ALBERT

Description: Neoplastic transformation requires the elimination of key tumor suppressors, which may result from E3 ligase-mediated proteasomal degradation. We previously demonstrated a key role for the E3 ubiquitin ligase E6AP in the regulation of promyelocytic leukemia protein (PML) stability and formation of PML nuclear bodies. Here, we report the involvement of the E6AP-PML axis in B-cell lymphoma development. A partial loss of E6AP attenuated Myc-induced B-cell lymphomagenesis. This tumor suppressive action was achieved by the induction of cellular senescence. B-cell lymphomas deficient for E6AP expressed elevated levels of PML and PML-nuclear bodies with a concomitant increase in markers of cellular senescence, including p21, H3K9me3, and p16. Consistently, PML deficiency accelerated the rate of Myc-induced B-cell lymphomagenesis. Importantly, E6AP expression was elevated in ∼ 60% of human Burkitt lymphomas, and down-regulation of E6AP in B-lymphoma cells restored PML expression with a concurrent induction of cellular senescence in these cells. Our findings demonstrate that E6AP-mediated down-regulation of PML-induced senescence is essential for B-cell lymphoma progression. This provides a molecular explanation for the down-regulation of PML observed in non-Hodgkin lymphomas, thereby suggesting a novel therapeutic approach for restoration of tumor suppression in B-cell lymphoma.

Description: Introduction: Leukemic sub-clones which survive chemotherapy during induction period may remain dormant and subsequently lead to relapse in patients with acute myeloid leukemia (AML). No mechanism linking clonal selection to a specific leukemia-associated genetic aberration is known. Clonal selection is a common phenomenon; therefore, any suggested mechanism should be applicable to all kinds of leukemia-specific genetic aberrations. We hypothesized that clonal selection might result from differences in differentiation capacity among sub-clones and that chemo-resistance is not necessarily directly affected by specific mutations. Patients and Methods: We tested this hypothesis in vitro using the kasumi-1 leukemia cell line, which undergoes limited differentiation while growing and is composed of both CD34+ and CD34- sub-populations. Kasumi-1 cells were sorted by fluorescence-activated cell sorting (FACS-Aria IIIu, BD Biosciences) to CD34+CD117+ and CD34-CD117+ sub-populations. Each sub-population was separately exposed to escalating doses of daunorubicin (DNR) and cytarabine (ARA-C) for 66 hours. Cell viability was determined by alamarBlue assay (Bio-Rad, USA); apoptosis was assessed by Annexin-V and propidium iodide (PI) staining. In vivo experiments included BM samples derived from three AML patients, with both NPM1 and FLT3 gene mutations, who received intensive induction and subsequently relapsed. Blast cells were sorted to CD34+CD117+ and CD34-CD117+ sub-populations. Targeted gene sequencing was conducted using Ion Torrent™ Personal Genome Machine® (PGM) System (Life Technologies, USA). Allelic frequency of NPM1 and DNMT3A mutations in CD34+ and CD34-sub-populations was measured. FLT3-ITD was sequenced using theGeneScan method and theallelic ratio (AR) was calculated. Results: Following sorting,cultured kasumi-1 CD34+ cells partly differentiated into CD34- cells and several weeks later, the phenotype became similar to the original kasumi-1 phenotype (a mixture of 34+/- cells). In contrast, the CD34- sub-population maintained its phenotype. Immediately after sorting by CD34 expression, both sub-populations were exposed to increasing doses of DNR or ARA-C. Only the CD34+ sub-population was found to be resistant to ARA-C (at all tested concentrations), as determined by the cell viability assay (fig 1a). The CD34- sub-population exhibited a 4-fold higher apoptosis rate than CD34+ cells after exposure to 0.16 µM ARA-C. DNR (0.01 µM) resulted in 2.5-fold higher apoptosis in the CD34- compared to CD34+ sub-population (fig 1b). The sub-clonal composition of BM blasts obtained from three AML patients at diagnosis and relapse was compared. NPM1 mutation was identified in all patients at diagnosis. FLT3-ITD mutation was identified in one patient at diagnosis and in the other two at relapse. To explore the differentiation capacity of each sub-clone, blasts were sorted to well-defined CD34+ and CD34- subgroups. At diagnosis, most of the leukemic blasts presented with the CD34- phenotype, while at relapse, the CD34+ population grew in all patients. The FLT3-ITD allelic ratio was higher in CD34+ cells. In the first patient, FLT3-ITD was not detected at diagnosis, while at relapse, the wild-type allele of FLT3 was lost and FLT3-ITD was solely present in the CD34+ sub-population. In CD34- cells, the AR was low (0.55) at relapse. In the second patient, the dominant clone at diagnosis was NPM1mutFLT3wtDNMT3Awt and 〉95% cells were CD34-. At relapse, the FLT3-ITD DNMT3Awt clone became dominant and was mostly located within the rising CD34+ population. Another sub-clone, exhibiting both FLT3-ITD and DNMT3A R882C mutation, was identified and was found to reside only within the CD34+ population. Similar findings were also observed in the third patient. We screened additional eight patients presenting with FLT3-ITD and in 4 of them, while both CD34+/- blasts were observed, the FLT3-ITD carrying clones resided only in one sub-population. Conclusions: Chemo-resistance and survival of a specific AML sub-clone correlate with its phenotype and differentiation level. Even genetic aberrations which have no direct interaction with chemotherapy biological effect, may give rise to clonal selection. Sub-clonal differentiation capacity may affect its sensitivity to chemotherapy. Disclosures No relevant conflicts of interest to declare.

