38 months) after allogeneic SCT. Underlying diagnoses were ALL (3 pts), AML (4 pts), CML (10 pts), MDS (2 pts), NHL (1 pt). In a prospective trial 20 pts were included. Skin biopsies were taken before conditioning, then at 3–6 months as well as 12 months after SCT. Median age before SCT was 32 years (range 16–49). Underlying diagnoses were ALL (4 pts), AML (3 pts), CML (7 pts), other CMPS (2 pts), MDS (3 pts), NHL (1 pt). Samples from all patients were cut into small pieces, placed at the bottom of three culture flasks and fibroblasts cultured according to standard techniques. Cytogenetic analysis was performed with trypsin-Giemsa banding technique. The number of cells with aberrant karyotypes, the number of clonal chromosome abnormalities, the number of breakpoints per aberrant metaphase, the preferential location of breakpoints in chromosome bands and the type of chromosomal lesions (e.g. translocations, deletions, duplications) were determined. In retrospective analysis, all 17 evaluable patients had cytogenetic aberrations, comprising 33–100% cells per patient. Aberrant cells had a median of 4 breakpoints per aberrant metaphase. In the prospective study, evaluable samples from 18 patients before SCT could be analysed and only 2/18 revealed cytogenetic aberrations. 3–6 months after allogeneic SCT, abnormal karyotypes were detected in 12/13 patients. 47–100% of analysed skin cells per patient had aberrant karyotypes (median 77%). Aberrant metaphases had 1–10 chromosomal breakpoints (median 3). 12 months after allogeneic SCT, all 11 evaluable skin samples demonstrated aberrant karyotypes, the number of abnormal cells being between 33–100% (median 63%). 2–5 clones were detected per patient. 1–9 breakpoints were found per aberrant metaphase. All chromosomal aberrations were structural (translocations, deletions, additions, inversion), most of them were balanced translocations; some aberrations were complex. The number of aberrant cells differed significantly before SCT on one hand and both time points after SCT (p

Description: Relative quiescence is a defining characteristic of hematopoietic stem cells. Reasoning that inhibitory tone dominates control of stem cell cycling, we previously showed that mice engineered to be deficient in the cyclin-dependent kinase inhibitor, p21Cip1/Waf1 (p21), have an increased stem cell pool under homeostatic conditions. Since p21 was necessary to maintain stem cell quiescence and its absence sufficient to permit increased murine stem cell cycling, we tested whether reduction of p21 alone in human adult–derived stem cells could affect stem cell proliferation. We demonstrate here that interrupting p21 expression ex vivo resulted in expanded stem cell number and in vivo stem cell function compared with control, manipulated cells. Further, we demonstrate full multilineage reconstitution capability in cells where p21 expression was knocked down. Therefore, lifting the brake on cell proliferation by altering cell cycle checkpoints provides an alternative paradigm for increasing hematopoietic stem cell numbers. This approach may be useful for relative ex vivo human stem cell expansion.