Description: Introduction: Activating mutation of FLT3 by internal tandem duplications (ITD) is the most common molecular aberration found in AML. FLT3-ITD mutation induces a proliferation signal and is associated with leukocytosis and poor prognosis. The co-existence of FLT3-ITD with translocation t(5:11) that yields fusion transcript of Nucleoporin98 and the nuclear receptor binding SET-domain protein 1 (NUP98/NSD1) was recently reported to be associated with chemo-resistance. Relapse risk of the majority of FLT3-ITD positive AML patients who are negative to NUP98/NSD1 is increased compared to other AML with normal karyotype, despite similar remission rates. The mechanism of FLT3-ITD contribution to relapse development in patients who achieved remission is unknown. Aims: To explore potential mechanisms that allow relapse in FLT3-ITD positive AML in the presence or absence of NUP98/NSD1 fusion gene. In addition, time sequence of the emergence of both mutations is suggested from a case with relapse. Methods: Leukemic blasts derived from newly diagnosed or relapsed FLT3-ITD positive AML patients were enriched for FLT3-ITD mutated sub-clones by sorting according to CD34 expression. Genomic DNA was extracted from each sorted fraction and FLT3-ITD mutation allele load was quantitatively determined by GeneScane. Total RNA was extracted from FLT3-ITD positive patients and RT-PCR was performed to detect NUP98/NSD1 fusion mRNA. All patients received induction with intensive chemotherapy combination of Daunorubicin and Cytarabine and their outcome was recorded. Chemo-sensitivity assays using Ara-C were conducted on different leukemia cell lines sub-populations divided by their CD34 expression. Results: NUP98/NSD1 was identified in 4 of 19 (21%) FLT3- ITD positive adult AML patients with normal karyotype who had a compatible donor and were considered transplant eligible. Patients harboring both NUP98/NSD1 and FLT3-ITD (75%) had higher rate of induction failure than FLT3-ITD patients without NUP98/NSD1 (40%). To explore the correlation between FLT3-ITD and differentiation capacity, primary AML FLT3-ITD positive blasts were sorted into two distinct sub-populations according to CD34 expression (example is shown in fig. 1). In 14/19 patients DNA from CD34+ and CD34- leukemic blasts was successfully extracted and FLT3- ITD allele load was tested and recorded as the ratio between FLT3 normal and mutated alleles. Of these 14 patients, 3 experienced induction failure and 8 (58%) achieved complete remission but unfortunately eventually relapsed. FLT3-ITD allelic ratio (AR) was equally measured in both CD34+ and CD34- sub-populations in 7 patients (50%) while in 5 patients (35.7%) the mutated allele was restricted to the CD34 positive cells and 4/5 (80%) patients experienced relapse. In two patients (14%) who also expressed NUP98/NSD1, the FLT3-ITD mutated allele was restricted to CD34 negative sub-population. In one patient, NUP98/NSD1 was detected in relapse but not in diagnostic specimen. Cytotoxic assay confirmed that differentiation stage as determined by CD34 expression is fundamental for chemo-sensitivity of leukemic cells regardless of their genetic profile. CD34 positive cell lines as well as CD34+ sub-population of Kasumi-1 cell line were resistant to the Ara-C compare to CD34 negative cell lines or the matched Kasumi-1 CD34- sub-population (fig. 2). Conclusions: FLT3-IDT mutation is a late event during leukemogenesis. We observed NUP98/NSD1 that emerged as a new mutation on relapse in a FLT3-IDT positive patient, assuming this may also be a later event. Our strategy to sort blasts according to their CD34 expression enable us to describe the accumulation of FLT3-ITD sub-clones at the primitive stage that express CD34, therefore, these leukemic sub-clones may be enriched with early leukemic precursors. Such differentiating blockage results a higher portion of cells which survive chemotherapy among FLT3-ITD and hence increases relapse risk as an indirect effect. Inducing differentiation in FLT3-ITD positive AML should be further studied as a therapeutic strategy. Figure 1. Figure 1. Figure 2. Figure 2. Disclosures No relevant conflicts of interest to declare.

Description: Introduction: Human leukocyte antigen (HLA) molecules are membrane-bound transporters carrying peptides from the cytoplasm to the cell surface where they become exposed to T lymphocytes. Soluble HLA molecules (sHLA) bound to their specific peptides are present in human plasma. Patient-based evidence that most of these peptides are derived from cancerous cells may lead to the identification of cancer-related sHLA peptidomes in the disease state. Leukemic cells in the bone marrow (BM) are usually composed of several malignant subclones even within the same patient. These leukemic subclones may vary in their sensitivity to chemotherapy. During the first days of chemotherapy, a dynamic change in the blast mass and subclonal cell composition is observed. We have thus hypothesized that the repertoire and amount of peptides bound to sHLA molecules derived from the BM plasma of acute myeloid leukemia (AML) patients will be changing during induction. Our study has aimed at identifying peptides derived from resistant subclones, assuming that they could represent potential immunogenic targets specific to these leukemic cells. Particular focus has been made on the peptides common among multiple AML patients. Methods: Mononuclear cells and plasma fractions obtained from AML patients at diagnosis (day 1), during induction chemotherapy (day 5) and on day 14 of induction were studied. HLA molecules loaded with peptides were purified using pretreated pan HLA-A, B and C monoclonal antibody W6/32 TopTip column, and sHLA class I molecules with their bound peptides were eluted with tri-fluoracetic acid. The eluted fraction containing sHLA was separated from its peptides on C18 Micro TipColumn with acetonitrile. The peptide fraction was then examined using liquid chromatography coupled with mass spectrometry (LC-MS/MS). Based on the LC-MS analysis, we excluded peptides longer than 15 amino-acids and shorter than 8 amino-acids, peptides known as contaminators and peptides highly expressed by healthy donors. Immunogenicity of the identified candidate peptides was evaluated by ɣ-IFN Elispot assay. Results: The total amount of sHLA-bound peptides in sequential BM plasma samples of 11 patients was significantly decreased at nadir (day 14) compared to diagnosis or day 5 samples (3 and 1.33 fold, respectively). About 17 percent (16.9%) of the peptides identified on day 5 of induction were not present at diagnosis. These peptides originate from the proteins known to be involved in cell proliferation, migration, death, cycle control, metastasis promotion, DNA damage, or mitochondria processes, and could thus reflect chemotherapy-induced effects. On day 14, 75% of identified peptides were similar to those observed on days 1 and 5, representing cancer-derived peptides expressed by leukemic cells that survived the first days of therapy. Analysis of whole peptidome sequences was performed in the plasma specimens obtained from 8 healthy donors (HLA A02) and collected at the above 3 time points from 11 patients (4 HLA A02, 5 HLA A01, 2 HLA A68). Three major peptides were found to be common across the tested patient samples. The peptide originating from GUCY1B3 protein was solely detected in 10 patients and in none of the healthy donors; one peptide derived from ETS1 protein and one peptide cut from STAG1 protein were identified in 7 and 6 patient samples, respectively, as well as in one healthy donor. Immunogenicity of the 3 peptides was examined against T lymphocytes derived from 5 AML patients (HLA 02) in remission. High T cell reactivity was confirmed in the peptide derived from GUCY1B3 protein. Additionally, similarities between membranal HLA (mHLA) and plasma sHLA peptidomes derived from the same patient were observed (87.7%) (n=3, HLA A01). Conclusions: The requirement of a large number of cells, limiting the application of membranal HLA peptidome analysis could be overcome using sHLA-bound peptides from the BM plasma. Plasma sHLA-originating peptides, detectable by a simple blood test, may serve as potential biomarkers of response to treatment or as targets for immunotherapy. Such peptides derived from resistant subclones during induction chemotherapy could be used to stimulate T cell clones in a peptide-restricted HLA manner and thus act as a personalized immunotherapy adjuvant in AML. Disclosures Ofran: Novartis: Other: Served on a Novartis advisory board.